Experimental ciliochoroidal detachment. Effect on intraocular pressure and aqueous humor flow. Ciliochoroidal detachments were produced in rhesus monkeys by injecting glutathione bicarbonate Ringer's solution, autologous serum, or silicone oil into the anterior suprachoroidal space. Silicone oil detachments, like sham operations, caused a transient 2 mm Hg reduction in intraocular pressure. Detachments with glutathione bicarbonate Ringer's solution or serum caused a 6 mm Hg reduction in intraocular pressure, which normalized in three weeks. Aqueous humor flow in serum-detached eyes was normal, indicating that the hypotonia does not result from reduced aqueous humor formation. Blood was present in Schlemm's canal in most eyes with hypotonia, which suggests that hypotonia is not the result of enhanced conventional aqueous humor outflow. Therefore, it is postulated that hypotonia might result from enhanced aqueous humor outflow from the anterior chamber into the suprachoroidal space and out of the eye through the emissarial channels of the sclera (uveoscleral outflow).


A longitudinal study of the effectiveness of self applied 10 per cent stannous fluoride paste for secondary school children. 1. Stannous fluoride toothpaste was used twice yearly for three years in supervised brush-ins by puplis at two Melbourne schools--one coeducational and one boys. 2. Of the original 492 pupils who formed the Test and Control Groups, 347 were available for the final examination. Of these, only the Test Group (167) had participated in four brush-ins. 3. The Test Group consisted of 119 boys and 48 girls, and the Control Group 129 boys and 51 girls. 4. The reduction in caries increment was 16 per cent for DMFS and 19 per cent for DMFT. A reduction of 34 per cent DMFS and 30 per cent DMFT was recorded for the girls. 5. The results for boys in the co-educational State school showed no reduction in caries increment. 6. Time taken for the brush-ins was not more than 20 minutes.


Sideroblastic anaemia and leukaemia in multiple myeloma. Two patients with IgA myeloma and one patient with kappa light chain disease developed sideroblastic anaemia from two to four years after the initial diagnosis. All had previously received radiotherapy and chemotherapy (melphalan and prednisone). In two patients the myeloma was quiescent when the sideroblastic change occurred. Leukaemia occurred in two patients two and seven months respectively after the diagnosis of sideroblastic anaemia was made. In one of them, the myeloma became active again at the same time. The development of sideroblastic anaemia may be a pre-leukaemic event and may be recognised by the appearance of a dimorphic blood film.


Brucella cultures typed by the Who Brucellosis Centre at the Commonwealth Serum Laboratories, Melbourne. Results of the typing of Brucella cultures received at the WHO Brucellosis Centre, CSL, Melbourne, from 1968-1976 are presented. The distribution of the biotypes of cultures recovered throughout Australia is shown on a host and State basis and atypical cultures are discussed. Cultures identified from Australia were Br. abortus, biotypes 1, 2 and 4 and Strain 19; Br. suis, biotype 1 and Br. ovis. Br. melitensis biotypes 1, 2 and 3 were recorded only as exotic human infections from the Mediterranean area and from laboratory infections. Br. abortus, biotype 1 was the most common bovine and human isolate and was found in horses, a goat and a sheep. There was a low incidence of Br. abortus, biotype 2 in cattle and it was found in 1 horse. Br. abortus, biotype 3 was not found in Australia but was submitted from Tanzania. Br. abortus, biotype 4 was rare in cattle and was found once in a horse. Br. abortus, Strain 19 was found occasionally in cattle and once from a test guinea pig. Br. suis, biotype 1 was found in both man and pigs in Queensland and New South Wales whereas biotype 3 was isolated only in New Guinea from pigs and cattle. Br. ovis was submitted from 3 States. Br. canis has not been found in Australia.


Tubal microsurgery--a review.

Tubal microsurgery--a review. Microsurgery is being applied with considerable advantage to tubal reconstruction, particularly in relation to improved pregnancy rates. These results justify the additional costs in equipment and time which are required.


Relationship between genetic variation in thermal stability and electrophoretic mobility of mouse beta-galactosidase. We have examined the relationships between the genetic determinants for mouse beta-galactosidase heat stability and electrophoretic mobility, in order to clarify previous reports indicating that a variation for enzyme heat stability is restricted to kidney while that for electrophoretic mobility is expressed in all tissues. We find that the two phenotypes show concordant strain distributions and cosegregate in genetic crosses. In contrast to a previous report, the thermal stability variation is expressed in all tissues, although the absolute rate of enzyme inactivation is tissue specific. The evidence supports the notion that a single beta-galactosidase structural locus is expressed in all tissues and that the differences in enzyme stability between tissues results from posttranslational enzyme modification.


Demonstration of 6-phosphogluconolactonase activity in Drosophila melanogaster using a null allele of 6-phosphogluconate dehydrogenase. Using a double mutant strain, Pgdn Zwn, we have developed an assay for 6-phosphogluconolactonase activity and have demonstrated its occurrence in adult Drosophila melanogaster.


An anthranilate synthase of the extreme aminase type in a species of blue-green bacteria (algae). Anthranilate synthase of Agmenellum quadruplicatum, a unicellular species of blue-green bacteria, consists of two nonidentical subunits. A 72,000 dalton protein has aminase activity but is incapable of reaction with glutamine (amidotransferase) unless a second protein (18,000 molecular weight) is present. The small subunit was first detected through its ability to complement a partially purified aminase subunit from Bacillus subtilis to produce a hybrid complex capable of amidotransferase function. Conditions for the function of the heterologous complex were less stringent than for the homologous A. quadruplicatum complex. A reducing agent such as dithiothreitol stabilizes the A. quadruplicatum aminase subunit and is obligatory for amidotransferase function. L-Tryptophan feedback inhibits both the aminase and amidotransferase reactions of anthranilate synthase; Ki values of 6 X 10(-8) M for the amidotransferase activity and 2 X 10(-6) M for the aminase activity were obtained. The Km value calculated for ammonia (2.2 mM) was more favorable than the Km value glutamine (13 mM). Likewise, the Vmax of anthranilate synthase was greater with ammonia than with glutamine. Starvation of a tryptophan auxotroph results in a threefold derepression of the aminase subunit, but no corresponding increase in the small 18,000 M subunit occurs. While microbial anthranilate synthase complexes are remarkably similar overall, the relatively good aminase activity of the A. quadruplicatum enzyme may be of physiological significance in nature.


Genetic and biochemical aspects of trehalase from Drosophila melanogaster. Only one molecular form of trehalase (E.C. 3.2.1.28) was detectable in adult Drosophila melanogaster by polyacrylamide gel electrophoresis and isoelectric focusing. An examination of duplication- and deletion-bearing aneuploids exhibiting dosage sensitivity indicated that the enzyme is encoded by a gene, Treh+, located between 55B and 55E of the second chromosome. The tissue-specific soluble and particulate forms of trehalase appear to be manifestations of a single protein encoded by a single gene.


The beta-glucosidase from Botryodiplodia theobromae Pat. Kinetics of enzyme-catalysed hydrolysis of o-nitrophenyl beta-D-glucopyranoside in dioxan/water. 1. The hydrolysis of o-nitrophenyl beta-D-glucopyranoside by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Botryodiplodia theobromae Pat. has been studied in the presence of added dioxan. 2. At donor saturation, the maximum rate of hydrolysis in the presence of up to 50%(v/v) dioxan was pH4.3-4.5 (pH of the buffer system in water) in McIlvaine's buffer. 3. Increasing dioxan concentrations progressively decreased the maximum rate of hydrolysis. 4. The rate of enzyme-catalysed reaction was enhanced at high donor concentrations, but inhibited at low donor concentrations in the presence of glycerol, methanol, fructose of sucrose. 5. The hydrolytic reaction was found to proceed with retention of configuration at the anomeric carbon atom. 6. The kinetics of the enzyme-catalysed process in the presence of added acceptors indicated that water was necessary for the maintenance of the active enzyme conformation apart from its acceptor function.


Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZ beta-galactosidase of Escherichia coli by beta-D-galactopyranosylmethyl-p-nitrophenyltriazene. 1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ beta-galactosidase from Escherichia coli. 2. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. 3. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached beta-D-galactopranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label per enzyme protomer. 4. When the beta-galactopyranosylmethyl cation is generated in the active site of Mg2+-enzyme, it is captured essentially completely by the protein, but in the active site of Mg2+-free enzyme it is only captured with an efficiency of 25%. 5. Labelled enzyme was carboxymethylated and digested with trypsin; acidic hydrolysis of the isolated tryptic peptide, and field-desorption mass spectrometry of the isolated radioactive derivative, showed it to be 2,5-dioxo-3[2-(beta-D-galactopyranosylmethylthio)ethyl]-1,6-trimethylenepiperazine. 6. This is considered to have arisen from labelling of the sulphur atom of a methionine residue adjacent to a proline residue. 7. The complete amino acid sequence of the molecule [Fowler & Zabin (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507-1510] enables the labelled methionine residue to be identified as either Met-421 or Met-500. 8. Sequence data [Fowler, Zabin, Sinnott & Smith (1978) J. Biol. Chem. in the press] show the site of attack to be Met-500.


The necessity of magnesium cation for acid assistance aglycone departure in catalysis by Escherichia coli (lacZ) beta-galactosidase. 1. Removal of Mg2+ from Escherichia coli (lacZ) beta-galactosidase slightly increases the rate of hydrolysis of galactosyl pyridinium salts, but decreases the rate of hydrolysis of arylgalactosides. 2. Fair correlation of logkcat. and log (Km) with the pKa of aglycone is now observed for arglygalactosides, as well as for glycosyl pyridinium salts. 3. Degalactosylation of Mg2+-free enzyme is the rate-limiting step in the hydrolysis of 2,4-dinitrophenyl galactoside. 4. alpha-Deuterium kinetic isotope effects for both sets of substrates are consistent with the rate-determining generation of a glycosyl cation. 5. The pH-independent, SNl hydrolysis of 3,4-dinitrophenyl galactoside has been measured: it is as fast as that of the galactosyl 3-chloropyridinium ion. 6. Hydrolysis of these two substrates by Mg2+-free enzyme proceeds at very similar rates. 7. It is concluded that loss of both types of aglycone takes place, without acid catalysis, from the first ES complex of substrate and apoenzyme. 8. Data for galactosyl azide and thiopicrate confirm that neither charge nor change of atom is the cause of the differences in behavior between aryl galactosides and galactosylpyridinium salts.


Analysis of glycosaminoglycans in urine by using acridine orange fluorescence. The fluorescence technique described here utilizes the electrostatic interaction between the polyanionic sites of glycosaminoglycans and the cationic dye Acridine Orange to analyse urinary glycosaminoglycans from patients suffering from mucopolysaccharidoses. The basis of the titration is the decrease in the fluorescence of free Acridine Orange that occurs when it is bound to polyanions. The effect of the presence of possible interfering materials such as salt, proteins and trace materials in urine was evaluated. This fluorescence technique is technically simple.


Relationship between biotin-binding proteins from chicken plasma and egg yolk. The plasma of laying hens contains a specific biotin-binding protein that appears to be identical with an egg-yolk biotin-binding protein. Both proteins are saturated with biotin and require elevated temperatures to effect the exchange of [14C]biotin for the protein-bound vitamin. The heat-exchange curve in each case is the same and differs sharply from that of avidin, the egg-white biotin-binding protein. On Sephadex G-100 gel filtration, plasma and yolk biotin-binding proteins were each eluted slightly ahead of avidin (mol.wt. 68,000), suggesting that they are of similar molecular weight. Plasma and yolk biotin-binding proteins required the same ionic strength to be eluted from a phosphocellulose ion-exchange column. Both the plasma and yolk biotin-binding proteins had a pI of 5; avidin has a pI of 10. Plasma biotin-binding protein cross-reacted with antiserum to yolk biotin-binding protein and showed a precipitin line of identity with purified yolk biotin-binding protein. It is suggested that biotin-binding plays an important role in mediating the transport of the vitamin from the bloodstream to the developing oocyte.


Substrate specificity of 5'-methylthioadenosine phosphorylase from human prostate. 5'-Methylthioadenosine phosphorylase was purified approx. 340-fold from human prostate by using affinity chromatography by Hg-coupled Sepharose. The enzyme, responsible for the breakdown of 5'-methylthioadenosine into adenine and methylthioribose 1-phosphate, was partially characterized. The apparent Km for 5'-methylthioadenosine is 25 microM. It is activated by thiols and shows an absolute requirement for phosphate ions. New analogues of 5'-methylthioadenosine were prepared and their activity as substrates or inhibitors of the reaction was investigated. The replacement of the 6-amino group of the adenine moiety by a hydroxy group, as well as the replacement of N-7 by a methinic radical, resulted in an almost complete loss of activity. Otherwise the replacement of sulphur by selenium, as well as that of the methyl group by an ethyl one, is compatible with the activity as substrate. The positively charged sulphonium group also prevents catalytic interaction with the enzyme. The inhibitory effect of 5'-methylthiotubercidin (competitive) and 5'-dimethylthioadenosine sulphonium salt (non-competitive) was also demonstrated. The reported results suggest three binding sites between the substrate and the enzyme.


An improved assay technique for uridine diphosphate glucuronosyltransferase activity towards 5-hydroxytryptamine and some properties of the enzyme. A simplified and sensitive procedure for the routine assay of UDP-glucuronosyltransferase activity towards 5-hydroxytryptamine (serotonin) was developed and the reaction product confirmed as the O-glucuronide of this substrate. The assay was used to study some properties of this UDP-glucuronosyltransferase activity. In mouse liver activity was stimulated by membrane-perturbation procedures and by UDP-N-acetylglucosamine. In rat liver it was stimulated by digitonin, but not by diethylnitrosamine. Mouse duodenum, kidney, and lung possessed activity that was less latent than in liver. No activity was found in homogenates of brain. The activity was present in Gunn rat liver, though only one-third of that in Wistar rat liver. Cat liver contained no UDP-glucuronosyltransferase activity towards 5-hydroxytryptamine.


The exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G. Interaction with beta-lactam antibiotics. Kinetically, the three-step model proposed for the interaction between beta-lactam antibiotics and the exocellular DD-carboxypeptidases-transpeptidases of Streptomyces R61 and Actinomadura R39 [Frère, Ghuysen & Iwatsubo (1975) Eur. J. Biochem. 57, 343--357; Fuad, Frère, Ghuysen, Duez & Iwatsubo (1976) Biochem. J. 155, 623--629] applies to the interaction between the much less penicillin-sensitive exocellular DD-carboxypeptidase-endopeptidase of Streptomyces albus G and at least phenoxymethylpenicillin, cephalothin and cephalosporin C. The penicillin resistance of the albus G enzyme is mainly due to the low efficiency with which the first reversible complex formed with the antibiotic (complex EI) undergoes transformation into a second more stable complex EI*. Analysis of the ternary interaction between enzyme, NalphaNepsilon-diacetyl-L-lysyl-D-alanyl-D-alanine (Ac2-L-Lys-D-Ala-D-Ala) and cephalosporin C indicates a non-competitive mechanism.


N-acetyl-beta-d-glucosaminidase in marmoset kidney, serum and urine. N-Acetyl-beta-D-glucosaminidase activities were determined in homogenates of marmoset kidney, in serum and in urine by using the 4-methylumbelliferyl substrate. The enzyme activity was separated into several components by DEAE-cellulose ion-exchange chromatography, starch-gel electrophoresis and isoelectric focusing. The kidney contained two major forms of the enzyme, A and B, which had similar pH optima and Km values. The A-form bound to DEAE-cellulose at pH 6.8, migrated towards the anode on starch-gel electrophoresis and had a pI of 5.0. The B-form did not bind to DEAE-cellulose at pH 6.8, remained near the origin on starch-gel electrophoresis and had a pI of 7.64. The isoenzymes also differed in heat stability, the B-form being the more stable. Serum  contained B-form activity and, in addition, two intermediate forms (I1 and I2) were loosely bound to DEAE-cellulose. The serum A-form activity was less firmly bound to DEAE-cellulose than was the tissue A-form and was designated As. Serum from a pregnant marmoset contained a form which may be analogous to the human P-isoenzyme. Urine contained only a small amount of B-form activity, the majority being present in the A-form. The kidney A- and B-forms both had mol.wts. of 96000--100000 and the activity was predominantly lysosomal. Partial purification of the kidney A isoenzyme was undertaken. Immunoprecipitation studies indicated a relationship between marmoset kidney A-form and human liver A-form activity.


Conformational studies on phospholipase C from Bacillus cereus. The effect of urea on the enzyme. 1. When heated in 8 M-urea, phospholipase C(EC 3.1.4.3) from Bacillus cereus undergoes conformational transitions depending on the temperatures used. These transitions were studied by examining protein fluorescence, iodide quenching of protein fluorescence, u.v. difference spectroscopy, chemical availability of histidine residues in the enzyme, circular dichroism and catalytic activity. 2. Unless simultaneously exposed to elevated temperatures the enzyme appears to be unaffected by 8 M-urea. Removal of the two zinc atoms from the enzyme renders phospholipase C very sensitive to denaturation by 8 M-urea as indicated by fluorescence emission spectra and circular dichroism. 3. Both the native and the zinc-free enzymes are markedly more resistant to irreversible thermal inactivation in the presence of 8 M-urea than in its absence. 4. The response of the enzyme to 8 M-urea and the role of zinc in stabilizing the enzyme are discussed.


The acidic ribosomal phosphoprotein of eukaryotes and its relationship to ribosomal proteins L7 and L12 of Escherichia coli. The acidic ribosomal phosphoprotein, Lgamma, of Krebs II ascites cells was further characterized and compared with proteins L7 and L12 of Escherichia coli. Ribosomal protein Lgamma was selectively removed from 60S sibosomal subunits by 50% ethanol and 1M-NH4Cl, and antibodies raised against protein Lgamma cross-reacted with E. coli protein L12 in immunodiffusion experiments. These and other, previously reported, data support the proposal that the uekaryotic counterpart of E. coli proteins L7 and L12 is phosphorylated.


Long-term chrysotherapy in rheumatoid arthritis. The efficacy of chrysotherapy appears to increase with prolonged administration. In patients who were followed for 3 years after gold therapy was started, remissions occurred in 73% of those who continued chrysotherapy. Only 6% of those who stopped gold injections during the first 18 months subsequently developed "spontaneous" remissions, although initially both groups had comparable moderately severe rheumatoid arthritis of 6 to 8 years' duration. Therefore, in the absence of definite toxicity, maintenance gold injections should be continued indefinitely.


Immunoelectrophoretic assay for synovial fluid C3 with correction for synovial fluid globulin. Synovial fluid C3 was measured by electroimmunoassay. When C3 was expressed as mg/ml, the amounts found in Reiter's disease, psoriatic arthritis, gout, and systemic lupus erythematosus were significantly different from degenerative arthritis. When C3 was corrected for total protein, the levels for rheumatoid arthritis, Reiter's disease, psoriatic arthritis, and systemic lupus were significantly different from degenerative arthritis. When C3 was corrected for synovial fluid globulin, only rheumatoid arthritis and systemic lupus were significantly different from degenerative arthritis. Correction of C3 for globulin increases the difference between rheumatoid arthritis and degenerative arthritis. A proportion of gouty fluids with a relative decrease in C3 is demonstrated. It is argued that correction of C3 for globulin is more meaningful than correction for total protein. While many nonrheumatoid inflammatory effusions demonstrate split products of C3, the majority of fluids from patients with systemic lupus have none.


Original method of showing some constitutive elements of the capillary wall. The ascorbic acid and the 1-epi-3', 4', 5', 5, 7-pentahydroxy-flavan-3-ol have been located in the blood capillary wall by an histochemical method for electron microscopy. The method consists in an intra-arterial administration of vegetal oxydases specific of the compound to put in evidence. Those oxydases fix themselves on their effectors, and the addition of osmium and lead salts gives co-precipitates opaque to electrons.


Physico-chemical interpretation with cells electrophoresis of some antigen -- antibodies reactions in immuno-hematology. The authors report the action of different antibodies of complete or incomplete type on the electrophoretic mobility of red-blood cells. They try to explain the results in terms of physicochemical stability. They make a distinction between three main possible classes concerning the mechanisms of action: a. direct diminution of the Zeta potential due to the antibody which in this case, is agglutinant. b. diminution of electrocinetic potential by "etching" of ionized groups responsible of the charge. To this mechanism, we could link the increase of the ionic force. c. Artificial increase of the dielectric constant by macromolecules.


Extracellular labeling of growing secreted polypeptide chains in Bacillus subtilis with diazoiodosulfanilic acid. Studies of the mechanism of protein secretion in a Gram-positive bacterium, Bacillus subtilis, yielded results very similar to those previously obtained with a Gram-negative organism: nascent chains protruding from protoplasts could be labeled extracellularly; the labeled chains could be recovered on polysomes isolated from the membrane--polysome fraction; they could be released by puromycin, low Mg2+, or chain completion; the completed chains include a known secreted protein (alpha-amylase); and their ribosomes appear to be attached to membrane solely by their nascent chains. The reagent used for extracellular labeling, [1252]diazoiodosulfanilic acid, yielded severalfold more specific labeling of the nascent chains (7--10% of the total cellular labeling and one-fourth to one-third of that of the membrane--polysome fraction) than was obtained earlier with another nonpenetrating reagent.


Complete amino acid sequence of the gamma chain from the major fetal hemoglobin of the pig-tailed macaque, Macaca nemestrina. The complete primary structure of the gamma chain of the major fetal hemoglobin from the pig-tailed macaque, Macaca nemestrina, was obtained by the automated sequencing of fragments produced by three nonenzymatic cleavage reactions. About two-thirds of the sequence was established from the amino terminus of the intact chain and two of the three fragments produced by cleavage at methionyl residues by cyanogen bromide. Acid clevage at the single aspartyl-prolyl linkage and cleavage at tryptophanyl residues in intact chains yielded the two fragments necessary to complete the sequence. This gamma chain, the first from a nonhuman primate to be sequenced, differes from the human G gamma and A gamma chains at but 4 and 5 positions, respectively. All substitutions are conservative and unlikely to produce alterations in the oxygen-binding properties of tetrameric fetal hemoglobin. Consideration of the data presented herein, together with published observations made on portions of other primate gamma chains, provides some insight into the evolutionary history of the multiple gamma-globin chains observed in several anthropoid primates.


Drosophila melanogaster lacks eye-pigment binding proteins. Drosophila melanogaster contains no detectable eye-pigment binding proteins, and the previous evidence for the presence of such protein in the cecropia moth is probably not valid. The major brown pigment of Drosophila (and of Cecropia), dihydroxanthommatin, behaves as a high molecular weight compound in Sephadex chromatography, thus leading to false conclusions.


Mutual homology of mouse immunoglobulin gamma-chain gene sequences. We have assessed the relative homology of mouse immunoglobulin heavy-chain gene sequence using complementary DNAs (cDNAs) synthesized against gamma-chain mRNAs (gamma 1, gamma 2a, gamma 2b, and gamma 3) purified from mouse myelomas. cDNAs complementary to the gamma-chain mRNAs did not cross-hybridize with the mu- and alpha-chain mRNAs, whereas they cross-hybridized to significant extents (22--66%) with the gamma-chain mRNAs of other subclasses. The heterologous hybrids formed, however, melt at 5--13 degrees C lower temperatures as compared to the homologous hybrids, indicating that significant portions of the heterologous hybrids are mismatched. The rates of the cross-hybridization reactions are 2- to 17-fold slower than those of the homologous hybridization reactions. Therefore, the gamma-chain gene sequences of four subclasses share a part of homology with each other, but they are different enough to be measured separately. Cross-hybridization analysis indicate that the gamma 2a and gamma 2b genes are the most closely related, while the gamma 1 and gamma 3 genes are the least related among the gamma subclass genes.


Structural and antigenic studies of an idiotype-bearing murine antibody to the arsonate hapten. Mice of strain A/J responded to repeated intraperitoneal injection of Limulus hemocyanin derivatized with arsanilic acid by producing large quantities (approximately 5 mg/mL of ascites fluid) of IgG antibodies specific for this hapten. The antibodies possessed a characteristic idiotypic determinant and exhibited restricted heterogeneity as demonstrated by isoelectric focusing and primary N-terminal amino acid sequence analysis of isolated light and heavy polypeptide chains. Both light- and heavy-chain sequences were comparable to those of myeloma proteins in lack of heterogeneity. The N terminus of the light chain exhibited V KI sequence and only one position in the first 30 residues showed more than one amino acid. No variability was observed in the first 10 N-terminal residues of the heavy chain. Rabbit antiserum to the idiotype blocked binding of hapten by the purified antibody. The presence of both light- and heavy-chain antigenic determinants was required for optimal formation of the idiotypic determinant.


Comparative in vivo nitrogen-15 nuclear magnetic resonance study of the cell wall components of five Gram-positive bacteria. The proton-decoupled 9.12 MHz 15N NMR spectra of 15N-labeled Bacillus subtilis, Bacillus licheniformis, Staphylococcus auresu, Streptococcus faecalis, and Micrococcus lysodeikticus intact cells, isolated cells walls, and cell wall digests have been examined. The general characteristics of Gram-positive bacteria 15N NMR spectra and described and spectral assignments are provided, which allow in vivo 15N NMR to be applied to a wide range of problems in bacterial cell wall research. The qualitative similarity of the intact cell and cell wall spectra found in each bacteria allowed the 15 N resonances observed in the proton broad-band noise-decoupled 15N NMR spectra of intact cells to be assigned to cell wall components. Each of the five Gram-positive bacteria displayed a unique set of cell wall 15N resonances, which reflected variations in the primary structure of peptidoglycans and the amounts of teichoic acid and teichuronic acid in the cell wall, as well as the dynamic properties of the cell wall polymers. Spectral assignments of cell wall 15 N resonances assigned to teichoic D-Ala residues, teichuronic acid and acetamido groups, and peptidoglycan acetamido, amide, peptide, and free amino groups have been made on the basis of specific isotopic labeling and dilution experiments, comparison of chemical shifts to literature values, determination of pH titration shifts, cell wall fractionation experiments, and comparative analysis of the cell wall lysozyme digest spectra in terms of the known primary sequences of peptide chains. All the peptidoglycan 15N peptide resonances observed in the intact cells and isolated cell walls could be accounted for by residues in the bridge or crossbar regions of the peptide chains, which indicated that only the cross-linking groups had a high degree of motional freedom. Thermal- and pH-induced conformational changes around the cross-linking D-Ala residues were detected in the B. licheniformis cell wall lysozyme digest products. Comparison of the proton broad-band noise-decoupled and gated decoupled intact cell and cell wall 15N spectra indicated that broad-band proton decoupling resulted in nulling of cytoplasmic resonances and enhancement of the cell wall resonances by the 15N [1H5 nuclear Overhauser effect.


Membrane changes during growth of Tetrahymena in the presence of ethanol. After a brief lag period for acclimation, Tetrahymena pyriformis, strain NT-1, is capable of growing in culture medium containing high levels of ethanol. When grown in a medium having 1.6% ethanol, the membrane phospholipid composition was significantly different from that of control cells. The principal changes included a reduction in hexadecenoic acids (16 : 1 and 16 : 2) from 23% to 5% and an increase in linoleic acid (18 : 2) from 14% to 25% Similar but less pronounced changes were observed in cells grown in lower ethanol concentrations. There was also a decrease in 2-aminoethylphosphonolipid in the ethanol-grown cells from 16% of the lipid phosphorus to 6% and a coincident rise in the phosphatidylethanolamine from 39% to 46%. The lipid pattern quickly reverted to normal when ethanol was removed. In order to ascertain the effects of ethanol on membrane physical properties, freeze-fracture electron microsocopy and fluorescence polarization studies were performed. Ethanol, as expected, had a clearly detectable fluidizing influence when present at 1.6%. However, changes induced in the membrane lipids by growth in the ethanol-containing medium led to a further fluidizing effect, whether they were tested in the presence or absence of ethanol. The alterations found here were qualitatively similar but quantitatively much more pronounced than those observed in mammals chronically exposed to ethanol.


Comparative studies on highly metabolically active histone acetylation. Histone acetate is hydrolyzed rapidly in logarithmically dividing hepatoma tissue culture cells (Jackson, V., Shires, A., Chalkley, R. and Granner, D.K. (1975) J. Biol. Chem. 250, 4856--4863). The phenomenon has been analyzed further in hepatoma tissue culture cells at various stages of the cell cycle, in stationary phase, and in the presence of actinomycin D. We also investigated the phenomenon in Tetrahymena pyriformis macronuclei, bovine thymocytes, and human foreskin fibroblasts. The data suggest that this highly metabolically active histone acetylation while altered in mitotic cells, is independent of the overall rate of cell division, and is only slightly sensitive to actinomycin D. Finally, we conclude that the same general phenomenon is found in both cancerous and normal cells and is apparently common to cells from various stages of the evolutionary scale.


Base composition heterogeneity of Euglena gracilis chloroplast DNA. Euglena gracilis chloroplast DNA has an average buoyant density of 1.685 gm/cm3, corresponding to 25 mol% G . C base pairs. To test for base compositional heterogeneity within this 130 kilobase pairs (kbp) genome, previously mapped restriction endonuclease fragments were isolated, and characterized by equilibrium buoyant density centrifugation. The chloroplast DNA can be characterized as containing two major buoyant density components. A segment of 17 kbp, representing 13% of the genome and containing the rRNA genes is 43--44 mol% G . C. The remaining 113 kbp, accounting for 87% of the genome, has an average 20--21 mol% G . C content.


Thyroid purine nucleoside phosphorylase. II. Kinetic model by alternate substrate and inhibition studies. Nucleoside analog inhibition studies have been conducted on thyroidal purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) which catalyzed an ordered bi-bi type mechanism where the first substrate is inorganic phosphate and the last product is ribose 1-phosphate. Heterocyclic- and carbohydrate-modified nucleoside inhibitors demonstrate mixed type inhibition suggesting such analogs show an affinity (Ki) for the free enzyme. A kinetic model is proposed which supports the observed inhibition patterns. These studies together with alternate substrate studies indicate that nucleoside binding requires a functional group capable of hydrogen bonding at the 6-position of the purine ring and that the orientation of the bound substrate may be syn. Proper geometry of the phosphate is dependent upon the 3'-substituent to the orientated below the furanose ring. The 5'-hydroxyl group is required for substrate activity. The proposed rate limiting step of the phosphorylase mechanism is the enzymatic protonation of the 7-N position of the nucleoside.


The cell wall-associated levansucrase of Actinomyces viscosus. Actinomyces viscosus produces both a soluble extracellular levansucrase and a cell wall-associated levansucrase. The enzyme from cell walls was solubilized by lysozyme digestion. The soluble extracellular and cell wall-associated forms of the enzyme were compared and appeared to be identical, based on molecular weight estimations, kinetic parameters, and reactions with antisera. The product of both forms of the enzyme was a high molecular weight, branched levan, as shown by its reactivity with myeloma proteins specific for beta(2 leads to 1) and for beta(2 leads to 6) linkages in fructosans. Although levansucrase remained tightly bound to the levan which it synthesized, the enzyme did not bind to exogeneously added levan. Regarding the potential pathogenicity of the levan product, pure levan, produced using purified levansucrase, did weakly activate complement by the alternative pathway. However, the pure levan did not directly cause bone resorption in an in vitro bone resorption assay.


[Immunochemical study of subtilisins obtained from Bacillus subtilis A-50 and some of its mutants]. Physico-chemical parameters of subtilisins from the original Bacillus subtilis A-50 strain (proteolytic activity, electrophoretic mobility, molecular weight, reactions with specific inhibitors) were similar to those mentioned in the literature for the enzymes of other strains. Immunological experiment has shown, that Bacillus subtilis A-50 subtilisins with various electrophoretic mobility do not differ in their antigenic properties. Enzymes with high electrophoretic mobility from mutant strains were similar to I--III subtilisin fractions from Bac. subtilis A-50 in the antigenic characteristics. However, the antigenic heterogeneity was observed in I, II and III enzyme fractions of some mutant strains. Subtilisins studied appear to form the isoenzyme system.


[Isolation and characterization of lipase from Pseudomonas fluorescens 533-5b]. The enzyme was isolated from the culture fluid of Pseudomonas fluorescens 533-5b and purified by precipitation with (NH4)SO4 and acetone and by gel filtration through Sephadex G-200. The enzyme was found homogeneous during polyacrylamide gel disc electrophoresis. The effects of metal ions, inhibitors, bile salts, temperature, pH and the substrate specificity of the enzyme were studied. It was shown that the enzyme from Ps. fluorescens 533-5b has a broad specificity. It can use as substrates many vegetable oils (olive, soybean, castor, sunflower, corn, mustard, linseed). In addition, the enzyme is capable to hydrolyze synthetic triglycerides consisting of short-chained saturated fatty acids (butyric and caproic) and solid triglycerides containing saturated fatty acids with long carbon chains (myristic, lauric, stearic). It is assumed that the enzyme is a glycoprotein; its molecular weight (320,000) and the amino acid composition were determined.


[Inner and outer immobilization of chloroplasts]. The effects of glutaric aldehyde on pea leave chloroplasts and their inactivation kinetics were studied. Optimization of the chloroplasts fixation by glutaric aldehyde resulted in a 5-fold increase of stability of the chloroplasts. Immobilization of the chloroplasts in agar-agar gels was performed; the ability of chloroplasts for photooxidation of H2O was thereby retained. Immobilization did not actually affect the stability of chloroplasts. The inactivation kinetics of fixed and immobilized chloroplasts are in good agreement with the previously described model for inactivation of native chloroplasts.


Radiosensitivity of bacteriophages of aerobic spore-forming microorganisms. Under conditions of the direct action of radiation on seven original phages of aerobic spore-forming bacteria and four standard phages of E. coli, it was established that they possessed different sensitivity to gamma radiation and were distributed into four groups. The phages of E. coli of the T series, especially T2 phage, proved the most sensitive. The phages of aerobic spore-forming bacilli were characterized by greater resistance to the action of gamma radiation, which is evidently associated with the lower content of DNA of the phages of Bacillus in comparison with T phages.


Neurofibrosarcoma of the duodenum in von Recklinghausen's disease. The clinical features and surgical management of 2 patients with neurofibrosacroma of the duodenum in association with von Recklinghausen's disease are described. The tumours were excised and the resulting duodenal defects closed satisfactorily using the jejunal serosal patch technique. Three such neurofibrosarcomas reported previously in English are reviewed.


Fats and atheroma: an inquest.

Fats and atheroma: an inquest. All well-controlled trials of cholesterol-reducing diets and drugs have failed to reduce coronary (CHD) mortality and morbidity. Nevertheless, commercial, professional, and even government-sponsored propaganda continues. Experimentally some vegetable oils and hardened fats can be more damaging to arteries than butter. There are other hazards to heart muscle from vegetable oils. Israelis consume a high polyunsaturated fat diet equal to that recommended for prevention of CHD in USA but their CHD incidence is very high. Urban Bedouins are also affected. The primary clofibrate prevention trial underlines unacceptable risks which could apply also to diets. Official medical endorsement of these cholesterol reducing measures should be withdrawn.


Early postoperative feeding with elemental diet. The value of early postoperative feeding with an elemental diet was assessed in 30 patients after major gastrointestinal operations. The patients were allocated at random to conventional treatment (control group) or feeding with the elemental diet (ED group). The clinical and metabolic course of the 15 patients in the ED group was significantly better than that of the controls. Patients in the ED group lost less weight and had a shorter stay in hospital. Negative nitrogen balance was more pronounced in the control group throughout the seven postoperative days. Energy intake was higher in the ED group. Provided elemental feeding is used with caution, it may be given from the first postoperative day. Patients do better metabolically and require shorter stays in hospital.


Malnutrition in infants receiving cult diets: a form of child abuse. Severe nutritional disorders, including kwashiorkor, marasmus, and rickets, were seen in four children and were due to parental food faddism, which should perhaps be regarded as a form of child abuse. All disorders were corrected with more normal diets and vitamin supplements. In view of the potentially serious consequences of restricted diets being fed to children, families at risk should be identified and acceptable nutritional advice given. When children are found to be suffering from undernutrition due to parental food faddism a court order will normally be a necessary step in providing adequate treatment and supervision.


Origin of the fornix system in the squirrel monkey. Wit the use of the horseradish peroxidase retrograde axonal transport technique this experiment attempted to identify the cell bodies of origin whose axons supply the septal area, mammillary bodies and the anterior thalamic complex in the squirrel monkey. It was noted that axons which innervate the anterior thalamic complex arise predominantly from the deepest cell layer of both the retrosplenial cortex and adjoining portions of subicular cortex. Hippocampal afferents to the mammillary bodies arise principally from all cell layers of the subiculum proper and to a lesser extent from the presubiculum. Afferents which innervate the septal area originate from all cell fields of the cornu Ammonis and adjacent prosubicular cortex. It was further noted that axons which are distributed to caudal levels of the septum probably arise from both the cornu Ammonis and adjacent prosubiculum, whereas axons which innervate more rostral levels of septum originate exclusively from the prosubicular cortex.


Multiple sclerosis: immunochemical studies on the demyelinating serum factor. Sera from patients with multiple sclerosis (MS) frequently produce demyelination of central nervous system tissue cultures. The nature of the factors responsible for demyelination is not as yet clearly established. However, several authors previously reported, in in vivo and in vitro models, demyelinating activity in IgG fractions isolated from sera and cerebrospinal fluid of MS patients14,42,48. We found the demyelinating activity of MS sera to be extremely labile to conventional biochemical treatments. Therefore, we isolated IgG from MS sera by absorption with staphylococcal protein A. Protein A binds specifically IgG1. IgG2 and IgG4. With this method we were able to remove most of the IgG, leaving only a small percentage, most probably IgG3, in the sera. Isolated IgG fractions from several sera of MS patients in the presence of human complement actively demyelinated central nervous system tissue cultures. Although a small, but significant, decrease in demyelinating activity could be observed in most of the sera absorbed in most of the sera absorbed with protein A, the majority of the demyelinating activity could not be removed by this treatment. From these studies, it appears that at least the majority of demyelinating activity in MS sera is not associated with IgG1, IgG2 or IgG4. If IgG is responsible for demyelination, IgG3 will most likely be the active factor.


The pars tuberalis of the rhesus monkey secretes luteinizing hormone. The presence of luteinizing hormone within the pars tuberalis of the adenohypophysis and its secretion into pituitary stalk vessels were investigated in adult rhesus monkeys. Portal blood was collected in 9 monkeys, after section of the pituitary stalk. In 22 out of 26 samples, portal to peripheral LH ratios, as measured by radioimmunoassay, ranged from 2 to 48. Portal LH levels were highest in 3 animals studied at the time of the midcycle surge. No differences between portal and peripheral growth hormone (GH) and prolactin levels were observed. Immunocytochemical studies in 4 normal and 3 ovariectomized female monkeys indicated that LH, but not GH, prolactin or thyroid stimulating hormone were present within the pars tuberalis. Cells containing these hormones were identified within the pars distalis. These results indicate that the pars tuberalis forms and secretes LH via the hypophyseal portal circulation.


Thermostable antigens from the bone marrow of several animal species. Studies on bone marrow extracts from ten animal species have revealed the presence of thermostable antigens. Similar antigens were found in other organs as shown by immunodiffusion and cross-reactivity was demonstrated between the thermostable proteins from horse and dog and between cow and sheep proteins. Using an immunoenzymatic reaction with glucose oxidase as the marker, the thermostable antigens were localized in the blood and bone marrow polymorphonuclear leukocytes and the myeloid cell line.


Therapeutic effect of topical antibiotic on untreated eye in experimental keratitis. We studied the systemic absorption ot topical tobramycin and amikacin in experimental Pseudomonas keratitis in guinea pigs. After giving two drops of tobramycin 40 mg/ml every 30 minutes for 24 hours to both infected eyes (the corneal epithelium having removed) the mean serum concentration was 1.5 mcg/ml. Treatment of one of the infected eyes with the same strength of tobramycin or amikacin drops did not alter the number of viable bacteria in contralateral eyes treated with saline. Tobramycin 400 mg/ml or amikacin 250 mg/ml however, decreased the number of viable bacteria in the contralateral eyes. We conclude that the therapeutic effect on the contralateral eye was the result of systemic absorption.


Anomalies of the human hyaloid system.

Anomalies of the human hyaloid system. We describe two asymptomatic cases of persistent anomalies of the human hyaloid system. Fluorescein angiography of a cyst of the hyaloid system shows leakage from the hyaloid vessels.


Isolation of meningococci in meningococcal endophthalmitis. We describe a case of bilateral hypopyon in a 7-year-old African male receiving systemic antibiotic therapy for meningococcal meningitis. Aqueous from paracentesis of the left eye contained intra- and extra-cellular Gram negative diplococci. We believe this is the first report of isolation of the organism since the advent of antibiotic treatment.


Lingeous conjunctivitis with tracheal obstruction. A case report, with light and electron microscopy findings. A white male infant of 1 year had unilateral membranous conjunctivitis and severe laryngotracheobronchitis which required tracheostomy. Cultures from eye and throat swabs and of fluid suctioned through the tracheostomy grew many organisms, including H. influenzae, adenovirus type 3, and Candida species, but he had no specific immunologic disturbance. Ligneous conjunctivitis was diagnosed. The infant's general condition responded slowly to intensive therapy but the membrane continued to slough off the regrow. The excised membrane contained massive subepithelial deposits of eosinophilic material and a moderately vascular chronic inflammatory-cell infiltrate with numerous mast cells in the perivascular spaces and the hyaline membrane. The conjunctivitis cleared when treated with topical sodium cromoglycate (Intal), a known inhibitor of mediator release from mast-cell granules. The success of Intal therapy in this case supports the theory that mast cells are involved in the pathogenesis of ligneous conjunctivitis.


Production and characterization of two hemolysins of Bacillus cereus. Bacillus cereus strain B-48 produced two hemolysins with molecular weights of 52,000 (H-I) and 31,000 (H-II). A mutant was isolated that produced only H-II but was identical with the wild type in all other respects. We exploited this mutant to produce H-II for study that was free of contamination by H-I. By manipulation of media composition, we produced H-I in the absence of H-II. The hemolysins were precipitated differently by ammonium sulfate, and both exhibited the Arrhenius effect when heated. Both hemolysins attached rapidly to erythrocytes; however, lysis by H-I was immediate, while lysis by H-II followed after a lag. Hemolysis by H-I and H-II increased in rate with increasing temperature and was absent at 0 degrees C. Only H-I was inhibited by cholesterol. The hemolysins of B. cereus appeared similar to the hemolysins of B. thuringiensis. H-I probably is identical with cereolysin.


Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae specific antibodies. An indirect enzyme-linked immunosorbent assay (ELISA) using rigid polystyrene microtiter plates was adapted to detect specific gonococcal antibodies against outer membrane-complex antigens extracted from Neisseria gonorrhoeae. The concentration of antigen to obtain maximum coating of the well was 10 micrograms protein per millilitre. The optimal binding of the primary antibody and enzyme-conjugated antimmunoglobulin was achieved after 1 h at 37 degrees C. Under these conditions using gonococcal antisera, no cross-reactivity was observed with outer membrane antigens extracted from Neisseria meningitidis serogroups B, C, X, Y, and W135. Neisseria meningitidis serogroup A demonstrated low levels of cross-reactivity. All the non-pathogenic Neisseria spp. tested were negative (absorbance value at 400 nm/30 min less than 0.15). The reaction of immune serum against outer membrane complex absorbed to the microwells was completely inhibited with soluble-specific antigen but not with purified N. gonorrhoeae lipopolysaccharide. Quantitative inhibition permitted the measurement of low levels of antigen (0.5 microgram/ml). The detection of N. gonorrhoeae antibody with ELISA is specific and highly sensitive.


Thermal analysis of the spores of Bacillus cereus with special reference to heat activation. The heat activation of bacterial spores was studied by means of differential thermal analysis in the temperature range 30-110 degrees C using the spores of Bacillus cereus. The thermogram showed three endothermic peaks at 56, 95, and 103 degrees C with one exothermic peak at 105 degrees C during the heating process. The spore coat separated from the native spores also showed a peak at 56 degrees C on its heating thermogram. The peak at 56 degrees C was reversible for both native spores and the spore coat. It was suggested that this peak at 56 degrees C might be related to the heat-activation process that takes place in the spore-coat region. It seems that the peak is due to the denaturation or the structural change of the spore-coat protein that might facilitate either the permeation of germination stimulators or the release of some germination inhibitor into or out of the spores.


Survival of microbial films in the microwave oven. Air-dried films of Escherichia coli, Saccharomyces cerevisiae, and Bacillus subtilis spores on membrane filters, exposed to 10 min full power (650 W, 2450 MHz) irradiation in a Toshiba model ER776BT microwave oven, showed a 5-, 2-, and 0-log reduction of viable organisms respectively. Suspensions of cells or spores in phosphate buffer treated under similar conditions showed 8 logs of killing within 30 s (S. cerevisiae), 45 s(E. coli), and by 10 min (B. subtilis spores) of exposure.


Clinical efficacy of valproic acid in relation to plasma levels. Valproic acid is a new antiepileptic drug recently introduced in the United States for the treatment of absence seizures. In this study on patients with absence and other seizure types, the majority of patients achieved optimal control within four weeks of therapy. No patient responded to valproic acid who did not show an initial clinical response by four weeks of active therapy. Optimal response was generally achieved when plasma levels were greater than 55 microgram/ml. Excellent clinical response was observed in the treatment of absence and myoclonic seizures. Twenty-two patients continued in a long term study have maintained the same degree of seizure control as observed at the time of optimal control.


Therapeutic experience with netilmicin.

Therapeutic experience with netilmicin. Netilmicin, a new aminoglycoside antibiotic, was used to treat 19 patients with urinary tract infection and 5 with systemic infection. The causal organisms were Escherichia coli (in 2), Klebsiella pneumoniae (in 4), Serratia marcescens (in 12) and Pseudomonas aeruginosa (in 7); 1 patient was infected with two of these organisms. All the isolates of causal organisms except one of Serratia were initially sensitive to netilmicin but many were resistant to other aminoglycosides. Sixteen of the urinary tract infections responded to netilmicin therapy, although relapse occurred in three patients. Two of the three patients with musculoskeletal infection responded to combined therapy with surgery and netilmicin; the other patient responded to the same regimen but with carbenicillin added. Netilmicin cured pneumonia in one patient but failed in the other patient with pneumonia, who had leukemia. Superinfection occurred in five patients with urinary tract infection. Adverse reactions to netilmicin were minor. Netilmicin may prove to be a useful agent, particularly for infections due to multiresistant Klebsiella or Serratia, or when prolonged aminoglycoside therapy is required.


Tumors of the pineal and suprasellar region: Childrens Cancer Study Group treatment results 1960--1975: a report from Childrens Cancer Study Group. Tumors of the pineal and suprasellar region form a rare and interesting group of lesions with germinomas accounting for over 50% of all lesions in this anatomic region. The Brain Tumor Committee of Childrens Cancer Study Group (CCSG) recently surveyed all CCSG member institutions to determine treatment parameters and assess the techniques. A total of 140 patients were seen during the period from 1960 to 1975; 118 patients were evaluable, having adequate treatment records. One hundred and one patients were less than 30 years of age with a 2:1 male predominance. Thirty-six of the 57 biopsied patients (63%) were found to have germinomas. The survival of patients in the germinoma group (72%) was comparable to that of the patients without biopsy (71%). The overall survival rate for all patients (biopsied and unbiopsied) was 65% with follow-up times ranging from 2 to 15 years. Nine patients developed spinal cord metastases (8%), two of whom also had simultaneous primary recurrence; none of these patients had received adjunctive spinal irradiation.


Adenocarcinoma of the prostate in perspective. Adenocarcinoma of the prostate is responsible for one of every nine deaths from cancer in Canada. In this review epidemiologic factors are considered and current staging systems are outlined. The American Urological System is recommended for staging because of its ability to reflect changes in the understanding of the biologic behaviour of this neoplasm. The adoption of a quantitative grading scheme is suggested to complement the information obtained from the staging assessment. The routes of spread of this disease, along with the procedures used to assess metastatic involvement, are described. Immunologic methods for the analysis of prostatic acid phosphatase have been shown to be superior to the enzymatic methods previously used, and the role of the new techniques is discussed. Emphasis is placed on radiotherapy and endocrine therapy for the treatment of this neoplasm, and the concept of withholding endocrine therapy until symptoms appear is discussed. Potential future developments in this field are considered.


Comparison of metabolism-mediated binding to DNA of 7-hydroxymethyl-12-methylbenz(a)anthracene and 7, 12-dimethylbenz(a)anthracene. Comparison of the binding to DNA of 7-hydroxymethyl-12-methylbenza(a)anthracene and 7, 12-dimethylbenz(a)anthracene (DMBA) catalyzed by mouse embryo cells in culture or by rat liver microsomes indicates that the products formed are different for the two hydrocarbons. Thus, the hydroxy compound is not an intermediate in the binding of DMBA to DNA in these systems. Binding of the hydroxy compound to DNA in mouse  embryo cells is less efficient than for DMBA and is inhibited by 1,1,1-trichloropropylene 2,3-oxide, an inhibitor of epoxide hydrase. This and the fluorescence spectra of the hydroxy compound-DNA adducts indicate that the hydroxy compound is activated for DNA binding through the formation of a diol-epoxide in the 1,2,3,4-ring. As previously found for DMBA, this is consistent with the activation of this compound through a bay-region diol-epoxide.


Systemic two-stage carcinogenesis in the epithelium of the forestomach of mice using 7,12-dimethylbenz(a)anthracene as initiator and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate as promoter. In a modified two-stage carcinogenesis experiment, the effectiveness of the initiator 7,12-dimethylbenz(a)anthracene (DMBA) and the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the epithelium of the forestomach of the mouse has been investigated. Fifty mice were treated intragastrically with a single dose of DMBA (50 mg/kg body weight), followed by repeated intragastric administration of TPA (10 mg/kg body weight) over a period of 35 weeks. In comparison with the corresponding control groups (no treatment, DMBA initiation only, and TPA treatment only), the initiated and promoted group clearly showed the highest tumor incidence in the target organ (45 tumor-bearing animals of 50 animals). No tumors of the forestomach were found in the untreated control group and the TPA-treated group, whereas in the DMBA-initiated group, ten animals had developed tumors of the forestomach. In addition to the mouse skin model for two-stage carcinogenesis, the mouse forestomach appears to respond to DMBA initiation-TPA promotion. This organ provides an additional tissue with which to investigate tumor promotion and further to ascertain specific parameters of the promotion step.


Animal models for the comparative assessment of neurotoxicity following repeated administration of vinca alkaloids. Neurotoxicity is the major side effect occurring during the clinical use of vincristine (VCR). Animal models predictive of potential neurotoxicity would be very useful in the preclinical development of new vinca compounds. To simulate conditions in which neurotoxicity is produced during the clinical use of VCR, experimental animals (except the guinea pig) were given the test compounds by the iv route over a prolonged time period. Doses were selected based on the production of leukopenia. Vindesine (VDS), a chemically modified vinblastine (VBL) product, was compared with VCR and VBL in animal studies. Definite neurotoxic manifestations developed when VCR was given to chickens, cats, and monkeys. The administration of VDS or VBL did not produce neurotoxic signs in these species. The mouse, rat, dog, and guinea pig were not found to be useful models. Thus, it would appear the chicken, cat, and monkey would be appropriate animal models for the preclinical testing of new vinca compounds.


A rapidly polymerizing polyurethane for transcatheter embolization. A polyurethane prepolymer was evaluated as a transcatheter embolizing agent. Low viscosity permits injection through small catheters. Polymerization is initated on contact with blood and completed within 10 seconds. Downstream propagation is better then that of cyanoacrylate and comparable to that of silicone rubber. No systemic toxicity was observed in acute animal experiments. However, dissolution of arterial walls and extravasation out of the vascular bed of chronically embolized organs suggest significant local tissue toxicity and make further evaluation necessary before clinical testing.


Nucleolar changes in response to dietary protein malnutrition in the neurons of the motor cerebral cortex and cerebellum of squirrel moneky Saimiri sciureus. Nucleolo-cytoplasmic relationships have been studied in healthy squirrel monkeys and those subjected to a known degree of protein malnutrition. In the latter group, thirty-two pregnant animals starting from 35 days of gestation and 24 young adult animals were given a diet containing 7.5% and 2.0% protein content, respectively, compared to a diet with 25% protein for the controls. The motor cortex and the cerebellum removed from neonates as well as young adult animals sacrificed after 9, 11, 13 and 15 weeks of feeding schedules were investigated. Four animals after 15 weeks of dietary protein deprivation were rehabilitated with a balanced diet over a year's period. Formaldehyde-fixed as well as fresh frozen tissues were used for the histological study and to employ histochemical techniques for the demonstration of lipids, carbohydrates, nucleic acids and enzymes of various metabolic cycles. As a result of protein malnutrition, the nucleolus in a majority of the neurons from the motor cortex and the Purkinje cells of the cerebellum undergoes a series of morphological and cytochemical transformations in response to cytoplasmic changes related to impaired protein metabolism. The greater the level of protein deprivation, the greater is the degree of cytoplasmic chromatolysis and more pronounced are the nucleolar transformation in terms of enlarged size, secretory activity and transfer of nucleolar material in the cytoplasm. The nucleolar buds located close to the periphery of the nuclear membrane and the nucleolar material in the cytoplasm show identical cytochemical nature except for the presence of DNA in the former. It appears that during migration through the nuclear membrane the nucleolar material loses its DNA component and only aggregates of ribosomes and protein pass into cytoplasm, which aid in the synthesis of specific proteins lost as a result of catabolic processes initiated by protein malnutrition. Most of the observed changes in the adult squirrel monkeys are reversed when they are rehabilitated with a balanced diet.


Epithelial differentiation and taste buds in the soft palate of the monkey, Macaca irus. A combination of light, transmission and scanning electron microscopy was employed to demonstrate the occurrence, arrangement and structure of taste buds in the oral mucosa of the soft palate of monkeys (Macaca irus). Taste buds are found in aggregates confined to 0.15 to 0.3 mm wide, round islands of keratinizing epithelium embedded in the normally non-keratinizing integument. Topography, configuration and structure of these epithelial islands and their taste buds are described, and the question of a developmental and functional interrelationship between epithelial differentiation and properties, and taste bud function is discussed.


[Reversal of human ophthalmic vein blood flow : selective cooling of the brain]. Direction of blood flow in angularis oculi veins was recorded in humans. In mild hypothermia, blood flow was weak and directed from brain to face. In hyperthermia, however, blood flowed rapidly in the opposite direction, angularis oculi vein collecting cool facial blood and supplying cavernous sinus. Therefore selective cooling of human brain is possible.


[Inhibition of post-hemicastration hypertrophy of the remaining testis by testosterone or aqueous testicular extract treatments in the young rat]. In the 10 day old hemicastrated Rat daily injections of testosterone (total doses of 0.04 to 4 mg/Rat) or of proteinaceous extract of Rat testis (4 mg/Rat) inhibit compensatory hypertrophy of the remaining testis. Both testosterone and testicular proteinaceous extract could be involved in the growing processes of the testis, locally or by means of modification of gonadotropin secretion. Results have been analysed by covariance analysis to eliminate the influence of body weight variations.


[Purfication and molecular weight of an RNA-dependant RNA polymerase from Brassicae oleracea var. Botrytis]. An RNA-dependent RNA polymerase has been completely purified from Cauliflower inflorescences. Analysis of the purified enzyme on SDS-polyacrylamide gels showed one polypeptide chain with a molecular weight of 140,000 dalton. The enzyme is monomeric in its native state. The in vitro activity was completely dependent on added RNA, the most efficient templates being poly (U), poly (U, C), poly (I) and viral RNA.


[Experimental cholera in monkeys].

[Experimental cholera in monkeys]. The immune response to Vibrio cholerae was studied in baboons, rhesus monkeys and marmosets inoculated by the oral, intraintestinal or intravenous route with various strains of V. cholerae. Sera were collected from all animals on days 2, 6, 10, 17, 24, 32, 45 and 60 after infection. Serum protein, immunoglobulins (IgA, IgG, IgM), GOT, and alkaline phosphatases were determined. The results showed that baboons and rhesus monkeys were not susceptible to cholera infection under these experimental conditions. However, we were able to induce a lethal cholera infection in cotton-topped marmosets. In baboons and rhesus monkeys serum IgG levels decreased significantly following inoculation with V. cholerae; however, the ratio of GOT, alkaline phosphatase and antibodies against toxin were only slightly modified.


[Serum iodothyronine concentrations and properties of mitochondria phosphorylative oxidations in old rats]. Serum concentrations of T4 and T3 in 2 year-old Rats were decreased by about 30% compared to 2 month-old animals, but rT3 was similar. Activities of old Rats' liver mitochondria were near those of young thyroidectomized animals. Low circulating T3 partly explains metabolic changes observed during senescence.


[Application of hydrophobic chromatography to the fractionation of wheat prolamins]. Hydrophobic chromatography is applied to the fractionation of wheat prolamins. Proteins are separated on "Phenyl Sepharose CL 4B" column. They are eluted by variations of pH and polarity of solvent. Components with the same electrophoretic mobility appear in several chromatographic fractions and gliadin groups, as indicated by Woychick classification, are heterogeneous. This method is excepted to give new information about interaction properties of gluten proteins.


[Effects of mescaline and lysergide on the mechanical responses of isolated organs of the rat to catecholamines and the role of calcium transfers in these phenomena]. On isolated Rat vas deferens the contracting responses of noradrenaline and dopamine are increased by mescaline (23 micromoles) and decreased by lysergide (15 micromoles). Similarly, on isolated Rat duodenum previously decalcified the hypertonic responses of recalcification are modified in the opposite direction by the two agonists. On the other hand, mescaline such as lysergide increase the fall of tonus which ensues the decalcification of isolated duodenum.


[Effect of blockade or stimulation of the reticuloendothelial system on the blood clearance kinetics of the anticholinesterase insecticide carbaryl in the rat]. The blood clearances of 14C-carbaryl and colloïdal carbon were studied in Rats with reticuloendothelial system (RES) inhibited or activated, and in control Rats. A correlation was established between the blood clearance kinetics of carbon particles and carbaryl; such data support the concept of the contribution of the RES in the disappearance of carbaryl from the circulation.


[Evidence for plasmidic localization of "beta-lactamase" gene in a Pseudomonas aeruginosa strain, highly resistant to carbenicillin]. Pseudomonas aeruginosa strain HL, isolated in Besançon (France), shows a high level of resistance for carbenicillin, as proved in preliminary studies. In this paper, we report the transfer of carbenicillin resistance from Pseudomonas aeruginosa HL1 carrying helper plasmids to other strains of the same species. Comparative study of beta-lactamase activity in the HL1 strain and in the conjugated strains allows confirmation of the transfer of an R-factor from the HL1 strain: in all these strains, the beta-lactamase has the same pI (5.3) and the same Vmax (relative to benzylpenicillin) for ampicillin, carbenicillin and cephaloridin.


[Structure-activity relationship of various ephedrine isomers on noradrenaline uptake by the isolated perfused rabbit heart]. (-)Ephedrine, (+) Ephedrine, (-) psi ephedrine and (+) psi ephedrine inhibit both the neuronal and extraneuronal uptakes of noradrenaline in isolated Rabbit heart. (-) ephedrine is the most active isomer for the inhibition of both neuronal and extraneuronal uptakes. The relative inhibition potential of the three other isomers is not the same when applied to one or other uptake.


A controlled clinical trial comparing disodium cromoglycate and ICI 74,917 in extrinsic asthma. A controlled clinical trial comparing inhalation of disodium cromoglycate (DSCG) and of ICI 74,917 was carried out using a double-placebo technique in thirty-two patients with extrinsic asthma already shown to be reasonably responsive to DSCG. In view of the fact that ICI 74,917 is 300 times more potent than DSCG in inhibiting antigenic challenge in animals a better effect was anticipated from the new drug in the human asthmatic subject. Whilst this was not obtained, there appeared to be no lesser effect than that of DSCG.


Pharyngocutaneous fistulae following total laryngectomy. Postoperative pharyngocutaneous fistula formation is a troublesome complication in head and neck surgery and occurred in 8 out of 53 patients following a total laryngectomy (15%). The incidence falls within the lower range of that reported in the literature (6-66%). Different factors which may influence the formation of fistulae were analysed. Factors which seem to increase fistula formation are previous full dose radiotherapy (Co60), residual carcinoma and surgical technique.


A supernumerary "G" like chromosome originating from a maternal 13;15 translocation in a nondysmorphic, retarded girl. This paper describes a retarded girl who had a severe seizure disorder and a few minor anomalies, and who was found to have a "G-sized" supernumerary chromosome in all her cells which were examined. Studies of the patient's and her parents' chromosomes by a variety of techniques suggest that the supernumerary chromosome arose following a prezygotic maternal 13;15 translocation.


Comparison of placebo, nitroglycerin, and isosorbide dinitrate for effectiveness of relief of angina and duration of action. Each of 13 patients with angina had either chewable isosorbide dinitrate, nitroglycerin (sublingual therapy), or placebo administered on each of three different days when mild anginal pain had been induced by walking on a treadmill. Both therapy with isosorbide dinitrate and therapy with nitroglycerin were effective in bringing about complete relief of the angina in less than ten minutes of uninterrupted continuous walking on the treadmill in 11 of the 13 subjects, while administration of placebo afforded complete relief in none. The duration of action of the drug was determined by following the first effort (during which the tested drug was given) by successive ten-minute walks at the same workload that first induced anginal pain. Half-hour resting periods separated the repeated periods of exercise, and the duration of action was taken to be the time from administration of the agent to the return of angina on one of the repetitive efforts. No prolonged protection was afforded by administration of the placebo. Nitroglycerin protected for slightly longer than one hour, while isosorbide dinitrate protected for 2 1/2 to 3 hours.


Treatment of variant angina pectoris with perhexilene maleate. Three patients with variant angina pectoris resistant to therapy with nitrates and propranolol were treated with perhexilene maleate. Two patients had normal coronary arteries with documented coronary artery spasm, while the third patient had a fixed coronary artery obstruction. In all three patients, attacks of variant angina pectoris disappeared following institution of therapy with perhexilene maleate. When the dose of this drug was decreased to 100 mg per day or less, symptoms reappeared in all patients. Reinstitution of therapeutic doses of perhexilene maleate once again resulted in complete control of symptoms. Perhexilene maleate is therefore a useful agent for the treatment of variant angina pectoris.


The effect of tinidazole on several ciliates. The effect of tinidazole on several ciliates (Stylonychia muscorum, Stylonychia mytilus, Tetrahymena pyriformis, Vorticella companula) has been studied. There is a very pronounced difference in sensitivity against this drug between the free-living ciliates here investigated and certain parasitic protozoa. A high resistance of the free-living ciliates against tinidazole, probably due to their aerobic condition, has been found. Nonetheless, tinidazole at high doses (1--3 mg/ml) caused severe alterations in 3 of the 4 aerobic ciliate species here studied.


Genome structure of Tetrahymena pyriformis. Reassociation kinetics of DNA from the macronucleus of the ciliate, Tetrahymena pyriformis GL, has been studied. The genome size determined by the kinetic complexity of DNA was found to be 2.0 X 10(8) base pairs (or 1.2 X 10(11) daltons). About 90% of the macronuclear DNA fragments 200-300 nucleotides in length reassociate at a rate corresponding to single-copy nucleotide sequences, and 7-9% at a rate corresponding to moderate repetitive sequences; 3-4% of such DNA fragments reassociate at C0t practically equal to zero. To investigate the linear distribution of repetitive sequences, DNA fragments of high molecular weight were reassociated and reassociation products were treated with S1-nuclease. DNA double-stranded fragments were then fractionated by size. It has been established that in the Tetrahymena genome long regions containing more than 2000 nucleotides make up about half of the DNA repetitive sequences. Another half of the DNA repetitive sequences (short DNA regions about 200-300 nucleotides long) intersperse with single-copy sequences about 1,000 nucleotides long. Thus, no more than 15% of the Tetrahymena genome is patterned on the principle of interspersing single-copy and short repetitive sequences. Most of the so called "zero time binding" or "foldback" DNA seem to be represented by inverted self-complementary (palindromic) nucleotide sequences. The conclusion has been drawn from the analysis of this fraction isolated preparatively by chromatography. About 75% of the foldback DNA is resistant to S1-nuclease treatment. The S1-nuclease resistance is independent of the original DNA concentration. Heat denaturation and renaturation are reversible and show both hyper- and hypochromic effects. The majority of the inverted sequences are unique and about 20% are repeated tens of times. According to the equilibrium distribution in CsCl density gradients the average nucleotide content of the palindromic fraction does not differ significantly from that of total macronuclear DNA. It was shown that the largest part of this fraction of the Tetrahymena genome are not fragments of ribosomal genes.


Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus. DNA in the macronucleus of Stylonychia mytilus exists as discrete gene-sized fragments which are derived from micronuclear DNA through a series of well-defined developmental events. It has been proposed that each of the DNA fragments might represent a gene and its controlling elements. We have investigated this possibility using genes which code for the five histone proteins. Macronuclear DNA fragments were fractionated according to size by agarose gel electrophoresis, the fragments transferred to nitrocellulose filters using the technique of Southern, and the filter-bound DNA hybridized with labeled cloned histone genes of the sea urchin, Psammechinus miliaris. Results indicate, first, that sequences homologous to the five individual histone gene probes are present in discrete macronuclear fragments which appear as bands in the gel hybridization assay. Secondly, for each of the five individual histone gene probes the homologous DNA fragments are several in number, ranging in size in from 7.6 Kb (Kilo base pairs) to 0.73 Kb. For example, the largest of six detected fragments hybridizing to the H3 gene probe contains approximately 10 times the amount of DNA required to code for a Stylonychia H3 histone. The smallest detected fragment hybridizing to the H3 probe contains enought DNA to code for approximately two copies of the histone. Finally, in general, no two histone approximately two copies of the histone. Finally, in general, no two histone gene probes hybridized to the same macronuclear DNA fragment. This result indicates that genes coding for the five histones in Stylonychia are not located together on the same macronuclear DNA fragments and implies that the five functionally related genes would not be transcribed together as a polycistronic unit.


[General anesthesia and long term psychotropic treatments]. Anesthesists often ask for withdrawal of psychotropic treatment a few days before surgery. Usually they do not differentiate among the various classes of psychotropic drugs. Some drugs, including MAO inhibitors and reserpine-like agents, can induce or precipitate accidents; however, this does not occur with other types of antidepressants, neuroleptics and minor tranquilizers. The authors review the psychiatric risks related to such withdrawals; these risks are to be considered. They report that in 8,210 ECT under general anesthesia (among which 5,688 were done without psychotropic withdrawal) there was no occurrence of severe accidents.


The arcuate nucleus and the control of gonadotropin and prolactin secretion in the female rhesus monkey (Macaca mulatta). Attempts were made to destroy selectively the arcuate nucleus with radiofrequency current in adult female rhesus monkeys as a first step in identifying the areas of the mediobasal hypothalamus (MBH) that are responsible for the neural control of gonadotropin secretion in this species. Extensive or complete destruction of the arcuate region was produced in three animals and in two of these the lesion was confined primarily to the arcuate region and the dorsal aspect of the posterior median eminence. These lesions resulted in the cessation of LH and FSH secretion and blocked the positive feedback action of estradiol on gonadotropin release but did not appear to influence grossly basal thyroid and adrenocortical function, or to abolish GH discharge in response to insulin hypoglycemia. Adenohypophysial infarcts were not observed and exogenous LHRH and TRH induced marked discharges of the appropriate anterior pituitary hormones. In two additional animals with large hypothalamic lesions, destruction of the arcuate region was incomplete. In this group only partial inhibition of gonadotropin secretion was observed. LH and FSH secretion did not appear to be influenced in one animal bearing a large MBH lesion that entirely spared the arcuate region. Although serum prolactin remained at pre-lesion control levels after placement of the two relatively discrete lesions confined to the arcuate region, unambiguous increases in the secretion of this hormone were observed when the area of destruction encompassed tissue anterior and/or dorsal to the arcuate region. These observations suggest that the arcuate region is the primary structure mediating the hypothalamic control of gonadotropin secretion in the rhesus monkey. They also suggest that, in this species, the regions of the MBH involved with the regulation of gonadotropin release and those which control prolactin secretion are anatomically distinct.


Plasma thyroxine and triiodothyronine levels in spontaneously metamorphosing Rana catesbeiana tadpoles and in adult anuran amphibia. We have developed sensitive and reliable radioimmunoassays for T4 and T3 in amphibian plasma and have used these procedures to measure plasma T4 and T3 levels in spontaneously developing Rana catesbeiana tadpoles at various stages of metamorphosis. During premetamorphosis circulating levels of both T4 and T3 were below the limits of detection of the RIA procedures (T4 less than 50 ng/100 ml, T3 less than 5 ng/100 ml). A gradual rise in plasma T3 and T4 became apparent during prometamorphosis, and at the onset of metamorphic climax the levels of both T4 and T3 increased sharply. Peak levels for both hormones were observed in the middle of metamorphic climax (stage XXIII). The circulating T3 level reached a mean peak of 78 ng/100 mg, at least 15 times greater than the level during premetamorphosis. The peak T4 level was 0.5 microgram/100 ml, about a 10-fold increase over the premetamorphosis level. The surge in thyroid hormone secretion lasted only for several days, and during the latter half of metamorphic climax there was a fairly rapid decrease in plasma T4 and T3. By 2 days post-climax the levels had declined to about 20% of their peak values. Free T4 and T3 levels in plasma followed the same general pattern as the total hormone levels during the various stages of tadpole development. In adult R. catesbeiana, plasma T4 and T3 levels were surprisingly low. Similarly low values were observed in Bufo marinus and in Rana pipiens. The very low levels of circulating T4 and T3 both in premetamorphosis tadpoles and in adults suggest that thyroid hormones in anuran Amphibia may be of importance only during the period of metamorphosis.


Effect of somatostatin on parathyroid hormone and calcitonin secretion. This study evaluated the effect of somatostatin on immunoreactive parathyroid hormone (iPTH) and calcitonin (iCT) secretion in vivo in rats and monkeys and on iPTH secretion in vitro by normal bovine parathyroid tissue and by a human parathyroid adenoma. Somatostatin infusion promptly (within 0.5 h) suppressed both iPTH and iCT in both species studied in vivo, the suppression being progressive during the infusion period. In in vitro studies, somatostatin caused significant dose-related decreases in basal, low Ca-stimulated, and high Ca-suppressed PTH secretion from normal bovine parathyroid tissue and from basal and low Ca-stimulated PTH secretion from a human parathyroid adenoma. Therefore, somatostatin 1) suppresses both PTH and CT secretion in vivo; 2) acts directly on the parathyroid cell and presumably directly on the C-cell also; 3) acts upon normal and adenomatous parathyroid tissue; 4) suppresses basal, low Ca-stimulated and high Ca-suppressed PTH secretion; and 5) has a dose-related effect. The possible role of somatostatin in the physiological control of PTH and CT secretion (and therefore in Ca homeostasis), and in the pathogenesis of abnormalities of Ca homeostasis, requires further evaluation.


On the sites of the negative and positive feedback actions of estradiol in the control of gonadotropin secretion in the rhesus monkey. Ovariectomized rhesus monkeys bearing hypothalamic lesions which had abolished endogenous LHRH production, as evidenced by a profound reduction in gonadotropin secretion, but in which LH and FSH secretion was reestablished by a chronic intermittent iv infusion of synthetic LHRH (1 microgram/min for 6 min every hour) were used to investigate the sites of the negative and positive feedback actions of estradiol in the control of gonadotropin secretion. The administration of estradiol to such animals, while continuing the LHRH replacement regimen, resulted in a decline in circulating LH and FSH levels, followed by an unambiguous discharge of these hormones. The time course of this biphasic pattern of gonadotropin secretion was remarkably similar to that observed in response to estradiol administration in otherwise intact ovariectomized animals. These results suggest that, in the rhesus monkey, estradiol can exert both its negative and positive feedback actions on gonadotropin secretion at the level of the pituitary gland.


The sites of action of estradiol and phentolamine in the inhibition of the pulsatile, circhoral discharges of LH in the rhesus monkey (Macaca mulatta). Pulsatile LH secretion was re-established in ovariectomized monkeys bearing hypothalamic lesions by an intermittent infusion of LHRH. The administration of estradiol to such animals resulted in a prompt cessation of these pulsatile discharges of LH and a resultant decline in the mean plasma concentration of the gonadotropin. The time course of this inhibition of LH secretion was indistinguishable from that observed after estrogen administration to ovariectomized animals with intact nervous systems. In contrast, phentolamine did not interrupt the pulsatile LH discharges occasioned by the hourly administration of exogenous LHRH to the lesioned animals. These results are consistent with the conclusion that the acute negative feedback action of estradiol on circhoral LH release in the monkey is at the level of the pituitary gland, whereas the inhibitory action of phentolamine on this mode of LH secretion is at a neural site.


Studies on the subsynaptosomal localization of luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the rat hypothalamus. In the current investigation, subcellular particles (synaptosomes) of hypothalamic homogenates were isolated by differential centrifugation and discontinuous sucrose density gradient fractionation and found to be rich in LHRH, TRH, and the neuronal marker, norepinephrine (NE). Of the total quantity of LHRH, TRH, or NE in the nuclei-free homogenate, 52-65% was recovered in synaptosomes, whereas the cytosol, myelin/microsomes, and mitochondria contained only 1-12%. To determine the subsynaptosomal localization of LHRH and TRH, purified synaptosomes were lysed and the resulting suspensions were fractionated on discontinuous sucrose density gradients. LHRH (30-40%) was found to be localized primarily in subsynaptosomal particles which banded at sucrose densities between 0.6-1.0 M. Electron micorscopic analysis of these particles revealed the presence of dense-cored granules (70-80 nm diameter) and synaptosomal membrane remnants. Norepinephrine was found in two pools within the isolated nerve endings: 15-25% of synaptosomal NE was associated with the synaptic vesicles (45-55-nm diameter); about 40% was in the cytosol. TRH was present primarily as a soluble component of the nerve ending. No apparent association of TRH with dense-cored granules was demonstrable in this study; however, there may be some TRH in synaptic vesicles.


Effect of estrogens on thyroid function. I. Alterations in rhesus plasma thyrotropin and its kinetics. The circulating levels of TSH, its metabolism, and its response to synthetic TRH were studied in five euthyroid menstruating rhesus monkeys before and during treatment with estradiol monobenzoate (E2B, 50 microgram/kg BW/day sc). The pre-E2B treatment mean plasma TSH level was 1.4 +/- 0.12 (SE) microunit/ml. A significant increase in mean plasma TSH (P less than 0.01) to 1.54 +/- 0.29 microunit/ml was observed as early as 48 h after intiation of E2B treatment; it continued to rise progressively to day 28 when it plateaued around a mean concentration of 3 microunit/ml. It normalized within 10 days after cessation of E2B therapy. After iv TRH (5 microgram/kg BW), a consistent rise in plasma TSH was observed before and on days 11 and 56 of E2B therapy. The peak TSH level and maximum rise over the basal level (deltaTSH) during the three tests were not significantly different. During E2B therapy there were remarkable changes in TSH kinetics. These alterations included a significant decrease (P less than 0.01) in metabolic clearance rate, contraction of the distribution space, and expansion of the extrapituitary TSH pool, but there was no appreciable change in TSH production rate. Although a definite trend towards the above alterations was discernible on day 17 of treatment, they were well established by day 66. These data suggest that the estrogen-induced rise in circulating TSH was caused mainly by decreased degradation and not by increased production.


Can the pituitary secrete directly to the brain? (Affirmative anatomical evidence). Vascular casts of 10 rhesus monkey pituitary glands and three vascular casts of the rhesus monkey cavernous sinus were examined by scanning electron microscopy. A continuous neurohypophyseal capillary bed was found uniting the infundibulum, infundibular stem, and infundibular process. The neurophypophysis was supplied by three groups of arteries: superior hypophyseal, middle hypophyseal, and inferior hypophyseal. Numerous anastomoses were found between individual arteries, and some hypophyseal arteries formed anastomotic links between different portions of the circle of Willis. Veins located at the caudal pole of the infundibular process, capillaries linking the infundibulum to the hypothalamus, and portal vessels extending from the infundibulum to the adenohypophysis provided efferent vascular pathways from the neurohypophysis. The adenohypophysis received no direct arterial supply; its entire afferent vascular supply was provided by portal vessels. Lateral hypophyseal veins were not found; small adenohypophyseal veins joined larger neurohypophyseal veins to form confluent pituitary veins which extended to the cavernous sinus. The capacity of the venous connections draining the adenohypophysis directly to the cavernous sinus appeared small when compared to that of of the long portal vessels supplying the adenohypophysis. However, many of the short portal vessels interposed between the adenohypophysis and the infundibular stem and process were well arranged to function as alternative efferent routes from the adenohypophysis. The limited potential for venous drainage directly to the cavernous sinus suggests that blood leaves the adenohypophysis by other routes; blood carried via long portal vessels from the infundibulum to the adenohypophysis may return to the neurohypophyseal capillary bed via short portal vessels. This anatomical study suggests that hypothalamic and adenohypophyseal secretions are conveyed to the capillary bed of the neurohypohysis. These secretions may leave the neurohypophysis via any of seven potential routes: one efferent route is directed to the adenohypophysis, another route is directed to the systemic circulation, but five of the potential efferent routes are directed toward the brain.


Heterologous radioimmunoassay for rabbit prolactin. A highly specific heterologous double-antibody RIA has been developed to measure rabbit PRL by using guinea pig antiserum to human PRL and ovine [125 I]iodo-PRL. Rabbit pituitary PRL and serum give parallel dose-response curves in the assay and no cross-reaction (less than 0.1%) occurs with GH, placental lactogens, LH, FSH, or TSH from several different species. The assay is suitable for the measurement of human, ovine, bovine, caprine, and canine PRL in addition to rabbit PRL, but shows no cross-reaction with rat PRL. Reproducibility and precision of the assay are within acceptable limits. Gel filtration of rabbit pituitary PRL and rabbit serum on Sephadex G-100 revealed coincident peaks of activity measured by RIA and by PRL radioreceptor assay. The molecular weight of rabbit PRL appeared similar to that of ovine PRL. Serum PRL levels increased after the injection of both TRH and chlorpromazine and were reduced by CB154 (Bromocriptine). Venepuncture stress caused an increase in PRL in nonpregnant or postpartum non-suckled animals, but small or no increases were seen in lactating female rabbits.


Synchronous, sustained oscillation of C-peptide and insulin in the plasma of fasting monkeys. The previously reported synchronous oscillations in plasma glucose and insulin levels have been further studied to determine whether the phenomenon can be attributed to cyclic secretion or degradation of the hormone. Plasma C-peptide concentrations were measured and found to cycle with the same period as plasma insulin suggesting that the oscillations of insulin arise from changing secretion rather than degradation. The amplitude of C-peptide oscillation was 50 percent that of insulin. Assuming first order kinetics and the same relative rates of disappearance as in humans, this difference in amplitudes in consistent with equal secretion rates for the two peptides.


Insulin and glucagon binding and degradation by kidney cell membranes. The binding and degradation of insulin and glucagon to kidney cell membranes was examined. Time- and temperature-dependent specific binding of [125I]iodo-insulin to kidney cell membranes was demonstrated. The membranes also degraded insulin in a time-, temperature-, and protein concentration-dependent manner. The apparent Km of the degradation was 2.7 x 10(-7) M. Glucagon degradation by the kidney membranes was extremely active. Per milligram of protein the kidney membrane was over 20 times as active as the liver membrane. Even at 4 C, significant glucagon degradation occurred. Because of this very active degradation, glucagon binding could not be accurately assessed. The kidney glucagon-degrading activity was inhibited by glutahione and EDTA but unaffected by N-ethylmaleimide, ACTH, or insulin, all potent inhibitors of liver glucagon degradation. The apparent Km for glucagon degradation by the kidney, however, was essentially identical with that for the liver, 2.4 x 10(-6) M.


Peripheral metabolism of homologous thyrotropin in euthyroid and hypothyroid rats: acute effects of thyrotropin-releasing hormone, triiodothyronine, and thyroxine. The peripheral metabolism and metabolic clearance rate (MCR) of homologous TSH was studied in euthyroid and hypothyroid rats. Incubation of freshly labeled [125I]iodo-TSH with rat serum revealed a labeled nonimmunoreactive protein in the void volume of a Sephadex G-100 column which could not be detected by conventional chromatographic purification. Removal of this contaminant from the tracer reduced the nonspecific binding in the absence of serum and increased the binding of tracer in the absence of added exogenous TSH. Injection of [125I]iodo-TSH into rats was followed within 15 min by the appearance of at least three labeled protein components. Gel filtration showed that these peaks were trichloroacetic acid (TCA)-precipitable proteins of larger molecular weight than TSH, but not all were precipitable by antibody to rat TSH. The disappearance rate of TCA-precipitable 125I (t1/2 = 28 min) was significantly longer than the disappearance rate of immunoprecipitable 125I (t1/2 = 22 min). The disappearance rate of immunoprecipitable [125I]iodo-TSH was identical to that of injected purified rat TSH and of the TRH-induced TSH increment in euthyroid rats. The disappearance rate os suppressible TSH (after 100 microgram T3) in hypothyroid animals was only slightly longer than the rate of disappearance of immunoprecipitable [125I]iodo-TSH (40 vs. 36 min) in the same rats. The calculated MCR of TSH was slightly lower (P less than 0.05) in hypothyroid rats (18.3 +/- 3.0 ml/h/100 g BW, mean +/- SD) than it was in euthyroid rats (22.6 +/- 2.1). The pituitary TSH concentration in hypothyroid rats was 29 mU/mg wet wt, similar to that of euthyroid animals. These results indicate that the turnover rate of pituitary TSH in hypothyroid rats with serum TSH concentrations of 1400-3000 microunit/ml is 7-14 times/day. Therefore, the significant increase we observed in pituitary TSH concentration 1 h after T4 (1.5 microgram/100 g BW) or T3 (0.15 microgram/100 g BW) administration indicates that the 35% decrease in plasma TSH at this interval is due to inhibition of TSH release, not to inhibition of TSH synthesis.


Effect of luteinizing hormone-releasing hormone on the secretion of luteinizing hormone, follicle-stimulating hormone, and testosterone in adult male rhesus monkeys. Plasma levels of radioimmunoreactive LH, FSH, and testosterone (T) were assayed before and after administration of synthetic gonadotropin-releasing hormone (GnRH) to five chair-restrained rhesus monkeys with chronic indwelling venous catheters. Intravenous injection of 1, 5, and 25 microgram or infusion of 1 microgram/min for 25 min of GnRH resulted in a significant increase in plasma levels of LH. However, no significant increases in plasma FSH levels were detected. Plasma levels of T were also elevated after administration of 25 microgram GnRH, but peak concentration of T lagged behind peak levels of LH by approximately 30 min. These studies indicate that the male rhesus responds to GnRH administration by increased secretion of LH, followed by an increase in T levels. A concomitant increase in plasma FSH was not observed after treatment with GnRH in the doses used.


Progesterone is not essential to the differentiative potential of mammary epithelium in the male mouse. In pursuit of a model system in which to determine whether or not exposure to progesterone is necessary for mammary epithelial cells to develop their differentiative potential, we explored hormone-dependent growth of the mammary epithelial rudiment in adult male mice. Initiation of the formation of ductal cells can be effected by administration of estradiol in the absence of endogenous progesterone and glucocorticoid using adrenalectomized-castrated animals. The resulting epithelium contains three times more lactose synthetase activity per epithelial cell than that in midpregnant mice. The blood spermidine level in these doubly operated animals was similar to the concentration of spermidine required to substitute effectively for glucocorticoid during mammary differentiation in vitro. It is suggested that spermidine can partially supplant glucocorticoid in vivo in milk protein synthesis. We also concluded that, unlike other secondary sex tissues, mammary cells do not require exposure to progesterone during their ontogeny in order to realize their differentiative potential. The positive role of this steroid in mammary development is apparently limited to its effect on the formation of alveolar structures.