The influence of fixation procedure, embedding medium and section thickness on morphometric data in thyroid gland. In this study, the effects of fixation procedures, embedding medium and section thickness on stereological measurements of normal thyroid were analysed. The following conclusions were drawn: A) the use of a single section for the analysis of a lobe is sufficient if this section is located in the central part of the lobe. B) fixation and embedding with glutaraldehyde-Epon leads to a larger shrinkage than Bouin-paraplast, but the difference between the two procedures is not significant. C) osmium post-fixation reduces the shrinkage induced by glutaraldehyde and lowers the axial deformation produced by sectioning. D) Bouin's fixative and paraplast embedding induce considerable shrinkage of the interstitial tissue. The shrinkage obtained with glutaraldehyde-Epon is less. However, it is still not known whether this difference is due to the fixative, or to the embedding procedure or to both. E) only in glutaraldehyde and osmium-fixed material, embedded in Epon, can follicles and colloids be assumed to be spherical in shape without significant errors.


Mucopolysaccharidosis in a domestic short-haired cat--a disease distinct from that seen in the Siamese cat. A 10-month-old male domestic short-haired cat was examined because of progressive lameness, a broad face with depressed nasal bridge, small ears, corneal clouding, and multiple bone dysplasia. The cat excreted excessive amounts of glycosaminoglycan (a component of connective tissue) in its urine and had evidence of lysosomal storage of glycosaminoglycans in fibroblasts and neurons. Activity of alpha-L-iduronidase, a lysosomal enzyme involved in glycosaminoglycan degradation, was deficient in cultured fibroblasts and leukocytes. The mucopolysaccharidosis was distinct from that seen in Siamese cats in terms of the pathologic changes and the specific enzyme deficiency.


Polioencephalomalacia in range cattle.

Polioencephalomalacia in range cattle. Polioencephalomalacia developed in 27 of 225 cattle grazing on 486 hectares of dry, short, grama grass pasture. Chemicals in drinking water, toxin from nitrate-utilizing ruminal bacteria, and documented poisonous plants were considered as etiologic agents. Attempts to reproduce the disease by injecting mice and dosing sheep with broth filtrate from nitrate-utilizing ruminal bacteria were not successful. Mushrooms collected from the pasture and fed to a cow did not reproduce the disease.


Apocrine secretion in the terminal bronchiole of mouse lung. The question of the existence of apocrine secretory activity by the Clara cells of the mouse lung terminal bronchiole has been investigated in depth. The overall superiority of 1--2 micrometer plastic sections for light microscopy was demonstrated. The preservation of the anatomy of the terminal bronchiole was shown to be adversely affected by slow killing methods, by post mortem delays before fixation, and by the instillation of fluids via the trachea. The use of collapsed lungs removed from rapidly killed animals is probably the best method for the study of the small bronchioles of the lung. Apocrine secretion takes place as originally described by Clara in 1937. The reason why the phenomenon has received so little attention in the literature is probably because the tracheal or vascular perfusion of fixative, and delays before fixation, all prevent apocrine droplets from being preserved.


The effect of triethylenetetramine dihydrochloride on the in vivo susceptibility of Pseudomonas aeruginosa to gentamicin. A chelating agent, triethylenetetramine dihydrochloride (TRIEN dihydrochloride) increased the efficacy of gentamicin in vivo against a clinical isolate of P. aeruginosa, designated Ps 15. Mice which were inoculated with 10 X LD50 of Ps 15 and treated with doses of 2 approximately 16 mg of gentamicin per kg per day all died. However, treatment with 8 mg of gentamicin per kg body weight per day plus 30 mg of Trien dihydrochloride per day markedly reduced the mortality. The combined therapy also reduced the number of viable organisms that accumulated in the kidney during a 24-hour period post inoculation. When a dosage level of 8 mg of gentamicin was exceeded in the combined treatment regimen, all of the infected mice died, and a high concentration of endotoxin could be detected in the mouse sera by the limulus assay.


Influence of neomycin and metronidazole on colonic microflora of volunteers. The influence on colonic microflora of neomycin and metronidazole alone, or in combination, was studied in volunteers taking a normal diet. Bacterial counts on daily faecal samples revealed that metronidazole had no influence on anaerobic faecal bacteria. Neomycin on the other hand, was effective against sensitive aerobes. However, the combination of neomycin and metronidazole profoundly reduced both aerobic and bacteria. Assay of faecal antibiotic concentrations showed that neomycin achieved high intraluminal levels while metronidazole was undetectable or present at low concentrations.


Effects of low ampicillin concentrations on penicillin sensitive and beta-lactamase producing strains of Neisseria gonorrhoeae. The effects of therapeutic concentrations of ampicillin on non-beta-lactamase and beta-lactamase producing strains of Neisseria gonorrhoeae were studied. A small but significant fraction of bacteria in a gonococcal population was found to respond in a bacteriostatic rather than a bactericidal way upon ampicillin treatment. In agreement with this was the finding of morphologically unaltered cells in the scanning electron microscope after ampicillin exposure. Ampicillin treatment of beta-lactamase producing gonococci caused a significant release of the enzyme into the surrounding growth media. However, initially all beta-lactamase activity was cellbound. The rate of initial ampicillin hydrolysis was much higher in intact cells of N. gonorrhoeae (TEM-1) than in cells of Escherichia coli K-12 (TEM-1). This suggests that the diffusion rate of ampicillin is much higher in the former organism. The viability of gonococci (TEM-1) was unlike E. coli (TEM-1) affected by low concentrations of ampicillin. However, after complete hydrolysis of ampicillin, viable gonococci (probably bacteriostatic reacting cells) were able to initiate new growth. This heterogeneity of the cell population to penicillin killing is probably one reason why beta-lactamase producing gonococci despite a rather low MIC-value to ampicillin cause infections that are not susceptible to therapy by this agent.


Bacteriological studies with cefsulodin (CGP 7174/E), the first antipseudomonal cephalosporin. The new cephalosporin, cefsulodin, has considerable antibacterial activity against Pseudomonas aeruginosa. When 217 strains of Ps. aeruginosa were tested against both azlocillin and cefsulodin, 26.3% were found to have the same minimal inhibitory concentration (MIC); the MIC for azlocillin was lower than that for cefsulodin in 16.6% of strains, but higher in 57.1%. 22 gentamicin-resistant strains were all susceptible to cefsulodin. Biophotometer investigations demonstrate less bactericidal effects for cefsulodin and azlocillin than for carbenicillin and ticarcillin using higher inocula than used in the agar of tube dilution test. Cefsulodin and gentamicin are synergistic against Ps. aeruginosa. Using high pressure liquid chromatography and biological techniques, cefsulodin is found to be moderately stable in solution and in standard solid laboratory media.


A pharmacological and in vitro comparison of three oral cephalosporins. The pharmacology of cephradine, cephalexin and a new oral cephalosporin, cefaclor, has been compared in six volunteers. Cefaclor was absorbed rapidly and was cleared from the serum more rapidly than the other two agents. This was probably partially due to its instability in serum at body temperature, which was investigated. Against a wide range of common pathogens cefaclor was the more active oral cephalosporin. In particular the activity against Neisseria gonorrhoeae and Haemophilus influenzae was of interest.


Induction of enzyme activity in cell culture: a rapid screen for detection of planar polychlorinated organic compounds. Induction of aryl hydrocarbon hydroxylase (AHH) activity in rat hepatoma cell line serves as a simple and rapid method to detect minute (pg) amounts of certain classes of compounds, e.g., dibenzo-p-dioxins, dibenzofurans, and biphenyls. This method may provide a quick screen for such substances in extracts from foods prior to chemical identification. AHH activity is measured by conversion of benzo[a]pyrene (BP) to 3-hydroxy BP in homogenized cell extracts from control and treated cultures and is reported as pmol product formed/mg protein/min. Substances screened by this method include polyhalogenated analogs of dibenzo-p-dioxin (24 compounds), dibenzofuran (11 compounds), biphenyl (7 compounds), and extracts from several food sources. Response of the most reactive compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was used to prepare a standard curve, and the AHH activity induced by mole doses of test substance is reported as an ED50 response (the estimated dose needed to produce 50% maximum enzyme induction). The AHH activity induced by food extracts is equated to the standard curve and reported as TCDD equivalents. A potent ED50 response in cell culture appears to correlate well with known toxic responses in other mammalian and avian systems for certain test substances. This correlation suggests that the cell culture enzyme induction method is a useful model for screening food extracts that are suspected to be contaminated with polychlorinated planar substances. A collaborative study would demonstrate the reproducibility of the method.


Developmental induction of Myxococcus xanthus myxospores. Myxospore differentiation during the developmental cycle of Myxococcus xanthus is characterized by several distinguishable morphological stages. Two experimentally useful criteria of myxospore induction are the conversion of vegetative rods to optically refractile short rods or ovoids and the development of resistance to sonic lysis. The use of optical refractility as the first morphological criterion of myxospore induction has facilitated an analysis of induction on developmental plates. The time-dependent changes in the cell population from vegetative rods to the final products of development, autolysed cells and myxospores, were determined in liquid suspension by interrupting cells from developmental plates before the first appearance of myxospores. The treatment of cells involved a two-step induction system. The cells were first aerated in buffer at 32 degrees C (preinduction) and then aerated in 1% tryptone (Difco) at 32 degrees C (induction). At early plate times (0 to 18 h) there was little or no response to these treatments. After 18 h, many of the cells undergoing development on plates responded to preinduction in buffer by subsequent induction to myxospores in tryptone medium (intermediate cells). After 32 h, cells induced to myxospores in tryptone medium and did not require preinduction (competent cells). After 36 h, cells begin to undergo differentiation to myxospores on plates. These results indicate that there was a sequence of physiological changes in developing cells that are defined by the differential response of cells to treatment in liquid suspension. The liquid induction system described here provides a means to analyze the regulation of developmental myxospore induction.


Cotransformation of temperature sensitivity and nutritional markers in Neisseria gonorrhoeae. Cotransformation remains the only tool for establishing linkage in Neisseria gonorrhoeae. Because of the difficulty of inducing auxotrophic markers via mutagenesis in this species, most previous studies have utilized antibiotic resistance and naturally occurring (auxotypic) auxotrophic markers. We have succeeded in isolating auxotrophic and temperature-sensitive mutants. The temperature-sensitive mutants have been characterized by their growth on complex and defined media at 31, 37, and 40 degrees C. Two of the mutants exhibited an unusual pattern of temperature sensitivity--growth on the defined medium but absence of growth on the complex medium at 37 degrees C. Both mutants, however, were temperature-sensitive on the two media at 40 degrees C. We have demonstrated linkages between markers isolated in our laboratory and the auxotypic markers of the clinical isolate RUG208. Ts-2 exhibited 85 to 95% linkage to Arg- and his-2 exhibited 40% linkage to Val-. In addition weak linkages were shown between his-2 and Arg- (2 to 6%) and between Arg- and Val- (3 to 5%). Linkages among his-2, Arg-, and Val- which could be demonstrated when deoxyribonucleic acid from strain F62 was used to transform RUG208 were absent when F62 was used as recipient for RUG208 DNA. Our data are consistent with a tentative map order of his-2, Val-, Arg-, Ts-2.


Transformation and transfection of Pseudomonas aeruginosa: effects of metal ions. The ability of different metal ions to promote transformation of Pseudomonas aeruginosa by deoxyribonucleic acid of the plasmid RP1 was examined. CaCl2, MgCl2, and MnCl2 were found to promote such transformation, although at different frequencies and with the optimum response at different concentrations. Only MgCl2 promoted transfection of P. aeruginosa by the linear deoxyribonucleic acid of phage F116. CaCl2 was demonstrated to allow adsorption and entry into the cell of F116 deoxyribonucleic acid such that it became resistant to exogenous deoxyribonuclease, but phage production occurred only when MgCl2 was provided. Inactivation of linear phage deoxyribonucleic acid taken up in the absence of MgCl2 was observed. The transfection frequencies at various concentrations of MgCl2 were compared, and the optimum response occurred at the concentration which promoted the highest frequency of transformation by RP1 deoxyribonucleic acid.


Macromolecular synthesis in synchronized cultures of Anacystis nidulans. Synchronous culture of Anacystis nidulans has been induced by the light-dark-light regimen. At various time intervals during synchronous growth, samples were pulsed with radioactive labels to determine phospholipid, protein, ribonucleic acid (RNA), and deoxyribonucleic acid (DNA) syntheses within the cell division cycle. A temporal order of protein, RNA, and DNA syntheses occurred within the cell division cycle, whereas phospholipid was characteristically synthesized during midcycle (during cell enlargement) and during the time of cell division. Chemically determined protein, RNA, and DNA syntheses were found to support the schedule of these macromolecules in cultures growing at an 8-h doubling time.


Restriction enzyme analysis of Bacillus subtilis ribosomal ribonucleic acid genes. The organization of the ribosomal ribonucleic acid (rRNA) genes (rDNA) of Bacillus subtilis was examined by cleaving the genome with several restriction endonucleases. The rDNA sequences were assayed by hybridization with purified radioactive rRNA's. Our interpretation of the resulting electrophoretic patterns is strengthened by an analysis of a fragment of B. subtilis rDNA cloned in Escherichia coli. The results indicated that there are eight rRNA operons in B. subtilis. Each operon contains one copy of the sequences coding for 16S, 23S, and 5S rRNA. The sequences coding for 5S rRNA were shown to be more closely linked to the 23S rRNA genes than to the 16S rRNA genes.


Mutants of Anabaena strain CA altered in their ability to grow under nitrogen-fixing conditions. Mutants of Anabaena strain CA impaired in nitrogenase activity and growth on N2 were isolated and characterized. One mutant was selected for resistance to L-methionine-DL-sulfoximine, and others were selected for resistance to DL-7-azatryptophan or for requirements for combined nitrogen. The mutants varied in sensitivity of growth and nitrogenase activity to atmospheric 02. Several of the mutants whose growth on N2 was impaired under aerobic conditions could grow and reduce acetylene at rates comparable to the wild type when grown microaerobically under N2-CO2 (99:1). The acetylene reduction activity of some of the strains grown under N2-CO2 was immediately and completely lost upon exposure to atmospheric O2, but in at least one strain this loss was reversed when the O2 concentration was lowered, even after 10 h of exposure to air. The characteristics of the O2-sensitive mutants suggest that there may be several sites sensitive to O2 and that the protective mechanism involves several different phenomena.


Transcriptional regulation of Escherichia coli K-12 major outer membrane protein 1b. Eleven independent insertion mutations were isolated that prevented expression of major outer membrane protein 1b. Seven of the mutations were Mucts insertions located at ombP. These ompB::Mucts strains fell into two phenotypic classes with regard to expression of proteins 1a and 1b. The remaining four mutants were comprised of one Tn5 and three Mucts insertions mapping at par. The Mucts insertions at par were used to construct fusions of the lac operon to the par promoter. Expression of beta-galactosidase in these fusion strains reflected known regulatory properties of protein 1b. When an ompB allele was introduced into the par-lac fusion strains, beta-galactosidase activity was reduced 14- to 31-fold. Transcriptional regulation of the par gene and the existence of two functions at ompB are discussed. The results suggest that par is the structural gene for protein 1b and that an ompB gene product is a diffusible, positive regulatory element controlling expression of par.


Function of modified nucleosides 7-methylguanosine, ribothymidine, and 2-thiomethyl-N6-(isopentenyl)adenosine in procaryotic transfer ribonucleic acid. To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis. The tRNA's differ in modifications in the anticodon loop, the extra arm, and the TUC loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNA(f) (Met) the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine-forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the V(max) value is decreased by one-half. (ii) tRNA(f) (Met) species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis. The formylated fMet-tRNA(f) (Met) species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated Met-tRNA(t) (Met) species, however, differ from each other: "Met-tRNA(f) (Met) rT" is inactive, whereas the U54 counter-upart effectively forms the initiation complex. (iii) Two isoacceptors, tRNA(1) (Phe) and tRNA(2) (Phe), were obtained from B. subtilis. tRNA(1) (Phe) accumulates only under special growth conditions and is an incompletely modified precursor oftRNA(2) (Phe): in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N(6)-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU(3)-directed formation of fMet-Phe, the undermodified tRNA(1) (Phe) is always less efficient at Mg(2+) concentrations between 5 and 15 mM than its mature counterpart.


Transfer ribonucleic acid synthesis during sporulation and spore outgrowth in Bacillus subtilis studied by two-dimensional polyacrylamide gel electrophoresis. The synthesis of transfer ribonucleic acid (tRNA) was examined during spore formation and spore outgrowth in Bacillus subtilis by two-dimensional polyacrylamide gel electrophoresis of in vivo 32P-labeled RNA. The two-dimensional gel system separated the B. subtilis tRNA's into 32 well-resolved spots, with the relative abundances ranging from 0.9 to 17% of the total. There were several spots (five to six) resolved which were not quantitated due to their low abundance. All of the tRNA species resolved by this gel system were synthesized at every stage examined, including vegetative growth, different stages of sporulation, and different stages of outgrowth. Quantitation of the separated tRNA's showed that in general the tRNA species were present in approximately the same relative abundances at the different developmental periods. tRNA turnover and compartmentation occurring during sporulation were examined by labeling during vegetative growth followed by the addition of excess phosphate to block further 32P incorporation. The two-dimensional gels of these samples showed the same tRNA's seen during vegetative growth, and they were in approximately the same relative abundances, indicating minimal differences in the rates of turnover of individual tRNA's. Vegetatively labeled samples, chased with excess phosphate into mature spores, also showed all of the tRNA species seen during vegetative growth, but an additional five to six minor spots were also observed. These are hypothesized to arise from the loss of 3'-terminal residues from preexisting tRNA's.


Regulatory nucleotides involved in the Rel function of Bacillus subtilis. We have examined the accumulation of polyphosphorylated nucleotides in Bacillus subtilis in relation to the function of the rel gene. Our results are as follows. (i) During inhibition of isoleucine activation by O-methylthreonine, wildtype B. subtilis cells accumulate unusual nucleotides with the chromatographic and chemical properties of pppApp, ppApp, pppGpp, ppGpp, pGpp, and ppGp. (ii) During the carbon source downshift elicited by inhibiting glucose uptake, we observed accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iii) At the end of long phase in sporulation medium, we observed a small transient accumulation of the polyphosphorylated guanosine but not adenosine nucleotides. (iv) We were unable to detect a nucleotide with chromatographic behavior expected for pppAppp under any conditions. (v) The rel mutant of Swanton and Edlin (Biochem. Biophys. Res. Commun. 46-583-588, 1972) did not accumulate any of these polyphosphorylated nucleotides under any of the conditions examined. (vi) the rel mutant is unimpaired in sporulation. We conclude that one or more of the nucleotides we have detected may be involved in controlling the specificity of transcription during the stringent response, but none of them are required for sporogenesis.


Completed Bacillus subtilis nucleoid as a doublet structure. When outgrowing spores of the temperature-sensitive dna initiation mutants of Bacillus subtilis, TsB134 and dna-1, were allowed to undergo a single round of replication by shifting to the restrictive temperature soon after its initiation, both segregating daughter nucleoids appeared as clearly defined doublet structures. The components of each doublet remained together as a discrete pair, even under conditions which resulted in the formation of deoxyribonucleic acid (DNA)-less cells. A doublet nucleoid was also observed at a high frequency when TsB134 spores were allowed to germinate and grow out in the complete absence of DNA synthesis at the permissive temperature. TsB134 spores were foud to contain the usual "haploid" amount of DNA. It is suggested that the doublet nucleoid reflects a folding of a single chromosome into two large domains which resolve from one another under conditions of cell extension in the absence of DNA synthesis.


Organization of transfer and ribosomal ribonucleic acid genes in Bacillus subtilis. The structural relationship between the transfer ribonucleic acid (tRNA) and the ribosomal RNA (rRNA) genes of Bacillus subtilis has been studied by restriction endonuclease analysis of total chromosomal deoxyribonucleic acid (DNA) and characterization of DNA fragments cloned in Escherichia coli. The DNA sequences encoding rRNA and tRNA were assayed by hybridization to radioactive RNA. The results support the conclusion that the tRNA genes are interspersed between and closely linked to the rRNA genes of B. subtilis. They probably do not appear between the 16S and 23S rRNA genes as in E. coli.


Nucleotide sequence of 5S ribosomal RNA from rainbow trout (Salmo gairdnerii) liver. Staring from low molecular weight RNA obtained from rainbow trout (Salmo gairdnerii) liver, 5S ribosomal RNA (rRNA) was highly purified by successive chromatography on columns of DEAE-Sephadex A50 and Sephadex G100. Products of complete and partial digestions on this RNA with pancreatic ribonuclease (RNase A) [EC 3.1.4.22] and RNase T [EC 3.1.4.8] were isolated and sequenced by conventional and high-performance liquid chromatography (HPLC) procedures. The nucleotide sequence of this RNA thus established was compared with those of five other vertebrae 5S rRNAs, and the rates of base substitution per site per year were found to be nearly constant in these RNAs. The analyses of the partial digests of the trout 5S rRNA revealed several sites susceptible to RNase attack, which could be accounted for by the secondary structure model for eukaryotic 5S rRNAs proposed by Nishikawa and Takemura (1).


Methods for sequencing oligoribonucleotides and RNA by high performance liquid chromatography. Conditions were established for the separation and quantitative determination of ribonucleosides, mono- and oligo-ribonucleotides by high-performance liquid chromatography (HPLC) on columns of AS-Pellionex SAX and AL-Pellionex WAX. By combining a high-speed UV spectrum monitor with an HPLC apparatus, products of RNase digestions of oligonucleotides and 5S ribosomal RNA (rRNA) were identified by measuring their UV spectra under continuous solvent flow, and also from their retention times on the columns (positions of elution). It took only 10 to 30 min for one chromatography run and required less than 0.01 A260 unit of sample per nucleotide material in each peak.


Extracellular hydrogenase from photosynthetic bacterium, Rhodospirillum rubrum. With Rhodospirillum rubrum, hydrogenase was found to exist partly as an extracellular enzyme in the culture medium. After 4-day cultivation, the total activity and the specific activity of the enzyme in the medium were about 10 times and 230 times as high as those in the crude extract obtained from disrupted cells. The time course for the production of hydrogenase during cultivation was studied.


The complete amino acid sequence of the beta-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa. The complete amono aicd sequence of the beta-subunit of protocatechuate 3,4-dioxygenase is presented. The beta-subunit contained 237 amino acid residues, 4 of which were methionines. Accordingly, cyanogen bromide cleavage of the S-carboxymethylated beta-subunit produced five peptides. The sequences of these peptides were determined by analyses of the peptides obtained by tryptic, staphyloccal protease and thermolysin digestions. The alignment of the cyanogen bromide peptides was deduced by the use of overlapping peptides containing methionine which were obtained by tryptic digestion of the S-carboxymethylated beta-subunit. The calculated molecular weight was 26,588, which is close to the value estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.


Purification and properties of reductases for aromatic aldehydes and ketones from guinea pig liver. NADPH-dependent enzymatic reduction of aromatic aldehydes and ketones observed in the cytosol of guinea pig liver was mediated by at least three distinct reductases (AR 1, AR 2, and AR 3), which were separated by DEAE-cellulose chromatography. By several procedures AR 2 and AR 3 were purified to homogeneity, but AR 1 could be purified only 30-fold because of the small amount. These enzymes were found to have similar molecular weights of 34,000 to 36,000 and similar Stokes radii of about 2.5 nm. AR 3 was identical to aldehyde reductase [EC 1.1.1.2] in substrate specificity for aromatic aldehydes and D-glucuronate and specific inhibition by barbiturates. AR 1 and AR 2 acted on aromatic ketones and cyclohexanone as well as aromatic aldehydes at optimal pHs of 5.4 and 6.0, respectively, and were immunochemically distinguished from AR 3. AR 1 was the most sensitive to sulfhydryl reagents, and AR 2 was more stable at 50 degrees C than the other enzymes. Similar heterogeneity was observed in the kidney enzymes, but other tissues had little aldehyde reductase activity and contained only AR 3. In addition, lung contained a high molecular weight aromatic ketone reductase different from the above reductases.


Nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8. The nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8, was determined by a combination of classical methods using unlabeled samples to determine the sequences of the oligonucleotides of RNase T1 and RNase A digests and a rapid sequencing gel technique using 5'-32P labeled samples to determine overlapping sequences. Formylmethionine tRNA from T. thermophilus is composed of two species, tRNAf1Met and tRNAf2Met. Their nucleotide sequences are almost identical, and are also almost identical with that of E. coli tRNAfMet, except for slight modifications and replacements. Both species have modifications at three points which do not exist in E. coli tRNAfMet: 2'-O-methylation at G19, N-1-methylation at A59 and 2-thiolation at T55. Moreover U51 in E. coli tRNAfMet is replaced by C51 in both species, so that a G-C pair is formed between this C51 and G65. tRNAf2Met has a reversed G-C pair at positions 52 and 64 compared with those in tRNAf1Met and E. coli tRNAfMet. Other regions are mostly the same as those in all prokaryotic initiator tRNAs so far reported. The thermostability of these thermophile initiator tRNAs is discussed in relation to their unique modifications.


Biochemical studies on the mechanism of difference in the renal toxicity of 5-hydroxy-L-tryptophan between Sprague Dawley and Wistar rats. The biochemical mechanisms of the renal toxicity of 5-hydroxy-L-tryptophan to rats were studied using Wistar and Sprague Dawley rats, which had different LD50 values. When the amino acid was injected intraperitoneally, Wistar rats, which had a low LD50 value of 5-hydroxy-L-tryptophan, excreted larger amonts of serotonin and smaller amounts of 5-hydroxyindole acetic acid into the urine than Spraque Dawley rats, which had a high LD50 value. The activity of renal aromatic L-amino acid decarboxylase was higher in Wistar rats than in Sprague Dawley rats, while the activity of renal aromatic amino acid transaminase was in an opposite relationship. The activity of renal monoamine oxidase was almost the same in both strains and the activity of renal UDP glucuronyltransferase in Wistar rats was higher than in Sprague Dawley rats. Since the renal damage caused in rats by 5-hydroxy-L-tryptophan was very similar to that caused by serotonin, the amine formed from the administered amino acid was thought to be an important factor for the renal necroses, and difference in serotonin formation from the administered precursor amino acid may be one of the important factors leading to the difference in LD50 values in the two strains of rats.


Purification of viral proteins from avian sarcoma virus QV2. A procedure was established whereby most of the major viral proteins were isolated to apparent homogeneity in biologically and immunologically active forms from a single batch of avian sarcoma virus QV2. For the initial step of purification, gently disrupted virions were fractionated by CsCl centrifugation into envelope proteins, RNA-dependent DNA polymerase, and viral core proteins. Further purification of envelope glycoproteins and DNA polymerase was performed by affinity chromatography on agarose columns cross-linked with plant lectins and poly(C), respectively. On the other hand, core proteins were fractionated by a combination of gel filtration and ion-exchange column chromatography into components p27, p19, and p15. The core protein p15 thus isolated retained proteolytic activity even after storage for 6 months. The present study also demonstrated that QV2 p19 is structurally altered from the corresponding protein of avian myeloblastosis virus (AMV), a reference avian leukosis-sarcoma virus having a well-characterized polypeptide composition.


Enzyme immunoassay of pancreatic glucagon at the picogram level using beta-D-galactosidase as a label. An enzyme immunoassay of pancreatic glucagon was established by using E. coli beta-D-galactosidease [EC 3.2.1.23] as a marker. In order to increase the sensitivity of the immunoassay, different peptides obtained from glucagon fragments were used to produce the enzyme conjugate and the immunogen. Antiserum N6E raised against C-terminal fragment peptide (15-29) could be diluted to more than 1 : 100,000 in the assay and was highly specific for pancreatic glucagon. The antiserum reacted well with the C-terminal fragment peptide (21-29) as well as another fragment peptide (15-29) and pancreatic glucagon. The enzyme immunoassay using antiserum N6E and fragment peptide (21-29)-enzyme conjugate could detect as little as 1 to 2 pg of glucagon. The mean recovery of glucagon added to serum specimens was 104% and the coefficients of variation were 3.7-14.5% (within assay) and 9.0-18.5% (between assay).


Isolation and characterization of major outer membrane proteins of Pseudomonas aeruginosa strain PAO with special reference to peptidoglycan-associated protein. The outer membrane of Pseudomonas aeruginosa PAO contains six major proteins (proteins D, E, F, G,H, and I). Two of them (protein F and protein H) were found to be retained by the peptidoglycan layer when cell envelopes were extracted with 2% sodium dodecyl sulfate (SDS) solution at 35 degrees C. At higher temperature (greater than 55 degrees C), no proteins were retained by peptidoglycan. By making use of this property, purification of protein F and protein H was achieved. Three other major outer membrane proteins, D, E, and I were also isolated and characterized. Their amino acids compositions were determined. Circular dichroism spectra of these isolated proteins were measured in SDS solution. Protein F was rich in beta-structure, while protein I was rich in alpha-helix. When isolated protein F was heated (100 degrees C-15 min) in SDS solution, the circular dichroism spectrum changed significantly. In parallel with the conformational change, the electrophoretic mobility of protein F on urea-SDS polyacrylamide gel also changed. These results indicate that protein F is a so-called heat-modifiable protein.


A novel peptidoglycan-associated lipoprotein found in the cell envelope of Pseudomonas aeruginosa and Escherichia coli. Protein H, one of the major outer membrane proteins Pseudomonas aeruginosa, shows an interesting interaction with the peptidoglycan layer of the cell. It is retained by peptidoglycan after extraction of the cell envelope with SDS solution at 35 degrees C. A protein of the same molecular weight (21,000) as protein H was found in the peptidoglycan-associated fraction of Escherichia coli K-12 prepared under the same conditions. This protein, designated here as protein 21K, was purified from the cell envelope of E. coli, and the properties of two proteins (protein H and protein 21K) were compared. By gas chromatographic analysis of purified protein 21K and protein H, it was found that both contained covalently linked fatty acid. In isotopic labeling experiments, [14C]palmitic acid and [2-3H]glycerol were incorported into both proteins H and 21K. These two proteins showed similar amino acid compositions, but no apparent correlation was found between protein 21K or protein H and Braun's lipoprotein. These results suggest that protein 21K of E. coli K-12 and protein H of P. aeruginosa PAO are a novel type of lipoprotein. All nine gram-negative bacteria tested, including enteric and non-enteric bacteria, contained a similar protein of apparent molecular weight 21,000 as a peptidoglycan-protein complex.


Mouse immunoglobulin mu heavy chain mRNA of Y5781, a high yield myeloma. The BALB/c myeloma tumor, Y5781, has a high level of mu heavy chain mRNA and kappa light chain mRNA, as suggested by denaturing gel analyses of poly(A)-rich, total polysomal mRNA, and confirmed for the mu heavy chain mRNA by kinetic complexity analyses. Both the mRNA coding for the heavy and light chains appear as very prominent and discrete peaks above the generally polydisperse background of the total polysomal mRNA. This mRNA level appears to be stable through a limited number of subcutaneous passages of this myeloma, providing a potentially useful system for mu heavy chain mRNA synthesis and processing. The mu heavy chain mRNA of this myeloma has been enriched to about 60% homogeneity by physicochemical means. In agreement with a previous report (Faust, C.H., Jr., Heim, I., and Moore, J. (1979) Biochemistry 18, 1106-1119), the following physical and biological properties were observed. The mature cytoplasmic mu heavy chain mRNA is 950,000 daltons, i.e. about 2800 nucleotides, and contains approximately 800 undefined, nontranslated bases. In an mRNA-dependent cell-free system, this mRNA stimulates the synthesis of a single, serologically reactive mu heavy chain-like protein, confirmed by tryptic peptide maps.


Site of action of RNase I on the 50 S ribosome of Escherichia coli and the association of the enzyme with the partially degraded subunit. The 50 S ribosome of Escherichia coli is partially degraded by RNase I in presence of a high concentration of Mg2+ (10 to 20 mM); the partially degraded subunit becomes resistant to the further action of RNase I. The latter remains latent in association with the subparticle as in case of 30 S ribosome (Neu, H.C., and Heppel, L.A. (1954) Proc. Natl. Acad. Sci. U.S.A. 51, 1267-1274). As a result of nucleolytic action, 23 S RNA is degraded to a smaller size and four proteins (L4, L10, L7/L12) are released from the subunit. From the location of these proteins, it appears that the primary site of action of RNase I is the central protuberance of the armchair model proposed for the subunit (Stoffler, G., and Whitman, H.G. (1977) in Molecular Mechanisms of Protein Biosynthesis (Weissbach, H., and Pestka, S., eds) pp. 117-144, Academic Press, New York).


Evidence for the presence of two separate protein activators for the enzymic hydrolysis of GM1 and GM2 gangliosides. Two different protein activators were isolated simultaneously from human liver for the enzymic hydrolysis of GM1 (Gal beta 1 leads to 3GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-galactosidase and GM2 (GalNAc beta 1 leads to 4Gal(3 comes from 2 alpha NeuAc)beta 1 leads to 4Glc-Cer) by beta-hexosaminidase A. The hydrolysis of GM1 is stimulated only by the GM1-specific activator which has very little effect on the hydrolysis of GM2. The same is also true for the hydrolysis of GM2. The antiserum raised against GM1 activator did not cross-react with GM2 activator and vice versa. These results suggest the presence of two different activators for the separate hydrolysis of GM1 and GM2. In connection with the enzymic hydrolysis of GM1 and GM2, we found that the hydrolysis of GM2 by human hepatic beta-N-acetylhexosaminidase A was severely inhibited by a buffer of high ionic strength, whereas no such inhibition was observed in the hydrolysis of GM1 by beta-galactosidase.


Characterization of "thyroliberin-deamidating enzyme" as a post-proline-cleaving enzyme. Partial purification and enzyme-chemical analysis of the enzyme from anterior pituitary tissue. An enzyme which catalyzes the deamidation of thyroliberin (TRF; less than Glu-His-Pro-NH2) has been purified 110-fold from extracts of bovine anterior pituitary by ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration. This enzyme of 76,000 molecular weight (as estimated by gel filtration) exhibits maximal activity at neutral pH (optimum pH 7.4 to 7.6) in buffers of high ionic strength supplemented with thiol-protecting agents. As indicated by the strong inhibition of the enzymatic activity by N-ethylmaleimide and Hg2+, as well as by the extreme sensitivity toward diisopropyl fluorophosphate, -SH, and -OH residues apparently represent essential functional groups of the enzyme. The stereospecific deamidation of TRF (Km = 4.1 . 10(-4) M) is inhibited competitively by TRF analogues which contain proline or by the proline containing biologically active peptides luliberin (LH-RF), oxytocin, vasopressin, angiotensin II, and Substance P. TRF analogues without proline or peptide amides without proline are ineffective. This enzyme cleaves the appropriate Pro-X bonds in luliberin, angiotensin II, pyroGlu-His-Pro-Gly-NH2, and the collagenase substrate Z-Gly-Pro-Leu-Gly-Pro. Thus, it may be characterized as a post-proline-cleaving enzyme.


The primer tRNA for Moloney murine leukemia virus DNA synthesis. Nucleotide sequence and aminoacylation of tRNAPro. The nucleotide sequence of a tRNA primer molecule for initiation of Moloney murine leukemia virus DNA synthesis has been determined. The sequence is heterogeneous in two positions but both forms, when drawn in a cloverleaf structure, have anticodon specificities for proline. We have termed these isoacceptors tRNA1Pro and tRNA2Pro. Aminoacylation studies confirmed the specificity for proline. The two forms occur in approximately equal amounts in uninfected mouse and chicken cells and in Moloney leukemia virus particles.


Interaction of rabbit secretory component with rabbit IgA dimer. Secretory component (SC), a glycoprotein with an apparent molecular weight of approximately 80,000, has been isolated from rabbit milk and found to be heterogenous in size and charge. Functionally intact IgA dimer has been dissociated from milk secretory IgA using a chaotropic agent and further purified to homogeneity. The interaction between SC and IgA dimer is a reversible time- and temperature-dependent process. At 23 degrees C, the association rate constant (2.4 x 10(5) M-1 min-1) and the dissociation rate constant (1.8 x 10(-3) min-1) have been measured independently and the affinity constant based on these rates (1.3 x 10(8) M-1) is similar to that calculated from Scatchard plots (1.9 x 10(8) M-1). One class of binding sites has been estimated from Scatchard plots in spite of the observed heterogeneity of SC. The interaction is tighter at low temperatures because the decrease in dissociation rate is greater than the decrease in association rate. The thermodynamic calculations reveal a delta G of -11.0 kcal . mol-1, a delta H of -8.9 kcal . mol-1 and a delta S of +7.0 cal. mol-1 degree-1. The pH range over which interaction occurs is rather large (5 to 8) with no significant differences in apparent Ka.


Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells. The receptor-mediated internalization and degradation of radiolabeled diphtheria toxin by cultured monkey kidney cells was studied. The ability of a number of enzymes and chemicals to remove cell surface-bound toxin was tested; the combination of pronase and inositol hexaphosphate (PIHP) proved most effective. Using PIHP, the kinetics of toxin-cell association at 37 degrees C was resolved into two compounds: surface binding and internalization. The PIHP assay also allowed estimation of the half-time of toxin internalization (about 25 min). An assay involving precipitation of culture supernatants with trichloroacetic acid was developed and used to measure the rate of degradation and excretion of cell-associated toxin. Agents which markedly inhibited toxin internalization similarly prevented degradation, implying an intracellular location for the degradative process. The primary radioactive product excreted by Vero cells was monoiodotyrosine. The extent and rate of toxin degradation indicated lysosomal involvement. Finally, agents which blocked internalization or degradation, or both, (e.g. antibody and concanavalin A), protected cells from the cytotoxin action of diphtheria toxin, suggesting that these processes are necessary for expression of biological effect.


Cathepsin D isozymes from porcine spleens. Large scale purification and polypeptide chain arrangements. Six cathepsin D isozymes have been purified from porcine spleen using a large scale purification procedure. Five isozymes, I to V, have an identical molecular weight of 50,000 and are similar in specific activity. Isozymes I to IV contained two polypeptide chains each. The light and heavy chains have Mr = 15,000 and 35,000, respectively. Isozyme V is a single polypeptide. The molecular weight of the sixth isozyme is about 100,000 and it has only 5% of the specific activity of the other isozymes. On Ouchterlony immunodiffusion, an antiserum formed precipitin lines against the urea-denatured isozyme with Mr = 100,000. This immunoreactivity showed immunoidentity with those formed against other isozymes. The NH2-terminal sequence of light chains was identical for the isozymes. This sequence is homologous to the NH2-terminal sequence of other acid proteases, especially near the region of the active center aspartate-32. The NH2-terminal sequence of the single chain, isozyme V, Is apparently the same as the light chain sequence. The NH2-terminal sequence analysis of the heavy chain from isozyme I produced two sets of related sequences, suggesting the prescene of structural microheterogeneity. The carbohydrate analysis of the isozymes, the light chain, and the heavy chain revealed the presence of possibly four attachment sites, with one in the light chain and three in the heavy chain. Each carbohydrate unit contains 2 residues of mannose and 1 residue of glucosamine. The results suggest that the high molecular weight cathepsin D (Mr = 100,000) is the probable precursor of the single chain (Mr = 50,000), which in turn produces the two-chain isozymes. These are likely in vivo processes.


Purification and characterization of hydroperoxidase II of Escherichia coli B. The second heme-containing hydroperoxidase isozyme (HP-II) has been isolated from aerobic cultures of Escherichia coli B. The protein exists as a stable tetramer of subunits of equal size, with a combined molecular weight of 312,000. The heme spectrum of HP-II is unusual, in that it exhibits two absorbance maxima at 407 and 591 nm; the alkaline pyridine hemochromogen spectrum shows maxima at 425, 559, and 609 nm. HP-II differs in several respects from the HP-I isozyme previously reported (Claiborne, A., and Fridovich, I. (1979) J. Biol. Chem. 254, 4245-4252). Thus HP-II is virtually devoid of peroxidatic activity toward o-dianisidine but has a 6-fold higher catalatic activity than HP-I. Antisera to HP-II do not cross-react with HP-I, and analyses of chymotryptic and cyanogen bromide digests suggest differences in primary structure between these two isozymes.


Anomeric configuration of N-acetylglucosaminyl phosphorylundecaprenols formed in Bacillus cereus Membranes. The structural difference was studied between two N-acetylglucosaminyl phosphorylundecaprenols formed by incubation of Bacillus cereus membranes with UDP-N-acetylglucosamine. On the treatment with 50% phenol, the major one of these glycolipids (Lipid 1) yielded a saccharide phosphate, while the other (Lipid 2) yielded N-acetylglucosamine along with a saccharide phosphate. The saccharide phosphates from Lipids 1 and 2 were identified as alpha-N-acetylglucosamine 1-phosphate and its beta-anomer, respectively, based on their susceptibility to acid, alpha- and beta-N-acetylglucosaminidases, and UDP-N-acetylglucosamine pyrophosphorylase. Thus, it seems most probable that Lipids 1 and 2 were alpha- and beta-N-acetylglucosaminyl phosphorylundecaprenols, respectively.


The interaction of divalent copper and the microsomal electron transport system. A re-examination of the effects of copper chelates on the function of cytochrome P-450. Lipophilic chelates of divalent copper, possessing superoxide dismutase-like activity, have been proposed to enhance the decay of oxycytochrome P-450 to explain their inhibitory effect on microsomal mixed-function oxidation reactions (Richter, C., Azzi, A., Weser, U., and Wendel, A. (1977) J. Biol. Chem. 252, 5061-5066). The present investigation, however, failed to provide evidence in favor of this hypothesis. In particular, it was found that the reported inhibition of cytochrome P-450-catalyzed hydroxylation reactions by copper-tyrosine is associated with an inhibition rather than a stimulation of the formation of hydrogen peroxide, the product of the dismutation of the superoxide radicals generated as a result of the decay of oxycytochrome P-450. The attenuation of both these reactions was shown to be the consequence of an impaired function of the NADPH-cytochrome P-450 reductase. Additional sites of interaction of copper chelates and the microsomal electron transport system appear to exist since divalent copper was found to undergo reduction reactions with NADPH and NADH as electron donors. These reduction reactions do not involve superoxide radicals and, therefore, are unrelated to the ability of copper chelates to function in a superoxide dismutase-like manner.


Covalent structure of protein A. A low molecular weight protein degraded during germination of Bacillus megaterium spores. The complete covalent structure of Protein A, a protein degraded during bacterial spore germination, has been determined. The intact protein was cleaved with a highly specific spore protease into two peptides, residues 1 to 21 and 22 to 61. The larger peptide was further cleaved into two fragments with either cyanogen bromide or by trypsin cleavage following arginine modification with cyclohexanedione. The peptides derived from cyanogen bromide fragmentation encompassed residues 22 to 53 and 54 to 61 while trypsin hydrolysis yielded overlapping fragments comprising residues 22 to 48 and 49 to 61. Automated sequenator analysis together with carboxypeptidase Y digestion of the intact protein and the peptide fragments provided data from which the following unique amino acid sequence was deduced. NH2-Ala-Asn-Thr-Asn-Lys-Leu-Val-Ala-Pro-Gly10-Ser-Ala-Ala-Ala-Ile-Asp-Gln-Met-Lys-Tyr20-Glu-Ile-Ala-Ser-Glu-Phe-Gly-Val-Asn-Leu30-Gly-Pro-Glu-Ala-Thr-Ala-Arg-Ala-Asn-Gly40-Ser-Val-Gly-Gly-Glu-Ile-Thr-Lys-Arg-Leu50-Val-Gln-Met-Ala-Glu-Gln-Gln-Leu-Gly-Gly60-Lys-COOH.


Purification of an adenosine deaminase complexing protein from human plasma. A protein which specifically complexes with adenosine deaminase (complexing protein) has been purified to homogeneity from human plasma. This protein was compared with complexing protein isolated from human kidney. The two proteins produce electrophoretically different forms of high molecular weight adenosine deaminase when combined with the Mr = 36,000 enzyme monomer from erythrocytes. This difference may, at least in part, be due to the greater sialic acid content of complexing protein from plasma. By other criteria, including amino acid composition, total carbohydrate content, and subunit structure, the two proteins are quite similar. In addition, plasma complexing protein shows complete cross-reactivity with anti-kidney complexing protein serum. These results suggest that plasma and kidney complexing proteins are products of the same gene.


Cephalosporin-sensitive penicillin-binding proteins of Staphylococcus aureus and Bacillus subtilis active in the conversion of [14C]penicillin G to [14C]phenylacetylglycine. Breakdown of the covalent complex formed between [14C]penicillin G and higher molecular weight, cephalosporin-sensitive penicillin-binding proteins was studied using mixtures of the purified proteins isolated from membranes of Staphylococcus aureus and Bacillus subtilis. These penicillin-binding proteins were found to release the bound 14C label in a first order process characterized by half-lives of 10 to 300 min at 37 degrees C. Denaturation of the penicilloyl.penicillin-binding proctein complex prevented this release, indicating that the process is enzyme-catalyzed. [14C]Phenylacetylglycine was identified as the major labeled fragmentation product, indicating that these cephalosporin-sensitive penicillin-binding proteins, for which no in vitro transpeptidase or carboxypeptidase activity has been found, catalyze the same fragmentation of the bound penicilloyl moiety previously described for several penicillin-sensitive D-alanine carboxypeptidases.


Synthesis of gamma-L-glutaminyl-[3,5-3H]4-hydroxybenzene and the study of reactions catalyzed by the tyrosinase of Agaricus bisporus. gamma-L-Glutaminyl-[3,5-3H]4-hydroxybenzene was synthesized in order to study the kinetics of its hydroxylation by tyrosinase purified from Agaricus bisporus and to explore its role in the induction of the dormant state in the spores of this species. It was found to be unique among the monophenolic substrates for tyrosinase in that the lag period for the hydroxylation reaction decreased with increasing substrate concentration. Unlike previously studied compounds, this phenol appeared to function as an electron donor, allowing it to act as its own co-substrate in the hydroxylation reaction. Its catechol product, gamma-L-glutaminyl-3,4-dihydroxybenzene, was found to be a superior co-substrate, yielding its electrons more readily (oxidation peak potential +0.18 V as compared with +0.65 V for the phenol). In situ periodate oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone confirmed the co-substrate role of the catechol in the hydroxylation reaction. The tyrosinase-mediated oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene to gamma-L-glutaminyl-3,4-benzoquinone occurred with an apparent Km = 1.54 mM and Vmax = 0.36 mmol/min/mg of enzyme. gamma-L-Glutaminyl-4-hydroxybenzene acted as an inhibitor of the oxidation reaction.


Carbohydrate structure of yeast invertase. Demonstration of a form with only core oligosaccharides and a form with completed polysaccharide chains. Invertase, extracted from broken cells of Saccharomyces cerevisiae X-2180 mm2 mannan mutant, was separated into a fraction insoluble in 75% ammonium sulfate (P75 invertase, 36% carbohydrate) and a soluble fraction (S75 invertase, 53% carbohydrate). The latter reacted with antibodies specific for the alpha 1 leads to 6-linked mannose of the mannoprotein outer chain, whereas the P75 invertase failed to react with this antiserum although it did react with serum against terminal alpha 1 leads to 3-linked mannose units that are characteristic of the mannoprotein core. A bacterial endo alpha 1 leads to 6-mannanase removed the outer chains from the S75 invertase and converted it to a form that was similar in electrophoretic and immunochemical properties to the P75 invertase, whereas the endomannanase had little effect on the latter invertase. The results suggest that the P75 invertase is a form of the enzyme to which only the core oligosaccharide units had been added, and the S75 invertase represents an enzyme fraction to which the polysaccharide outer chains were also attached. A strong anomeric PMR signal for unsubstituted alpha 1 leads to 6-linked mannose in the S75 invertase, and a much reduced signal in the P75 invertase and endomannanase-digested S75 invertase, support these conclusions. Endo-N-acetyl-beta-glucosaminidase digestion of the S75 and P75 invertases, as well as of a purified wild type yeast invertase, produced an apparently identical series of 3 to 4 carbohydrate-containing proteins that were separable by polyacrylamide gel electrophoresis in sodium dodecyl sulfate but that migrated as a single band on isoelectric focusing. The bands ranged from about 63,000 to 69,000 daltons and differed by the size of one or more carbohydrate core units each of 15 mannoses and 1 N-acetylglucosamine. The results suggest that the external invertase molecules contain some core units without attached outer chains, and that the cells contain a precursor form of the enzyme to which only the core units have been added. In support of this conclusion, PMR spectra and chromatographic patterns show that the core fragments from the P75, S75, and wild type invertases are essentially identical.


Thymidylate synthetase and 2'-deoxyuridylate form a tight complex in the presence of pteroyltriglutamate. Thymidylate synthetases of human and bacterial origin form a tightly bound complex with the substrate dUMP in the presence of pteroyltriglutamate. This complex and the weaker enzyme . dUMP binary complex can be isolated and conveniently assayed by nitrocellulose disc filtration using [6-3H]dUMP as the radioactive ligand. Intact thymidylate synthetase . dUMP . pteroyltriglutamate complex can be obtained by gel filtration chromatography on Sephadex G-25, but the binary enzyme . dUMP complex dissociates under the same conditions. Scatchard plots show the presence of two nonequivalent dUMP binding sites on the enzyme for the pteroyltriglutamate complex, with dissociation constants of 5 and 95 nM compared to 730 nM for the binary complex. The implications of these findings for folate analog inhibition of thymidylate synthetase are discussed.


Characterization and biosynthesis of proteoglycans of corneal stroma from rhesus monkey. The proteoglycans of the Rhesus monkey corneal stroma were characterized by analyzing both radiolabeled proteoglycans synthesized by corneas in organ culture and native corneal proteoglycans obtained by large scale preparations. The analyses indicate that the proteoglycans synthesized in organ culture were similar to, if not identical with, their counterparts in the stroma although they are synthesized in different prportions in vitro than they acumulate in vivo. The corneal stroma contains two proteoglycans. The chondroitin-dermatan sulfate proteoglycan consists of approximately 70% protein and has a Mr = approximately 100,000 to 150,000. It contains one chondroitin-dermatan sulfate side chain of Mr = approximately 55,000. The keratan sulfate proteoglycan consists of approximately 74% protein and has a Mr = approximately -40,000 to 70,000. It contains one or two keratan sulfate side chains with a Mr = approximately 7,000 each. Radiolabeling indicates that both proteoglycans contain glycoprotein-type oligosaccharides as part of their structure.


The relationship between human serum and human pancreatic DNase I. Deoxyribonuclease (DNase) activities have been partially purified from human serum and pancreas. Several of their physical and enzymatic characteristics were determined and compared in order to evaluate their relatedness. Human serum deoxyribonuclease has an isoelectric point in the range of 3.9 to 4.3 and a molecular weight of 33,000 to 38,000. Optimal enzymatic activity at pH 7.0 was dependent on both Mg2+ and Ca2+, whereas a pH optimum of from 5.5 to 5.8 was observed in the presence of Mg2+ and ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). The proportion of single strand or double strand breakage products at early stages of DNA digestion were variable functions of the composition of the buffers employed for the reactions. Single strand break age was predominant under all reaction conditions. Double strand breakage occurred with greatest frequency under neutral conditions in the presence of Mg2+ and Ca2+, was inhibited by the inclusion of 0.15 M NaCl, and did not occur at pH 5.8 in the presence of Mg2+, EGTA, and 0.15 M NaCl. Human pancreas deoxyribonuclease exhibited essentially the same physical properties and enzymatic characteristics as those of the human serum enzyme. Thus, human serum deoxyribonuclease may originate in this pancreas.


Transcription termination in the Escherichia coli ribosomal RNA operon rrnC. The distal portion of the Escherichia coli ribosomal RNA operon rrnC has been analyzed to determine (a) the transcription boundaries of an operon containing rRNA genes distal to rDNA, and (b) the sequence elements which dictate efficient termination of RNA synthesis. I have sequenced a 505-base pair DNA restriction fragment containing the genes which specify 5 S rRNA, TRNA1Asp, and tRNATrp, in that order. Segments of a spacer DNA separate these genes: the 5 S-tRNA1Asp and tRNA1Asp-tRNATrp spacers are 52 and 8 base pairs long, respectively. In vitro transcription of restriction endonuclease fragments containing the distal portion of rrnC indicates that termination of transcription occurs at a locus beginning 28 base pairs after the tRNATrp gene. The rrnC terminator shares several features with many other known sites of transcription termination in E. coli.


Serum alkaline phosphatase in heterotopic para-articular ossification after total hip replacement. Estimations of serum alkaline phosphatase were carried out prospectively on a series of patients having a total hip replacement. The levels of serum alkaline phosphatase before operation indicated a group of patients who subsequently developed heterotopic ossification. Levels of this enzyme after operation did not indicate those patients who were developing heterotopic ossification.


Plasma cell immunoglobulin M molecules. Their biosynthesis, assembly, and intracellular transport. Immunoglobulin M (IgM)-secreting murine plasmablasts have been used to explore the cytologic site(s) of the successive modifications of the polypeptide H and L chains (steps of glycosylation, chain assembly, and polymerization) which occur during intracellular transport (ICT) and the interrelationships between these events. A combination of pulse-chase biosynthetic labeling protocols (using amino acids and sugars), subcellular fractionation, and electron microscope autoradiography was used in conjunction with inhibitors of glycosylation and agents (carboxyl cyanide m-chlorophenyl hydrazone [CCCP] and monensin) which block Ig exit from the rough endoplasmic reticulum (RER) or Golgi cisternae. The data are consistent with the following conclusions: (1) Sugar addition and modification occur in three main steps: (a) en bloc addition of core sugars to nascent H chains, (b) partial trimming of these oligosaccharide chains in the RER, (c) quasiconcerted addition of terminal sugars (galactose, fucose, and sialic acid) in a very distal compartment between monensin-sensitive Golgi cisternae and the cell surface. (2) H and L chain assembly occurs between nascent H chains and a pool of free light chains present in the RER, followed by interchain disulfide bonding and rapid assembly of monomers into J chain-containing pentamers in the RER. Small amounts of various apparently non-obligatory intermediates in polymerization are also formed. (3) Carbohydrate addition is not required for chain assembly, polymerization, and secretion since completely unglycosylated chains (synthesized in the presence of deoxyglucose or tunicamycin) undergo polymerization and are secreted (although at a reduced rate). (4) Surface 8s IgM molecules do not represent a step in the IgM secretory pathway.


Covalent attachment of enzyme as a membrane-label for viable eucaryotic cells. An enzyme, beta-D-galactosidase, was covalently coupled to mammalian cells by means of a bifunctional reagent. The coupling procedure did not cause appreciable loss of cell viability (less than 6%) as measured by plating efficiently and membrane integrity. After 24 h in culture, the cells exhibited an average of 2.6 x 10(4) molecules of beta-D-galactosidase per cell. Histological evidence indicated that the enzyme was localized on the cell surface and distributed uniformly among the cell population. Considerations for choosing enzyme-label include sensitivity of assay by enzymatic, immunologic and histochemical methods, and the possibility of isolating labeled membrane components by enzyme-specific affinity chromatography.


Preparation of reference antisera for laboratory diagnosis of blastomycosis. Antiserum was prepared in rabbits against the A antigen and a new antigen (labeled the D antigen) of yeast-phase cells of Blastomyces dermatitidis by using immunoelectrophoretic precipitin arcs as vaccine. The antiserum can be used as a reference serum in the immunodiffusion test for identyfying antibodies to the A antigen in sera from patients thought to have blastomycosis. Antibodies to the D antigen have not yet been found in human sera. The rabbit antiserum that was absorbed with Histoplasma capsulatum yeast-phase cells did not react in the complement fixation test with either H. capsulatum yeast-phase or mycelial-phase complement fixation antigen. Therefore, it could also be used as a positive control serum in the complement fixation test. A fluorescent-antibody conjugate prepared from the absorbed serum was found to be valuable for the identification of yeast-phase cells of B. dermatitidis.


New serotypes of group B streptococci isolated from human sources. Antisera raised in rabbits against two nontypable group B streptococci, which were not agglutinable in a specific group B antiserum, were tested with acid extracts of 78 nontypable human group B streptococci. One antiserum (12351) reacted with 15 strains, and the other (7271) reacted with only 2 strains. Antiserum to Wilkinson's strain SS 1169 (NT 1) reacted with three strains. Antiserum against strain 12351 appears to be a useful antiserum against a new type antigen, which is probably polysaccharide in nature.


Radial immunodiffusion test with a Brucella polysaccharide antigen for differentiating infected from vaccinated cattle. A Brucella antigen containing polysaccharide but lacking smooth lipopolysaccharide was employed in a rapid radial immunodiffusion test. With this serological test, cattle infected with Brucella abortus could be identified in recently vaccinated herds which had high numbers of reactors to standard diagnostic tests.


Production of cells and viruses in a new multiple-tube tissue culture propagator. A novel method for large-scale culture of anchorage-dependent cells is described. Culture vessels were constructed by using roller bottles and Pyrex tubing. Parallel glass tubes, equally spaced by silicone rings and inserted into the roller bottles, provided an increased culture area. The roller bottles were fitted with a second opening for medium recirculation. Culture vessels of 1,000-, 4,000-, 8,000- and 16,000-cm2 growth area were constructed to propagate Vero, BS-C-1, primary chicken embryo, and IMH-P cells. These cultured cells were used to efficiently produce several viruses: rubella virus, measles virus, poliovirus, cytomegalovirus, and herpesvirus of turkey.


Characterization of a screening test for diphtherial toxin antigen produced by individual plaques of corynebacteriophages. A passive immune hemolysis assay has been developed to detect diphtherial toxin produced in individual plaques of tox+ corynebacteriophages. This assay permits rapid screening of large numbers of corynebacteriophages for their ability to code for diphtherial toxin or related antigens. The specificity of the assay and its potential usefulness for genetic studies of toxinogenesis have been demonstrated with well-characterized tox+ and tox- laboratory strains of corynebacteriophages.


Enzyme-linked immunosorbent assay for Pseudomonas aeruginosa exotoxin A. An enzyme-linked immunosorbent assay (ELISA) is described for Pseudomonas aeruginosa exotoxin A. A double antibody sandwich method was used, employing polyvinyl microtiter plates as the solid phase, a primary coat of monospecific rabbit antitoxin serum, an outer layer composed of a horseradish peroxidase-sheep antitoxin immunoglobulin G conjugate, and an ortho-phenylene-diamine substrate. Absorbance (optical density) of hydrolyzed end product was read spectrophotometrically at 492 nm. ELISA detected as little as 30 pg (0.3 ng/ml) of purified toxin, and absorbance was linear over a 20-fold or greater concentration range. Toxin was demonstrated in culture filtrates from 42 of 48 (88%) consecutive clinical P. aeruginosa isolates compared with 37 of 48 (77%) positive by hemagglutination inhibition. Results of the two assays correlated closely (r = 0.82, P less than 0.001). Specificity was confirmed by neutralizability of ELISA activity with monospecific antitoxin. ELISA was thus a sensitive, specific, and quantifiable technique for the assay of P. aeruginosa exotoxin A in both purified and crude culture materials.


Radioimmunoassay of human intestinal goblet cell mucin. Investigation of mucus from different organs and species. We have developed a double-antibody radioimmunoassay for the quantitative measurement of human goblet cell mucin (GCM) in order to study intestinal mucus in human and other species. The assay used 3H-labeled mucin as the antigen, rabbit antisera, and sheep anti-rabbit IgG antisera as the second antibody. A number of applications of the assay were investigated. A survey of human tissues revealed that mucins of the rectum, colon, and small intestine had identical affinity for the rabbit antibody, whereas lung eyelid conjunctiva, esophagus, and stomach reacted less strongly. GCM concentration ranged from 1.9 to 14 microgram mucin protein/mg tissue protein in the small and large intestine, respectively. The radioimmunoassay was also found to be useful as a marker during the isolation of GCM from human ileal extracts, where it indicated that a 10,000-fold purification had been achieved. Antigenic determinants of the mucin did not rely upon ABH blood group-specific terminal sugars in oligosaccharide chains. A comparison of mucins among various species revealed a partial species specificity of the GCM antibody. Human GCM cross-reacted with dog, monkey, and rabbit mucins, but not with mucins of rat, pig, toad, and oyster. Organ distributions of cross-reactive mucins in rabbit tissues indicated a pattern that was qualitatively similar to that seen in human tissues. Possible implications of these findings for autoimmune diseases are briefly discussed.


Purinogenic immunodeficiency diseases. Differential effects of deoxyadenosine and deoxyguanosine on DNA synthesis in human T lymphoblasts. Deoxyadenosine and deoxyguanosine are toxic to human lymphoid cells in culture and have been implicated in the pathogenesis of the immunodeficiency states associated with adenosine deaminase and purine nucleoside phosphorylase deficiency, respectively. We have studied the relative incorporation of several labeled nucleosides into DNA and into nucleotide pools to further elucidate the mechanism of deoxyribonucleoside toxicity. In the presence of an inhibitor of adenosine deaminase [erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA], 5 muM], deoxyadenosine (1-50 muM) progressively decreased the incorporation of thymidine, uridine, and deoxyuridine into DNA, but did not affect uridine incorporation into RNA. This decrease in DNA synthesis was associated with increasing dATP and decreasing dCTP pools. Likewise, incubation of cells with deoxyguanosine caused an elevation of dGTP, depletion of dCTP, and inhibition of DNA synthesis. To test the hypothesis that dATP and dGTP accumulation inhibit DNA synthesis by inhibiting the enzyme ribonucleotide reductase, simultaneous rates of incorporation of [(3)H]uridine and [(14)C]thymidine into DNA were measured in the presence of deoxyadenosine plus EHNA or deoxyguanosine, and in the presence of hydroxyurea, a known inhibitor of ribonucleotide reductase. Hydroxyurea (100 muM) and deoxyguanosine (10 muM) decreased the incorporation of [(3)H]uridine but not of [(14)C]thymidine into DNA; both compounds also substantially increased [(3)H]cytidine incorporation into the ribonucleotide pool while reducing incorporation into the deoxyribonucleotide pool. In contrast, deoxyadenosine plus EHNA did not show this differential inhibition of [(3)H]uridine incorporation into DNA, and the alteration in [(3)H]cytidine incorporation into nucleotide pools was less impressive. These data show an association between accumulation of dATP or dGTP and a primary inhibition of DNA synthesis, and they provide support for ribonucleotide reductase inhibition as the mechanism responsible for deoxyguanosine toxicity. Deoxyadenosine toxicity, however, appears to result from another, or perhaps a combination of, molecular event(s).


Erythropoiesis in vitro. Role of calcium. The in vitro plasma clot technique was employed to examine the role of calcium during the interaction of erythropoietin and mouse erythroid progenitor cells. Erythropoietin-induced erythroid colony formation was increased 24% by the carboxylic ionophore A23187 (10 nM), whereas a 35% increase was produced by the carboxylic ionophore Ro 2-2985/1 (1 nM). EGTA (3 mM) inhibited erythropoietin-induced erythroid colony formation. Inhibition of erythroid colony formation by EGTA could be reversed by Ca2+, but not by Mn2+, Mg2+, Zn2+, or Fe2+. At least 30 min exposure of marrow cells to erythropoietin in vitro was required for production of erythroid colonies. EGTA substantially inhibited erythropoietin-induced erythroid colony formation even when the marrow cells were exposed to the hormone for up to 2 h before addition of the chelator. Marrow cells incubated first in calcium-free medium with erythropoietin and then cultured in the presence of calcium but not erythropoietin, failed to form erythroid colonies although colony formation occurred when erythropoietin was provided. Taken together, the data indicate that calcium is required for both extracellular and intracellular events during the interaction of erythropoietin with its target cells.


Biotin-response organicaciduria. Multiple carboxylase defects and complementation studies with propionicacidemia in cultured fibroblasts. Fibroblast cultures from two individuals with biotin-responsive organicacidemia were found to have a pleiotropic deficiency of propionyl-CoA carboxylase, beta-methylcrotonyl-CoA carboxylase, and pyruvate carboxylase activities after growth in biotin limited culture medium, conditions which do not affect the carboxylase activities of normal cells. All three enzyme activities were restored to normal levels after transferring the mutant strains to biotin-rich medium. Both patients excreted abnormal levels of an array of metabolic intermediates, including beta-methylcrotonate, beta-hydroxyisovalerate, beta-hydroxypropionate, and lactate, which reflect metabolic blocks at all three carboxylase sites.14 mutants deficient in only propionyl-CoA carboxylase activity from patients with propionicacidemia and the two biotin-responsive strains were examined for complementation with seven previously mapped pcc mutants. No new pcc complementation groups were identified. Nine of the mutants were mapped to group pccA. The remaining 12 mutants mapped to pccBC or its B or C subgroups, confirming the complex nature of this group. The biotin-responsive mutants failed to complement each other but did complement mutants from all the pcc groups. Thus biotin-responsive organicacidemia is defined by a new complementation group, bio. The results obtained in this study suggest that the bio mutants have a defect of either biotin transport or a common holocarboxylase synthetase required for the biotin activation of all three mitochondrial carboxylases.


Characteristics of Pseudomonas aeruginosa in relation to laboratory-induced resistance to gentamicin. Pseudomonas aeruginosa strain C2 was habituated to gentamicin by serial passage in broth containing increasing concentrations of the antibiotic and up to 250 microgram/ml. The resistant progenies differed from the parent strain in antibiotic susceptibility to two other aminoglycosides, colonial morphology, lytic phage patterns, phage adsorption, and agglutination with the seven Fisher's antisera. All the progenies failed to grow at 42 degrees C and oxidised glucose in O/F tubes after incubation at 37 degrees C for three days but were catalase- and oxidase-positive. Reversion to the original properties of the parent strain was demonstrated in all cases after 10 serial subcultures in antibiotic-free broth.


Commissural columns in the sensory-motor cortex of monkeys. Callosally projecting cells and the terminal ramifications of their axons were identified in the monkey sensory-motor cortex by retrograde and anterograde labeling techniques, often by double labeling cells and axons in the same animal. Bundles of callosal fibers terminate in small column-like zones 0.5-1 mm wide in the motor cortex (area 4) and in the first (SI) and second (SII) somatic sensory areas. Such columns are aligned in register to form elongated strips extending mediolaterally in the long axes of the pre- and postcentral gyri. Significant portions of area 4, SI and SII, in regions corresponding to the representations of the hand and foot, are not callosally connected. The cells of origin of callosal fibers in SI are largely confined to layer IIIB and form columns and strips corresponding to the above. In connected zones of SI, the callosal connection is reciprocal and precisely point-to point. This and the laminar distribution of the terminal ramifications of callosal fibers (to layers I-IV) suggest that callosal fibers may arise from the terminate upon exactly homotopic, column-like groups of layer IIIB pyramidal cells. Commissurally projecting cells and their terminal ramifications are not limited to particular architectonic fields or particular parts of fields in SI. All architectonic fields of SI project heterotopically to the contralateral SII.


Commissural projections of the cortical hand motor area in monkeys. The topographical organization of the contralateral cortical projections of the motor hand area was studied with autoradiographic methods in 11 macaque monkeys. Two general observations were noted in the material studied. (1) The commissural cortico-cortical connections of the motor hand area were directed to the contralateral precentral gyrus. The projections were preferentially directed to both homotopic and non-homotopic areas. (2) Focally labeled areas of motor cortex (diameter 900 micrometers) gave rise to individual terminal columns of label (diameter 600-900 micrometers). Larger areas of labeled motor cortex, (3,000 micrometers in diameter) gave rise to contralateral terminal bands of label. These bands (600-1,000 micrometers in width) were oriented in an antero-posterior direction and appeared to be formed by a sequence of adjacent labeled columns.


The structural organization of the inferior and lateral subdivisions of the Macaca monkey pulvinar. Previous light microscopic studies of Macaca pulvinar have demonstrated that both the inferior and adjacent portion of the lateral pulvinar subdivisions are reciprocally connected to the entire occipital lobe, including striate cortex. They differ in that inferior but not lateral pulvinar receives a projection from the superficial layers of the superior colliculus. In this study, the internal organization of these two subdivisions in compared by relating light microscopic Golgi morphology to the synaptic organization observed by electron microscopy. The Golgi impregnated neurons in inferior and lateral pulvinar are typical of other thalamic nuclei and are not qualitatively different in the two subdivisions. Projections neurons (PN) vary in cell body (15--40 micrometers) and dendritic tree (150--600 micrometers) diameters but bear the same varieties of dendritic appendages; spine-like, hair-like, and knot-like. Local circuit neurons (LCN) have smaller cell body diameters (10--20 micrometers) but can have very large dendritic field diameters (150--600 micrometers). They are best distinguished from PNs by their elaborate dendritic appendages, which have been identified as pre-synaptic dendrites in the EM. LCN axons are infrequently seen. In the EM both subdivisions contain four types of synaptic terminals. RS and RL terminal both contain round symaptic vesicles and make asymmetric synaptic contacts, but are subdivided on the basis of small (RS = 0.09 micrometers) versus large (RL = 2.2 micrometers) cross sectional diameters and organelle content. RLs contact larger caliber dendrites and frequently form synaptic complexes with presynaptic dendrites of LCNs, while RSs contact fine caliber dendrites and rarely take part in synaptic complexes. F terminal and P boutons both contain flat and pleomorphis vesicles and make symmetric synaptic contacts. They are characterized by vesicle number and cytoplasmic density. Fs are infrequently observed in pulvinar compared to P boutons and are of uncertain origin. P boutons can be equated with LCN dendritic appendages and have been identified as pre-synaptic dendrites. The quantitative distribution of each type is very similar in both subdivisions, avveraging RS 85%, RL 5%, F 0.3%, P 8% and unidentified 2%.


The morphology and distribution of striate cortex terminals in the inferior and lateral subdivisions of the Macaca monkey pulvinar. The origin of the various types of axon terminals in Macaca pulvinar remains uncertain because of the contradictory results obtained in EM degeneration studies. We have used EM-autoradiography to determine the morphology of terminals in the inferior and lateral pulvinar which originate from neurons in visual cortex. After injections of H3 proline into area 17, both the small diameter (RS) and the large diameter (RL) terminals containing round vesicles and making asymmetric contacts are labeled in the two pulvinar subdivisions. Labeled and unlabeled terminals are intermixed within the pulvinar focus which suggests that the dendrites of the same pulvinar neuron receive overlapping inputs from several cortical areas. Because only 5% of the pulvinar terminals are RLs (Ogren and Hendrickson, '79), and this small number of RLs originates from at least two visual cortical areas plus the superior colliculus (Partlow et al., '77), superior colliculus input to inferior pulvinar is small compared to the combined RS and RL cortical input. Together the findings from this study and the preceding paper (Ogren and Henderickson, '79), show that while pulvinar is typical of other thalamic nuclei in the structure of its neurons and synapses, it differs in that the input from subcortical structures is minimal. It is suggested that inferior and lateral pulvinar function principally as integrators of visula cortical information.


Projections of the cerebellar and dorsal column nuclei upon the inferior olive in the rhesus monkey: an autoradiographic study. Projections from the cerebellar and dorsal column nuclei to the inferior olive of the rhesus monkey were traced with anterograde autoradiographic methods. The cerebellar nuclei give rise to a massive projection which reaches the contralateral inferior olivary complex by way of the descending limb of the superior cerebellar peduncle. Dentato-olivary fibers project exclusively upon the principal olivary nucleus (PO) and observe a strict topography. The dorsal, lateral, and ventral dentate project respectively to the dorsal, lateral, and ventral lamellae of the PO. Within the lamellae, the dentato-olivary fibers are related point for point in the medio-lateral axis. By contrast, the rostro-caudal topography is reversed so that the rostral pole of the dentate projects to the caudal PO and the caudal dentate to the rostral PO. These connections are predominantly crossed but a small ipsilateral component recrosses the midline at the olivary commissure and mirrors the topography on the opposite side. The anterior interpositus projects only to the medial half of the DAO and the posterior interpositus projects only to the rostral two thirds of the MAO. The ipsilateral component is minor in comparison with the contralateral projection, but appears to be more substantial than the ipsilateral projection to the PO arising from the dentate nucleus. The fastigial nucleus does not project upon the olivary complex. The dorsal column nuclei project topographically upon the contralateral accessory nuclei with the gracile nucleus sending fibers primarily to the lateral half of the DAO and the cuneate nucleus projecting to rostral cell groups of the MAO. The present results when compared with other olivary connections described by previous studies in a variety of species suggest that regions of the MAO and DAO receiving sensory information from the periphery may lie outside the influence of cerebellar feedback loops.


The distribution of oxytocin- and neurophysin-stained fibers in the spinal cord of the rat and monkey. Immunohistochemical methods have been used to describe the distribution of neurophysin I- and oxytocin-stained fibers in the spinal cord. In albino rats of either sex, such fibers descend through the dorsal part of the lateral funiculus to the caudal end of the cord. Fibers leave the lateral funiculus to innervate the marginal zone of the dorsal horn at all levels, and the intermediolateral column at thoracic, lumbar and sacral levels. Some fibers course medially through the intermediate gray to end in the central gray, which is innervated at all levels, and also appears to be innervated by fibers descending in the central gray itself. A semiquantitative analysis shows that the density of terminal fields in the intermediolateral column, marginal zone, and central gray varies significantly at different levels. The intermediolateral column is preferentially innervated between T1-T3, T9-T11, and T13-L2. The nucleus intercalatusspinalis and the dorsal commissural nucleus (between T13 and L2), both of which contain preganglionic neurons, are densely innervated. In the monkey (Macaca fascicularis), neurophysin I- and oxytocin-stained fibers descend through the lateral funiculus to the filum terminale, where some of them enter the pia-arachnoid surrounding the filum. The intermediolateral column is not innervated between T1-T3, is sparsely innervated at sacral levels, and is moderately innervated between T4 and L3; the greatest density of fibers occurs at L3. At most levels, few if any fibers are found in the central gray and marginal zone; the central gray is sparsely innervated between T8 and L2 at S2, and between Co3-Co6, and the marginal zone contains a small number of fibers in the region of S2 and Co6. Neurophysin I- and oxytocin-stained fibers were also found in the intermediolateral column, marginal zone, and central gray of homozygous Brattleboro, spontaneously hypertensive, and Egyptian sand rats. The results suggest that the paraventriculo-spinal pathway is particularly related to specific groups of sympathetic and parasympathetic preganglionic neurons, and to the marginal zone, whick is involved in the relay of ascending nociceptive information through the spinothalamic tract.


Improved methodology for quantitative determination of serum and milk proteins by single radial immunodiffusion. Single radial immunodiffusion is a simple inexpensive method for quantitating specific proteins in a heterogenous mixture. However, it has been criticized for its lack of accuracy and repeatability; coefficients of variation of 25% have been reported by some workers. We have adopted several refinements to the single radial immunodiffusion techniques which have appeared in the literature and have made several innovations of our own. Also, as an integral part of the technique, we have developed a computer program that determines the best-fitting curve for a given set of standards, applies this curve to the raw ring-diameters, and corrects the data for plate variation and sample dilution. This program has reduced errors greatly in calculating protein concentration since all data handling beyond the recording of the precipitant ring diameter is by computer. It also allows for the storage, retrieval, and analysis of large volumes of data on many different proteins simultaneously. The overall result of this system has been to decrease the coefficients of variation to below 5% for all the milk and serum proteins tested.


Effect of diet on the metabolism and toxicology of drugs. The human organism is continuously exposed to a variety of xenobiotics, as drugs, pesticides, food additives and many environmental contaminants, all of which are potentially toxic. The body metabolizes these highly reactive chemicals to pharmacologically-inert compounds, which are readily eliminated from the body. This process of deactivation is dependent on nutritional status. Malnutrition, almost invariably, leads to a reduced capacity to deactivate these exogenous materials with consequent increase in toxicity.


Genetic control of experimental autoimmune myasthenia gravis in mice. I. Lymphocyte proliferative response to acetylcholine receptors is under H-2-linked Ir gene control. As a first step in determining the genetic control of experimental autoimmune myasthenia gravis in mice, we tested the proliferative responses of lymph node cells to torpedo acetylcholine receptors (TAR). Studies with congenic and recombinant inbred strains of mice revealed that T-cell responses to TAR are controlled by an H-2 linked Ir gene, mapping in the I-A subregion of mouse major histocompatibility complex.


Macrophages in resistance to rickettsial infection: macrophage activation in vitro for killing of Rickettsia tsutsugamushi. Rickettsia tsutsugamushi, strain Gilliam, replicates in cultures of resident peritoneal macrophages from BALB/c mice. Macrophage cultures treated with culture supernatants of spleen cells from rickettsial-infected mice stimulated with heat-killed rickettsiae markedly suppressed macrophage infection by rickettsiae. Rickettsiacidal activity of activated macrophages was dependent upon both lymphokine concentration and time of incubation in lymphokines. Treatment of macrophage cultures with lymphokines before exposure to viable rickettsiae resulted in an immediate decrease in percent macrophages infected and numbers of viable intracellular rickettsiae. In these cultures, enhanced intracellular killing was also apparent with further incubation (24 hr). The immediate effect of lymphokine-pretreated macrophages was dissociated from intracellular killing by infecting macrophage cultures first and adding lymphokines after infection. In these cultures, both percent macrophages infected and titers of viable intracellular rickettsiae were dramatically reduced as well.


Lipid A from endotoxin: antigenic activities of purified fractions in liposomes. Isolation of lipid A by acid hydrolysis of Shigella flexneri lipopolysaccharide resulted in a product that consisted of a heterogeneous mixture of bands when visualized by thin layer chromatography. Differential extraction with ethyl acetate and chloroform, or extraction with EDTA, followed by chloroform-methanol-water (Bligh-Dyer extraction), or a combination of both extraction schemes, resulted in partial purification of immunologically active lipid A. Eight fractions were purified further by preparative thin layer chromatography, and each of the fractions had phosphate, carbohydrate, and esterified fatty acids. Upon incorporation into liposomes, five of the eight purified fractions reacted with anti-lipid A serum, but the three fractions with the most number of esterified fatty acids failed to react with anti-lipid A serum. At least one fraction that originally was unreactive with anti-lipid A serum became reactive as a hapten inhibitor upon removal of esterified fatty acids by alkaline hydrolysis. Alkali-treated fractions from "unreactive" and "reactive" lipid A had similar activities as hapten inhibitors. Our data suggest that lipid A can exist in multiple forms that differ by the number and placement, and possibly by the type, of fatty acids linked to the carbohydrate of lipid A. Highly acylated forms of lipid A do not react with antiserum against the unpurified lipid A mixture, but removal of fatty acids does expose immunoreactive groups.


Expression of a sheep red blood cell receptor by a murine lymphoma. Murine 6C3HED lymphoma cells were found to rosette with sheep red blood cells (SRBC). Normal C3H lymphocytes did not exhibit this property. This rosetting capacity of 6C3HED cells was found to be an accurate and reproducible means for discriminating between normal and tumor cells. The SRBC receptor on these lymphoma cells appeared to be serologically distinct from that expressed by normal human T lymphocytes since reciprocal blocking experiments demonstrated that inhibitory antisera were not cross-reactive. The expression of the SRBC receptor by the 6C3HED cells appeared to correlate with the expression of a tumor-associated antigen and was spatially related to an antigen expressed by 6C3HED and normal neonatal but not adult mouse cells.


Cross-reactivity of rat, mouse, and human IgD. Highly purified sheep anti-rat lymphocyte membrane IgD (mIgD) was used to detect cross-reactivity with the putative murine-delta chain on mouse lymphocytes. Cross-reactivity is demonstrated by indirect immunofluorescent staining and by immunoprecipitation of 125I-labeled lymphocyte membrane extracts followed by electrophoresis on 10% polyacrylamide gels. In addition, cross-reactivity of anti-rat-delta with human IgD is shown by gel diffusion analysis. The anti-rat-delta reagent stained both Ig5a+ and Ig5b+ lymphocytes. Preincubation of Ig5b+ (but not Ig5a+) cells with monoclonal allotype-specific antibodies (anti-Ig5b) under capping conditions caused inhibition of staining by the sheep anti-rat-delta reagent, indicating that it is the delta-chain that is recognized on mouse lymphocytes and that the anti-rat-delta reagents does not distinguish between mouse-delta allotypes. Furthermore, absorption of the sheep anti-rat-delta serum with purified human IgD reduced subsequent staining of mouse lymphocytes by approximately 50%; staining was not affected by absorption with human IgM. This xenogeneic anti-delta antiserum appears to detect determinants on the delta-heavy chain, which are shared by at least three species of mammals, suggesting that these determinants represent important molecular features conserved during evolution.


Brucella abortus lipopolysaccharide is mitogenic for spleen cells of endotoxin-resistant C3H/HeJ mice. Preparations of lipopolysaccharide (LPS) from rough and smooth strains of Brucella abortus were mitogenic for spleen cells of athymic nude mice, C3H/HeAU mice, and the endotoxin-resistant C3H/Hej mice. The mitogenic response induced by crude smooth-LPS (f5) was greater than that produced by purified smooth-LPS (f5p); however, the dose-response curves were similar for both preparations. The mitogenic activity of mouse spleen cells to both f5 and f5p was higher than that produced by stimulation with purified rough-LPS. The dose-response curves with rough-LPS were also qualitatively different from those produced with the preparations of smooth-LPS.


A simple two-step method for purification of secretory IgA from human colostrum. This paper describes an extremely simple method for the purification of secretory IgA in two steps from a colostrum pool. The first step of this technique utilizes the agar transparency effect produced by high concentrations of secretory IgA contained in colostrum pool when it is subjected to electrophoresis in this support. The secretory IgA is obtained by extraction and elution of the transparent zone adjacent to the well. In the second step, in order to obtain the maximum degree of purity, the eluate is subjected to precipitation with ammonium sulfate (1.33 M) or chromatographed through Sephadex G-200 gel. The simplicity of the method and the purity of the protein obtained make this procedure suitable for purification of macromolecular substances in concentrated state.


Immunohistological staining of antigens on semithin sections of specimens embedded in plastic (GMA-Quetol 523). Glycol methacrylate-Quetol 523, introduced by Kushida (1977) for combined light and electronmicroscopy studies at low magnification, also permits application of immunofluorescence methods to semithin sections. To recover the antigenicity of proteins fixed with formaldehyde, abrupt dehydration before embedding and subsequent treatment of the semithin sections with protease were essential. Post-staining with suitable histological stains allows exact correlation of antigen localization with tissue structure.


Immunological studies on Pityrosporum genus and Malassezia furfur. The antigenicity of Malassezia furfur from patients with tinea versicolor and 3 species of Pityrosporum was investigated using the antiserum against P. orbiculare. The Ouchterlony gel diffusion test revealed a considerable similarity between the antigenicities of P. orbiculare and P. ovale, and little similarity between P. orbiculare andP. canis. Similar results were obtained by immunofluorescence staining with the FITC-labeled P. orbiculare antiserum. Hyphae and round spores of M. furfur in the scales and biopsy specimens from the lesions in patients with tinea versicolor revealed specific fluorescence much the same as seen in cases of P. orbiculare or P. ovale. The FITC-labeled antiserum absorbed with P. orbiculare or P. ovale failed to give a positive reaction with hyphae and round spores of M. furfur. These findings suggest a similar antigenicity among P. orbiculare, P. ovale and M. furfur.


Response to therapy in an experimental rabbit model of meningitis due to Listeria monocytogenes. A uniformly fatal, reproducible model of experimental meningitis due to Listeria monocytogenes was developed in rabbits for study of the natural progression of the disease and was used to evaluate treatment regimens. Bacterial titers in cerebrospinal fluid and pleocytosis approximated those found in humans. Therapeutic studies in vivo revealed that rifampin was less rapidly bactericidal than ampicillin or penicillin, the agents usually recommended for treatment of meningitis; that penicillin plus rifampin was no more efficacious than penicillin alone; that ampicillin demonstrated greater bactericidal activity in vivo than did penicillin; and that addition of an aminoglycoside (gentamicin) to either penicillin or ampicillin significantly enhanced their bactericidal activity in vivo. Ampicillin plus gentamicin was the most effective combination in vivo and may represent the preferred mode of therapy for listeria meningitis.