Covalent structure of collagen: amino acid sequence of cyanogen bromide peptides from the amino-terminal segment of type III collagen of human liver. Human liver type III collagen was prepared by limited pepsin digestion, differential salt precipitation, and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type III collagen chains yielded nine distinct peptides. Three peptides, alpha1(III)-CB3, alpha1(III)-CB7, and alpha1(III)-CB6, were isolated by carboxymethylcellulose chromatography and Sephadex G-50 SF gel filtration. Automated Edman degradation together with selective hydroxylamine cleavage and chymotrypsin and trypsin digestion enabled determination of their complete amino acid sequence. Compared with type I collagen, the data show tentative homology of alpha1(III)-CB3 with alpha1(I)-CB1, alpha1(I)-CB2, and alpha1(I)-CB4; alpha1(III)-CB7 with alpha1(I)-CB5; and alpha1(III)-CB6 with the amino-terminal portion of alpha1(I)-CB8. Close interspecies homology was found between the sequences presented here with 90 residues of alpha1(III)-CB3 and 26 of alpha1(III)-CB8 of calf aorta. The present study establishes the amino acid sequence of 229 residues near the amino terminus or nearly one-quarter of the type III collagen chains. The disaccharide, Glc-Gal, was convalently bound to hydroxylysine at a position corresponding to the same location in the alpha1(I) chain.


Photochemical functionality of rhodopsin-phospholipid recombinant membranes. Purified rhodopsin was incorporated into phospholipid bilayers to give recombinant membranes. The photochemical functionality in these systems was examined by low-temperature spectroscopy and by kinetic spectrophotometry. Changes in the absorption spectra of glycerol-water mixtures of rhodopsin-egg phosphatidylcholine and rhodopsin-asolectin recombinants were monitored after the sample was cooled to -196 degrees C, presented with light of wavelength greater than 440 nm, and then warmed gradually to room temperature. Absorption characteristics indicative of the spectral intermediates prelumirhodopsin, lumirhodopsin, metarhodopsin I, and metarhodopsin II were observed. The kinetics of the metarhodopsin I -o metarhodopsin II transition in these recombinants was studied by flash photolytic observation of the decay of meta I and the formation of meta II. Recombinants prepared from unsaturated phospholipids, e.g., asolectin, egg phosphatidylcholine, egg phosphatidylethanolamine, and dioleoylphosphatidylcholine, showed first-order kinetics for the transition with rates comparable to that of rod outer segment membranes. Recombinants prepared from saturated phosphatidylcholines have a retarded rate of conversion from meta I to meta II and are considered to be nonfunctional. The photochemical functionality of rhodopsin-phospholipid recombinants is dependent upon the presence of phospholipid unsaturation and the fluidity of the phospholipid hydrocarbon chains, and is independent of the polar head group of the phospholipid.


Specificity of the phosphatidylcholine exchange protein from bovine liver. The phosphatidylcholine exchange protein from bovine liver stimulates the specific transfer of phosphatidylcholine (PC) from rat liver microsomes to mitochondria or phospholipid vesicles (Wirtz, K.W.A., Kamp, H.H., and van Deenen, L.L.M. (1972), Biochim. Biophys. Acta 274, 606). In the present study, it has been established which components of the PC molecule are essential to the specific interaction with the protein. Radiochemically labeled analogues of PC have been synthesized with modifications in the polar and apolar moiety, and their transfer was measured between donor and acceptor vesicles. Relative to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (egg yolk PC), transfer is inhibited or abolished when (a) the distance between phosphorus and nitrogen is decreased or increased and (b) a methyl group on the quaternary nitrogen is removed or substituted by an ethyl or propyl group. Transfer is much less affected when (a) the ester bonds are replaced by ether or carbon-carbon bonds, (b) the PC molecule contains two saturated fatty acids, and (c) the D stereoisomer is used. It is concluded that the protein has a binding site which interacts specifically with the phosphorylcholine head group and which cannot accommodate substantial configurational changes. Interaction with the apolar moiety of PC is less specific. However, lyso-PC is not transferred, suggesting that two hydrocarbon chains are required to stabilize the exchange protein-phospholipid complex. Interaction of [14C]PC-labeled exchange protein with vesicles of different phospholipid compositon has been analyzed by measuring the release of [14C]PC into these vesicles. Vesicles of egg PC or dimethylphosphatidylethanolamine function as acceptors, in contrast to vesicles of sphingomyelin or phosphatidylethanolamine.


Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase. The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.


Synthesis of elastin in aortas from chick embryos. Conversion of newly secreted elastin to cross-linked elastin without apparent proteolysis of the molecule. The biosynthesis of elastin was examined in matrix-free cells isolated by enzymic digestion of aortas from 17-day old chick embryos. After the cells were incubated with [14C]proline and then were rapidly boiled in buffer containing high concentrations of protease inhibitors and sodiumdodecyl sulfate, about one-quarter of the intracellular 14C-labeled protein was recovered as an elastin component with an apparent molecular weight of about 72 000. Examination of the medium from the cell suspension indicated that the largest elastin component secreted by the cells also had an apparent molecular weight of about 72 000. Pulse-chase experiments with intact aortas demonstrated that about two-thirds of the 72 000-dalton component disappeared in 2 h, apparently because it was converted to cross-linked fibers. When cross-linking was inhibited with penicillamine, the 72 000-dalton component persisted in the tissue 5 h. When cross-linking was inhibited with beta-aminopropionitrile, the elastin component of 72 000 daltons persisted for about 2 h, but thereafter it was gradually degraded to small peptides which were recovered in the incubation medium. The results suggest that elastin is secreted by cells in chick aorta as a polypeptide of about 72 000 daltons and that the secreted protein is incorporated into elastin fibers without cleavage to a protein of considerably smaller size.


Crystallographic studies of protein denaturation and renaturation. 1. Effects of denaturants on volume and X-ray pattern of cross-linked triclinic lysozyme crystals. Triclinic crystals of hen egg-white lysozyme cross-linked with glutaraldehyde have been treated with various denaturants and found to be susceptible to x-ray structure analysis even after major conformational changes in the protein. Cross-linked crystals were isomorphous with the native form, and electron density difference maps indicated the locations of intermolecular corss-links, but showed no appreciable differences in the protein conformation. Soaking of the cross-linked crystals in danaturant solutions of increasing concentrations caused corresponding increases in crystal volume and decreases in minimum observable x-ray spacings. These changes proved partly reversible on diluting the solutions, and measurements of crystal volume and minimums x-ray spacing were used to follow denaturation and renaturation as a function of concentration for several denaturants. Some of these, including bromoethanol and sodium dodecyl sulfate, had little effect on the crystals below critical concentrations at which there was a sharp volume increase and loss of x-ray pattern, which could, however, be regenerated to about 3.2-A resolution. Others, including KCNS and urea, caused more gradual changes, but with a smaller degree of recovery. It is suggested that at least two different denaturation mechanisms are involved with detergent-like reagents disrupting the hydrophobic interactions joining the two wings of the lysozyme molecule and hydrophilic denaturants interacting primarily with polar groups on the molecular surface.


Nucleoprotein chromatin subunit from Physarum polycephalum. The nucleoproteins resulting from digestion of the nuclei of the true slime mold Pysarum polycephalum with micrococcal nuclease have been resolved according to the size classes in linear sucrose gradients containg 0.5 M NaCl, and analysed for DNA, RNA and protein content. The basic nucleoprotein subunit has been found to contain a DNA fragment of about 150--170 base pairs complexed with an approximately equal amount, on a weight basis, of basic proteins and a relatively small amount of non-histone proteins (about 35% of the amount of DNA). Higher nucleoprotein oligomers were shown to contain spacer DNA fragments between adjacent subunits and a considerably higher ratio of non-histone proteins to DNA than the basic subunit. Both the basic subunit and higher nucleoprotein oligomers of Physarum chromatin contain some amount of tightly bound RNA. However, in contrast to the distribution of the non-histone proteins, the ratio of RNA to RNA is similar in both fractions.


Protein composition of chromatin subfractions prepared from chick embryos. Chromatins of four embryonic chick tissues were digested with DNAase II and the MgCl2-soluble and insoluble chromatin fractions were isolated. The MgCl2-soluble fractions displayed a high protein/DNA ratio and enrichment of nascent RNA as compared to the MgCl2-insoluble fractions. The recovery of DNA and protein in the MgC12-soluble chromatin of erythrocytes was much lower than that in the other cell types. The difference is likely to be associated with the low transcriptional activity of the erythrocyte chromatin. In polyacrylamide gels histones appeared to be the predominant protein constituent of the MgCl2-insoluble chromatins. Brain, skin and muscle displayed an apparently similar group of actively labelled nonhistones at 70 000 to 100 000 daltons. These nonhistones were not observed in erythrocytes. The MgCl2-soluble chromatins of erythrocytes, brain, skin and muscle had a prominent group of polypeptides at approx. 40 000 daltons. In all tissues except for erythrocytes the group of proteins was actively labelled. These polypeptides are suggested to be the major structural protein constituent of the MgCl-soluble chromatin.


Preparation and enzymatic hydrolysis of dinucleoside monophosphates and DNA modified with aromatic residues. The following procedures have been used to prepare fifteen modified dinucleoside monophosphates: (a) bisulfite-catalyzed transamination with aniline to give an N4-phenylcytidine (CPh), (b) bisulfite-catalyzed transamination with beta-naphthylamine to give an N4-beta-naphthylcytidine (CbetaN), (c) alkylation with 7-bromomethylbenz[a] anthracene to afford a 7(benz[a]anthryl-7-methyl)guanosine (GMBA), and (d) reaction with N-acetoxy-2-acetylaminofluorene to give an 8-(N-2-fluorenylacetamido)guanosine (GAAF). The compounds prepared were A-CPh, CPh-A, CPh-G, U-CPh, CPh-U, A-CbetaN, CbetaN-A, G-CbetaN, CbetaN-G, U-CbetaN, CbetaN-U, GMBA-U, U-GMBA, GAAF-U, and U-GAAF. All of the modified compounds were hydrolyzed to the expected monomers with venom and spleen exonucleases. Hydrolysis by micrococcal nuclease was inhibited in the following cases: A-CPh, A-CbetaN, U-GMBA, and U-GAAF. The first three reactions above were applied to denatured calf thymus DNA to prepare modified DNA samples containing from 0.3 to 2.0% bound aromatic residues. The modified nucleic acids were completely hydrolyzed to nucleosides by the combination of venom exonuclease, deoxyribonuclease I and alkaline phosphatase. The same results were obtained with a combination of spleen exonuclease, deoxyribonuclease II, and alkaline phosphatase. Hydrolysis of the modified nucleic acids by micrococcal nuclease and alkaline phosphatase afforded primarily nucleosides, with some dinucleoside monophosphates. The amount of the latter did not exceed that found in the hydrolysis of control DNA, however. Other workers have observed inhibition of enzymatic hydrolysis of nucleic acids modified by aromatic carcinogens. We postulated that their results may have been caused by cross-links, which were avoided in our studies.


On the relative location of the inhibitor-and calcium-binding sites in bovine trypsin as determined by nuclear magnetic resonance. Possible ambiguities in paramagnetic probe mapping studies. Possible pitfalls in mapping studies utilizing the nuclear relaxation rates induced by paramagnetic probes are pointed out. In cases in which a distance is sought between a paramagnetic ion and a small molecule (e.g. substrate, inhibitor, etc.), both bound non-covalently to a macromolecule, heterogeneity in the system with respect to the binding of either of them may result in ambiguous conclusions. It is shown that the trypsin-gadolinium (III)-inhibitor system is heterogeneous, as revealed in the dependence of the water and inhibitor proton line-widths upon both the Gd3+ and the enzyme concentrations and in the effects of added Ca2+ on the line-widths. The results imply that in published work (Abbott et al. (1975) Biochemistry 14, 4935) the distance from a weak rather than from the strong metal ion binding site of trypsin (EC 3.4.21.4) may have been determined.


Sulfhydryl groups in relation to the structure and catalytic activity of 2-oxo-4-hydroxyglutarate aldolase from bovine liver. Bovine liver 2-oxo-4-hydroxyglutarate aldolase (suggested name: 2-oxo-4-hydroxyglutarate glyoxylate-lyase catalyzing the reaction: 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate) contains eight to ten sulfhydryl groups as determined by titration of the enzyme with either 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or p-mercuribenzoate in the presence of 1% sodium dodecyl sulfate. In the absence of a denaturant, all of the cysteinyl residues react with p-mercuribenzoate whereas only four are accessible to titration with Nbs2. No differences in -SH group reactivity can be detected during titration of the aldolase with p-mercuribenzoate. In contrast, two classes of sulfhydryls can be differentiated in the disulfide exchange reaction with Nbs2 in the absence of a denaturant; one -SH group (Class I) reacts rapidly whereas three additional thiols (Class II) titrate at approx. 0.1 the rate of the Class I-SH residue. Both pyruvate and glyoxylate protect one of the three -SH residues in Class II from reaction with Nbs2. Either substrate also prevents titration of one to two thiol groups by p-mercuribenzoate and decreases the rate of reaction of aldolase -SH groups with Nbs2 in 8 M urea. These ligand-induced changes in -SH reactivity provide a sensitive indication that the enzyme exists in an altered conformational state in the presence of either of its cosubstrates. Titration of the enzyme with either Nbs2 or p-mercuribenzoate results in a progressive loss of aldolase activity which is not proportional to the number of -SH groups modified. The enzyme retains 50% of the activity of the native enzyme when Class I and Class II thiols (i.e. four -SH groups total) are modified with Nbs2; 15% residual activity is still observed following titration of all of the cysteinyl residues with p-mercuribenzoate. Pyruvate and glyoxylate provide partial protection against inactivation. It is concluded that inactivation of 2-oxo-4-hydroxyglutarate aldolase by Nbs2 or p-mercuribenzoate is a consequence of alterations in protein structure which accompany modification of -SH groups. The data argue against the direct participation of an active-site thiol group in the catalytic mechanism of 2-oxo-4-hydroxyglutarate aldolase, be that aldol cleavage and condensation or beta-decarboxylation.


Interaction of glucagon with dimyristoyl glycerophosphocholine. Glucagon can form amphipathic helices and can interact with dimyristoyl glycerophosphocholine at temperatures below the phase transition leading to a shift in the fluorescence emission maximum of tryptophan from 350 to 338 nm and a 3-fold enhancement of fluorescence intensity as well as a change in the polarization of fluorescence. The circular dichroism properties of the lipid-associated glucagon indicates that it has an increased content of alpha-helix. The phase transition temperature of the lipid as monitored by pyrene excimer fluorescence is not altered by interaction with glucagon although at higher glucagon/lipid ratios a decrease in excimer formation is noted at low temperature. Above the phase transition temperature, the addition of lipid has no effect on the fluorescence emission or circular dichroism of glucagon. Thus this hormone can interact with dimyristoyl glycerophosphocholine and this interaction is stronger below the phase transition temperature than above it.


Incorporation of galactose into galactosyltransferase. Bovine skim milk galactosyltransferase (EC 2.4.1.22) retained its catalytic activity after partial enzymatic removal of sialic acid and galactose. Desialylated and degalactosylated galactosyltransferase was a galactosyl acceptor in the galactosyltransferase reaction. [14C]Galactose from UDP-[14C]galactose was incorporated into the carbohydrate-depleted galactosyltransferase by native galactosyltransferase. The results suggest that galactosyltransferase participates in the biosynthesis of its glycopeptides of the sialic acid-galactose-N-acetylglucosamine type.


Uptake and degradation of formaldehyde-treated 125I-labelled human serum albumin in rat liver cells in vivo and in vitro. The uptake of formaldehyde-treated 125I-labelled human serum albumin in rat hepatocytes and nonparenchymal liver cells was measured in vivo and in vitro. Isolated liver cells were prepared by treating the perfused liver with collagenase. Purified hepatocytes and nonparenchymal cells were obtained by differential centrifugation. Human serum albumin was found to be taken up exclusively or almost exclusively by nonparenchymal cells in vitro and in vivo (after intravenous injection). The maximal rate of human serum albumin-uptake in vitro was comparable to that in vivo. Nonparenchymal cells degraded human serum albumin in vitro as indicated by release of trichloroacetic acid-soluble radioactivity. Degradation started about 20-30 min after addition of human serum albumin to cells and rate of degradation was proportional to rate of uptake. Human serum albumin-degradation could be studied without interference of concurrent uptake by separating cells that had been preincubated with human serum albumin from the medium and then reincubating them with human serum albumin-free medium. The lag phase before human serum albumin-degradation starts and the inhibitory effect of chloroquine on degradation indicate that human serum albumin is degraded in lysosomes. The data obtained show that enzymatically prepared nonparenchymal liver cells retain their endocytic activity in vitro. Denatured human serum albumin should be useful both as a marker for rat liver macrophages and for the study of intracellular proteolysis in these cells.


Glutathione dependent control of protein disulfide-sulfhydryl content by subcellular fractions of hepatic tissue. The disulfide-sulfhydryl (SS/SH) ratios of subcellular fractions of rat hepatic tissue were found to vary diurnally with the ratio lowest in the early morning and highest in the early evening. These changes were found in the nuclear, microsomal and cytosol fractions. The primary reaction is the reversible formation of mixed disulfides of glutathione with proteins. This formation is controlled by the activity of thiol transferase and the level of oxidized glutathione (GSSG) as substrate. Several enzymes including mitochondrial and microsomal oxidases, glutathione reductase and peroxidase and glucose-6-phosphate dehydrogenase were found to control the levels of GSSG. An NADPH-dependent microsomal oxidase system, inhibited by GSSG, was found to produce activated oxygen which served as substrate for flutathione peroxidase. Evidence is presented for the concept that the formation of mixed disulfides of proteins with glutathione is a mechanism for maintenance of a disulfide-sulfhydryl ratio such that the integrity of particulate membranes is maintaine during oxidative and reductive stresses on the hepatic cells.


Agent-determined type of maternal influence on induced cleft palate in mice. The maternal influence on induced cleft palate frequency, as revealed by reciprocal crosses, was investigated after treatment with triamcinolone or cortisone. Mouse blastocysts from the CBA and A/Jax strains were transferred to pseudopregnant A/Jax and CBA foster mothers and in addition CBA X A/Jax and A/Jax X CBA embryos were raised in pseudopregnant CBA foster mothers. According to the period of maximum sensitivity revealed by a time response study triamcinolone was injected as a single dose (2 mg/kg, i.m.) on day 11 at 8 a.m. when the precocious development of transferred fetuses had been taken into account. A predominant uterine factor slightly modified by the fetal genome was found. This was in contrast to the effect of the 4-day treatment with cortisone (62.5 mg/kg i.m.) where, as also previously has been shown, cytoplasmic factors in the embryos were accountable for the magnitude of the teratogenic response. An increased corticoid elimination from the resistant CBA fetuses might explain the maternal influence on triamcinolone treatment but would not be responsible for the influence on cortisone-induced cleft palate frequency.


The release of prolactin by medroxy-progesterone acetate in human subjects. Medroxyprogesterone acetate an injectable contraceptive when administered to four lactating women at a dose of 150 mg every three months significantly raised prolactin levels over those observed in four control women. Prolactin levels were higher at all times during 4-17 weeks in the drug-treated women, irrespective of whether the samples were collected before or after suckling. It was also observed that while suckling clearly released prolactin in both groups the difference was greater in the women receiving the injection, indicating that the drug enhanced the release of prolactin in response to the suckling stimulus.


Studies of daily recordings from the Moos Menstrual Distress Questionnaire. Nineteen volunteers completed a Moos Menstrual Distress Questionnaire daily for a period exceeding one menstrual cycle. The data were analysed, using a least mean square method of fitting sine waves. The fact that the results obtained on this group are essentially those found by other workers looking at the menstrual cycle suggests that this may be a useful method for assessing menstrual distress.


Vulnerability and conditioning in relation to the human menstrual cycle. An experiment aimed at testing some implications of a model of symptom acquisition is reported. In particular, the effect conditioning and extinction is examined. Sixteen female phobic patients were tested either immediately pre-menstrually or inter-menstrually. Using white noise as the UCS and a blue light as the CS, conditioned GSR responses were readily established in members of the pre-menstrual group, who also showed resistance to extinction. Subjects tested inter-menstrually were less susceptible to these effects. Those subjects taking the contraceptive pill showed increased sensitivity to the acquisition of conditioned responses.


Significance of leucocytes in smears taken from the female urethral meatus. A prospective study of 86 women attending the clinic for the first time showed that nine patients had more than 10 leucocytes per highpower microscope field (HPF) in smears taken from the urethral meatus, even though six of them had micturated less than two hours before examination. Six of these patients had urethral gonorrhoea, one had trichomoniasis, and two were contacts of men with non-specific urethritis. It is concluded that women with more than 10 leucocytes per HPF of urethral secretion at their first visit and in whom a diagnosis has not been made, should strongly be urged to return for further assessment.


Clinical characteristics of familial acute anterior uveitis. Of 24 patients with familial acute anterior uveitis (AAU) 22 cases were non-granulomatous and 2 granulomatous. Of the 23 HLA tested patients 19 had the antigen HLA-B27. The 2 granulomatous and 2 non-granulomatous AAU cases were HLA-B27 negative. The iritis associated with the antigen HLA-B27 was unilateral, acute and recurrent, with cells and flare in the aqueous humor, and it lasted on an average 5 weeks. Keratic precipitates were never mutton fat, if the antigen B27 was present. X-ray examination of 8 patients revealed sacroilittis in 5. It is concluded that certain factors like trauma or infective agents may provoke familial AAU which is in close relationship with the HLA-B27 associated rheumatic group of diseases.


Immunological studies of human anterior uveitis. Immunofluorescent studies revealed extracellular deposits of IgG and C3 as well as immunoglobulin containing inflammatory cells in the affected iris of a patient suffering from endogenous acute uveitis. Antibodies to uveal components were detected in the serum of 4 out of a total of 23 endogenous uveitis patients examined. Examination of these 23 sera also revealed antinuclear activity in one, antismooth muscle activity in one, and anti-mitochondrial activity in three. None of the sera showed anti-thyroglobulin activity. Antibodies to uveal components or immune complexes containing 'auto-antibodies' do not appear to be a common source of tissue damage in human endogenous anterior uveitis.


Effect of local antilymphocyte serum on antibody forming cells. Locally administered antilymphocyte serum was irritating to the eye and failed to alter the production of antibody-forming cells in the homolateral lymph nodes, corneas, and uveal tracts of rabbits that had received intracorneal injections of bovine gamma globulin.


Mechanism of uptake of nitrosoureas by L5178Y lymphoblasts in vitro. The mechanism of uptake of nitrosoureas by L5178Y cells in vitro was investigated. A time course of the uptake of radioactivity on incubation of L5178Y lymphoblast with [14C]-1,3-bis(2-chloroethyl)-1-nitrosourea was linear for 30 min and then entered a plateau phase; it was markedly temperature dependent. A similar time course for cells incubated with [14C]ethylene-labeled 1-(2-chlorethyl)-3-cyclohexyl-1-nitrosourea reached equilibrium rapidly, was temperature independent, and resulted in a relatively low level of uptake of radioactivity. However, cells treated with 3-[cyclohexyl-14C]-1-(2-chlorethyl)-1-nitrosourea had a time course that was linear for 30 min, resulted in much higher levels of uptake of radioactivity, and was strongly temperature dependent. These findings, at least for 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, suggest that some drug decomposition precedes uptake. The percentage of radioactivity found in the cell sap fraction was at least 85% of total cell activity when cells were incubated with any of the three 14C-labeled nitrosoureas. Furthermore, thin-layer chromatography of the cell sap fraction revealed the presence of free intact drug. These findings indicate that intracellular uptake of intact nitrosoureas occurred. A time course of uptake of intact 1,3-bis(2-chloroethyl)-1-nitrosourea reached equilibrium rapidly with cell/medium distribution ratios of 0.2 to 0.6 and was temperature independent. The addition of excess unlabeled 1,3-bis(2-chlorethyl)-1-nitrosourea or 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea had no effect on uptake of [14C]-1,3-bis(2-chloroethyl)-1-nitrosourea, These findings suggest that uptake of intact 1,3-bis(2-chloroethyl)-1-nitrosourea was by passive diffusion. A time course of the uptake of intact 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with either [14C]ethylene- or ring-labeled drug rapidly reached equilibrium, was temperature independent, and attained a cell/medium ratio greater than unity. Uptake of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was sodium independent and was unaffected by the metabolic inhibitors (sodium fluoride, sodium cyanide, or 2,4-dinitrophenol) or by urea, a potential physiological competitor. Furthermore, addition of unlabeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea or 1,3-bis(2-chlorethyl)-1-nitrosourea had no effect on uptake of labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. These findings suggest that uptake of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea also occurs by passive diffusion.


Microfilament bundles and cell shape are related to adhesiveness to substratum and are dissociable from growth control in cultured fibroblasts. The distribution of microfilament bundles in cells was examined using antibodies to fibroblast myosin and indirect immunofluorescence microscopy. There is no correlation between the presence of bundles of microfilaments and normal growth control. A normal cell line (Balb/c 3T3) cultured on a poorly adhesive substratum showed no microfilament bundles. Similarly, a mutant cell line (AD6) with normal growth, but a rounded shape due to defective adhesiveness to substratum, showed no bundle formation. On the other hand, two transformed cell lines with a flat morphology (Swiss SV3T3 and Balb MSV-85) showed extensive bundle formation. When a transformed cell line with poor adhesiveness (MC5-5) was treated with CSP (a major surface glycoprotein of normal cells) which increases adhesiveness to substratum, the cells formed extensive microfilament bundles without any decrease in growth. We conclude that the distribution of microfilament bundles is related to adhesiveness to substratum and cell shape but not to growth properties.


Isolation and characterization of trout testis protamine mRNAs lacking poly (A). Poly(A)+ protamine mRNA was isolated from trout testis cells in a very pure form, and artificial poly(A)- protamine mRNA molecules were derived from it by enzymatic deadenylation with RNAase H from calf thymus after hybridization with oligo(dT). The deadenylated protamine mRNA was found to be active in a wheat germ cell-free system and yielded a labeled product which co-migrated with authentic protamine. These deadenylated mRNA molecules were subsequently used as markers on denaturing polyacrylamide gels to identify and allow the purification of the poly(A)- protamine components known to exist in vivo in the total cellular poly(A)- RNA. RNA species of molecular weights similar to the enzymatically deadenylated subcomponents of protamine mRNA were observed in the natural poly(A)-RNA population of the testis cells. These naturally occurring poly(A)- protamine mRNAs were isolated by preparative  gel electrophoresis and further characterized by 3H-poly(U) hybridization assay, by hybridization to complementary DNA made against highly purified poly(A)+ protamine mRNA, and by their ability to direct protamine synthesis in a cell-free system.


Pancreatic DNAase cleavage sites in nuclei. The DNA of nuclei is cleaved by a variety of nucleases in such a way that the cuts on a given strand are always separated by an integral multiple of 10 nucleotides. However, the spacing between cutting sites on opposite strands is not known for any nuclease. In this paper, we describe the determination of the spacing, or stagger, between cuts on opposite strands produced by the action of pancreatic DNAase (DNAase I) on nuclei. When nuclei are digested with DNAase I and the resultant DNA is analyzed by gel electrophoresis without prior denaturation, a complex pattern of bands is observed. A method which gives better than 90% recovery of DNA from polyacrylamide gels was used to isolate the individual fractions corresponding to these bands. The structure of the fractions was then determined using single-strand-specific nuclease to digest single-stranded "tails" and using DNA polymerases to extend recessed 3'-OH termini of partially duplex regions. Our results show that each component consists of a double-stranded region terminating in single-stranded tails at both ends. Although both chains of every duplex are 10-n nucleotides long (n integer), the chains are never completely paired. The experiments with DNA polymerase show an abundance of structures in which the 3'-OH termini of these duplexes are recessed by 8 nucleotides, and by inference, there must be structures with 5'-P termini recessed by 2 or 12 nucleotides. Thus DNAase I acts on nuclei to produce DNA with staggered cuts on opposite strands, separated by (10-n + 8) and (10-n + 2) base pairs (with 5'-P and 3'-OH termini extending, respectively). Two classes of models of DNA folding in the nucleosome have been proposed by other investigators to account  for the presence of DNAase I cleavage sites at 10-n intervals along each DNA chain. One class of models leads to the prediction that cuts should either be unstaggered or separated by 10 nucleotides, while the other class is consistent with staggers of 6 and 4 nucleotides. Neither prediction is verified by our data; however, all these models may be made consistent with the results by assuming that the enzyme's site of recognition on nucleosomal DNA is not the same as its site of cleavage.


Thallium 201 imaging and gated cardiac blood pool scans in patients with ischemic and idiopathic congestive cardiomyopathy. A clinical and pathologic study. In ischemic cardiomyopathy (CM) fibrosis replaces large segments of myocardium, but in idiopathic congestive CM the myocardium contains only small foci of fibrosis or is morphologically normal. As coronary disease and myocardial infarction may be clinically silent, it is not always possible to distinguish ischemic from idiopathic congestive CM during life without cardiac catheterization. To determine whether noninvasive methods, thallium 201 myocardial (Tl) imaging and technetium 99m gated cardiac blood pool scans (GCBPS), could separate the entities, we evaluated radioisotope images of the heart in 13 patients with ischemic, and eight patients with idiopathic congestive CM, and 14 patients with normal hearts. Diagnosis was setablished by cardiac catherterization and/or autopsy in each of the 35 patients. The 14 normals could be readily distinguished from CM, and ischemic could be distinguished from idiopathic dilated CM in 20 of 21 patients. All patients with myocardiopathy showed hypokinetic and dilated left ventricles, but right ventricular dilatation was evident mainly in those with idiopathic CM. Tl images in the ischemic type had defects of greater than 40% of image circumference which corresponded to segmental wall motion abnormalities on GCBPS, whereas those with the idiopathic congestive form were homogeneous or had defects of less than 20% of image circumference. Autopsy studies in 7 of 35 patients correlated Tl defects of greater than 20% of circumference with transmural myocardial fibrosis.


Genetic variability of clinical chemical values. A study of cardiovascular risk factors in middle-aged twin men provided an opportunity to test for genetic variability in the SMA 12/60 (Technicon) battery of clinical chemistry tests. Classical twin methodology was used to analyze the variation of monozygotic and dizygotic twins. In addition, frequency of co-twin contact was used to control for effects of differences in shared environment. Genetic variability played a definite role in controlling four of the 11 reported tests: one-hour serum glucose, serum urea nitrogen, uric acid, and bilirubin. No genetic variation was found for lactate dehydrogenase, phosphorus, and alkaline phosphatase. Significantly higher means for calcium, total protein, albumin, and aspartate aminotransferase in monozygotic twins precluded any statement about heredity and environment for these tests.


A radioimmunoassay for serum ferritin.

A radioimmunoassay for serum ferritin. We describe the development and evaluation of a serum ferritin radioimmunoassay, in which 125l-labeled ferritin and rabbit anti-ferritin antibody are used. Goat anti-rabbit gamma-globulin antibody, together with polyethylene glycol, is used as the separating reagent. The assay has a working range up to 500 mug of ferritin per litre, and a sample requirement of 75 mul of serum for assay at two dilutions. The assay requires 24 h. it has a sensitivity of 1.5 mug of ferritin per litre and a long-term precision (CV) of 13%. Reference intervals for a population of men were 18-330 mug/litre, with no marked age dependence, while those for a population of women older than 50 years were 18-200 mug/litre. Many apparently healthy women in the 20-50 year age group have much lower concentrations. Serum ferritin concentrations of less than 18 mug/litre are indicative of iron deficiency, defined as the absence of stainable iron in an aspirate of bone marrow.


Determination of serum bilirubin concentration during phototherapy of newborns and in vitro: results compared by the direct spectrometric method and the diazo method. During direct illumination of a serum bilirubin solution the bilirubin concentration decreased markedly, both as determined by the direct spectrometric method and (even more so) by the diazo method. In contrast, I found the same values for serum bilirubin concentrations as determined by these two methods for serum from untreated, "single light", and "double light" treated full-term infants with neonatal hyperbilirubinemia without blood type immunization. The same was true for untreated and "single light" treated premature infants with this disease. Furthermore, no difference was found in the above-mentioned relationship between "single light" treated infants with the same disease, and untreated infants with neonatal hyperbilirubinemia without immunization, all born at  term. This is important, because the direct spectrometric method is simpler and requires less serum than does the diazo method.


Rapid antibody production and sample preparation for radioimmunoassay of prostaglandin A. A dialysis equilibrium method for plasma or tissue preparation for radioimmunoassay of prostaglandins is presented and compared to the silica gel column technique. The plasma or tissue is extracted, dried, redissolved in buffer and dialyzed for further purification before assay. Results comparing both methods shows that dialysis equilibrium greatly simplifies the silica gel column technique yet preserves specificity, sensitivity and reproducibility. In addition a rapid micromethod for the development of a specific antibody to prostaglandin A is detailed.


Diurnal variations in plasma prolactin, growth hormone, cortisol and blood glucose in labile diabetes mellitus. In labile diabetes mellitus wihout ketoacidosis we have studied plasma prolactin levels and a possible causal connection between fluctuation in blood glucose concentration and plasma prolactin, growth hormone and cortisol levels. The hormone concentrations in plasma and blood glucose concentration were determined at 20 min intervals for a 24 h period in six male patients with insulin treated diabetes mellitus. Prolactin varied within the normal range but without any significant rise in relation to sleep in five out of the six patients. Growth hormone levels were low with superimposed secretory peaks. Plasma cortisol showed a normal diurnal rhythm. Blood glucose fluctuated as expected, but the variations and especially the falls in blood glucose to non-hypoglycaemic levels were not followed by increases in plasma hormone concentrations. No relationship could be demonstrated between the changes in the plasma concentration of prolactin, growth hormone and cortisol.


The urinary excretion of catecholamines and their derivatives in primary hypertension in man. 1. The 24 h urinary excretion of adrenaline, noradrenaline, normetadrenaline, metadrenaline and vanilloylmandelic acid has been compared in 17 male normotensive subjects and 25 age-matched male hypertensive patients studied under similar in-patient conditions. 2. 24 h urinary metadrenaline was significantly lower in the hypertensive patients. With this exception, no significant differences were found between the two groups when the total 24 h excretion of free catecholamines and their metabolites was analysed. 3. Diurnal variation in free catecholamine excretion was found in both normotensive and hypertensive subjects. There was no corresponding variation in metabolite excretion. 4. No correlation could be established between systolic or diastolic blood pressure and the amounts of the catecholamines or their metabolites in the urine of either group. 5. The results are considered in the light of recent work demonstrating high plasma catecholamine concentrations in hypertension. They lend no support to the concept that excessive circulating catecholamines are responsible for the elevated blood pressure in essential hypertension.


Androgen production by theca and granulosa isolated from proestrous rat follicles. Theca and granulosa isolated from proestrous rat ovarian follicles were cultured for 72 h in the presence or absence of highly purified luteinizing hormone (LH, 0.1 microng/ml) and/or follicle-stimulating hormone (FSH, 0.1 microng/ml). Medium was collected and replaced at 3, 6, 12, 24, 48, and 72 h of culture and measured for testosterone by radioimmunoassay. Pieces of isolated theca secreted androgen; androgen production was greatest during the first 12 h of culture. Addition of highly purified LH to the culture medium produced a significant increase (P less than 0.001) in the thecal androgen secretion, while addition of highly purified FSH had no significant effect. Addition of LH + FSH to culture medium produced the same effect as addition of LH alone. The response of the theca to LH was dose-dependent with doses of 0.01 microng/ml or greater eliciting a maximum response. The addition of eoxgenous progesterone (5 x 10(-7)M) to culture medium had no effect on thecal androgen production. Thecal androgen secretion was the same in the presence or absence of fetal calf serum. Since the testosterone antibody used was not entirely specific for testosterone, testosterone and the cross-reacting androgen, 17beta-hydroxy-5alpha-androstan-3-one (DHT), were chromatographically isolated from samples and assayed separately. The androgen measured in culture medium was found to consist primarily of testosterone; DHT was present in much lower concentrations. Granulosa cells isolated from the same follicles as the theca and grown in monolayer culture produced negligible amounts of androgen. It is concluded that the theca is the site of follicular androgen production and that LH regulates androgen secretion by rat ovarian follicles. The results suggest that the theca provides the androgen precursor needed for follicular estradiol-17beta synthesis.


Repetitive sequences in protein A from Staphylococcus aureus. Arrangement of five regions within the protein, four being highly homologous and Fc-binding. After tryptic digestion of intact Staphylococcus aureus the residual portion of protein A that was still bound to the cell wall was cleaved off with lysostaphin. From the two digests Fc-binding fragments were isolated and the following characteristics observed. (a) There are four Fc-binding, highly homologous regions, each consisting of 58--62 amino acid residues. (b) These regions are consecutively arranged from the N-terminal part of the protein. (c) The residual C-terminal part, approximately 150-residues long, differs to a great extent with respect to primary and secondary structures from the four active regions. Furthermore, it is suggested that the protein is bound to the bacterial cell wall structure through this C-terminal part.


Supersensitivity to apomorphine following destruction of the ascending dopamine neurons: quantification using the rotational model. A new surgical preparation is described with which it is possible to quantify the degree of supersensitivity to dopamine receptor-stimulating agents using the rotational model. One group of rats received a unilateral injection of 6-hydroxydopamine which destroys the dopamine-containing neurons in one hemisphere, followed by a diencephalic electrocoagulation which interrupts both afferents and efferents of the striato-pallidal complex in the opposite hemisphere. Another series of animals received only the unilateral electrocoagulation. When given appomorphine both groups of animals rotated toward the side of the electrocoagulation. However, the 6-hdroxydopamine-treated animals were 10-40 times more sensitive to the behavioral effect of the drug. These results contrast with previous reports in which behavioral sensitivity to apomorphine was increased 2- to 7-fold following partial degeneration of central dopamine neurons of following the chronic adminstration of dopamine synthesis inhibitors of receptor blocking agents. The extent of loss of dopamine appears to be a critical factor in determining the degree of supersensitivity which will develop.


Effects of 1694 and other dopaminergic agents on circling behavior. One hour after the administration of 40 mg/kg of amineptine chlorydrate (1694) the HVA concentration in the striatum was increased but the concentrations of DA, NA, 5HT and 5-HIAA in the striatum, cortex, thalamus-hypothalamus and pons-midbrain of rats were not significantly altered. Unilateral lesioning at the level of the entopeduncular nucleus in cats and rats resulted in spontaneously occurring ipsiversive circling behavior in the two species. However circling was more sustained in cats than in rats. Apomorphine, d-amphetamine, methamphetamine, L-dopa and piribedil (ET-495) exaggerated the ipsiversive circling. 1694 (amineptine chlorydrate), a new agent, was comparatively more active than L-dopa and ET-495 and less active than apomorphine, d-amphetamine and methamphetamine. Although in higher doses (30--40 mg/kg), 1694 caused increased exploratory activity it was not associated with any stereotypy. Its biochemical and pharmacological effects are comparable to those of d-amphetamine and methamphetamine. Removal of the contralateral (with respect to the side of the entopeduncular lesion) motor cortex in the lesioned cat abolished spontaneous and drug-induced circling movements. The results of this and of previous studies support the idea that these dopaminergic agents act on the striopallidal system of the intact side which is no longer properly counterbalanced by the corresponding system of the lesioned side. Although this experimental model is useful to determine the degree of dopaminergic activity of various chemical agents it does not duplicate the motor disorders encountered in parkinsonism which are associated with a decreased concentration of dopamine.


The effect of Mycoplasma pulmonis on fertilization and preimplantation development in vitro of mouse eggs. The effect of Mycoplasma pulmonis suspension on mouse on mouse fertilization and preimplantation development in vitro was examined. When sperm were preincubated with M. pulmonis, fertilization of eggs occured less consistently than when untreated sperm were used. There was also a highly significant and consistent reduction in embryonic develoment in the treated group, with relatively few embryos reaching the blastocyst stage. The adverse effects were not seen with M. fermentans or with M. pulmonis organisms inactivated by heating or sonication before incubation with gametes. Likewise, preincubation of M. pulmonis in medium containing tetracycline, an antibiotic which interferes with protein synthesis, resulted in embryonic development similar to that seen in the untreated group. These results suggest that the deleterious effects obtained with viable mycoplasmas are due to some substance produced by their active metabolism. Other results indicate that the egg is susceptible to M. pulmonis for a limited time, since incubation of two-cell embryos with mycoplasmas had no effect on development to the blastocyst stage. The findings are discussed in relation to the possible association between human infertility and mycoplasma infection.


Action of bull seminal vesicle ribonuclease on mouse leukaemic cells BP-8 and EL-4. Bull seminal vesicle ribonuclease, when added in vitro to the suspension of ascitic leukaemic cells EL-4 and BP-8 for 2 hours at 37 degrees C and then transplanted to the abdominal cavities of mice, inhibitied the proliferation of these cells in vivo. When the temperature of the medium was decreased to 25 degrees C, AS RNase was bound to the leukaemic cell surfaces, but it exerted no inhibitory effect on BP-8 cells and showed a partial effect on EL-4 cells even after 24 hours. AS RNase administered to mice with leukaemic cells in diffusion chambers significantly decreased the number of cells inside the chambers as compared to the controls. The number of ascites cells was also significantly reduced after the injection of AS RNase for 1 week into mice with leukaemic cells implanted in the abdominal cavities. The reduction of cells in ascitic fluid did not cause a significant prolongation of survival of experimental mice. Only mice with transplanted BP-8 cells had significantly less cells in ascitic fluid and their survival was prolonged to 48 days in comparison with 25 days' survival in control mice after administration of pure AS RNase. In addition, AS RNase depressed the growth of solid tumours BP-8 and EL-4. AS RNase did not change the weight of body organs except the testes. Absorption experiments showed increased absorption of AS RNase by leukaemic cells BP-8 and EL-4 and by testes and spleen homogenates as compared with other body organs.


Melanosomes of pigmented basal cell epithelioma. Electron microscopic examination of a pigmented basal cell epithelioma of scrotal skin revealed many tumor cells and melanocytes which were laden with large packages of melanosomes. In tumor cells these packages were delimited by either single- or double-limiting membranes or were not membrane bound. The matrix of these large complexes was less electron dense and homogeneous than normal, containing electrontranslucent droplets besides melanosomes. No remnants of intracellular organelles such as nucleus, mitochondria or ribosomes could be found. The basal cell epithelioma cells in this case appear to have differentiated toward fetal basal cells and have developed the ability to phagocytize large packages of melanosomes.


Cyclic kinetics and mathematical expression of the primary immune response to soluble antigen. VII. The conveyer hypothesis and its mathematical expression. The conveyer hypothesis is based on the fact that because of clone predetermination, antibody production takes place in an organism without the presence of antigen as a result of natural cell differentiation. Soluble antigen is an analogue of a specific mitogen which gives rise to reproduction mainly of cells carrying on their surface the immunoglobulin receptors to the given antigen. The mathematical model of the conveyer hypothesis takes into account the initial conditions, among them the background level of antibody-producing cells before injection of a soluble antigen, migration of precursor cells in the draining lymphoid organ, and the rate of precursor differentiation, including the rate of the change of the immunoglobulin receptor number on the cell surface. Changes of antigen concentration in blood determine the intensity of precursor proliferation. Comparison of real experiments (intraperitoneal injections of capsular antigen of Pasteurella pestis into inbred mice) with calculations done on the basis of the developed mathematical model shows a definite qualitative resemblance with the kinetics of antibody-producing cells and free antibodies as well as with the decrease of free antigen concentration in blood. In spite of some differences between model experiments and real experiments the conveyer hypothesis and its mathematical model appear suitable for describing the primary immune response of mice immunized with low doses of capsular antigen of Pasteurella pestis.


The recent rise to the alpha-rank in a population of free-living chimpanzees. The recent rise of a high-ranking adult male chimpanzee to the alpha male position of the Gombe National Park's Kasakela chimpanzee community is reported. The male Figan is the fourth individual to assume this status in the wild chimpanzees' social hierarchy during Goodall's 16 year study in Tanzania. The paper describes the overthrow of the previous top-ranking male, and the manner in which Figan has maintained his new position after the take-over. Emphasis is placed upon his relationship with his elder male sibling, Faben, and the second highest-ranking male in the community, Evered.


[Neonatal jaundice, pathogenesis and clinical aspects]. This report is a short summary of a scientific subject titled: "Studies of the pathogenesis and clinical course of the icterus neonatorum." Medical records of 805 deliveries were evaluated for factors that influence icterus of newborns. Further investigations should answer the question, if there are at the same time other disturbances of the function of the liver. Bilirubin is a cytotoxic substance; every pediatrician knows the problems about kernicterus. In this study the toxic effects on teeth and kidneys were investigated. Finally all methods of present therapy were discussed. Especially the situation of very small premature babies requires prophylactic phototherapy.


[The optimal protein quantity in infant nutrition]. Nowadays, the trend of recommendations to prepare infant formulas with only 2 per cent or less of protein does not correspond with the optimal protein demand and protein utilization in infants. Such formulas often prompt mothers to add carbohydrates to the protein half-strength milks in order to satisfy the infant's hunger. This often leads to the unwanted effect to obesity. There is no evidence to prove that infants above the age of 1 month should not receive 2.2 or 2.3% of protein in their formulas. This quantity is fully utilized by the infants; it has no disadvantages and is safe. Hence, the recommendations should not be based on the minimum daily requirements but on the optimal utilization, whereby the tendency to obesity can also be reduced.


[Genetic control of the ethanol effect on the human EEG]. 26 identical and 26 fraternal twin pairs were subjected to 1,2 ml/kg alcohol loading under standardized conditions; furthermore, 13 non-twin persons were repeatedly subjected to the same procedure in order to test the intraindividual variability. The alcohol effect was pursued by repeated EEG recordings. The EEGs of identical twins proved to react identically to alcohol loading, whereas the EEGs of fraternal twins became more dissimilar during the course of the experiment. The identical reaction of the brain wave pattern in monozygotic twins cannot be attributed to more similar blood alcohol concentrations; instead, this finding is a pharmacogenetic phenomenon of the central nervous system.


Comparative cytogenetic and histologic studies on early malignant transformation in mesothelial tumors of the ovary. Comparative cytogenetic and histologic studies on 18 mesothelial ovarian tumors revealed a normal chromosome complement in benign lesions, and the well-known cytogenetic pattern in cystadenocarcinomas. But all borderline tumors of the series evidenced an abnormal stem line and a more or less marked tendency to polyploidization. Serous papillary cystadenomas of this group showed in five out of six cases a stem line with the karyotype 47,XX+C10 (identified by Q-banding), present in both sides of bilateral lesions. It is evidenced that malignant change on the chromosomal level precedes histologically detectable features of malignancy. Histologic equivalents appeared, when the abnormal cell line was established. The initiation of malignant transformation therefore may be signalized by karyotype abnormalities before structural changes can be detected in the corresponding histologic specimens. The results discussed include the concepts of multicentric origin and clonal evolution of malignancy.


Genetic control of ethanol action on the central nervous system. An EEG study in twins. The purpose of the investigation is to claify the genetic contribution to the interindividual variability of ethanol action on the central nervous system. The 52 adult male healthy twin pairs (26 MZ, 26 DZ) got 1.2 ml/kg ethanol p.o. under standardized conditions; furthermore, 13 non-twin subjects were repeatedly subjected to the same procedure in order to test the intraindividual variability. The EEG was recorded before and 60, 120, 180, and 240 min after alcohol intake. The EEGs were off-line analyzed by means of a computer program for time domain analysis. As was already known, on the average alcohol led to a better synchronisation of the EEG, i.e., the number of beta-waves decreased whereas the number of alpha- and theta-waves increased. The extent of the alcohol effect on the EEG varied enormously between individuals; however, the EEGs of MZ twins proved to react indentically to alcohol loading, whereas the EEGs of DZ twins became mor dissimilar during the course of the experiment. The low-voltage EEG presumably is resistant to alchohol; furthermore, it is supposed that there exists a special beta-prone EEG-type which is also genetic in origin. The identical EEG reaction of MZ twins to alcohol loading could not be attributed to more similar blood alcohol concentrations. It is hypothesized that the differences in the extent of the alcohol effect on the EEG between individuals might reflect differences in the sensitivity of the ascending reticular activating system. In the literature it has frequently been reported that alcoholics have preferentially brain wave patterns which are poorly synchronized. These findings are discussed in the light of the present results.


Dominance and environmental variances: their effect on heritabilities estimated from twin data. A method for partitioning genetic variance estimated from twin data into additive and dominance variances was presented using Falconer's variance component model. The effects of dominance and environmental variances on a number of heritability estimates were also reviewed. A heritability estimate, based on the analysis of variance and the genetic variance estimates presented by HASEMAN and ELSTON and CHRISTIAN et al. which utilizes all available information from twin data, was proposed and discussed. This estimate seems to be the least affected by fluctuations in the magnitudes of dominance and environmental variances.


[A highly sensitive determination of immunoglobulin G and M with sensibilized latex particles (author's transl)]. Latexparticles, sensitized with heavy chain specific anti-human IgG and IgM, were used for the detection of IgG and IgM in sera of adults and newborn infants. Heat-inactivated sera showed an IgG-reactive component of high density near the band of the IgM after centrifugation in a sucrose gradient. The test is specific and highly sensitive.


Effect of extracts of the bursa of Fabricius on IgG antibody production in hormonally bursectomized chickens. The effects of extracts of the bursa of Fabricius on the return to normal and greater than normal of antibody production in hormonally bursectomized chickens and on the switch-over of IgM antibody production to IgG antibody production was studied. Antibody production in hormonally bursectomized chickens treated with bursal extract was markedly increased as compared with bursectomized birds not given bursal extract and the IgG antibodies at three weeks was similar to that in normal immune chickens.


[Preoperative infusion therapy in childhood. I. Initial conditions and principles]. Infusion therapy for purposes of surgery differs significantly from the infusion requirements for "conservative" purposes, in adults as well as in infants and children. The preoperative situation of infants and children undergoing usual surgical procedures is particularly characterized by 1. the physiological conditions of the water-, electrolyte- and acid-base-balances; 2. the effects of the preoperative food-and fluid-restrictions; 3. the intraoperative fluid- and volumen-losses; 4. the combination between 1--3 and the postoperative food- and fluid-restriction and the postoperative metabolic conditions respectively. The problems, summarized under 1--4, have been discussed within this article. Further two papers will deal with studies on the use of "basic solutions" for the substitution of fluid- and electrolyte losses in pediatric surgery.


[Flavor improvement of the Vivasorb --elementary diet]. By addition of special flavours and spices and making use of fruit juices and soups we were able to achieve an improvement in the taste of the "astronaut diet" Vivasorb. Experiences in the case of 15 patients which allowed long-term treatment with a reduced degree of difficulty were put forward. In some cases, however, methods had to be applied which do not quite adhere to the principle of feeding completely elementary nutrients.


[The geriatric patient and his nutrition]. The nutrition of geriatric patients should observe the following characteristics of advanced age: diminished calorie requirement, impaired glucose tolerance, tendency to elevated serum cholesterin and triglyceride levels, increased requirements for protein of high biological value, absence of thirst and reduced total body water. Considering these metabolic changes recommendations for an adequate nutrition of senescent people are discussed.


Determination of serum proteins in the presence of dextran by means of the biuret reaction. 264 biuret reagents for total protein determination in serum containing dextran were investigated. The usefulness of the method is dependent on proper ratio of the components of the reagent: concentrations of NaOH and copper sulphate, and the ratio of sodium-potassium tartrate to copper sulphate.


[Flavor improvement of a balanced synthetic diet]. By improving the flavour of an elementary diet rich in aminoacids it was possible to treat 33 patients with inflammatory bowel diseases over longer periods compared to the duration of treatment with Vivasorb elementary diet. This was accomplished by addition of certain fruit juices, several kinds of teas, soups and relishes. Difficulties by taste of BSD could largely be overcome.


Effect of squamous metaplasia on infection of hamster trachea organ cultures with Mycoplasma pneumoniae. An organ culture system for hamster trachea was developed for maintenance of the ciliated respiratory epithelium during periods of extended cultivation (i.e., greater than 20 days). Evaluation of five serum types showed that horse serum and fetal calf serum were best for the maintenance of epithelial ciliary activity and morphology. Rings that were opened on one side ("split rings") had the best maintenance of the ciliated epithelium as judged by the retention of ciliary activity and normal histological appearance after 3 to 4 weeks in culture. The in vitro induction of squamous metaplasia was achieved by cultivating explants in Waymouth MAB 87/3 (vitamin A-free) medium, without serum. This system allowed a direct comparison of the effects of Mycoplasma pneumoniae infection in two epithelial types, ciliated pseudostratified columnar and keratinizing squamous. Attachment of 14C-labeled mycoplasmas was more than twofold greater in the normal epithelium. Pretreatment of explants with neuraminidase decreased attachment for both squamous and pseudostratified epithelial surfaces to a similar basal level. Recovery of viable organisms from infected tissue of both epithelial types indicated that the organism titer remained essentially constant during the infection period, but was significantly higher for the pseudostratified ciliated epithelium. These results suggest that specific receptor sites for M. pneumoniae are markedly reduced by the induction of squamous metaplasia and, hence, appear to be specific for the normal respiratory surface containing goblet cells and pseudostratified, ciliated epithelial cells.


Ocular effects of scrapie agent in hamsters: preliminary observations. Scrapie is caused by one of a group of so-called slow viruses responsible for the subacute spongiform encephalopathies. In the present study, young hamsters were inoculated intracerebrally with hamster-adapted scrapie agent. At termination, all inoculated animals showed signs and central nervous system pathology compatible with scrapie infection. The eyes appeared well developed grossly, but histologically the retina and optic nerve were abnormal. There were varying degrees of thinning of the retina, with the photoreceptor layer being most severely affected. Although the ganglion cell layer was not much different from the controls, the optic nerve appeared more cellular than that of the controls.


In vitro propagation of prostate adenocarcinoma cells from rats. Two rat adenocarcinomas were physically dispersed and propagated in vitro. Epithelial and fibroblast cell lines were cloned from them and the monolayer cell lines derived therof were further characterized. The cells produced acid phosphatase in early in vitro cell passages, and later they turned negative. Fibroblast-like cells produced no tumors when implanted in syngeneic Lobund Wistar rats, but as few as 10 epithelial cells produced metastasizing adenocarcinomas in them. A third prostate tumor has yielded a line of epithelial cells which reproduced the original tumor type in inoculated rats, but the cells have not yet been characterized. Rat prostate adenocarcinomas provided a useful model system for in vitro and in vivo studies on prostate cancer and on metatasis.


Histochemistry of 3beta-hydroxysteroid dehydrogenase in rat ovary. I. Amethodological study. By recording the incubation time needed for initial appearance of the red and blue formazans the reliability of the histochemical method for 3beta-HSD was investigated: 1. Prefixation of small tissue blocks with 1% W/V methanol-free formaldehyde (pH=7.2) for up to 30 min preserved morphological integrity as well as maximal enzyme activity. Moreover, the substantivity of formazans and lipids was enhanced. 2. Commercial available glutaraldehyde (pH=7.2) induced SH groups in the tissue (even at 0.1% W/V for 5 min) thereby enhancing the Nothing dehydrogenase reaction. 3. Preextraction of lipids with acetone for 20 min at -30 degree C caused no loss of activity and was an inevitable step if a reliable activity pattern had to be achieved (e.g. in interstitial cells). 4. No diffusion of enzyme was noticed within 30 min of preincubation in phosphate buffer (0.2 M, pH=7.2) at 20 degree C. 5. By using the double-section incubation method no diffusion of 3beta-HSD or rediffusion of NADH or PMSH could be noticed withn 45 min of incubation, provided that low concentrations of NAD (0.1 mg/ml) and PMS (0.003 mg/ml) were balanced against the concentration of Nitro BT (0.5 mg/ml) or Tetranitro BT (1.0mg/ml). 6. The utlity of different inhibitors of alkaline phosphomonoesterase was tested and discussed. 7. By inhibiting alkaline phosphomonoesterase with 0.1 mM of L-p-bromotetramisole or 16 mM of beta-glycerophosphate, 3beta-HSD was shown to be exclusively NAD-linked. 8. Levamisole was a potent inhibitor of NADH-tetrazolium reductase as well as 3 beta-HSD, but not of NADPH-tetrazolium reductase. 9. 3beta-HSD possess SH groups requisite for the activity as this enzyme was totally inhibited by N-ethyl maleimide. 10. Whether alcohol dehydrogenases may use steroids as substrate is discussed; It is concluded that preextraction (by acetone) and/or the use of an inhibitor of alcohol dehydrogenase (1,10-phenanthroline) has to be performed. 11. Propylene glycol was a poor solvent for all substrates and was itself an excellent substrate for alcohol dehydrogenase. 12. Specifications for the ideal solvent of steroid substrates in the histochemical practice are proposed. DMSO showed to be promising as a steroid solvent (e.g. extraction of formazans was considerably lower as compared to DMF). 13. The utilization of substrates was descending in the following order (using 1 mM and 0.1 ml/ml of either DMF or DMSO): epiandrosterone, methandriol, dehydroepiandrosterone and pregnenolone. 14. If DMSO was used as solvent for pregnenolone (but not for the other substrates tested) an evident increase of activity was recorded as compared to DMF.


Congenital pericardial diaphragmitic hernia and multiple cardiac defects in a litter of collies. Four Collie littermates had multiple cardiac and diaphragmatic anomalies. Ventricular septal defect was found in 3, pulmonic stenosis in 2, atrial septal defect in 1, mitral regurgitaion in 1, and pericardial diaphragmatic hernia in 3. Five additional littermates died within 72 hours of birth but were not necropsied. On the basis of available evidence, it was believed that the defects were congenital but not hereditary.


Attempted transmission of Toxoplasma gondii infection from pregnant cats to their kittens. Sixteen 1- to 7-week-old pregnant specific-pathogen free cats were inoculated orally with Toxoplasma gondii cysts. Fetuses and neonatal kittens were examined for toxoplasma infection by inoculating suspensions of their tissues into mice. Toxoplasma gondii was not isolated from 23 fetuses and 16 newborn kittens from 13 queens. Six (3 litters) of the 15 kittens from the 3 remaining queens were killed on the day of or a day after birth, and the remaining 9 kittens were housed with their mothers for 7 to 33 days. None of the 9 kittens from the 2 litters examined between 0 and 33 days of age was infected with T gondii. In the other litter, T gondii was isolated from 3 kittens killed at 9, 16, and 22 days of age but not from 3 littermates killed on days 1, 1, and 22. Internal organs from the 3 kittens with proved toxoplasma infectivity in mice were examined histologically. Multifocal granulomatous encephalitis, hepatitis, nephritis, myocarditis, myositis, and interstitial pneumonia were found in all 3 kittens. Toxoplasma forms were demonstrated histologically in the tissues of 2 of these kittens. The mode of toxoplasma infection in newborn kittens was not determined but did not appear to be either transplacental or via fecal contamination from oocysts excreted by the mother cat. Evidence obtained in these experiments suggests that transplacental toxoplasma infection in the cat is not an important epidemiologic factor in perpetuation of the disease in the feline population.


Attempeted transmission of feline coccidia from chronically infected queens to their kittens. Eight female, 12- to 34-month-old, specific-pathogen free cats were inoculated orally with Toxoplasma gondii cysts on day 0, then with Isospora felis and Isospora rivolta oocysts on day 39, and cysts of Hammondia hammondi on day 86 after inoculation with Toxoplasma. All cats shed oocysts of all 4 of these coccidia within 11 postinoculation days. The female cats were caged with 4 male Toxoplasma-free cats, starting 66 days after inoculation with Toxoplasma, until they were 5 to 6 weeks pregnant. Kittens that were born were housed with their mothers until necropsied or weaned. One 42-day-old kitten shed T gondii oocysts in feces. It was necropsied 2 days later and asexual stages of Toxoplasma (types D and E), gametocytes, and oocysts were demonstrated in sections of superficial epithelial cells of its small intestine. Lesions or forms of Toxoplasma were not demonstrated histologically in tis extraintestinal organs. Toxoplasma was not isolated from feces or tissues of the remaining 47 kittens born to these 8 queens. Toxoplasma was not isolated from the 4 male cats that were caged with infected females for 53, 59, 217, and 217 days. The source of toxoplasma infection in the kitten remained unknown but was considered unlikely to be congenital or through fecal contamination. Oocysts of I felis, I rivolta, and H hammondi were not found in the feces of any kittens, indicating that these coccidia are unlikely to be transmitted congenitally.


Partial deficiency of antidiuretic hormone in a cat. Marked polydipsia and polyuria developed subsequent to trauma in a 1 1/2-year-old male Abyssinian cat. Diabetes insipidus was suspected, inasmuch as intramuscualr vasopressin administration resulted in amelioration of polydipsia and polyuria. However, hypertonic (3%) saline solution given intravneously resulted in anuria, an indication of antidiuretic hormone activity. Polyuria and polydipsia were abolished by oral chlorpropamide therapy, which was indirect evidence for partial deficiency of antidiuretic hormone.


Architecture and chemistry of microconidial walls of Trichophyton mentagrophytes. The ultrastructure and chemical composition of the walls of Trichophyton mentagrophytes microconidia were investigated with particular emphasis on the localization of the major structural components within the walls. The walls consisted of carbohydrate (56.1% neutral polysaccharide, and 16.0% chitin), protein (22.6%), lipid (6.5%), ash (1.7%), and trace amounts of melanin (0.2%) and phosphorus (0.2%). in thin sections, three distince layers were recognized. The electron-transparent pellicle (15 to 20 nm thick) covering the outermost surface of the wall consisted of a glycoprotein-lipid complex and was mostly extracted by sodium phosphate buffer (0.1 M, pH 6.5) containing 8 M urea, 1% (vol/vol) mercaptoethanol, and 1% (wt/vol) sodium dodecyl sulfate. The middle electron-dense layer (30 to 50 nm thick) represented the proteinaceous rodlet layer embedded in polysaccharides and could be completely solubilized by hot alkali extraction (1 N NaOH, 100 DEGREES C, 1 h). The thick inner layer (200 to 300 nm thick) was relatively resistant to the above treatments and was found to consist of amorphous glucans and microfibrillar chitin. Approximately half of the inner wall glucans was susceptible to (1 leads to 3)-beta-glucanase.


Inhibition of Dictyostelium discoideum beta-glucosidase by purines. beta-Glucosidase of Dictyostelium discoideum is inhibited by purines in the following order: adenine greater than adenosine greater than 6-methylaminopurine greater than hypoxanthine greater than inosine greater than purine greater than guanosine. Adenine inhibits activity by 50% at 1 to 2 mM. The kinetics are complex because the enzyme is stimulated by substrate and inhibited by glucose.


Death of microbial cells: rate constant calculations. The previously published procedure for calculation of rate constants associated with the death of microbial cells is shown to be so sensitive to variation in experimental data as to render it impractical for this application. The only obvious modification to the published procedure met with only limited success. It is concluded that a fresh search should be undertaken for the solution to this fundamental problem. The success of such further attempts may depend upon an alternative description of the normal probability integral rather than upon refined experimentation.


Decrease in nitrate reductase activity in extracts of Trichoderma viride Incubated with chlorides. Reduced nicotinamide adenine dinucleotide phosphate-dependent nitrate reductase activity in crude extracts of Trichoderma virde was significantly inhibited by physiological concentrations of ammonium chloride, sodium chloride, and potassium chloride, but not by ammonium or sodium sulfate. The chloride inhibition of nitrate reductase activity increased in a linear manner with chloride concentration.


Effect of temperature on Mg-ITP-induced superprecipitation of actomyosin. Superprecipitation of an actomyosin suspension was measured at various temperatures (2.5 degrees - 20 degrees) using Mg-ITP as substrate. Superprecipitation was induced by the addition of Mg-ITP at all temperatures, but decreased in extent with decrease in temperature. The predominant intermediate in the Mg-ITP hydrolysis of myosin depends on the temperature; at 20 degrees it is the myosin-IDP-Pi complex, while below 8 degrees it is the myosin-ITP complex (Hozumi, T. (1976) Eur. J. Biochem. 63, 241). Therefore, the occurrence of superprecipitation below 8 degrees is not compatible with muscle models in which formation of a myosin-product complex is the rate-limiting intermediate.


Substrate specificities of cathepsin A,L and A,S from pig kidney. The substrate specificities of two different molecular sizes of cathepsin A, A,L (large form) and A,S (small form), for synthetic substrates were examined kinetically. Both enzymes showed a similar broad substrate specificity against various acyl dipeptides, amino acid esters, and amino acid amides. Z-Phe-Ala and Ac-Phe-OEt were good substrates. Peptides containing hydrophobic amino acids were hydrolyzed rapidly. The presence of hydrophobic amino acid residues, not only at the C-terminal position but also at the second position and probably the third position from the C-terminal, resulted in an increase in the rate of hydrolysis. Peptides containing glycine and proline were hydrolyzed slowly. Inhibition studies with Z-D-Phe-D-Ala and Z-Phe suggested that the peptidase and esterase activities of the enzymes are both catalyzed by the same site of the enzyme molecule, but it remains to be elucidated whether or not the binding sites for peptides and esters are the same.


Effects of polymer surface molecular structure and force-field characteristics on blood interfacial phenomena. To quantify the effects of major surface structural factors influencing interfacial reactions induced by polymers in native blood, model surfaces of solvent-cast films of two analogous poly(ether urethanes) and three homologous polyamides (nylon 4, 6/6, and 12) were exposed ex vivo to canine blood under the well-defined hemodynamic conditions of the Stagnation Point Flow Experiment. The selected surfaces allow for incremental changes in properties and were characterized by their "Composite Surface Free ENergy Function," gamma'S, which describes the surface force field as the sum of the mean dispersion (gammaSd) and polar (gammaSp) contributions and is computed from wettability spectra obtained with ultrapure diagnostic liquids. Blood interfacial effects were measured by the shear-limited diameter of the white cell circle formed around the stagnation point, the flow parameter at which symmetric aggregation occurred, and the surface-number density of platelets, [P s], remaining adherent under fixed conditions. At identical flows, within each group of polymers, both the WBC-circle diameter and [P s] scale with gamma Sp/gamma'S, implying that 1) only the magnitude but not the interaction mechanism varies as a function of incremental structural and surface changes, 2) the primary determinant of surface-induced effects is the polar force contribution, and 3) the magnitude of gamma'S is secondary if gammaSd/gamma'S is sufficiently great.


Use of stable, sensitized cells in an indirect microhemagglutination test for melioidosis. Two evaluations were carried out in this study. The first was a comparison of the standard tube test with the automated microtitration test for the detection of antibodies to Pseudomonas pseudomallei by the indirect hemagglutination method. Data from this comparison indicated that the tests were equivalent. The second evaluation consisted of reproducibility studies on two lots of pyruvic aldehyde-stabilized sensitized erythrocytes in comparison with freshly prepared sensitized erythrocytes in the automated microtitration test. The influence of different types of the microtitration plates used was also examined. Results indicated that the use of stabilized antigens is feasible, and these antigens offer the advantage of being ready for immediate use.


Comparison of rabbit and pig plasma in the tube coagulase test. A total of 627 clinical isolates of Micrococcaceae were characterized accoridng to their ability to coagulate rabbit and pig plasma, produce thermostable nuclease, and anaerobically ferment glucose and mannitol. By using these characteristics, 416 of the isolates were classified as Staphylococcus aureus and 211 as non-S. aureus. All 416 strains produced a 3 to 4+ clot formation in heparinized pig plasma, whereas 415 isolates produced a similar reaction in citrated rabbit plasma. All of the S. aureus strains possessed a thermostable nuclease, whereas only four of the non-S. aureus isolates exhibited this  characteristic. The results obtained using heparinized pig plasma were almost identical to those obtained with commercial rabbit plasma.


Platelet interaction with modified articular cartilage. Its possible relevance to joint repair. During studies concerned with the platelet-collagen interaction, it was observed that platelets did not adhere to bovine or human articular cartilage and that cartilage did not induce platelet aggregation in vivo or in vitro. To study the mechanism responsible for this observation, the role of proteoglycans was examined. Purified cartilage collagen proved to be fully active as a platelet aggregant. Addition of small amounts of proteoglycan subunit (PGS) blocked platelet aggregation, whereas chondroitin sulfate, a major glycosaminoglycan component of cartilage matrix, impaired platelet aggregation only at concentrations which resulted in a marked increase in viscosity. Moreover, PGS abolished aggregation of platelets by polylysine but did not prevent aggregation by ADP, suggesting that PGS may block strategically placed lysine sites on the collagen molecule. Treatment of fresh articular cartilage with proteolytic enzymes rendered the tissue active as a platelet aggregant. In vivo experiments demonstrated that surgical scarification of rabbit articular cartilage does not result in adhesion of autologous platelets. Treatment of rabbit knee joints with intraarticular trypsin 1 wk before the injection of blood resulted in adhesion and aggregation of platelets on the surface of the lesions. Since there is evidence from other studies that some degree of cartilage healing may take place after initiation of an inflammatory response, it is postulated that induction of platelet-cartilage interaction may eventuate in cartilage repair.


Adenylate cyclase of human fat cell ghosts. Stimulation of enzyme activity by parathyroid hormone. Some of the effects of native bovine parathyroid hormone and of the synthetic aminoterminal 1-34 fragment on the adenylate cyclase activity of human fat cell ghosts were studied. Saturating concentrations of both hormone preparations caused a significant increase of enzyme activity by about 200-300%. Guanosine 5'-triphosphate (0.1 mM) inhibited basal enzyme activity but had no substantial effect on parathyroid hormone-stimulated enzyme activity. The guanosine 5'-triphosphate analogue, 5'-guanylyl-imidodiphosphate, produced about a threefold enhancement of basal and parathyroid hormone-stimulated enzyme activities under standard conditions (5 mM Mg+2, 1mM ATP, pH 8.0, 30 degrees C). Activation by parathyroid hormone was not influenced by beta-adrenergic blockade in contrast to stimulation by epinephrine. The sensitivity of the enzyme system to the native and the synthetic parathyroid hormone was, however, abolished after pretreatment of the fat cells with trypsin (1 mg/ml). The stimulatory effects of epinephrine and NaF were not affected by pretreatment with trypsin. The results suggest that human fat cells, like rat adipocytes, contain a multireceptor-coupled adenylate cyclase.


Features of prognostic significance in testicular germ cell tumours. One hundred and one testicular tumours previously diagnosed as "teratoma" were examined and 93 of the patients were followed up. These neoplasm were assigned to one of three groups. Those compared exclusively of somatic tissues were the only tumours referred to as teratoma. The second group was exclusively extra embryonic and consisted of either yolk sac or choriocarcinoma. Neoplasms in the third group were called mixed germ cell tumours and incorporated somatic and extra embryonic tissue and occasionally seminoma. The patients with teratomas showed a very low mortality whereas pure yolk sac tumours proved highly malignant. In mixed germ cells tumours the malignant nature of the yolk sac components was maintained even when combined with somatic elements, but when seminoma was also present the survival rate was significantly improved. It would appear the yolk sac tumour tissue occurs more frequently in adult testicular neoplasms than was previously suspected and in mixed germ cell tumours it can be expected to dictate behaviour except when combined with seminoma.


Processing urinary endoscopes in a low-temperature steam and formaldehyde autoclave. Methods of disinfection and sterilisation of urinary endoscopes are considered. A small mobile low-temperature steam and formaldehyde autoclave (Miniclave 80) is evaluated and shown to be satisfactory for this purpose as judged by a variety of relevant microbiological test pieces.


Prediction of the reading and spelling performances of normal and retarded readers: a four-year follow-up. The purpose of this study was to determine the relative predictive accuracy of a number of measures employed in a 4-year longitudinal study of the neuropsychological abilities of normal and retarded readers. The results indicated that there were some very accurate predictive measures of reading and spelling achievement levels over the 4-year age span studied, and that the relative accuracy of these measures differed markedly for normal and retarded readers. Especially in the case of retarded readers, performance of the Underlining Test was a much more accurate predictor of eventual achievement levels in reading and spelling than were tests of reading, spelling, or psychometric intelligence.


Characterization of IgC receptors of the fetal rabbit yolk sac membrane: localization to subcellular fraction and effects of chemical agents and enzymes on binding. Previous studies have established the existence of IgG receptors on the endodermal cells of the fetal rabbit yolk sac membrane (YSM). The present study partially characterizes these cell-associated receptors. The specific binding of rabbit IgG (IgGR) to freshly prepared cell homogenates, nuclei-free brush border preparations, and plasma membrane-rich fractions confirms that receptor function is associated with the endodermal cell, and indicates that this function is localized on its apical brush border, specifically on its plasma membrane. The protein nature of the receptor is demonstrated by the loss of specific binding capacity after treatment of formalin-fixed YSM with papain and trypsin. Evidence has also been adduced which indicates that membrane carbohydrate is not involved in receptor function. Thus, treatment of YSM with neuraminidase, beta-galactosidase, mixed glycosidases, as well as oxidation of YSM with periodate all are without effect on its capacity to bind IgGR. The interaction of IgGR with the receptor elements of formalin-fixed YSM is partially ionic in character. NaCl reversibly inhibits binding of IbGR by 60%. Divalent ions such as Ca++ are not involved in this receptor-ligand interaction since EDTA-treated YSM binds IgGR to the same extent as do controls. Receptor material on fixed YSM resists extraction by non-ionic detergents, deoxycholate, and chaotropic agents.


Solubilization and partial characterization of cell membrane Fc receptors. We assayed detergent cell lysates and culture supernatant fluid of mouse cells for the presence of soluble FcR by means of a radioligand bioassay. Strict correlation was found between the amount of FcR present in soluble form and that found on intact cells. The soluble FcR of all cells tested, except mouse macrophages, behaved as lipoprotein by buoyant density centrifugation analysis and enzyme treatment. In the absence of detergent, soluble FcR sedimented as a 20S macromolecule; in detergent the material was 7 to 9S. It was readily bound by insolubilized IgG but not by insolubilized BSA or F(ab')2 fragments of IgG.


Modulation of macrophage C3b receptor function by cytochalasin-sensitive structures. A quantitative rosette assay was employed in order to determine if through pharmacologic probes we could gain an insight into the nature of the interaction between C3b-coated particles and the macrophage C3b receptor. Rabbit alveolar macrophage monolayers were challenged with chromium-labeled, complement-coated (via cold agglutinin) human erythrocytes (HEC3b) and the per cent of bound counts determined in the distilled water lysate. With this assay system in which ingestion is negligible, the cytochalasins (A greater than E greater than D greater than B) produced the most marked inhibition of rosette formation compared to control treated monolayers. No agent examined produced consistent augmentation. Cytochalasin A at 10(-5), 10(-6), and 10(-7) M inhibited rosette formation by 77+/- 2, 44 +/- 4 and 15 +/- 7 (S.E.), per cent, respectively. Cytochalasin E was also markedly inhibitory, Cytochalasins B and D produced approximately 30% inhibition at 10(-5) M. The cytochalasin effect was not secondary to an interaction between these agents and complement-sensitized erythrocytes, although cytochalasin E was also able to reduce erythrocyte-bouund C3b reactivity. Cytochalasin A and E modulation of the macrophage C3b reactivity occurred within a few minutes and was only slightly reversible. Cytochalasins A and E could also disrupt performed rosettes but the effect was not as pronounced as when these agents were present before and/or during the actual adherence phenomenon. Vinblastine and colchicine (10(-5) and 10(-6) M) also produced significant inhibition of rosette formation, although the magnitude of the effect was less than that for cytochalasins A and E. Further characterization of the vinblastine and colchicine effect demonstrated that the inhibition was rapid, irreversible over a 60-min incubation, and not explained by an alteration in macrophage attachment or in HEC3b reactivity. Agents producing insignificant inhibition of rosette formation included the following: dibutyryl cAMP and cAMP agonists (PGE1, theophylline), 8-bromo cGMP and cGMP agonists (carbachol, asorbic acid), dimethylsulfoxide, heparin, ethanol, dextran sulfate, DEAE-dextran, and poly-L-lysine. The data suggest that cytochalasin, vinblastine and colchicine sensitive membrane structures, most likely microfilaments and microtubules, are important in the interaction of C3b-coated particles with the macrophage C3b receptor.


Zinc protoporphyrin level in blood determined by a portable hematofluorometer: a screening device for lead poisoning. An inexpensive portable fluorometer has been designed which can be used to determine the concentration of zinc protoporphyrin (ZPP) in a drop of unprocessed blood deposited on a disposable glass slide. No volume measurement of the blood sample need be made. The instrument displays the ZPP level in micrograms per deciliter of blood accurately and reproducibly, automatically calibrating each determination by comparing the blood fluorescence with that of a stable dye. The instrument, which we call a hematofluorometer, is ideally suited for use in primary screening for childhood and occupational lead intoxication. It can be used by public health personnel after a few minutes of instruction. Comparisons of ZPP levels obtained with the use of the hematofluorometer with erythrocyte protoporphyrin levels obtained using accepted extraction methods are presented.


Dissimilarity of in vitro colon-forming cells and erythrocyte-enhanced colony-forming cells from mouse marrow. In vitro colony-forming cells (CFU-C's) differed from erythrocyte-enhanced colony-forming cells (EE-CFU-C's) in their liquid culture kinetics, adherence properties, and sedimentation velocities. Murine marrow cells stimulated with colony-stimulating activity (CSA) were grown in liquid culture in the presence or absence of washed erythrocytes. A seven to eightfold increase in the number of CFU-C's occurred on day 2 of culture. A somewhat greater increase of nine to 11-fold was observed for EE-CFU-C's which reached a maximum on day 4. When CSA was omitted from the liquid cultures, no increase in CFU-C's or EE-CFU-C's was observed. Marrow cell separation by glass bead adherence columns disclosed a greater degree of adherence for the EE-CFU-C population. Nonadherent cell fractions exhibited a decreased EE-CFU-C/CFU-C ratio (1.19 to 1.24) when compared to controls (3.33) whereas an increase was observed for the eluted, adherent cell population (5.06-6.02). Velocity sedimentation experiments demonstrated a size difference for the marrow-derived CFU-C and EE-CFU-C. CFU-C's sedimented with a velocity of 4.5 mm./hour and EE-CFU-C's sedimented at the rate of 5.7 mm./hour. These observations indicate that EE-CFU-C's appear to represent a population of cells different from the CFU-C's. The later time of peak appearance of EE-CFU-C's in liquid culture suggests that the EE-CFU-C may be a progeny of the CFU-C.


An analysis of specific stimuli causing the release of prolactin and growth hormone at milking in the goat. Experiments to investigate the relative importance of the tactile, conditioned and possible metabolic components of the milking stimulus on the release of prolactin and growth hormone (GH) from the anterior pituitary of the goat are described. A comparison of the hormonal responses to milking the auto-transplanted mammary gland (i.e. denervated gland) with that obtained by milking the intact mammary gland of the same goat showed that the concentration of prolactin in the plasma increased only after milking the intact gland, whereas in two out of four goats an increase in plasma GH was detected several minutes after milking the transplanted gland. In the intact animal significantly more prolactin (P less than 0-01) was released in response to milking both teats for 6 min as compared with that released by milking only one teat for the same time. No significant difference (P less than 0-1) was found for GH. Similar quantities of prolactin and GH were released by goats milked when conscious or under anaesthesia. A comparison of the hormonal responses to teat stimulation in the same anaesthetized goats with and without the removal of milk from the mammary gland showed a significant reduction (P less than 0-001) in the quantity of prolactin and GH released in the absence of milk removal. The significance of these results is discussed.


[Androgen binding in human plasma and its physiological significance (author's transl)]. The literature on the determination and the meaning of androgen binding to plasma proteins is reviewed. Because of the high affinity for the active androgens the sex hormone binding globulin (SHBG) is of considerable importance. The blood level of this globulin varies according to sex, age, medicamentation and pathological conditions. The influence of protein binding on the metabolism and mechanism of action of androgens is discussed and the hypotheses of several authors are compared.


Activation of hamster eggs by pricking.

Activation of hamster eggs by pricking. When mature unfertilized eggs of the golden hamster were picked with a glass needle, nearly 80% of them were activated. The diameter of needle was of critical importance for the successful induction of activation. It appeared that extracellular calcium was not necessary for the establishment (reception) of stimulation by pricking but was necessary for later phases of activation. The possible mechanisms by which pricking with a needle triggers the activation of the egg were compared with the mechanisms by which divalent ionophore induces activation.