Glycogen synthesis by rat hepatocytes.

Glycogen synthesis by rat hepatocytes. 1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12--15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50--60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.


Recognition of human urine alpha-N-acetylglucosaminidase by rat hepatocytes. Involvement of receptors specific for galactose, mannose 6-phosphate and mannose. Adsorptive endocytosis of alpha-N-acetylglucosaminidase from human urine by isolated rat hepatocytes is inhibited by glycoproteins, polysaccharides and sugars that are known to bind to cell-surface receptors specific for either terminal galactose/N-acetylgalactosamine residues, terminal mannose residues or mannose 6-phosphate residues. Recognition of alpha-N-acetylglucosaminidase by a cell-surface receptor specific for terminal galactose/N-acetylgalactosamine residues is supported by the observations (a) that neuraminidase pretreatment of the enzyme enhances endocytosis, (b) that beta-galactosidase treatment decreases endocytosis and (c) that neuraminidase pretreatment of hepatocytes decreases alpha-N-acetylglucosaminidase endocytosis. Recognition of alpha-N-acetylglucosaminidase via receptors recognizing mannose 6-phosphate residues is lost after treatment of the enzyme with alkaline phosphatase and endoglucosaminidase H. The effect of endoglucosaminidase H supports the view that the mannose 6-phosphate residues reside in N-glycosidically linked oligosaccharide side chains of the high-mannose type. The weak inhibition of endocytosis produced by compounds known to interact with cell-surface receptors specific for mannose residues suggests that this recognition system plays only a minor role in the endocytosis of lysosomal alpha-N-acetylglucosaminidase by hepatocytes.


Assay of adenosine 3'-phosphate 5'-sulphatophosphate in hepatic tissues. A fluorimetric assay, based on the ability of boiled hepatic extracts to support the sulphO-conjugation of harmol, was used to demonstrate and quantify PAdoPS (adenosine 3'-phosphate 5'-sulphatophosphate) present in liver. A stoicheiometric relationship was established between the sulphate conjugate formed and the 'active sulphate' utilized. Guinea-pig, rat, mouse and rabbit livers contain 3.3, 2.9, 0.8 and 0.5 mumol of PAdoPS/100 G wet wt. respectively.


Immunochemical characterization of monoamine oxidase from human liver, placenta, platelets and brain cortex. 1. Antiserum raised to purified human liver monoamine oxidase was used to characterize the monoamine oxidase from human liver, brain cortex, placenta and platelets. 2. Antibodies to monoamine oxidase were purified by adsorption with a mitochondrial preparation. 3. Monoamine oxidase was present in liver particle-free supernatant as measured by enzyme activity and immunodiffusion. 4. Multiple precipitin lines were obtained on immunodiffusion analysis against the purified liver enzyme. It is proposed that this is due to either aggregation or to differential lipid binding. 5. The results suggest that the functionally different enzymes found in liver, brain cortex, platelets and placenta are immunochemically related and may be identical.


Biosynthesis of proline in Pseudomonas aeruginosa. Properties of gamma-glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase. gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.


The chemical composition and stability of bacillomycin S. Bacillomycin S, an antifungal antibiotic from culture filtrates of Bacillus subtilis, was shown to consist of proteinaceous matter (83.6 g/100 g) and lipid material (16 g/100 g). The amino acid and fatty acid components were determined qualitatively and quantitatively. It was shown that bacillomycin S is stable at physiological pH and it is suggested that its antifungal activity resides in the intact molecule.


Pharmacological studies on 3-[gamma-(p-fluorobenzoyl)propyl]-2,3,4,4a,5,6-hexahydro-1-(H)-pyrazino (1,2-a) quinoline (compound 69/183). Part I: Hypotensive activity. 3-[gamma-(p-Fluorobenzoyl)propyl]-2,3,4,4a,5,6-hexahydro-1-(H)-pyrazinol(1,2-a)quinoline (compound 69/183, centpyraquin) has been found to possess promising hypotensive activity in anaesthetised cat, dog and monkey- It also lowers the blood pressure of unanaesthetised cat, dog and hypertensive rat. The effective doses are between 0.5 to 2.0 mg/kg in all the species except rat, in which doses of 10.0 and 20.0 mg/kg are effective. The compound potentiates epinephrine and norepinephrine pressor responses but inhibits carotid occlusion, tyramine and DMPP induced pressor responses. The contraction of the nictitating membrane due to pre- as well as post-ganglionic sympathetic nerve stimulation is blocked equally. In mice the compound produces ptosis which is antagonised by N-benzyl-N-methylguanidine. Localisation of the compound either to the superior cervical ganglion of cat or to the central cardiovascular loci has no effect on the activities of either of them. No evidence of an initial catecholamine release by the compound could be obtained. It has weak smooth muscle relaxant activity. The mechanism of hypotensive action seems to be the blockade of adrenergic neurones along with direct smooth muscle relaxation.


Pharmacological investigations on asclepin--a new cardenolide from Asclepius curassavica. Part I: Cardiotonic activity and acute toxicity. 3'-O-Acetylcalotropin (asclepin), a new glycoside was evaluated for its cardiotonic activity by various in vitro and in vivo procedures in cat, guinea pig, dog, monkey, pigeon and mouse. It was found to posses the properties of a cardenolide. It was more potent than digoxin and had a wider safety margin. It increased the myocardial contractility both in normal and hypodynamic heart muscles.


Investigations on antibacterial activity of cefazedone against Neisseria gonorrhoeae. The sensitivity of 67 strains of Neisseria gonorrhoeae isolated from 1977 to 1978 were tested against the new cephalosporin derivative (6R,7R)-7-(2-[3,5-dichloro-4-oxo-1(4H)-pyridyl]-acetamido)-3-([(5-methyl-1,3,4-thiadiazol-2-yl)-thio]methyl)-8-oxo-5-thia-1-azabicyclo[4,2,0]oct-2-ene-2-carboxylic acid (cefazedone, Refosporen). All strains including three penicillin-resistant beta-lactamase producing Neisseria were sensitive at a range of 0.0078 to 2.0 micrograms/ml cefazedone.


Pharmacological studies on a new thymoleptic antidepressant, 1-[3-(dimethylamino)propyl]-5-methyl-3-phenyl-1H-indazole (FS-32). Some pharmacological actions of 1-[3-(dimethylamino)propyl]-5-methyl-3-phenyl-1H-indazole (FS-32), a newly synthesized indazole derivative, were investigated in comparison with imipramine. FS-32 showed anti-reserpine activity in a dose-dependent manner, whereas imipramine exhibited a bell-shaped dose-response pattern. Catecholaminergic potentiation was demonstrated with FS-32. The results obtained from a norepinephrine potentiation test in vitro suggest that FS-32 may act in a manner qualitatively different from the tricyclic antidepressant. FS-32 produced a definite suppressive effect on isolation-induced fighting without affecting coordinated motor activity and on the duration of afterdischarge elicited by electrical stimulation to the amygdala or the hippocampus without producing a slow wave pattern in the EEG. Similar effects on fighting behavior and the afterdischarge were shown under imipramine with a slight motor incoordination and with a slow wave pattern, respectively. FS-32 produced practically no peripheral anti-cholinergic action, while it antagonized central cholinergic activity. FS-32 tended to produce an increase in chatecholamine content in the brain without MAO or COMT inhibitory activity. Norepinephrine uptake was inhibited by FS-32, but less than by imipramine. These pharmacological properties suggest a potential clinical utility of FS-32 as an antidepressant possessing thymoleptic activities.


Correlation between antiinflammatory activity and inhibition of prostaglandin biosynthesis induced by various non-steroidal antiinflammatory agents. The effect of several non-steroidal antiinflammatory agents (some of them original from our laboratory) on prostaglandin biosynthesis in guinea pig lung (biological method) and in bovine seminal vesicles (spectrophotometric method) has been studied. When this effect is compared with the in vivo antiinflammatory activity determined in the rat carragenin foot edema and with the recommended clinical daily dose in rheumatoid arthritis, a good correlation between these properties was observed. Only mefenamic acid showed a clear discordance. The experience shows that inhibitory effect of antiinflammatory drugs on prostaglandin synthetase in vitro may be used to predict, in most cases, their antiinflammatory activity in vivo in the pharmacological screening of the new compounds. Structure-activity relationship in the UR-series is also discussed.


Acrolein, the causative factor of urotoxic side-effects of cyclophosphamide, ifosfamide, trofosfamide and sufosfamide. The urotoxicity of oxazaphosphorine cytostatics is not based on their alkylating activity but on the presence of acrolein, which is spontaneously formed in the urine from the primary metabolites eliminated via the kidneys. Thus, acrolein proved to be the causative factor in the urotoxicity of oxazaphosphorines. The mechanism of action of the uroprotector sodium 2-mercaptoethane-sulfonate (mesnum, Mitexan) is mainly based on the formation of a non-toxic additive compound with acrolein.


[Bioavailability and local anesthetic effect of the combination of tyrothricin, fomocain, diphenhydramine and 8-hydroxyquinoline in a gel preparation for burns and wounds]. The biological availability was investigated of diphenhydramine and fomocaine, two of the active principles of the new formulation "Herit" used as a jelly against burns and small wounds. Fomocaine and diphenhydramine penetrate deeply into stratum corneum. The addition of fomocaine guarantees a quick pain relieving effect on the damaged skin. The jelly does not retain diphenhydramine and fomocaine.


Varied effects of dietary sucrose and cholesterol on serum lipids, lipoproteins and apolipoproteins in rhesus monkeys. Serum lipid, lipoproteins, apolipoproteins and plasma insulin and glucose were studied in rhesus monkeys (Macaca mulatta) fed high sucrose diets (69%, w/w), with and without added cholesterol. When compared to basal diet, a high sucrose diet with no added cholesterol fed for 6 weeks increased serum total cholesterol and triglycerides by factors of 1.2 and 2.8, respectively. Cholesterol supplementation of sucrose diets increased the serum total cholesterol levels by a factor of 2.2 and decreased the serum triglycerides by 0.47. The serum cholesterol response to experimental diets was reflected predominantly in beta-lipoprotein and to a lesser extent in alpha-lipoprotein. Sucrose diets without cholesterol enriched the beta- and pre-beta-lipoproteins with triglycerides and protein at the expense of cholesterol. On the same diet, the protein content of alpha-lipoprotein increased at the expense of cholesterol and triglycerides. In contrast, dietary cholesterol decreased the triglyceride content and increased the cholesterol content of all the lipoprotein classes. Sucrose feeding seems to increase ApoB more than non-ApoB proteins. The proportion of ApoC-II relative to ApcoC-III increased in each animal on a sucrose diet; exogenous cholesterol further increased this trend. While sucrose diet decreased ApoA-I/ApoA-II ratios, cholesterol supplementation reversed this trend. Dietary sucrose increased the plasma glucose, insulin, and insulin-glucose ratios. The addition of cholesterol also tended to decrease plasma glucose and insulin levels. These observations indicate varied responses of serum lipoproteins and apoproteins to dietary sucrose with and without cholesterol supplementation.


Studies of ciliated epithelia of the human genital tract. 3: Mucociliary wave activity in organ cultures of human Fallopian tubes challenged with Neisseria gonorrhoeae and gonococcal endotoxin. Quantative determinations of the mucociliary activity of human Fallopian tube epithelium maintained as organ cultures were performed using a light beam reflex method. In non-infected organ cultures the mucociliary wave (MCW) frequency slowly decreased during the first 54 hours of culture maintenance. In organ cultures experimentally infected with fresh isolates of Neisseria gonorrhoeae producing T1/T2 colonies the MCW frequency either decreased to subnormal values or completely ceased whereas in organ cultured infected with a laboratory-adapted gonococcal strain the MCW frequencies remained within normal range. In organ cultures exposed to gonococcal endotoxin prepared from the laboratory-adapted strain, as well as in cultures in which cell-free filtrates of medium from organ cultures infected with N. gonorrhoeae (producing T1/T2 colonies) were added to the culture medium, the ciliary activity decreased and subsequently ceased. The same phenomenon occurred when organ cultures were challenged with Escherichia coli endotoxin. The ciliostatic effect appeared before any morphological changes in the surface epithelium, including the cilia, were demonstrable by scanning electron microscopy.


Secretory antibody response of the cervix to infection with Neisseria gonorrhoeae. Cervical secretions from 157 women were examined for antibody against Neisseria gonorrhoeae by an indirect immunofluorescent antibody test. Antigonococcal antibody was detected in 73 (97%) of 75 infected women, being of the IgG class in 73 (97%), of the IgA class in 71 (95%), and of the IgM class in 29 (39%). IgM antibody was nearly always associated with infections of less than 15 days' duration. Immunoglobulin G, reactive with N. gonorrhoeae, was found in 23 (33%) of 70 non-infected women; of these, 19 had non-gonococcal cervicitis. Neither IgA nor IgM antibodies were detected in these women. Antigonoccal IgA and IgG was found in each of 12 women who had no evidence of infection but were contacts of infected men. Successful treatment resulted in a rapid decline in IgA antibody activity but a more gradual decrease in IgG reactivity.


Evaluation of urethral culture for Neisseria gonorrhoeae in the routine investigation of men attending a STD clinic. Urethral gonorrhoea was diagnosed by culture in 957 (18.8%) of 5076 new male patients attending a sexually transmitted diseases clinic; the infection was asymptomatic in 38 (4%) of these patients. The diagnosis in only six (0.3%) out of 2159 men with urethral gonorrhoea but with no symptoms or signs of urethritis would have been missed if urethral cultures had not been performed.


Double-blind comparison of two regimens in the treatment of nongonococcal urethritis. Seven-day vs 21-day course of triple tetracyclinc (Deteclo). In a double-blind comparison of two regimens of triple tetracycline (Deteclo, Lederle) in the treatment of nongonococcal urethritis, 68 (88.6%) of 70 patients treated with one tablet twice for 21 days and seen four weeks after starting therapy had satisfactory results. This was significantly better than the findings among the 73 patients treated with one tablet twice daily for seven days and followed for four weeks, among whom only 47 (64.4%) had satisfactory results. Results were also better for the group treated with the 21-day regimen at three months afer the start of treatment. When analysed individually at four and 12 weeks, urethral discharge, urethral Gram-stained smears, and first-glass urine test all gave similar results, which were markedly better than those before treatment. It appears that the longer course of treatment it indicated where any regular partner may not be treated. Slightly fewer patients had satisfactory results among those who admitted consuming alcohol than among those who did not. Chlamydiae-negative patients, treated for seven days, had fewer clinically satisfactory results than other sub-groups.


[Behavior of some enzymes of nucleoside catabolism in circulating lymphocytes during paraproteinemia]. The circulating lymphocytes of 16 normal subjects and of 18 patients with paraproteinemia have been characterized by measuring the levels of ADA, AMPA, PNPase, CDA. The results obtained reveal a highly significant increase in PNPase of subjects affected by paraproteinemia as compared to that of normal subjects (p less than 0.005). The fact that the increased enzymatic levels are found mainly in patients not affected by Bence-Jones proteinuria seems to indicate that the evolution of the paraproteinemia disease is in some way related to the intralymphocyte levels of PNPase.


Comparison of mutagenesis and malignant transformation by dihydrodiols from benz[a]anthracene and 7,12-dimethylbenz[a]anthracene. Five dihydrodiols derived from benz[a]anthracene (BA) and 4 dihydrodiols derived from 7,12-dimethylbenz[a]anthracene (DMBA) have been tested, together with the parent hydrocarbons, for their abilities to induce mutations to 8-azaguanine resistance in V79 (Chinese hamster cells and malignant transformation in M2 mouse fibroblasts. The syn- and anti-isomers of benz[a]anthracene 8,9-diol 10,11-oxide were also tested for biological activity in these two systems. The non-K-region 1,2- and 3,4-dihydrodiols of BA induced mutations but the non-K-region 8,9-dihydrodiol and the K-region 5,6-dihydrodiol were inactive as mutagens; none of these BA diols transformed M2 mouse fibroblasts. The 3,4- and the 8,9-dihydrodiols derived from 7,12-dimethylbenz[a]anthracene induced mutations in V79 cells and malignant transformation in M2 mouse fibroblasts and both were more active than the hydrocarbon itself. The K-region 5,6-dihydrodiol and the non-K-region 10,11-dihydrodiol of DMBA were inactive in both test systems. The results are not inconsistent with other data suggesting that the metabolic activation of both BA and DMBA occurs through conversion of the respective 3,4-dihydrodiols into the related vicinal diol-epoxides, although other dihydrodiols may also be involved in vivo. Both the BA diol-epoxides tested were mutagenic, but although the anti-isomer transformed M2 fibroblasts, the syn-isomer was inactive.


Interactions of 4-nitroquinoline 1-oxide with deoxyribodinucleotides. The interactions of 4-nitroquinoline 1-oxide (NQO), a potent mutagen and carcinogen, with several self- and non-self-complementary deoxydinucleotides were probed by using absorption spectra of the charge transfer bands and 1H and 13C NMR spectra. Absorption spectra were analyzed by using Benesi-Hildebrand-type equations to yield stoichiometries and equilibrium constants of complex formation. Non-self complementary dimers form weak l:1 complexes [dpTpG:NQO, K(25 degrees C) = 22 M-1] while self-complementary dimers form strong 2:1 complexes [dpCpG)2:NQO, K(25 degrees C) = 2.2 X 10(4) M-2]. A mixture of dpTpG and dpCpA with NQO gives a 2:1 complexes [dpCpG)2:NQO, K(25 degrees C) = 2.2 X 10(4) M-2]. A mixture of dpTpG and dpCpA, K(25 degrees C) = 8.6 X 10(3) M-2]. Analyses of the changes in 13C and 1H NMR chemical shifts with complex formation gave approximate orientations for the intercalation of NQO with self-complementary dimer minihelixes. In the (dpCpG)2:NQO and (dpGpC)2:NQO complexes, the NO2 group of NQO probably lies in the major grove and the NO2, NO containing NQO ring is stacked near the purine imidazole ring. In the (dpTpA)2:NQO and (dpApT)2NQO complexes, the NO2 seems to project into the minor grove and the NQO benzenoid ring is over the purine imidazole ring.


Nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase. The resonances of the aromatic protons of trimethoprim [2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine] in its complexes with dihydrofolate reductases from Lactobacillus casei and Escherichia coli cannot be directly observed. Their chemical shifts have been determined by transfer of saturation experiments and by difference spectroscopy using [2',6'-2H2]trimethoprim. The complex of 2,4-diamino-5-(3',4'-dimethoxy-5'-bromobenzyl)pyrimidine with the L. casei enzyme has also been examined. At room temperature, the 2',6'-proton resonance of bound trimethoprim is very broad (line width great than 30 Hz); with the E. coli enzyme, the resonance sharpens with increasing temperature so as to be clearly visible by difference spectroscopy at 45 degrees C. This line broadening is attributed to an exchange contribution, arising from the slow rate of "flipping" about the C7-C1' bond of bound trimethoprim. The transfer of saturation measurements were also used to determine the dissociation rate constants of the complexes. In the course of these experiments, a decrease in intensity of the resonance of the 2',6'-proton resonance of free trimethoprim on irradiation at the resonance of the 6 proton of free trimethoprim was observed, which only occurred in the presence of the enzyme. This is interpreted as a nuclear Overhauser effect between two protons of the bound ligand transferred to those of the free ligand by the exchange of the ligand between the two states. The chemical shift changes observed on the binding of trimethoprim to dihydrofolate reductase are interpreted in terms of the ring-current shift contributions from the two aromatic rings of trimethoprim and from that of phenylalanine-30. On the basis of this analysis of the chemical shifts, a model for the structure of the enzyme-trimethoprim complex is proposed. This model is consistent with the (indirect) observation of a nuclear Overhauser effect between the 2',6' and 6 protons of bound trimethoprim.


Isolation of a 70 000 molecular weight antigen of the Novikoff hepatoma. Previous reports from this laboratory have indicated that a number of cytosol and nuclear proteins of Novikoff hepatoma cells were immunologically related [Yeoman, L. C., Jordan, J. J., Busch, R. K., Taylor, C. W., Savage, H., & Busch, H. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3258; Busch, R. K., & Busch, H. (1977) Tumori 63, 347]. In preparation for analysis of their structure and function, studies were undertaken to purify nuclear antigen 2 from the cytosol of Novikoff hepatoma cells in high yield and purity. It was shown on Ouchterlony gels that cytosol nuclear antigen 2 formed a single immunoprecipitin band of identity with one of the bands extracted from Novikoff nuclear chromatin. In this study, a 70 000 molecular weight antigen was isolated from the cytosol of Novikoff hepatoma cells by ammonium sulfate fractionation, ion-exchange chromatography, and isoelectric focusing in a granulated gel bed. This protein which focused at a pI of 6.3 was labeled with 125I-labeled Bolton-Hunter reagent and purified on an Ultrogel AcA-44 column. As shown by electrophoresis on NaDodSO4-polyacrylamide gels, the antigen in the excluded volume migrated as a single protein with a molecular weight of 70 000. The overall purification over the starting material was 2890-fold.


The crystallizable human myeloma protein Dob has a hinge-region deletion. During experiments to prepare heavy-metal derivatives of the crystallizable human IgG1 (k) immunoglobulin Dob, it became apparent that this protein has several unusual features. (1) Instead of the four labile interchain disulfide bridges ordinarily found in IgG1, the Dob protein has only a single interchain disulfide bridge, which connects its two light chains. (2) The Dob heavy chain appears to be slightly smaller than a control gamma1 chain, as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration in guanidine. (3) The Dob heavy chain has three fewer residues of half-cystine than expected in gamma1 chains. (4) The Dob IgG is relatively resistant to digestion with papain and trypsin; however, it is readily digested with pepsin, although at an unusual site. These findings suggest that some or all of the gamma1 hinge region is missing in Dob. To localize the deletion, we prepared an F(ab')2 fragment consisting of two heavy-chain pieces (Fd') noncovalently associated with the light-chain dimer. The Fd' piece was isolated and digested with trypsin. The sequence of the C-terminal tryptic peptide was Val-Ala-Pro-Glu-Leu-Leu-Gly-Gly-Pro-Ser-Val. Positions 2-11 of this peptide are identical with residue positions 231-240 of the gamma1 chain. The N-terminal valine could be either Val-211 or Val-215 of the gamma1 sequence. A tryptic peptide, Val-Asp-Lys-Lys, was also isolated from Dob Fd'; this sequence is not found in the variable region of the Dob heavy chain [Steiner, L. A., Garcia Pardo, A., & Margolies, M. N. (1979) Biochemistry (following paper in this issue)] but corresponds to positions 211-214 of the gamma1 constant region. Therefore, the deletion cannot include these residues and must begin after Val-215; normal gamma1 sequence resumes at Ala-231. The same 15-residue deletion has been found in two other IgG1 proteins, Mcg [Fett, J. W., Deutsch, H. F., & Smithies, O. (1973) Immunochemistry 10, 115] and Lec [Rivat, C., Schiff, C., Rivat, L., Ropartz, C., & Fougereau, M. (1976) Eur. J. Immunol. 6, 545]. Possible explanations for the occurrence of identical hinge-region deletions in three different immunoglobulins are suggested by recent experiments demonstrating that the three constant domains and the hinge region of mouse gamma1 chains are each encoded by separate segments of DNA [Sakano, H., Rogers, J. H., Hüppi, K., Brack, C., Traunecker, A., Maki, R., Wall, R., & Tonegawa, S. (1979) Nature (London) 277, 627].


Amino acid sequence of the heavy-chain variable region of the crystallizable human myeloma protein Dob. The amino acid sequence of the heavy-chain variable region of the crystallizable human myeloma protein Dob has been determined. This protein has previously been shown to have a deletion in the hinge region [Lopes, A. D., & Steiner, L. A. (1973) Fed. Proc., Fed. Am. Soc. Exp. Biol. 32, 1003; Steiner, L. A., & Lopes, A. D. (1979) Biochemistry (preceding paper in this issue)]. The complete sequence was established by analysis, in the automated sequenator, of the intact Fd' piece and of three large overlapping fragments prepared from Fd' by digestion with cyanogen bromide, by tryptic digestion of the citraconylated Fd', and by cleavage with hydroxylamine. Portions of the sequence were confirmed by examination of the amino acid composition and the partial sequence of a variety of small peptides obtained by enzymatic degradation. The Dob heavy-chain variable region appears to belong to the VHIII subgroup, but there are several unusual substitutions. Residue 45 in the Dob sequence is proline, although all other known heavy-chain sequences in man, mouse, rabbit, and guinea pig have leucine at this position. Positions 10 (aspartic acid), 68 (alanine), and 82 (leucine) in the Dob sequence are also atypical. There is no deleted segment in the variable region of the Dob heavy chain nor any abnormality in the variable-constant joining region. The hinge-region deletion appears to be the only gross structural anomaly in the Dob heavy chain.


Interaction of divalent cations with beta-galactosidase (Escherichia coli). Although the addition of various divalent metals to beta-galactosidase resulted in apparent activation, only Mg2+ and Mn2+ actually did activate. The apparent activation by the other divalent metals was shown to be due to Mg2+ impurities. Calcium did not activate, but experiments suggested that it did bind. Other divalent metals which were studied failed to bind. The dissociation constants for Mg2+ and Mn2+ were 2.8 X 10(-7) and 1.1 X 10(-8) M, respectively, and in each case one ion bound per monomer. These constants corresponded very closely to apparent values which were obtained from activation studies. The apparent binding constant for Ca2+, obtained from competition studies, was 1.5 X 10(-5) M. Data were obtained which showed that Mg2+, Mn2+, and Ca2+ all compete for binding at a single site. Of interest and of possible molecular biological importance was the observation that, while Mg2+ bound noncooperatively (n = 1.0), Mn2+ did so in a highly cooperative manner (n = 3.4). The binding of Mn2+ (as compared to Mg2+) resulted in a twofold drop in the Vmax for the hydrolysis and transgalactosylis reactions of lactose but had little effect on the Vmax of hydrolysis of allolactose, p-nitrophenyl beta-D-galactopyranoside (PNPG), or o-nitrophenyl beta-D-galactopyranoside (ONPG); Km values were not effected differently for any of the substrates by Mn2+ as compared to Mg2+. When very low levels of divalent metal ions were present (0.01 M EDTA added) or when Ca2+ was bound with lactose as the substrate, a greater decrease was observed in the rate of the transgalactosylic reaction than in the rate of the hydrolytic reaction, and the Km values for lactose and ONPG were increased. Of the three divalent metal ions which bound to beta-galactosidase, only Mn2+ had significant stabilizing effects toward denaturing urea and heat conditions.


Purification of the messenger ribonucleic acid for the lipoprotein of the Escherichia coli outer membrane. The mRNA for the lipoprotein of the Escherichia coli outer membrane has been purified to 85% homogeneity. The purification procedure involved phenol extraction, NaCl extraction, gel filtration on Sephadex G-100 and Sephadex G-200, and reversed-phase column chromatography on RPC-5. The purity of the final product was estimated to be 85% by analysis of the ribonuclease T1 fingerprint of the mRNA. The purified mRNA was able to direct the synthesis of cross-reactive material with antilipoprotein serum in both the E. coli and the wheat germ cell-free protein-synthesizing systems. The size of the mRNA was determined to be 8.2 S from its mobility in polyacrylamide--agrose gels. During the purification, two other RNA species, similar in size to the lipoprotein mRNA, were also isolated. Their sizes were determined to be 8.7 and 9.1 S. They both were inactive in an E. coli cell-free protein-synthesizing system.


Photoreaction center of photosynthetic bacteria. 1. Further chemical characterization of the photoreaction center from Rhodospirillum rubrum. The photoreaction center from Rhodospirillum rubrum contains about 90% protein, 6% pigment, mere traces of lipids, and no cytochromes. It also contains at least 1 mol of ubiquinone and 1 iron atom per mol. Its three-component polypeptide chains were isolated by preparative electrophoresis, and their molar stoichiometry was established as 1:1:1. The amino acid composition of the photoreaction center from strain S1 and from its subunits is reported. The protein as a whole contains about 65% nonpolar residues, and the degree of hydrophobicity of its subunits is alpha less than beta less than gamma. The minimal molecular weight based on the extinction coefficient and on the amino acid content is 90 000. This corresponds to a half-cystine mole number of 6.


Photoreaction center of photosynthetic bacteria. 2. Size and quaternary structure of the photoreaction centers from Rhodospirillum rubrum strain G9 and from Rhodopseudomonas sphaeroides strain 2.4.1. The photoreaction center from Rhodospirillum rubrum strain G9 binds about 6 times as much sodium dodecyl sulfate as certain proteins commonly used as molecular weight markers for sodium dodecyl sulfate--polyacrylamide gel electrophoresis. This presumably explains the apparent discrepancy between the molecular weight of the photoreaction center determined by electrophoresis (76 000) and its minimal molecular weight (87 000). The molecular weight of the photoreaction center solubilized with Triton X-100 was determined by three different methods: conventional sedimentation equilibrium, a combination of sedimentation velocity and gel filtration measurements, and sedimentation equilibrium in H2O and in D2O. Each technique required a determination of the amount of bound detergent. All three methods gave molecular weight values close to 60 000. A similar molecular weight was found for the photoactive beta gamma dimer obtained from the photoreaction center of Rhodopseudomonas sphaeroides strain 2.4.1 which, as a whole, had a molecular weight of 87 000. These results indicate that the photoreaction center from Rp. sphaeroides is an oligomer of the type alpha 1 beta 1 gamma 1. In contrast, the photoreaction center from Rs. rubrum appears to be dissociated, in solution, into a photoactive beta gamma dimer and a free polypeptide alpha.


Isolation and characterization of an active variable domain from a homogeneous rabbit antibody light chain. The variable domain (VL) of allotype b4 light chains of rabbit IgG was isolated from both nonimmune heterogeneous IgG and a homogeneous antibody directed against type III pneumococcal polysaccharide. Light chains were first isolated and then cleaved under mild acidic conditions between residues 109 and 110. Reduction with dithiothreitol in guanidine hydrochloride cleaved both intradomain disulfide bridges as well as the interdomain disulfide bridge joining the variable and constant domain. The sulfhydryl groups were protected after reduction by p-chloromercuribenzoate. VL was isolated from this mixture of variable and constant domains by affinity chromatography, utilizing sheep antibodies directed against a peptide including residues 110--211 from nonimmune IgG light chain. The isolated VL domain was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and automated Edman degradation. VL from a homogeneous antibody was treated with dithiothreitol to remove p-chloromercuribenzoate, reoxidized, and recombined with homologous heavy chain. The binding of this recombinant to type III pneumococcal polysaccharide was identical with that of the light-chain--heavy-chain recombinant.


Nitrogenase from Rhodospirillum rubrum. Relation between 'switch-off' effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios. Nitrogenase activity of 'membrane-free' extracts, produced from nitrogen-starved Rhodospirillum rubrum to which 4 mM NH4+ had been added is only about 10% of the activity in the control. The activity could be restored to 80% by including the membrane component, earlier found to activate R. rubrum nitrogenase, in the reaction mixture. The relation between this 'switch-off/switch-on' effect and the function of the membrane component is discussed. Hydrogen production catalyzed by R. rubrum nitrogenase is also dependent on activation by the membrane component. Hydrogen production is inhibited by acetylene but the degree of inhibition is dependent on the nitrogenase component ratio. The strongest inhibition is achieved at low MoFe protein/Fe protein rations. The ATP/2E- values are 4-5 at the component ratios giving the highest activity and increase at high MoFe protein/Fe protein ratios. CO inhibits acetylene reduction but has no effect on the hydrogen production.


Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells. Techniques are described for the isolation of plastid thylakoid membranes from light-grown and dark-grown cells of Euglena gracilis var. bacillaris, and from mutants affecting plastid development. These membranes, which have minimal contamination with other cell fractions, are localized in sucrose gradients by using the thylakoid membrane sulfolipid as a specific marker. The plastid thylakoid membrane polypeptides isolated from these membranes were separated on SDS polyacrylamide gels and yielded patterns containing 30-40 polypeptides. Light-grown strain Z gave patterns identical with bacillaris. Since the plastid thylakoid polypeptide patterns obtained from dark-grown wild-type cells and from a bleached mutant W3BUL in which plastid DNA is undetectable are identical, it appears that the proplastid thylakoid polypeptides of wild-type cannot be coded in plastid DNA and are probably coded in nuclear DNA. The plastid thylakoid polypeptide patterns obtained from various dark-grown mutants, making large but abnormal chloroplasts, show a correlation between the amount of chlorophyll formed and the amount of a plastid thylakoid polypeptide thought to be associated wtth one of the pigment-protein light-harvesting complexes. Treatment with SAN 9789 (4-chloro-5-(methylamino)-2(alpha, alpha, alpha,-trifluoro-m-tolyl)-3-(2H(pyridazinone) known to block carotenoid synthesis at the level of phytoene, causes a progressive loss of all plastid thylakoid polypeptides during growth in darkness and results in the establishment of a new, lowere steady-state level of sulfolipid. At least ten of the plastid thylakoid polypeptides become labeled when isolated chloroplasts are supplied with radioactive amono acids; of these six are undectable in W3BUL and are, therefore, candidates for coding by plastid DNA.


Events surrounding the early development of Euglena chloroplasts. 16. Plastid thylakoid polypeptides during greening. Using sulfolipid to locate plastid thylakoid membranes in gradients from dark-grown resting cells it has been possible to study the plastid thylakoid membrane polypeptides of Euglena gracilis var. bacillaris undergoing light-induced chloroplast development. All plastid thylakoid bands seen in dark-growing wild-type cells and in mutant W3BUL in which plastid DNA is undetectable, are observed to increase in amount during plastid development. Others, which are undetectable in dark-grown wild-type and W3BUL increase greatly during plastid development and appear to be those associated with pigment-protein complexes. The data obtained from experiments where the polypeptides were labeled with 35S during development, either continuously or in pulses, were consistent with these findings. Cycloheximide strongly inhibited the increases in amount in all bands and chloramphenicol or streptomycin produced a lower level of inhibition in all bands indicating tight control of theformation of each plastid membrane constituent by the others. The formation of a polypeptide band of 25 000 molecular weight, thought to be a part of a pigment-protein complex of the thylakoid, and chlorophyll synthesis were inhibited identically by these antibiotics.


Identification of the protein producing transmembrane diffusion pores in the outer membrane of Pseudomonas aeruginosa PA01. The outer membrane of Pseudomonas aeruginosa PA01 is permeable to saccharides of molecular weights lower than about 6000. Triton X-100/EDTA-soluble outer membrane proteins were fractionated by ion-exchange chromatography in the presence of Triton X-100 and EDTA, and the protein contents of the various fractions analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Each of the major protein bands present in the Triton X-100/EDTA soluble outer membrane was separated from one another. Adjacent fractions were pooled, concentrated and extensively dialyzed to reduce the Triton X-100 concentration. Vesicles were reconstituted from lipopolysaccharide, phospholipids and each of these dialyzed fractions, and examined for their ability to retain [14C]sucrose. Control experiments indicated that the residual levels of Triton X-100 remaining in the dialyzed fractions had no effect on the formation or permeability to saccharides of the reconstituted vesicles. It was concluded that a major outer membrane polypeptide with an apparent weight of 35,000 is a porin, responsible for the size-dependent permeability of the outer membrane.


Calcium translocation and storage of isolated intact cattle rod outer segments in darkness. Bovine rod outer segments (rods), isolated with an intact plasma membrane and a stable calcium exchange and storage capacity, contain 2-3 mol endogenous calcium/mol rhodopsin. By means of 45Ca accumulation experiments and concomitant 40Ca analysis, the calcium metabolism of these organelles has been studied with the following results: 1. The majority of endogenous calcium is localized within disks. 2. In the presence of the ionophore A23187 the intradiskal binding sites can be titrated with external calcium. 3. The Scatchard plot of calcium binding of rods indicates the presence of a single set of intradiskal binding sites with a maximal capacity of 8-9 mol calcium/mol rhodopsin and an affinity constant of 55 microM to calcium. 4. Without A23187 more than 99% of the rod calcium appears in a bound state in equilibrium with a free calcium concentration of 15-25 microM. 5. External calcium exchanges with endogenous calcium in a fast (t 1/2 = 12 s) process with a uniform rate constant, whereas net calcium transport is very slow (t 1/2 greater than 2 h). 6. Intact rods contain a calcium translocation system, presumably located in the plasma membrane, which performs Ca-Ca exchange with a high unidirectional flux of 2 . 10(6) calcium ions/rod per s. 7. This translocation system can be saturated by external calcium (Km = 0.5 -1 microM) and has a low Q10 (1.08). Both the calcium translocation system and the calcium binding system appear to depend on the structural integrity of the stacked disks and are very sensitive to the experimental conditions. The relevance of these findings is discussed in relation to the proposed role of calcium ions as the intracellular transmitter in vertebrate rod photoreceptor cells.


Evoluation of Drosophila mitochondrial DNAs. Analysis of heteroduplex molecules. We have mapped the single block of non-homologous sequences and measured the extent and distribution of base-pair substitutions within the homologous sequences in Drosophila melanogaster: Drosophila virilis heteroduplex mitochondrial DNAs (mtDNAs). Of the 4.8 kilobases long, unusually (A + T)-rich region in D. melanogaster mtDNA, only 0.5 kilobases can react with related, but not identical sequences in D. virilis mtDNA, while the rest (4.3 kilobases in the long arm of a heteroduplex loop) is replaced by a shorter, non-homologous region (1.0 kilobases in the short arm of the loop). No additional heterologous regions are evident. Homologous sequences have accumulated on the average 15.5% base-pair changes. Regionally, these substitutions are relatively uniformly distributed (14.5--16.5%) except for a single, more conserved region (10--13%), which presumably represents the ribosomal cistrons. The lack of general sequence stability suggests that the invariant topographic organization of the nucleotide sequence, previously recognized among Drosophila mtDNAs, is under more stringent selection than the sequence per se.


Multiple carbohydrate-cleaving specificities in human acidic and neutral glycosidases. The common identity of human acidic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) and beta-D-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) as one enzyme and that of acidic beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), beta-D-fucosidase (no allotted EC number) and alpha-L-arabinosidase (alpha-L-arabinofuranoside arabinohydrolase, EC 3.2.1.55) as another enzyme is indicated by similar binding patterns of glycosidase activities of each enzyme to various lectins. by similar ratios between their intra- and extracellular levels in normal and I-cell fibroblasts and by their deficiencies in liver tissues from patients with Gaucher disease and GM1 gangliosidosis, respectively. A third enzyme, neutral beta-D-galactosidase, purified to homogeneity from human liver has been shown to possess all these five glycosidase activities at neutral pH. These neutral enzymic activities were not bound by any of the lectins examined and found to be reduced in liver and spleen of a patient with neutral beta-D-galactosidase deficiency. An additional form of beta-D-xylosidase with optimal activity at pH 7.4 was bound by the fucose-binding lectin from Ulex eurpaeus while no binding was observed for the acidic (pH 4.8) and neutral (pH 7.0) beta-D-xylosidase activities of the multiple glycosidase enzymes.


Immobilization of L-glutamate dehydrogenase into soluble cross-linked polymers. ADP effect and electron microscopy studies. Active soluble cross-linked L-glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) albumin polymers were produced. Electron microscopic studies and kinetic properties were studied with the polymer in solution and compared with previous published data about the enzyme immobilized inside proteic films (Barbotin, J.N. and Breuil, M. (1978)  Biochim. Biophys. Acta 525, 18--27). The glutaraldehyde effect on activity yield, ADP and beta-NAD+ protection, stability and pH rate profile were studied and discussed. Apparent Michaelis constants were determined with soluble polymers produced with or without ADP during the grafting process. Experiments were performed on the regulatory properties of immobilized glutamate dehydrogeanse showing the decrease of ADP activation and GTP inhibition as compared to the free form. In other respects, electron microscopy observations showed morphological differences between the two populations of soluble polymers produced in presence of ADP, obtained after gel filtration on Sepharose 6B. Linear aggregates of high molecular weight and classical soluble polymers were obtained. Similar Km values and regulatory properties were exhibited by the two forms, demonstrating the absence of interdependence between the allosteric control and the polymerization of enzyme monomers.


5-methylthioribose 1-phosphate: a product of partially purified, rat liver 5'-methylthioadenosine phosphorylase activity. 5'-Methylthioadenosine phosphorylase from rat liver has been purified 112-fold. A molecular weight of 90 000 for the enzyme was estimated from gel filtration on Sephadex G-150. The Km for 5'-methylthioadenosine was 4.7 . 10(-7) M, while the Km for phosphate was 2 . 10(-4) M. The products of the reaction were isolated and identified as adenine and 5-methylthioribose 1-phosphate. In addition to 5'-methylthioadenosine the nucleoside analogues 5'-ethylthioadenosine and 5'-n-propylthioadenosine also served as substrates for the enzyme. The 7-deaza analogue 5'-methylthiotubercidin was found to be an inhibitor of the reaction, but was inactive as a substrate.


Circular dichroism of lipoxygenase-1 from soybeans. The circular dichroism spectra of the three forms of lipoxygenase-1 from soybeans show characteristic differences in the region between 300 and 600 nm. Native lipoxygenase-1 only shows a negative dichroic band around 330 nm. Yellow lipoxygenase-1, obtained by addition of an equimolar amount of 13-F-hydroperoxylinoleic acid to the native enzyme, shows a positive Cotton effect at 425 nm, while the negative band band at 330 nm has increased in intensity. The blue enzyme, representing a complex of yellow enzyme with 13-L-hydroperoxylinoleic acid exhibits a negative dichroic band at 580 nm and positive bands at 410 and 391 nm. The near-ultraviolet CD spectra of the three forms of lipoxygenase are very similar, showing several well resolved positive dichroic bands at 0 degrees C. Using the method of Chen et al. (Chen, Y.-H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131) the contents of alpha-helix, beta- and unordered form of native lipoxygenase-1 were estimated to be 34, 27 and 39% respectively.


The high autoantibody activity of antibodies to rat skeletal muscle glycogen phosphorylase b produced in rabbits. Antibodies to rat skeletal muscle glycogen phosphorylase b were prepared in six rabbits by weekly injection of the enzyme emulsified with complete Freund's adjuvant. All the antiserum preparations showed high autoantibody activities to react with the rabbit muscle enzyme in both inhibition of enzyme activity and precipitation. In Ouchterlony double diffusion in agar, the antiserum preparations were precipitable to give a distinct spur between the two precipitin lines formed with rat and rabbit enzymes. When the autoantibody index was taken as per cent cross-reactivity of rabbit enzyme with rat enzyme, the autoantibody indices of inhibition and precipitation of one of the antiserum preparations were as high as 98% and 52.3%, respectively.


Inhibition of tyrosinase by indole compounds and reaction products. Protection by albumin. Tyrosinase activity decreases as the reaction proceeds and is inhibited by L-3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-product(s) inhibition or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.


Parvalbumins. Distribution and physical state inside the muscle cell. Single skinned muscle fibres (frog) have been submitted to double Ouchterlony immunodiffusion assays with antibodies directed against the two species of frog parvalbumins. The antigenic material which diffuses out of each fibre contains the two parvalbumins. Their presence in each cell is thus demonstrated. The amount of parvalbumins having diffused out of the fibre has been quantified. It corresponds to the parvalbumin content of the cell. This implies that these proteins are freely soluble in the muscle sarcoplasm.


[Comparative study of microsomal enzymic activities in adult and foetal monkey hepatocytes (author's transl)]. Differential centrifugation was applied to adult and foetal liver of monkey. Obtained fractions were: F1 (800 X g); F2 (12 500 X g); F3 (200 000 X g); and cell sap. Analysis of chemical compounds of these fractions shows that: (1) adult and foetal nucleic acids levels are similar; (2) there are more proteins in adult than in foetal hepatocytes; (3) most of the glycogen is located in F3; the foetal level is twenty times higher than the adult level. Plasma membrane enzymes (5'-nucleotidase, adenylate cyclase) show a nucleomicrosomic distribution. The distribution of alkaline phosphatase is not significant. Mitochondrial enzymes (monoamine oxydase, succinate cytochrome c reductase, cytochrome oxydase) are enriched in F2 without any sedimentation in F3. There is more malate dehydrogenase liberated in cell sap during foetal liver fractionation. This indicates the foetal mitochondria are more sensitive to the homogenisation method. Lysosomal enzymes (acid phosphatase, N-acetylglucosaminidase) are enriched in F2. The same observation for N-acetylglucosaminidase as for malate dehydrogenase leads to the same conclusion for foetal lysosomes. Endoplasmic reticulum and Golgi enzymes (glucose-6-phosphatase and related phosphotransferase activity, NADPH-cytochrome c reductase and sialytransferase) are much enriched in F3. Thus this fraction F3 is pure enough to allow the observation of the modification produced on endoplasmic reticulum and Golgi apparatus during foetal and neonatal development.


Response of heterogenous rat liver lysosome populations to starvation and refeeding. Starvation-induced alterations in liver lysosomes and their recovery pattern following refeeding were investigated. Fasting of adult rats for five days caused an increase in 'free' activities of acid hydrolyses in liver homogenates and loss in sedimentation of one of the heterogenous populations of lysosomes that could be isolated by differential centrifugation. Isopycnic sucrose gradient centrifugation revealed a decrease in the median and modal equilibration densities of all the forms of lysosomes in response to the dietary deprivation. Further, starvation also evoked a distinct bimodal distribution in a population that was rich in acid phosphatases, beta-galactosidase and N-acetyl-beta-glucosaminidase. Realimentation of starved animals for 10 days was found to restore the enzyme levels and the sedimentation characteristics to normal profiles.


Selective modification of positive chemotaxis in the true slime mold Physarum polycephalum by ethylenediaminetetraacetic acid treatment. The plasmodium of the true slime mold Physarum polycephalum was treated with EDTA or EGTA and the effect of the treatment on the chemotactic response was examined by measuring the chemotactic motive force with the double-chamber method. The results obtained were as follows: (1) The treatment of the plasmodium with 5 mM EDTA (pH 7.0, 20 min) did not give any significant effect on the protoplasmic streaming or motility. (2) The plasmodium treated with EDTA exhibited no chemotactic response to non-electrolyte attractants (D-glucose, D-galactose, D-mannose, and maltose) and negative chemotaxis to electrolyte attractants (cyclic AMP and NaH2PO4). (3) The EDTA treatment gave no effect on the chemotactic response to repellents (D-fructose, NaCl, CaCl2, MgCl2). (4) The EDTA-treated plasmodium exhibited changes in the membrane potential in response to both attractants and repellents as similar to the untreated plasmodium. (5) The treatment of the plasmodium with 5 mM EGTA (pH 7.0, 20 min) gave results similar to those obtained with the EDTA treatment. The results obtained suggested that EDTA (or EGTA) treatment did not affect the receptor sites but modified the transduction mechanism from reception into tactic movement.


Size measurements on isolated rat heart cells using Coulter analysis and light scatter flow cytometry. Isolated ventricular muscle cells from the adult rat heart have been examined by both Coulter analysis and light scatter flow cytometry. The dispersed cell preparations contain two main cell types: viable, rod-shaped cells and damaged, round cells. Coulter analytical techniques provided statistical data on cell volume for both cell types. The contribution of each population to the Coulter pulse height distributions were separated by a subtraction method using data obtained from digitonin-treated preparations that contain only round cells. A shape factor for cells aligned with the flow direction was computed from light microscope measurements and the effects of cell orientation within the Coulter aperture were approximately assessed. The estimated volumes for intact myocytes compare favourably with those reported in the literature. No significant size difference was observed between fresh and fixed cells. Narrow angle, forward light scatter measurements were made on individual cells flowing across a focused laser beam. Both scatter pulse height and pulse width (pulse duration) distributions were collected. Values for myocyte length calculated from pulse width information agree well with published data and confirm that the hydrodynamic forces in the flow system produced alignment of the cells with the flow direction. Scatter pulse width distributions reveal two distinct peaks assignable to either rod or round cells. Preliminary electronic gating experiments, using pulse height signals, suggest that signals derived from round cells could be eliminated entirely using a gating regime based on pulse width. This would enable flow cytometric measurements to be made on only the intact myocytes present in heterogeneous preparations.


The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis. Massive sporulation of Bacillus subtilis normally begins when carbon, nitrogen or phosphorus sources able to support rapid growth are no longer available. Sporulation can also be induced in exponentially growing cultures, in the presence of rapidly utilizable ammonia, glucose and phosphate if growth is partially but not completely inhibited either by inhibitors of nucleotide synthesis (hadacidin, decoyinine or 6-azauracil) or by purine deprivation in purine and especially in guanine auxotrophs. All these conditions allowing sporulation result in a decrease in the intracellular concentration of guanosine di- and tri-phosphates and usually uridine di- and triphosphates while other nucleotides decrease in some but increase in other cases. A decrease of uracil nucleotides alone, in a uracil auxotroph, does not produce massive sporulation. Our results demonstrate that the partial reduction of a guanine nucleotide, probably relative to some other compound, suffices to initiate sporulation. This reduction may always play a decisive role in the initiation of sporulation, as we have observed it under all conditions so far known to produce massive sporulation.


Homeostatic regulation of free retinol-binding protein and free thyroxine pools of plasma by their plasma carrier proteins in chicken. The mechanism underlying homeostatic regulation of the plasma levels of free retinol-binding protein and free thyroxine, the systemic distribution of which is of great importance, has been investigated. A simple method has been developed to determine the rate of dissociation of a ligand from the binding protein. Analysis of the dissociation process of retinol-binding protein from prealbumin-2 reveals that the free retinol-binding protein pool undergoes massive flux, and that prealbumin-2 participates in homeostatic regulation of the free retinol-binding protein pool. Studies on the dissociation process of thyroxine from its plasma carrier proteins show that the various plasma carrier proteins share two roles. Of the two types of protein, the thyroxine-binding globulin (the high affinity binding protein) contributes only 27% of the free thyroxine in a rapid transition process, despite its being the major binding protein. But prealbumin-2, which has lower affinity towards thyroxine, participates mainly in a rapid flux of the free thyroxine pool. Thus thyroxine-binding globulin acts predominantly as a plasma reservoir of thyroxine, and also probably in the 'buffering' action on plasma free thyroxine level, in the long term, while prealbumin-2 participates mainly in the maintenance of constancy of free thyroxine levels even in the short term. The existence of these two types of binding protein facilitates compensation for the metabolic flux of the free ligand and maintenance of the thyroxine pool within a very narrow range.


Purification and characterization of an estrogen-binding peroxidase from human fetuses. A peroxidase found under two forms with a molecular weight of 220,000 and 170,000 respectively, was purified from human fetuses. The purification procedure included ammonium sulfate precipitation, ion exchange chromatography, gel filtration and hydrophobic interaction chromatography. The purification factor approximated 400. These two forms of peroxidase were found to be immunologically identical as shown when utilizing immunodiffusion. They were able to bind estradiol in the presence of H2O2. This bond resisted to denaturation and solvent extraction therefore suggesting a covalent binding of estradiol to the enzyme.


Light adaptation in cone photoreceptors: the occurrence and significance of unitary adaptive strength. The behavior of "peak response-log intensity" functions generated by the "dark glasses model" is examined and is shown to describe previously observed light adapted behavior in cone photoreceptors. The models of Boynton and Whitten (1970) and Norman and Werblin (1974) are closely related to the dark glasses model--the Boynton-Whitten model being more specific and the Normann-Werblin model more general. For the models, a certain parameter relationship will produce systems which have optimal intensity discriminative capacities. When the data are fitted, this parameter relationship--unitary adaptive strength--seems to emerge. Possible evolutionary and psychophysical implications are discussed.


Dynamics of a long-latency reflex pathway in the monkey. Angular wrist displacements in the monkey result in a short-latency (20--25 ms) "reflex" response of motor cortical cells in area 4 and separate peaks of activity in the gross EMG of the stretched muscles. Frequency domain analysis was carried out between (1) wrist position as input and motor cortical neuron response as output, (2) position as input and EMG response as output, and (3) motor cortical neuron response as input and EMG response as output. The results show that the dynamics of primary spindle afferents characterize the dynamics of the pathway to the motor cortex and that of the reflex loop. Results are discussed in terms of "long-loop" reflexes.


Glucose and galactose infusions in newborns of diabetic and healthy mothers. The changes of insulin, blood sugars, lactate and free fatty acid levels were studied in 12 newborns of diabetic mothers and in 21 newborns of healthy mothers in the course of intravenous infusions of glucose and galactose at a dose of 0.5 g.kg-1.h-1. During glucose infusion a striking increase of insulin levels took place, which was higher in the infants of diabetic mothers. Galactose administration caused only a mild insulinemia increase in the 1st h of infusion. In the infants of healthy mothers blood glucose increased during the 1 st h of infusion but decreased afterwards. In the infants of diabetic mothers the increase lasted for the whole time of infusion. The decrease of free fatty acid levels was present in the course of infusions of both sugars. The results show that galactose is quickly metabolized by the newborn and provokes minimal stimulation of the insular apparatus.


[Effect of Ca2+ ions on thermal denaturation of chromatin in an aqueous medium]. Thermal denaturation of chromatin has been investigated in the presence of Ca2+ ions by adiabatic scanning microcalorimetryc and thermomechanical methods. In contrast to EDTA treated chromatin with two melting bands (74 degrees C and 82,5 degrees C) on differential melting curve, the only denaturation peak (82 degrees C) was observed in the presence of a very low Ca2+ level (0,1 mM). Chromatin melting temperature has been shown to decrease with Ca2+ concentration and in the region of 65-67 degrees C a heatless transition due to chromatin repacking in a more compact form occurred.


Development of jejunoileal differences of activity of lactase, sucrase and acid beta-galactosidase in isografts of fetal rat intestine. Jejunal and ileal segments from preterm rat fetuses were implanted under the kidney capsula of adult rats. Sucrase, lactase and acid beta-galactosidase activities were determined in the isografts at different times after implantation, and in corresponding segments developing in situ. Whereas fetal intestine contains considerable activity of acid beta-galactosidase and lactase, no sucrase activity is detectable. Similarly -- as in situ -- 4 weeks after the implantation the jejunal segment exhibited higher activity of sucrase and lactase than the ileal segment. Acid beta-galactosidase was more active in ileal than in jejunal segments -- both growing in situ as well as isografts. Experiments have thus demonstrated that the expression of the jejunoileal gradient of activity of the 3 enzymes studied does not depend on direct contact with food or gastric, pancreatic and biliary juices. This gives validity to the suggestion that the gradient may already be programmed in fetal intestinal tissue, but other factors active in situ might be responsible for its magnitude.


Double antibody specificity of lymphoid cells isolated from normal human intestinal mucosa. Lymphoid cells were isolated from normal human mucosal specimens. A certain fraction of these cells exhibited double antibody specificities at the levels of both light kappa and lambda chains and heavy mu and gamma chains. The variable frequency with which these cells were detected appears to be a characteristic of this population. They represent a very low proportion of the total number of cells isolated.


Mass spectral characterization of the K-region and non K-region dihydrodiols of 7,12-dimethylbenz (a) anthracene. Dihydrodiols are derived from intermediary arene oxides during the metabolism of polycyclic aromatic hydrocarbons. In this study, the mass spectra of the trimethylsilyl ether derivatives obtained from the 5,6-dihydrodiol and 8,9-dihydrodiol of 7,12-dimethylbenz [a] anthracene were compared. The mass spectral fragmentation patterns indicate some key features in distinguishing a K-region dihydrodiol from its non K-region isomer. The 8,9-dihydrodiol, a non K-region metabolite, exhibits a greater tendency to undergo a ring contraction fragmentation process while the K-region 5,6-dihydrodiol participates preferably in other fragmentation pathways. The data presented in this report will be useful in charcterizing K-region and nonK-region metabolic epoxidation of polycyclic aromatic hydrocarbons.


Use of an enzyme thermistor in continuous measurements and enzyme reactor control. The enzyme thermistor measures the heat produced by the action of an immobilized enzyme on a substrate present in the sample. Its application in analysis of discrete samples, e.g., in clinical chemistry, is well documented, but it has not been used so far for continuous measurements. We decribe here the application of the enzyme thermistor for continuous monitoring and control of enzyme reactors. An enzyme thermistor filled with coimmobilized glucose oxidase and catalase was used to measure the amount of glucose in the outflow from a column reactor containing immobilized lactase acting on a lactose solution pumped through the reactor. The lactose conversion was kept on a constant level, irrespective of the actual enzymatic activity in the reactor, by regulating the flow through the reactor. The experiments were carried out with aqueous solutions of lactose as well as with whey from cow's milk.


[Correlation between differentiation and malignancy in human malignant melanocytes "in vivo" and "in vitro" (author's transl)]. The relationships between differentiation and malignant transformation were studied in human malignant melanomas in vivo and in vitro. Melanocyte differentiation was assessed by ultrastructural morphological characteristis (the appearance of the melanosomes and related structures) localization of dopa-oxidase and assay of 5-S-cysteinyldopa, a specific metabolite. The transformed characteristic of the cells in vitro was evaluated by their ability to give rise to established cell lines, karyological modifications and heterotransplantation in Nude mice and Syrian hamsters. Morphological variability of the cells in malignant melanomas is accompanied by variability in the localization of dopa-oxidase, the level of 5-S-cysteinyldopa, chromosome pattern and their heterotransplantibility. The lack of pigmentation in some malignant melanoma lines can result from either an irreversible loss of some functions which give rise in melanization and the malignancy in maintained, or by phenomenon of regulation determined by intra or extra-cellular factors with the loss of heterotransplantability. Modulation phenomena affecting tumorigenicity and pigmentation although sometimes concomitant are not identical.


Elevation of blood lead concentration by confinement in the rhesus monkey. Rhesus monkeys were exposed to lead (Pb) acetate under various regimens during the first 12 months of life. At 30 months of age, these animals and unexposed controls were confined to an unfamiliar experimental chamber for one week. Serum cortisol concentration and Pb concentration in whole blood (PbB) were measured prior to, during, and after this confinement. Cortisol concentrations rose 60--90% within 2 hours of confinement, and declined to baseline levels after 98 hours of confinement. Mean baseline PbB levels reflected the state of clearance of the previously-ingested lead, rose 25--35% within 2 hours of confinement, and reached mean maximum levels as much as 100% above baseline after 98 hours of confinement. The data are discussed in terms of hormonal mobilization of Pb stored in bone, and subgest (1) that this storage is multicompartmental, (2) that more than one process is probably involved in its mobilization, (3) that cortisol probably does not directly affect PbB levels, and (4) that stress should be considered a potential factor in determining the PbB in studies of Pb metabolism.


Reduction of epileptic seizures by reinforcement of bladder continence. Epileptic seizures and resulting incontinence of urine were effectively reduced by reinforcement of incompatible behaviour with individual programming within a general token economy system. Two treatment phases, including token and social reinforcement, during an 11 week period resulted in significant seizure reduction. Therapeutic gains were maintained in a 6 month follow-up.


Two gas-gangrene-like infections due to Bacillus cereus. Two cases of postoperative gas-gangrene-like infection due to Bacillus cereus are reported, drawing attention to the fact that Bacillus cereus, a common environmental bacterium, can occasionally give rise to severe post-operative infection. Characteristics of the organism related to the epidemiology and pathogenesis of such infections are discussed.


Configurational prey selection by Bufo, Alytes, Bombina and Hyla. The aim of the present study is to answer the following questions; (1) Is the ability of toads to distinguish between different configurational prey stimuli based on learning during ontogeny? (2) Is the configurational meaning of a stimulus influenced by its distance from the animal? (3) Are there differences in configurational prey selection among different anuran species? (1) Experiments with postmetamorphic juveniles of Alytes obstetricans indicate that preferences for worm-like objects can be obtained immediately after metamorphosis. This preference undergoes some specialization during ontogeny. (2) The configurational meaning of 'worm-like' and 'antiworm-like' objects is -- within broad ranges -- independent of the distance from the animal so far as has been investigated in A. obstetricans and Bufo bufo. (3) The configurational discrimination ability (between worm-like and antiworm-like objects) has common basic components in different anuran species, so far investigated in the following species: B. bufo, Hyla arborea, Hyla cinerea, A. obstetricans. Bombina variegata. There are remarkable species differences for preferred absolute prey sizes, which may depend on different body sizes and in particular upon the size of the mouth.


An anatomical asymmetry in the baboon brain. A group of 7 baboon brains was examined for the presence of an asymmetry of the frontal poles. 6 of the 7 brains exhibited an asymmetry in which the right frontal pole was longer than the left by a mean of 1.4 mm. The seventh brain had frontal poles of equal length.


Organization of somatic thalamus in monkeys with and without section of dorsal spinal tracts. In an attempt to understand the modifications which appears at the thalamic level when dorsal cord sections are performed, peripheral fields of thalamic units were studied in normal and dorsal-cord sectional monkeys, totally awake and implanted with glass micropipettes. Six normal Macaca cynomolgus and 7 having received spinal sections, were studied. Ventricular radiography was performed and all the coordinates were related to new stereotaxic coordinates using the posterior commissure as the origin. Cell-bodies and axon units were recognized on the basis of the shape of the spikes. In normal animals, 972 units were studied; 307 were thalamic units with peripheral fields and 177 were derived from cell bodies. Localization of these cells was studied as a function of their peripheral field and response characteristics. The majority of ventralis posterior (VP) cell bodies were only activated by contralateral stimulation, their peripheral field being frequently found on the extremities. Somatotopic organization consisted of concentric layers rather than zones in apposition. Dorsoventral segregation of afferent modality sensitivity (movement, pressure, light touch) was observed. Somatosensory convergence was found in VP inferior (VPi) as well as convergence of different types of afferents on a few VP cells. Units responding to bilateral or ipsilateral stimulations were found only in posterior VP and in surrounding nuclei. A particular somatotopy was shown to exist in n. reticularis. A total of 838 units were studied in animals having had dorsal columns and Morin's bundle served. Only a few cells (13) responded in VPl to contralateral hindlimb stimulation. Their characteristics recall those noted in layer V spinal cord cells. The other cells (55) still driven by a hindlimb were dispersed in the nuclei just adjacent to VPl. The majority of cells found in the VPl were activated from the forelimb. They were observed in their normal VPl localization as well as in areas where hindlimb representation was found in normal animals. This change of afferent input is attributed to a reinnervation of hindlimb cells (probably at the gracilis level) by sprouting from forelimb afferent.


Function of caudate neurons during limb movements in awake primates. Recordings from single neurons were made in the head of the contralateral caudate nucleus in awake primates during performance of visually and somatically guided hand movements. Populations of cells showed temporal correlation of discharge patterns with visual or somatic disturbances or both. However these discharges occurred about or after the onset of the compensating movement triggered by the disturbances. These findings indicate that the observed cells do not participate in the initiation of these compensating movements, and may instead act as part of an internal feedback loop whose function is unrelated to movement onset.


Enhancement of self-stimulation behavior in rats and monkeys after chronic neuroleptic treatment: evidence for mesolimbic supersensitivity. The effect of chronic neuroleptic drug treatment on self-stimulation of the mesolimbic dopamine system was tested. Rats with electrodes implanted into the ventral tegmental nucleus (A10 cell body area) were treated with haloperidol for three weeks. Afterwards, the rats showed a 35% increase in self-stimulation rate, as compared to pre-drug control rates. This increase persisted for three weeks after drug withdrawal before returning to baseline rates. Rats treated for three weeks with the atypical neuroleptic, clozapine, also showed an increase, the duration and magnitude of which was similar to that seen in the haloperidol group. In addition, four rhesus monkeys with electrodes in the nucleus accumbens (one of the terminal projection areas of the A10 mesolimbic dopamine system) were given a three week treatment with haloperidol, after which all animals showed a significant, long-lasting decrease in self-stimulation threshold, as measured by a rate-independent reward paradigm. Taken together, these results suggest the induction of receptor supersensitivity in the mesolimbic dopamine system by long-term treatment with neuroleptic drugs.


Behavioral functions of the reticular formation. Studies of the behavioral correlates of activity in reticular formation cells, usually performed in restrained animals, have found units whose discharge relates to sensory stimuli, pain and escape behavior, conditioning and habituation, arousal, complex motivational states, REM sleep, eye movements, respiration and locomotion. Units with these different behavioral correlates were found in the same anatomical areas. Most studies report that a large proportion of encountered cells related to the behavior being studied. If one adds up the reported percentages, the total far exceeds 100%. Therefore it appears that many investigators are looking at the same cells and reaching very different conclusions about their behavioral roles. On the basis of observations in unrestrained cats, it is hypothesized that discharge in most RF cells is primarily related to the excitation of small groups of muscles. This hypothesis can parsimoniously explain many previous observations on the behavioral correlates of these cells, and is consistent with anatomical, physiological and phylogenetic studies of the reticular formation. The hypothesized simplicity of reticular formation unit function is contrasted with the complexity of the behavioral functions mediated by the RF, and the implications of this contrast discussed.


Intestinal calcium binding protein in uremia. In order to assess the usefulness of intestinal biopsies as indicators of end-organ responsiveness to vitamin D in uremic patients, calcium binding activity and calcium binding protein (CaBP) content were measured in intestinal biopsies from 12 uremic patients (glomerular filtration rate less than 5.0 ml/min) and 12 adult controls. Values for both were found to vary with the site of biopsy, highest values being obtained in the duodenal bulb, with lower values distally. Values for activity correlated with values for CaBP content in both normals and uremics and no difference was observed between groups. Levels of calcium binding activity and content of CaBP did not correlate with serum immunoreactive parathormone levels, but were directly related to circulating 25-hydroxycholecalciferol (25-OHD) levels. The data show that intestinal CaBP is normal in activity, quantity, and affinity for calcium in malabsorbing uremic patients, and are consistent with the hypothesis that calcium malabsorption in uremia is unrelated to deficiency of intestinal calcium binding protein.


Subcellular localization of immunoreactive alpha-melanocyte stimulating hormone in human brain. The regional and subcellular distribution of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHi) in the post mortem adult human brain was investigated. alpha-MSHi was highly concentrated in medial basal hypothalamic tissue (1.02 ng/mg protein). Lower levels of alpha-MSHi were present in the optic chiasm and mammillary bodies, 0.08 and 0.11 ng/mg protein, respectively. The concentrations of alpha-MSHi in cerebellum and frontal cerebral cortex were 1/1,000th that of the medial basal hypothalamus. When medial basal hypothalamic homogenates were subjected to discontinuous or continuous sucrose density gradients, alpha-MSHi was found to be associated primarily with subcellular particles which resembled isolated nerve terminals, i.e., synaptosomes. Low to undetectable amounts of alpha-MSHi were found in the cytosol or the myelin/microsome fraction of the gradients. The results of these studies are consistent with the view that alpha-MSH is a neuronal peptide in the human brain.


Purification and partial characterization of two acid phosphatases from rat bone. Acid phosphatase activity in homogenized tibiae and femora of suckling rats was extracted with 0.3M KCl and 0.1% Triton X-100. A high-speed supernatant was treated with protamine sulfate, dialyzed, and chromatographed on CM-52 cellulose. All of the acid phosphatase activity was eluted with a sodium acetate buffer and combined ionic strength-pH gradient into two peaks (E1 and E2). Both enzyme peaks were further purified with Sephadex G-200, which resulted in 700- and 1000-fold purification for E2 and E1, respectively. A total of 220 units (mumoles substrate/min) of E2 with a specific activity of 160 units/mg protein has been obtained in one run by this procedure. E1 has a high molecular weight (greater than 100,000) and shows preference for monophosphate ester substrates, is markedly inhibited by tartrate, and has a pH optimum near 5. E2 has a lower molecular weight (greater than 40,000) and shows negligible activity with monophosphate esters [except with p-nitrophenyl phosphate (p-NPP)], but high activity with ADP and ATP. E2 is unaffected by tartrate and shows a pH optimum near 6. Both enzymes are competitively inhibited by inorganic phosphate, and E2, but not E1, is markedly inhibited by p-chloromercuribenzoate. With p-NPP as substrate, E1 and E2 have distinctly different values for Km. E1 appears similar to the high molecular weight acid phosphatases of soft tissues. However, E2 appears to differ from the low molecular weight phosphatases in soft tissues with regard to substrate specificity.


Isolation of specific bone cell types by free-flow electrophoresis. The bone cells and fibroblasts from fetal rat calvaria can be isolated by collagenase digestion of the collagen matrix and separated into specific cell types by free-flow electrophoresis. The method involves injection of a specially prepared suspension of cells into a stream of buffer across which is maintained an electric field of 60 V/cm. The fetal bone cell types are differentially deflected toward the anode where they can be collected. Free-flow electrophoresis of this heterogenous cell preparation yields three distinguishable peaks which can be identified by morphologic, morphometric, and enzymatic characteristics. All three cell peaks have greater than 95% viability as judged by trypan blue exclusion and will grow to confluent monolayers in culture. The data indicate that these cell peaks may be comprised of osteoclasts and/or preosteoclasts, osteoblasts and/or preosteoblasts, and fibroblasts.


Chondroitinase-resistant sulfated glycosaminoglycans synthesized by cartilages of chick embryos and of newborn chickens and rats. Biosynthesis of chondroitinase-resistant glycosaminoglycans as minor components was studied in the cartilages of chick embryos and of newborn chickens and rats. Sternal and knee cartilages were labeled in vitro with 35SO42-, and then 35S-labeled glycosaminoglycans were analyzed. In rats up to 2 weeks old, only one glycosaminoglycan could be detected as heparan sulfate. In the chick embryos and the newborn chickens, however, keratan sulfate as well as heparan sulfate could be detected. As chondroitinase-sensitive glycosaminoglycans, large amounts of both chrondroitin 4- and 6-sulfates were synthesized in the chick cartilage, but the synthesis of chondroitin 6-sulfate could scarcely be seen in the rat cartilage. The results seem to indicate that the biosynthesis of keratan sulfate has some relation to that of chondroitin 6-sulfate.


Changes in proline synthetic and degradative enzymes during matrix-induced cartilage and bone formation. Proline biosynthetic and degradative enzymes are unevenly distributed in differentiated mammalian tissues. Activities of the synthetic enzymes are relatively high in collagenous tissues, whereas activities of the degradative enzymes are high in noncollagenous tissues. In order to further characterize tissue-specific proline biosynthesis and degradation, we have determined proline enzyme activities during cartilage and bone formation induced by demineralized bone matrix. We can thus follow temporal changes in enzyme activity in a single tissue as different cell types develop. Ornithine aminotransferase and pyrroline-5-carboxylate reductase have peaks of activity which correlate with maximal type II collagen synthesis by chondrocytes. Both enzymes also are active during bone formation. In contrast, proline oxidase and pyrroline-5-carboxylate dehydrogenase are present at low levels and do not change as new cell types appear. Arginase activity peaks during the first 3  days and then rapidly decreases by the time cartilage and bone formation begin. These observations further substantiate the importance of proline biosynthesis in collagenous tissues. The close correlation between ornithine aminotransferase activity and type II collagen synthesis suggests that the pathway from ornithine to proline may be especially important during formation of type II collagen.


Phosphopeptides and gamma-carboxyglutamic acid-containing peptides in calcified turkey tendon: their absence in uncalcified tendon. Uncalcified samples of turkey tendon obtained prior to calcification, and other samples from areas of tendon that never calcify, contain little or no O-phosphoserine [Ser(P)], O-phosphothreonine [Thr(P)] and gamma-carboxyglutamic acid (Gla). Significant amounts of all three of these Ca2+-binding amino acids, which are found in EDTA-extractable, non-collagenous proteins, are detected coincident with the onset of mineralization of a tendon and increase in concentration as mineralization proceeds.


Cemento-ossifying fibroma of the orbit.

Cemento-ossifying fibroma of the orbit. Cemento-ossifying fibroma presents with ophthalmic symptoms and signs rarely. We report the clinical and pathological findings in a case of cemento-ossifying fibroma of the right maxilla with extension into the orbital floor causing intermittent vertical diplopia, proptosis, and upward displacement of the globe. Compression of the nasolacrimal duct produced epiphora early in the course of the disease. Fibrous dysplasia has often been diagnosed in other cases of benign monostotic fibro-osseous conditions. Ossifying fibroma is easily confused with fibrous dysplasia. The histopathological difference between the two lesions is described.


[Transmissible modification induced in Mycobacterium tuberculosis and Mycobacterium bovis in vitro]. A biologically active material (fraction "S") is isolated from cultures of scotochromogenic mycobacteria. Mycobacterium tuberculosis, or Mycobacterium bovis by disrupting the cells, sedimentation through 2.2 M sucrose, and ultrafiltration. The fraction "S" induces the modification of tubercle bacilli into non acid-fast bacteria forming smooth colonies on nutritive glycerol agar within 24-36 h of incubation. Three new phenotypes are thus obtained; two proved to be stable upon subculturing. Frequently the phenomenon occurs with a very large part of the Koch's bacillus population exposed to the inducing agent effect. It can be reproduced with crude preparations of DNA obtained from the fraction "S." It is inhibited by concanavalin A. The observed modification does not correspond to a transfer of characteristics of the inducing agent from the donor mycobacteria; furthermore it can be manifested even in the strain used for the preparation of the fraction "S."


[Carbon assimilation and taxonomic study of Bacillus subtillis and B. licheniformis]. All 14 strains of B. subtilis can use the following 17 sources of carbon and energy: D-glucose, D-mannose, D-glucosamine, salicin, D-ribose, maltose, sucrose, cellobiose, trehalose, arbutin, starch, mannitol, glycerol, glycerate, pyruvate, fumarate, and L-proline. All 15 strains of B. licheniformis can use the following 41 sources of carbon and energy: D-glucose, D-galactose, D-mannose, D-fructose, D-glucosamine, alpha-methyl-D-glucoside, beta-methyl-D-glucoside, salicin, D-gluconate, saccharate, D-xylose, L-arabinose, L-rhamnose, D-ribose, maltose, sucrose, cellobiose, melibiose, trehalose, arbutin, raffinose, starch, inulin, mannitol, D-sorbitol, glycerol, glycerate, citrate, L-malate, D-malate, mucate, pyruvate, fumarate, alpha-L-alanine, alpha-D-alanine, asparagine, L-glutamate, L-arginine, DL-ornithine, L-proline, and 4-amino-n-butyrate. The 29 strains form two distinct groups. Group A includes the 15 strains of B. licheniformis and 2 strains of B. subtilis; group B is formed of 11 strains of B. subtilis; the remaining strain of B. subtilis belongs to neither group. Bacillus licheniformis is a more homogeneous species than B. subtilis. The percentage of guanine + cytosine in the DNA of all 29 strains was determined. In the 14 strains of B. subtilis the average is 46.3% +/- 1.5. In the 15 strains of B. licheniformis the average is 46.4% +/- 0.9.


Auxanographic grouping and typing of Neisseria gonorrhoeae. Nearly 96% of 1297 Neisseria gonorrhoeae isolates in Hamilton were assigned to six major auxanographic groups (non-requiring or NR, Pro-, Orn-, Pro-Cit-Ura-, Orn-Ura-Hyx-, Cit-Ura-Hyx-) as established by requirements for none, or any one or more of proline, uracil, hypoxanthine, citrulline (Cit-), or citrulline replaceable by ornithine (Orn-) on chemically defined medium modified from Catlin (1973). Seven other groups, and strains not growing, accounted for 4.2%. The most common groups were Orn-Ura-Hyx- (25.6%) and Pro-Cit-Ura (31.8%). This latter group has not been previously described. These "Pro Cit/Orn Ura Hyx" criteria were among the most unequivocal to interpret; with rare exceptions for proline, a requirement was shown by absence of growth at any time in the zones of inoculation from a replicator. For some strains, some of the possible additional requirements (leucine, valine, isoleucine, glutamine, threonine, serine, histidine, etc.) could be less readily ascertained, because an occasional manifestation was to reduce the amount of growth, or to slow down the rate of growth, compared to complete medium. The first four of the six groups were the least fastidious, in that few strains had any additional requirements. About 2% of strains were inhibited by 0.25 mM phenylalanine.


Photically induced seizures in the yellow baboon, Papio cynocephalus. Significant susceptibility to photically induced seizures has in the past been observed only in Senegales baboons (Papio papio) and epileptic humans. However, we have unexpectedly observed a photomyoclonic response to intermittent photic stimulation in 5 of a sample of 6 yellow baboons (Papio cynocephalus).


Taurine and beta-alanine uptake in cultured human skin fibroblasts from patients with Friedreich's ataxia. Taurine and beta-alanine uptake kinetics were studied in cultured skin fibroblasts from 9 patients with Friedreich's Ataxia and 8 controls. No significant difference was observed. The data support the presence of normal beta-amino acid carrier protein in Friedreich's Ataxia cell membrane.


The ineffectiveness of analogs of D-galactal as competitive inhibitors of, and substrates for, beta-D-galactosidase from Escherichia coli. 2,6-Anhydro-3-deoxy-aldehydo-D-lyxo-hept-2-enose (7) and 2,6-anhydro-3-deoxy-D-lyxo-hept-2-enitol (8) were synthesized as half-chair analogs of D-galactal (1). As 1 is a strong inhibitor of, as well as a substrate for, beta-D-galactosidase from Escherichia coli, the same properties were expected for 7 and 8; however, both were ineffective. This result, together with those of other authors, allows speculative conclusions on the tight binding of 1 to the enzyme only, when water or an alcohol is bound as a co-substrate.


Dissociation of effects of nitroglycerin on regional refractoriness and regional myocardial blood flow following acute coronary occlusion. Nitroglycerin is known to affect the electrophysiological properties of the ischaemic ventricle, possibly by altering regional myocardial blood flow. This study correlated the effects of nitroglycerin, given after acute coronary occlusion, on regional ventricular refractoriness and regional myocardial blood flow. Nitroglycerin returned ventricular refractory periods to their pre-occlusion values in spite of no significant effect on regional myocardial blood flow. Although the beneficial electrophysiological effects of nitroglycerin were not explained by increased regional flow to the ischaemic myocardium, an improved myocardial oxygen supply-demand balance may have produced these favourable effects. This study emphasises the need for electrophysiological evaluation of the effects of interventions intended to limit infarct size.


Ultrastructure of an excitatory synapse. The ultrastructure of the afferent synapse in hair cells of the lateral line-canal organ was studied using different fixation and staining techniques. Glutaraldehyde-fixed tissue without post-osmication, contrasted by section staining with uranyl acetate and lead citrate, was compared with (a) osmium tetroxide-fixed tissue followed by the same staining procedure, and with (b) glutaraldehyde-fixed tissue, block-impregnated with phosphotungstic acid (PTA). The results reveal a pronounced heterogeneity in the composition of the synaptic body, reflecting regional differences it the "intracleft substance", the synaptic structure defined by the PTA staining technique, is actually due to the glutaraldehyde fixation procedure and is apparently the outer leaflet of the postsynaptic membrane. A special technique that allows alternate sections of a series to be differentially stained for electron microscopy is proposed.


[Development of thyroid peroxidase activity during the early stage of iodine deficiency in the hypophysectomized rat]. The iodide-peroxidase activity of the thyroid gland remains, in hypophysectomized Rats maintained on a low iodine diet, below normal value. This result suggests that the early increase of peroxidase activity observed in control Rats maintained on a low iodine diet is TSH dependent.


[Existence of a non-retinotopic contralateral retino-tectal visual projection in the normal frog Rana esculenta L]. In the Frog, after unilateral optic nerve and tract section, contralateral visual responses were recorded in the tectum ipsilateral to the section. These responses were elicited by stimulation of a unique spatial region located near the projection of the contralateral eye optic axis and could be randomly recorded on the tectal surface. The possible pathways and the role of such retino-tectal afferences are discussed.


[Ultrastructure of gonadotropic cells of the normal eel and after estradiol injection]. Male and female silver eels, in fresh water or sea water, have few gonadotrophs (GTH) poorly differentiated. An oestradiol (E2) treatment induces their development: a well developed Golgi area, abundant secretory granules (200-500 nm), dilated cisterna and large globules 1.2-2.2 micron) are observed. A positive feedback may control gonadotropin synthesis in immature eels. No macroscopic effect is discernible on the gonad.


[Role and origin of the centriole].

[Role and origin of the centriole]. The centriole could constitute a system of inertia in the cell, providing the system of references controlling the movement of the cell. It would ensure equally the coherence of cell metabolism and its stability. The origin and functioning of the centriole would be linked to a singel physical phenomenon.


[Specific binding of 3H-progesterone to a plasma protein in the guinea pig fetus]. The presence of a protein that binds specifically and with high affinity to progesterone is found in the plasma of fetal guinea Pig. Progesterone and 5 alpha-dihydroxyprogesterone strongly compete for the 3H-progesterone-protein complex. 5 beta-dihydroxy progesterone, 20 beta-dihydroxyprogesterone, testosterone and synthetic progestagen, R-5020 (17,21-dimethyl-19-norpregna-4, 9-diene-3,20 dione), also compete but less intensely. 17-hydroxyprogesterone, aldosterone, cortisol, oestradiol and oestrone have no effect. The 3H-progesterone-protein, complex has an affinity of Kd = 8.8 +/- 3.5 x 10(-10) M and in sucrose density gradient the sedimentation coefficient is 4.6 S.


[Type of interactions between agarose and Aspergillus niger endopolygalacturonases]. Endopolygalacturonase has been purified by binding on Sepharose 6 B gel; studies on interactions between agarose and the enzymes have shown that endopolygalacturonase chromatography on Sepharose 6 B was on ion-exchange chromatography and did not involve biospecific interactions.


[Study on the distribution of methyl-mercury (203Hg) by whole body autoradiography of macaque monkeys (Macaca irus)]. The distribution of methyl-mercury labelled with mercury-203 has been studied in Monkeys by whole body autoradiography technique. The mercury is rapidly absorbed. It is localized in all the body and mainly in well defined areas of the central nervous system.