The alteration of membrane proteins in human erythrocyte membranes induced by quinolinic acid, an endogenous neurotoxin. Correlation of effect with structure

Mixtures containing subfractions of human plasma high-density lipoproteins (HDL) and human lipoprotein-free plasma were incubated in vitro at 37 degrees C. Esterification of cholesterol was observed both in incubations containing HDL-subfraction 3 (HDL3) and in those containing HDL-subfraction 2 (HDL2). The implication that the lecithin: cholesterol acyltransferase in lipoprotein-free plasma may therefore interact with lipoproteins in both HDL subfractions was developed further by proposing a simple model in which the two HDL subfractions may compete for interactions with the enzyme. This model was described mathematically and tested in experiments in which a constant amount of the enzyme was incubated with a wide range of concentrations of HDL2 and HDL3 present either alone or in combination. The model was able to predict experimentally observed rates of cholesterol esterification with great accuracy. The best fit was obtained with a Vmax for HDL3 that was 2.4-4-times greater than that for HDL2 and values of the apparent Km for HDL3 free cholesterol and HDL2 free cholesterol of 43-60 nmol/ml and 167-391 nmol/ml, respectively. The model thus predicts that, at physiological concentrations of lipoproteins, HDL2 will function as a competitive inhibitor of the cholesterol esterification reaction by displacing lecithin: cholesterol acyltransferase from a more effective substrate, HDL3, to a less effective substrate, HDL2.     

Studies with a human plasma-derived immunosuppressive,anti-lymphoma factor

Summary A low-molecular-weight (1,400) factor isolated from a human plasma -globulin concentrate by acid-salt dissociation and ultrafiltration inhibits proliferation of mitogen-stimulated T cells and L1210 leukemia cells. The factor (UM05R) inhibits DNA, RNA, and protein synthesis in sensitive cells, acts in G1 of the cell cycle, and appears to suppress mitogen-responsive T cells without an accessory cell requirement. UM05R activity is enhanced by known cAMP-elevating agents and by sulfhydryl compounds. The results of the present study are consistent with the hypothesis that the plasma-derived agent inhibits lympho-proliferation as a result of elevation of intracellular cAMP.     

Effect of diamide administration on longevity, oxygen consumption, superoxide dismutase, catalase, inorganic peroxides and glutathione in the adult housefly, Musca domestica

The effects of various levels of tetracycline hydrochloride on the feeding habits, larval growth, and the metabolism of lipids, carbohydrates and proteins of Heliothis virescens were studied. Regression analysis showed that larval weight, fatty acids and glycogen decreased exponentially with increasing concentration of antibiotic, whereas protein showed a linear decrease. Larval feeding was initially decreased when antibiotics were added to the diet but there was no difference after 24 hr.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

Heterogeneity of Intermediate Filament Expression in Vascular Smooth Muscle: A Gradient in Desmin Positive Cells from the Rat Aortic Arch to the Level of the Arteria Iliaca Communis

The display of the two distinct intermediate filament proteins, desmin and vimentin, in rat vascular smooth muscle tissue was studied by immunofluorescence microscopy on frozen sections of aorta and other blood vessels. Vascular smooth muscle cells present in these vessels always appeared rich in vimentin. However, staining of sections covering six distinct but contiguous parts of the aorta showed that the number of desmin containing cells was low distal to the truncus brachiocephalicus, but increases until in distal parts of the aorta and in the arteria iliaca communis almost all cells appear positive for desmin. Thus blood vessels show heterogeneity of intermediate filament expression not only in cross-section but can also display heterogeneity along their length. Muscular arteries such as the renal artery and the arteria femoralis, as well as arterioles and veins including the vena jugularis and the vena cava also contain desmin. Thus it may be that low numbers of desmin-positive cells are typical of elastic arteries, while muscular arteries and other blood vessels are characterized by large numbers of desmin-positive cells. We discuss whether desmin-positive and desmin-negative vascular smooth muscle cells may perform different functions and raise the possibility that desmin expression may coincide with the turn on of a specially regulated contractility program.     

Isolation and characterization of adult rat hearts cells.

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca²⁺ at concentrations greater than 10^?5m.     

In vivo growth hormone (GH) response to human GH-releasing factor (GRF) or somatostatin (SRIF) in foetal pigs.

The short-term effect of hypothalamic GRF and SRIF on the pituitary release of GH at different stages of gestation has been studied. In the present experiment eighteen gilts were used, six at each of 66, 88 and 110 days of gestation. Ventral laparotomy was performed under general anaesthesia and a section of uterus was exteriorized. Blood samples were obtained from the umbilical vein of three foetuses per gilt just prior to the injection of each foetus with either saline, 5 micrograms/kg of hGRF (1-44)NH2 or 50 micrograms/kg of SRIF into the umbilical vein. Additional blood samples were obtained 15, 30, 45 and 60 min post-injection. Serum samples were radioimmuno-assayed for GH (porcine). There was a treatment by gestational age interaction (P less than 0.01) on mean GH concentrations, area under the GH curve and GH peaks. While treatments had no effect (P greater than 0.1) on GH variables at 66 days of gestation, the area under the GH curve was slightly increased by GRF (P = 0.14) at day 88 and all GH variables were significantly increased (P less than 0.01)) by GRF at 110 days of gestation. There was a quadratic effect of time post-injection on GH concentrations at 88 (P less than 0.05) and 110 (P less than 0.001) days of gestation. There was no effect of SRIF injection (P greater than 0.1) on GH concentrations at any gestational age. In conclusion, the foetal pituitary responsiveness to GRF develops with foetal age and is maximal at the end of gestation, whereas there is no short-term response to a bolus of SRIF at any stage of gestation.     

A continuous fluorescent assay for measuring protease activity using natural protein substrate.

A continuous caseinolytic activity assay has been developed and characterized with trypsin, a serine protease, and transin, a metalloproteinase. Beta-casein labeled with both N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and fluorescein isothiocyanate (FITC) is used as the substrate in this assay. The effect of proteolysis of the substrate is a reduction of the intermolecular energy transfer from DACM to FITC. The caseinolytic activity is then monitored by the fluorescence increase. The activities of both proteases obey Michaelis-Menten kinetics with Km = 1.6 +/- 0.2 microM for trypsin and Km = 13.2 +/- 1.9 microM for transin. Protease concentrations as low as 10 ng/mL can be utilized. The pH dependence of the caseinolytic activity has been determined for both enzymes.     

Nucleotide sequence and evolution of a mammalian α-Tubulin messenger RNA

Bacterial clones containing complementary DNA sequences specific for rat brain α-tubulin messenger RNA were constructed. One plasmid, pILαTl, contains >95% of the sequences found in the mRNA: the entire coding sequence as well as extensive 5′ and 3′ untranslated sequences. Comparison of the rat amino acid sequence with the known chicken α-tubulin sequence (Valenzuela et al., 1981) reveals the extraordinary evolutionary stability of α-tubulin protein. The presence of only two interspecies amino acid differences within analogous 411 amino acid sequences predicts that amino acid substitutions in this protein are fixed with a unit evolutionary period (Wilson et al., 1977) of 550 million years (i.e. the time required for a 1% difference to arise within a specific protein in two diverging evolutionary lineages). An analysis of the silent nucleotide differences, permissible because of the degeneracy of the genetic code, demonstrates that these might not occur in a random fashion. The high guanine-cytosine bias in silent codon positions within the chicken α-tubulin sequence, previously noted by Valenzuela et al. (1981), is not conserved within the rat sequence. This decrease in guanine-cytosine bias is accompanied by a selective loss of CpG dinucleotides in the rat sequence.     

Human plasma-derived immunosuppressive factors having anti-lymphoma activity

Summary Extraction of Cohn IV-1, an -globulin enriched fraction of human plasma, with a high-salt, low-pH solution, followed by sequential ultrafiltration steps yielded an immunosuppressive preparation (UM05R) of mol.wt. 500–10,000. UM05R inhibited antibody formation in the mouse in vivo and transformation in vitro of lymphocytes treated with either T-or B-cell stimulants. Suppression of lymphocyte transformation, indicated by inhibition of ³H-thymidine incorporation into DNA, was confirmed by inhibition of blast cell formation. From dose-response curves the UM05R concentration to produce 50% suppression of lymphocyte blast transformation was 15–50 g protein/ml. Selectivity for lymphoid cells was suggested by growth inhibition in vitro of L1210 and P1798 leukemias but not murine neuroblastoma or human fetal fibroblasts. This observation also rules out the presence of an agent which is broadly cytotoxic. Fractionation of UM05R on Sephadex G-25 in 10% acetic acid yielded an early-emerging fraction, mol. wt. 5,000–10,000, containing B-cell inhibitor, and a late fraction, mol. wt. 1,400, inhibitory for both T- and B-cell transformation and growth of L1210. The inhibitory activity for B cells was removed from the other two activities by 5% trichloroacetic acid (TCA). The possibility is raised that the inhibitory activity for T cells and L1210 may reside in the same molecule. Sensitivity of the early-emerging B-cell inhibitor to carboxypeptidase B suggests that it is a polypeptide, but resistance of the T-cell inhibitor to various treatments leaves its nature uncertain. The properties of these factors suggest consideration of them as lymphocyte chalones occurring in plasma complexed to high-molecular-weight components.