Bacteria-derived human growth hormone lacks lipolytic activity in rat adipose tissue

Bacteria-derived human growth hormone (hGH) shows little $\text{in}\text{vitro}$ lipolytic activity in adipose tissue from fed rats. In adipose tissue from fasted rats no lipolytic activity is observed. However, bacteria-derived hGH increased serum free fatty acids after intraperitoneal administration to hypophysectomized rats to the same extent as purified pituitary hGH. The dose response of the bacteria-derived hGH tested for $\text{in}\text{vitro}$ insulin-like activity was very similar to the pituitary extracted material. Thus bacteria-derived hGH behaves in a manner indistinguishable from highly purified preparations of pituitary hGH.     

Relationship between growth and meiotic maturation of the mouse oocyte

Oocytes of various sizes were isolated from trypsinized ovaries of juvenile mice, cultured in a chemically defined medium, and scored for the resumption and completion of meiotic maturation. Oocytes recovered from mice younger than 15 days remained in the germinal vesicle stage, whereas those from mice 15 days or older resumed meiosis at a frequency which increased with the age of the mice. The mean diameter of the oocytes recovered also increased with the age of the mice. Within individual litters, the mean diameter of oocytes which failed to mature (incompetent oocytes) was significantly less than that of oocytes which matured (competent oocytes). The frequency of premature metaphase I arrest decreased markedly as the age of the mice and oocyte volume increased. These results suggest that the ability to resume meiosis is acquired at a specific stage of oocyte growth in the juvenile mouse, and that the ability to complete meiotic maturation is acquired subsequently. These oocytes provide an in vitro system with which to study the control of meiosis in the mammal.     

Serum-independent human lymphoblastoid cells. Effects of cell density on growth

A serum-independent (SI) line of human lymphoblastoid cells has been developed from a clone of the serum-dependent (SD) line RPMI 8866. The SI cells have been growing in serum-free culture for more than one year. In high serum concentrations the growth of the SI cells is identical to that of the SD cells. At low serum concentrations the SD cells die while the SI cells survive and grow. The growth of SI cells is density-dependent and can be overcome by the addition of serum, conditioned medium, or daily feeding with fresh medium.     

Investigation of the role of lysine in the subunit contact regions of rabbit muscle aldolase.

Rabbit muscle aldolase (E.C. 4. 1. 2. 13) was guanidinated by reaction with O-methylisourea. Up to 60% of the lysine residues can be guanidinated without any dissociation of the tetramer but with a complete loss of enzymatic activity. Native and guanidinated aldolase can be dissociated into monomers in 2.4 m MgCl₂ with only slight change in conformation of the subunit. Nitrotroponylation of guanidinated aldolase in dilute buffer gives no reaction whereas in 2.4 m MgCl₂ nitrotroponlylation modifies another 8–12% of the lysine residues. Removal of MgCl₂ by dialysis affords 100% recovery of activity and tetrameric structure for native aldolase and 100% recovery of tetrameric structure for guanidinated aldolase. In contrast nitrotroponylated and guanidinated aldolase remains monomeric before precipitating as the MgCl₂ concentration is lowered. It is concluded that lysine may be involved in the protein-protein interaction of the subunit contact domains of muscle aldolase.     

Program of early development in the mammal: changes in the patterns and absolute rates of tubulin and total protein synthesis during oocyte growth in the mouse

Oocytes at several stages of growth have been isolated by enzymatic digestion and/or physical disruption of ovaries excised from juvenile and adult mice. The absolute rates of total protein synthesis and tubulin synthesis in these isolated oocytes were determined by measuring sizes of the endogenous methionine pool and apparent rates of incorporation of [³⁵S]methionine into total protein and tubulin using methods described previously (R. M. Schultz, M. J. LaMarca, and P. M. Wassarman, 1978,Proc. Nat. Acad. Sci. USA,75, 4160;R. M. Schultz, G. E. Letourneau, and P. M. Wassarman, 1979,Develop. Biol.,68, 341). The size of the endogenous methionine pool increases approximately 350-fold during oocyte growth, from 0.16 fmole in nongrowing oocytes (12 μm) to 56 fmole in fully grown oocytes (85 μm). Since the volume of mouse oocytes also increases about 350-fold during growth, the concentration of intracellular free methionine remains constant at approximately 170 μM. The absolute rate of protein synthesis increases from 1.1 to 41.8 pg/hr/oocyte for nongrowing and fully grown mouse oocytes, respectively. Since this represents about a 38-fold increase in the absolute rate of protein synthesis, the rate of synthesis per picoliter of cytoplasm actually decreases nearly 10-fold during oocyte growth. These measurements indicate that the growing mouse oocyte itself is capable of synthesizing only about 50% of the protein found in fully grown oocytes. Tubulin is one of the major proteins synthesized by growing mouse oocytes since the absolute rate of tubulin synthesis is, on the average, 1.8% of total protein synthesis. The absolute rate of tubulin synthesis increases from 0.4 to 0.6 pg/hr/oocyte as the oocyte grows from 40 to 85 μm in diameter. However, overall, the percentage of total protein synthesis devoted to the synthesis of tubulin actually declines somewhat during this phase of growth, from 2 to 1.5%. Although equimolar amounts of tubulin subunits are present in microtubules, the ratio of absolute rate of synthesis of the β subunit to that of the α subunit varies from 1.3 to 2.0 throughout oocyte growth. High-resolution two-dimensional gel electrophoretic analyses of [³⁵S]methionine-labeled proteins reveal that many changes take place in the pattern of protein synthesis during oocyte growth.     

Direct hypophysial inhibition of luteinizing hormone release by dopamine in the rabbit.

The role and site of action of dopamine in regulating gonadotropin secretion remain unclear. In the present study, we investigated the possibility that dopamine regulates LH secretion by acting directly on the pituitary gland of the rabbit. The effect of dopamine infusion on LHRH-evoked LH release was determined in intact and pituitary stalk sectioned animals. Intravenous injection of LHRH (1 μg) in intact and acutely stalk sectioned rabbits increased peripheral plasma LH levels from a resting value of 0.2 ng/ml to maximal values of 12–14 ng/ml within 10–20 min. When dopamine was infused iv at a dose of 6.6 μg/min/kg BW from 30 min before LHRH injection until 120 min after, the rise in plasma LH levels in intact and stalk sectioned animals was decreased by 50–70%. However, dopamine infused at a lower dose (0.66 μg/min/kg BW) or at a higher dose (66.0 μg/min/kg BW), did not affect the LHRH-induced secretion of LH. These results suggest that dopamine can exert a direct hypophysial inhibitory effect on release of LH. They also demonstrate that dopamine is inhibitory only within a restricted dose-range, extending to the pituitary an established property of dopamine in the cardiovascular system.     

Purification and properties of cytochrome P-450(11beta) from adrenocortical mitochondria.

A cytochrome P-450, which is functional in the steroid methylene 11β-hydroxylation (P-450_11β), has been purified to a protein weight of 85 kg per heme from bovine adrenocortical mitochondria. The purification is accomplished in the presence of deoxycorticosterone as a substrate stabilizer. The procedure involved solubilization of sonicated mitochondrial pellets, ammonium sulfate fractionation, alumina Cγ gel treatment and aniline-substituted Sepharose 4B chromatography.The purified preparation when freed from deoxycorticosterone, has a low spin type absorption spectrum which can rapidly be converted into a typical high spin substrate-bound form by the addition of an 11β-hydroxylatable steroid, either deoxycorticosterone or testosterone. The preparation exhibits high 11β-hydroxylase activity and is free from the cholesterol side-chain cleavage cytochrome P-450 (P-450_scc).The purified P-450_11β, when submitted to SDS-polyacrylamide gel electrophoresis, exhibits a single protein band (molecular weight of 46 kilodaltons) which is clearly distinguished from P-450_scc. As determined by the sedimentation equilibrium method, the molecular weight of the guanidine-treated P-450_11β is estimated to be 43 kilodaltons.     

Studies on a possible pituitary effect of monoamines on luteinizing hormone release in ovariectomized rats

Incubation of anterior pituitaries from ovariectomized rats with LHRH and various concentrations of dopamine, norepinephrine or serotonin indicated that none of these neurotransmitters could decrease pituitary LH secretion in response to the releasing hormone. This indicated that the inhibitions of pulsatile LH release previously observed in our laboratory in ovariectomized rats in response to intraventricularly administered catecholamines or stimulation of brain serotoninergic neurons are due to central rather than pituitary effects of these transmitters.     

Morphological observations on cultured lampbrush-stage Rana pipiens oocytes.

Early lampbrush-stage oocytes are characterized by small lampbrush chromosome loops, a small amount of ribonucleoprotein (RNP) matrix on the loops, small nucleoli, few RNP particles in the nucleoplasm, and a smooth germinal vesicle contour. In vitro culture of these oocytes in serum-free culture medium for 24 hr at 18°C promotes a number of morphological changes in the oocytes: The lampbrush loops increase in diameter and acquire extensive RNP matrix, the nucleoli increase in size and complexity, the nucleoplasm accumulates numerous polymorphic RNP particles, and the germinal vesicle envelope acquires a sacculated contour. These characteristics are typical of the in vivo maximum lampbrush stage, and their appearance is due to an apparent in vitro acceleration of the lampbrush phase. Two possible interpretations of these observations are discussed.     

Regulation of palmitoyl-CoA inhibition of mitochondrial adenine nucleotide transport by cytosolic fatty acid binding protein.

Defatted liver fatty acid binding protein (FABP) reverses the inhibitory effect of palmitoyl-CoA on adenine nucleotide transport in rat liver mitochondria; addition of titrating amounts of FABP to mitochondria pretreated with palmitoyl-CoA stimulates nucleotide transport and that activation parallels the removal of the inhibitor from mitochondria. This effect is specific only for FABP; all other cytosolic proteins which do not bind fatty acids do not influence nucleotide transport activity. Addition of free fatty acids (which can compete for ligand binding sites on FABP) to mitochondria pretreated with palmitoyl-CoA interferes with the reversal activity of FABP. Adding FABP alone to freshly isolated mitochondria also activates nucleotide transport activity suggesting that the originally submaximal activity is probably due to the presence of endogenous long-chain acyl-CoA esters in the mitochondrial preparation. Because FABP is present in relatively high concentration in most mammalian cells, these observations offer a likely explanation of why the potent inhibitory effects of long-chain acyl-CoA esters on adenine nucleotide transport in isolated mitochondria are not seen in the intact cell.     

Evidence for an essential role for arginyl residues for yeast phosphoglycerate kinase.

Reaction of yeast phosphoglycerate kinase with either butanedione or cyclohexanedione can result in modification of up to all 13 arginyl residues with total loss of activity; however, extrapolation to zero activity for partially modified preparations indicates that up to 7 arginyls are essential. Whereas 20 mm 3-phosphoglycerate affords partial protection of activity toward both reagents, 20 mm MgATP affords complete protection of activity and protects 2 arginyls against modification by butanedione and 1 arginyl against modification by cyclohexanedione. With butanedione the modification could be reversed with total recovery of activity, suggesting that only arginyl groups were modified, which is consistent with the amino acid analysis of the modified protein. Only at high cyclohexanedione concentrations or long reaction times was a yellow product obtained that showed loss of lysyl residues. Circular dichroism spectra show that even when all the arginyls are modified by butanedione or up to 10 modified by cyclohexanedione there is no change observed in the far or near ultraviolet, indicating that there is no detectable conformational change concomitant with modification, which is confirmed by hydrodynamic studies. It is concluded that at least one, possibly two, arginyls of yeast phosphoglycerate kinase are essential for its action on ATP.     

Oxytocin: anti-insulin-like effects in isolated fat cells

Oxytocin has both insulin-like and insulin antagonistic actions in fat cells in vitro. The anti-insulin-like effects of oxytocin in dispersed rat fat cells have been studied. The magnitude of the anti-insulin-like activity varies with the metabolic pathway of glucose utilization; oxidation [14CO2 production], 32%; glycogen synthesis (D-[U-14C] glucose incorporation into glycogen), 77%. In addition, direct inhibition of the activation of fat cell glycogen synthase has been shown. These inhibitory effects depend upon an intact disulfide ring, since the ability of N-ethylmaleimide-reacted oxytocin to inhibit insulin-stimulated processes was reduced by more than 90% when compared to the intact molecule.     

Isolation and properties of OSCP and an F1-ATPase binding protein from rat liver mitochondria - evidence against OSCP as the linking "stalk" between F1 and the membrane.

Oligomycin Sensitivity Conferral Protein (OSCP) and an F₁-ATPase Binding Protein were isolated from F₁-depleted rat liver mitochondrial membrane. Their molecular weights on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea were 22,500 and 8,500 respectively. When incubated with liver TUA (trypsin, urea and ammonia-treated) submitochondrial particles, the binding protein was effective in the binding of F₁ to the particles with the resultant particle-bound ATPase activity not oligomycin sensitive. When OSCP was then incubated with the reconstituted membrane-bound ATPase, its activity became oligomycin sensitive. These results suggest that, first; the binding protein, but not OSCP, connects F₁-ATPase to the membrane of rat liver mitochondria and maybe to the “stalk”, if indeed there is a stalk in mitochondrial membrane ATPase complex; and second; the function of OSCP is solely to render the ATPase activity sensitive to oligomycin and other similar inhibitors.     

Anomalous behavior of certain poliovirus polypeptides during SDS-gel electrophoresis.

Urea is shown to be a useful additive to sodium dodecyl sulfate gels prior to electrophoresis and offers an alternative means for the resolution of some SDS-protein complexes. Two types of effect are described, one of which causes increases in the relative mobilities of certain polypeptides found in poliovirus-infected cells. It is postulated that urea plus SDS is able to achieve a more complete denaturation of some polypeptides, which leads to faster electrophoretic mobilities. The molecular weight estimations for such proteins in these conditions are therefore lower than those determined in the presence of SDS alone. A second effect of some urea solutions is to cause the multiple banding of the structural polypeptide VP3 when included in gels at high concentrations. This effect is variable and appears to be unrelated to the presence of cyanate ions.     

Studies on the immunological properties of complex V (mitochondrial ATP synthetase complex)

Antibody raised in rabbits against Complex V (miochondrial ATP synthetase complex) purified from beef heart mitochondria cross-reacted with Complex V and submitochondrial particles from beef heart, beef adrenals, and rat liver as shown by double-diffusion and rocket immunoelectrophoresis analysis. Of the various isolated and purified components of Complex V, only the oligomycin sensitivity-conferring protein showed strong reactivity with the anti-Complex V antibody, soluble F₁-ATPase reacted very faintly, while F₆ and ATPase inhibitor protein showed no precipitin lines. Crossed immunoelectrophoresis indicated that antigenic determinants recognized by the antibody were present on OSCP and possibly on the dicyclohexylcarbodiimide-binding protein. The components of Complex V could be precipitated from beef heart submitochondrial particles dissolved in Triton X-100 and pretreated with control IgG. When the composition of the immunoprecipitate was compared to that of purified Complex V, all the constituent polypeptides of the latter were present in the immunoprecipitate, except for one polypeptide in the low-molecular-weight region. Incubation of Complex V or submitochondrial particles with the anti-Complex V antibody in the absence of Triton X-100 caused inhibition of ATP-P_i exchange but not of ATPase activity. In the presence of Triton X-100, oligomycin sensitivity of Complex V was lost and the antibody was able to inhibit also the ATPase activity. The enzymic activity of soluble F₁-ATPase was unaffected by the antibody in the absence or presence of Triton X-100. These results suggest that the anti-Complex V antibody might be a useful tool for identifying and probing the role of Complex V components involved in energy transduction.     

Correlation of (Na---K)-ATPase activity with growth of normal and transformed cells

The (Na⁺---K⁺)-stimulated Mg²⁺-dependent ATPase activities of 3T3 and SV40 transformed 3T3 cells were compared as a function of cell population density. For normal cells the enzyme activity remained relatively constant during exponential growth, but decreased sharply coincident with contact inhibition of growth at confluence. This decrease in activity could be reversed by stimulating contact-inhibited cultures to undertake renewed short-term growth either by adding fetal calf serum or changing the medium completely. Transformed cells did not experience a decrease in (Na⁺---K⁺)-ATPase activity upon reaching confluence, but this is consistent with the fact that they were still growing exponentially at this stage. However, non-confluent cultures of both normal and transformed cells incurred a marked decrease in levels of the enzyme when growth was inhibited by serum depletion. The results have been interpreted as indicating that levels of (Na⁺---K⁺)-ATPase in both normal and transformed cells are correlated with growth. Hence the different patterns of ATPase activity displayed by malignant cells and their normal counterparts with increase in cell number appear to be a reflection of their dissimilar growth behaviours rather than of any innate difference between them.     

Polyadnylic acid sequences in RNA able to transfer specific delayed type hypersensitivity in vitro.

Antibody-depedent cell-mediated cytotoxicity (ADCC) could be initiated without protein synthesis [human peripheral blood lymphocytes as effector cells incubated with 10^?3M cycloheximide, (Cy)], although the reaction did not achieve its full lytic ability. This partial inhibition of ADCC was dependent on the dose of Cy. Both ADCC and protein synthesis returned to normal values after removal of the inhibitor. The kinetics of the reaction carried out by Cy-treated effector cells for short periods was similar to that of controls. After this time, the percentage of lysed target cells increased continuously in controls, while the cytotoxiciy of Cy-treated effector cells reached a plateau. When effector cells carried out ADCC in the presence of Cy, their lytic mechanism was “wasted,” and it could be recovered only by removal of the inhibitor. Our results indicate that effector cells have a preformed lytic mechanism operative in ADCC. This lytic mechanism is consumed during the reaction and its recovery requires protein synthesis.     

Separation of bases, ribonucleosides and deoxyribonucleosides by anion-exclusion and partition chromatography on cation-exchange resin: application to the assay of ribonucleotide reductase, deaminase and nucleosidase.

[5-³H]CDP and CTP are used as substrates in the assay of ribonucleotide reductase, deaminase and nucleosidase activity in crude enzyme preparations. After incubation, the nucleotides are hydrolyzed to nucleosides by sequential treatment with potato apyrase and alkaline phosphatase. An aliquot is then chromatographed on a cation-exchange column at 50°C with 0.1 m boric acid, adjusted to pH 7.4 with ammonia, used as eluant. The pyrimidines Ura, Urd, dUrd, Cyt, Cyd and dCyd are separated and eluted in about 50 min in small volumes. Assays by this procedure of CTP reductase activity in crude fractions of ribonucleotide reductase from Euglena gracilis gave results comparable to those obtained by the standard method. The new procedure is also applicable when adenine or guanine nucleotides are used as substrates. The adenine derivatives Ade, Ado, dAdo, Hyp, Ino, dIno as well as the guanine derivatives Gua, Guo, dGuo, Xan, Xao are separated from each other in this chromatographic system in about an hour.