Differential effect of growth factors on growth stimulation and phenotypic stability of glutamine-synthetase-positive and -negative hepatocytes in primary culture

In rat liver parenchyma, two subpopulations of hepatocytes can be distinguished by the absence or presence of the marker enzyme, glutamine synthetase (GS). Hepatocytes in the perivenous zone immediately adjacent to the hepatic venules in the liver acinus are positive for GS. Using autoradiography in combination with immunocytochemistry, the response of these two hepatocyte populations (GS positive and GS negative) to a variety of growth factors (defined compounds or complex stimuli) was investigated in vitro. Irrespective of the individual growth-promoting activity (which varied considerably), all stimuli led to much higher labeling indices in GS-negative cells as compared to GS-positive cells. In GS-negative cells, the strongest effect was exerted by serum obtained from partially hepatectomized rats (labeling index, 67%) and the conditioned media of JM1 and JM2 hepatoma cells (63%-82%), followed by a combination of insulin and either norepinephrine (46%) or epidermal growth factor (EGF; 42%). In contrast, serum had the weakest influence on GS-positive cells (0.3%), while the other potent stimuli enhanced the labeling index of these cells by between 6% and 15% within 48 h. The percentage of labeled nuclei was higher in mononucleated than in binucleated GS-positive hepatocytes. The time course of thymidine incorporation was also different for the two subpopulations. Under all growth-promoting conditions, the stimulation of GS-negative cells peaked between 72 and 96 h, while it increased continuously in GS-positive cells for at least 120 h, particularly in the case of serum. In proliferating cultures, both the absolute and the relative number of GS-positive hepatocytes decreased, while no such effect was found in various nonproliferating control cultures maintained at low and high cell density. Similar results were found for GS activity. In contrast, the hormonal induction of tyrosine aminotransferase (TAT) was not affected. It is suggested that these differences in the growth response of GS-positive and -negative cells contribute to the acinar gradient in hepatocyte proliferation that occurs during liver regeneration. Furthermore, the striking phenotypic instability of GS-positive cells that have undergone DNA synthesis and mitosis supports the hypothesis that cellular reprogramming depends on passage through the cell cycle.     

Reduced cerebrospinal fluid levels of immunoreactive pro-opiomelanocortin related peptides (including beta-endorphin) in anorexia nervosa

The discovery that the endogenous opioid peptides contribute to the modulation of appetitive behavior and neuroendocrine function has raised questions as to whether disturbances of opioids contributes to the pathophysiology of eating disorders. To assess central nervous system (CNS) beta-endorphin in patients with anorexia nervosa we measured cerebrospinal fluid (CSF) beta-endorphin concentrations before, and at intervals after weight correction. In addition, we measured three sister peptides (beta-lipotropin, adrenocorticotropic hormone (ACTH), and the N-terminal fragment) derived from the same precursor molecule, pro-opiomelanocortin (POMC) to determine whether possible disturbances might extend to sister peptides. Underweight anorectics (58 +/- 5% of average body weight (ABW), n = 10) had significantly lower CSF concentrations of all 4 peptides compared to healthy controls (102 +/- 10% ABW, n = 11). CSF concentrations of all 4 POMC-related peptides were found to be significantly increased when the same anorectics were restudied 4 to 6 weeks after weight gain (83 +/- 4% ABW). After weight gain, levels of CSF beta-endorphin, beta-lipotropin, and ACTH were similar to controls, whereas levels of CSF N-POMC remained significantly less than controls. Another group of women, previously underweight with anorexia nervosa, but weight-restored (93 +/- 11% ABW, n = 12) for greater than 1 year had CSF concentrations of all 4 POMC-related peptides that were similar to controls. We conclude that underweight anorectics have state-associated disturbances of CNS beta-endorphin as well as other POMC-related peptides. These abnormalities are part of the neurobiological syndrome of anorexia nervosa and may contribute to the characteristic alterations in behavior and neuroendocrine function.     

Dehydrodolichyl diphosphate synthetase from rat seminiferous tubules

Homogenates of seminiferous tubules from rat testes catalyzed the incorporation of label from [14C]isopentenyl diphosphate into a variety of polyprenyl products. Long chain polyprenyl mono- and diphosphates were formed as major products when undesirable side reactions were minimized. The long chain polyprenyl diphosphate synthetase was measured as a sum of the mono- and diphosphate derivatives formed and was dependent on the addition of t,t-farnesyl diphosphate, isopentenyl diphosphate, and divalent cation. The highest activity was associated with the membranous fractions, whereas activity was negligible in the cytosolic fraction. The products of this prenyl transferase were labile to acid and yielded petroleum ether soluble products which indicated that the alpha-isoprene unit was unsaturated. Hydrolysis of either the polyprenyl mono-or diphosphates with a testicular phosphatase in the absence of NaF yielded C75, C80, C85, and C90 polyprenols. The chain lengths of the products of the synthetase suggest that this enzyme is responsible for the de novo biosynthesis of dehydrodolichyl diphosphates which are precursors of the dolichyl derivatives found in testes.     

Human axillary secretions influence women''s menstrual cycles: The role of donor extract of females

Menstrual synchrony in human females has previously been demonstrated among women attending a predominantly female university as well as among women attending coeducational universities. In each of these studies, women who spent the most time together were most likely to show the menstrual synchrony. In this experiment, the possibility that substances in axillary secretions might mediate this effect was tested using a prospective, double-blind research design and a combined axillary extract from a group of female donors. Female subjects who reported themselves to have normal (29.5 +/- 3 day) cycles were exposed to the axillary extracts or blank/ethanol for 10 to 13 weeks. Recipients of the axillary extracts showed a significant reduction in "days' difference in menses onset" relative to the donor cycle, no change was evident for recipients of blank/ethanol. These results demonstrate that constituents from the axillary region of donor females can shift the time of menstrual onset of another group to conform with the donors' cycle and that this effect can occur even in the absence of social contact.     

Comparative analysis of galactoside-binding lectins isolated from mammalian spleens

Galactoside-inhibitable lectins have been isolated from rabbit, rat, mouse, pig, lamb, calf, and human spleens. Native molecular mass, subunit structure, pI, and hemagglutinating activity have been compared for these lectins. The yields of lectin varied from 1.8 mg/kg for rabbit spleen to 79 mg/kg for lamb spleen. Pig, lamb, calf, and human spleen lectins yielded single protein peaks when subjected to Superose 12 fast-protein liquid chromatography. The apparent molecular mass for these lectins was 33-34 kDa. In contrast, rat and mouse spleen lectin preparations were separated into three components ranging from 8.4 to 34 kDa. Superose 12 chromatography of rabbit spleen lectin revealed the presence of at least six components. Gradient slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of single polypeptides for pig, calf, lamb, and human lectins corresponding to a molecular mass of 14-14.5 kDa. Multiple polypeptides were detected for the mouse, rat, and rabbit lectins. The molecular mass of the major polypeptides were 15, 15, and 17 kDa for rat, mouse, and rabbit, respectively. The presence of isolectins in all preparations was shown by isoelectric focusing. The major isolectins were acidic proteins with pI 4.38-4.80. Hemagglutination and hemagglutination inhibition assays demonstrated similarities as well as differences among the lectin preparations. Hemagglutinating activity could not be demonstrated in rabbit spleen extracts nor for isolated putative lectin. Human buffy coat cells were reversibly agglutinated by calf and human spleen lectins, demonstrating the presence of leucocyte cell surface lectin receptors.     

Stretch-induced nitric oxide modulates mechanical properties of skeletal muscle cells

In this study, we examined the hypothesis that stretch-induced (nitric oxide) NO modulates the mechanical properties of skeletal muscles by increasing accumulation of protein levels of talin and vinculin and by inhibiting calpain-induced proteolysis, thereby stabilizing the focal contacts and the cytoskeleton. Differentiating C₂C₁₂ myotubes were subjected to a single 10% step stretch for 0–4 days. The apparent elastic modulus of the cells, E_app, was subsequently determined by atomic force microscopy. Static stretch led to significant increases (P < 0.01) in E_app beginning at 2 days. These increases were correlated with increases in NO activity and neuronal NO synthase (nNOS) protein expression. Expression of talin was upregulated throughout, whereas expression of vinculin was significantly increased only on days 3 and 4. Addition of the NO donor L-arginine onto stretched cells further enhanced E_app, NOS activity, and nNOS expression, whereas the presence of the NO inhibitor N-nitro-L-arginine methyl ester (L-NAME) reversed the effects of mechanical stimulation and of L-arginine. Overall, viscous dissipation, as determined by the value of hysteresis, was not significantly altered. For assessment of the role of vinculin and talin stability, cells treated with L-NAME showed a significant decrease in E_app, whereas addition of a calpain inhibitor abolished the effect. Thus our results show that NO inhibition of calpain-initiated cleavage of cytoskeleton proteins was correlated with the changes in E_app. Together, our data suggest that NO modulates the mechanical behavior of skeletal muscle cells through the combined action of increased talin and vinculin levels and a decrease in calpain-mediated talin proteolysis. mechanical stimulation; apparent elastic modulus; skeletal muscle cells; nitric oxide; stretch