Nuclear location of phosphoglycerate mutase BB isozyme in rat tissues

Summary We have previously reported (Ureña et al. Eur. J. Cell Biol. 1990) that in skeletal muscle, type MM phosphoglycerate mutase isozyme is present in the nucleus as well as in the cytosol. To determine whether type BB phosphoglycerate mutase isozyme is also present in nucleus, the subcellular location of this isozyme was studied in different rat tissues by cell fractionation and immunogold techniques. With the aid of high affinity-purified anti-phosphoglycerate mutase BB isozyme antibodies, the isozyme was located in the nucleus of neuronal, astroglial and liver cells but not in the nucleus of oligodendroglial and endothelial cells. Biochemical studies on purified nuclear fractions also demonstrated the presence of phosphoglycerate mutase activity in the nucleus. Both immunocytochemical and biochemical techniques showed that nuclear phosphoglycerate mutase-specific activity depended on the type of cell.Abbreviations PGAM phosphoglycerate mutase - PGAM-M(M) muscle specific subunit (isozyme) of PGAM - PGAM-B(B) brain type subunit (isozyme) of PGAM - ssDNA single stranded DNA - PBS 0.001 M phosphate buffer, pH 7.4, containing 0.15 M NaCl - kDa kilodalton     

The plasma membrane of epididymal epithelial cells has a specific receptor which binds to androgen-binding protein and sex steroid-binding protein.

The binding of [3H] delta 6-testosterone photoaffinity-labelled rat androgen-binding protein (rABP) has been studied in an enriched fraction of plasma membranes of epithelial epididymal cells in immature (15 days) and adult rats (40 days). The binding was maximal in less than 30 min and more rapid at 4 degrees C than at 34 degrees C. It was calcium and pH dependent. Scatchard plots of the binding data gave curvilinear plots with two types of binding sites corresponding to a K(ass1) of 18.2 nM-1 and K(ass2) of 1.6 nM-1 (2.2 x 10(11) sites/mg protein and 5.4 x 10(11) sites/mg protein, respectively). In adult rats, only one type of binding site was found, with a K(ass) of 3.7 nM-1 (4.5 x 10(11) sites/mg protein). The number of receptors was 5-fold lower in the cauda than in the caput of the epididymis. The pretreatment of the isolated intact cells with streptozotocin induced a 45% reduction of the binding. Only unlabelled rABP and hSBP (human sex steroid-binding protein) but not other proteins (lactotransferrin, serotransferrin, asialofetuine, fetuine and bovine serum albumin) competed with the labelled ligand to bind plasma membranes. The membrane fraction was solubilized by triton X-100. Its incubation with labelled rABP and hSBP provoked the elution of the tracer as an aggregate into the void volume fraction of superose 6B mini-gel filtration columns. Structural homology between hSBP and rABP could be responsible for the common behaviour of the steroid-carrier molecules for the ABP receptor of rat epididymal epithelial cells.     

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.     

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

Summary The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO) — a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.     

Distribution of salmon gonadotrophin releasing-hormone in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Summary The distribution of salmon gonadotrophin-releasing hormone (sGnRH) was studied in the brain and pituitary of two-year-old immature sea bass (Dicentrarchus labrax) by means of an enzymoimmunoassay (EIA) for sGnRH and immunocytochemistry. The EIA for sGnRH is a competitive assay using a tracer made of sGnRH coupled to acetylcholinesterase from an electric eel. The separation of free and bound tracer is achieved by coating the plates with mouse anti-rabbit IgG monoclonal antibodies. Displacement curves generated by sGnRH and extracts from pituitary and different brain regions showed a good parallelism allowing the assay to be used for sGnRH measurements in this species. Although all parts of the brain contained measurable levels of sGnRH, the highest concentrations were found in the pituitary, the olfactory bulbs and the telencephalon. These data were confirmed by immunocytochemistry. Cell bodies were found in the olfactory bulbs, ventral telencephalon, preoptic region and mediobasal hypothalamus. Immunoreactive fibers could be observed in all parts of the brain including the optic tectum, the cerebellum (corpus and valvula), the vagal lobe, the medulla oblongata and the rostral spinal cord. In most cases, these fibers do not form well defined bundles; however, there was clearly a continuum of immunoreactive fibers, extending from the olfactory bulbs to the pituitary, and along which all the cell bodies described above were located. In the ventral telencephalon and the preoptic region, clear pictures of varicose positive fibers contacting immunoreactive perikarya could be observed. These data indicate that sGnRH is most likely an endogenous peptide in the brain of the sea bass, although the presence of other forms of GnRH cannot be excluded at this point. This study also demonstrates that the general organization of the GnRH systems in the sea bass is highly similar to what has been described in most freshwater teleost species, and provides basis for further studies on the neuroendocrine control of gonadotrophin release in this commercially important species.     

Palmar flexion creases in schizophrenia

Palmar flexion creases have been studied in schizophrenics with a family history of schizophrenia or other psychiatric disorders and without such a background, and compared to a control population. Palmar flexion creases have been analyzed according to the method suggested byBali & Chaube (1971). When compared to controls, differences in the DRBC and TRBC frequencies are significant in the subgroup with no family history, supporting the existence of biological heterogeneity in schizophrenia, and of congenital factors when there is no known genetic background.     

Correlation between serum enzymes and serum unsaturated vitamin B12 binding proteins in primary liver carcinoma

The serum unsaturated vitamin B12-binding capacity (UBBC), unsaturated transcobalamin (UTC) I, UTC II, UTC III levels, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase activities and bilirubin concentration were estimated in 61 patients with liver diseases (31 with hepatoma, 30 with viral hepatitis). The levels of serum cobalamin, UTC I, UTC III, UBBC, alanine and aspartate aminotransferases, and bilirubin were raised in both hepatoma and viral hepatitis patients. Serum UTC II was reduced in both conditions. Alkaline phosphatase activity was significantly increased in hepatoma. Four significant correlations were observed among these parameters in the hepatoma patients while only one significant correlation was observed in viral hepatitis.     

Aromatase activity in human skin fibroblasts: characterization by an enzymatic method

Human skin fibroblasts were incubated for 24 h with 10(-6) M androstenedione and the estrone + estradiol released in the culture medium were measured by an enzymatic assay. Aromatase activity was expressed as pmol (estrone + estradiol) formed in the medium per mg cell protein per day. Using this method we were able to investigate the kinetic properties of aromatase in different cell strains and its stimulation by dexamethasone. Values of 92 nM and 9.1 pmol/mg protein/day were obtained respectively for Km and Vmax in cultured fibroblasts derived from genital skin of normal prepubertal subjects. In patients with complete androgen insensitivity syndrome CAIS, the Km was 156 nM and the Vmax 42 pmol/mg protein/day. Aromatase activity varied from 7.9 +/- 1.2 pmol/mg protein/day (mean +/- SD; n = 19) in normal prepubertal boys to 24.5 +/- 4.7 pmol/mg protein/day (mean +/- SD; n = 11) in those from normal postpubertal boys. The values were even higher in fibroblasts cultured from genital skin of prepubertal patients with CAIS. Cell concentrations did not modify the pattern of estrogen formation and aromatase activity did not vary with serial subcultures. The stimulatory effect of dexamethasone on aromatase activity in cultured fibroblasts was measured after preincubation of the cells for 48 h with dexamethasone, by determining estrogen formation after 24 h incubation of the cells with androstenedione 10(-6) M using this enzymatic method. This data suggest that aromatase activity measured in cultured fibroblasts could be a useful tool for studying extraglandular estrogen formation in physiological and pathological conditions.