Epitope analysis and molecular modeling reveal the topography of the C-terminal peptide of the beta-subunit of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) belongs to a family of heterodimeric glycoprotein hormones that share a common alpha-subunit and a hormone-specific beta-subunit. Among the gonadotropin beta-subunits, greater than 85% homology exists between lutropin (hLH)beta and hCGbeta in their first 114 amino acid residues. However, unlike hLHbeta, hCGbeta contains a 31-amino acid hydrophilic stretch at its carboxyl end (CTPbeta: C-terminal peptide). Although the crystal structure of deglycosylated hCG has been solved, the topography of CTPbeta remains unknown. In this study, we have attempted to define the topology of CTPbeta using mAb probes. We investigated three epitopes on hCGalpha, which are hidden in the hCGalphabeta dimer. However, these epitopes are not hidden in hLH, which has a similar subunit interface to that of hCG, but lacks CTPbeta. This suggested that these epitopes are not masked at the subunit interface of hLH or hCG. Hence, we hypothesized that, in the case of hCG, these epitopes are masked by the CTPbeta. Consistent with this view, several treatments of hCG that removed CTPbeta unmasked these epitopes and enhanced their reactivity with the corresponding mAbs. In order to localise the position of CTPbeta on the alpha-subunit, we used an epitope-mapping strategy [N. Venkatesh & G. S. Murthy (1997) J. Immunol. Methods 202, 173-182] based on differential susceptibility of epitopes to covalent modifications. This enabled us to predict the possible topography of CTPbeta. Further, we were also able to build a model of CTPbeta, completely independently of the epitope-mapping studies, using a homology-based modeling approach [S. Krishnaswamy, I. Lakshminarayanan & S. Bhattacharya (1995) Protein Sci. 4 (Suppl. 2), 86-97]. Results obtained from these two different approaches (epitope analysis and homology modeling) agree with each other and indicate that portions of CTPbeta are in contact with hCGalpha in the native hCG dimer.     

Haploid plant regeneration from unpollinated ovule cultures of niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.)

Haploid plants were regenerated in vitro from unpollinated ovules of niger (Guizotia abyssinica (L. f.) (Cass.) on Murashige and Skoog nutrient medium (MS) supplemented with 10 μM naphthaleneacetic acid or 10 μM NAA + 1.5 μM kinetin and 30 g/l sucrose. Gamborg (B5) medium was the best for plant regeneration (in comparison with MS, Nitsch and Nitsch (NN), and Chu (N6) media) from cultured ovules, and 6.66 and 7.33 ovules of JNC-6 and Ootacamund cultivars were involved in direct plant regeneration on this medium. Matured ovules (ovules collected one day before anthesis or on the day of anthesis) only responded to cultural regimes and involved in direct plantlet development. Cytological preparation of root tips and chloroplast counts in the guard cells of leaf stomata of regenerated plants confirmed their haploid nature. This text was submitted by the authors in English.     

Biodistribution and pharmacokinetics of vanadium following intraperitoneal administration of vanadocene dichloride to mice

The biodistribution and pharmacokinetics of vanadium following i.p. administration of vanadocene dichloride (VDC), a representative of a new class of organometallic anticancer agents, is reported for Strain A mice. A convenient flameless atomic absorption spectroscopic assay is described and is used to determine kinetic profiles for vanadium in blood, kidney, liver, small intestine and brain tissue for times up to 24 h after administration. For a VDC dose of 80 mg/kg, vanadium concentration decreases rapidly from both the blood and small intestine, and the data can be fit to a phenomenological exponential function (blood: t1/2 = 118 +/- 43 min; small intestine: t1/2(alpha) = 18.10 +/- 0.14 min, t1/2(beta) = 341 +/- 45 min). In contrast, vanadium accumulates in both the kidney and liver up to a maximal concentration (1.12 +/- 0.06 mM and 0.56 +/- 0.06 mM after 12 and 8 h, respectively), and is then excreted with estimated half-lives of 7.9 +/- 0.7 and 12.1 +/- 0.1 h, respectively. No detectable levels of vanadium are found in the brain tissue over the temporal course of the experiment. These results are compared to previous mammalian studies with cis-dichlorodiammineplatinum(II) (CDDP) and related 'second generation' platinum derivatives; there are both qualitative similarities between the vanadium and platinum systems as well as important quantitative differences.     

A comparative study of the soil zinc fractions determined by chemical methods and electro-ultrafiltration (EUF) and their relations to zinc nutrition in rice

Rice (IR 42) was grown on two soils differing in zinc status for 30 days with and without Zn under submerged conditions in pots. The fate of soil zinc was characterized by extraction of the soil successively with copper acetate and sodium hypochlorite and by EUF extraction. Most of the applied zinc was extracted by copper acetate and represented as complexed fraction. There exists a close and significnat relation between Cu(OAc)₂-extractable zinc and Zn extracted by EUF for 5 minutes at 50 volts (r=0.98). The EUF-extractable zinc and Cu(OAc)₂-extractable zinc were significantly correlated with the zinc content in the plant (r=0.82). The data from this investigation suggest the possibility of Zn fractionation with the EUF technique and the fractions obtained agree closely to those determined by chemical methods. The results obtained indicate that Zn in soil is held by weak organic bonding and that the extractions by Cu(OAc)₂ and/or EUF-5 minutes serve as a useful basis for extimating zinc availability in rice soils.     

Some differences in the properties of carnitine palmitoyltransferase activities of the mitochondrial outer and inner membranes.

Recent evidence has shown that the outer, overt, malonyl-CoA-inhibitable carnitine palmitoyltransferase (CPTo) activity resides in the mitochondrial outer membrane [Murthy & Pande (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 378-382]. A comparison of CPTo activity of rat liver mitochondria with the inner, initially latent, carnitine palmitoyltransferase (CPTi) of the mitochondrial inner membrane has revealed that the presence of digitonin and several other detergents inactivates CPTo activity. The CPTi activity, in contrast, was markedly stimulated by various detergents and phospholipid liposomes. These findings explain why in previous studies, which used digitonin or other detergents to expose, separate and purify the CPT activities, the inferences were drawn that (a) the ratio of latent to overt CPT was quite high, (b) both the CPT activities could be ascribed to one active protein recovered, and (c) the observed lack of malonyl-CoA inhibition indicated possible loss/separation of a putative malonyl-CoA-inhibition-conferring protein. Although both CPTo and CPTi were found to catalyse the forward and the backward reactions, CPTo showed greater capacity for the forward reaction and CPTi for the backward reaction. The easily solubilizable CPT, released on sonication of mitoplasts or of intact mitochondria under hypo-osmotic conditions, resembled CPTi in its properties. When octyl glucoside was used under appropriate conditions, 40-50% of the CPTo of outer membranes became solubilized, but it showed limited stability and decreased malonyl-CoA sensitivity. Malonyl-CoA-inhibitability of CPTo was decreased also on exposure of outer membranes to phospholipase C. When outer membranes that had been exposed to octyl glucoside or to phospholipase C were subjected to a reconstitution procedure using asolectin liposomes, the malonyl-CoA-inhibitability of CPTo was restored. A role of phospholipids in the malonyl-CoA sensitivity of CPTo is thus indicated.     

Acute Effect of Ammonia on Branched-Chain Amino Acid Oxidation and Incorporation into Proteins in Astrocytes and in Neurons in Primary Cultures

14CO2 production and incorporation of label into proteins from the labeled branched-chain amino acids, leucine, valine, and isoleucine, were determined in primary cultures of neurons and of undifferentiated and differentiated astrocytes from mouse cerebral cortex in the absence and presence of 3 mM ammonium chloride. Production of 14CO2 from [1-14C]leucine and [1-14C]valine was larger than 14CO2 production from [U-14C]leucine and [U-14C]valine in both astrocytes and neurons. In most cases more 14CO2 was produced in astrocytes than in neurons. Incorporation of labeled branched-chain amino acids into proteins varied with the cell type and with the amino acid. Addition of 3 mM ammonium chloride greatly suppressed 14CO2 production from [1-14C]-labeled branched chain amino acids but had little effect on 14CO2 production from [U-14C]-labeled branched-chain amino acids in astrocytes. Ammonium ion, at this concentration, suppressed the incorporation of label from all three branched-chain amino acids into proteins of astrocytes. In contrast, ammonium ion had very little effect on the metabolism (oxidation and incorporation into proteins) of these amino acids in neurons. The possible implications of these findings are discussed, especially regarding whether they signify variations in metabolic fluxes and/or in magnitudes of precursor pools.     

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.     

Mercury-Induced Inhibition of Photosystem II Activity and Changes in the Emission of Fluorescence from Phycobilisomes in Intact Cells of the Cyanobacterium, Spirulina platensis

Addition of low concentrations of mercury chloride (HgCl₂ tointact cells of the cyanobacterium, Spirulina platensis causedan enhancement in the intensity of fluorescence emitted fromphycocyanin at room temperature and induced blue shifts in theemission peak suggestive of changes in energy transfer withinthe phycobilisomes. HgCl₂ also suppressed the whole-chain electrontransport activity (H₂O methylviologen) at much lower concentrationsthan that required to inhibit Hill activity supported by para-benzoquinone.The extent of inhibition of Hill activity was much higher underhigh-intensity light than that under low-intensity light. Ourresults indicate that mercury ions at low concentrations affectthe transfer of energy within phycobilisomes and at high concentrationsthey inhibit electron transport in this cyanobacterium. (Received February 21, 1989; Accepted October 2, 1989)     

Radioimmunoassay of polypeptide hormones using immunochemically coated plastic tubes

A method has been developed for immobilisation of antisera on fresh plastic tubes through an immunochemical bridge. This type of immobilisation has been shown to be more consistent than direct adsorption on plastic. Such immunochemically coated antisera on plastic tube has been used in the development of a noncentrifugation radioimmunoassay. This assay system has been found to be technically as sound as the conventional method.