25-Hydroxylation of vitamin D3 in the human hepatoma cell lines Hep G2 and Hep 3B

Two human hepatoma cell lines, Hep G2 and Hep 3B, were screened for vitamin D3-25-hydroxylase enzyme activity by incubation with radioactive vitamin D3. A compound co-chromatographing with 25-OH-D3 was synthesized in both cell lines but its rate of synthesis was tenfold greater in Hep 3B than in Hep G2 cells. The identity of the compound was confirmed by comparing its chromatographic properties with authentic 25-OH-D3 on three different high pressure liquid chromatography systems. Its production was suppressed by adding fetal calf serum (10%), lipoprotein-deficient fetal calf serum, or pure vitamin D-binding globulin to the medium. The mechanism of action of these plasma proteins appears to involve retardation of uptake of the substrate. These two cell lines offer considerable potential as defined in vitro models for studying the effects of physiological factors on the 25-hydroxylation of vitamin D3.     

Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3−C4 intermediate species

Immunogold labelling has been used to determine the cellular distribution of glycine decarboxylase in leaves of C₃, C₃–C₄ intermediate and C₄ species in the genera Moricandia, Panicum, Flaveria and Mollugo. In the C₃ species Moricandia foleyi and Panicum laxum, glycine decarboxylase was present in the mitochondria of both mesophyll and bundle-sheath cells. However, in all the C₃–C₄ intermediate (M. arvensis var. garamatum, M. nitens, M. sinaica, M. spinosa, M. suffruticosa, P. milioides, Flaveria floridana, F. linearis, Mollugo verticillata) and C₄ (P. prionitis, F. trinervia) species studied glycine decarboxylase was present in the mitochondria of only the bundle-sheath cells. The bundle-sheath cells of all the C₃–C₄ intermediate species have on their centripetal faces numerous mitochondria which are larger in profile area than those in mesophyll cells and are in close association with chloroplasts and peroxisomes. Confinement of glycine decarboxylase to the bundle-sheath cells is likely to improve the potential for recapture of photorespired CO₂ via the Calvin cycle and could account for the low rate of photorespiration in all C₃–C₄ intermediate species.Abbreviation and symbol kDa kilodaltons - CO₂ compensation point     

Evidence for precursor forms of the low isoelectric point alpha-amylase isozymes secreted by barley aleurone cells

Gibberellin-treated barley (Hordeum vulgare L.) aleurone cell protoplasts have been shown previously to contain two α-amylase isozymes which are not secreted (JV Jacobsen, JA Zwar, PM Chandler 1985 Planta 13: 430-438). This report shows that these intracellular forms are immunochemically related to the low isoelectric point but not the high isoelectric point group of α-amylase isozymes and that they arise by new synthesis like the secreted forms. Pulse-chase studies show that the intracellular isozymes are precursors to the secreted isozymes. Conversion of the intra- to the extracellular forms involves decreases in isoelectric points with no change in size detectable by SDS-PAGE. The precursor isozymes were also detected in aleurone layer homogenates but they were unstable. They could be stabilized by various treatments including heating the homogenate to 70°C for 10 minutes indicating that the instability was enzymically mediated. Using purified radioactive precursor isozymes, it was shown that instability did not involve inactivation but the conversion to secreted forms. The nature of the covalent modification associated with conversion was not determined but available data indicate that it does not involve glycosylation.     

Monensin inhibits the secretion of α-amylase but not polysaccharide slime from seedling tissues ofZea mays

Summary The effect of monensin on polysaccharide slime secretion by root tips of corn (Zea mays) was studied. Various treatment times and ionophore concentrations were tested: none resulted in inhibition of slime secretion. Because monensin changes the pH of the medium, its effect was also monitored in strongly buffered media and at different pH's. Even in such media, monensin did not inhibit slime secretion. We also measured the effect of the drug after a pulse with [³H]fucose or a pulse followed by a chase. The amount of labeled slimed secreted was not altered by the ionophore. However, 10M monensin affected the development of root tips and drastically reduced their growth. We showed that monensin inhibits the secretion of -amylase by the scutellum of the same plantlet. The importance of the nature of the secretory compound in relation to monensin inhibition of its secretion is discussed.Abbrevations Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sul-fonic acid - Mes 2-(N-morpholino)ethane-sulfonic acid     

Production of somatic hybrids by electrofusion in Solanum

Summary Conditions are described for large scale electrofusion of mesophyll protoplasts of dihaploid S. tuberosum with those of diploid S. brevidens. Overall fusion frequencies of 20%–30% were achieved, and following fusion, large numbers of protoplast-derived calli were obtained. Putative somatic hybrid plants were selected from the regenerated shoots by examining their morphological characteristics. Twenty-one somatic hybrids were confirmed by isoenzyme analysis and six somatic hybrids were further confirmed by Southern hybridization. Tetraploid hybrids were obtained, but cytogenetic studies indicated that more of the regenerated hybrids were hexaploid than had previously been found following chemical fusion of the same partners. Some advantages of electrofusion over chemical fusion are discussed.     

The effect of feeding tall fescue seed infected by Acremonium coenophialum on pregnancy and parturition in female rats

1. Female Sprague-Dawley rats (Rattus norvegicus) were randomly assigned to various dietary treatments containing: (1) 100% Purina rodent chow, ad libitum; (2) same as 1, but restricted to daily intake of 7; (3) 50% rodent chow (w/w) and 50% endophyte-free tall fescue (Festuca arundinacea) seed; (4) same as 3, but restricted to intake of 5; (5) 50% rodent chow, 25% endophyte-free tall fescue seed and 25% endophyte-infected (Acremonium coenophialum) tall fescue seed; (6) 50% rodent chow, 12.5% endophyte-free and 37.5% endophyte-infected tall fescue seed; and (7) 50% rodent chow and 50% endophyte infected tall fescue seed. 2. Average daily feed intakes and average daily weight gains decreased with higher levels of endophyte infected seed. 3. Frequency of litter production was affected by all endophyte-infected containing diets. 4. Conception was reduced only in dietary treatment (7). 5. Litter weights, number of pups per litter and weight per pup were proportionally reduced as higher levels of infected seed were incorporated in the ingested diets.     

Glycosaminoglycan accumulation by normal and malignant human mammary epithelial cells in primary culture

The effects of glycosaminoglycans (GAGs) on cell growth and differentiation appear to vary with cell type and stage of development. This study describes the types and distribution of GAGs accumulated by normal and malignant human mammary epithelial cells in primary culture during their exponential and stationary phases of growth. Cultures incubated with [3H]glucosamine or [35S]sulfate were separated into medium, extracellular matrix (ECM), and cell fractions. Labelled GAGs were identified by chemical and enzymatic degradations and cellulose acetate electrophoresis. Cultures of normal cells in the exponential phase of growth released the most labelled GAGs into the medium fraction, the majority of which was hyaluronic acid (HA). The increase in labelled GAG accumulation, the increase in sulfated GAGs localized in the ECM fraction correlated with the reduced proliferative activity and increased cell density of cells in stationary cultures. In contrast, cultures of mammary tumour cells had the same labelled GAG profile, regardless of their growth status. Although there was variation among tumours, in general, the majority of the labelled GAGs were secreted into the medium fraction and the predominant GAG was HA. The results are comparable with those obtained from studies on mammary tissue in vivo. Primary cultures of human mammary epithelial cells should be useful for determining how modulations of GAGs affect growth and differentiation of these cells.     

Refined structure of human carbonic anhydrase II at 2.0 Å resolution

The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure–factor least-squares refinement at 2.0 Å resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc-bound water, seven more water molecules are identified in the active site. Assuming that Glu-106 is deprotonated at pH 8.5, some of the hydrogen bond donor–acceptor relations in the active site can be assigned and are described here in detail. The Oγ1 atom of Thr-199 donates its proton to the Oε1 atom of Glu-106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.     

Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: a pentacoordinated binding of the SCN- ion to the zinc at high pH

The binding of four inhibitors--mercuric ion, 3-acetoxymercuri-4-aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion--to human carbonic anhydrase II (HCA II) has been studied with X-ray crystallography. The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 A resolution. Mercuric ions are observed at both nitrogens in the His-64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys-206. The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 A, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closest to the zinc ion is 3.1 A away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc. The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 A resolution using the least-squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN- ion is 1.9 A from the zinc ion but shifted 1.3 A with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the O gamma l atom of Thr-199. This is due to the inability of the O gamma l atom of Thr-199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN- molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so-called "deep" water molecule of the native enzyme. The zinc-bound water molecule is 2.2 A from the zinc ion and 2.4 A from the SCN- nitrogen. In addition, this water is hydrogen bonded to the O gamma l atom of Thr-199 and to another water molecule. We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3- ion are ligated to the zinc ion.