Reactors for continuous primary beer fermentation using immobilised yeast

A continuous multistage column bioreactor with fluidised beds and continuous gas-lift bioreactor system with immobilised yeast Saccharomyces cerevisiae was developed for the first step of wort fermentation. The system of gas-lift reactor with yeast entrapped in calcium pectate beads was stable for 5 weeks by the optimal residence time of 12.75h and produced beer with a composition and flavour profile similar to that of beer produced by batch fermentation. Concentration of diacetyl was less than 0.1mg/l.     

Antitumor agents-CLXXV. Anti-tubulin action of (+)-thiocolchicine prepared by partial synthesis

(+)-Thiocolchicine (2b) was prepared from (±)-colchicine (1) in a five-step reaction sequence that included chromatographic separation of appropriate camphanylated diastereomers. Acid hydrolysis of the (+)-diastereomer, followed by acetylation, yielded the desired product 2b. (+)-Thiocolchicine has 15-fold lower inhibitory activity against tubulin polymerization than (−)-thiocolchicine, and is 29-fold less potent for inhibiting growth of human Burkitt lymphoma cells. The enantiomer 2a, prepared from the (−)-camphanylated diastereomer, had potent activity in all assays comparable to that of (−)-thiocolchicine prepared by other methods. These results support the hypothesis that the proper configuration of colchicine-related compounds is an important requirement for their anti-tubulin action.     

Knowledge-based modeling of the D-lactate dehydrogenase three-dimensional structure

A three-dimensional structure of the NAD-dependent D -lactate dehydrogenase of Lactobacillus bulgaricus is modeled using the structure of the formate dehydrogenase of Pseudomonas sp. as template. Both sequences share only 22% of identical residues. Regions for knowledge-based modeling are defined from the structurally conserved regions predicted by multiple alignment of a set of related protein sequences with low homology. The model of the D -LDH subunit shows, as for the formate dehydrogenase, an α/β structure, with a catalytic domain and a coenzyme binding domain. It points out the catalytic histidine (His-296) and supports the hypothetical catalytic mechanism. It also suggests that the other residues involved in the active site are Arg-235, possibly involved in the binding of the carboxyl group of the pyruvate, and Phe-299, a candidate for stabilizing the methyl group of the substrate. © 1995 Wiley-Liss, Inc.     

Asymmetry of the Na+-succinate cotransporter in rabbit renal brush-border membranes

We have investigated the symmetry of Na⁺-succinate cotransport in rabbit renal brush-border membrane vesicles. Succinate influx and efflux kinetics were measured under voltage-clamped conditions using [¹⁴C]succinate and a rapid filtration procedure. Both influx and efflux were Na⁺-dependent, saturable, temperature-sensitive, and influenced by the trans Na⁺ and succinate concentrations. The system was judged to be asymmetric, since the maximal velocity for influx was 3-fold higher than that for efflux, and trans Na⁺ inhibited influx more than efflux. This may be due to the asymmetrical insertion of the transporter in the brush-border membrane, which leads to differences in either the forward and backward translocation rates of the fully loaded carrier or the Na⁺ and succinate binding constants at the inner and outer faces of the membrane.     

Distribution of fibres reacting with an α-endorphin antiserum in the neurohypophysis of Carassius auratus and Cyprinus carpio

Summary A strong positive immunoreaction with an -endorphin antiserum occurs in two distinct sites of the goldfish and carp neurohypophysis. Fluorescent nerve terminals are found in the laminar nerve processes located in the rostral pars distalis, but the immunocytological reaction is mainly localised on the nerve processes of the posterior neurohypophysis lying between the intermediate lobe cells. Almost all the digitations of the neurohypophysis are strongly fluorescent. The immunoreactive fibres probably originate from the hypothalamus, where perikarya displaying the same immunoreaction have been found in the pars lateralis of the nucleus lateralis tuberis and in some minor centres. The possibility that the immunoreactive substances revealed on the neurohypophyseal processes may originate in the intermediate lobe cells is also discussed. It has now to be established if this hypothalamo-hypophyseal system contains a substance with endorphic properties or only some immunologically related substance devoid of the corresponding physiological activities.     

Volume flows across gallbladder epithelium induced by small hydrostatic and osmotic gradients

Summary The hydraulic conductivity of rabbit gallbladder epithelium has been studied using a continuous volumetric method based on capacitance measurements. The time resolution for measuring osmotic flows is in the range of seconds. Volume flows have been induced by osmotic gradients between 0 and 100 mosmol. In this range the flow-force relation is linear and theP _f value is 9.3×10^–3 cm/sec. After correction for solute polarization effects, theP _f value amounts to 0.05 cm/sec. The observed flow is constant between 5 sec up to 20 min after a sudden increase in the osmolarity of the mucosal solution. The wet weight of the gallbladder tissue decreases by 22% and increases by 30% during osmotic flows from serosa to mucosa and from mucosa to serosa, respectively. Volume flows induced by hydrostatic pressure gradients on the mucosal surface are linearly related to the driving forces between 0 and 40 mbar. TheP _f value is 0.15 cm/sec. The volume flows are constant between 2 sec and 15 min after pressure application. The flow-force relation for pressure gradients on the serosal surface is markedly nonlinear for gradients greater than 5 mbar. Below 5 mbar theP _f value is 4.5 cm/sec. From electrical measurements, e.g., resistance and streaming potentials, and from flux studies with inulin and polyethylene glycol 4000, it is concluded that hydrostatic and osmotic gradients are not comparable when they are applied to gallbladder epithelium. They induce volume flows across different pathways, e.g., osmosis predominantly across the cellular route and pressure filtration predominantly across paracellular routes.     

Genetic studies of the Macushi and Wapishana Indians

Summary Blood samples from 509 Macushi and 623 Wapishana Amerindians of Northern Brazil and Southern Guyana have been analyzed with reference to the occurrence of rare variants and genetic polymorphisms of the following 25 systems: (i) Erythrocyte enzymes: acid phosphatase-1, adenosine deaminase, adenylate kinase-k, carbonic anhydrase-1, carbonic anhydrase-2, esterase A_1,2,3, esterase D, galactose-1-phosphate uridyltransferase, isocitrate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, nucleoside phosphorylase, peptidase A, peptidase B, phosphoglucomutase 1, phosphoglucomutase 2, phosphogluconate dehydrogenase, phosphohexoseisomerase, triosephosphate isomerase and (ii) Serum proteins: albumin, ceruloplasmin, haptoglobin, hemoglobin A, hemoglobin A₂ and transferrin. Fifteen different rare variants were detected, involving 11 of these systems. In addition, a previously undescribed variant of ESA_1,2,3 which achieves polymorphic proportions in both these tribes is described. Excluding this variant, the frequency of rare variants is 1.1/1000 in 12510 determinations in the Macushi and 4.7/1000 in 15 396 determinations in the Wapishana. The ESA_1,2,3, polymorphism was not observed in 382 Makiritare, 232 Yanomama, 146 Piaroa, 404 Cayapo, 190 Kraho and 112 Moro. Irregularities in the intratribal distribution of this polymorphism in the Macushi and Wapishana render a decision as to the tribe of origin impossible at present. Gene frequencies are also given for previosly described polymorphisms of 5 systems: haptoglobin, phosphoglucomutase 1, erythrocyte acid phosphatase, esterase D, and galactose-1-phosphate-uridyl-transferase.Research supported by the National Science Foundation and the Energy Research and Development Administration.     

Studies on the mode of action of calciferol: Biological activity of 1α,24R,25-trihydroxyvitamin D3 in the chick

The biological activity of 1α,24R,25-trihydroxyvitamin D₃ [1α,24R,25(OH)₃D₃] was elevated in comparison to the hormonally active form of vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], in the rachitic chick in terms of its ability to (a) stimulate intestinal calcium absorption, (b) mobilize bone calcium, (c) induce intestinal calcium binding protein, (d) modulate the level of enzyme activity of the renal 25-OH-D₃-1-hydroxylase system, and (e) interact with the intestinal cystosol-chromatin receptor system for the 1α,25(OH)₂D₃ receptor system. In each of these assays, the relative ratio of activity of 1α,24R,25(OH)₃D₃ to 1α,25(OH)₂D₃was (a) 25–50, (b) ca. 20, (c) 10, (d) 50, and (e) 36%, respectively.     

Expression of a full-length cDNA coding for human intestinal lactase-phlorizin hydrolase reveals an uncleaved, enzymatically active, and transport-competent protein

Lactase-phlorizin hydrolase (LPH) (EC 3.2.1.23/62) is a major intestinal microvillar membrane glycoprotein that digests lactose, the main carbohydrate of milk. To investigate structure/function relationships of LPH and to assess the impact of intracellular processing on the function of LPH and on its transport to the cell surface, we have expressed a full-length cDNA encoding LPH in mammalian COS-1 cells. Analysis of the expressed protein by immunoprecipitation with monoclonal anti-LPH antibodies and treatments with endo-beta-N-acetylglucosaminidase H and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two polypeptides with apparent molecular masses of 215 and 230 kDa, representing the mannose-rich (pro-LPHh) and complex (pro-LPHc) glycosylated forms of the precursor. By contrast to pro-LPH in human enterocytes, the expressed pro-LPH in COS-1 cells does not undergo intracellular proteolytic cleavage to generate a form similar to the mature enzyme of the brush-border membrane. Intracellular cleavage, however, is not essential for the molecule to acquire its enzymatic activity since pro-LPH in COS-1 cells is enzymatically as active as LPH isolated from intestinal brush-border membranes. Indirect immunofluorescent staining of transfected cells demonstrated that pro-LPH is expressed at the cell surface. This was further corroborated by the sensitivity of the complex glycosylated form (pro-LPHc) to trypsin in the medium. Our results provide the first conclusive evidence that pro-LPH is an enzymatically active molecule and that the intracellular proteolysis of pro-LPH is not essential for the generation of transport-competent forms of LPH.