Altered regulation of glycogen metabolism by vasopressin and phenylephrine in hepatocytes from insulin-resistant obese (fa/fa) rats. Role of protein kinase C.

The hormonal control of glycogen synthase and phosphorylase interconversion was investigated in hepatocytes isolated from lean and genetically obese (fa/fa) rats. In cells from obese animals, the inactivation of synthase by 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), phospholipase C, vasopressin and the alpha 1-adrenergic agonist phenylephrine was markedly impaired, and the property of PMA to counteract phosphorylase activation by phenylephrine was attenuated. The maximal response of phosphorylase activation to phenylephrine and vasopressin was increased in obese-rat hepatocytes, but the sensitivity to these hormones was similar to that in lean-rat hepatocytes. These observations indicate that the defect in protein kinase C that we reported previously in heart of insulin-resistant fa/fa rats [van de Werve, Zaninetti, Lang, Vallotton & Jeanrenaud (1987) Diabetes 36, 310-319] is probably also expressed in liver.     

Domains involved in multimer assembly of von willebrand factor (vWF): multimerization is independent of dimerization.

The precursor protein of von Willebrand factor (pro-vWF) consist of four repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2. The domains D1 and D2 constitute the amino-terminal pro-polypeptide and the remaining domains mature vWF, generated upon proteolytic processing. We have shown previously that the pro-polypeptide of pro-vWF is obligatory for assembly of pro-vWF dimers into multimers, a process vital for efficient adhesion of platelets to an injured vessel wall. Here, we have employed full length vWF cDNA to construct a series of deletion mutants, based on the homology between the various domains. Specifically, the domains D', D3 or both were deleted and the multimeric pattern of the mutant vWF proteins was analysed after transient expression in COS-1 cells. It is demonstrated that in addition to the pro-polypeptide, both the D' and the D3 domain are required for multimer assembly. Furthermore, by analysing a construct containing only the domains D' and D3 next to the pro-polypeptide it is shown that this is the only part of the vWF protein involved in multimer assembly. Since, the formation of pro-vWF dimers relies on the carboxy-terminal area of mature vWF, it is concluded that multimer assembly is a process independent of dimerization.     

T-cell specific rearrangement of T-cell receptor beta transgenes in mice.

To study rearrangement of T cell receptor (TCR) genes, transgenic mice were generated with a TCR beta minilocus in germline configuration, containing three V beta, two D beta, fourteen J beta and two C beta gene segments and the TCR beta enhancer. Using the polymerase chain reaction as an analytical tool both partial DJ as well as complete VDJ rearrangements were seen, indicating that the minilocus contained all sequence elements required for rearrangment. Rearrangements of minilocus gene segments were restricted to T cells in the thymus and the periphery and did not occur in B cells. V beta 8.3 and V beta 5 sequences encoded by the minilocus were expressed on the surface of peripheral T cells at high frequencies. Transgenic mice with TCR minilocus genes will be a useful system to identify DNA sequence elements required for regulation of rearrangement in vivo.     

Quantification of human hepatic glutathione S-transferases.

Human hepatic glutathione S-transferase (GST) subunits were characterized and quantified with the aid of a recently developed h.p.l.c. method. In 20 hepatic tissue specimens the absolute amounts of the basic Class Alpha subunits B1 and B2, the near-neutral Class Mu subunits mu and psi and the acidic subunit pi were determined. The average total amount of GST was 37 micrograms/mg of cytosolic protein, with the Class Alpha GST being the predominant class (84% of total GSTs), and pi as the sole representative of the Class Pi GSTs present in the lowest concentration (4% of total GSTs). Large interindividual differences were observed for all subunits, with variations up to 27-fold, depending on the subunit. For the Class Alpha GST-subunits B1 and B2, a biphasic ratio was observed. The genetic polymorphism of the subunits mu and psi was confirmed by h.p.l.c. analysis, and correlated with the enzymic glutathione conjugation of trans-stilbene oxide and with Western blotting of cytosols, using a monoclonal anti-(Class Mu GST) antibody. Of the 20 livers examined, ten contained only mu, whereas the occurrence of psi alone, and the combination of mu and psi, were found in only one liver each.     

Craniosynostosis and low middle frequency perceptive deafness in mother and son. A distinct entity?

A distinct entity?: In this report we describe the hitherto unreported association of low and middle frequency perceptive deafness and craniosynostosis in mother and son. The occurrence of hearing loss in the craniosynostosis syndromes is briefly reviewed.     

Depletion flocculation and depletion stabilization of erythrocytes

At dextran (Mw ≈ 500,000) concentrations from 2 to ≈10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations > 10%, the erythrocytes resegregate in a stable monodisperse suspension. At all these dextran concentrations, the erythrocytes are coated with considerable amounts of dextran. It can be argued that at dextran concentrations from 2 to 10%, as well as at dextran concentrations > 10%, there is a thin layer, which is depleted of dextran, between the dextran layer adsorbed onto the erythrocytes and the bulk dextran solution. It can also be shown that there is a repulsive interaction between the two layers of dextran: one adsorbed and one free. When the adsorbed dextran layer is the most concentrated, stability must ensue, and when the dextran in free solution is the most concentrated, flocculation should occur. Below 7% dextran, the concentration of free dextran is higher than the adsorbed concentration; above 10% dextran that situation is reversed. These data correlate well with the depletion flocculation predicted for the lower concentration and the depletion stabilization predicted for the higher dextran concentration.

     

Butanol recovery from fermentations by liquid-liquid extraction and membrane solvent extraction

Extraction can successfully be used for in-situ alcohol recovery in butanol fermentations to increase the substrate conversion. An advantage of extraction over other recovery methods may be the high capacity of the solvent and the high selectivity of the alcohol/water separation. Extraction, however, is a comprehensive operation, and the design of an extraction apparatus can be complex. The aim of this study is to assess the practical applicability of liquid-liquid extraction and membrane solvent extraction in butanol fermentations. In this view various aspects of extraction processes were investigated.Thirty-six chemicals were tested for the distribution coefficient for butanol, the selectivity of alcohol/water separation and the toxicity towards Clostridia. Convenient extractants were found in the group of esters with high molar mass.Liquid-liquid extraction was carried out in a stirred fermenter and a spray column. The formation of emulsions and the fouling of the solvent in a fermentation broth causes problems with the operation of this type of equipment. With membrane solvent extraction, in which the solvent is separated from the broth by a membrane, a dispersion-free extraction is possible, leading to an easy operation of the equipment. In this case the mass transfer in the membrane becomes important.With membrane solvent extraction the development of a process is emphasized in which the extraction characteristics of the solvent are combined with the property of silicone rubber membranes to separate butanol from water. In the case of apolar solvents with a high molar mass, the characteristics of the membrane process are determined completely by the solvent. In the case of polar solvents (e.g. ethylene glycol), the permselectivity of the membrane can profitably be used. This concept leads to a novel type of extraction process in which alcohol is extracted with a water-soluble solvent via a hydrophobic semipermeable membrane. This extraction process has been investigated for the recovery of butanol and ethanol from water. A major drawback in all processes with membrane solvent extraction was the permeation of part of the solvent to the aqueous phase.The extraction processes were coupled to batch, fed batch and continuous butanol fermentations to affirm the applicability of the recovery techniques in the actual process. In the batch and fed batch fermentations a three-fold increase in the substrate consumption could be achieved, in the continuous fermentation about 30% increase.     

The CR1 and CR3 domains of the adenovirus type 5 E1A proteins can independently mediate activation of ATF-2.

The adenovirus 12S E1A protein can stimulate the activity of the c-jun promoter through a conserved region 1 (CR1)-dependent mechanism. The effect is mediated by two AP-1/ATF-like elements, jun1 and jun2, that preferentially bind c-Jun-ATF-2 heterodimers. In this study, we show that the ATF-2 component of the c-Jun-ATF-2 heterodimer is the primary target for 12S E1A: 12S E1A can enhance the transactivating activity of the N terminus of ATF-2 when fused to a heterologous DNA-binding domain, whereas the transactivating activity of the c-Jun N terminus is not significantly affected. Activation of the ATF-2 N terminus by 12S E1A is dependent on CR1. In the context of the 13S E1A protein, CR1 and CR3 can both contribute to activation of ATF-2, and their relative contributions are dependent on the cell type. In contrast to activation of ATF-2 by stress-inducing agents, CR1-dependent activation of ATF-2 was found not to depend strictly on the presence of threonines 69 and 71 in the N terminus of ATF-2, which are targets for phosphorylation by stress-activated protein kinases (SAPKs). In agreement with this observation, we did not observe phosphorylation of threonines 69 and 71 or constitutively enhanced SAPK activity in E1A- plus E1B-transformed cell lines. These data suggest that CR1-dependent activation of ATF-2 by 12S E1A does not require phosphorylation of threonines 69 and 71 by SAPK.     

FECAL METHANOGENS AND VERTEBRATE EVOLUTION

It has been assumed that the feeding habits of vertebrates predispose the variety of intestinal differentiations and the composition of the microbial biota living in their intestinal tracts. Consequently, the presence of methanogenic bacteria in the various differentiations of the large intestine and the foregut of herbivorous vertebrates had been attributed primarily to the existence of anaerobic habitats and the availability of carbon dioxide and hydrogen originating from the fermentative microbial digestion of plant-based diets. However, Australian ratites, many murids, and several New World primates lack methanogens, despite their intestinal differentiations and their vegetarian feeding habits. Crocodiles, giant snakes, aardvarks, and ant-eaters on the other hand release significant amounts of methane. A determination of methane emissions by 253 vertebrate species confirmed that competence for intestinal methanogenic bacteria is shared by related species and higher taxa, irrespective of different feeding habits. In “methanogenic” branches of the evolutionary tree, a variety of differentiations of the large intestine evolved and, in some cases, differentiations of the foregut. In contrast, the lack of competence for methanogens in chiropterans/insectivores and carnivores apparently has precluded the evolution of specialized fermenting differentiations of the digestive tract. Our observations reveal that the presence of intestinal methanogenic bacteria is under phylogenetic rather than dietary control: competence for intestinal methanogenic bacteria is a plesiomorphic (primitive-shared) character among reptiles, birds, and mammals. This competence for methanogenic bacteria has been crucial for the evolution of the amniotes.