The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Neuropeptide-γ: A Peptide Isolated from Rabbit Intestine that Is Derived from γ-Preprotachykinin

The neurokinin A-like immunoreactivity in an extract of rabbit small intestine was resolved into two molecular forms by gel permeation chromatography. These components were purified to apparent homogeneity by reverse-phase HPLC. The primary structure of the larger component was established as the following: Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val- Gly-Leu - Met.NH2. This amino acid sequence represents residues (72-92) of gamma-preprotachykinin, as predicted from the nucleotide sequence of a cloned cDNA from the rat. The peptide, termed neuropeptide-gamma, lacks residues (3-17) of neuropeptide K, and this segment is specified exactly by exon 4 in the preprotachykinin gene. The smaller form of neurokinin A-like immunoreactivity was identical to neurokinin A. Neuropeptide K was not present in the extract, demonstrating that the pathways of post-translational processing of beta- and gamma-preprotachykinins in the rabbit gut are different.     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.     

Batch production and growth kinetics of hybridomas

Batch cultures of mouse-mouse hybridoma cell lines were carried out and their growth and production kinetics investigated. Three main cell specific production patterns (expressed as pg IgG/cell x hour) were found, which can be used as a classification system for hybridoma cell lines (groups I–III). Cells showing the highest IgG-production at the beginning of the batch culture (during the lag and the onset of the log-phase) were classified as either group I and II. The difference was that cell lines of group II showed a second high cell specific production at the onset of the stationary and death phases. Cell lines of group III had a quite constant production of antibodies during their growth; but IgG secretion completely stopped after the beginning of the stationary phase. The implications of these three production patterns on the design of a production process are discussed.     

Characterization of macrophage-like cells in the external layers of human small and large intestine

Summary In the external layers of human small and large intestine macrophage-like cells were characterized by immunohistochemical, histochemical and electronmicroscopical methods. Using immunohistochemistry and a number of monoclonal antibodies, the presence and distribution of phenotypic subpopulations of macrophages were evaluated. In all locations macrophage-like cells were identified with antibody EBM11, which recognizes CD68 antigen, C3bi which recognizes CD11b, and partly with an antibody which recognizes protein 150,95 (CD11c). Macrophage-like cells in the external muscle layer were HLA-DR-positive (expressing the MHC class-II antigen), in contrast to macrophage-like cells in the subserosa and submucosa. Macrophage-like cells in the external muscle layer were mostly acid phosphatase-negative, and at the electron-microscopic level they were found to have features of macrophages: primary lysosomes, coated vesicles and pits. However, very few secondary lysosomes were present. Birbeck granules were not observed. It is concluded that in the external muscle layer of human small and large intestine numerous macrophages of a special type are present. It is discussed whether this cell type plays a role in gastrointestinal motility and/or has an immunological function.     

Effects of tumour necrosis factor-α (cachectin) on glucose metabolism in the rat

Intravenous administration of a single dose (20 g) of recombinant tumour necrosis factor- (TNF, cachectin) to rats decreased the rate of intestinal glucose absorption. In vivo, the oxidation of [U-¹⁴C]glucose to ¹⁴CO₂ was significantly increased by the cytokine. In addition, [¹⁴C]lipid accumulation from [U-¹⁴C]glucose was increased both in liver and brown adipose tissue of the TNF-injected animals. The decrease observed in intestinal glucose absorption was not associated with changes in intestinal metabolism. There was no difference in glucose metabolism by isolated enterocytes from either control or TNF-injected rats whether in the absence or presence of different concentrations of the cytokine in the incubation medium. In contrast, tumour necrosis factor altered the rate of gastric emptying as measured by the gastrointestinal distribution of [³H]inulin following an intragastric glucose load. These results suggest that the cytokine profoundly alters glucose metabolism by increasing its whole-body oxidation rate and delaying intestinal absorption through a reduced gastric emptying.     

几种药物抑制马传染性贫血病毒和人免疫缺陷病毒的实验研究

用马传染性贫血病毒—驴胚肺二倍体细胞(EIAV-DELDC)为实验体系,以细胞中病毒逆转录酶活性及病毒相关抗原的表达为观察指标,检测了叠氮胸苷(AZT)、三氮唑核苷(Ribavirin,病毒唑)、磷羧基甲酸钠(PFA)和苏拉明等4种已知抗人免疫缺陷病毒(HIV-1)药物对马传染性贫血病毒的抑制作用。结果表明,PFA、AZTTP(三磷酸AZT)和苏拉明均能抑制病毒相关抗原的表达,AZT虽无此作用,但能抑制细胞内逆转录酶活性。用~3H-TMP掺入法比较了PFA、AZTTP、苏拉明对体外无细胞系EIAV逆转录酶粗提物和HIV-1基因工程产物逆转录酶活性的抑制作用表明,两种逆转录酶对苏拉明的敏感性相近,而HIV-1逆转录酶对PFA和AZTTP的敏感性较EIAV者高约100倍。又以无细胞系中逆转录酶活性测定法,检测了12种中药提取物的抑制作用,其中小柴胡汤对EIAV和HIV-1逆转录酶活性都有抑制作用,IC_(50)为717μg/ml和700μg/ml(生药浓度)。小柴胡汤对两种病毒感染细胞中抗原的表达和HIV引起细胞病变都有抑制作用,对HIV-1的抑制比EIAV强。这些结果表明,EIAV-DELDC体系可考虑作为抗HIV-1药物筛选模型。     

Techniques for encapsulating bioactive agents into liposomes

As a prerequisite for the use of liposomes for delivery of biologically active agents, techniques are required for the efficient and rapid entrapment of such agents in liposomes. Here we review the variety of procedures available for trapping hydrophilic and hydrophobic compounds. Considerations which are addressed include factors influencing the choice of a particular liposomal system and techniques for the passive entrapment of drugs in multilamellar vesicles and unilamellar vesicles. Attention is also paid to active trapping procedures relying on the presence of (negatively) charged lipid or transmembrane ion gradients. Such gradients are particularly useful for concentrating lipophilic cationic drugs inside liposomes, allowing trapping efficiencies approaching 100%.     

On the origin of neoblasts in freshwater planarians (Turbellaria)

Experiments on 1) regeneration of the cave-adapted planarian, Sphalloplana zeschi, 2) induction of sexuality in an asexual strain of Dugesia japonica japonica by feeding, and 3) culture of dissociated planarian cells, show that neoblasts originate from intestinal cells, i.e. phagocytic cells and granular clubs.