Modulation of glycolysis induction in primary cultures of rabbit kidney proximal tubule cells: the role of shaking,glucose and insulin.

We have assessed the impact of increasing oxygen availability on cellular phenotype expression of rabbit proximal tubule cells in primary culture developed with variable glucose and/or insulin contents. To mitigate hypoxia at the cell/medium interface, cells were shaken for the whole culture duration and their expressed phenotype was compared with those expressed by static cultures. O₂ and CO₂ tensions were kept constant in the incubator atmosphere. Glycolysis and gluconeogenesis pathways, detoxication system, and mitochondrial, apical and basolateral membrane marker enzyme activities were assessed. This study showed that the induction of glycolysis which appear in primary cultures of proximal tubule cells may be partially prevented by continuously shaking the cultures. This effect was more marked in the presence of glucose, suggesting better substrate oxidation in shaken cultures.     

Influence of age,castration, and testosterone on T cell subsets in healthy and leukemia grafted mice

The distribution of T cell subsets in pubertal (2 months) and post-pubertal (10 months) mice showed a significant decrease in the percentage of CD4+ splenocytes and peripheral blood lymphocytes (PBL) with age, unlike the percentage of CD8+ cells in PBL, which remained unchanged. The change in the distribution of T cell subsets in the spleen and blood occurred in 2 months old castrated mice, as in 10 months old animals. P388 tumor grew better in post-pubertal and in castrated mice than in young mice. The intact mice survived longer than the castrated ones. The relative number of CD4+, CD8+ and CD2+ splenocytes was lower in transplanted intact mice than that in controls. The CD8+ and CD2+ subsets in the blood of 2 months transplanted mice were higher than those in controls, whereas in PBL, in 10 months old and castrated mice, the T lymphocyte subsets remain unchanged. Depo-testosterone (DT) injection strongly reduced weight and tumor growth in all the intact and castrated animals. A significant correlation is observed between the tumor weight and testosterone level in the plasma of the 2 months old DT treated mice. Moreover, DT injection induced a significant increase in the percentage of blood CD8+ cells in all the batches. These data indicate that physiologically, androgens affect the age-related distribution of lymphocyte T subsets and suggest that they slow down tumor growth, besides causing a direct effect, through an immunological process.     

Cross-Reactions between the Cytotoxic T-Lymphocyte Responses of Human Immunodeficiency Virus-Infected African and European Patients

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.     

Index of names of syntaxa typified in 1994

Following the “Index of new names” (Theurillat & Moravec 1998), the present work collects the names of syntaxa (in the sense of the Code of phytosociological nomenclature,Barkman et al. 1986) above subassociation rank typified in 1994. The list comprises 101 names of syntaxa; three names are added to the Index 1992 (Theurillat & Moravec 1995).     

Hepatocytes from newborn and weanling rats in monolayer culture: Isolation by perfusion,fibronectin-mediated adhesion,spreading, and functional activities

Summary The two-step collagenase perfusion method originally developed for the high yield isolation of parenchymal cells from adult rat livers has been adapted to rats of 1 day, 1 week, and 3 weeks of age. The use of this method to isolate hepatocytes from five or six rats of the respective ages demonstrated its reliability in terms of cell yield, percentage of single cells, and cell viability. In all cases, hepatocytes attach with high efficiency to fibronectin precoated dishes using serum-free culture medium. The dynamics of spreading is faster for newborn hepatocytes than adult ones. The functional integrity of these parenchymal liver cells was assessed by their capacity to secrete albumin and α-fetoprotein in serum-free medium and to express lactate dehydrogenase activity over a 24-hr period in primary culture. Part of this work was presented at the 30th Annual Meeting of the Tissue Culture Association, Seattle, June, 1979.     

Reprogramming in nuclear transfer

Reprogramming a nucleus by transferring it to oocyte cytoplasm triggers epigenetic changes that eventually lead to the restoration of a totipotent state and the birth of a viable animal. A simplified way of studying this complex process is to study cell hybrids. These studies suggest that the pluripotent character of one nucleus is dominant over that of the other nucleus. The development of nuclear transfer embryos shows that there are several restriction points and that the extra-embryonic lineages may be the primary source of death. Successful reprogramming depends on the balance between epigenetic modifications and the regulative properties of development.     

Trafficking through Rab11 endosomes is required for cellularization during Drosophila embryogenesis

BACKGROUND: Embryonic cleavage leads to the formation of an epithelial layer during development. In Drosophila, the process is specialized and called cellularization. The trafficking pathways that underlie this process and that are responsible for the mobilization of membrane pools, however, remain poorly understood. RESULTS: We provide functional evidence for the role of endocytic trafficking through Rab11 endosomes in remobilizing vesicular membrane pools to ensure lateral membrane growth. Part of the membrane stems from endocytosed apical material. Mutants in the endocytic regulators rab5 and shibire/dynamin inhibit basal-lateral membrane growth, and apical endocytosis is blocked in shibire mutants. In addition, shibire controls vesicular trafficking through Rab11-positive endosomes. In shibire mutants, the transmembrane protein Neurotactin follows the secretory pathway normally but is not properly inserted in the plasma membrane and accumulates instead in Rab11 subapical endosomes. Consistent with a direct role of shibire in vesicular trafficking through Rab11 endosomes, Shibire is enriched in this compartment. Moreover, we show by electron microscopy the large accumulation of intracellular coated pits on subapical endocytic structures in shibire mutants. Finally, we show that Rab11 is essential for membrane growth and invagination during cellularization. CONCLUSION: Together, the data show that endocytic trafficking is required for basal-lateral membrane growth during cellularization. We identify Rab11 endosomes as key trafficking intermediates that control vesicle exocytosis and membrane growth during cellularization. This pathway may be required in other morphogenetic processes characterized by the growth of a membrane domain.     

Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells

BACKGROUND: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations.Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha. MATERIALS AND METHODS: The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN. RESULTS: Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa. CONCLUSION: In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.     

Body protein does not vary despite seasonal changes in fat in the White Stork Ciconia ciconia

To understand how a large soaring bird, the White Stork Ciconia ciconia , copes with energy constraints, we compared changes in body mass in 14 captive adult storks with the body composition of 12 free-ranging adult storks found dead from accidents. The captive storks, already in an enclosure for several years, were fed ad libitum . They were weighed daily for 1.53.5 years using an automatic device. The bodies of the accidentally killed storks were analysed to determine total water, lipid, protein and ash contents, and to assess the biochemical composition of certain organs. Females were on average 20% lighter and 24% smaller than males, but the body mass of the sexes varied in parallel throughout the year. Body mass peaked in December and January (2530% above minimal body mass), due essentially to large fat stores in subcutaneous and abdominal adipose tissues. Body mass and body lipid rapidly decreased from February to June, whether the storks reared chicks successfully or not, and remained minimal for a few days into July. In contrast to birds using flapping flight, no variation in body protein or pectoral muscle protein was observed while breeding, even though the moult occurred then, nor in August, before the time when wild storks migrate. An endogenous regulation of body fuels is discussed.