Single Na+ channel currents observed in cultured rat muscle cells

The voltage- and time-dependent conductance of membrane Na+ channels is responsible for the propagation of action potentials in nerve and muscle cells. In voltage-step-clamp experiments on neurone preparations containing 10(4)-10(7) Na+ channels the membrane conductance shows smooth variations in time, but analysis of fluctuations and other eivdence suggest that the underlying single-channel conductance changes are stochastic, rapid transitions between 'closed' and 'open' states as seen in other channel types. We report here the first observations of currents through individual Na+ channels under physiological conditions using an improved version of the extracellular patch-clamp technique on cultured rat muscle cells. Our observations support earlier inferences about channel gating and show a single-channel conductance of approximately 18 pS.     

Habitat-induced reciprocal transformation in the root phenotype of Oriental ginseng is associated with alteration in DNA methylation

Oriental ginseng is an important medicinal plant that grows in 2 major forms or ecotypes, wild and domesticated. Each form differs conspicuously in root phenotype, but can be converted from one type to another by habitat. Here we show that the habitat-induced transformation of ginseng root phenotype was accompanied by alteration in cytosine methylation at a large number of 5′-CCGG-3′ sites detected by the methylation-sensitive polymorphism (MSAP) marker. The collective CG and CHG methylation levels of all 4 landraces of the domesticated form were significantly lower than those of the wild form. Interestingly, artificially transplanted ginseng plants recreated in both directions the methylation levels (at least in CHG) of their natural counterparts. The methylation differences between the 2 ginseng ecotypes were validated at 2 isolated MSAP loci bearing homology to a 5S rRNA gene or a copia retrotransposon. Our results implicate a link between epigenetic variation and habitat-induced phenotypic flexibility in Oriental ginseng.     

Clonal expansion of p53 mutant cells is associated with brain tumour progression.

Tumour progression is a fundamental feature of the biology of cancer. Cancers do not arise de novo in their final form, but begin as small, indolent growths, which gradually acquire characteristics associated with malignancy. In the brain, for example, low-grade tumours (astrocytomas) evolve into faster growing, more dysplastic and invasive high-grade tumours (glioblastomas). To define the genetic events underlying brain tumour progression, we analysed the p53 gene in ten primary brain tumour pairs. Seven pairs consisted of tumours that were high grade both at presentation and recurrence (group A) and three pairs consisted of low-grade tumours that had progressed to higher grade tumours (group B). In group A pairs, four of the recurrent tumours contained a p53 gene mutation; in three of them, the same mutation was found in the primary tumour. In group B pairs, progression to high grade was associated with a p53 gene mutation. A subpopulation of cells were present in the low-grade tumours that contained the same p53 gene mutation predominant in the cells of the recurrent tumours that had progressed to glioblastoma. Thus, the histological progression of brain tumours was associated with a clonal expansion of cells that had previously acquired a mutation in the p53 gene, endowing them with a selective growth advantage. These experimental observations strongly support Nowell's clonal evolution model of tumour progression.     

PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression

Functional impairment of T cells is characteristic of many chronic mouse and human viral infections. The inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, is markedly upregulated on the surface of exhausted virus-specific CD8 T cells in mice. Blockade of this pathway using antibodies against the PD ligand 1 (PD-L1, also known as CD274) restores CD8 T-cell function and reduces viral load. To investigate the role of PD-1 in a chronic human viral infection, we examined PD-1 expression on human immunodeficiency virus (HIV)-specific CD8 T cells in 71 clade-C-infected people who were naive to anti-HIV treatments, using ten major histocompatibility complex (MHC) class I tetramers specific for frequently targeted epitopes. Here we report that PD-1 is significantly upregulated on these cells, and expression correlates with impaired HIV-specific CD8 T-cell function as well as predictors of disease progression: positively with plasma viral load and inversely with CD4 T-cell count. PD-1 expression on CD4 T cells likewise showed a positive correlation with viral load and an inverse correlation with CD4 T-cell count, and blockade of the pathway augmented HIV-specific CD4 and CD8 T-cell function. These data indicate that the immunoregulatory PD-1/PD-L1 pathway is operative during a persistent viral infection in humans, and define a reversible defect in HIV-specific T-cell function. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T cells in chronic HIV infection.     

The impaired development of innate lymphoid cells by preterm birth is associated with the infant disease

Innate lymphoid cells (ILCs) are immune cells newly defined in recent decades and are recognized as the innate counterpart of the T helper cells, including IFNγ...     

Surface expression of MHC class II in dendritic cells is controlled by regulated ubiquitination

Dendritic cells have a unique function in the immune response owing to their ability to stimulate immunologically naive T lymphocytes. In response to microbial and inflammatory stimuli, dendritic cells enhance their capacity for antigen presentation by a process of terminal differentiation, termed maturation. The conversion of immature to mature dendritic cells is accompanied by a marked cellular reorganization, including the redistribution of major histocompatibility complex class II molecules (MHC II) from late endosomal and lysosomal compartments to the plasma membrane and the downregulation of some forms of endocytosis, which has been thought to slow the clearance of MHC II from the surface. The relative extent to which these or other mechanisms contribute to the regulation of surface MHC II remains unclear, however. Here we find that the MHC II beta-chain cytoplasmic tail is ubiquitinated in mouse immature dendritic cells. Although only partly required for the sequestration of MHC II in multivesicular bodies, this modification is essential for endocytosis. Notably, ubiquitination of MHC II ceased upon maturation, resulting in the accumulation of MHC II at the cell surface. Dendritic cells thus exhibit a unique ability to regulate MHC II surface expression by selectively controlling MHC II ubiquitination.     

Deficiency in catechol-O-methyltransferase and 2-methoxyoestradiol is associated with pre-eclampsia

Despite intense investigation, mechanisms that facilitate the emergence of the pre-eclampsia phenotype in women are still unknown. Placental hypoxia, hypertension, proteinuria and oedema are the principal clinical features of this disease. It is speculated that hypoxia-driven disruption of the angiogenic balance involving vascular endothelial growth factor (VEGF)/placenta-derived growth factor (PLGF) and soluble Fms-like tyrosine kinase-1 (sFLT-1, the soluble form of VEGF receptor 1) might contribute to some of the maternal symptoms of pre-eclampsia. However, pre-eclampsia does not develop in all women with high sFLT-1 or low PLGF levels, and it also occurs in some women with low sFLT-1 and high PLGF levels. Moreover, recent experiments strongly suggest that several soluble factors affecting the vasculature are probably elevated because of placental hypoxia in the pre-eclamptic women, indicating that upstream molecular defect(s) may contribute to pre-eclampsia. Here we show that pregnant mice deficient in catechol-O-methyltransferase (COMT) show a pre-eclampsia-like phenotype resulting from an absence of 2-methoxyoestradiol (2-ME), a natural metabolite of oestradiol that is elevated during the third trimester of normal human pregnancy. 2-ME ameliorates all pre-eclampsia-like features without toxicity in the Comt(-/-) pregnant mice and suppresses placental hypoxia, hypoxia-inducible factor-1alpha expression and sFLT-1 elevation. The levels of COMT and 2-ME are significantly lower in women with severe pre-eclampsia. Our studies identify a genetic mouse model for pre-eclampsia and suggest that 2-ME may have utility as a plasma and urine diagnostic marker for this disease, and may also serve as a therapeutic supplement to prevent or treat this disorder.     

Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11-Rad50-Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.     

Adrenomedullin inhibits the endothelin production induced by urotensin II in rat vascular smooth muscle cells

The aim of this study was to observe the effects of adrenomedullin (ADM) on endothelin (ET) production induced by urotensin Ⅱ (UⅡ) in rat vascular smooth muscle cells (VSMCs). Cultured VSMCs which were incubated with UⅡ (10^-8 mol/L) and with various concentrations of ADM were used to measure the VSMCs ³H-TdR incorpora- tion, the activity of extracellular signal-regulated kinase (ERK), the amount of ET mRNA and ET production in VSMCs. In this work we found that incubation with UⅡ(10^-8 mol/L) increased obviously the amount of ET mRNA in VSMCs and ET production in medium, however, coincubation with ADM (10^-10—10^-8 mol/L) and UⅡ(10^-8 mol/L) reduced the amount of ET mRNA by 15%, 24% and 45% (P< 0.01) respectively, compared with UⅡ alone. The content of ET in medium was 14.13, 11.38 and 11.00 pg/mL. ADM alone (10^-8 mol/L) had no effect on ET production in VSMCs. UⅡ (10^-8 mol/L) promoted the 3H-TdR incorpo- ration and activity of ERK in VSMCs. ADM inhibited VSMCs 3H-TdR incorporation and activation of ERK in a concentration-dependent manner. Compared with UⅡ group, after coincubation with ADM (10^-10—10^-8 mol/L) and UⅡ (10^-8 mol/L) the VSMCs 3H-TdR incorporation was decreased by 7% (P > 0.05), 32% (P < 0.05) and 41% (P < 0.01), respectively, and the activity of ERK was decreased by 24% (P > 0.05), 32% (P < 0.05) and 36% (P < 0.05), re- spectively, in a concentration-dependent manner. The results show that in cultured VSMCs ADM inhibits ET mRNA expression, ET production and proliferation stimulated by UⅡ, and that inhibitory effect of ADM on UⅡ bioaction could be mediated through inhibiting MAPK pathway.     

Neurotransmitter turnover in rat striatum is correlated with morphine self-administration

Drugs of abuse probably exert their reinforcing effects through 'reward' pathways in the central nervous system (CNS). Neuronal systems mediating opiate reinforcement have been investigated using pharmacological and electrolytic lesion procedures. Drugs that interfere with catecholaminergic and cholinergic neuronal activity decrease intravenous (i.v.) morphine self-administration in monkeys and rats. Electrolytic lesion procedures in rats have demonstrated that the medial forebrain bundle and caudate nucleus are important in maintaining i.v. morphine self-administration. We have now carried out a direct investigation of striatal (caudate nucleus, putamen and globus pallidus) neuronal systems. We show here that striatal catecholaminergic systems are important in mediating opiate reinforcement, and present direct evidence for the involvement of neurotransmitter systems in morphine reward.     

Phospholipid flippase associates with cisplatin resistance in plasma membrane of lung adenocarcinoma A549 cells

The fusion of the liposomes containing N-(7-nitro-2, 1, 3-benzoxadiazol-4-yl)-i ,2-hexadecanoylSn-glycero-3-1abeled phosphatidylethanolamine (NBD-PE) with A549 and A549/DDP cells was performed, and the activity of the phospholipid flippase in the plasma membrane of the cells was measured by fluorescence intensity change of NBDPE in the outer membrane. When A549 or A549/DDP cells containing N BD-PE were incubated at 37 C for 0, 30, 60 and 90 min, the fluorescence intensities in the outer membrane of the cells were 0%, 1.4%, 2.9% and 7.8% for A59cells, and 0%, 10.5 %, 15. 5 % and 18.3 % for A549/DDP cells respectively, demonstrating that the phospholipid flippase was distributed in the plasma membrane of As49 cells, but its activity in the drug-resistant A549/DDP cells was much higher than that in the A549 cells. When the A549/DDP cells were incubated with a multidrug resistance reverse agent, verapamil, for 60 min at 37C, the results showed that the NBD-PE in outer membrane decreased by 25.0% compared with the control's. Furthermore, when A549/DDP cells were incubated with 25 μmol/L cisplatin, which is a specific anticancer drug, the flippase activity decreased by 31.6%, and it further decreased with the increase of cisplatin concentration, suggesting that phospholipid flippase in the membrane might be related to the cisplatin-resistance of human lung adenocarcinoma cancer cells.     

Expression of the T-cell-specific gamma gene is unnecessary in T cells recognizing class II MHC determinants

Subtractive complementary DNA cloning combined with partial protein sequencing has allowed identification of the genes encoding the alpha and beta subunits of T-cell receptors. The subtractive cDNA library prepared from the cytotoxic T lymphocyte (Tc) clone 2C has been found to contain a third type of clone encoding the gamma chain. The gamma gene shares several features with the alpha and beta genes: (1) assembly from gene segments resembling immunoglobulin V, J and C (respectively variable, joining and constant region) DNA segments; (2) rearrangement and expression in T cells and not in B cells; (3) sequences reminiscent of transmembrane and intracytoplasmic regions of integral membrane proteins; (4) a cysteine residue at the position expected for an interchain disulphide bond. The alpha and beta genes are expressed at equivalent levels in both Tc cells and helper T cells (TH). The gamma gene, obtained from 2C, has been found to be expressed in all Tc cells studied. Here we present evidence that strongly suggests that TH cells do not require gamma gene expression.