Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio)

In the skin of zebrafish embryo, the vacuolar H(+)-ATPase (V-ATPase, H(+) pump) distributed mainly in the apical membrane of H(+)-pump-rich cells, which pump internal acid out of the embryo and function similarly to acid-secreting intercalated cells in mammalian kidney. In addition to acid excretion, the electrogenic H(+) efflux via the H(+)-ATPases in the gill apical membrane of freshwater fish was proposed to act as a driving force for Na(+) entry through the apical Na(+) channels. However, convincing molecular physiological evidence in vivo for this model is still lacking. In this study, we used morpholino-modified antisense oligonucleotides to knockdown the gene product of H(+)-ATPase subunit A (atp6v1a) and examined the phenotype of the mutants. The H(+)-ATPase knockdown embryos revealed several abnormalities, including suppression of acid-secretion from skin, growth retardation, trunk deformation, and loss of internal Ca(2+) and Na(+). This finding reveals the critical role of H(+)-ATPase in embryonic acid -secretion and ion balance, as well.     

RacGTPase-activating protein 1 interacts with hepatitis C virus polymerase NS5B to regulate viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus responsible for chronic liver disease and hepatocellular carcinoma (HCC). RacGTPase-activating protein 1 (RacGAP1) plays an important role during GTP hydrolysis to GDP in Rac1 and CDC42 protein and has been demonstrated to be upregulated in several cancers, including HCC. However, the molecular mechanism leading to the upregulation of RacGAP1 remains poorly understood. Here, we showed that RacGAP1 levels were enhanced in HCV cell-culture-derived (HCVcc) infection. More importantly, we illustrated that RacGAP1 interacts with the viral protein NS5B in mammalian cells. The small interfering RNA (siRNA)-mediated knockdown of RacGAP1 in human hepatoma cell lines inhibited replication of HCV RNA, protein, and production of infectious particles of HCV genotype 2a strain JFH1. Conversely, these were reversed by the expression of a siRNA-resistant RacGAP1 recombinant protein. In addition, viral protein NS5B polymerase activity was significantly reduced by silencing RacGAP1 and, vice versa, was increased by overexpression of RacGAP1 in a cell-based reporter assay. Our results suggest that RacGAP1 plays a crucial role in HCV replication by affecting viral protein NS5B polymerase activity and holds importance for antiviral drug development.     

Replication and temperature-sensitive maintenance functions of lactose plasmid pSK11L from Lactococcus lactis subsp. cremoris.

The replication region of pSK11L, the lactose plasmid of Lactococcus lactis subsp. cremoris (L. cremoris) SK11, was isolated on a 14.8-kbp PvuII fragment by shotgun cloning into an Escherichia coli vector encoding erythromycin resistance and selection for erythromycin-resistant transformants of L. lactis subsp. lactis (L. lactis) LM0230. Deletion analysis and Tn5 mutagenesis of the resulting plasmid (pKMP1) further localized the replication region to a 2.3-kbp ScaI-SpeI fragment. DNA sequence analysis of this 2.3-kbp fragment revealed a 1,155-bp open reading frame encoding the putative replication protein, Rep. The replication origin was located upstream of rep and consisted of an 11-bp imperfect direct repeat and a 22-bp sequence tandemly repeated three and one-half times. The overall organization of the pSK11L replicon was remarkably similar to that of pCI305, suggesting that pSK11L does not replicate by the rolling-circle mechanism. Like pSK11L, pKMP1 was unstable in L. lactis LM0230. Deletion analysis allowed identification of several regions which appeared to contribute to the maintenance of pKMP1 in L. lactis LM0230. pKMP1 was significantly more stable in L. cremoris EB5 than in L. lactis LM0230 at all of the temperatures compared. This stability was lost by deletion of a 3.1-kbp PvuII-XbaI fragment which had no effect on stability in L. lactis LM0230. Other regions affecting stability in L. cremoris EB5 but not in L. lactis LM0230 were also identified. Stability assays conducted at various temperatures showed that pKMP1 maintenance was temperature sensitive in both L. lactis LM0230 and L. cremoris EB5, although the plasmid was more unstable in L. lactis LM0230. The region responsible for the temperature sensitivity phenotype in L. lactis LM0230 was tentatively localized to a 1.2-kbp ClaI-HindIII fragment which was distinct from the replication region of pSK11L. Our results suggest that the closely related L. lactis and L. cremoris subspecies behave differently regarding maintenance of plasmids.     

Experimental mild renal insufficiency mediates early cardiac apoptosis, fibrosis, and diastolic dysfunction: a kidney-heart connection

Impaired renal function with loss of nephron number in chronic renal disease (CKD) is associated with increased cardiovascular morbidity and mortality. However, the structural and functional cardiac response to early and mild reduction in renal mass is poorly defined. We hypothesized that mild renal impairment produced by unilateral nephrectomy (UNX) would result in early cardiac fibrosis and impaired diastolic function, which would progress to a more global left ventricular (LV) dysfunction. Cardiorenal function and structure were assessed in rats at 4 and 16 wk following UNX or sham operation (Sham); (n = 10 per group). At 4 wk, blood pressure (BP), aldosterone, glomerular filtration rate (GFR), proteinuria, and plasma B-type natriuretic peptide (BNP) were not altered by UNX, representing a model of mild early CKD. However, UNX was associated with significantly greater LV myocardial fibrosis compared with Sham. Importantly, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining revealed increased apoptosis in the LV myocardium. Further, diastolic dysfunction, assessed by strain echocardiography, but with preserved LVEF, was observed. Changes in genes related to the TGF-β and apoptosis pathways in the LV myocardium were also observed. At 16 wk post-UNX, we observed persistent LV fibrosis and impairment in LV diastolic function. In addition, LV mass significantly increased, as did LVEDd, while there was a reduction in LVEF. Aldosterone, BNP, and proteinuria were increased, while GFR was decreased. The myocardial, structural, and functional alterations were associated with persistent changes in the TGF-β pathway and even more widespread changes in the LV apoptotic pathway. These studies demonstrate that mild renal insufficiency in the rat results in early cardiac fibrosis and impaired diastolic function, which progresses to more global LV remodeling and dysfunction. Thus, these studies importantly advance the concept of a kidney-heart connection in the control of myocardial structure and function.     

ADP Ribosylation Factor 1 Is Required for Synaptic Vesicle Budding in PC12 Cells

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein–T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O''Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041–1049). A peptide spanning the effector domain of human ARF1 (2–17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.Generation of carrier vesicles from plasma membrane or intracellular membranous compartments involves at least two components, GTP-binding proteins and coats (Rothman and Wieland, 1996; Schekman and Orci, 1996). A particularly widespread protein that regulates coat assembly on intracellular membranes is ADP ribosylation factor (ARF)¹ 1, a small GTP-binding protein (Donaldson and Klausner, 1994; Boman and Kahn, 1995). The budding of vesicles from Golgi cisternae can be fully reconstituted in the presence of ARF1 and coatomer (COPI) (Ostermann et al., 1993; Donaldson and Klausner, 1994; Boman and Kahn, 1995; Rothman and Wieland, 1996). ARF1 recruits coatomers to the budding vesicle and couples uncoating to fusion with target membranes (Ostermann et al., 1993; Tanigawa et al., 1993). ARF1 is also required for the recruitment of COPI to vesicles budding from the ER (Bednarek et al., 1995). A hallmark of ARF1-mediated processes is their sensitivity to the fungal metabolite brefeldin A (BFA).The GDP–GTP exchange activity that replaces GDP bound to ARF proteins with GTP is inhibited by BFA (Donaldson et al., 1992; Helms and Rothman, 1992). The GDP form of ARF1 is unable to bind membranes and consequently, to recruit coats (Robinson and Kreis, 1992; Donaldson and Klausner, 1994). The selectivity of BFA is such that, if a membrane traffic event is sensitive to BFA, it is predicted to require ARF proteins. Inhibition of intra-Golgi and ER to Golgi traffic by BFA probably involves the COPI coatomers. BFA also interferes with coats other than COPI, especially those involved in budding from TGN. Thus it inhibits the formation of vesicles from the TGN (Simon et al., 1996) and causes the redistribution of assembly protein 1 and clathrin to the cytosol (Robinson and Kreis, 1992). Post-Golgi trafficking of the mannose-6-phosphate receptor (Wood et al., 1991) and the maturation of secretory granules (Dittie et al., 1996) are also sensitive to BFA. In addition to clathrin and COPI, BFA affects the recruitment of other “coating” molecules, such as the p47–βNAP complex (Simpson et al., 1996; Dell''Angelica et al., 1997) and p200 (Narula and Stow, 1995) to TGN membranes.Some endocytotic pathways are also sensitive to BFA. For example, the delivery of polyimmunoglobulin A (pIgA) to plasma membrane from the specialized apical endosome in epithelial MDCK cells, or from dendritic endosomes in hippocampal neurons, is inhibited by BFA (Hunziker et al., 1991; Barroso and Sztul, 1994). BFA-sensitive recruitment of COP1-related proteins and ARF proteins to endosomes has also been reported (Whitney et al., 1995; Cavenagh et al., 1996).The formation of synaptic vesicles at nerve terminals is a specialized endocytotic pathway that has many similarities to the formation of carrier vesicles from Golgi membranes. In this case, the donor membrane for synaptic vesicle formation is the plasma membrane or the endosome (De Camilli and Takei, 1996). Morphological evidence strongly suggests that synaptic vesicles are generated in nerve terminals through a coat-dependent mechanism (Shupliakov et al., 1997). In lysed nerve terminals, recruitment of dynamin and clathrin coats to membranous organelles is modulated by nonhydrolyzable GTP analogues (Takei et al., 1996). Cell-free reconstitution assays of neuroendocrine synaptic vesicle (SV) formation in PC12 cell extracts showed that GTPγS blocks the generation of properly sized SVs (Desnos et al., 1995), but the identity of the GTP-binding protein or proteins was not determined.In this paper, we show that reagents that interfere with the cycling of ARF1 between cytosol and membranes block SV formation in neuroendocrine PC12 cells. SV formation was reconstituted in vitro using recombinant ARF1 and a cytosol-derived high molecular weight fraction. Since SV production in vitro is from an endocytotic pool, these results suggest that coating mechanisms associated with ER and Golgi biosynthetic pathways are also associated with at least one endocytotic pathway.     

Lethal and mutagenic effects of radiation and alkylating agents on two strains of mouse L5178Y cells

In previous studies, the two closely related strains of L5178Y (LY) mouse lymphoma cells, LY-R and LY-S, have been shown to differ in their sensitivity to UV and ionizing radiation. Thus, in comparison to strain LY-R, strain LY-S has been found to be more sensitive to the lethal effects of ionizing radiation, more resistant to the lethal effects of UV radiation, but less mutable at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus by both UV and X-radiation. In the present work, the lethal and mutagenic effects of ethyl methanesulfonate (EMS), methyl nitrosourea (MNU) and UV radiation (254 nm) were compared in the two strains. Mutability at the Na+/K+-ATPase locus as well as the HGPRT locus was determined. As previously reported, we found strain LY-S to be more resistant than strain LY-R to the lethal effects of UV radiation. In contrast, strain LY-S was more sensitive to the cytotoxic effects of the two alkylating agents. In spite of these differences in sensitivity, we found strain LY-S to be less mutable than strain LY-R by all 3 agents at the HGPRT locus. At the Na+/K+-ATPase locus, strain LY-S was also less mutable than strain LY-R by equal concentrations of EMS and UV radiation and by equitoxic concentrations of MNU. However, the difference between the strains was much more pronounced at the HGPRT locus than at the Na+/K+-ATPase locus. We have suggested that the interaction of unrepaired lesions in strain LY-S tends to cause an excess of deletions and multilocus effects, which in turn result in a locus-dependent decrease in the recovery of viable LY-S mutant cells.     

Cloning and characterization of the lipase and lipase activator protein from Vibrio vulnificus CKM-1

The gene (lipA) encoding the extracellular lipase and its downstream gene (lipB) from Vibrio vulnificus CKM-1 were cloned and sequenced. Nucleotide sequence analysis and alignments of amino acid sequences suggest that Lip Ais a member of bacterial lipase family I.1 and that LipB is a lipase activator of LipA. The active LipA was produced in recombinant Escherichia coli cells only in the presence of the lipB. In the hydrolysis of p-nitrophenyl esters and triacylglycerols, using the reactivated LipA, the optimum chain lengths for the acyl moiety on the substrate were C14 for ester hydrolysis and C10 to C12 for triacylglycerol hydrolysis.     

The influence of dose rate on the lethal and mutagenic effects of X-rays in proliferating L5178Y cells differing in radiation sensitivity

The lethal and mutagenic effects of ionizing radiation delivered at high (53 Gy/h) and low (0.02 Gy/h) dose rates were measured in two closely related strains of mouse lymphoma L5178Y cells differing in radiation sensitivity (LY-R and LY-S). Strain LY-R was more resistant to the lethal effects of radiation than strain LY-S when exposed at either the high or low dose rate. The survival of strain LY-R was markedly enhanced by the reduction in dose rate. The dose-rate dependence of the survival of strain LY-S was less clear, because of the biphasic nature of its survival curve following low dose-rate radiation. However, if the initial slope of the low dose-rate survival curve is compared to the slope of the high dose-rate survival curve for strain LY-S, only a slight increase in survival at the low dose rate is apparent. Although more sensitive to the lethal effects of radiation, strain LY-S was less mutable at the hypoxanthine/guanine phosphoribosyl transferase locus by both low dose-rate and high dose-rate radiation than strain LY-R. Little dose-rate dependence was exhibited by either strain with regard to the mutagenic effects of radiation. Thus, for strain LY-R, which showed marked dose-rate dependence for survival but not for mutation, the ratio of mutational to lethal lesions was much greater following exposure to low dose-rate than to high dose-rate radiation.     

Primary suspension culture of calf anterior pituitary cells on a microcarrier surface

In order to achieve a steady-state primary culture system for mammalian cells, with the potential to eventually correlate and control cell function and growth, a serious evaluation of various suspension systems was made. Calf anterior pituitary cells were employed as a differentiated cell type and successfully cultivated in a microcarrier suspension culture system. DEAE-Sephadex was demonstrated to be a satisfactory type of microcarrier. The cells readily attached to the bead and, after a short lag period, they actively proliferated on the bead surface to yield growth of a predominantly epithelial cell type. Under specific conditions the microcarrier supported primary cell growth up to levels of 2 × 10⁶ cells per ml. High bead concentrations inhibited cell growth. The inhibition could be overcome by using proportionately higher cell inoculum so that a concentrated culture with 5 × 10⁶ cells per ml was achieved. The inhibitory effect of high bead concentration was found to be due to the absorption of serum protein and certain growth enhancing factors. The fact that the growth enhancing factors were released from cells during the period of trypsinization and were both thermostable and nondialyzable, seems to suggest one approach to a dialysis culture system. In addition, relatively trauma-free primary cell cultures can be achieved by using explant culture without prior trypsinization. In microcarrier suspensions direct growth of primary rat mammary tumor cells was also demonstrated.