Molecular basis of hypoxia-induced pulmonary vasoconstriction: role of voltage-gated K+ channels

The hypoxia-induced membrane depolarization and subsequent constriction of small resistance pulmonary arteries occurs, in part, via inhibition of vascular smooth muscle cell voltage-gated K+ (KV) channels open at the resting membrane potential. Pulmonary arterial smooth muscle cell KV channel expression, antibody-based dissection of the pulmonary arterial smooth muscle cell K+ current, and the O2 sensitivity of cloned KV channels expressed in heterologous expression systems have all been examined to identify the molecular components of the pulmonary arterial O2-sensitive KV current. Likely components include Kv2.1/Kv9.3 and Kv1.2/Kv1.5 heteromeric channels and the Kv3.1b alpha-subunit. Although the mechanism of KV channel inhibition by hypoxia is unknown, it appears that KV alpha-subunits do not sense O2 directly. Rather, they are most likely inhibited through interaction with an unidentified O2 sensor and/or beta-subunit. This review summarizes the role of KV channels in hypoxic pulmonary vasoconstriction, the recent progress toward the identification of KV channel subunits involved in this response, and the possible mechanisms of KV channel regulation by hypoxia.     

Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses

The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogeneous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.     

Targeted disruption of the mouse gene encoding the V-ATPase accessory subunit Ac45

Acidification of organelles of the eukaryotic vacuolar system is important for multiple intracellular processes including receptor-mediated endocytosis, proteolytic activity in lysosomes, and prohormone sorting and processing in secretory granules. Responsible for the generation of a proton gradient across a membrane is vacuolar H(+)-ATPase (V-ATPase). How the activity of this multisubunit enzyme is regulated remains to be established. Accessory subunits of the V-ATPase may be involved in the organelle-specific regulation, one candidate being the chromaffin granular V-ATPase-associated protein Ac45. To assess the function of Ac45, we disrupted its gene by gene targeting in male mouse embryonic stem cells. We have successfully generated Ac45 null mutant (-IY) embryonic stem cells and injected them into C57BL/6 recipient blastocysts. The blastocysts were replaced into pseudopregnant foster mothers, giving rise to 16 littermates. One of these appeared to be a low-chimeric female mouse that died 6 weeks after birth. No signs of late abortion were detected in the foster mothers. The results suggest that the injected Ac45 null mutant embryonic stem cells affect the normal development of the blastocyst and are in line with knockout studies on other V-ATPase subunits that point to an essential role for the V-ATPase in early embryonic development.     

Controls on methane production in a tidal freshwater estuary and a peatland: methane production via acetate fermentation and CO2 reduction

Rates of total methane production, acetate fermentation andCO₂ reduction were compared for two different wetland sites. On aper-liter basis, sediments from the White Oak River estuary, a tidal freshwatersite in eastern North Carolina, had an annual methane production rate (53.3mMyr^–1) an order of magnitude higher thanthat ofBuck Hollow Bog (5.5 mMyr^–1), a peatlandinMichigan. Methane was produced in the White Oak River site on an annual basisbyboth acetate fermentation (72%) and CO₂ reduction (28%) in a ratiotypical of freshwater methanogenic sites. Competition for acetate bynon-methanogenic microorganisms in Buck Hollow peat limited methane productionfrom acetate to only a few months a year, severely impacting annual methaneproduction rates. However, when acetate was available to the methanogens in thepeat during early spring, the percentage of methane production from acetatefermentation (84%) and CO₂ reduction (16%) and rates of totalmethaneproduction were similar to those of the White Oak River sediments at the sametemperature. Rates of CO₂ reduction and acetate fermentationconducted at both sites at various temperatures showed that Buck Hollow peatmethane production was also limited by a colder temperature regime as well asdifferences in the response of the CO₂ reducing and aceticlasticmethanogens to temperature variations.     

Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells

The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 +/- 0.14 to 0.76 +/- 0.06 mM, corresponding to a Mg2+ uptake rate of 15 microM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 +/- 19%) in the presence of CO2/HCO(-)3. In contrast, Mg2+ uptake was strongly diminished if REC were suspended in HCO(-)3-buffered high-KCl medium (22.3 +/- 4 microM/min) rather than in HEPES-buffered KCl medium (37.5 +/- 6 microM/min). After switching from high- to low-Cl- solution, [Mg2+]i was reduced from 0.64 +/- 0.09 to 0.32 +/- 0.16 mM and the CO2/HCO(-)3-stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl- and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.     

Effect of low inoculation density in the scale-up of insect cell cultures

The scale-up of insect cell cultures and the production of baculovirus with these cultures is dependent on the inoculation density applied. The effect of applying a low inoculation on the specific growth rate and on the duration of the lag phase was tested. Three different cell lines, HzAm1, Ha2302, and Sf21 were tested in a total of five cell line/medium combinations. Growth in suspension culture was examined, and data obtained were fitted with the Gompertz equation. A significant decline in specific growth rate with decreasing inoculation density was observed in all cell line/medium combinations, except for HzAm1. No critical inoculation density, below which no growth would occur, was found. In suspension culture in shake flasks, an inoculation density of 5 x 10(4) cells/mL is achievable, without severely influencing the overall growth rate. A lower inoculation density in suspension culture results in less steps in the scale-up process and might be a tool in bypassing the viral passage effect.     

Multicomponent synthesis of novel amino acid-nucleobase chimeras: a versatile approach to PNA-monomers

This paper describes a multicomponent approach to novel totally protected precursors of PNA-monomers via Ugi 4CC. The obtained bisamides are converted into several partially protected PNA-monomers or derivatives thereof using three different procedures. Methods for hydrolysis are shown to be dependent on the nature of the isocyano component required for Ugi 4CC. Several novel monomers suitable for oligomer synthesis are prepared demonstrating the high versatility of the reaction sequence.     

Induction of proopiomelanocortin mRNA expression in animal caps of Xenopus laevis embryos

To convert animal pole cells of a frog embryo from an ectodermal fate into a neural one, inductive signals are necessary. The alkalizing agent NH4Cl induces the expression of several anterior brain markers and the early pituitary marker XANF-2 in Xenopus animal caps. Here it is demonstrated that NH4Cl also induced proopiomelanocortin (POMC)-expressing cells (the first fully differentiated pituitary cell type) in stage 9 and 10 Xenopus animal caps, and that all-trans retinoic acid, a posteriorizing agent, was able to block this induction when it was administered within 2 h after the start of NH4Cl incubation. Thus, after 2 h, the fate of Xenopus animal cap cells was determined. Microinjection of ribonucleic acid (RNA) encoding noggin, an endogenous neural inducer, led to the induction of POMC gene expression in animal caps of stage 10 embryos, suggesting that noggin represents a candidate mesodermal signal leading to the POMC messenger (m) RNA producing cell type in uncommitted ectoderm. Hence, an alkalizing agent and a neural inducer can generate a fully differentiated POMC cell lineage from Xenopus animal caps.     

Accumulation of short telomeres in human fibroblasts prior to replicative senescence

The loss of telomere repeats has been causally linked to in vitro replicative senescence of human diploid fibroblasts (HDFs). In order to study the mechanism(s) by which telomere shortening signals cell senescence, we analyzed the telomere length at specific chromosome ends at cumulative population doublings in polyclonal and clonal HDFs by quantitative fluorescence in situ hybridization. The rate of telomere shortening at individual telomeres varied between 50 and 150 bp per population doubling and short telomeres with an estimated 1-2 kb of telomere repeats accumulated prior to senescence. The average telomere length in specific chromosome ends was remarkably similar between clones. However, some exceptions with individual telomeres measuring 0.5-1 kb were observed. In the fibroblast clones, the onset of replicative senescence was significantly correlated with the mean telomere fluorescence but, strikingly, not with chromosomes with the shortest telomere length. The accumulation of short telomeres in late passages of cultured HDFs is compatible with selection of cells on the basis of telomere length and limited recombination between telomeres prior to senescence.