Pisum sativum mitochondrial pyruvate dehydrogenase can be assembled as a functional alpha(2)beta(2) heterotetramer in the cytoplasm of Pichia pastoris

Pea (Pisum sativum) mitochondrial pyruvate dehydrogenase (E1) was produced by coexpression of the mature alpha and beta subunits in the cytoplasm of the yeast Pichia pastoris. Size-exclusion chromatography of recombinant E1, using a Superose 12 column, yielded a peak at M(r) 160,000 that contained both alpha and beta subunits as well as E1 activity. This corresponds to the size of native alpha(2)beta(2) E1. Recombinant E1 alpha (His(6))-E1 beta was purified by affinity chromatography using immobilized Ni(+), with a yield of 2.8 mg L(-1). The pyruvate-decarboxylating activity of recombinant E1 was dependent upon added Mg(2+) and thiamin-pyrophosphate and was enhanced by the oxidant potassium ferricyanide. Native pea mitochondrial E1-kinase catalyzed phosphorylation of Ser residues in the alpha-subunit of recombinant E1, with concomitant loss of enzymatic activity. Thus, mitochondrial pyruvate dehydrogenase can be assembled in the cytoplasm of P. pastoris into an alpha(2)beta(2) heterotetramer that is both catalytically active and competent for regulatory phosphorylation.     

Effects of sex, body size, temperature, and location on the antipredator tactics of free-ranging gartersnakes (Thamnophis sirtalis, Colubridae)

Gartersnakes (Thamnophis sirtalis parietalis) in southern Manitoba are subject to intense predation (primarily by crows) duringtheir spring breeding season. The huge numbers of snakes providea unique opportunity to quantify behavioral traits. We simulatedpredator attacks by "pecking" more than 500 free-ranging snakes,to explore the determinants of snake response. Snakes respondedto a human finger in the same way as they did to a more realisticstimulus (a model crow). A snake's response to attack dependedon several factors, which interacted in complex ways. The primaryinfluences on response were body temperature (warmer snakes tended to flee, whereas colder snakes remained cryptic or flattenedand/or gaped and struck) and sex (males were more likely toflee). Responses also depended on microhabitat (i.e., insidethe winter den versus in adjacent grassland) and on the snake'sprior activity (e.g., courting snakes often ignored our closeapproach). These factors interacted in significant ways; for example, snakes outside the den were smaller and warmer thanthose inside, male snakes were smaller and warmer than females,and mean body temperatures were higher in larger snakes withineach sex. Thus, a snake's body size and its location affectedits defensive response indirectly (via their influence on bodytemperature). Our results differ from those of previous studiesand suggest that antipredator responses in these animals dependin a flexible and complex way upon biotic and abiotic variables.Interactions among these variables also must be consideredbefore we can identify underlying causal processes.     

Intron recognition comes of AGe

The molecular basis for the consensus sequence at the 3' ends of introns in higher eukaryotes has now been elucidated. However, this discovery does not explain all aspects of 3' splice site selection.     

Chronic hypoxia, pregnancy, and endothelium-mediated relaxation in guinea pig uterine and thoracic arteries

Vasodilation that occurs during normal pregnancy is associated with enhanced relaxation and decreased contractile response to agonists, which are in part due to increased stimulated and basal nitric oxide (NO). In preeclampsia and/or pregnancies carried at high altitude (HA), this normal vascular adjustment is reversed or diminished. We previously reported that HA exposure did not inhibit the pregnancy-associated decrease in contractile response to agonist or basal NO in guinea pig uterine arteries (UA). We therefore sought to determine whether altitude interfered with effects of pregnancy on endothelium-dependent relaxation through a reduction in stimulated NO. We examined the relaxation response to ACh in UA and bradykinin in thoracic arteries (TA) and effects of NO inhibition with 200 microM N(G)-nitro-L-arginine (L-NNA) in arterial rings isolated from nonpregnant and pregnant guinea pigs exposed throughout gestation to low altitude (LA, 1,600 m, n = 26) or HA (3,962 m, n = 22). In pregnant UA, relaxation to ACh was enhanced (P < 0.05) at both altitudes and NO inhibition diminished, but did not reverse, ACh relaxation. The effect of L-NNA on the relaxation response to ACh was less in HA than in LA animals (P = 0.0021). In nonpregnant UA, relaxation to ACh was similar in LA and HA animals. L-NNA reversed the relaxation response to ACh at HA but not at LA. In TA, relaxation to bradykinin was unaltered by pregnancy or altitude and was completely reversed by NO inhibition. These data suggest that effects of NO inhibition are diminished in UA during pregnancy at HA. Additional studies are needed to confirm whether these effects are mediated through inhibition of stimulated NO. HA exposure did not inhibit relaxation to ACh, perhaps because of stimulation of other vasodilators.     

Soluble P-selectin moderates complement-dependent reperfusion injury of ischemic skeletal muscle

P-selectin is an adhesion molecule expressed on activated endothelial and platelet surfaces. The function of the short consensus repeats (SCRs) of P-selectin, homologous with the SCRs of complement regulatory proteins is largely unknown. In a model of murine hindlimb ischemia where local reperfusion injury is partly mediated by IgM natural antibody and classical complement pathway activation, we hypothesized that human soluble P-selectin (sP-sel) would moderate the complement component of the inflammatory response. Infusion of sP-sel supernatant or purified (p) sP-sel prepared from activated human platelets, reduced ischemic muscle vascular permeability by 48% and 43%, respectively, following reperfusion. Hindlimb immunohistochemistry demonstrated negligible C3 staining colocalized with IgM in these groups compared with intense staining in the untreated injured mice. In vitro studies of mouse serum complement hemolytic activity showed that psP-sel inhibited the classical but not alternative complement pathway. Flow cytometry demonstrated that psP-sel inhibited C1q adherence to sensitized red blood cells. From these data we conclude that sP-sel moderates skeletal muscle reperfusion injury by inhibition of the classical complement pathway.     

Hydrogenophaga intermedia sp. nov., a 4-aminobenzenesulfonate degrading organism

The taxonomic status of a gram-negative, oxidase positive rod (strain S1) able to degrade 4-aminobenzenesulfonate was studied using a polyphasic approach. Chemotaxonomic investigations of quinones and polar lipids established the allocation of this strain to the beta-subclass of the Proteobacteria and revealed similarities to Hydrogenophaga palleronii. 16S rRNA sequence comparisons demonstrated that this strain clusters phylogenetically with H. palleronii and H. taeniospiralis, but clearly represents a new species. The fatty acid patterns and substrate utilization profile displayed similarity to the characteristics of the four validly published species of Hydrogenophaga, although clear differentiating characters were also observed. No close similarities between the type strains of H. palleronii and H. taeniospiralis were detected in hybridization experiments with the genomic DNAs. On basis of these results, the new species Hydrogenophaga intermedia sp. nov. is proposed, with the type strain S1T (= DSM 5680).     

Comparative evolution of the mitochondrial cytochrome b gene and nuclear beta-fibrinogen intron 7 in woodpeckers

Most molecular phylogenetic studies of vertebrates have been based on DNA sequences of mitochondrial-encoded genes. MtDNA evolves rapidly and is thus particularly useful for resolving relationships among recently evolved groups. However, it has the disadvantage that all of the mitochondrial genes are inherited as a single linkage group so that only one independent gene tree can be inferred regardless of the number of genes sequenced. Introns of nuclear genes are attractive candidates for independent sources of rapidly evolving DNA: they are pervasive, most of their nucleotides appear to be unconstrained by selection, and PCR primers can be designed for sequences in adjacent exons where nucleotide sequences are conserved. We sequenced intron 7 of the beta-fibrinogen gene (beta-fibint7) for a diversity of woodpeckers and compared the phylogenetic signal and nucleotide substitution properties of this DNA sequence with that of mitochondrial-encoded cytochrome b (cyt b) from a previous study. A few indels (insertions and deletions) were found in the beta-fibint7 sequences, but alignment was not difficult, and the indels were phylogentically informative. The beta-fibint7 and cyt b gene trees were nearly identical to each other but differed in significant ways from the traditional woodpecker classification. Cyt b evolves 2.8 times as fast as beta-fibint7 (14. 0 times as fast at third codon positions). Despite its relatively slow substitution rate, the phylogenetic signal in beta-fibint7 is comparable to that in cyt b for woodpeckers, because beta-fibint7 has less base composition bias and more uniform nucleotide substitution probabilities. As a consequence, compared with cyt b, beta-fibint7 nucleotide sites are expected to enter more distinct character states over the course of evolution and have fewer multiple substitutions and lower levels of homoplasy. Moreover, in contrast to cyt b, in which nearly two thirds of nucleotide sites rarely vary among closely related taxa, virtually all beta-fibint7 nucleotide sites appear free of selective constraints, which increases informative sites per unit sequenced. However, the estimated gamma distribution used to model rate variation among sites suggests constraints on some beta-fibint7 sites. This study suggests that introns will be useful for phylogenetic studies of recently evolved groups.     

Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line

Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, RyR1 and RyR2 have well-defined roles in contraction. However, studies on mammalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free amplicon system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1- or RyR3-transduced cells expressed the appropriate RyR isoform of the correct molecular mass. Although RyR1 channels exhibited the expected unitary conductance for Cs(+) in bilayer lipid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance behavior. Western blot analysis with an FKBP12/12.6-selective antibody reveals that differences in gating behavior exhibited by RyR1 and RyR3 may be, in part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, whereas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1, they were much less sensitive to 4-chloro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillations were observed in response to caffeine and CMC but were never seen in RyR3-expressing 1B5 cells. In [(3)H]ryanodine binding studies, only RyR1 exhibited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian muscle context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding sites.     

alpha-actinin-2 couples to cardiac Kv1.5 channels, regulating current density and channel localization in HEK cells

Voltage-gated K(+) (Kv) channels are particularly important in the physiology of excitable cells in the heart and the brain. PSD-95 is known to cluster Shaker channels and NMDA receptors and the latter is known to couple through alpha-actinin-2 to the post-synaptic cytoskeleton [Wyszynski et al. (1997) Nature 385, 439-442], but the mechanisms by which Kv channels are linked to the actin cytoskeleton and clustered at specific sites in the heart are unknown. Here we provide evidence that Kv1.5 channels, widely expressed in the cardiovascular system, bind with alpha-actinin-2. Human Kv1.5 interacts via its N-terminus/core region and can be immunoprecipitated with alpha-actinin-2 both after in vitro translation and from HEK cells expressing both proteins. The ion channels and alpha-actinin-2 co-localize at the membrane in HEK cells, where disruption of the actin cytoskeleton and antisense constructs to alpha-actinin-2 modulate the ion and gating current density.