Role of Cav1.2 L-type Ca2+ channels in vascular tone: effects of nifedipine and Mg2+

Ca(2+) entry via L-type voltage-gated Ca(2+) channels (LVGCs) is a key factor in generating myogenic tone (MT), as dihydropyridines (DHPs) and other LVGC blockers, including Mg(2+), markedly reduce MT. Recent reports suggest, however, that elevated external Mg(2+) concentration and DHPs may also inhibit other Ca(2+)-entry pathways. Here, we explore the contribution of LVGCs to MT in intact, pressurized mesenteric small arteries using mutant mice (DHP(R/R)) expressing functional but DHP-insensitive Ca(v)1.2 channels. In wild-type (WT), but not DHP(R/R), mouse arteries, nifedipine (0.3-1.0 microM) markedly reduced MT and vasoconstriction induced by high external K(+) concentrations ([K(+)](o)), a measure of LVGC-mediated Ca(2+) entry. Blocking MT and high [K(+)](o)-induced vasoconstriction by <1 microM nifedipine in WT but not in DHP(R/R) arteries implies that Ca(2+) entry via Ca(v)1.2 LVGCs is obligatory for MT and that nifedipine inhibits MT exclusively by blocking LVGCs. We also examined the effects of Mg(2+) on MT and LVGCs. High external Mg(2+) concentration (10 mM) blocked MT, slowed the high [K(+)](o)-induced vasoconstrictions, and decreased their amplitude in WT and DHP(R/R) arteries. To verify that these effects of Mg(2+) are due to block of LVGCs, we characterized the effects of extracellular and intracellular Mg(2+) on LVGC currents in isolated mesenteric artery myocytes. DHP-sensitive LVGC currents are inhibited by both external and internal Mg(2+). The results indicate that Mg(2+) relaxes MT by inhibiting Ca(2+) influx through LVGCs. These data provide new information about the central role of Ca(v)1.2 LVGCs in generating and maintaining MT in mouse mesenteric small arteries.     

Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology

We investigated the origins of greater clot rigidity associated with FXIIIa-dependent cross-linking. Fibrin clots were examined in which cross-linking was controlled through the use of two inhibitors: a highly specific active-center-directed synthetic inhibitor of FXIIIa, 1,3-dimethyl-4,5-diphenyl-2[2(oxopropyl)thio]imidazolium trifluoromethylsulfonate, and a patient-derived immunoglobulin directed mainly against the thrombin-activated catalytic A subunits of thrombin-activated FXIII. Cross-linked fibrin chains were identified and quantified by one- and two-dimensional gel electrophoresis and immunostaining with antibodies specific for the alpha- and gamma-chains of fibrin. Gamma-dimers, gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrids were detected. The synthetic inhibitor was highly effective in preventing the production of all cross-linked species. In contrast, the autoimmune antibody of the patient caused primarily an inhibition of alpha-chain cross-linking. Clot rigidities (storage moduli, G') were measured with a cone and plate rheometer and correlated with the distributions of the various cross-linked species found in the clots. Our findings indicate that the FXIIIa-induced dimeric cross-linking of gamma-chains by itself is not sufficient to stiffen the fibrin networks. Instead, the augmentation of clot rigidity was more strongly correlated with the formation of gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrid cross-links. A mechanism is proposed to explain how these cross-linked species may enhance clot rigidity.     

Household and climate factors influence Aedes aegypti presence in the arid city of Huaquillas,Ecuador

Arboviruses transmitted by Aedes aegypti (e.g., dengue, chikungunya, Zika) are of major public health concern on the arid coastal border of Ecuador and Peru. This high transit border is a critical disease surveillance site due to human movement-associated risk of transmission. Local level studies are thus integral to capturing the dynamics and distribution of vector populations and social-ecological drivers of risk, to inform targeted public health interventions. Our study examines factors associated with household-level Ae. aegypti presence in Huaquillas, Ecuador, while accounting for spatial and temporal effects. From January to May of 2017, adult mosquitoes were collected from a cohort of households (n = 63) in clusters (n = 10), across the city of Huaquillas, using aspirator backpacks. Household surveys describing housing conditions, demographics, economics, travel, disease prevention, and city services were conducted by local enumerators. This study was conducted during the normal arbovirus transmission season (January—May), but during an exceptionally dry year. Household level Ae. aegypti presence peaked in February, and counts were highest in weeks with high temperatures and a week after increased rainfall. Univariate analyses with proportional odds logistic regression were used to explore household social-ecological variables and female Ae. aegypti presence. We found that homes were more likely to have Ae. aegypti when households had interruptions in piped water service. Ae. aegypti presence was less likely in households with septic systems. Based on our findings, infrastructure access and seasonal climate are important considerations for vector control in this city, and even in dry years, the arid environment of Huaquillas supports Ae. aegypti breeding habitat.     

Molecular engineering of cyclic α-diketone aromatic ring bichromophoric molecules for studies of intramolecular electronic energy transfer

The design, structures and spectral properties of a number of bichromophoric molecules are presented. These bichromophoric molecules are composed of an aromatic ring connected by two methylene chains to an -diketone moiety. Both absorption and emission spectra can be attributed to a superposition of the individual spectra of the separate chromophores. The critical transfer radius for electronic energy transfer from the aromatic (donor) chromophore to the -diketone (acceptor) chromophore was calculated from the spectral overlap between the fluorescence spectrum of the aromatic moiety and the absorption spectrum of the -diketone moiety. The results show that this series of molecules is well suited for a mechanistic study of short-range intramolecular electronic energy transfer.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

Comparative Effect of Transient Global Ischemia on Extracellular Levels of Glutamate, Glycine, and γ-Aminobutyric Acid in Vulnerable and Nonvulnerable Brain Regions in the Rat

We evaluated whether regional differences in the magnitude of glutamate, gamma-aminobutyric acid (GABA), and glycine release could explain why some regions are vulnerable to ischemia whereas others are spared. By means of the microdialysis technique, the temporal profile of ischemia-induced changes in extracellular levels of glutamate, GABA, and glycine was compared in regions that demonstrate differing susceptibilities to a 10- and 20-min ischemic insult (dorsal hippocampus, anterior thalamus, somatosensory cortex, and dorsolateral striatum). The degree of ischemia (as established by local cerebral blood flow reduction) and the magnitude of histopathological neuronal damage were also evaluated in these regions. The blood flow reduction was severe and uniform in all regions; however, the histopathological outcome illustrated a different pattern. Whereas the CA1 sector of the hippocampus was severely damaged, the thalamus and cortex were relatively spared from both 10 and 20 min of ischemia. Striatal neurons were resistant to a 10-min insult but severely damaged after 20 min of ischemia. Ischemia-induced increase in glutamate and GABA content were of a similar magnitude and temporal profile in all four brain regions. A uniform increase in extracellular glycine levels was also observed in all four brain structures. The postischemic response, however, was different. Glycine levels remained twofold higher than baseline in the hippocampus but fell to baseline in the cortex and thalamus after both 10- and 20-min insults. In the striatum, glycine levels returned to baseline after 10 min of ischemia but remained relatively high after a 20-min insult.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA efflux from synaptosomes: Effects of membrane potential,and external GABA and cations

Summary Presynaptic GABAergic nerve terminals accumulate -aminobutyric acid (GABA) by a sodium-dependent carrier mechanism in which two Na⁺ are co-transported with one GABA. We have examined the influence of external GABA and cations on GABA efflux from³H-GABA loaded rat brain synaptosomes, to determine whether or not the carriers can also mediate GABA efflux. We observed that, in Ca-free media (to minimize Ca-dependent evoked release), external GABA promotes GABA efflux when the medium contains Na⁺, butinhibits GABA efflux in the absence of Na⁺. The efflux of GABA into Ca-free media is stimulated by depolarization (either with veratridine or increased external K⁺). These data, and published data on the internal Na⁺ dependence of GABA efflux into Ca-free media, indicate that exiting GABA is cotransported with Na⁺. The stimulatory effect of depolarization is consistent with efflux of Na⁺ along with the uncharged GABA. The (carrier-mediated) efflux is also stimulated when the carriers cycle inward with Na⁺+GABA. The inhibitory effect of GABA in Na⁺-free media implies that GABA can bind to unloaded carriers and that the carriers loaded only with GABA cycle very slowly, if at all. Our data, and data from the literature, can be fitted to a simple model with sequential binding of solutes: external GABA binds to the carrier first, and only the free or fully-loaded (with 2Na⁺+1GABA) carriers can cycle. Other binding sequences and random binding, do not fit the experimental observations.     

Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.