Effects of caffeine on crayfish muscle fibers. II. Refractoriness and factors influencing recovery (repriming) of contractile responses

When caffeine evokes a contraction, and only then, crayfish muscle fibers become refractory to a second challenge with caffeine for up to 20 min in the standard saline (5 mM K_o). However, the fibers still respond with contraction to an increase in K_o, though with diminished tension. Addition of Mn slows recovery, but the latter is greatly accelerated during exposure of the fiber to high K_o, or after a brief challenge with high K_o. Neither the depolarization induced by the K, nor the repolarization after its removal accounts for the acceleration, which occurs only if the challenge with K had itself activated the contractile system; acceleration is blocked when contractile responses to K are blocked by reducing the Ca in the bath or by adding Mn. Recovery is accelerated by redistribution of intracellular Cl and by trains of intracellularly applied depolarizing pulses, but not by hyperpolarization. The findings indicate that two sources of Ca can be mobilized to activate the contractile system. Caffeine mobilizes principally the Ca store of the SR. Depolarizations that are induced by high K_o, by transient efflux of Cl, or by intracellularly applied currents mobilize another source of Ca which is strongly dependent upon the entry of Ca from the bathing medium. The sequestering mechanism of the SR apparently can utilize this second source of Ca to replenish its own store so as to accelerate recovery of responsiveness to a new challenge with caffeine.     

The genetic contribution to heart rate and heart rate variability in quiescent mice

Recent studies have suggested a genetic component to heart rate (HR) and HR variability (HRV). However, a systematic examination of the genetic contribution to the variation in HR and HRV has not been performed. This study investigated the genetic contribution to HR and HRV using a wide range of inbred and recombinant inbred (RI) mouse strains. Electrocardiogram data were recorded from 30 strains of inbred mice and 29 RI strains. Significant differences in mean HR and total power (TP) HRV were identified between inbred strains and RI strains. Multiple significant differences within the strain sets in mean low-frequency (LF) and high-frequency (HF) power were also found. No statistically significant concordance was found between strain distribution patterns for HR and HRV phenotypes. Genomewide interval mapping identified a significant quantitative trait locus (QTL) for HR [LOD (likelihood of the odds) score = 3.763] on chromosome 6 [peak at 53.69 megabases (Mb); designated HR 1 (Hr1)]. Suggestive QTLs for TP were found on chromosomes 2, 4, 5, 6, and 14. A suggestive QTL for LF was found on chromosome 16; for HF, we found one significant QTL on chromosome 5 (LOD score = 3.107) [peak at 53.56 Mb; designated HRV-high-frequency 1 (Hrvhf1)] and three suggestive QTLs on chromosomes 2, 11 and 15. In conclusion, the results demonstrate a strong genetic component in the regulation of resting HR and HRV evidenced by the significant differences between strains. A lack of correlation between HR and HRV phenotypes in some inbred strains suggests that different sets of genes control the phenotypes. Furthermore, QTLs were found that will provide important insight to the genetic regulation of HR and HRV at rest.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

Epidemiology and risk factors associated with Anaplasma marginale infection of cattle in Peninsular Malaysia

Bovine anaplasmosis is a major concern to cattle farming in most parts of the world. Anaplasmosis negatively impacts the profitability of cattle farming by reducing the production, reproduction, and draft ability of cattle. Here, we report results from a one-year cross sectional study to determine the epidemiology and the risk factors for Anaplasma marginale infection of cattle in Peninsular Malaysia. Examination of one thousand and forty five blood samples of apparently healthy cattle from forty-three farms in all the states of Peninsular Malaysia by polymerase chain reaction (PCR) assay revealed an overall prevalence of A. marginale infection of cattle of 72.6%, showing high endemicity of this heamoprotozoan among cattle in the country. Cattle breeds, production type, herd owner, herd size, management system, farm size, farm age, prophylactic treatment against blood parasites, presence of ticks, frequency of deticking, zones, closeness to forest, closeness to waste area, closeness to human settlement and closeness to body of water were the risk factors significantly associated (P?<?0.05) with the detection of A. marginale in cattle. Results of this first molecular study on the epidemiology and risk factors for A. marginale infection of cattle from all the states of Peninsular Malaysia suggest policies and strategies for the prevention and control of the parasite to improve profitability of cattle farming in the country.     

Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg²⁺ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.     

Phase I clinical trial of systemically administered TUSC2(FUS1)-nanoparticles mediating functional gene transfer in humans

Background

Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2.

Methods

Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks.

Results

Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in post-treatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high post-treatment levels of TUSC2 mRNA and protein showed significant post-treatment changes in the intrinsic apoptotic pathway. Twenty-nine genes of the 82 tested in the apoptosis array were identified by Igenuity Pathway Analysis to be significantly altered post-treatment in both patients (Pearson correlation coefficient 0.519; p<0.01).

Conclusions

DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in uptake of the gene by human primary and metastatic tumors, transgene and gene product expression, specific alterations in TUSC2-regulated pathways, and anti-tumor effects (to our knowledge for the first time for systemic DOTAP:cholesterol nanoparticle gene therapy).

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00059605","term_id":"NCT00059605"}}NCT00059605     

Developmental dynamics of myogenesis in the shipworm <Emphasis Type="Italic">Lyrodus pedicellatus</Emphasis> (Mollusca: Bivalvia)

Background

The shipworm Lyrodus pedicellatus is a wood-boring bivalve with an unusual vermiform body. Although its larvae are brooded, they retain the general appearance of a typical bivalve veliger-type larva. Here, we describe myogenesis of L. pedicellatus revealed by filamentous actin labelling and discuss the data in a comparative framework in order to test for homologous structures that might be part of the bivalve (larval) muscular ground pattern.

Results

Five major muscle systems were identified: a velum retractor, foot retractor, larval retractor, a distinct mantle musculature and an adductor system. For a short period of larval life, an additional ventral larval retractor is present. Early in development, a velum muscle ring and an oral velum musculature emerge. In late stages the lateral and dorsal mantle musculature, paired finger-shaped muscles, an accessory adductor and a pedal plexus are formed. Similar to other bivalve larvae, L. pedicellatus exhibits three velum retractor muscles, but in contrast to other species, one of them disappears in early stages of L. pedicellatus. The remaining two velum retractors are considerably remodelled during late larval development and are most likely incorporated into the elaborate mantle musculature of the adult.

Conclusions

To our knowledge, this is the first account of any larval retractor system that might contribute to the adult bodyplan of a (conchiferan) mollusk. A comparative analysis shows that a pedal plexus, adductors, a larval velum ring, velum retractors and a ventral larval retractor are commonly found among bivalve larvae, and thus most likely belong to the ground pattern of the bivalve larval musculature.

     

Conservation of blood plasma fluids in hamadryas baboons after thermal dehydration

After 2 days of water deprivation in a warm climate, Papio hamadryas baboons lost 10% of their body mass, 12.5% of their total body water (3H2O) space, but only 4% of their plasma volume [Evans blue (EB) space]. Hematocrit and hemoglobin concentration as well as blood viscosity and blood pressure were not affected by thermal dehydration. Plasma colloid osmotic pressure (COP) in the dehydrated animals was, however, 8 Torr higher than in fully hydrated baboons. Total mass and concentration of plasma albumin, and protein concentration increased after dehydration. Both half times (T 1/2) of EB and T 1/2 of 131I-serum albumin were twice as high as in the dehydrated animal than in the fully hydrated ones. Incorporation rate of L-[3H]leucine in the plasma proteins was similarly higher in the dehydrated animals. The capacity of the P. hamadryas baboon to maintain its plasma volume at the expense of losses from other body fluid compartments is related to an increase in the blood COP that is brought about by a more efficient retention of albumin and an increase in its rate of synthesis.