RhoA/ROCK signaling is critical to FAK activation by cyclic stretch in cardiac myocytes

Focal adhesion kinase (FAK) has been shown to be activated in cardiac myocytes exposed to mechanical stress. However, details of how mechanical stimuli induce FAK activation are unknown. We investigated whether signaling events mediated by the RhoA/Rho-associated coiled coil-containing kinase (ROCK) pathway are involved in regulation of stretch-induced FAK phosphorylation at Tyr(397) in neonatal rat ventricular myocytes (NRVMs). Immunostaining showed that RhoA localized to regions of myofilaments alternated with phalloidin (actin) staining. The results of coimmunoprecipitation assays indicated that FAK and RhoA are associated in nonstretched NRVMs, but cyclic stretch significantly reduced the amount of RhoA recovered from anti-FAK immunoprecipitates. Cyclic stretch induced rapid and sustained (up to 2 h) increases in phosphorylation of FAK at Tyr(397) and ERK1/2 at Thr(202)/Tyr(204). Blockade of RhoA/ROCK signaling by pharmacological inhibitors of RhoA (Clostridium botulinum C3 exoenzyme) or ROCK (Y-27632, 10 micromol/l, 1 h) markedly attenuated stretch-induced FAK and ERK1/2 phosphorylation. Similar effects were observed in cells treated with the inhibitor of actin polymerization cytochalasin D. Transfection of NRVMs with RhoA antisense oligonucleotide attenuated stretch-induced FAK and ERK1/2 phosphorylation and expression of beta-myosin heavy chain mRNA. Similar results were seen in cells transfected with FAK antisense oligonucleotide. These findings demonstrate that RhoA/ROCK signaling plays a crucial role in stretch-induced FAK phosphorylation, presumably by coordinating upstream events operationally linked to the actin cytoskeleton.     

Corticosterone 21-acetate in vivo induces acute stress in chicken thymus: cell proliferation, apoptosis and cytokine responses

In vivo effects of acute stress induced by corticosterone 21-acetate in male Gallus domesticus thymus are studied and the steroid actions are evaluated in terms of cell proliferation, apoptosis and cytokine response in 10- and 21-day-old chickens. Steroid treatment induced thymocyte apoptosis and cell death decreased in the cortical-medullar direction and was more evident in younger animals. 24 h after treatment, the observed effect was reversed. The mitotic activity and thymic cells containing cytokine-like molecules were also affected. Indeed, the acute stress stimulated cytokine immunoreactivity to anti-IL-1alpha, IL-6 and TNF-alpha antibodies both in epithelial cells and interdigitating cells located in medullar and cortical-medullar regions. The increased cytokine expression observed after 12 h was maintained after 24 h. The comparison between 10- and 21-day-old chickens showed a lower number of cells containing cytokine-like molecules in younger specimens. The present findings suggest that cytokines activated by acute stress in vivo could contribute to restoring immunological homeostasis and influence thymic glucocorticoid-mediated functions.     

Psycho-physiological aspects of small combats in taekwondo: impact of area size and within-round sparring partners

The study investigated physiological and psychological responses to taekwondo combat sessions as a function of different area sizes and within-round sparring partners. Twenty-four adolescent (age: 17 ± 1years) male (n = 12) and female (n = 12) taekwondo athletes participated in the study. Each athlete confronted 1 (1vs.1; no sparring partner change) or 2 (1vs.2; within-round sparring partner change every minute) opponents in different area sizes (i.e., 4 × 4 m, 6 × 6 m, and 8 × 8 m) for 2 min. Blood lactate concentration ([La]) was measured before and after bouts. Heart rate (HR) was measured throughout the contests and rating of perceived exertion was assessed after bouts. Mean HR (HRmean) and percentage of maximum HR (%HRmax) determined during a 20-m multistage shuttle run test were used for analysis. Mood states were assessed before and after bouts and physical enjoyment was analyzed after bouts. The results showed higher HRmean and %HRmax values for the 1vs.1 compared to the 1vs.2 condition (p < 0.001) and [La] values were higher at post-combat measurements (p < 0.001). Moreover, tension and fatigue were higher in 6 × 6 m compared with 8 × 8 m (p = 0.022 and p = 0.023, respectively) and anger was higher in 6 × 6 m and 8 × 8 m in comparison with 4 × 4 m (p = 0.012 and p = 0.043, respectively). Confusion increased from before to after bouts (p < 0.001), from 4 × 4 m and 6 × 6 m area sizes to 8 × 8 m (p = 0.001 and p = 0.018, respectively), and from 1vs.1 to 1vs.2 (p < 0.001). Furthermore, vigour decreased from before to after bouts (p < 0.01). Taekwondo combat sessions are a specific conditioning exercise for athletes. Thus, coaches can use the 1vs.1 condition to elicit higher HR responses and 6 × 6 m area size to induce higher psychological stress, mimicking what occurs during a competition.     

THE INFLUENCE OF KARATE PRACTICE LEVEL AND SEX ON PHYSIOLOGICAL AND PERCEPTUAL RESPONSES IN THREE MODERN KARATE TRAINING MODALITIES

Objective

The aim of the present study was to examine the influence of karate practice level (national vs international level) and sex (women vs men) on physiological and perceptual responses in three modern karate training modalities (tactical-technical (TT), technical-development (TD), and randori).

Method

The study included 18 karatekas participating in an eight-session training camp of four TT, two TD, and two randori. During each session, the percentage of maximal heart rate (HR), blood lactate concentration [La^-], and rating of perceived exertion (RPE) were assessed.

Results

The main results showed that the percentage of maximal HR was significantly higher in women than in men regardless of practice level or training modality (70.3 ± 4.1 vs 66.2 ± 6.3, respectively). Moreover, [La^-] and RPE were significantly lower in international-level karatekas compared with their national-level counterparts whatever the sex or training modality ([La^-] = 11.4 ± 2.6 vs 8.3 ± 2.4 mmol · L^-1 and RPE = 3.6 ± 1.2 vs 4.3 ± 1.5, respectively). Last, physiological and perceptual responses were significantly higher during randori in comparison with TT and TD for both sexes.

Conclusion

The combination of [La^-] and RPE thus seems to be a good indicator for discriminating between national- and international-level karatekas, and randori seems to be an effective means to reproduce official karate sparring.     

Palmitoylation and p8-Mediated Human T-Cell Leukemia Virus Type 1 Transmission

The orf-I gene of human T-cell leukemia type 1 (HTLV-1) encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers, and only the monomeric forms of p8 and p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.     

Early activation of p160ROCK by pressure overload in rat heart

We investigated the mechanisms underlying regulation of contraction with measurements of isometric force and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in NIH 3T3 fibroblast reconstituted into fibers with the use of a collagen matrix. Treatment with the major phospholipids, neurotransmitters, and growth factors had little effect on baseline isometric force. However, U-46619, a thromboxane A₂ (TxA₂) analog, increased force and [Ca²⁺]_i; EC₅₀ values were 11.0 and 10.0 nM, respectively. The time courses were similar to those induced by calf serum (CS), and the maximal force was 65% of a CS-mediated contraction. The selective TxA₂ receptor antagonist SQ-29548 abolished the U-46619-induced responses. CS-induced contractions are dependent on an intracellular Ca²⁺ store function; however, the U-46619 response depended not only on intracellular Ca²⁺ stores, but also on Ca²⁺ influx from the extracellular medium. Inhibition of Rho kinase suppressed U-46619- and CS-induced responses; in contrast, inhibition of C kinase (PKC) reduced only the U-46619 response. Moreover, addition of U-46619 to a CS contracture enhanced force and [Ca²⁺]_i responses. These results indicate that U-46619-induced responses involve PKC and Rho kinase pathways, in contrast to activation by CS. Thus TxA₂ may have a role in not only the initial step of wound repair as an activator of blood coagulation, but also in fibroblast contractility in later stages. collagen matrix; signal transduction; wound repair     

Ancient exaptation of a CORE-SINE retroposon into a highly conserved mammalian neuronal enhancer of the proopiomelanocortin gene

The proopiomelanocortin gene (POMC) is expressed in the pituitary gland and the ventral hypothalamus of all jawed vertebrates, producing several bioactive peptides that function as peripheral hormones or central neuropeptides, respectively. We have recently determined that mouse and human POMC expression in the hypothalamus is conferred by the action of two 5′ distal and unrelated enhancers, nPE1 and nPE2. To investigate the evolutionary origin of the neuronal enhancer nPE2, we searched available vertebrate genome databases and determined that nPE2 is a highly conserved element in placentals, marsupials, and monotremes, whereas it is absent in nonmammalian vertebrates. Following an in silico paleogenomic strategy based on genome-wide searches for paralog sequences, we discovered that opossum and wallaby nPE2 sequences are highly similar to members of the superfamily of CORE-short interspersed nucleotide element (SINE) retroposons, in particular to MAR1 retroposons that are widely present in marsupial genomes. Thus, the neuronal enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon in the lineage leading to mammals and remained under purifying selection in all mammalian orders for the last 170 million years. Expression studies performed in transgenic mice showed that two nonadjacent nPE2 subregions are essential to drive reporter gene expression into POMC hypothalamic neurons, providing the first functional example of an exapted enhancer derived from an ancient CORE-SINE retroposon. In addition, we found that this CORE-SINE family of retroposons is likely to still be active in American and Australian marsupial genomes and that several highly conserved exonic, intronic and intergenic sequences in the human genome originated from the exaptation of CORE-SINE retroposons. Together, our results provide clear evidence of the functional novelties that transposed elements contributed to their host genomes throughout evolution.     

Original Articles: Sources of Dissolved Organic Carbon Supporting Planktonic Bacterial Production in the Tidal Freshwater Hudson River

Planktonic bacterial production in the tidal freshwater Hudson River is a major component of secondary productivity and is uncoupled from planktonic primary productivity. There are several major sources of allochthonous dissolved organic carbon (DOC) whose potential contribution to heterotrophic bacterial growth was examined with bioassays. Supply of DOC from the upper Hudson drainage basin and a large tributary in the mid-Hudson together comprise 70 kT DOC/year, which is the bulk of the DOC load to the tidal freshwater Hudson River. Two contrasting tidal wetlands contribute DOC to the main-stem river but were only a few percent of the tributary load even during summer low-flow conditions. The quantity of DOC released from fine sediments was intermediate to the other two loadings considered. Bacterial growth in bioassays receiving water from the sources varied, but differences in thymidine incorporation between reference and DOC sources were small, usually less than 2 nmol/L/h. Similarity in thymidine incorporation suggests that all sources of DOC were capable of supporting bacterial growth at approximately equal rates. Seasonal shifts in carbon availability were clear in several cases, for example, greater growth on wetland-derived DOC at times of peak plant productivity. Seasonal differences in tributary DOC bioavailability were not large despite the well-known seasonality of tributary inputs. Activities of a suite of extracellular enzymes were used as a biologically based characterization of DOC from the various sources. Shifts in allocation among enzymes were apparent, indicating that there are biologically relevant differences in composition among the sources. Fluorescence characteristics and absorbance per unit carbon also varied among sources, providing an independent confirmation of compositional differences among sources. The absence of large differences in bacterial productivity among sources suggests that growth is supported by a wide range of DOC, and the relative importance of the sources is probably related to the quantitative differences in inputs. Efforts to classify carbon supplies to ecosystems must recognize that organism plasticity in carbon use and physical mixing processes will both act to homogenize what might initially appear to be quite distinctive carbon inputs. Received 15 April 1997; accepted 17 February 1998     

Ultrastructural Study of Haemocytes of the Freshwater Snail Planorbarius corneus (L.) (Gastropoda, Pulmonata)

Ultrastructural studies were performed on the blood cells of Planorbarius corneus. Spreading and round cell forms were observed. The spreading form can be distinguished from the round form by its abundant cytoplasm, pseudopods and irregularly shaped nucleus. Electron dense granules in the mitochondrial matrix are found in the round form and are lacking in the spreading form. Moreover, cellular apoptosis is observed in the round form. The ultrastructural findings and the previously described characteristics support the idea that the two cell forms belong to two basic cell types.