Glycogen synthase binds to sarcoplasmic reticulum and is phosphorylated by CaMKII in fast-twitch skeletal muscle

We investigated the subcellular localization of glycogen synthase (GS) in the adductor muscle of anesthetized rabbits injected intravenously with propranolol. Under these experimental conditions, glycogen content was about 10 mmol/kg of fresh tissue. Immunofluorescent and fractionation studies showed that GS associated with sarcoplasmic reticulum (SR) membranes. Glycogen and GS always co-sedimented, suggesting a predominant role of glycogen in targeting of GS to SR. SR-associated GS was phosphorylated in vitro by SR-bound Ca2+-calmodulin dependent protein kinase (CaMKII) and dephosphorylated by endogenous protein phosphatase 1 (PP1c). Based on measurements of GS activity ratio, in vitro phosphorylation of GS by CaMKII did not significantly affect GS activity per se. However, GS activity ratio was slightly reduced, when SR membranes were further incubated with ATP after prior phosphorylation by CaMKII, suggesting that CaMKII might act sinergistically with other protein kinases. We propose that SR-bound CaMKII plays a role in regulation of glycogen metabolism in skeletal muscle, when intracellular Ca2+ is raised.     

Skeletal muscle performance determined by modulation of number of myosin motors rather than motor force or stroke size

Skeletal muscle can bear a high load at constant length, or shorten rapidly when the load is low. This force-velocity relationship is the primary determinant of muscle performance in vivo. Here we exploited the quasi-crystalline order of myosin II motors in muscle filaments to determine the molecular basis of this relationship by X-ray interference and mechanical measurements on intact single cells. We found that, during muscle shortening at a wide range of velocities, individual myosin motors maintain a force of about 6 pN while pulling an actin filament through a 6 nm stroke, then quickly detach when the motor reaches a critical conformation. Thus we show that the force-velocity relationship is primarily a result of a reduction in the number of motors attached to actin in each filament in proportion to the filament load. These results explain muscle performance and efficiency in terms of the molecular mechanism of the myosin motor.     

Amazonian Amerindians exhibit high variability of KIR profiles

Natural killer cell immunoglobulin-like receptors (KIRs) mediate cell lysis through the recognition of human leukocyte antigen class I complexes in target cells, playing an important role in innate immune response. In this context, disease-based selective pressures could be relevant, leaving signatures detected by population studies. However, most population studies on KIR variability have focused on Europe and Asia, while Americas, Oceania, and Africa remain poorly studied. The aim of this study was to analyze the variability of KIR genes in Amerindian tribes from the Amazon region to infer about their evolutionary history. KIR profiles were estimated in 40 individuals from six Amazonian Amerindian tribes using single specific primer polymerase chain reaction. Twenty-five different profiles were identified, and surprisingly, the haplogroup A frequency was the lowest observed in human populations (16%). Results showed also that KIR variability was higher in this group in contrast to Venezuelan Amerindians. Principal components analysis evidenced that Amerindians formed a separated group from other worldwide populations and showed a higher intraethnic differentiation in comparison to other ethnic groups. Such pattern may reflect small effective size and intense genetic drift. However, because of the role of KIR in immune response, selective pressures cannot be entirely ruled out.     

Infection by <Emphasis Type="Italic">Rickettsia bellii</Emphasis> and <Emphasis Type="Italic">Candidatus “Rickettsia amblyommii”</Emphasis> in <Emphasis Type="Italic">Amblyomma neumanni</Emphasis> Ticks from Argentina

The tick species, Amblyomma neumanni (Acari: Ixodidae) is the most frequent tick parasitizing humans in northwestern Argentina. The present study evaluated the rickettsial infection among 55 A. neumanni adult free-living ticks collected in Dean Funes, Córdoba Province. Ticks were individually processed by the hemolymph test with Gimenez staining, isolation of rickettsia in Vero cell culture by the shell vial technique, and polymerase chain reaction (PCR) targeting the citrate synthase rickettsial gene. Through the shell vial technique, rickettsiae were successfully isolated and established in Vero cell culture from two ticks (ticks 4 and 13), which previously showed to contain Rickettsia-like organisms by the hemolymph test. These two Rickettsia isolates were designated as An4 and An13. Molecular characterization (partial DNA sequences of two to three rickettsial genes were determined) of these two isolates and phylogenetic analyses identified them as Rickettsia bellii (isolate An4) and Candidatus “Rickettsia amblyommii” (isolate An13). After testing all A. neumanni ticks by PCR, the prevalence of Candidatus R. amblyommii and R. bellii was 23.6% (13/55) and 3.6% (2/55), respectively. These two rickettsiae have been considered of unknown pathogenicity and appropriate studies to test their pathogenicity to humans or animals need to be conducted. This is the first report of Rickettsia in ticks from Argentina, and also in the species A. neumanni. The results reinforce previous findings that R. bellii (and probably Candidatus R. amblyommii) are widespread among some Neotropical Amblyomma species, suggesting that these ticks gained these bacterial agents from a common ancestor and/or by recent horizontal transmission of rickettsiae between ticks.     

Simulation of working conditions by maximum work load on firefighters

The aim of this research is to find out whether the firefighter manpower is adequate to the requirements in the field under the most severe conditions. It also attempts to test whether firefighters' working ability corresponds to their age. To that purpose 220 Croatian firefighters from the Littoral Mountainous county, 99 professional firefighters from the city of Rijeka, 45 professional firefighters from its suburbs and 76 volunteer firefighters from the suburbs were submitted to load test, fitness test--stepping on the bench for three minutes, repetitive power test--sit-ups lasting 1 minute, and a leap-explosive power test. The fitness test was repeated carrying Drd?er's respiratory apparatus PSS 100 with compressed air, so respiratory values were compared before and after the burden of the respiratory apparatus. The results have shown that professional firefighters from the city have the mean increased body mass index (BMI) 26, and professionals from the suburbs BMI 27. In spite of the increased body mass they showed good fitness, spirometric values before and after the load showed neither restrictive nor obstructive ventilation difficulties, which indicates a good condition of cardio respiratory system and also adequate protective equipment. The initial hypothesis has been confirmed: with age, equipped with personnel does not necessarily mean operative equipment, because linear regressive analyses have shown a negative correlation coefficient in relation to repetitive and explosive power. Also, on the average somewhat younger volunteer firefighters are stronger in performing the repetitive power test (p < 0.05) compared to professional firefighters. Occupational medicine should suggest administrative health measures to improve the accelerated retirement plan and shorten the shifts so that all available firefighters could instantaneously be included in the field intervention.     

Examining psychic consequences in firefighters exposed to stress

The total of 178 firefighters from the Littoral Mountainous County, the Republic of Croatia, have undergone psychotestings in three groups--99 professionals from city Rijeka, 44 professionals from suburb and 35 volunteers from suburb. The scores of PSS 10 Cohen Stress Perception Scale, COPE questionnaire, STAI X1 anxiety test as condition and STAI X2 anxiety test as trait personality and Beck Depression Inventory were analyzed. The results have shown that firefighters do not suffer from negative effects of stress. There is slight anxiety as condition in city firefighters and medium anxiety as trait personality in all firemen. All firefighters are often problem-oriented, need only occasionally compassion of environment and sometimes react by avoiding to face stress. They differ significantly by depression (p < 0.001), which is negligible in relation to ordinary population. "Working anxiety", i.e. medium anxiety which is a distinctive trait of their personality becomes the necessary initiator of functioning in the most difficult working conditions.     

Conserved features of imprinted differentially methylated domains

Genomic imprinting is a conserved epigenetic phenomenon in eutherian mammals, with regards both to the genes that are imprinted and the mechanism underlying the expression of just one of the parental alleles. Epigenetic modifications of alleles of imprinted genes are established during oogenesis and spermatogenesis, and these modifications are then inherited. Differentially methylated domains (DMDs) of imprinted genes are the genomic sites of these inherited epigenetic imprints. We previously showed that CpG-rich imperfect tandem direct repeats within three different mouse DMDs (Snurf/Snrpn, Kcnq1 and Igf2r), each with a unique sequence, play a central role in maintaining the differential methylation. This finding implicates repeat-related DNA structure, not sequence, in the imprinting mechanism. To better define the important features of this signal, we compared sequences of these three DMD tandem repeats among mammalian species. All DMD repeats contain short indirect repeats, many of which are organized into larger unit repeats. Even though the larger repeat units undergo deletion and addition during evolution (most likely through unequal crossovers during meiosis), the size of DMD tandem repeated regions has remained remarkably stable during mammalian evolution. Moreover, all three DMD tandem repeats have a high-CpG content, an ordered arrangement of CpG dinucleotides, and similar predicted secondary structures. These observations suggest that a structural feature or features of these DMD tandem repeats is the conserved DMD imprinting signal.     

Simvastatin and atorvastatin enhance gene expression of collagen type 1 and osteocalcin in primary human osteoblasts and MG-63 cultures

To clarify the mechanism of the stimulatory effect of statins on bone formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, as well as collagen, osteocalcin, and bone morphogenetic protein-2 (BMP2) gene expression in primary human osteoblast (hOB) and MG-63 cell line cultures. Explants of bone from patients without any metabolic disease under orthopedic hip procedures were used to obtain hOB. Cell cultures were established, synchronized, and different concentrations of simvastatin or atorvastatin were added (10(-9) M, 10(-8) M, 10(-7) M, 10(-6) M) during the experiment. Cell proliferation was analyzed after 24 h. Collagen polypeptide alpha1 type 1 (COL1A1) gene expression, osteocalcin, and BMP2 expression levels were quantified by real-time PCR after 24 h incubation with statins. There was a statistically significant decrease in cell proliferation related to simvastatin or atorvastatin addition at all concentrations in primary hOB compared with those not treated. A significant increase in COL1A1, osteocalcin, and BMP2 gene expression was detected when hOB cultures were treated with simvastatin or atorvastatin at different concentrations. Similar but less significant effects were found on MG-63 cells. After statin treatment we observed both an arrest of proliferation in hOB cells and an increase in collagen, osteocalcin, and BMP2 gene expression, consistent with a stimulatory effect towards mature osteoblast differentiation. These findings support the bone-forming effect of statins, probably through the BMP2 pathway.     

General health of elderly institutionalised and community-dwelling Brazilians

Objective: To evaluate the heart rate, blood pressure, blood glucose and other important indicators of the general health of an elderly population of São José dos Campos, Brazil. Materials and methods: A cross‐sectional study was conducted among 150 institutionalised and 150 community‐dwelling individuals aged 60 years or more. The parameters evaluated were heart rate, blood pressure and blood glucose. The elderly also answered a questionnaire about osteoporosis, falls, physical exercising, participation in social events, type of foods ingested, Alzheimer and Parkinson’s diseases and medication. Results: The institutionalised elderly showed an average heart rate of 75.1, while that of the community‐dwelling elderly was 76.7. The percentage of systolic hypertension in the institutionalised and community‐dwelling groups was 36% and 30% respectively, while diastolic hypertension showed a percentage of 40% and 57%, and diabetes was 32% and 30%. Among the institutionalised and community‐dwelling groups, 13.3% and 21.3% respectively, reported osteoporosis, 31.3% and 42.7% falls, 7.3% and 24% exercised regularly, 4% and 69.3% participated regularly in social events, 58.7% and 51.3% reported eating solid foods, and 13.3% and 2%, respectively, suffered from Alzheimer’s disease. Parkinson’s disease was reported by 2% in both groups. The institutionalised elderly reported taking an average of 3.2 medications, while among community‐dwelling elderly this number was 1.8. Conclusion: The entire sample presented a high prevalence of hypertension, diabetes and osteoporosis. No significant differences were found in the systemic health of the elderly institutionalised and community‐dwelling groups. However, the latter group reported more frequent falls, participation in social events and exercised regularly.