The ischemic rat heart releases S100B

S100B is an astrocytic protein assessed in cerebrospinal fluid and serum as a biochemical marker of cerebral injuries. However, increasing evidences suggest the influence of extra cerebral sources on its serum levels. Since it was reported that the injured myocardium expresses S100B, we investigated whether the isolated heart releases this protein. The rat hearts were excised and perfused by the Langendorff technique of isolated heart perfusion. After stabilization, 10 hearts (ischemic group) were submitted to 20 minutes of ischemia and 30 minutes of reperfusion, and 5 hearts (control group) were submitted to 50 minutes of perfusion. The perfusion fluid was collected at pre-ischemia, and 0, 5, 10, 15 and 30 min after ischemia (or equivalent in controls) for S100B and cardiac troponin T (a heart injury marker) assays. In the ischemic group, S100B and troponin T levels increased significantly at time 0 min: S100B values [mug/L, median (IQ25/IQ75)] increased from < or = 0.02 (< or = 0.02/0.03) to 0.38 (0.22/0.84), while troponin T values [mug/L, median (IQ25/IQ75)] increased from 0.31 (0.15/0.45) to 2.84 (2.00/3.63). Our results point to the ischemic heart as an extra cerebral source of S100B.     

Glucose-dependent activation of protein kinase A activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic subunit

Protein kinase A (PKA), in yeast, plays a major role in controlling metabolism and gene expression in connection with the available nutrient conditions. We here measure, for the first time, a transient change in the in vivo PKA activity, along a cAMP peak produced by 100 mM glucose addition to glycerol-growing cells as well as a change in the phosphorylation state of its catalytic subunit (Tpk1p) following PKA activation. PKA activity was measured in situ in permeabilized cells, preserving its intracellular localization. Comparison of total PKA activity, measured in situ in permeabilized cells with data obtained from in vitro assays in crude extracts, underscores the inhibitory potency of the regulatory subunit within the cell. Tpk1p phosphorylation was detected through non-denaturing gel electrophoresis. Phosphorylation of Tpk1p increases its specificity constant toward kemptide substrate. The use of mutants of the cAMP pathway showed that phosphorylation depends on the activation of PKA via the G-protein coupled receptor pathway triggered by glucose. The phosphorylation state of Tpk1p was followed during the diauxic shift. Tpk1p phosphorylation is dynamic and reversible: its up-regulation correlates with a fully fermentative metabolism, while its down-regulation with stationary phase or respiratory metabolism. Reversible phosphorylation can thus be considered a new control mechanism possibly pointing to a fine-tuning of PKA activity in response to environmental conditions.     

Influence of maternal dietary calcium levels on milk zinc, calcium and phosphorus contents and milk production in rats

The aim of this study was to analyze zinc (Zn), calcium (Ca) and phosphorus (P) contents in milk and the lactational performance in rats fed different Ca levels. Female Wistar rats were fed during pregnancy and lactation with experimental diets containing 20% protein and high (0.90%, HCa), normal (0.60%, NCa) or low (0.20%, LCa) Ca levels. Milk samples were collected after 15 days to determine the milk mineral composition. Pup weight was recorded from birth to weaning (litter size: 6-8 pups) to determine weight gain and calculate milk production. At delivery there were no significant differences in the body weight of the pups between the groups, but at day 15, the LCa group showed lower values than both NCa and HCa groups (p<0.05). The weight gain of the LCa group was significantly lower than of the HCa and NCa groups, between delivery and day 5 (p<0.05). This reduced rate of weight gain led to the LCa group reaching weaning weight later than the other groups. Milk production (g/pup/day) was significantly lower when dams were fed the LCa than the NCa and HCa diets (p<0.05). There were no significant differences among the groups in milk Ca, P and Zn levels and Ca/P ratio. The body mineral composition of the pups at birth did not differ between the groups; at weaning, however, both LCa and HCa groups had lower element contents than the NCa group (p<0.05). In conclusion, dams fed with a diet containing low Ca levels produced smaller volumes of milk and their pups reached weaning weights later than the other groups. As the milk mineral composition was not affected, it can be hypothesized that in dams fed low dietary Ca, the smaller milk yield might have been a way of maintaining milk quality. High Ca levels affected neither pregnancy outcome nor lactational performance.     

Serine/threonine protein kinase PrkA of the human pathogen Listeria monocytogenes: biochemical characterization and identification of interacting partners through proteomic approaches

Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thr-phosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His(6)-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes.     

Long-term cyclosporine treatment in non-transplanted rats and metabolic risk factors of vascular diseases

Cyclosporine (CsA) is an immunosuppressive agent frequently used in the clinic for prevention of allograft rejection and for the treatment of autoimmune diseases. Despite its desired action on the immune system, CsA treatment may present serious adverse effects, which are masked by the concomitant use of other drugs. The search for effective immunosuppression protocols which does not affect the quality of life of patients is driving research to investigate the CsA involvement in vascular diseases, frequent in patients under immunosuppression. Thus, 45 non-transplanted Wistar rats were treated for 8 weeks with vehicle or 5 or 15 mg/kg CsA (n = 15/group) by gavage administration to evaluate the specific influence of cyclosporine on the levels of risk factors (metabolic and inflammatory) of vascular disease and its mechanism of action. Therefore, serum insulin levels, glucose tolerance test, serum lipids profile, total homocysteine and fibrinogen levels were assessed. The biochemical alterations reported here suggest the development of a framework straight to diabetes. Glucose homeostasis was affected as indicated by decreased insulin levels and altered glucose tolerance test in CsA 15 mg/kg group compared to other groups. Serum insulin and total homocysteine levels presented a significant negative correlation (R = ? 0.76, P < 0.0001). Fibrinogen and serum lipids profiles were significantly increased in CsA 15 mg/kg group compared to other groups and correlated positively with total homocysteine levels. Considering the well-established correlation among insulin resistance, lipid and total homocysteine levels, hypercoagulability and atherosclerosis, we can assume that this protocol of long-term CsA treatment in non-transplanted rats alter biochemical parameters related to cardiovascular and cerebrovascular risk, mainly in CsA 15 mg/kg group. Insulin and tHcy serum levels appear to be central in this process.     

Reproductive biology of Patagonotothen ramsayi (Regan, 1913) (Pisces: Nototheniidae) around the Falkland Islands

The reproductive biology of one of the most abundant notothenioids, Patagonotothen ramsayi, was investigated between February 2003 and November 2004 on the Falkland and Patagonian Shelves (Southwest Atlantic). Male and female P. ramsayi were mature at 27.6 and 24.8 cm L _T, respectively. P. ramsayi is a total spawner, with a total fecundity ranging between 24,300 and 76,700 eggs. Spawning occurs on the shelf breaks between June and August with the peak in gonado-somatic indices in June. Analysis of length frequency distributions over the year and sex ratios of mature fish during the spawning season may indicate the presence of a nesting and nest guarding behaviour in male fish, similar to other rockcods. Features of its reproductive strategy, which enabled P. ramsayi to dominate the medium-sized demersal fishes on the Patagonian shelf are discussed and compared with those of other nototheniids and cottid sculpins from the Northern Hemisphere.