Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [γ-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.     

ANTHROPOMETRIC DETERMINANTS OF ROWING ERGOMETER PERFORMANCE IN PHYSICALLY INACTIVE COLLEGIATE FEMALES

The aim of the study was to evaluate anthropometric characteristics as determinants of 500 m rowing ergometer performance in physically inactive collegiate females. In this cross-sectional study, which included 196 collegiate females aged 19-23 years not participating in regular physical activities, body mass (BM), body height (BH), length of upper limbs (LA), length of lower limbs (LL), body mass index (BMI), slenderness index (SI), and the Choszcz-Podstawski index (CPI) were measured and a stepwise multiple regression analysis was performed. Participants performed 500 m maximal effort on a Concept II rowing ergometer. BM, BH, LA, LL, and the BMI, SI and CPI indices were found to be statistically significant determinants of 500 m performance. The best results (T) were achieved by females whose BH ranged from 170 to 180 cm, with LA and LL ranging from 75 to 80 cm and 85 to 90 cm, respectively. The best fitting statistical model was identified as: T = 11.6793 L_R – 0.1130 L_R² – 0.0589 L_N² + 29.2157 CPI² + 0.1370 LR·LN - 2.6926 LR·CPI – 211.7796. This study supports a need for additional studies focusing on understanding the importance of anthropometric differences in rowing ergometer performance, which could lead to establishing a better quality reference for evaluation of cardiorespiratory fitness tested using a rowing ergometer in collegiate females.     

The Emerging Role of Copeptin

Direct measurement of the nonapeptide vasopressin has been limited by analyte instability ex vivo and in vivo rapid degradation, low serum concentrations requiring a sensitive assay and inherent secretory pulsatility. Copeptin is a 39 amino acid glycopeptide cleavage product of vasopressin synthesis with high stability, providing a marker of vasopressin secretion. Copeptin measurement has applications in diagnosis of diabetes insipidus and other diseases with altered vasopressin secretion. This review summarises our current understanding of serum copeptin measurement in diabetes insipidus and possible future applications of copeptin assays. As vasopressin is a stress hormone, there is emerging evidence on the use of copeptin for diagnosis and prognostication of disorders such as syndrome of inappropriate anti-diuretic hormone secretion, diabetes mellitus, critical illness, stroke, cardiovascular disease, respiratory disease, renal disease and thermal stress. Copeptin concentration measurement is likely to improve the diagnostic reliability of diabetes insipidus and, as a marker of stress, may have diagnostic or prognostic utility in specific clinical circumstances. Further studies are needed to determine if goal-directed therapy using plasma copeptin concentrations may improve patient outcomes.     

Isolation and analysis of particulate detritus as an aid to studies on the nutritional ecology of deposit feeders

By submitting detrituss.l., consisting of the fractions particulate detrituss.s., mineral particles, mesofaunal elements, cyanobacterial colonies and other microalgae such as diatoms, to density gradient centrifugation (DGC) in Percoll and Ludox-TM the major part of detrituss.s. could be separated. Qualitative analysis of the fractions proved that the organic carbon content and (to a small extent) the nitrogen content of the fractions distinguished after DGC were higher than those of the detrituss.l. before DGC, probably due to contamination with the gradient material. These results as well as the problems of quantifying the natural food of deposit feeders are discussed.     

Application of phage display to high throughput antibody generation and characterization

We have created a high quality phage display library containing over 10¹⁰ human antibodies and describe its use in the generation of antibodies on an unprecedented scale. We have selected, screened and sequenced over 38,000 recombinant antibodies to 292 antigens, yielding over 7,200 unique clones. 4,400 antibodies were characterized by specificity testing and detailed sequence analysis and the data/clones are available online. Sensitive detection was demonstrated in a bead based flow cytometry assay. Furthermore, positive staining by immunohistochemistry on tissue microarrays was found for 37% (143/381) of antibodies. Thus, we have demonstrated the potential of and illuminated the issues associated with genome-wide monoclonal antibody generation.     

Changes in the surface charge properties of isolated cardiac sarcolemmal vesicles measured by light scattering. II. Characteristics of rabbit preparations

Numerous studies suggest that cation-sarcolemmal interactions play an essential role in the excitation/contraction/relaxation cycles of cardiac muscle cells. To help elucidate the molecular mechanisms involved in these processes the cation binding characteristics of isolated rabbit cardiac sarcolemmal vesicles were investigated. Cation-membrane interactions were studied by examining the cation-induced aggregation properties of the vesicles. The results obtained were qualitatively very similar to those previously reported for rat and canine cardiac sarcolemmal vesicle preparations (Leonards, K.S. (1988) Biochim. Biophys. Acta 938, 293-309), indicating that all three species have a shared set of basic membrane characteristics. Specifically the results indicate that cations, such as Ca2+, bind to the sarcolemmal surface, and suggest that two (or more) interacting sites are involved in the process. The selectivity series for the cation-induced aggregation of the sarcolemmal vesicles was: La3+ greater than or equal to Cd2+ much greater than Mn2+ greater than Ca2+ greater than Ba2+ = Sr2+ = Mg2+. Protons (H+) could also induce massive vesicle aggregation at pH 5.60-5.75. However, the results obtained also show that the interactions of cations with the rabbit cardiac sarcolemmal membrane surface are quantitatively distinct from those obtained in either rat or canine sarcolemmal vesicle preparations, thereby confirming the species specific nature of cation-sarcolemmal interactions in cardiac cells.