Intracellular calcium responses of circadian pacemaker neurons measured with fura-2

The circadian pacemaker in the eye of the mollusk Bulla gouldiana is located within basal retinal neurons (BRNs) that express a circadian rhythm in cell culture. Light and other depolarizing stimuli shift the phase of the pacemaker in the eye through a process that requires extracellular calcium and is blocked by Ni2+. To test directly if an influx of Ca2+ is present throughout depolarizing treatments that produce phase shifts, dissociated BRNs in cell culture were loaded with a membrane-permeable form of the calcium-sensitive dye fura-2, and then depolarized with elevated levels of extracellular K+. Calcium levels in the BRNs remained elevated during treatments with 50 mM K+ lasting 1 h, a sufficient duration to phase shift the circadian pacemaker. Lowering extracellular free Ca2+ (approx. 1.7 x 10(-7) M) during depolarization blocked the rise in intracellular Ca2+, verifying that a Ca2+ influx is required. The sustained Ca2+ elevation during depolarization was also prevented with 50 mM Ni2+, which blocks phase shifts of the rhythm to depolarization, but not with 5 mM Ni2+, which does not block phase shifts. The initial rise in [Ca2+]i in response to 50 mM K+ was largest on average during the subjective night. The results show that a critical portion of the entrainment pathway persists in pacemaker neurons during cell culture, and that the phase-shifting stimulus may depend on a prolonged Ca2+ signal.     

Cloning and sequencing of a cDNA encoding bovine intercellular adhesion molecule 3 (ICAM-3)

A cDNA encoding a putative bovine intercellular adhesion molecule (ICAM)-3, a ligand of the leukocyte integrin LFA-1 (CD11a/CD18), was sequenced and compared with human ICAM sequences. The 1635-bp bovine sequence codes for a protein of 544 amino acids (aa). This putative bovine ICAM-3 has five immunoglobulin (Ig)-like domains similar to human ICAM-1 and ICAM-3, and belongs to the Ig gene superfamily. The overall identities of the deduced aa sequence with those of human ICAM-3 and ICAM-1 are 61% and 58%, respectively. The predicted number and positions of Cys residues are all conserved between the bovine and human ICAM 3 aa sequences.     

Contribution of phosphocreatine and aerobic metabolism to energy supply during repeated sprint exercise

This study examined the contribution of phosphocreatine (PCr) and aerobic metabolism during repeated bouts of sprint exercise. Eight male subjects performed two cycle ergometer sprints separated by 4 min of recovery during two separate main trials. Sprint 1 lasted 30 s during both main trials, whereas sprint 2 lasted either 10 or 30 s. Muscle biopsies were obtained at rest, immediately after the first 30-s sprint, after 3.8 min of recovery, and after the second 10- and 30-s sprints. At the end of sprint 1, PCr was 16.9 +/- 1.4% of the resting value, and muscle pH dropped to 6.69 +/- 0.02. After 3.8 min of recovery, muscle pH remained unchanged (6.80 +/- 0.03), but PCr was resynthesized to 78.7 +/- 3.3% of the resting value. PCr during sprint 2 was almost completely utilized in the first 10 s and remained unchanged thereafter. High correlations were found between the percentage of PCr resynthesis and the percentage recovery of power output and pedaling speed during the initial 10 s of sprint 2 (r = 0.84, P < 0.05 and r = 0.91, P < 0.01). The anaerobic ATP turnover, as calculated from changes in ATP, PCr, and lactate, was 235 +/- 9 mmol/kg dry muscle during the first sprint but was decreased to 139 +/- 7 mmol/kg dry muscle during the second 30-s sprint, mainly as a result of a approximately 45% decrease in glycolysis. Despite this approximately 41% reduction in anaerobic energy, the total work done during the second 30-s sprint was reduced by only approximately 18%. This mismatch between anaerobic energy release and power output during sprint 2 was partly compensated for by an increased contribution of aerobic metabolism, as calculated from the increase in oxygen uptake during sprint 2 (2.68 +/- 0.10 vs. 3.17 +/- 0.13 l/min; sprint 1 vs. sprint 2; P < 0.01). These data suggest that aerobic metabolism provides a significant part (approximately 49%) of the energy during the second sprint, whereas PCr availability is important for high power output during the initial 10 s.     

Abnormalities in the p34cdc2-related PITSLRE protein kinase gene complex (CDC2L) on chromosome band 1p36 in melanoma

Recently, several clusters of hepatitis A have been observed among hemophiliacs linked to factor VIII concentrates treated for virus inactivation solely with the solvent/detergent (S/D) method, a procedure that does not affect nonenveloped viruses such as the hepatitis A virus (HAV). A new outbreak of hepatitis A in six hemophiliacs treated with the same lot of a factor VIII preparation occurred recently in Germany. The objective of the study was to clarify whether these diseases were caused by the administration of the S/D-treated plasma product, rather than a community-acquired infection. Polymerase chain reactions designed to detect HAV nucleic acid have been carried out in the implicated factor VIII lots, in the corresponding plasma pools, and in serum samples of four out of six infected individuals. The nucleic acid sequences were determined in samples that resulted in positive amplification products. HAV sequences were found in one of the two plasma pools used for manufacture of the incriminated product, in the incriminated lot itself, and in all recipient sera tested so far, although the latter were collected up to 7 weeks after the onset of jaundice. The sequences obtained were completely identical, revealing a unique HAV strain of genotype IA. This study provides conclusive evidence that hepatitis A can be transmitted by factor VIII concentrates treated solely by the S/D procedure for virus inactivation. This inactivation method is not effective against nonenveloped viruses. Since a number of hepatitis A transmission episodes have been described with such preparations during the past 10 years, their continued use seems to be questionable unless additional virus removal or inactivation steps are introduced to prevent the transmission of nonenveloped viruses. Molecular approaches again proved to be reliable tools for elucidating the chain of virus transmission.     

Crucial role of FcgammaRIIa (CD32) in assessment of functional anti-Streptococcus pneumoniae antibody activity in human sera

Immunoglobulin G (IgG)-mediated phagocytosis by polymorphonuclear leukocytes (PMNL) constitutes the main defense against Streptococcus pneumoniae. Two leukocyte IgG receptors, FcgammaRIIa and FcgammaRIIIb, are constitutively expressed on PMNL. Blocking experiments showed FcgammaRIIa is crucial for opsonophagocytosis of serum-opsonized S. pneumoniae. The biallelic, genetically determined FcgammaRIIa polymorphism (FcgammaRIIa-R131 vs. IIa-H131) determines the capacity of IgG2-mediated phagocytosis via this receptor. Comparative studies with PMNL from donors either homozygous for FcgammaRIIa-R131 or IIa-H131 showed the latter had higher phagocytic capacity. These results were confirmed in FcgammaRIIa-R131- and FcgammaRIIa-H131-transfected IIA1.6 cells. The performance of FcgammaRIIa-transfected cells in S. pneumoniae phagocytosis was validated using sera from adults and children. Serum-induced phagocytic activity depended mainly on anti-pneumococcal IgG2 antibodies. Results obtained with PMNL and IIA1.6 cells showed high correlation (r=0.94; P<.001), and support that FcgammaRIIa transfectants are a good alternative to PMNL as effector cells in opsonophagocytosis assays.     

Histochemical study of magellanic penguin (Spheniscus magellanicus) minor salivary glands during postnatal growth

The histological and histochemical features of the minor salivary glands during postnatal development have been generally associated with the type of food ingested. However, recent studies support the fact that these salivary glands develop independently of the diet; in fact, minor salivary glands have similar morphological and histochemical characteristics in adult individuals of species with different diet regimens. Thus, the aim of this study was to characterize the developmental morphology of the penguin minor salivary glands and to contrast them with minor salivary glands of other species. The tongue, palatine, and mouth cavity (bottom) minor salivary glands of newborn, 1- to 20-day-old, and adult magellanic penguins were studied with hematoxylin-eosin, periodic acid-Schiff, alcian blue, toluidine blue, and lectin histochemistry. Minor salivary glands were present at all ages, although they were only moderately developed in animals less than 15 days old. After this age, glands were abundant in all age groups; in addition, cells from the glandular epithelium were functionally mature and secreted mucins. Nevertheless, in newborn to 15-day-old penguins, mucins were located only at the apical cytoplasm of mucous cells. In all ages, mucous cells displayed periodic acid-Schiff-positive, alcianophilic, and metachromatic reactions; among mucous cells, other orthochromatic cells appeared interspersed. From 15 days on, histochemical reactions became more intense until adulthood, and the cytoplasm of secretory cells was filled with glycoproteins and sulfomucins. Moreover, lectins bound to different oligosaccharides in mucous cells, depending on the stage of maturation of the glands. In conclusion, penguin minor salivary glands are already present at birth, and show progressive and quantitative increases in mucous secretion during postnatal development. These changes are necessary not only for nutrient ingestion, but also for nonimmune protection of the buccal cavity.     

Can retrograde cardioplegia alone provide adequate protection for cardiac valve surgery?

Pretreatment of rats with the extract of Ginkgo biloba termed EGb761 reduced the behavioral sensitization induced by successive D-amphetamine administrations (0.5 mg/kg) as estimated by increasing values of locomotor activity. EGb761 pretreatment also prevented the reduced density of [3H]dexamethasone binding sites in the dentate gyrus and the CA1 hippocampal regions of D-amphetamine treated animals. These observations suggest that EGb761, by reducing glucocorticoid levels, could modulate the activity of the neuronal systems involved in the expression of the behavioral sensitization.