Diurnal, circadian and photic regulation of calcium/calmodulin-dependent kinase II and neuronal nitric oxide synthase in the hamster suprachiasmatic nuclei

Mammalian circadian rhythms are entrained by light pulses that induce phosphorylation events in the suprachiasmatic nuclei (SCN). Ca²⁺-dependent enzymes are known to be involved in circadian phase shifting. In this paper, we show that calcium/calmodulin-dependent kinase II (CaMKII) is rhythmically phosphorylated in the SCN both under entrained and free-running (constant dark) conditions while neuronal nitric oxide synthase (nNOS) is rhythmically phosphorylated in the SCN only under entrained conditions. Both p-CaMKII and p-NOS (specifically phosphorylated by CaMKII) levels peak during the day or subjective day. Light pulses administered during the subjective night, but not during the day, induced rapid phosphorylation of both enzymes. Moreover, we found an inhibitory effect of KN-62 and KN-93, both CaMKII inhibitors, on light-induced nNOS activity and nNOS phosphorylation respectively, suggesting a direct pathway between both enzymes which is at least partially responsible of photic circadian entrainment.     

Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.     

Microlaparoscopy in sex reassignment surgery

Sex reassignment (male to female surgery) is a standard operation which is aimed at constructing female genitalia and obtaining a cosmetic and functional result that is similar to that of a normal female subject. The ideal surgical procedure has not yet been described, but the various techniques which have been proposed in the literature are similar. The most cumbersome maneuver of the procedure is that of creating a neovaginal cavity inside the perineum. This step is generally carried out by means of blunt dissection between the rectal wall and the prostate, but most of the surgery is blindly performed without visual control. In these conditions, the risk of rectal injury is high, and may lead to severe intraoperative complications. Microlaparoscopy allows for a direct observation of the perineal dissection from inside the peritoneal cavity, thus avoiding risk of rectal injury. The technique is simple to perform, is non-invasive, and only 15 minutes are added to the operation.     

Angiogenesis and nerve regeneration in a model of human skin equivalent transplant

The angiogenesis and reinnervation were studied in a porcine model of human skin equivalent (SE) graft and the relationship between the two processes was investigated. Confocal laser scanning microscopy was used to monitor, during the healing process, the pattern of vascularization and reinnervation at different time points. The SE was obtained by co-culturing fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan biopolymer and grafted on dorsal wounds generated by full-thickness resection in 25/30 Kg Large white pigs. Frozen sections were obtained from biopsies performed in autograft and xenograft, then were immunolabeled by using the endothelial marker lectin Lactifolia and with the neuronal marker gene product PGP9.5. Cajal staining was also used to visualize the nerve fibers. The results show that the vascularization precedes the innervation process. These data are consistent with the view that the development of nervous tissue is driven by nutritional and trophic factors provided by the vascular system. The arborization of the two systems observed during the third week from the graft might play a key role in maintaining the healing process and the graft survival.     

Crystal structure and anion binding in the prokaryotic hydrogenase maturation factor HypF acylphosphatase-like domain

[NiFe]-hydrogenases require a set of complementary and regulatory proteins for correct folding and maturation processes. One of the essential regulatory proteins, HypF (82kDa) contains a N-terminal acylphosphatase (ACT)-like domain, a sequence motif shared with enzymes catalyzing O-carbamoylation, and two zinc finger motifs similar to those found in the DnaJ chaperone. The HypF acylphosphatase domain is thought to support the conversion of carbamoylphosphate into CO and CN(-), promoting coordination of these ligands to the hydrogenase metal cluster. It has been shown recently that the HypF N-terminal domain can aggregate in vitro to yield fibrils matching those formed by proteins linked to amyloid diseases. The 1.27A resolution HypF acylphosphatase domain crystal structure (residues 1-91; R-factor 13.1%) shows a domain fold of betaalphabetabetaalphabeta topology, as observed in mammalian acylphosphatases specifically catalyzing the hydrolysis of the carboxyl-phosphate bonds in acylphosphates. The HypF N-terminal domain can be assigned to the ferredoxin structural superfamily, to which RNA-binding domains of small nuclear ribonucleoproteins and some metallochaperone proteins belong. Additionally, the HypF N-terminal domain displays an intriguing structural relationship to the recently discovered ACT domains. The structures of different HypF acylphosphatase domain complexes show a phosphate binding cradle comparable to the P-loop observed in unrelated phosphatase families. On the basis of the catalytic mechanism proposed for acylphosphatases, whereby residues Arg23 and Asn41 would support substrate orientation and the nucleophilic attack of a water molecule on the phosphate group, fine structural features of the HypF N-terminal domain putative active site region may account for the lack of acylphosphatase activity observed for the expressed domain. The crystallographic analyses here reported were undertaken to shed light on the molecular bases of inactivity, folding, misfolding and aggregation of the HypF N-terminal acylphosphatase domain.     

Pancreatic hepatocytes in hamsters (Mesocricetus auratus) infected with Trypanosoma cruzi

Hepatocytic metaplasia may be induced in hamsters by carcinogens, and associated with aging, diabetes or chronic pancreatitis. By means of histopathologic and immunohistochemic studies, we observed pancreatic hepatocytes in hamsters infected and reinfected with Trypanosoma cruzi. The change was seen in 18 (19%) out of 94 infected animals, and was not found among 53 controls, Normal islet cells were immunoreactive for neuron-specific enolase and not reactive for NCL-HAS. Metaplastic cells were immunoreactive for NCL-HAS and not reactive for islet hormones and enolase. No relationship was observed between number of inoculations and metaplasia; however, the intensity of the inflammatory process and sequels seems to favor the development of metaplastic cells. Hamsters infected with T. cruzi may be useful to study hepatocytic metaplasia, and contribute to clarify aspects of Chagas' disease and pancreatic changes. Our data indicate that aging, in addition to inflammation and atrophy, plays a role in this change.     

The MAPK pathway triggers activation of Nek2 during chromosome condensation in mouse spermatocytes

Chromosome condensation during the G2/M progression of mouse pachytene spermatocytes induced by the phosphatase inhibitor okadaic acid (OA) requires the activation of the MAPK Erk1. In many cell systems, p90Rsks are the main effectors of Erk1/2 function. We have identified p90Rsk2 as the isoform that is specifically expressed in mouse spermatocytes and have shown that it is activated during the OA-triggered meiotic G2/M progression. By using the MEK inhibitor U0126, we have demonstrated that activation of p90Rsk2 during meiotic progression requires activation of the MAPK pathway. Immunofluorescence analysis indicates that activated Erks and p90Rsk2 are tightly associated with condensed chromosomes during the G2/M transition in meiotic cells. We also found that active p90Rsk2 was able to phosphorylate histone H3 at Ser10 in vitro, but that the activation of the Erk1/p90Rsk2 pathway was not necessary for phosphorylation of H3 in vivo. Furthermore, phosphorylation of H3 was not sufficient to cause condensation of meiotic chromosomes in mouse spermatocytes. Other proteins known to associate with chromatin may represent effectors of Erk1 and p90Rsk2 during chromosome condensation. Nek2 (NIMA-related kinase 2), which associates with chromosomes, plays an active role in chromatin condensation and is stimulated by treatment of pachytene spermatocytes with okadaic acid. We show that inhibition of the MAPK pathway by preincubation of spermatocytes with U0126 suppresses Nek2 activation, and that incubation of spermatocyte cell extracts with activated p90Rsk2 causes stimulation of Nek2 kinase activity. Furthermore, we show that the Nek2 kinase domain is a substrate for p90Rsk2 phosphorylation in vitro. These data establish a connection between the Erk1/p90Rsk2 pathway, Nek2 activation and chromosome condensation during the G2/M transition of the first meiotic prophase.     

Magnetic resonance microscopy for the quantitative analysis of trabecular bone architecture

A major concern for long-term spaceflight is the effect of microgravity on bone structure and mass as a loss of cortical and trabecular bone volume and density, both of which can lead to decreased bone strength and an increased risk of bone fracture. Detailed analysis of the three-dimensional structure of trabecular bone, and its relation to bone strength has become feasible only recently using high-resolution 3D imaging techniques. In particular, magnetic resonance microscopy (MRM) has proved to be particularly useful for the ex vivo evaluation of the complex architecture of trabecular bone. In this study, we describe the use of two different MRM-based methods for the quantitative evaluation of the three-dimensional structure of trabecular bone explants and for the prediction of their biomechanical properties. The in vivo application of such methods is also discussed.     

Conservation of the mosaic structure of the four internal transcribed spacers and localisation of the rrn operons on the Streptococcus pneumoniae genome

The detection of heterogeneity of the 16S-23S ribosomal intergenic transcribed spacer (ITS) region has become rather common over the past years for identification and typing purposes of bacteria. The ITS not only varies in sequence and length, but also in number of alleles per genome and in their position on the chromosome together with the ribosomal clusters. The ITS characterisation has allowed discrimination of several species within a genus and variation in ITS sequences between the multiple rrn operons present within a genome may be as high or greater than between strains of the same species or subspecies. It is important to understand the variability of ITS sequences in a given genome to gain insights into bacterial physiology and taxonomy. The present study describes the possibility to type Streptococcus pneumoniae by PCR-ribotyping of the spacer region, the determination of the molecular structure of the ITS, and the determination of the number and localisation of rrn operons in this microorganism. Our results show that the genome of S. pneumoniae contains four ribosomal operons, showing the same genomic organisation among strains, each containing a single ITS allele of 270 bp. The ITS sequence presents a mosaic organisation of blocks highly conserved intra- and inter-species within the genus Streptococcus, giving no possibility for variations to arise.