Bacterial and Archaeal 16S rRNA Genes in Late Pleistocene to Holocene Muddy Sediments from the Kanto Plain of Japan

Microbial communities in ancient marine sediments composed of clay and silt obtained from the terrestrial subsurface were phylogenetically analyzed based on their 16S rRNA gene sequences. Chloroflexi and Miscellaneous Crenarchaeotic Group were predominant in bacterial and archaeal clone libraries, respectively. Of 44 operational taxonomic units (OTUs) that had close relatives in the database, 30 were close to sequences obtained from marine environments. Some sequences belonged to the candidate groups JS1, ANME-I, and Marine Benthic Group-C, which are typically found in marine sediments. Low chloride concentrations in the sediments suggest that these marine-affiliated sequences may not reflect currently active microbial communities. Our results indicate the existence of long-term preserved DNA or descendants of ancient oceanic microbial components in subsurface muddy sediments in a temperate region, which may reflect indigenous population of paleoenvironments.     

Primary culture and cryopreservation of mouse astrocytes under serumfree conditions

The methods of primary culture and cryopreservation of mouse astrocytes under serum-free conditions were examined. Cerebra from newborn C3H/He mice were employed as the source of astrocytes. The cultured cells were able to grow in a serum-free, chemically defined medium containing transferrin, hydrocortisone, biotin, sodium selenite, insulin, fibroblast growth factor and epidermal growth factor. After the culture was maintained in the medium for 3 weeks, purity was assessed using immunofluorescence staining. The great majority of the cells (>98%) contained glial fibrillary acidic protein and S-100 protein which are cell markers of astrocytes. To cryopreserve the enriched astrocytes under serum-free conditions, various cryoprotectants were examined. The combination of 10% dimethylsulfoxide and 0.1% methylcellulose gave the highest survival rate. These methods of primary culture and cryopreservation will be useful in physiological and biochemical studies which require mouse astrocytes.     

Social stress decreases marking behavior independently of testosterone in Mongolian gerbils

This study examined the endogenous androgen regulation of the marking behavior in Mongolian gerbils (Meriones unguiculatus). In the first experiment, developmental changes of fecal testosterone levels, ventral gland growth, and the marking frequency of male gerbils were investigated. From 9 weeks of age, marking frequency increased with increases in fecal testosterone levels and ventral gland size. The ventral gland size and marking frequency were significantly correlated to the fecal testosterone level. In the second experiment, we hypothesized that reduction in the marking frequency of subordinate males after social confrontations was controlled by a decrease in the circulating testosterone level, and we followed changes in marking frequency, endocrine status, and ventral gland size after social confrontations in which two adult male gerbils established their social ranks by fighting. As expected, marking frequency and ventral gland size were significantly related to social rank, that is, marking frequency was higher among dominant gerbils and lower among subordinates. In addition, fecal corticosterone levels among subordinates were higher than those of dominant animals. However, neither the fecal and plasma testosterone levels, nor testis size, differed between dominant and subordinate gerbils. These results revealed that endogenous androgen played a role in regulating marking behavior and ventral gland size during the developmental stage and that the reductions in marking frequency and ventral gland size occurring in subordinate males after social confrontations were not directly regulated by androgen changes.     

Destabilization of transthyretin by pathogenic mutations in the DE loop

Transthyretin single-amino-acid variants are responsible for familial amyloidotic polyneuropathy, in which transthyretin variants accumulate extracellularly in the form of fibrillar aggregates. We studied the structural stabilities of four transthyretin variants (L58H, L58R, T59K, and E61K), in which a positively charged amino acid is introduced in a loop region between the D- and E-strands. In addition to being located in the DE-loop, L58 and T59 are involved in the core of the transthyretin monomer. The L58H, L58R, and T59K substitutions destabilized transthyretin more than the E61K mutation did, indicating that transthyretin is substantially destabilized by the substitution of residues located in both the DE-loop and the monomer core. By utilizing hydrogen-deuterium exchange and nuclear magnetic resonance, we demonstrated that residues in the G-strand and the loop between the A- and B-strands were destabilized by these pathogenic mutations in the DE loop. At the quaternary structural level, the DE-loop mutations destabilized the dimer-dimer contact area, which may lead to transient dissociation into a dimer. Our results suggest that the destabilization of the dimer-dimer interface and the monomer core is important for the amyloidogenesis of transthyretin.     

TGF-beta isoform signaling regulates secondary transition and mesenchymal-induced endocrine development in the embryonic mouse pancreas

Transforming growth factor-beta (TGF-beta) superfamily signaling has been implicated in many developmental processes, including pancreatic development. Previous studies are conflicting with regard to an exact role for TGF-beta signaling in various aspects of pancreatic organogenesis. Here we have investigated the role of TGF-beta isoform signaling in embryonic pancreas differentiation and lineage selection. The TGF-beta isoform receptors (RI, RII and ALK1) were localized mainly to both the pancreatic epithelium and mesenchyme at early stages of development, but then with increasing age localized to the pancreatic islets and ducts. To determine the specific role of TGF-beta isoforms, we functionally inactivated TGF-beta signaling at different points in the signaling cascade. Disruption of TGF-beta signaling at the receptor level using mice overexpressing the dominant-negative TGF-beta type II receptor showed an increase in endocrine precursors and proliferating endocrine cells, with an abnormal accumulation of endocrine cells around the developing ducts of mid-late stage embryonic pancreas. This pattern suggested that TGF-beta isoform signaling may suppress the origination of secondary transition endocrine cells from the ducts. Secondly, TGF-beta isoform ligand inhibition with neutralizing antibody in pancreatic organ culture also led to an increase in the number of endocrine-positive cells. Thirdly, hybrid mix-and-match in vitro recombinations of transgenic pancreatic mesenchyme and wild-type epithelium also led to increased endocrine cell differentiation, but with different patterns depending on the directionality of the epithelial-mesenchymal signaling. Together these results suggest that TGF-beta signaling is important for restraining the growth and differentiation of pancreatic epithelial cells, particularly away from the endocrine lineage. Inhibition of TGF-beta signaling in the embryonic period may thus allow pancreatic epithelial cells to progress towards the endocrine lineage unchecked, particularly as part of the secondary transition of pancreatic endocrine cell development. TGF-beta RII in the ducts and islets may normally serve to downregulate the production of beta cells from embryonic ducts.     

Diversity of Helicobacter pylori cagA and vacA genes in Costa Rica: its relationship with atrophic gastritis and gastric cancer

BACKGROUND: Associations between Helicobacter pylori gene diversity and gastric cancer have not been reported on in Costa Rica, despite its being one of the countries with the highest gastric cancer incidence and mortality rates in the world. The aim of this study was to determine the prevalence of H. pylori cagA and vacA genes and investigate whether it could be correlated with atrophic gastritis (AG) and gastric cancer (GC) in Costa Rica. MATERIALS AND METHODS: Genomic DNAs from isolates of 104 patients classified into two groups: non-atrophic gastritis group (n = 68) and atrophic gastritis group (n = 36), were subjected to PCR-based genotyping of cagA and vacA genes and their correlation with clinical outcome was investigated. Total DNA extractions from gastric tissues of 25 H. pylori-infected gastric cancer patients were utilized for comparative purposes. RESULTS: The presence of cagA (75.3%), vacA s1b (75.3%), and vacA m1 (74.2%) was detected, and colonization by strains with different vacA genotypes in the same stomach was found in 9.7% of the patients. Age- and sex-adjusted vacA s1b and vacA m1 were associated with GC while only vacA m1 was significantly associated with AG. A tendency for association between cagA and vacA s1b, and AG was reported. CONCLUSIONS: The prevalence status of the cagA and vacA (s1/m1) genes in Costa Rica seems to fall between that found in European/North American and East Asian countries, and both cagA and vacA seem to have clinical relevance in this country.     

Past Japanese successes show the way to accomplish future goals

Many parasitic diseases have been eradicated in industrialized countries and well-proven tools and techniques exist to control them. However, the same diseases still cause incalculable ill health and suffering in the developing world. The difficulty remains how best to apply existing solutions where they are most needed. Within a period of 25 years following World War II, Japan eliminated many parasitic diseases and raised national health and living standards to world-leading levels. Gradually, the predominantly community-driven and intersectoral collaborative partnership systems (i.e. private sector, public sector, general public, etc.) and practices that worked in Japan are being extended to Asia and now Africa. These are backed by the provision of substantial human and financial resources from a nation whose population retains the reputation as being the healthiest and longest living in the world.     

Modulatory role of testosterone in alarm pheromone release by male rats

An alarm pheromone released from stressed conspecifics evokes behavioral and autonomic responses in rats. We have previously reported that male Wistar rats show behavioral changes including increased sniffing, walking and rearing, and decreased resting as well as exaggerated response of body temperature to a novel environment [known as stress-induced hyperthermia (SIH)] when they are exposed to an alarm pheromone released from other male rats receiving foot shocks. The purpose of the present study was to examine the role of testosterone in the production and release of the alarm pheromone using these behavioral and autonomic responses in recipient rats. Three groups of alarm pheromone donors were presented, namely, intact males, castrated males, and testosterone-implanted castrated males. The effects of the alarm pheromone on the autonomic responses did not differ among the three groups, regardless of the donor's steroidal milieu, whereas behavioral responses were altered by castrating the donor males and the effects were restored by testosterone implantation. These results suggest that the alarm pheromone released from stressed male rats can be classified into at least two categories according to the androgen dependency of their production and/or release.     

Unfolding and aggregation of transthyretin by the truncation of 50 N-terminal amino acids

Senile systemic amyloidosis (SSA) is caused by amyloid deposits of wild-type transthyretin in various organs. Amyloid deposits from SSA contain large amounts of the C-terminal fragments starting near amino acid residue 50 as well as full-length transthyretin. Although a number of previous studies suggest the importance of the C-terminal fragments in the pathogenesis of SSA, little is known about the structure and aggregation properties of the C-terminal fragments of transthyretin. To understand the role of C-terminal fragments in SSA, we examined the effects of the truncation of the N-terminal portions on the structure and aggregation properties of wild-type transthyretin. The deletion mutant lacking 50 N-terminal residues was largely unfolded in terms of secondary and tertiary structure, leading to self-assembly into spherical aggregations under nearly physiological conditions. By contrast, the deletion mutant lacking 37 N-terminal residues did not have a strong tendency to aggregate, although it also adopted a largely unfolded conformation. These results suggest that global unfolding of transthyretin by proteolysis near amino acid residue 50 is an important step of self-assembly into aggregations in SSA.