Expression profiles of PERIOD1, 2, and 3 in peripheral blood mononuclear cells from older subjects

AimsCircadian clocks regulate daily rhythms of behavior and physiology such as the sleep–wake cycle and hormonal secretion. Numerous characteristics of the behavioral and physiological processes change with age. In this study, we evaluated the circadian clockwork in older people by measuring daily profiles of PERIOD (PER) gene expression in peripheral blood mononuclear cells (PBMCs).Main methodsBlood samples were collected from 6 healthy older subjects (mean age 62 years) at 2-h intervals over a 24-h period under a semi-constant routine condition where masking effects are minimized. PBMCs were isolated from whole blood and temporal mRNA expression profiles of PER1, PER2, and PER3 were determined by RT-PCR. Phases of the PER rhythms, and times of sleep onset and offset were determined using data from those subjects who showed significant 24-h rhythms. The values for the parameters were compared between the older subjects and 8 young control subjects (mean age 21 years).Key findingsProminent daily rhythms of PER1, PER2, and PER3 mRNA levels, advanced sleep–wake timing and advanced phases of PER rhythms were observed in the older subjects compared to the young controls. There was no significant age-related phase difference in PER1 or PER2 rhythm with respect to sleep timing; however, PER3 expression pattern was altered in the older subjects.SignificanceThis preliminary study shows that human circadian clockwork in PBMCs remains intact at least until the presenile stage and suggests that the altered PER3 expression pattern may reflect decreased homeostatic sleep drive in older people.     

Specialization and medical mycology in the US, Britain and Japan

This paper attempts to bring new insights to a long-standing historical debate over medical specialization by analyzing the formation of medical mycology, a somewhat marginal biomedical discipline that emerged in the mid-twentieth century around studies of fungal disease in humans. The study of fungi predates that of bacteria and viruses, but from the 1880s it became eclipsed by bacteriology. However, in the postwar period, there were moves to establish medical mycology as an independent speciality. I trace the processes that led to the launch of professional societies in the United States, Britain and Japan, three major players in medical mycology, and more broadly in biomedicine. The analysis of the three different national contexts illustrates how geographical, medico-technological, epidemiological, political and social conditions gave the specialty a distinctive character in each country; this was further complicated by the different and changing medical fields in which fungal diseases were studied and treated. The three case studies show medical specialization as a process that is not simply cumulative but responds to specific historical events and developments.     

Identification of a novel intracellular interaction domain essential for Bves function

While Blood vessel epicardial substance (Bves) confers adhesive properties, the molecular mechanism of regulating this activity is unknown. No predicted functional motifs in this highly conserved integral membrane protein, other than the transmembrane domain, have been identified. Here, we report for the first time that Bves interacts with itself through an intracellular interaction domain that is essential for its intercellular adhesion activity. Glutathione-S-transferase (GST) pull-down and SPOTs analyses mapped this domain to amino acids 268-274 in the intracellular C-terminus. Site-directed mutagenesis revealed that lysines 272 and 273 are essential for homodimerization and cell adhesion. Human corneal cells transfected with wild-type Bves trafficked the protein to the cell surface, assembled junction complexes and formed epithelial sheets. In contrast, cells expressing Bves mutated at these positions did not form continuous epithelial sheets or maintain junctional proteins such as ZO-1 and E-cadherin at the membrane. A dramatic reduction in transepithelial electrical resistance was also observed indicating a functional loss of tight junctions. Importantly, expression of mutated Bves in epithelial cells promoted the transformation of cells from an epithelial to a mesenchymal phenotype. This study is the first to demonstrate the essential nature of any domain within Bves for maintenance of epithelial phenotype and function.     

Aspergillus nidulans ChiA is a glycosylphosphatidylinositol (GPI)-anchored chitinase specifically localized at polarized growth sites

It is believed that chitinases play important physiological roles in filamentous fungi since chitin is one of the major cell wall components in these organisms. In this paper we investigated a chitinase gene, chiA, of Aspergillus nidulans and found that the gene product of chiA consists of a signal sequence, a region including chitinase consensus motifs, a Ser/Thr/Pro-rich region and a glycosylphosphatidylinositol (GPI)-anchor attachment motif. Phosphatidylinositol-specific phospholipase C treatment of the fusion protein of ChiA and enhanced green fluorescent protein (EGFP)-ChiA-EGFP-caused a change in its hydrophobicity, indicating that ChiA is a GPI-anchored protein. ChiA-EGFP localized at the germ tubes of conidia, at hyphal branching sites and hyphal tips. chiA expression was specifically high during conidia germination and in the marginal growth regions of colonies. These results suggest that ChiA functions as a GPI-anchored chitinase at the sites where cell wall remodeling and/or cell wall maturation actively take place.     

Defense mechanism of the termite, Coptotermes formosanus Shiraki, to entomopathogenic fungi

Termites, Coptotermes formosanus Shiraki, reared individually, were highly susceptible to entomopathogenic fungi, Paecilomyces fumosoroseus and Beauveria brongniartii and Metarhizium anisopliae, while termites reared in groups were highly resistant. Quantitative assays with an epifluoresent microscope revealed a significant difference in the number of conidia attachments among three entomopathogenic fungi. The conidia of B. brongniartii and P. fumosoroseus bound to termite cuticles more effectively than M. anisopliae conidia. Our results also suggested that self-grooming behavior is less effective, but mutual grooming is very effective in the removal of conidia from cuticles of their nestmates. Statistical analysis of removal rates indicated that conidia of P. fumosoroseus and B. brongniartii were removed more rapidly than M. anisopliae conidia from termite cuticles.     

A colorimetric assay for studying effector secretion through the bacterial type III secretion system

We have devised a colorimetric method that monitors secretion of effector proteins into host cytoplasm through the bacterial type III secretion machinery. Here we used constructs of effectors fused with Bordetella adenylate cyclase as a reporter, but evaluated the effector translocation by quantifying cell viability, rather than by measuring the intracellular cAMP concentration. This is based on our findings that cells infected by a secretion-competent bacterium expressing the fusion protein lost their viability under our experimental conditions. Cell death was quantified using commercially available reagents and basic research equipment. An observation that cell death was potentiated when the infected cells were treated with 2-deoxyglucose and sodium azide suggests that the depletion of intracellular ATP is partly involved in the process. Using enteropathogenic Escherichia coli, we demonstrated that the method was applicable to at least three effectors of bacteria, Tir, EspF, and Map, and was useful for studying a secretion signal sequence for Tir. This technically simple and inexpensive method is a good alternative to the existing procedure for studying the mechanism by which effectors are secreted through the type III secretion system in a high-throughput format.     

Synthesis of nuclease-resistant siRNAs possessing benzene-phosphate backbones in their 3'-overhang regions

We describe the synthesis and silencing activities of siRNA possessing N(1)-[3,5-bis(hydroxymethyl)phenyl]thymine (b(t)) in their 3'-overhang regions. We found that an siRNA possessing b(t) in the 3'-overhang region was more effective than an siRNA with natural nucleosides and the siRNA possessing 1,3-bis(hydroxymethyl)benzene (b) without a nucleobase at the 3'-overhang region in in vitro experiment using HeLa cells system. Furthermore, the RNA possessing b(t) at its 3'-end was more resistant to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3'-exonuclease) than the RNA possessing the natural nucleoside 2'-deoxythymidine at the 3'-end. Thus, the compound b(t) will be a novel 3'-overhang moiety that can enhance the silencing activity and nuclease-resistant property of siRNAs.