Functional conservation between fission yeast moc1/sds23 and its two orthologs, budding yeast SDS23 and SDS24, and phenotypic differences in their disruptants

The moc1/sds23 gene was isolated to induce sexual development of a sterile strain due to overexpression of adenylate cyclase in Schizosaccharomyces pombe. Here, we studied the functional conservation between moc1/sds23 and its two orthologs SDS23 and SDS24 in Saccharomyces cerevisiae. We observed that the temperature sensitivity, salt tolerance, cell morphology, and sterility of the Deltamoc1 mutant in S. pombe were recovered by expressing either S. cerevisiae SDS23 or SDS24. We found that deletion of both SDS23 and SDS24 resulted in the production of a large vacuole that was reversed by the expression of S. pombe moc1/sds23. In these ways we found that S. pombe Moc1/Sds23 and S. cerevisiae SDS23p or SDS24p are functional homologs. In addition we found that the Deltasds23 Deltasds24 diploid strain reduces cell separation in forming pseudohyphal-like growth in S. cerevisiae. Thus S. pombe moc1/sds23 and S. cerevisiae SDS23 or SDS24 are interchangeable with each other, but their disruptants are phenotypically dissimilar.     

Mitochondrial ABC transporter Atm1p is required for protection against oxidative stress and vacuolar functions in Schizosaccharomyces pombe

A potential correlation between mitochondrial and vacuolar functions is known to exit in yeast. Fission yeast atm1(+), SPAC15A10.01, encodes a putative half-type ABC transporter with an N-terminal mitochondrial-targeting signal. In an attempt to evaluate the possible involvement of mitochondrion in vacuole function, a functional analysis of atm1(+) was performed by gene disruption. Growth of the atm1 mutant was inhibited in the presence of oxidizing agents, and S. cerevisiae Atm1p was found to complement this growth defect. atm1Delta cells exhibited defects in fluid-phase endocytosis and vacuolar fusion under hypotonic stress. GFP-tagged Atm1p was observed to be localized in the mitochondria. These data strongly suggest that fission yeast Atm1p was not only involved in protection against oxidative stress, but also played a role in vacuolar functions.     

Influence of surface anesthesia on the pressure pain threshold measured with different-sized probes

Transcutaneous pressure with pressure probes of arbitrary diameters have been commonly used for measuring the threshold and magnitude of muscle pain, yet this procedure lacks scientific validation. To examine the valid probe dimensions, we conducted physiological experiments using 34 human subjects. Pin-prick pain, pressure pain threshold (PPT) to pressure probes of various diameters, heat pain threshold, and electrical pain threshold of deep tissues were measured before and after application of surface lidocaine anesthesia to the skin surface over the brachioradial muscle in a double-blinded manner. The anesthesia neither affected PPT with larger probes (diameters: 1.6 and 15 mm) nor increased electric pain threshold of deep structures, whereas it diminished pain count in pin-prick test and PPT with a 1.0 mm diameter probe, suggesting that mechanical pain thresholds measured with 1.6 and 15 mm probes reflect the pain threshold of deep tissues, possibly muscle. Pain thresholds to heat did not change after application of the anesthesia. These results suggest that larger pressure probes can give a better estimation of muscular pain threshold.     

Microtubule-cyclodextrin conjugate: functionalization of motile filament with molecular inclusion ability

A microtubule-beta-cyclodextrin conjugate was prepared on a kinesin-adsorbed glass surface by chemical and biochemical means. Fluorescence microscope observation and a motility assay indicated that the conjugate simultaneously expressed an inherent motor function and an inclusion property.     

XBtg2 is required for notochord differentiation during early Xenopus development

The notochord is essential for normal vertebrate development, serving as both a structural support for the embryo and a signaling source for the patterning of adjacent tissues. Previous studies on the notochord have mostly focused on its formation and function in early organogenesis but gene regulation in the differentiation of notochord cells itself remains poorly defined. In the course of screening for genes expressed in developing notochord, we have isolated Xenopus homolog of Btg2 (XBtg2). The mammalian Btg2 genes, Btg2/PC3/TIS21, have been reported to have multiple functions in the regulation of cell proliferation and differentiation but their roles in early development are still unclear. Here we characterized XBtg2 in early Xenopus laevis embryogenesis with focus on notochord development. Translational inhibition of XBtg2 resulted in a shortened and bent axis phenotype and the abnormal structures in the notochord tissue, which did not undergo vacuolation. The XBtg2-depleted notochord cells expressed early notochord markers such as chordin and Xnot at the early tailbud stage, but failed to express differentiation markers of notochord such as Tor70 and 5-D-4 antigens in the later stages. These results suggest that XBtg2 is required for the differentiation of notochord cells such as the process of vacuolar formation after determination of notochord cell fate.     

Laminin-5 suppresses chondrogenic differentiation of murine teratocarcinoma cell line ATDC5

Laminin-5 is an important basement membrane protein that regulates cell adhesion and motility. It was previously found that the gamma2 chain of laminin-5 is transiently expressed in embryonic cartilage. This suggests a possible role of laminin-5 in chondrogenesis. Here, we examined this possibility using the murine teratocarcinoma cell line ATDC5. ATDC5 cells transiently and weakly expressed laminin-5 when they were stimulated for differentiation. Exogenous laminin-5 in either insoluble or soluble form strongly inhibited the differentiation phenotypes, i.e. formation of cartilaginous cell aggregates and production of chondrogenic marker proteins through its integrin-binding domain LG3 in the alpha3 chain. Laminin-5 had no effect on cell growth. In addition, we found that the laminin-5 with the 105-kDa, processed gamma2 chain suppressed differentiation more strongly than one with the 150-kDa gamma2 chain. This indicated that the proteolytic processing of gamma2 chain regulated the activity of laminin-5. However, a gamma2 chain short arm fragment had no effect on the chondrogenesis, and it rather suppressed the differentiation at excessive concentrations. These results suggest that laminin-5 and its processing modulate chondrogenic differentiation during development.     

Induction of morphological changes in model lipid membranes and the mechanism of membrane disruption by a large scorpion-derived pore-forming peptide

The membrane disruption mechanism of pandinin 1 (pin1), an antimicrobial peptide isolated from the venom of the African scorpion, was studied using 31P, 13C, 1H solid-state and multidimensional solution-state NMR spectroscopy. A high-resolution NMR solution structure of pin1 showed that the two distinct alpha-helical regions move around the central hinge region, which contains Pro19. 31P NMR spectra of lipid membrane in the presence of pin1, at various temperatures, showed that pin1 induces various lipid phase behaviors depending on the acyl chain length and charge of phospholipids. Notably, it was found that pin1 induced formation of the cubic phase in shorter lipid membranes above Tm. Further, the 13C NMR spectra of pin1 labeled at Leu28 under magic angle spinning (MAS) indicated that the motion of pin1 bound to the lipid bilayer was very slow, with a correlation time of the order of 10(-3) s. 31P NMR spectra of dispersions of four saturated phosphatidyl-cholines in the presence of three types of pin1 derivatives, [W4A, W6A, W15A]-pin1, pin1(1-18), and pin1(20-44), at various temperatures demonstrated that all three pin1 derivatives have a reduced ability to trigger the cubic phase. 13C chemical shift values for pin1(1-18) labeled at Val3, Ala10, or Ala11 under static or slow MAS conditions indicate that pin1(1-18) rapidly rotates around the average helical axis, and the helical rods are inclined at approximately 30 degrees to the lipid long axis. 13C chemical shift values for pin1(20-44) labeled at Gly25, Leu28, or Ala31 under static conditions indicate that pin1(20-44) may be isotropically tumbling. 1H MAS chemical shift measurements suggest that pin1 is located at the membrane-water interface approximately parallel to the bilayer surface. Solid-state NMR results correlated well with the observed biological activity of pin1 in red blood cells and bacteria.