Polarized morphogenesis regulator Spa2 is required for the function of putative stretch-activated Ca2+-permeable channel component Mid1 in Saccharomyces cerevisiae

Mid1 is a putative stretch-activated Ca2+ channel component and is required for the maintenance of viability in the mating process. In response to mating pheromone, the mid1 mutant normally forms a pointed mating projection but eventually dies. This phenotype is called the mid phenotype. To identify a protein regulating Mid1 or regulated by Mid1, we isolated a multicopy suppressor that rescues the mid1-1 mutant from mating pheromone-induced death and found that it encodes a truncated Spa2 protein lacking an amino-terminal region responsible for interaction with components of the mitogen-activated protein kinase cascades. One of these SPA2 alleles was SPA2DeltaN, whose product lacked the region from Ser5 to Leu230. SPA2DeltaN on a multicopy plasmid (YEpSPA2DeltaN) complemented the mid phenotype but not another phenotype, low Ca2+ accumulation, of the mid1-1 mutant. Neither SPA2DeltaN on a low-copy plasmid nor wild-type SPA2 on a multicopy plasmid had suppressive activity. The SPA2 gene is involved in the formation of a pointed mating projection, and cells of the spa2Delta mutant lacking Spa2 are viable and develop a peanut shell-like structure when exposed to mating pheromone. Like the spa2Delta mutant, the mid1-1 spa2Delta double mutant and the mid1-1/YEpSPA2DeltaN strain developed the peanut shell-like structure. The mid1-1 spa2Delta double mutant did not have the mid phenotype, indicating that SPA2 is epistatic to MID1. Overexpression of Spa2DeltaN abolished the localization of Spa2-green fluorescent protein to the tip of the mating projection. These results suggest that the Spa2DeltaN protein interferes with the localization of the normal Spa2 protein and thereby prevents cells from entering the mating process. Therefore, we suggest that Mid1 function is influenced by Spa2 function through polarized morphogenesis.     

Flow-through anterior thigh flaps with a short pedicle for reconstruction of lower leg and foot defects

New flow-through perforator flaps with a large, short vascular pedicle are proposed because of their clinical significance and a high success rate for reconstruction of the lower legs. Of 13 consecutive cases, the authors describe two cases of successful transfer of a new short-pedicle anterolateral or anteromedial thigh flow-through flap for coverage of soft-tissue defects in the legs. This new flap has a thin fatty layer and a small fascial component, and is vascularized with a perforator originating from a short segment of the descending branch of the lateral circumflex femoral system. The advantages of this flap are as follows: flow-through anastomosis ensures a high success rate for free flaps and preserves the recipient arterial flow; there is no need for dissecting throughout the lateral circumflex femoral system as the pedicle vessel; minimal time is required for flap elevation; there is minimal donor-site morbidity; and the flap is obtained from a thin portion of the thigh. Even in obese patients, thinning of the flap with primary defatting is possible, and the donor scar is concealed. This flap is suitable for coverage of defects in legs where a single arterial flow remains. It is also suitable for chronic lower leg ulcers, osteomyelitis, and plantar coverage.     

Biological half time of deposited nickel oxide aerosol in rat lung by inhalation

Wistar male rats were exposed to nickel oxide (NiO) aerosols (mass median aerodynamic diameter, 1.2 and 4.0 μm). The average exposure concentration was controlled from a low level of 0.6 mg/m³ to a high level of 70 mg/m³ and total exposure time was 140 h. Some rats were sacrificed just after the exposure, whereas others were exposed for 1 mo and kept for 12 and 20 mo clearance periods before sacrifice. There were no differences in body weight gain between NiO exposure groups and controls. Nickel concentrations in lungs of exposure groups were much higher than those of controls and decreased with the passing of the clearance time. No apparent deposition of nickel was observed in the liver, kidney, spleen, and blood immediately after the exposure, but in the case of the high exposure groups, the nickel concentration in the liver, spleen, and blood slightly increased with the increasing time of clearance. The biological half time of NiO deposited in the lungs was estimated by the assumption that the amount of the clearance is proportional to the amount of the NiO deposited. This resulted in a biological half time of 11.5 and 21 mo for 1.2 and 4.0 μm, respectively.     

Genetic linkage analyses of Romano-Ward syndrome (RWS) in 13 Japanese families

Romano-Ward syndrome (RWS) is an autosomal dominant disorder characterized by prolongation of the electrocardiographic QT interval, with clinical manifestations that include recurrent syncope and sudden death from ventricular arrhythmias. Presymptomatic diagnosis is difficult because of the variability in these signs among carriers, but it is important for clinical management to prevent sudden cardiac death. To find an LQT (long QT) locus in Japanese patients and to identify DNA markers useful for presymptomatic diagnosis, linkage analyses were undertaken in 13 Japanese families with RWS patients by means of two DNA markers located on 11p15.5. One of these marker loci, HRAS, was previously reported to be tightly linked to the LQT locus in another ethnic group. Our analyses of homogeneity suggest evidence for genetic heterogeneity of RWS within the Japanese population.     

One-step purification of proteins from chicken egg white using counter-current chromatography

Proteins present in chicken egg white are separated by counter-current chromatography (CCC) in one step using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% (w/w) poly(ethylene glycol) 1000 and 12.5% (w/w) dibasic potassium phosphate by eluting the lower phase at a flow-rate of 1.0 ml/min. From about 20 g of the crude egg white solution, lysozyme, ovalbumin, and ovotransferrin were resolved within 5.5 h. Each component was identified by 12% SDS gel electrophoresis with Coomassie brilliant blue staining.     

Special smooth muscle cells along the submucosal surface of the guinea pig colon with reference to its spontaneous contractions

Antiperistalses occur from the flexure region of the guinea pig colon. We previously demonstrated that the circular muscle at the mesenteric border of the flexure region produced spontaneous regular contractions and found special smooth muscle cells believed to be pacemakers along the submucosal surface of the circular muscle layer. In this study, we revealed bipolar- and multipolar-type special smooth muscle cells along the submucosal surface of the muscle layer. Their slender cell processes contacted each other and formed a cellular network. Caveolae, filament structures expressing smooth muscle actin, vimentin, some desmin, and basal lamina were prominent features. The special smooth muscle cells corresponded to c-Kit-immunopositive cells and so-called interstitial cells or interstitial cells of Cajal in other reports. Their population was larger in the flexure region and the proximal colon than in the distal colon. The circular muscle layer at the flexure region was thicker than in other regions. The contraction in the flexure region showed the highest frequency and regularity. The dense population of special smooth muscle cells at the flexure region and thicker muscle layer may make the mechanical contraction more regular. The antiperistalsis from the flexure region could be explained in relation to the highest frequency of the pulsating contraction.     

Stimulated rapid expression in vitro for early detection of in vivo T-cell receptor mutations induced by radiation exposure

The T-cell receptor (TCR) mutation assay for in vivo somatic mutations is a sensitive indicator of exposure to ionizing radiation. However, this assay cannot be immediately applied after radiation exposure because expression of a mutant phenotype may require as long as several months. In the present study, we eliminate this time lag by stimulating lymphocytes with a mitogen that can accelerate the turnover of TCR protein expression in T-cells. When lymphocytes obtained from healthy donors were irradiated with various doses of X-rays and cultured with human interleukin-2 after phytohemagglutinin (PHA) pulse stimulation, the mutant frequency (MF) of CD4⁺ T-cells increased dose dependently during the first 7 days, then decreased rapidly due to the growth disadvantage of mutant cells. This suggests that PHA stimulation can shorten the expression time of a mutant phenotype to within a week after radiation exposure. The relationship between radiation dose and TCR MF on the seventh day was best fitted by a linear-quadratic dose–response model. We applied this improved TCR mutation assay to gynecological cancer patients who received 5 days of localized radiotherapy, totaling about 10 Gy. The in vivo TCR MF in the patients did not change within a week after radiotherapy, whereas the in vitro TCR MF of PHA-stimulated lymphocytes from the same patients significantly increased 7 days after initiating culture. The estimated mean radiation dose to the peripheral blood lymphocytes of the cancer patients was about 0.9 Gy, based on the in vitro linear-quadratic dose–response curve. This estimated dose was close to that described in a previous report on unstable-type chromosome aberrations from cervical cancer patients after receiving the same course of radiotherapy. On the basis of these findings, we propose that the improved TCR mutation assay is a useful biological dosimeter for recent radiation exposure.