Founder effect of the <Emphasis Type="Italic">C9</Emphasis> R95X mutation in Orientals

A nonsense mutation at codon 95 (R95X) in the C9 gene is responsible for most Japanese C9 deficiency (C9D) cases, with a carrier frequency of 6.7%. Upon analysis of microsatellite markers and newly identified dinucleotide repeat number polymorphisms in the 3' flanking region of the C9 gene, a founder effect was demonstrated for the R95X mutation of the C9 gene in Japanese. Screening for the R95X mutation in Korean and Chinese individuals showed that the R95X carrier frequencies in Koreans and Chinese were 2.0% and 1.0%, respectively. Although homozygotes for the R95X mutation were not found in Korea or China, the shared haplotype of the dinucleotide repeat number polymorphisms appeared to be associated with the R95X mutation in the heterozygotes in Korea and China. The founder effect found in East Asians (Japanese, Koreans and Chinese) but not in Caucasians, as well as the haplotype sharing in only a small chromosomal interval, suggested that the R95X mutation of C9 gene was ancient and had occurred after the divergence of East Asians and Caucasians, and before migration of the Yayoi people to Japan. Since the mortality of meningococcal infections in complement-deficient patients is lower than that in normal individuals, a founder effect and a selective advantage in isolation might be the main reasons for the high frequency of the R95X mutation in Japan.     

ik3-2, a relative to ik3-1/Cables, is involved in both p53-mediated and p53-independent apoptotic pathways

ik3-2 is a close relative to ik3-1/Cables, an associator with cdk3 and cdk5. ik3-1/Cables has been identified to be a candidate tumor suppressor for colon and head/neck cancers. In agreement, it has been pointed out that ik3-1/Cables is a regulator for both p53- and p73-induced apoptosis [J. Biol. Chem. 277 (2002) 2951] although ectopic expression of ik3-1/Cables does not induce apoptosis. Here we show that adenovirus-mediated overexpression of ik3-2 results in apoptosis of p53-intact U2OS cells. ik3-2 binds to p53 in vivo and ectopic coexpression of ik3-2 enhances apoptosis induced by adenovirus-mediated expression of p53. Furthermore, ectopic expression of ik3-2 results in apoptosis of primary p53/Mdm2- and p53/ARF-null mouse embryo fibroblasts, indicating that ik3-2-induced apoptosis is partially p53-independent. Both the highly conserved C-terminal cyclin box-homologous domain (ik3-2-C) and the N-terminal region consisting of 70 amino acids (ik3-2-N) are responsible for ik3-2-mediated enhancement of p53-induced apoptosis. In contrast, ik3-2-induced p53-independent apoptosis is mediated through ik3-2-N. We thus identified ik3-2 as a proapoptotic factor involved in both p53-mediated and p53-independent apoptotic pathways.     

Offspring derived from intracytoplasmic injection of transgenic rat sperm

The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at –20°C until use. When the sperm heads were aspirated into injection pipettes 7–10m in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2–4m in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague–Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed.     

The dominance of alleles controlling self-incompatibility in Brassica pollen is regulated at the RNA level

Self-incompatibility (SI) in Brassica is controlled sporophytically by the multiallelic S-locus. The SI phenotype of pollen in an S-heterozygote is determined by the relationship between the two S-haplotypes it carries, and dominant/recessive relationships often are observed between the two S-haplotypes. The S-locus protein 11 (SP11, also known as the S-locus cysteine-rich protein) gene has been cloned from many pollen-dominant S-haplotypes (class I) and shown to encode the pollen S-determinant. However, SP11 from pollen-recessive S-haplotypes (class II) has never been identified by homology-based cloning strategies, and how the dominant/recessive interactions between the two classes occur was not known. We report here the identification and molecular characterization of SP11s from six class II S-haplotypes of B. rapa and B. oleracea. Phylogenetic analysis revealed that the class II SP11s form a distinct group separated from class I SP11s. The promoter sequences and expression patterns of SP11s also were different between the two classes. The mRNA of class II SP11, which was detected predominantly in the anther tapetum in homozygotes, was not detected in the heterozygotes of class I and class II S-haplotypes, suggesting that the dominant/recessive relationships of pollen are regulated at the mRNA level of SP11s.     

Eosinophil migration induced by mast cell chymase is mediated by extracellular signal-regulated kinase pathway

Mast cell chymase is known to induce eosinophil migration in vivo and in vitro. In the present study, we investigated possible involvement of mitogen-activated protein (MAP) kinases; extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, in the chymase-induced eosinophil migration. Human chymase induced a rapid phosphorylation of ERK1/2 and p38 in human eosinophilic leukemia EoL-1 cells, while no phosphorylation was detected in JNK. The chymase-induced phosphorylation of ERK and p38 was inhibited by pertussis toxin. Similar results were obtained in the experiments using mouse chymase and eosinophils. U0126 (the inhibitor for MAP/ERK kinase) suppressed chymase-induced migration of EoL-1 cells and mouse eosinophils. However, SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) showed little effect on the migration. It is suggested therefore that chymase activates ERK and p38 probably through G-protein-coupled receptor, and that ERK but not p38 cascade may have a crucial role in chymase-induced migration of eosinophils.     

Effects of ambient temperature steps on thermal comfort requirements

The purpose of this study was to determine the thermal comfort requirements for steps in temperature. Thirty male subjects were exposed for 50 min to a 34 or 37°C condition, and then quickly transferred to a cooler environment of 31, 28, 25, and 22°C for 50 min. Mean skin temperature was continuously measured, and the subjects reported their thermal sensation and comfort sensation every 2 min. Just after the step changes, the mean skin temperature immediately decreased, while the thermal sensation overshot and gradually rose again. Both the skin temperature and the thermal sensation seemed to reach a constant level within about 20 min. However, there were differences in the mean skin temperature and the neutral temperature derived from the correlation between the ambient temperature and the thermal sensation even 50 min after the steps, due to the thermal environmental condition before the changes of temperature. The change in the neutral temperature with time was expressed as two attenuating equations. These equations indicate that there is an obvious difference between the neutral temperatures due to the thermal condition before step changes, and that it takes >50 min after the step changes to reach the steady state. It is expected that these equations predict in quantitative terms the thermal comfort requirements within a given experimental condition.     

Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro

Although possible biological functions of whey acidic protein (WAP) have been suggested, few studies have focused on investigating the function of WAP. This paper describes evidence for WAP function in lobulo-alveolar development in mammary glands in vivo and in the cell cycle progression of mammary epithelial cells in vitro. Ubiquitous overexpression of WAP transgene impaired only lobulo-alveolar development in the mammary glands of transgenic female mice but not other physiological functions, indicating that the inhibitory function of WAP is specific to mammary alveolar cells. The forced expression of WAP significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 cells and EpH4/K6 cells), whereas it did not affect that of NIH3T3 cells. Co-culturing of WAP-clonal cells and control cells using a transwell insert demonstrated that WAP inhibited the proliferation of HC11 cells through a paracrine action but not that of NIH3T3 cells, and that WAP was able to bind to HC11 cells but not to NIH3T3 cells. Apoptosis was not enhanced in the HC11 cells with stable WAP expression (WAP-clonal HC11 cells). BrdU incorporation and FACScan analyses revealed that cell cycle progression from the G0/G1 to the S phase was inhibited in the WAP-clonal HC11 cells. Among G1 cyclins, the expression of cyclin D1 and D3 was significantly decreased in the WAP-clonal HC11 cells. The present results provide the first documented evidence that WAP plays a negative regulatory role in the cell cycle progression of mammary epithelial cells through an autocrine or paracrine mechanism in vivo.