Signal Sequence and Keyword Trap in silico for Selection of Full-Length Human cDNAs Encoding Secretion or Membrane Proteins from Oligo-Capped cDNA Libraries

We have developed an in silico method of selection of humanfull-length cDNAs encoding secretion or membrane proteins fromoligo-capped cDNA libraries. Fullness rates were increased toabout 80% by combination of the oligo-capping method and ATGpr,software for prediction of translation start point and the codingpotential. Then, using 5'-end single-pass sequences, cDNAs havingthe signal sequence were selected by PSORT (‘signal sequencetrap’). We also applied ‘secretion or membrane protein-relatedkeyword trap’ based on the result of BLAST search againstthe SWISS-PROT database for the cDNAs which could not be selectedby PSORT. Using the above procedures, 789 cDNAs were primarilyselected and subjected to full-length sequencing, and 334 ofthese cDNAs were finally selected as novel. Most of the cDNAs(295 cDNAs: 88.3%) were predicted to encode secretion or membraneproteins. In particular, 165(80.5%) of the 205 cDNAs selectedby PSORT were predicted to have signal sequences, while 70 (54.2%)of the 129 cDNAs selected by ‘keyword trap’ preservedthe secretion or membrane protein-related keywords. Many importantcDNAs were obtained, including transporters, receptors, andligands, involved in significant cellular functions. Thus, anefficient method of selecting secretion or membrane protein-encodingcDNAs was developed by combining the above four procedures.     

Role of nerve growth factor-tyrosine kinase receptor A signaling in paclitaxel-induced peripheral neuropathy in rats

The mechanisms underlying paclitaxel-induced peripheral neuropathy remain unknown. Nerve growth factor (NGF) is a representative neurotrophic factor that maintains neuronal function, promotes survival, and mediates neuropathic pain. We investigated expression levels of NGF and its receptors in the dorsal root ganglia (DRG) and spinal dorsal horn (DH) following paclitaxel treatment. Intraperitoneal (I.P.) administration of paclitaxel induced significant mechanical hypersensitivity and cold allodynia in rats, significantly increased the expression of NGF and its receptor tyrosine kinase receptor A (trkA) in the DRG, and increased NGF expression in the DH. In contrast, paclitaxel treatment did not alter the mRNA levels of NGF or its receptors in the DRG, DH, sciatic nerve, or hindpaw skin. Moreover, expression of NEDD4-2, a negative regulator of trkA, was significantly increased in the DRG of paclitaxel-treated rats. Intrathecal (I.T.) administration of the tyrosine kinase receptor inhibitor k252a significantly alleviated mechanical hypersensitivity in paclitaxel-treated rats. Our results suggest that NGF–trkA signaling is involved in mechanical allodynia in paclitaxel-induced neuropathy.     

Morphological abnormalities in the spermatozoa of fertile and infertile men

The morphological analysis of the spermatozoa from fertile and infertile men was performed using light and electron microscopy to clarify the relationship between sperm morphology and fertility. Semen samples obtained from 22 partners of pregnant women were prepared according to the protocol standardized in an international collaborative study. Semen samples from 17 patients with asthenozoospermia or varicocele were collected in a hospital. Abnormalities in the spermatozoa were classified into three types for the tails, two for the midpieces, and six for the heads according to the criteria adapted from WHO guidelines (World Health Organization, 1999: WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction (4th edition)). Approximately 14% of the spermatozoa from the fertile men had abnormal tails at the light microscopic level while approximately 44% had abnormal heads. Most types of abnormalities found in the spermatozoa from the asthenozoospermic and varicocele patients were encountered in those from the fertile men, although the semen from the fertile men contained a higher percentage of normal spermatozoa than that from the patients. These results were also confirmed at the ultrastructural level. Most abnormal cell types are encountered in semen from fertile men, although the incidence of abnormalities is low.     

Sensitive periods for wing development and precocious metamorphosis after precocene treatment of the brown planthopper,Nilaparvata lugens

The critical periods for juvenile hormone suppression of wing development and metamorphosis were examined in a pure brachypterous line of Nilaparvata lugens following topical application of Precocene II (PII) to various stages of the third and fourth nymphal stadia. When PII, in doses ranging from 10 pg to 100 ng, was applied to 12-h-old third or 6-h-old fourth stadium nymphs, long-wing formation (macroptery) was induced. Macropter induction ranged from 5 to 50% in females and from 30 to 50% in males, the effect being more prominent in males. The sensitive period for macropter induction lasted from the early second stadium through to day one of the fourth (penultimate) stadium. Beyond that period nymphs required sensitivity to PII and the numbers of macropters increased towards the final nymphal molt, again males were more sensitive. On the other hand, periods sensitive to PII for precocious metamorphosis induction appeared from early second stadium to 18 h after molting to the third nymphal stadium. Overlapping application of JH-III with PII at early third stadium could totally inhibit the occurrence of precocious metamorphosis and significantly rescue the macropter induction. From the data described, a scheme is presented for the control of wing development and metamorphosis by juvenile hormone, assuming two types of threshold value determining wing form and metamorphosis.     

CD44 directs membrane-type 1 matrix metalloproteinase to lamellipodia by associating with its hemopexin-like domain

Membrane-type 1 matrix metalloproteinase (MT1- MMP) localizes at the front of migrating cells and degrades the extracellular matrix barrier during cancer invasion. However, it is poorly understood how the polarized distribution of MT1-MMP at the migration front is regulated. Here, we demonstrate that MT1-MMP forms a complex with CD44H via the hemopexin-like (PEX) domain. A mutant MT1-MMP lacking the PEX domain failed to bind CD44H and did not localize at the lamellipodia. The cytoplasmic tail of CD44H, which comprises interfaces that associate with the actin cytoskeleton, was important for its localization at lamellipodia. Overexpression of a CD44H mutant lacking the cytoplasmic tail also prevented MT1-MMP from localizing at the lamellipodia. Modulation of F-actin with cytochalasin D revealed that both CD44H and MT1-MMP co-localize closely with the actin cytoskeleton, dependent on the cytoplasmic tail of CD44H. Thus, CD44H appears to act as a linker that connects MT1-MMP to the actin cytoskeleton and to play a role in directing MT1-MMP to the migration front. The PEX domain of MT1-MMP was indispensable in promoting cell migration and CD44H shedding.     

Identification of 6-methylsulfinylhexyl isothiocyanate as an apoptosis-inducing component in wasabi

The ethanol extract from Japanese horseradish wasabi was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptotic cell death. Separation by methods including silica gel chromatography and preparative HPLC gave an active compound, which was identified as 6-methylsulfinylhexyl isothiocyanate (6-HITC). Several lines of evidence indicated that 6-HITC induced apoptosis in U937 cells and human stomach cancer MKN45 cells. Thus, 6-HITC is potentially useful as a natural anti-cancer agent.     

Second analysis of mortality of nuclear industry workers in Japan, 1986-1997

IA cohort study of nuclear industry workers was initiated in 1990 to determine the possible health effects of low-level radiation. A total of 5,527 deaths were ascertained among 176,000 male workers who had been retrospectively and/or prospectively followed for an average of 7.9 years during the observation period 1986-1997. Statistical analyses were made mainly on the prospective follow-up outcome of 120,000 workers followed for an average of 4.5 years. The standardized mortality ratio (and its 95% confidence interval) was 0.94 (0.90, 0.97) for 2,934 cases of all causes combined and 0.86 (0.82, 0.91) for 1,305 cases of non-malignant diseases combined, which suggested a healthy worker effect. For 1,191 cases of all cancers combined, it was 0.98 (0.93, 1.04), indicating no difference in mortality from that of the general population. In tests for trend of death rate with increasing radiation dose, no significant correlation was found for all cancers combined. For site-specific cancers, most cancers including leukemia showed no positive correlation with dose, except for cancers of the esophagus, stomach and rectum and multiple myeloma. External causes showed a significant correlation with dose. A separate questionnaire study indicated that these positive findings could be ascribed in part to lifestyle characteristics of the workers. For leukemia only, we attempted to estimate the excess relative risk per unit dose of radiation, which, with reservations because of its wide confidence interval, was within the range of variation of the risks reported in other radiation epidemiological studies. This population must be studied for a longer time and with a consideration of the possible effects of confounding factors.     

Angiogenic network formation in the developing vertebrate trunk

We have used time-lapse multiphoton microscopy of living Tg(fli1:EGFP)y1 zebrafish embryos to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Angiogenic vascular sprouts emerge from the longitudinal trunk axial vessels (the dorsal aorta and posterior cardinal vein) in two spatially and temporally distinct steps. Dorsal aorta-derived sprouts form an initial primary network of vascular segments, followed by emergence of vein-derived secondary vascular sprouts that interact and interconnect dynamically with the primary network to initiate vascular flow. Using transgenic silent heart mutant embryos, we show that the gross anatomical patterning of this network of vessels does not require blood circulation. However, our results suggest that circulatory flow dynamics play an important role in helping to determine the pattern of interconnections between the primary network and secondary sprouts, and thus the final arterial or venous identity of the vessels in the functional network. We discuss a model to explain our results combining genetic programming of overall vascular architecture with hemodynamic determination of circulatory flow patterns.