Blood System Formation in the Urochordate Ciona intestinalis Requires the Variable Receptor vCRL1

Adaptive immune systems are present only in vertebrates. How do all the remaining animals withstand continuous attacks of permanently evolving pathogens? Even in the absence of adaptive immunity, every organism must be able to unambiguously distinguish "self" cells from any imaginable "nonself." Here, we analyzed the function of highly polymorphic gene vCRL1, which is expressed in follicle and blood cells of Ciona intestinalis, pointing to possible recognition roles either during fertilization or in immune reactions. By using segregation analysis, we demonstrate that vCRL1 locus is not involved in the control of self-sterility. Interestingly, genetic knockdown of vCRL1 in all tissues or specifically in hemocytes results in a drastic developmental arrest during metamorphosis exactly when blood system formation in Ciona normally occurs. Our data demonstrate that vCRL1 gene might be essential for the establishment of a functional blood system in Ciona. Presumably, presence of the vCRL1 receptor on the surface of blood cells renders them as self, whereas any cell lacking it is referred to as nonself and will be consequently destroyed. We propose that individual-specific receptor vCRL1 might be utilized to facilitate somatic self/nonself discrimination.     

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.     

A gene, SMP2, involved in plasmid maintenance and respiration in Saccharomyces cerevisiae encodes a highly charged protein

Summary The smp2 mutant of Saccharomyces cerevisiae shows increased stability of the heterologous plasmid pSR1 and YRp plasmids. A DNA fragment bearing the SMP2 gene was cloned by its ability to complement the slow growth of the smp2 smp3 double mutant (smp3 is another mutation conferring increased stability of plasmid pSR1). The nucleotide sequence of SMP2 indicated that it encodes a highly charged 95 kDa protein. Disruption of the genomic SMP2 gene resulted in a respiration-deficient phenotype, although the cells retained mitochondrial DNA, and showed increased stability of pSR1 like the original smp2 mutant. The fact that the smp2 mutant is not always respiration deficient and shows increased pSR1 stability even in a rho ⁰ strain lacking mitochondrial DNA suggested that the function of the Smp2 protein in plasmid maintenance is independent of respiration. The SMP2 locus was mapped at a site 71 cM from lys7 and 21 cM from ilv2/SMR1 on the right arm of chromosome XIII.     

Effect of Caffeine-Containing Beverage Consumption on Serum Alanine Aminotransferase Levels in Patients with Chronic Hepatitis C Virus Infection: A Hospital-Based Cohort Study

Introduction

To date, there have been no prospective studies examining the effect of coffee consumption on serum alanine aminotransferase (ALT) level among individuals infected with the hepatitis C virus (HCV). We conducted a hospital-based cohort study among patients with chronic HCV infection to assess an association between baseline coffee consumption and subsequent ALT levels for 12 months.

Materials and Methods

From 1 August 2005 to 31 July 2006, total 376 HCV-RNA positive patients were recruited. A baseline questionnaire elicited information on the frequency of coffee consumption and other caffeine-containing beverages. ALT level as a study outcome was followed through the patients’ medical records during 12 months. The association between baseline beverage consumption and subsequent ALT levels was evaluated separately among patients with baseline ALT levels within normal range (≤45 IU/L) and among those with higher ALT levels (>45 IU/L).

Results

Among 229 patients with baseline ALT levels within normal range, 186 (81%) retained normal ALT levels at 12 months after recruitment. Daily drinkers of filtered coffee were three times more likely to preserve a normal ALT level than non-drinkers (OR=2.74; P=0.037). However, decaffeinated coffee drinkers had a somewhat inverse effect for sustained normal ALT levels, with marginal significance (OR=0.26; P=0.076). In addition, among 147 patients with higher baseline ALT levels, 39 patients (27%) had ALT reductions of ≥20 IU/L at 12 months after recruitment. Daily drinkers of filtered coffee had a significantly increased OR for ALT reduction (OR=3.79; P=0.034). However, in decaffeinated coffee drinkers, OR could not be calculated because no patients had ALT reduction.

Conclusion

Among patients with chronic HCV infection, daily consumption of filtered coffee may have a beneficial effect on the stabilization of ALT levels.     

Identification of Cryptosporidium parvum Oocysts by an Artificial Neural Network Approach

Microscopic detection of Cryptosporidium parvum oocysts is time-consuming, requires trained analysts, and is frequently subject to significant human errors. Artificial neural networks (ANN) were developed to help identify immunofluorescently labeled C. parvum oocysts. A total of 525 digitized images of immunofluorescently labeled oocysts, fluorescent microspheres, and other miscellaneous nonoocyst images were employed in the training of the ANN. The images were cropped to a 36- by 36-pixel image, and the cropped images were placed into two categories, oocyst and nonoocyst images. The images were converted to grayscale and processed into a histogram of gray color pixel intensity. Commercially available software was used to develop and train the ANN. The networks were optimized by varying the number of training images, number of hidden neurons, and a combination of these two parameters. The network performance was then evaluated using a set of 362 unique testing images which the network had never “seen” before. Under optimized conditions, the correct identification of authentic oocyst images ranged from 81 to 97%, and the correct identification of nonoocyst images ranged from 78 to 82%, depending on the type of fluorescent antibody that was employed. The results indicate that the ANN developed were able to generalize the training images and subsequently discern previously unseen oocyst images efficiently and reproducibly. Thus, ANN can be used to reduce human errors associated with the microscopic detection of Cryptosporidium oocysts.     

Mutations in a Saccharomyces cerevisme host showing increased holding stability of the heterologous plasmid pSRI

Summary We have isolated Saccharomyces cerevisiae mutants, smp, showing stable maintenance of plasmid pSRI, a Zygosaccharomyces rouxii plasmid. The smp mutants were recessive and were classified into at least three different complementation groups. The three mutants also showed increased stability of YRp plasmids and the mutations are additive for plasmid stability. One mutation, smp1, confers a respiration-deficient (rho ⁰) phenotype and several Rho^– mutants independently isolated by ethidium bromide treatment of the same yeast strain also showed increased stabilities of pSR1 and YRp plasmids. The wild-type S. cerevisiae cells showed a strongly biased distribution of pSR1 molecules as well as YRp plasmids to the mother cells at mitosis, while the smpf mutant did not show this bias. Another mutation, smp3, at a locus linked to ade2 on chromosome XV, confers temperature-sensitive growth. The SMP3 gene encodes a 59.9 kDa hydrophobic protein and disruption of the gene is lethal.     

MRGD, a MAS-related G-protein coupled receptor, promotes tumorigenisis and is highly expressed in lung cancer

To elucidate the function of MAS-related GPCR, member D (MRGD) in cancers, we investigated the in vitro and in vivo oncogenic function of MRGD using murine fibroblast cell line NIH3T3 in which MRGD is stably expressed. The expression pattern of MRGD in clinical samples was also analyzed. We found that overexpression of MRGD in NIH3T3 induced focus formation and multi-cellular spheroid formation, and promoted tumors in nude mice. In other words, overexpression of MRGD in NIH3T3 induced the loss of contact inhibition, anchorage-independent growth and in vivo tumorigenesis. Furthermore, it was found that the ligand of MRGD, beta-alanine, enhanced spheroid formation in MRGD-expressing NIH3T3 cells. From investigation of clinical cancer tissues, we found high expression of MRGD in several lung cancers by immunohistochemistry as well as real time PCR. Based on these results, MRGD could be involved in tumorigenesis and could also be a novel anticancer drug target.     

How is autonomic nervous system activity in subjects who are sleepy but are unable to sleep in the daytime?

We investigated changes in the activity of the autonomic nervous system (ANS) in the relaxed condition in subjects who felt sleepy, but were unable to sleep. A total of 1021 subjects underwent daytime polysomnography. The sleep latency (SL) and the visual analog scale (VAS) were used to assess “immediate” objective and subjective sleepiness, respectively. The subjects were assigned to an “Alert-Alert” group (VAS ≤ 25 mm, SL ≥ 8 min), a “Sleepy-Alert” group (VAS ≥ 75 mm, SL ≥ 8 min), or a “Sleepy-Sleepy” group (VAS ≥ 75 mm, SL ≤ 4 min). In order to assess the ANS, the spectral analysis and the geometric method were used. The ANS data collected during the relaxed condition (after lights off, post-LO) was compared to that obtained during the control condition (before lights off, pre-LO). From the spectral analysis, a significant decrease of sympathetic function and an increase of parasympathetic function at post-LO in the Sleepy-Sleepy group, a tendency for sympathetic function decrease at post-LO in the Alert-Alert group, and no significant changes to sympathetic and parasympathetic function in the Sleepy-Alert group were observed. The results from the geometric method supported the results of the spectral analysis in the Alert-Alert group and the Sleepy-Sleepy group. The results of this study suggest that the ANS plays a role in individuals who are unable to sleep even though they feel sleepy and are given the opportunity to sleep.

     

Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB

Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.