A systematic investigation identifies a significant number of probable pseudogenes in the Escherichia coli genome

Pseudogenes are open reading frames (ORFs) encoding dysfunctional proteins with high homology to known protein-coding genes. Although pseudogenes were reported to exist in the genomes of many eukaryotes and bacteria, no systematic search for pseudogenes in the Escherichia coli genome has been carried out. Genome comparisons of E. coli strains K-12 and O157 revealed that many protein-coding sequences have prematurely terminated orthologs encoding unstable proteins. To systematically screen for pseudogenes, we selected ORFs generated by premature termination of the orthologous protein-coding genes and subsequently excluded those possibly arising from sequence errors. Lastly we eliminated those with close homologs in this and other species, as these shortened ORFs may actually have functions. The process produced 95 and 101 pseudogene candidates in K-12 and O157, respectively. The assigned three-dimensional structures suggest that most of the encoded proteins cannot fold properly and thus are dysfunctional, indicating that they are probably pseudogenes. Therefore, the existence of a significant number of probable pseudogenes in E. coli is predicted, awaiting experimental verification. Most of them were found to be genes with paralogs or horizontally transferred genes or both. We suggest that pseudogenes constitute a small fraction of the genomes of free-living bacteria in general, reflecting the faster elimination than production of pseudogenes.     

Sulfite induces adherence of polymorphonuclear neutrophils to immobilized fibrinogen through activation of Mac-1 beta2-integrin (CD11b/CD18)

Sulfite is a major air pollutant which can cause respiratory tract inflammation characterized by an influx of polymorphonuclear neutrophils (PMN). We have previously shown that human PMN can produce sulfite either spontaneously or in response to stimulation with lipopolysaccharide. We now demonstrate that sulfite activates PMN to adhere to immobilized fibrinogen via the beta2-integrin Mac-1 (CD11b/CD18). Mac-1 expression is not altered by treatment with this agent. Although unaffected by pertussis toxin, sulfite-triggered PMN adhesion was abrogated by pretreating cells with the membrane-impermeant sulfhydryl reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a modifier of thiol groups on the cell surface. These results suggest that sulfite-induced PMN adhesion is dependent on a modification of thiols at the cell surface. Given its potent antioxidant and antimicrobial activities, sulfite may act as an endogenous mediator in host defense and/or inflammation.     

Larger genetic differences within africans than between Africans and Eurasians

The worldwide pattern of single nucleotide polymorphism (SNP) variation is of great interest to human geneticists, population geneticists, and evolutionists, but remains incompletely understood. We studied the pattern in noncoding regions, because they are less affected by natural selection than are coding regions. Thus, it can reflect better the history of human evolution and can serve as a baseline for understanding the maintenance of SNPs in human populations. We sequenced 50 noncoding DNA segments each approximately 500 bp long in 10 Africans, 10 Europeans, and 10 Asians. An analysis of the data suggests that the sampling scheme is adequate for our purpose. The average nucleotide diversity (pi) for the 50 segments is only 0.061% +/- 0.010% among Asians and 0.064% +/- 0.011% among Europeans but almost twice as high (0.115% +/- 0.016%) among Africans. The African diversity estimate is even higher than that between Africans and Eurasians (0.096% +/- 0.012%). From available data for noncoding autosomal regions (total length = 47,038 bp) and X-linked regions (47,421 bp), we estimated the pi-values for autosomal regions to be 0.105, 0.070, 0.069, and 0.097% for Africans, Asians, Europeans, and between Africans and Eurasians, and the corresponding values for X-linked regions to be 0.088, 0.042, 0.053, and 0.082%. Thus, Africans differ from one another slightly more than from Eurasians, and the genetic diversity in Eurasians is largely a subset of that in Africans, supporting the out of Africa model of human evolution. Clearly, one must specify the geographic origins of the individuals sampled when studying pi or SNP density.     

Influences of a dietary fatty acid composition on the emergence of glutathione S-transferase-P (GST-P) positive foci in the liver of carcinogen-treated rats

The rats treated with a single i.p. injection of diethylnitrosoamine (DEN) and percial hepatectomy were fed for 11 weeks with a high fat diet mixed with 10% lard, eicosapentaenoic-acid-rich oil (EPA-oil) or arachidonic-acid-rich oil (AA-oil) and the emergence of glutathione S-transferase placental form (GST-P) in the liver was evaluated. There were no significant differences in the serum aminotransferase activities. The molar ratio of n-6 and n-3 fatty acid in the liver phospholipids was significantly low in the EPA-oil group compared with the other groups. In the EPA-oil group, the area percent and the unit area of GST-P positive foci were significantly smaller than the other groups. In the AA-oil group, no significant differences were recognized in the quantitative values for GST-P positive foci compared with the control and lard groups. In conclusion, a hepatic neoplasmic lesion induced by DEN was suppressed with EPA-rich fish oil, and arachidonic-acid-rich oil showed no effect of suppression or acceleration.     

Roles for the two-hybrid system in exploration of the yeast protein interactome

Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.     

Susceptibility gene for non-obstructive azoospermia located near HLA-DR and -DQ loci in the HLA class II region

The technical developments and expanded indications for testicular sperm extraction (TESE) with intracytoplasmic sperm injection (ICSI) provide great advantages for patients with non-obstructive azoospermia. Such success, however, also means that genetic abnormalities in non-obstructive azoospermia can be transmitted to the next generation, demonstrating the importance of being able to understand the genetic background of non-obstructive azoospermia. We have previously reported that human leukocyte antigens (HLA)-A33 and -B44 in the HLA class I region and the HLA-DRB1*1302 allele in the HLA class II region are linked to susceptibility to non-obstructive azoospermia in Japanese men. However, strong linkage of HLA-DRB1*1302 with HLA-A33 and -B44 is also evident in the Japanese population. Thus, uncertainty prevails as to whether the HLA class I or class II molecule is more directly associated with non-obstructive azoospermia. In the present study, we performed association analysis with 21 polymorphic microsatellite markers identified near the HLA genes to map the gene involved in the development of non-obstructive azoospermia more precisely. Microsatellite markers located in the HLA class I region or the class III region showed no statistically significant association with this disorder, although once again the HLA-A33 and -B44 alleles showed a significant association. In contrast, some of the microsatellite markers in the HLA class II region and at the HLA-DRB1 and -DQB1 loci displayed strong associations with non-obstructive azoospermia. Taken together, our previous and present data suggest that the critical region for development of non-obstructive azoospermia is near the HLA-DRB1 and -DQB1 segments in the HLA class II region.     

Negative regulation of EphA2 receptor by Cbl

The c-Cbl proto-oncogene product Cbl has emerged as a negative regulator of receptor and non-receptor tyrosine kinases, a function dependent on its recently identified ubiquitin ligase activity. Here, we report that EphA2, a member of Eph receptor tyrosine kinases is negatively regulated by Cbl. The negative regulation of EphA2 mediated by Cbl is dependent on the activity of EphA2, as the kinase inactive mutant of EphA2 cannot be regulated by Cbl. Moreover, a point mutation (G306E-Cbl) in TKB region of Cbl that has been reported to abolish Cbl binding to RTKs and non-receptor tyrosine kinases impaired the binding to active EphA2. The dominant negative mutant 70Z-Cbl, which has a 17-amino acids deletion in the N-boundary of the RING finger domain, defuncted negative regulatory function of Cbl to EphA2. These results demonstrate that the TKB domain and RING finger domain of Cbl are essential for this negative regulation.