Generation of progeny from embryonic stem cells by microinsemination of male germ cells from chimeric mice

Mice chimeric for embryonic stem (ES) cells have not always successfully produced ES-derived offspring. Here we show that the male gametes from ES cells could be selected in male chimeric mice testes by labeling donor ES cells or host blastocytes with GFP. Male GFP-expressing ES-derived germ cells occurred as colonies in the chimeric testes, where the seminiferous tubules were separated into green and non-green regions. When mature spermatozoa from green tubules were used for microinsemination, GFP-expressing offspring were efficiently obtained. Using a reverse study, we also obtained ES-derived progeny from GFP-negative ES cells in GFP-labeled host chimeras. Furthermore, we showed this approach could be accelerated by using round spermatids from the testes of 20-day-old chimeric mice. Thus, this technique allowed us to generate the ES cell-derived progeny even from the low contributed chimeric mice, which cannot produce ES-origin offspring by natural mating.     

Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium

The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50 degrees C, lower than that of E. coli APase by 30 degrees C. The specific activity of SIB1 APase at 50 degrees C was 3.1 fold higher than that of E. coli APase at 80 degrees C. SIB1 APase lost activity with a half-life of 3.9 min at 70 degrees C, whereas E. coli APase lost activity with a half-life of >6 h even at 80 degrees C. Thus SIB1 APase is well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase.     

Protein-splicing reaction via a thiazolidine intermediate: crystal structure of the VMA1-derived endonuclease bearing the N and C-terminal propeptides

Protein splicing excises an internal intein segment from a protein precursor precisely, and concomitantly ligates flanking N and C-extein polypeptides at the respective sides of the precursor. Here, a series of precursor recombinants bearing 11 N-extein and ten C-extein residues is prepared for the intein of the Saccharomyces cerevisiae VMA1-derived homing endonuclease referred to as VDE and as PI-SceI. The recombinant with replacements of C284S, H362N, N737S, and C738S is chosen as a spliceable precursor model and is then subjected to a 2.1A resolution crystallographic analysis. The crystal structure shows that the introduced extein polypeptides are located in the vicinity of the splicing site, and that each of their peptide bonds is in the trans conformation. The S284 O(gamma) atom located at a distance of 3.1A from the G283 C atom in the N-terminal junction suggests that a nucleophilic attack of the C284 S(gamma) atom on the G283 C atom forms a tetrahedral intermediate containing a five-membered thiazolidine ring. The tetrahedral intermediate is supposedly resolved into a thioester acyl group upon the cleavage of the linkage between the G283 C and C284 N atoms, and this thioester acyl formation completes the initial steps of Nright arrowS acyl shift at the junction between the N-extein and intein. The S738 O(gamma) atom in the C-terminal junction is placed in close proximity to the S284 O(gamma) atom at a distance of 3.6A, and is well suited for another nucleophilic attack on the resultant thioester acyl group that is then subjected to the transesterification in the next step. The reaction steps proposed for the acyl shift are driven entirely by protonation and deprotonation, in which proton ingress and egress is balanced within the splicing site.     

Localization of porcine trappin-2 (SKALP/elafin) in trachea and large intestine by in situ hybridization and immunohistochemistry

Trappin-2 (SKALP/elafin), an elastase inhibitor, belongs to a unique family of proteinase inhibitors that are covalently anchored at the site of action through their transglutaminase substrate domain and are collectively called trappins. The transglutaminase substrate domain is therefore called "cementoin moiety". Currently, human, porcine, and bovine trappin-2 (SKALP/elafin) have been characterized. Previously, we showed that porcine trappin-2 (SKALP/elafin) occurs mainly in the trachea and large intestine. To determine the localization of trappin-2 (SKALP/elafin) at the cellular level, we carried out in situ hybridization and immunohistochemistry using the porcine trachea and large intestine and found that trappin-2 (SKALP/elafin) is produced in the goblet cells of the tracheal epithelium and of the large intestinal crypts. These locations suggest that trappin-2 (SKALP/elafin) is secreted onto the luminal surface of the trachea and crypts of Lieberkuhn and plays a protective role against destructive bacterial proteinases.     

BMPs are necessary for stomach gland formation in the chicken embryo: a study using virally induced BMP-2 and Noggin expression

Epithelial-mesenchymal interactions are necessary for the normal development of various digestive organs. In chicken proventriculus (glandular stomach), morphogenesis and differentiation of the epithelium depend upon the inductive signals coming from underlying mesenchyme. However, the nature of such signals is still unclear despite extensive analyses carried out using experimental tissue recombinations. In this study we have examined the possible involvement of bone morphogenetic proteins (BMPs) in the formation of stomach glands in the chicken embryo. Analysis of the expression patterns of BMP-2, -4 and -7 showed that these BMPs were present in the proventricular mesenchyme prior to the initiation of the proventricular gland formation. BMP-2 expression, in particular, was restricted to the proventriculus among anterior digestive organs. Virus-mediated BMP-2 overexpression resulted in an increase in the number of glands formed. Moreover, ectopic expression of Noggin, which antagonizes the effect of BMPs, in the proventricular mesenchyme or epithelium, led to the complete inhibition of gland formation, indicating that BMP signals are necessary for the proventricular gland formation. These findings suggest that BMPs are of prime importance as mesenchymal signals for inducing proventricular glands.     

Detection of hantaviral antibodies among patients with hepatitis of unknown etiology in Japan

Hantaviral antibodies were detected in the sera from patients with hepatic disease of unknown etiology in Japan by several different serological diagnostic methods. A total of 105 sera from diseased patients which were negative to A-G hepatitis virus infections in the Tokyo area were tested. Among them, 3 out of 73 sera from patients with chronic hepatic disease were positive to hantaviral antibody by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent antibody assay (IFA) and Western blot analysis (WB). Neutralizing antibody titers of the 3 sera to Seoul virus (SEO) were 4 to 8 times higher than those to Hantaan virus (HTN). However, all of the 32 sera from patients with acute hepatitis were negative for hantaviral antibody. Among the 60 patients with chronic hepatitis in Hokkaido which were serologically negative to B and C hepatitis virus infection, one was positive for hantaviral antibody by ELISA and WB. In contrast, the sera from healthy adults in Japan, 550 from the Honshu and Kyushu regions, and 1,000 from the Hokkaido region, were negative for hantavirus antibody. These results show that hantaviral antibodies are more frequently detected in patients with hepatic disease than in healthy adults. However, the observation that no positive sera were detected from patients with acute hepatitis implies that hantavirus might not be directly related to hepatitis.     

A comparative study of the immunity region of lambdoid phages including Shiga-toxin-converting phages: molecular basis for cross immunity

Comparison of eight lambdoid phages, including three Shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and CRO proteins on the one hand, and operator sequences on the other. Some as yet unassigned components of the regulatory circuits have been inferred by computer search. The cross immunity phenomenon shown by phages VT2-Sa and lambda is explained on the basis of similarity in their sequences. In addition, the similarity of 933W and HK022 in the sequences of their recognition helices of repressor and CRO, on the one hand, and operators, on the other, has led us to predict that they will have identical or similar immunity specificity. This homology has enabled us also to locate the OL (and consequently PL) of phage 933W that has been thought to be non-existent.