Alterations in gastric mucosal lineages induced by acute oxyntic atrophy in wild-type and gastrin-deficient mice

In addition to their role in gastric acid secretion, parietal cells secrete a number of growth factors that may influence the differentiation of other gastric lineages. Indeed, oxyntic atrophy is considered the most significant correlate with increased risk for gastric adenocarcinoma. We studied the alterations in gastric mucosal lineages elicited by acute oxyntic atrophy induced by treatment of C57BL/6 and gastrin-deficient mice with the parietal cell protonophore [S-(R*,S*)]-N-[1-(1,3-benzodioxol-5-yl)butyl]-3,3-diethyl-2-[4-[(4-methyl-1-piperazinyl)carbonyl]phenoxy]-4-oxo-1-azetidinecarboxamide (DMP-777). In both wild-type and gastrin knockout mice, DMP-777 elicited the rapid loss of parietal cells within 2 days of treatment. In wild-type mice, oxyntic atrophy was accompanied by a rapid increase in 5-bromo-2'-deoxyuridine-labeled proliferative cells and attendant increase in surface cell numbers. However, gastrin knockout mice did not demonstrate significant foveolar hyperplasia and showed a blunted proliferative response. After 7 days of treatment in wild-type mice, a second proliferative population emerged at the base of fundic glands along with the development of a mucous cell metaplasia expressing TFF2/spasmolytic polypeptide (SPEM). However, in gastrin knockout mice, SPEM expressing both TFF2 mRNA and protein developed after only 1 day of DMP-777 treatment. In wild-type mice, all changes induced by DMP-777 were reversed 14 days after cessation of treatment. In gastrin-deficient mice, significant SPEM was still present 14 days after the cessation of treatment. The results indicate that foveolar hyperplasia requires the influence of gastrin, whereas SPEM develops in response to oxyntic atrophy independent of gastrin, likely through transdifferentiation of chief cells.     

Overexpression of calreticulin sensitizes SERCA2a to oxidative stress

Calreticulin (CRT), a Ca(2+)-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac disorder in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In this study, the effect of overexpression of CRT on sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2a under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. The in vitro activity of SERCA2a and uptake of (45)Ca(2+) into isolated microsomes were suppressed by H(2)O(2) in CRT-overexpressing cells compared with controls. Moreover, SERCA2a protein was degraded via a proteasome-dependent pathway following the formation of a complex with CRT under the stress with H(2)O(2). Thus, we conclude that overexpression of CRT enhances the inactivation and degradation of SERCA2a in the cells under oxidative stress, suggesting some pathophysiological functions of CRT in Ca(2+) homeostasis of myocardiac disease.     

Overall carbohydrate-binding properties of Castanea crenata agglutinin (CCA)

The carbohydrate-binding properties of Castanea crenata agglutinin (CCA) were investigated by an enzyme-linked lectin absorbent assay. The binding ability of each carbohydrate was compared using IC(50) values. CCA exhibited mannose/glucose specificity, as observed with many mannose-binding jacalin-related lectins. For oligosaccharides containing glucose, it has been shown that the degree of polymerization and the linkage mode of glucose residues have no effect on CCA-carbohydrate interaction; thus, only the non-reducing end glucose unit in glucooligosaccharides may be involved in the interaction with CCA. Among mannooligosaccharides, CCA strongly recognized alpha-(1-->3)-D-Man-[alpha-D-Man-(1-->6)]-D-Man, which is a core in N-linked carbohydrate chains. By considering the results with glycoproteins, it is likely that CCA binds preferentially to mono- or non-sialylated biantennary carbohydrate chains. We also obtained K(d) values by analysis of the dependency of the IC(50) on CCA concentration, based on the hypothesis that CCA has a single binding site or two equivalent binding sites. The estimated K(d) values for mannose, glucose and alpha-(1-->3)-D-Man-[alpha-D-Man-(1-->6)]-D-Man were 2.39, 7.19 and 0.483 mM, respectively. The relative binding abilities showed good agreement with the relative inhibition intensities. Isothermal calorimetric titration was carried out to directly estimate the dissociation constants of CCA for mannose and for alpha-D-Man-(1-->3)-D-Man. The values were 2.34 mM for mannose and 0.507 mM alpha-D-Man-(1-->3)-D-Man. These results suggest that the relative inhibition intensity represents the ratio of K(d) values and that CCA has a single or two equivalent binding sites.     

Expression of 3-ketoacyl-acyl carrier protein reductase (fabG) genes enhances production of polyhydroxyalkanoate copolymer from glucose in recombinant Escherichia coli JM109

Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene (fabG) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vitro. The current study also presents the first evidence that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes can enhance the production of SCL-MCL PHA copolymers from nonrelated carbon sources. Differences in the substrate specificities of the FabG proteins were reflected in the monomer composition of the polymers produced by recombinant E. coli. SCL-MCL PHA copolymer isolated from a recombinant E. coli strain had improved physical properties compared to the SCL homopolymer poly-3-hydroxybutyrate. This study defines a pathway to produce SCL-MCL PHA copolymer from the fatty acid biosynthesis that may impact on PHA production in recombinant organisms.     

Microsatellite mutations show no increases in the children of the Chernobyl liquidators

We performed a study on Belarusian "liquidators", exploring whether increase in the frequencies of germline mutations at microsatellite loci could be found in their progeny. The liquidators, mostly young males, were those involved (during 1986 and 1987) in clean-up operations in the radioactively contaminated area following the Chernobyl nuclear power plant accident in 1986. Many liquidators fathered children during the clean-up period and after the work had been terminated. The numbers of families studied were 64 (liquidators) and 66 (controls). A total of 72 loci (31 autosomal, one X-linked and 40 Y-linked) were used. DNA was isolated from peripheral blood lymphocytes and the microsatellite loci were amplified by the polymerase chain reaction with fluorescence-labelled primers. Mutations were detected as variations in the length of the loci. At the Y-linked loci, the mutation rates (expressed as number of mutations among the total number of loci for the individuals included) are 2.9 x 10(-3) (4/1392) and 2.1 x 10(-3) (3/1458) in the children of exposed and control parents, respectively. This difference is not statistically significant. At the autosomal loci, the corresponding estimates are 5.9 x 10(-3) (11/1862; exposed group) and 8.5 x 10(-3) (18/2108; control). Again, the difference is not significant. The possibility that the Belarusian population might have been unexpectedly exposed to some chemical contaminants in the environment appears unlikely in view of the finding that the spontaneous mutation rates at the same set of loci in several non-Belarusian populations were similar to those in Belarus. The estimated mean radiation dose to the liquidators was small, being about 39 mSv, and this might be one reason why no increases in mutation rates due to radiation could be found.     

Facilitated release of substrate protein from prefoldin by chaperonin

Prefoldin is a chaperone that captures a protein-folding intermediate and transfers it to the group II chaperonin for correct folding. However, kinetics of interactions between prefoldin and substrate proteins have not been investigated. In this study, dissociation constants and dissociation rate constants of unfolded proteins with prefoldin were firstly measured using fluorescence microscopy. Our results suggest that binding and release of prefoldin from hyperthermophilic archaea with substrate proteins were in a dynamic equilibrium. Interestingly, the release of substrate proteins from prefoldin was facilitated when chaperonin was present, supporting a handoff mechanism of substrate proteins from prefoldin to the chaperonin.     

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 degrees C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35x10(10) pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.     

Accumulation of infectious mutants in stocks during the propagation of fiber-modified recombinant adenoviruses

In infected cells, replication errors during viral proliferation generate mutations in adenoviruses (Ads), and the mutant Ads proliferate and evolve in the intracellular environment. Genetically fiber-modified recombinant Ads (rAd variants) were generated, by modification of the fiber gene, for therapeutic applications in host cells that lack or express reduced levels of the Coxsackievirus and adenovirus receptor. To assess the genetic modifications of rAd variants that might induce the instability of Ad virions, we examined the frequencies of mutants that accumulated in propagated stocks. Seven of 41 lines of Ad variants generated mutants in the stocks and all mutants were infectious. Moreover, all the mutations occurred in the modified region that had been added at the 3' end of the fiber gene. Our results show that some genetic modifications at the carboxyl terminus of Ad fiber protein lead to the instability of Ad virions.     

Orientation and pore-forming mechanism of a scorpion pore-forming peptide bound to magnetically oriented lipid bilayers

The orientation and pore-forming mechanisms of pandinin 2 (pin2), an antimicrobial peptide isolated from venom of the African scorpion Pandinus imperator, bound to magnetically oriented lipid bilayers were examined by 31P and 13C solid-state, and 15N liquid-state NMR spectroscopy. 31P NMR measurements at various temperatures, under neutral and acidic conditions, showed that membrane lysis occurred only under acidic conditions, and at temperatures below the liquid crystal-gel phase transition of the lipid bilayers, after incubation for two days in the magnet. Differential scanning calorimetry measurements showed that pin2 induced negative curvature strain in lipid bilayers. The 13C chemical shift values of synthetic pin2 labeled at Gly3, Gly8, Leu12, Phe17, or Ser18 under static or slow magic-angle spinning conditions, indicate that pin2 penetrates the membrane with its average helical axis perpendicular to the membrane surface. Furthermore, amide H-D exchange experiments of 15N-Ala4, Gly8, and Ala9 triply-labeled pin2 suggest that this peptide forms oligomers and confirms that the N-terminal region creates membrane pores.     

Loss-of-function mutations at the rim of the funnel of mechanosensitive channel MscL

MscL is a bacterial mechanosensitive channel that is activated directly by membrane stretch. Although the gene has been cloned and the crystal structure of the closed channel has been defined, how membrane tension causes conformational changes in MscL remains largely unknown. To identify the site where MscL senses membrane tension, we examined the function of the mutants generated by random and scanning mutagenesis. In vitro (patch-clamp) and in vivo (hypoosmotic-shock) experiments showed that when a hydrophilic amino acid replaces one of the hydrophobic residues that are thought to make contact with the membrane lipid near the periplasmic end of the M1 or M2 transmembrane domain, MscL loses the ability to open in response to membrane tension. Hydrophilic (asparagine) substitution of the other residues in the lipid-protein interface did not impair the channel's mechanosensitivity. These observations suggest that the disturbance of the hydrophobic interaction between the membrane lipid and the periplasmic rim of the channel's funnel impairs the function of MscL.