Classification of hybrid and altered Vibrio cholerae strains by CTX prophage and RS1 element structure

Analysis of the CTX prophage and RS1 element in hybrid and altered Vibrio cholera O1 strains showed two classifiable groups. Group I strains contain a tandem repeat of classical CTX prophage on the small chromosome. Strains in this group either contain no element(s) or an additional CTX prophage or RS1 element(s) on the large chromosome. Group II strains harbor RS1 and CTX prophage, which has an E1 Tor type rstR and classical ctxB on the large chromosome.     

Influence of broth dilution on the disruption of Escherichia coli

Summary The effect of cell concentration (5 to 150 g/L wet wt after broth dilution) on homogenizer disruption efficiency and homogenate viscosity is reported for E. coli. Broth dilution increases homogenizer efficiency and decreases feed and homogenate viscosity. However, this increase in disruption efficiency is not sufficient to warrant dilution of the broth prior to homogenization. The optimal feed concentration is the maximum possible that does not lead to practical handling difficulties due to high viscosity.     

Reagents for astatination of biomolecules. 5. Evaluation of hydrazone linkers in (211)At- and (125)I-labeled closo-decaborate(2-) conjugates of Fab' as a means of decreasing kidney retention

Evaluation of monoclonal antibody (mAb) fragments (e.g., Fab', Fab, or engineered fragments) as cancer-targeting reagents for therapy with the α-particle emitting radionuclide astatine-211 ((211)At) has been hampered by low in vivo stability of the label and a propensity of these proteins localize to kidneys. Fortunately, our group has shown that the low stability of the (211)At label, generally a meta- or para-[(211)At]astatobenzoyl conjugate, on mAb Fab' fragments can be dramatically improved by the use of closo-decaborate(2-) conjugates. However, the higher stability of radiolabeled mAb Fab' conjugates appears to result in retention of radioactivity in the kidneys. This investigation was conducted to evaluate whether the retention of radioactivity in kidney might be decreased by the use of an acid-cleavable hydrazone between the Fab' and the radiolabeled closo-decaborate(2-) moiety. Five conjugation reagents containing sulfhydryl-reactive maleimide groups, a hydrazone functionality, and a closo-decaborate(2-) moiety were prepared. In four of the five conjugation reagents, a discrete poly(ethylene glycol) (PEG) linker was used, and one substituent adjacent to the hydrazone was varied (phenyl, benzoate, anisole, or methyl) to provide varying acid sensitivity. In the initial studies, the five maleimido-closo-decaborate(2-) conjugation reagents were radioiodinated ((125)I or (131)I), then conjugated with an anti-PSMA Fab' (107-1A4 Fab'). Biodistributions of the five radioiodinated Fab' conjugates were obtained in nude mice at 1, 4, and 24 h post injection (pi). In contrast to closo-decaborate(2-) conjugated to 107-1A4 Fab' through a noncleavable linker, two conjugates containing either a benzoate or a methyl substituent on the hydrazone functionality displayed clearance rates from kidney, liver, and spleen that were similar to those obtained with directly radioiodinated Fab' (i.e., no conjugate). The maleimido-closo-decaborate(2-) conjugation reagent containing a benzoate substituent on the hydrazone was chosen for study with (211)At. That reagent was conjugated with 107-1A4 Fab', then labeled (separately) with (125)I and (211)At. The radiolabeled Fab' conjugates were coinjected into nude mice bearing LNCaP human tumor xenografts, and biodistribution data were obtained at 1, 4, and 24 h pi. Tumor targeting was achieved with both (125)I- and (211)At-labeled Fab', but the (211)At-labeled Fab' reached a higher concentration (25.56 ± 11.20 vs 11.97 ± 1.31%ID/g). Surprisingly, while the (125)I-labeled Fab' was cleared from kidney similar to earlier studies, the (211)At-labeled Fab'was not (i.e., kidney conc. for (125)I vs (211)At; 4 h, 13.14 ± 2.03 ID/g vs 42.28 ± 16.38%D/g; 24 h, 4.23 ± 1.57 ID/g vs 39.52 ± 15.87%ID/g). Since the Fab' conjugate is identical in both cases except for the radionuclide, it seems likely that the difference in tissue clearance seen is due to an effect that (211)At has on either the hydrazone cleavage or on the retention of a metabolite. Results from other studies in our laboratory suggest that the latter case is most likely. The hydrazone linkers tested do not provide the tissue clearance sought for (211)At, so additional hydrazones linkers will be evaluated. However, the results support the use of hydrazone linkers when Fab' conjugated with closo-decaborate(2-) reagents are radioiodinated.     

Effect of 1-methylcyclopropene on postharvest physiological changes of ‘Zaohong’ plum

Plum is a highly perishable fruit and postharvest fruit softening limits its shelf life. The aim of this work was to study the specific effects of 1-methylcyclopropene (1-MCP) treatment on physiological changes in ‘Zaohong’ plums. Plums were treated with 500 nL L⁻¹ 1-MCP at 20°C for 18 h followed by 20°C storage. The results showed that 1-MCP treatment significantly reduced endogenous ethylene production and the activities of ethylene biosynthetic enzymes’ (1-aminocyclopropane-1-carboxylic acid synthase, ACS and 1-aminocyclopropane-1-carboxylic acid oxidase, ACO) in plum fruit during storage when compared with untreated fruit. Although 1-MCP treatment inhibited ethylene production and 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation, it did not inhibit the accumulation of N-malonyl-ACC (MACC). Higher firmness was also found in 1-MCP-treated plums than in controls. During storage, superoxide anion (O₂^−·) and hydrogen peroxide (H₂O₂) levels decreased in 1-MCP-treated fruit. 1-MCP treatment also regulated superoxide dismutase (SOD) and catalase (CAT) activities during storage. Xylanase activity was upregulated while activities of polygalacturonase (PG), pectin methyl esterase (PME) and cellulase enzymes in the fruit were downregulated by 1-MCP treatment. In conclusion, 1-MCP might be a potent compound for extending both storage period and shelf life of ‘Zaohong’ plums by suppressing ethylene biosynthesis, regulating cell wall degradation enzymes and reducing fruit softening.     

Temporal and Spatial Analysis of Clinical and Molecular Parameters in Dextran Sodium Sulfate Induced Colitis

Background

Inflammatory bowel diseases (IBD), including mainly ulcerative colitis (UC) and Crohn''s disease (CD), are inflammatory disorders of the gastrointestinal tract caused by an interplay of genetic and environmental factors. Murine colitis model induced by Dextran Sulfate Sodium (DSS) is an animal model of IBD that is commonly used to address the pathogenesis of IBD as well as to test efficacy of therapies. In this study we systematically analyzed clinical parameters, histological changes, intestinal barrier properties and cytokine profile during the colitic and recovery phase.

Methods

C57BL/6 mice were administered with 3.5% of DSS in drinking water for various times. Clinical and histological features were determined using standard criteria. Myeloperoxidase (MPO) activity, transepithelial permeability and proinflammatory mediators were determined in whole colon or proximal and distal parts of colon.

Results

As expected after administration of DSS, mice manifest loss of body weight, shortening of colon length and bloody feces. Histological manifestations included shortening and loss of crypts, infiltration of lymphocytes and neutrophil, symptoms attenuated after DSS withdrawal. The MPO value, as inflammation indicator, also increases significantly at all periods of DSS treatment, and even after DSS withdrawal, it still held at very high levels. Trans-mucosal permeability increased during DSS treatment, but recovered to almost control level after DSS withdrawal. The production of proinflammatory mediators by colonic mucosa were enhanced during DSS treatment, and then recovered to pre-treated level after DSS withdrawal. Finally, enhanced expression of proinflammatory mediators also revealed a different profile feature in proximal and distal parts of the colon.

Conclusion

Experimental colitis induced by DSS is a good animal model to study the mechanisms underlying the pathogenesis and intervention against IBD, especially UC.     

Limited chloroplast gene transfer via recombination overcomes plastomegenome incompatibility between Nicotiana tabacum and Solanum tuberosum

Green cybrids with a new nucleus-chloroplast combination cannot be selected after protoplast fusion in the intersubfamilial Nicotiana-Solanum combination. As an approach to overcome the supposed plastomegenome incompatibility, a partial plastome transfer by genetic recombination has been considered. After fusions of protoplasts of a light-sensitive Nicotiana tabacum (tobacco) plastome mutant and lethally irradiated protoplasts of wild-type Solanum tuberosum (potato), a single green colony was recovered among 2.5×10⁴ colonies. The regenerated plants had tobacco-like (although abnormal) morphology, but were normally green, and sensitive to tentoxin, demonstrating chloroplast markers of the potato parent. Restriction enzyme analysis of the chloroplast DNA (cpDNA) revealed recombinant, nonparental patterns. A comparison with physical maps of the parental cpDNA demonstrated the presence of a considerable part of the potato plastome flanked by tobacco-specific regions. This potacco plastome proved to be stable in backcross and backfusion experiments, and normally functional in the presence solely of N. tabacum nucleus.