A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.     

An application of β-glycosidase to transformation of ginsenosides for the effective production of specific ginsenosides with biological efficacy

Over the past several decades, the pharmacological effects of ginsenosides in Panax ginseng roots have been extensively investigated. Here, we developed a method for producing specific ginsenosides (F1 and F2) with good yields (F1:162 mg/g, F2:305 mg/g) using ??-glycosidase purified from Aspergillus niger. In addition, each ginsenoside (at least 25 species) was separated and purified by high performance liquid chromatography (HPLC) using five different types of solvents and different purification steps. In addition, the Rg3:Rh2 mixture (1:1, w/w) was shown to inhibit a specific lung cancer cell line (NCI-H232) in vivo, displaying an anticancer effect at a dose lower than achieved using treatments with single Rg3 or Rh2. This finding suggests that the combination of ginsenosides for targeting anticancer is more effective than the use of a single ginsenoside from ginseng or red ginseng.     

Ecology and Transmission of Listeria monocytogenes Infecting Ruminants and in the Farm Environment

A case-control study involving 24 case farms with at least one recent case of listeriosis and 28 matched control farms with no listeriosis cases was conducted to probe the transmission and ecology of Listeria monocytogenes on farms. A total of 528 fecal, 516 feed, and 1,012 environmental soil and water samples were cultured for L. monocytogenes. While the overall prevalence of L. monocytogenes in cattle case farms (24.4%) was similar to that in control farms (20.2%), small-ruminant (goat and sheep) farms showed a significantly (P < 0.0001) higher prevalence in case farms (32.9%) than in control farms (5.9%). EcoRI ribotyping of clinical (n = 17) and farm (n = 414) isolates differentiated 51 ribotypes. L. monocytogenes ribotypes isolated from clinical cases and fecal samples were more frequent in environmental than in feed samples, indicating that infected animals may contribute to L. monocytogenes dispersal into the farm environment. Ribotype DUP-1038B was significantly (P < 0.05) associated with fecal samples compared with farm environment and animal feedstuff samples. Ribotype DUP-1045A was significantly (P < 0.05) associated with soil compared to feces and with control farms compared to case farms. Our data indicate that (i) the epidemiology and transmission of L. monocytogenes differ between small-ruminant and cattle farms; (ii) cattle contribute to amplification and dispersal of L. monocytogenes into the farm environment, (iii) the bovine farm ecosystem maintains a high prevalence of L. monocytogenes, including subtypes linked to human listeriosis cases and outbreaks, and (iv) L. monocytogenes subtypes may differ in their abilities to infect animals and to survive in farm environments.     

The interplay between hMLH1 and hMRE11: role in MMR and the effect of hMLH1 mutations

Our previous studies indicate that hMRE11 plays a role in MMR, and this function of hMRE11 is most likely mediated by the hMLH1-hMRE11 interaction. Here, we explored the functional implications of the hMLH1-hMRE11 interaction in MMR and the effects of hMLH1 mutations on their interaction. Our in vitro MMR assay demonstrated that the dominant-negative hMRE11^452-634 mutant peptide (i.e., harboring only the hMLH1-interacting domain) imparted a significant reduction in both 3′ excision and 3′-directed MMR activities. Furthermore, the expression of hMRE11^452-634, and to a lesser extent hMRE11^1-634 (ATLD1), impaired G2/M checkpoint control in response to MNU and cisplatin treatments, rendering cells resistant to killings by these two anticancer drugs. Analysis of 38 hMLH1 missense mutations showed that the majority of mutations caused significant (>50%) reductions in their interaction with hMRE11, suggesting a potential link between aberrant protein interaction and the pathogenic effects of hMLH1 variants.     

Differential effects of secreted frizzled-related proteins (sFRPs) on osteoblastic differentiation of mouse mesenchymal cells and apoptosis of osteoblasts

Secreted frizzled-related proteins (sFRPs) are modulators of Wnt signaling. This study was undertaken for definitive assessment of contribution of different sFRPs in osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. Treatment of C3H10T1/2 cells with sFRP-2 at concentrations of 10, 50, and 100 nM and sFRP-4 at low concentrations (5 nM) significantly increased Wnt-3A-induced alkaline phosphatase (ALP) activities, whereas sFRP-1 or 3 did not. Retroviral transduction of the sFRP-2 but not other sFRPs also significantly enhanced ALP activity induced by β-glycerophosphate and ascorbic acid. Furthermore, transfection of all the sFRP expression vectors significantly increased β-catenin/TCF reporter activity and the effects were most prominent with sFRP-2 and -4. In osteoblast apoptosis assay, only sFRP-3 increased etoposide-induced apoptosis in MC3T3-E1 mouse osteoblasts. In conclusion, we found that different repertoires of sFRPs exert differential effects on osteoblastic differentiation of mouse mesenchymal cells and cellular apoptosis of mouse osteoblasts in vitro.     

Über den Zusammenhang zwischen Wasserstoffionenkonzentration,Oberflächenspannung und Wachstumsgeschwindigkeit

Zusammenfassung Die Oberflächenspannung des Preßsaftes wird durch p_H in charakteristischer Weise verändert. Im p_H 4,7 wird ein Maximum der Oberflächenspannung beobachtet, so daß in dieser Wasserstoffionenkonzentration der isoelektrische Punkt des Pflanzenpreßsaftes liegen dürfte. Es wird der Zusammenhang zwischen p_H, Oberflächenspannung und Wachstum besprochen.Mit 3 Textabbildungen.     

LTW4 Protein on Mouse Chromosome 1 Is a Member of a Family of Antioxidant Proteins

Based on its map position, polymorphism pattern, and expression in the kidney, the gene encoding liver 20,000–30,000 MW protein 4 (LTW4) can be considered a potential candidate for theJckm2modifying locus, which mediates the severity of polycystic kidney disease in thejuvenile cystic kidneymouse. Using two-dimensional gel electrophoresis, we identified variants of a 26-kDa polypeptide that differed in their isoelectric points between the C57BL/6J and the DBA/2J inbred strains in a pattern similar to that originally described for LTW4 protein. N-terminal amino acid sequence was obtained by microsequencing analysis, and full-length clones were obtained by RT-PCR amplification and characterized. The map position of the cloned gene was determined and corresponded to that previously described forLtw4.The gene has homology to a class of proteins characterized as thiol-specific antioxidants that are protective against damage caused by oxidative stress. The murine MER5 gene is also a member of this gene family and has recently been renamedAntioxidant protein 1 (Aop1),based on its functional characterization. We therefore propose that the gene encoding LTW4 be calledAop2.     

Life span and reproductive cost explain interspecific variation in the optimal onset of reproduction

Fitness can be profoundly influenced by the age at first reproduction (AFR), but to date the AFR–fitness relationship only has been investigated intraspecifically. Here, we investigated the relationship between AFR and average lifetime reproductive success (LRS) across 34 bird species. We assessed differences in the deviation of the Optimal AFR (i.e., the species‐specific AFR associated with the highest LRS) from the age at sexual maturity, considering potential effects of life history as well as social and ecological factors. Most individuals adopted the species‐specific Optimal AFR and both the mean and Optimal AFR of species correlated positively with life span. Interspecific deviations of the Optimal AFR were associated with indices reflecting a change in LRS or survival as a function of AFR: a delayed AFR was beneficial in species where early AFR was associated with a decrease in subsequent survival or reproductive output. Overall, our results suggest that a delayed onset of reproduction beyond maturity is an optimal strategy explained by a long life span and costs of early reproduction. By providing the first empirical confirmations of key predictions of life‐history theory across species, this study contributes to a better understanding of life‐history evolution.     

Mucosal T lymphocyte numbers are selectively reduced in integrin alpha E (CD103)-deficient mice.

The mucosal lymphocyte integrin alpha E(CD103)beta 7 is thought to be important for intraepithelial lymphocyte (IEL) localization or function. We cloned the murine integrin gene encoding alpha E, localized it to chromosome 11, and generated integrin alpha E-deficient mice. In alpha E-/- mice, intestinal and vaginal IEL numbers were reduced, consistent with the known binding of alpha E beta 7 to E-cadherin expressed on epithelial cells. However, it was surprising that lamina propria T lymphocyte numbers were diminished, as E-cadherin is not expressed in the lamina propria. In contrast, peribronchial, intrapulmonary, Peyer's patch, and splenic T lymphocyte numbers were not reduced in alpha E-deficient mice. Thus, alpha E beta 7 was important for generating or maintaining the gut and vaginal T lymphocytes located diffusely within the epithelium or lamina propria but not for generating the gut-associated organized lymphoid tissues. Finally, the impact of alpha E deficiency upon intestinal IEL numbers was greater at 3-4 wk of life than in younger animals, and affected the TCR alpha beta+ CD8+ T cells more than the gamma delta T cells or the TCR alpha beta+ CD4+CD8- population. These findings suggest that alpha E beta 7 is involved in the expansion/recruitment of TCR alpha beta+ CD8+ IEL following microbial colonization. Integrin alpha E-deficient mice will provide an important tool for studying the role of alpha E beta 7 and of alpha E beta 7-expressing mucosal T lymphocytes in vivo.