Fanconi anemia: genetic analysis of a human disease using chicken system

Fanconi anemia (FA) is a rare hereditary disorder characterized by skeletal abnormalities, bone marrow failure, and an increased incidence of cancer. The basic cellular abnormality in FA has been postulated to lie in the DNA repair mechanisms because cells from FA patients display chromosomal breakage, which is particularly remarkable following induction of DNA crosslinks. However, experimental evidence for this hypothesis has been lacking. To test whether DNA repair is really defective in FA cells, we disrupted several FA genes in chicken B cell line DT40. By measuring efficiency of gene conversion and hypermutation at the Immunoglobulin locus, we have shown that DT40 FA mutant cell lines exhibited defects in homologous DNA recombination, and possibly, translesion synthesis. However, levels of sister chromatid exchange, another important cellular event mediated by HR, were not reduced, possibly indicating the role of FA genes only in a subpathway of HR. Our results indicate that chicken DT40 cells could be highly useful in molecular dissection of basic biochemical processes, which are deficient in a human genetic disorder.     

Characterization of Mesorhizobium loti L-rhamnose isomerase and its application to L-talose production

The L-rhamnose isomerase gene (rhi) of Mesorhizobium loti was cloned and expressed in Escherichia coli, and then characterized. The enzyme exhibited activity with respect to various aldoses, including D-allose and L-talose. Application of it in L-talose production from galactitol was achieved by a two-step reaction, indicating that it can be utilized in the large-scale production of L-talose.     

Regulation of monocyte subset proinflammatory responses within the lung microvasculature by the p38 MAPK/MK2 pathway

Margination and activation of monocytes within the pulmonary microcirculation contribute substantially to the development of acute lung injury in mice. The enhanced LPS-induced TNF expression exhibited by Gr-1(high) compared with Gr-1(low) monocytes within the lung microvasculature suggests differential roles for these subsets. We investigated the mechanisms responsible for such heterogeneity of lung-marginated monocyte proinflammatory response using a combined in vitro and in vivo approach. The monocyte subset inflammatory response was studied in vitro in mouse peripheral blood mononuclear cell-lung endothelial cell coculture and in vivo in a two-hit model of intravenous LPS-induced monocyte margination and lung inflammation in mice, by flow cytometry-based quantification of proinflammatory genes and intracellular phospho-kinases. With LPS stimulation in vitro, TNF expression was consistently higher in Gr-1(high) than Gr-1(low) monocytes, markedly enhanced by coculture with endothelial cells, and abrogated by p38 MAPK inhibitors. Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was only detectable under coculture conditions, was substantially higher in Gr-1(high) monocytes, and was attenuated by p38 inhibition. Consistent with these differential responses, phosphorylation of p38 and its substrate MAPK-activated protein kinase 2 (MK2) was significantly higher in the Gr-1(high) subset. In vivo, p38 inhibitor treatment significantly attenuated LPS-induced TNF expression in "lung-marginated" Gr-1(high) monocytes. LPS-induced p38/MK2 phosphorylation was higher in lung-marginated Gr-1(high) than Gr-1(low) monocytes and neutrophils, mirroring TNF expression. These results indicate that the p38/MK2 pathway is a critical determinant of elevated Gr-1(high) subset responsiveness within the lung microvasculature, producing a coordinated proinflammatory response that places Gr-1(high) monocytes as key orchestrators of pulmonary microvascular inflammation and injury.     

The lactic acid bacterium Pediococcus acidilactici suppresses autoimmune encephalomyelitis by inducing IL-10-producing regulatory T cells

Background

Certain intestinal microflora are thought to regulate the systemic immune response. Lactic acid bacteria are one of the most studied bacteria in terms of their beneficial effects on health and autoimmune diseases; one of which is Multiple sclerosis (MS) which affects the central nervous system. We investigated whether the lactic acid bacterium Pediococcus acidilactici, which comprises human commensal bacteria, has beneficial effects on experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Methodology/Principal Findings

P. acidilactici R037 was orally administered to EAE mice to investigate the effects of R037. R037 treatment suppressed clinical EAE severity as prophylaxis and therapy. The antigen-specific production of inflammatory cytokines was inhibited in R037-treated mice. A significant increase in the number of CD4⁺ Interleukin (IL)-10-producing cells was observed in the mesenteric lymph nodes (MLNs) and spleens isolated from R037-treated naive mice, while no increase was observed in the number of these cells in the lamina propria. Because only a slight increase in the CD4⁺Foxp3⁺ cells was observed in MLNs, R037 may primarily induce Foxp3⁻ IL10-producing T regulatory type 1 (Tr1) cells in MLNs, which contribute to the beneficial effect of R037 on EAE.

Conclusions/Significance

An orally administered single strain of P. acidilactici R037 ameliorates EAE by inducing IL10-producing Tr1 cells. Our findings indicate the therapeutic potential of the oral administration of R037 for treating multiple sclerosis.     

Total and high molecular weight adiponectin and hepatocellular carcinoma with HCV infection

Background

Adiponectin is shown to be inversely associated with development and progression of various cancers. We evaluated whether adiponectin level was associated with the prevalence and histological grade of hepatocellular carcinoma (HCC), and liver fibrosis in patients with hepatitis C virus (HCV) infection.

Methods

A case-control study was conducted on 97 HCC patients (cases) and 97 patients (controls) matched for sex, Child-Pugh grade and platelet count in patients with HCV infection. The serum total and high molecular weight (HMW) adiponectin levels were measured by enzyme-linked immunosorbent assays and examined in their association with the prevalence of HCC. In addition, the relationship between these adiponectin levels and body mass index (BMI), progression of liver fibrosis, and histological grade of HCC was also evaluated. Liver fibrosis was assessed using the aspartate aminotransferase to platelet ratio index (APRI).

Results

There were no significant differences in the serum total and HMW adiponectin levels between cases and controls. Moreover, there were no inverse associations between serum total and HMW adiponectin levels and BMI in both cases and controls. On the other hand, serum total and HMW adiponectin levels are positively correlated with APRI in both cases (r = 0.491, P<0.001 and r = 0.485, P<0.001, respectively) and controls (r = 0.482, P<0.001 and r = 0.476, P<0.001, respectively). Interestingly, lower serum total (OR 11.76, 95% CI: 2.97–46.66 [P<0.001]) and HMW (OR 10.24, CI: 2.80–37.40 [P<0.001] adiponectin levels were independent risk factors of worse histological grade of HCC.

Conclusions

Our results suggested that serum total and HMW adiponectin levels were predictors of liver fibrosis, but not prevalence of HCC in patients with HCV infection. Moreover, low these adiponectin levels were significantly associated with worse histological grades.     

Relationship of number of remaining teeth to health-related quality of life in community-dwelling elderly

Objective: The aim of this study was to evaluate the relationship between number of remaining teeth and health‐related quality of life in community‐dwelling elderly. Subjects: A total of 207 participants who were community‐dwelling, 85 years of age. Data were from a population‐based study of age‐related general and oral health in Fukuoka Prefecture, Japan. Measurements: The Japanese version of the Short Form 36 Health Survey (SF‐36). Results: The mental component score for the participants, from the SF‐36, was higher than the Japanese national norm for those aged ≥70 years. There were no significant differences in the mean of any scores on the SF‐36 by having spouse, living with family, or education level. The mean of the SF‐36 scores of physical functioning (PF) and of the physical component scores were significantly higher in the 85‐year‐old participants with ≥20 teeth than in those with ≤19 teeth (p < 0.05 and p < 0.01 respectively). In addition, a significant difference (p < 0.05) was observed between the mean of participants with ≥20 teeth and those with ≤19 teeth after adjustment for region where the participant lived, activities of daily living (ADL), and sex. The PF (p < 0.001), role‐physical (p < 0.005), bodily pain (p < 0.001), vitality (p < 0.001), social functioning (p < 0.05), and physical component (p < 0.001) scores were significantly higher in participants with a good activities of daily living (ADL) assessment. However, ADL was not associated with the number of teeth. Conclusions: The findings of the present study indicated that 85‐year‐old participants with ≥20 teeth had better subjective physical health than those with ≤19 teeth.     

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.     

A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair

In DNA damage responses, the Fanconi anemia (FA) protein, FancD2, is targeted to chromatin and forms nuclear foci following its monoubiquitination, a process likely catalyzed by the FA core complex. Here, we show that a chicken FancD2-ubiquitin fusion protein, carrying a Lys-Arg substitution removing the natural monoubiquitination site (D2KR-Ub), could reverse cisplatin hypersensitivity and localize to chromatin in FANCD2-deficient DT40 cells. Importantly, the chromatin targeting was dependent on three core complex components as well as the hydrophobic surface of ubiquitin that may direct protein-protein interactions. Furthermore, a constitutively chromatin bound fusion of D2KR-histone H2B could complement cisplatin sensitivity in FANCD2- but not FANCC-, FANCG-, or FANCL-deficient cells. Thus these core complex components have an additional function in the DNA repair, which is independent of the monoubiquitination and chromatin targeting of FancD2. These results define functional consequences of FancD2 monoubiquitination and reveal previously hidden functions for the FA protein core complex.     

Wheat cultivar-specific proteins in grain revealed by 2-DE and their application to cultivar identification of flour

Wheat flour proteins were studied to identify the cultivar-specific proteins and use them to identify cultivars in flours. Proteins extracted from flours of Japanese wheat (cultivars Hokushin, Horoshirikomugi, Kitanokaori and Kachikei 33) and Canadian wheat (Canada Western Red Spring Wheat No. 1; 1CW) were analyzed by 2-DE with IEF gels over three pH ranges: pH 4-7, pH 5-8, and pH 6-11. This system enabled detection of more than 1600 protein spots. We recognized that among 50 protein spots showing cultivar-dependent qualitative changes, 25 proteins were wheat cultivar specific. These 50 protein spots were analyzed by N-terminal Edman degradation microsequencing and MALDI-TOF-MS; 21 protein spots were storage proteins, such as gliadin and low-molecular mass glutenin subunit. Five protein spots were identified as dehydroascorbate reductase (Triticum aestivum), triticin precursor (T. aestivum), alpha-amylase inhibitor (Oryza sativa), DNA-binding with one finger (Dof) zinc family protein (O. sativa), and nonphototropic hypocotyl 1 (NPH1) protein (Avena sativa). The other protein spots appeared to be hypothetical proteins (O. sativa or Arabidopsis thaliana) or functional unknown proteins. These specific proteins can be used as markers to identify wheat cultivars in blended flour composed of two or three flours.     

ATPase activity of p97/valosin-containing protein is regulated by oxidative modification of the evolutionally conserved cysteine 522 residue in Walker A motif

Valosin-containing protein (p97/VCP) has been proposed as playing crucial roles in a variety of physiological and pathological processes such as cancer and neurodegeneration. We previously showed that VCP(K524A), an ATPase activity-negative VCP mutant, induced vacuolization, accumulation of ubiquitinated proteins, and cell death, phenotypes commonly observed in neurodegenerative disorders. However, any regulatory mechanism of its ATPase activity has not yet been clarified. Here, we show that oxidative stress readily inactivates VCP ATPase activity. With liquid chromatography/tandem mass spectrometry, we found that at least three cysteine residues were modified by oxidative stress. Of them, the 522nd cysteine (Cys-522) was identified as the site responsible for the oxidative inactivation of VCP. VCP(C522T), a single-amino acid substitution mutant from cysteine to threonine, conferred almost complete resistance to the oxidative inactivation. In response to oxidative stress, VCP strengthened the interaction with Npl4 and Ufd1, both of which are essential in endoplasmic reticulum-associated protein degradation. Cys-522 is located in the second ATP binding motif and is highly conserved in multicellular but not unicellular organisms. Cdc48p (yeast VCP) has threonine in the corresponding amino acid, and it showed resistance to the oxidative inactivation in vitro. Furthermore, a yeast mutant (delta cdc48 + cdc48[T532C]) was shown to be susceptible to oxidants-induced growth inhibition and cell death. These results clearly demonstrate that VCP ATPase activity is regulated by the oxidative modification of the Cys-522 residue. This regulatory mechanism may play a key role in the conversion of oxidative stress to endoplasmic reticulum stress response in multicellular organisms and also in the pathological process of various neurodegenerative disorders.