Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).     

Structure of Bovine κ-Casein: Spectrophotometric Titration and Molecular Size Changes in Alkaline Solution

The ionization of tyrosyl groups in bovine κ-casein and S-carboxyamidomethyl-κ-casein (CAM-κ) was studied by spectrophotometric titration at 295 mµ. In the denaturing solvent 8 m urea, the titration curves are reversible and the pK_app values of eight tyrosyl groups both in κ-casein and in CMA-κ-casein are 10.7. In 0.2 m KCl solution, κ-casein has six tyrosyl groups with normal pK_app value of 10.5 and two groups with higher pK_app value of 11.4. CAM-κ-casein has eight tyrosyl groups with pK_app value of 10.6 in 0.2 m KCl solution. These observations suggest that -S-S- bondings in κ-casein are concerned with the ‘masking’ of the tyrosyl groups. The evidence of the rupture of intermolecular -S-S- bondings of κ-casein in alkaline solution was shown by the study of gel Chromatograph y on Sephadex G–150. One of the possible explanation is that the ionization of tyrosyl groups with higher pK_app value is associated with the destruction of hydrophobic regions, and this destruction is due to the rupture of intermolecular -S-S- bondings under alkaline conditions.     

Assessment of the salt tolerance and environmental biosafety of Eucalyptus camaldulensis harboring a mangrin transgene

Increasing soil salinization of arable land has a major impact on the global ecosystem. One approach to increase the usable global forest area is to develop transgenic trees with higher tolerance to conditions of salt stress. An allene oxide cyclase homolog, mangrin, contains a core protein domain that enhances the salt tolerance of its host. We utilized this feature to develop improved salt-tolerant eucalyptus trees, by using transgenic Eucalyptus camaldulensis carrying the mangrin gene as a model. Since the Japanese government requires an environmental biosafety assessment for the surrounding biosphere, we performed experiments on trees grown in a special netted-house. This study examined the transgenic E. camaldulensis carrying the mangrin gene to assess the feasibility of using these transformants, and assessed their salt tolerance and environmental biosafety. We found that seven of 36 transgenic genotypes had significantly higher salt tolerance than non-transformants, and more importantly, that these plants had no significant impact on environmental biosafety. These results suggest that introduction of the mangrin gene may be one approach to safely enhance salt tolerance in genetically modified Eucalyptus species, and that the transformants have no apparent risks in terms of environmental biosafety. Thus, this study provides valuable information regarding the use of transgenic trees in situ.     

A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes

A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.     

Drug-Related Hospital Visits and Admissions Associated with Laboratory or Physiologic Abnormalities—A Systematic-Review

Countless studies have demonstrated that many emergency-room visits and hospital admissions are drug-related and that a significant proportion of these drug-related visits (DRVs) are preventable. It has not been previously studied which DRVs could be prevented through enhanced monitoring of therapy. The objective of the study was to determine the incidence of DRVs attributed to laboratory or physiologic abnormalities. Three authors independently performed comprehensive searches in relevant health care databases using pre-determined search terms. Articles discussing DRV associated with poisoning, substance abuse, or studied among existing in-patient populations were excluded. Study country, year, sample, design, duration, DRV identification method, proportion of DRVs associated with laboratory or physiologic abnormalities and associated medications were extracted. The three authors independently assessed selected relevant articles according to the Strengthening the reporting of observational studies in epidemiology (STROBE) as applicable according to the studies'' methodology. The initial literature search yielded a total of 1,524 articles of which 30 articles meeting inclusion criteria and reporting sufficient laboratory or physiologic data were included in the overall analysis. Half employed prospective methodologies, which included both chart review and patient interview; however, the overwhelming majority of identified studies assessed only adverse drug reactions (ADRs) as a drug-related cause for DRV. The mean (range) prevalence of DRVs found in all studies was 15.4% (0.44%–66.7%) of which an association with laboratory or physiologic abnormalities could be attributed to a mean (range) of 29.4% (4.3%–78.1%) of cases. Most laboratory-associated DRVs could be linked to immunosuppressant, antineoplastic, anticoagulant and diabetes therapy, while physiologic-associated DRVs were attributed to cardiovascular therapies and NSAIDs. Significant proportions of laboratory and physiologic abnormalities contribute to DRVs and are consistently linked to specific drugs. These therapies are potential targets for enhanced medication monitoring initiatives to proactively avert potential DRVs.     

Pathogenicity of Genetically Similar,H5N1 Highly Pathogenic Avian Influenza Virus Strains in Chicken and the Differences in Sensitivity among Different Chicken Breeds

Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.     

Age at Transition from Pediatric to Adult Care Has No Relationship with Mortality for Childhood-Onset Type 1 Diabetes in Japan: Diabetes Epidemiology Research International (DERI) Mortality Study

Objective

To follow up Japanese patients with type 1 diabetes for a maximum of 40 years to examine when they transitioned from pediatric care to adult care and to explore whether the attending physician, i.e., pediatrician or internist, was associated with prognosis.

Methods

Participants consisted of 1,299 patients who had been diagnosed as having type 1 diabetes at less than 15 years old between 1965 and 1979 identified through two nationwide surveys. Patients were classified as having received either pediatric care or adult care at the age of 15 and 30, and were compared for differences in mortality associated with the attending physician.

Results

The attending physicians were confirmed for a total of 1,093 patients at the age of 15. Of these patients, 43.8% and 40.3% received pediatric care and adult care, respectively. Of the 569 patients receiving pediatric care, 74.2%, 56.6%, 53.4%, and 51.3% continued with pediatric care at 20, 30, 40, and 50 years old, respectively. The attending physicians (pediatrician or internist) at the age of 15 and 30 had no significant impact on their survival (P = 0. 892, 0.411, respectively).

Conclusions

More than half of the patients who had received pediatric care at the age of 15 continued to receive pediatric care even after the age of 30, suggesting that their transition was far from smooth, while the attending physician at the age of both 15 and 30 was not a prognostic factor for mortality. Thus, the timing for transition to adult care in these patients has no relationship with mortality in Japan.     

Human Leukocyte Antigen and Systemic Sclerosis in Japanese: The Sign of the Four Independent Protective Alleles,DRB1*13:02, DRB1*14:06, DQB1*03:01, and DPB1*02:01

ObjectiveSeveral studies on associations between human leukocyte antigen (HLA) allele frequencies and susceptibility to systemic sclerosis (SSc) have been reported. Anti-centromere antibodies (ACA) and anti-topoisomerase I antibodies (ATA) are found in SSc patients. Here, we sought to identify HLA alleles associated with SSc in Japanese, and explored their associations with SSc phenotypes including the presence of autoantibodies.MethodsAssociations of HLA-DRB1, DQB1, and DPB1 were analyzed in 463 Japanese SSc patients and 413 controls.ResultsWe found that DRB1*13:02 (P = 0.0011, Pc = 0.0319, odds ratio [OR] 0.46, 95% confidence interval [CI] 0.29–0.73), DRB1*14:06 (P = 6.60X10^-5, Pc = 0.0020, OR 0.05, 95%CI 0.01–0.41), DQB1*03:01 (P = 0.0009, Pc = 0.0150, OR 0.56, 95%CI 0.40–0.79), and DPB1*02:01 (P = 5.16X10^-6, Pc = 8.77X10^-5, OR 0.52, 95%CI 0.39–0.69) were protectively associated with SSc. In addition, these four alleles seemed to be independently associated with the protection against the susceptibility of SSc. On the other hand, we could not find predisposing alleles for overall SSc. With respect to SSc subsets, a tendency for these four alleles to be protectively associated was observed. However, there was a significant association between DRB1*01:01, DRB1*10:01, DQB1*05:01, and DPB1*04:02 and the susceptibility to SSc with ACA. On the other hand, the presence of DRB1*15:02, DQB1*06:01, DPB1*03:01, and DPB1*09:01 was associated with SSc with ATA.ConclusionThus, the present study has identified protective associations of the four HLA class II alleles with overall Japanese SSc and predisposing associations of HLA class II alleles with Japanese SSc subsets.     

IL-25, IL-33 and TSLP receptor are not critical for development of experimental murine malaria

IL-25, IL-33 and TSLP, which are produced predominantly by epithelial cells, can induce production of Th2-type cytokines such as IL-4, IL-5 and/or IL-13 by various types of cells, suggesting their involvement in induction of Th2-type cytokine-associated immune responses. It is known that Th2-type cytokines contribute to host defense against malaria parasite infection in mice. However, the roles of IL-25, IL-33 and TSLP in malaria parasite infection remain unclear. Thus, to elucidate this, we infected wild-type, IL-25^?/?, IL-33^?/? and TSLP receptor (TSLPR)^?/? mice with Plasmodium berghei (P. berghei) ANKA, a murine malaria strain. The expression levels of IL-25, IL-33 and TSLP mRNA were changed in the brain, liver, lung and spleen of wild-type mice after infection, suggesting that these cytokines are involved in host defense against P. berghei ANKA. However, the incidence of parasitemia and survival in the mutant mice were comparable to in the wild-type mice. These findings indicate that IL-25, IL-33 and TSLP are not critical for host defense against P. berghei ANKA.