Mutation of RNA polymerase beta subunit (rpoB) promotes hVISA-to-VISA phenotypic conversion of strain Mu3

The clinical vancomycin-intermediate Staphylococcus aureus (VISA) strain Mu50 carries two mutations in the vraSR and graRS two-component regulatory systems (TCRSs), namely, vraS(I5N) and graR(N197S) (hereinafter designated graR). The clinical heterogeneously vancomycin-intermediate S. aureus (hVISA) strain Mu3 shares with Mu50 the mutation in vraS that encodes the VraS two-component histidine kinase. Previously, we showed that introduction of the plasmid pgraR, carrying the mutated two-component response regulator graR, converted the hVISA strain Mu3 into VISA (vancomycin MIC = 4 mg/liter). Subsequently, however, we found that the introduction of a single copy of graR into the Mu3 chromosome by a gene replacement method did not confer on Mu3 the VISA phenotype. The gene-replaced strain Mu3graR thus obtained remained hVISA (MIC ≤ 2 mg/liter), although a small increase in vancomycin MIC was observed compared to that of the parent strain Mu3. Reevaluation of the Mu3 and Mu50 genomes revealed the presence of another mutation responsible for the expression of the VISA phenotype in Mu50. Here, we demonstrate that in addition to the two regulator mutations, a third mutation found in the Mu50 rpoB gene, encoding the RNA polymerase β subunit, was required for Mu3 to achieve the level of vancomycin resistance of Mu50. The selection of strain Mu3graR with rifampin gave rise to rpoB mutants with various levels of increased vancomycin resistance. Furthermore, 3 (33%) of 10 independently isolated VISA strains established from the heterogeneous subpopulations of Mu3graR were found to possess rpoB mutations with or without an accompanying rifampin-resistance phenotype. The data indicate that a sizable proportion of the resistant hVISA cell subpopulations is composed of spontaneous rpoB mutants with various degrees of increased vancomycin resistance.     

Oncofetal protein glypican-3 in testicular germ-cell tumor

The expression of an oncofetal protein, the glypican-3 (GPC3), was immunohistochemically evaluated in a wide variety of primary testicular germ-cell tumors (GCTs) in comparison with other markers, alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG)-beta, and OCT3/4. Eighty-nine cases of GCT including 22 cases of mixed GCT were evaluated with reference to each tumor component. GPC3 expression was observed in neoplastic cells of yolk-sac tumor (YST) (25/25), teratoma (2/10), components of syncytiotrophoblastic giant cells (STGCs) (10/14), and choriocarcinoma (1/3), but none in intratubular germ-cell neoplasias, unclassified type (0/33), seminomas (0/61), or embryonal carcinoma (0/19). All cases of YST showed diffuse labeling of neoplastic cells in cytoplasmic and membranous patterns, and the positive area of GPC3 was much larger than that of AFP. Glandular structures in teratomas showed GPC3 immunostaining as well as AFP. Although the number of GPC3-positive cells was smaller in STGC components and choriocarcinoma, there was no diffusion artifact in GPC3 immunostaining, as was frequently encountered in hCG-beta staining. Thus, GPC3 is a unique oncofetal protein, which is useful as an immunohistochemical marker for GCT differentiated to extraembryonic tissue, especially YST.     

Haplotype analysis reveals founder effects of thyroglobulin gene mutations C1058R and C1977S in Japan

CONTEXT: Thyroglobulin (Tg) mutations were previously believed to be rare, resulting in congenital goitrous hypothyroidism. However, an increasing number of patients with Tg mutations, who are euthyroid to mildly hypothyroid, have been identified in Japan. OBJECTIVES: The purpose of this study was to investigate whether the three frequently found Tg mutations, namely C1058R, C1245R, and C1977S, were caused by a founder effect. RESULTS: We found 26 different mutations within the Tg gene in 52 patients from 41 families. Thirty-five patients were homozygous for the mutations, whereas the others were compound heterozygous. The occurrence of Tg mutation within the general Japanese population is one in 67,000. Patients with the C1245R mutation were found throughout Japan, whereas those with the C1058R mutation were confined to a small village on a southern island, and those with the C1977S mutation were restricted to a city. The eight patients with the C1058R mutation and the seven patients with the C1977S mutation all showed the same combinations of 18 single-nucleotide polymorphisms in the coding region of the Tg gene, which would appear in one in 810 million and one in 37 billion, respectively, control subjects. CONCLUSIONS: The frequently found mutations, C1058R and C1977S, were caused by founder effects. This result suggests that Tg mutations may provide a genetic basis for the cause of familial euthyroid goiter.     

New Delhi metallo -beta-lactamase-1 (NDM-1) producing bacteria

New Delhi metallo-beta-lactamase-1 (NDM-1) producing organisms are now becoming highly prevalent worldwide. We detected first two Japanese cases infected by Escherichia coli in Dokkyo Medical University Hospital and Klebsiella pneumoniae in Saitama Citizen Medical Center. These two NDM-1-producing bacteria are resistant to multiple antibiotic drugs including carbapenems, aminoglycosides, and fluoroquinolones. The genetic analysis showed that these two bacteria possessed different NDM-1 plasmids. Although the patient with E. coli had previously been treated in India, MLST typing showed that these bacteria are distinct from the reported ST clones elsewhere in the world. These results suggest that NDM-1 plasmids are highly variable and transferable to wide-range organisms. We warrant rigorous surveillance and strict infection control.     

Emergence of NDM-1-positive capsulated Escherichia coli with high resistance to serum killing in Japan

The New Delhi metallo-β-lactamase-1 (NDM-1) gene, bla _NDM-1, is an emerging plasmid-borne drug resistance gene, which encodes for exceptionally broad-spectrum β-lactamase, being able to hydrolyze a wide variety of β-lactams, including carbapenems, and was first reported in Klebsiella pneumoniae from a Swedish patient of Indian origin in 2009. It is widely distributed among Enterobacteriacae and has geographically exhibited extremely rapid and global spread. In this study, we characterized the bla _NDM-1-positive ST38 Escherichia coli strain NDM-1 Dok01 (which was isolated from the blood of a 54-year-old Japanese inpatient, who had previously visited India), focusing on bacterial surface structures related to virulence. The E. coli culture contained colony variants, which developed a transparent smooth colony and a rough colony on blood agar plates. The smooth colony-forming cells (substrain M1) possessed a surface capsule and were resistant to serum killing, whereas rough colony-forming mutants (substrain B2) lacked a capsule (and a 5.3-kb plasmid) and were highly susceptible to serum killing. Reflecting the surface structural difference, substrain M1 was more flagellated and motile, whereas substrain B2 was less flagellated and apparently possessed straight pili 5 nm wide, which played a role in adherence to human intestinal cells and bacterial autoaggregation. Data suggest that the bla _NDM-1-positive ST38 E. coli has emerged in Japan and that it is a capsulated bacterial pathogen with virulence potential in the blood stream.     

Association between reported education and intellectual functioning in an ethnically diverse adult psychiatric inpatient sample

Patients' pre-existing survival skills, educational attainment, and intellectual functioning should be included in the development and implementation of treatment planning for adult psychiatric inpatients. When considering culturally diverse inpatient populations with possible cultural and language barriers, these variables may attain additional importance. Utilizing a sample (N = 60) primarily consisting of Asian/Pacific Islander inpatients, the present study investigated: (a) the rate at which professionals from different disciplines inquired about educational attainment; (b) the association between self-reported education and standardized measures of intelligence; and (c) the correspondence between different IQ scores. Axis 1 diagnoses included schizophrenia (N = 19; 32%), schizoaffective disorder or bipolar disorder (N = 23; 38%), and organic or substance-related disorders (N = 18; 30%). Thirty-five percent of the sample (N = 21) had dual diagnoses. The results indicated that only psychologists who administered IQ tests consistently inquired about educational attainment. An expected overall positive association between self-reported education and standardized intelligence measures was found. High correlations between the Test of Nonverbal Intelligence, 2nd edition (TONI-2) and Wechsler Adult Intelligence Test-Revised (WAISQ-R) IQs suggested that both tests were valid in the assessment of intellectual functioning. Implications included the need for more systematic assessment and incorporation of pre-existing skill-based information and the utility of self-reported education and different measures of intellectual functioning (including TONI-2).     

Rapid screening of antineoplastic candidates for the human organic anion transporter OATP1B3 substrates using fluorescent probes

A rapid screening system has been established to extract novel candidates that exhibit potent inhibition of the transport of fluorescent substrate by organic anion transporting polypeptide (OATP) 1B3. OATP1B3 is abundantly expressed in solid digestive organ cancers. Thus, the identification of new substrates leads to novel strategies for effective cancer chemotherapy with minimal adverse effects. We used an automated image acquisition and analysis system (IN Cell Analyzer 1000) to visualize the transport and subsequent accumulation of the fluorescent substrate chenodeoxycholyl-(Nε-NBD)-lysine (CDCA-NBD). Antineoplastic screening demonstrated that five candidates agents, docetaxel, actinomycin D, mitoxantrone, paclitaxel, and SN-38, exhibited potent inhibitory effects on OATP1B3-mediated transport of CDCA-NBD. To clarify if these antineoplastic drugs are substrates for OATP1B3, we performed transport assays in OATP1B3-expressing cells. We determined that SN-38 is a novel substrate for OATP1B3. In conclusion, our results demonstrate that the screening system established in this study is a useful method for the rapid extraction of candidate therapeutic agents from the large numbers of compounds.     

Dietary restriction induces microcytic change and shortened life span of erythrocytes without anemia in mice

Dietary restriction is well known to increase mean and maximal life span in rodents and other species, although the mechanisms remain unclear. To investigate the effects of dietary restriction on erythrocyte cellular life span, mice were fed 40% restricted diet and erythrocyte properties were examined. At the end of the 8th week of a feeding period, erythrocytes in the restricted mice showed strikingly shorter life span than those in the unrestricted mice (restricted; T1/2 = 15.4 +/- 1.1 days, unrestricted; T1/2 = 18.9 +/- 1.2 days). However, the restricted mice never showed anemic conditions although they had microcytes, in addition. Significant increase in the erythrocyte count was observed in the restricted mice. These results strongly suggest that increased erythropoiesis should exist in the restricted mice, and that some unknown biophysical significance made shortened erythrocyte life span in mice fed restricted amount of diet.