Frequent overexpression of CADM1/IGSF4 in lung adenocarcinoma

We reported comprehensive screening for antigens (Ags) overexpressed on various carcinomas via isolation of human monoclonal antibodies (mAbs) that may be therapeutic in a previous paper (Proc. Natl. Acad. Sci. USA 105, 7287-7292, 2008). Twenty-one distinct Ags highly expressed on several carcinomas were identified and 356 mAbs with unique sequences turned out to bind to one of the 21 Ags. Among them CADM1/IGSF4 which had been originally referred to as tumor suppressor lung cancer 1 (TSLC1) was included. Therefore we examined the expression of CADM1 in lung cancers in this study. Eight different anti CADM1 mAbs were used for immunohistochemical analysis of 29 fresh lung cancer specimens. Staining patterns were categorized to six groups based on the extent of positive staining and the localization of stained portions. While overexpression of CADM1 was observed on the cell surface of adenocarcinomas at a high frequency, around 60%, positive stainings were rarely observed on that of other lung carcinomas including squamous cell carcinomas. Moreover, some clones among the eight mAbs gave different staining patterns from those by the other clones against the same fresh specimen, suggesting presence of variant forms of CADM1 differentiated by mAbs.     

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

A culture system for the live analysis of successive developmental processes and the morphological control of mammalian vertebral cartilage

The mesoderm-derived segmental somite differentiates into dermomyotome and sclerotome, the latter of which undergoes vertebrogenesis to spinal cartilage and ultimately to vertebral bones. However, analysis and manipulation of the developing mammalian vertebrae in the same embryo has been infeasible because of their placental-dependent embryogenesis. Here, we report a novel culture system of the mouse embryonic tailbud, by which the developmental processes of mammalian vertebral cartilage are traceable and manipulatable in the same sample. The anaplastic segmental somites/sclerotomes in the tailbud of 13 gestational day (g.d.) embryo that are structurally continuous to the vertebral column underwent progressive vertebrogenesis when (1) the ectoderm-derived nascent epidermis was microsurgically removed prior to cultivation, and (2) the sample was incubated at the air-medium interface. After cultivation for 5 days, the size and shape of the instructed vertebral cartilage showed features comparable to well-differentiated body vertebra along with the expression of the cartilage marker collagen type II, suggesting that aggressive differentiation of the sclerotomal cell lineage was achieved. In the presence of recombinant bone morphogenic protein (BMP) and Noggin, or adenoviral particles for extracellular epimorphin, dramatic alteration of the vertebral morphology ensued in the explants. Thus, this model system provides an approach to study the detailed molecular mechanisms of mammalian vertebrogenesis and enables pretreatment strategies of precartilagious fragments for improving the efficacy of subsequent transplantation.     

Discovery of potent, selective, orally active benzoxazepine-based Orexin-2 receptor antagonists

During our efforts to identify a series of potent, selective, orally active human Orexin-2 Receptor (OX2R) antagonists, we elucidated structure-activity relationship (SAR) on the 7-position of a benzoxazepine scaffold by utilizing Hammett σ(p) and Hansch-Fujita π value as aromatic substituent constants. The attempts led to the discovery of compound 1m, possessing good in vitro potency with over 100-fold selectivity against OX1R, good metabolic stability in human and rat liver microsome, good oral bioavailability in rats, and in vivo antagonistic activity in rats by oral administration.     

Hybrid Transverse Magneto-Thermoelectric Cooling in Artificially Tilted Multilayers

In artificially tilted multilayers comprising two different conductors that are alternately and obliquely stacked, transverse thermoelectric conversion occurs, in which charge and heat currents are interconverted in the orthogonal direction. Although transverse thermoelectric conversion also occurs in homogeneous materials as an intrinsic transport phenomenon owing to the effects of magnetic fields, magnetization, and spins on conduction carriers, such magneto-thermoelectric effects are investigated independently of thermoelectrics for artificially tilted multilayers. Here, this study shows that the synergy of these different principles improves the performance of transverse thermoelectric conversion. Using lock-in thermography techniques, transverse thermoelectric conversion processes are visualized in artificially tilted multilayers and the experiments clarify how nonuniform charge currents are converted into orthogonal heat currents. Through the measurements of temperature change under magnetic fields, the contributions of the magneto-thermoelectric effects are quantified in the artificially tilted multilayers and magnetically enhanced hybrid transverse thermoelectric cooling is demonstrated. By replacing one of the conductors in the multilayer with permanent magnets, the same functionality is obtained even in the absence of magnetic fields, paving the way for the creation of “thermoelectric permanent magnets” that exhibit efficient transverse thermoelectric conversion together with spontaneous magnetization. This study provides a new material design guideline for transverse thermoelectrics.     

Mechanistic insights into tRNA cleavage by a contact-dependent growth inhibitor protein and translation factors

Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA–CT) into neighboring bacteria. The CdiA–CT of enterohemorrhagic Escherichia coli EC869 (CdiA–CT^EC869) cleaves the 3′-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA–CT^EC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA–CT^EC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA–CT^EC869 to the same extent in the presence of Tu/Ts, and the CdiA–CT^EC869:Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA–CT^EC869 interacts with domain II of Tu, thereby preventing the 3′-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA–CT^EC869:Tu structure suggests that the 3′-portion of tRNA relocates into the CdiA–CT^EC869 active site, located on the opposite side to the CdiA–CT^EC869 :Tu interface, for tRNA cleavage. Thus, CdiA–CT^EC869 is recruited to Tu:GTP:Ts, and CdiA–CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA–CT^EC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA–CT^EC869.     

Mycobacterium tuberculosis Bacteremia in a Cohort of HIV-Infected Patients Hospitalized with Severe Sepsis in Uganda–High Frequency,Low Clinical Sand Derivation of a Clinical Prediction Score

Background

When manifested as Mycobacterium tuberculosis (MTB) bacteremia, disseminated MTB infection clinically mimics other serious blood stream infections often hindering early diagnosis and initiation of potentially life-saving anti-tuberculosis therapy. In a cohort of hospitalized HIV-infected Ugandan patients with severe sepsis, we report the frequency, management and outcomes of patients with MTB bacteremia and propose a risk score based on clinical predictors of MTB bacteremia.

Methods

We prospectively enrolled adult patients with severe sepsis at two Ugandan hospitals and obtained blood cultures for MTB identification. Multivariable logistic regression modeling was used to determine predictors of MTB bacteremia and to inform the stratification of patients into MTB bacteremia risk categories based on relevant patient characteristics.

Results

Among 368 HIV-infected patients with a syndrome of severe sepsis, eighty-six (23%) had MTB bacteremia. Patients with MTB bacteremia had a significantly lower median CD4 count (17 vs 64 lymphocytes/mm³, p<0.001) and a higher 30-day mortality (53% vs 32%, p = 0.001) than patients without MTB bacteremia. A minority of patients with MTB bacteremia underwent standard MTB diagnostic testing (24%) or received empiric anti-tuberculosis therapy (15%). Independent factors associated with MTB bacteremia included male sex, increased heart rate, low CD4 count, absence of highly active anti-retroviral therapy, chief complaint of fever, low serum sodium and low hemoglobin. A risk score derived from a model containing these independent predictors had good predictive accuracy [area under the curve = 0.85, 95% CI 0.80–0.89].

Conclusions

Nearly 1 in 4 adult HIV-infected patients hospitalized with severe sepsis in 2 Ugandan hospitals had MTB bacteremia. Among patients in whom MTB was suspected, standard tests for diagnosing pulmonary MTB were inaccurate for correctly classifying patients with or without bloodstream MTB infection. A MTB bacteremia risk score can improve early diagnosis of MTB bacteremia particularly in settings with increased HIV and MTB co-infection.     

Dual subcellular localization in the endoplasmic reticulum and peroxisomes and a vital role in protecting against oxidative stress of fatty aldehyde dehydrogenase are achieved by alternative splicing

Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.     

Molecular taxonomy of dermatophytes and related fungi by chitin synthase 1 (CHS1) gene sequences

In the present study, the nucleotide sequences of the CHS1 gene from dermatophytes and related fungi in the genera Chrysosporium, Epidermophyton, Microsporum and Trichophyton were investigated using molecular methods. About 440-bp genomic DNA fragments of the CHS1 gene from 21 species were amplified by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these fungi showed more than 83% similarity. The molecular taxonomy of the CHS1 gene sequences revealed that Microsporum was genetically distinct from Chrysosporium and Trichophyton, as classified by morphological characteristics. This revised version was published online in June 2006 with corrections to the Cover Date.     

Short tandem repeat polymorphism in the flanking region of the human phosphoglycerate kinase gene in a Japanese population

The human phosphoglycerate kinase (PGK1) gene is located within Xqll-Xql3 and is closely linked to the androgen receptor gene within a region implicated in a number of X-chromosome-linked urologic disorders. A polymorphism of a TATC short tandem repeat (STR) is present downstream from the PGK1 3' nuclease-sensitive site. We present the PGK1 flanking STR sequence and population genetic data for 190 Japanese males and 83 Japanese females. Ten STR alleles and 29 genotypes were identified in the population. Five alleles--*10, *11, *12, *13, and *14--were common in the Japanese with frequencies greater than 10%. No significant deviations from Hardy-Weinberg equilibrium were established. The power of discrimination was 0.993 for females and 0.819 for males; heterozygosity was 0.759 for females; and the polymorphic information content was 0.936. These data indicate that this STR locus shows a high degree of polymorphism in this Japanese population and may prove to be a useful genetic marker in forensic medicine, in determining the clonality of neoplasms, and potentially in studying predisposition to prostate cancer and other urologic diseases.