Senescence marker protein-30 regulates Akt activity and contributes to cell survival in Hep G2 cells

Senescence marker protein-30 (SMP30) is highly expressed in cytosol of hepatocytes, and its amount decreases with aging. Human hepatocellular carcinoma cell line was transfected with pcDNA3/SMP30 (SMP30 transfectants), or as a control with pcDNA3 (mock transfectants). When cells were exposed to 20 ng/ml tumor necrosis factor-alpha (TNF-alpha) plus 10 ng/ml actinomycin D (Act-D) for 15 h, the viability of cells was decreased in both SMP30 and mock transfectants. However, the viability of cells was threefold higher in SMP30 transfectants than mock transfectants. Cell death was confirmed as apoptosis by TUNEL assay. The presence of trifluoperazine, a calmodulin (CaM) inhibitor, attenuated anti-apoptotic effect of SMP30 in both transfectants, but the effect was more prominent in SMP30 transfectants. Western blot analyses revealed that Akt, which acts as a survival factor in cells, was activated in SMP30, but not mock, transfectants either in the presence or absence of TNF-alpha plus Act-D. Further, trifluoperazine inhibited Akt activation in SMP30 transfectants. We therefore propose that interplay between CaM and SMP30 regulates Akt activity, and thus SMP30 acts as a survival factor in hepatocytes.     

Early differential gene expression of rat lung after exposure to paraquat

Paraquat (PQ), a quaternary nitrogen herbicide, is highly toxic to humans and animals. Acute poisoning and death due to PQ exposure have been reported over the past few decades. Excessive production of oxygen free radicals has been proposed to play an important role in the pulmonary pathology. The aim of the present work was to evaluate the implications for genes that are regulated by oxidative stress at the early stage of PQ exposure in rat lungs. We performed differential display RT-PCR (DD-PCR) on total RNA extracted from rat lungs after injection of 20mg per kg body weight. The experimental DD-PCR conditions, primer length and annealing temperature, were adjusted to improve reproducibility, and 19 differentiated clones were isolated. Sequence analysis followed by conventional RT-PCR and real-time RT-PCR analyses were used to confirm the results. Four clones were finally determined to be significantly affected. These genes were mRNAs for plasma phospholipid transfer protein (PLTP), CL1BA protein, (latrophilin: LPH), and alphaII-spectrin as well as one unknown gene. We demonstrated the distribution of mRNA expression of one gene, LPH, in lung tissues. The present study suggests that 20mg per kg intraperitoneal PQ affects the expression of numerous genes in the lung at 3 h, the onset of pulmonary injury, and that the four genes specified may be major contributors to serious lung injury due to PQ exposure.     

Prospective surveillance of community-onset and healthcare-associated methicillin-resistant Staphylococcus aureus isolated from a university-affiliated hospital in Japan

We conducted a prospective comparative study of community-onset (CO) and healthcare-associated (HA) methicillin-resistant Staphylococcus aureus(MRSA) strains between 2000 and 2001 at Tokyo Women's Medical University Hospital (1,500 beds) in Japan. Of the 172 consecutive MRSA isolates analyzed, 13 (8%) were categorized as CO-MRSA. The mean age of patients with CO-MRSA was significantly younger than that of patients with HA-MRSA. Most CO-MRSA strains were isolated from skin and more likely to be susceptible to erythromycin, clindamycin, tetracycline, levofloxacin, and spectinomycin compared to HA-MRSA isolates. Pulsed-field gel electrophoresis (PFGE) analysis, staphylococcal cassette chromosome mec(SCCmec) typing, and multi-locus sequence typing (MLST) revealed that CO-MRSA strains were divided into the following multi-clones: 3 clone A: II: ST5 (PFGE type: SCCmec type: MLST sequence type); 1 L: II: ST5; 1 H: IV: ST1; 1 I: IV: ST81; 2 D: IV: ST8; 1 B: IV: ST89; 1 B: IV: ST379; and 3 B: IV: ST91. Of the 159 HAMRSA strains, 124 (78%) belonged to a single clone (PFGE clone A: SCCmec type II: tst and sec positive: coagulase type II: multi-drug resistance). Four CO-MRSA strains belonging to PFGE clone B: SCCmec type IV: MLST clonal complex 509 (ST89, 91, 379) had the exfoliative toxin B (etb) genes, but all CO-MRSA and HA-MRSA strains did not possess the Panton-Valentine leukocidin (pvl) genes. These results demonstrate that multiple lineages of CO-MRSA have the potential for dissemination in the community in Japan.     

Discovery of a novel, potent and selective human beta3-adrenergic receptor agonist

The discovery of a novel, potent and selective beta(3)-adrenergic receptor (AR) agonist is described. SAR studies demonstrated the structural requirements for activity and selectivity. Compound 1c, which showed good beta(3)-AR activity and selectivity, was identified and pharmacokinetics were investigated.     

Isohumulones modulate blood lipid status through the activation of PPAR alpha

Isohumulones derived from hops are the major bitter compounds in beer. It was recently reported that isohumulones activated peroxisome proliferator-activated receptors (PPARs) alpha and gamma in vitro and modulated glucose and lipid metabolism in vivo. In this study, we examined the effects of isomerized hop extract (IHE) primarily containing isohumulones in C57BL/6N male mice and found that such treatment increased their liver weight and reduced their plasma triglyceride and free fatty acid levels. Microarray analysis and quantitative real time PCR (QPCR) showed that IHE dose-dependently upregulated the expression of a battery of hepatic genes that are involved in microsomal omega-oxidation and peroxisomal and mitochondrial beta-oxidation. These effects were common in both genders and very similar to those found with the PPARalpha agonist, fenofibrate (FF). Moreover, these effects were not found in PPARalpha-deficient mice. Thus, our results strongly suggest that IHE intake upregulates the expression of key genes that are involved in hepatic fatty acid oxidation, and that it ameliorates the blood lipid profile by activating PPARalpha.     

Aspiration cytology of malignant fibrous histiocytoma after radiation therapy for carcinoma of the uterine cervix: a case report

BACKGROUND: Cytologic reports on malignant fibrous histiocytoma (MFH) following radiation therapy for carcinoma of the uterine cervix are very rare. CASE: A 59-year-old woman presented with slowly increasing pain in the left hip joint. Eight years earlier, she had received radiotherapy at a dosage of 5,000 cGy to the whole pelvis for carcinoma of the uterine cervix. An osteolytic lesion of the pelvic bone was revealed on computed tomography, and a hard tumor was palpable in the left pelvic cavity. Fine needle aspiration (FNA) of the tumor via the left vaginal wall obtained 0.5 mL of yellow fluid consisting of markedly anaplastic and pleomorphic giant cells. Frequent multinucleation and mitoses were observed, although no atypical spindle cells were observed. Immunocytochemistry disclosed vimentin reactivity. An open biopsy of the tumor revealed the histologic and immunohistochemical features of MFH arising in the pelvic cavity. CONCLUSION: FNA of the pelvic lesion via the vaginal wall revealed an MFH in the radiation therapy field. This is one of the few reports dealing with FNA cytology of a postradiation sarcoma in the pelvic cavity.     

Effects of habitual smoking on cardiorespiratory responses to sub-maximal exercise

The effects of habitual cigarette smoking on cardiorespiratory responses to sub-maximal and maximal work were evaluated in nine adult nonsmokers and nine smokers with a mean age of 33 yr. A maximal treadmill test was followed by three tests at 45, 60 and 75% of each subject's VO(2)max. Compared to nonsmokers, the habitual smokers had a non-significantly lower VO(2)max in L/min and per lean body mass (9 and 6%, respectively), but had higher %fat (p<0.01), resulting in a significantly lower VO(2)max per kg body wt (13%, p<0.03). Maximal exercise ventilation (V(E)) was 16% lower in smokers. During sub-maximal work at equivalent exercise stress levels in the two groups, the V(E)/VO(2) ratio was higher in smokers by an average of 11% because VO(2) was lower and the respiratory exchange ratio values were significantly elevated in smokers at 75% of VO(2)max. Blood lactate concentrations in smokers were higher as workloads increased and O(2) pulse (VO(2)/HR) was significantly lower throughout, indicating reduced O(2) extraction, probably due to carbon monoxide. The resting HR was significantly higher in smokers and the HR recovery following all three submaximal exercises was significantly slower in smokers. These results show that detrimental cardiorespiratory effects of chronic cigarette smoking in apparently healthy individuals are evident at moderate exercise levels as reduced gas exchange efficiency in lungs and muscles.     

Nucleotide sequence variation is frequent in the mitochondrial DNA displacement loop region of individual human tumor cells

The mitochondrial DNA (mtDNA) displacement loop (D-loop) regions of 76 various tumor cell lines were examined to investigate the existence of a specific relationship between a somatic mtDNA sequence and initiation and/or progression of a tumor. Based on molecular cloning-sequencing analysis, a nucleotide sequence in the D-loop region in each cell line was found to be homoplasmic. Several site-specific nucleotide variations were found in stomach and liver tumor cell lines more frequently than those in other tumor cell lines. Subsequently, 20 pairs of noncancerous and cancerous parts from stomach and liver tumor tissues were examined. In the liver tumor tissue, 80% of the noncancerous parts exhibited slightly higher heterogeneity than the corresponding cancerous parts. Several site-specific nucleotide variations found in 76 tumor cell lines were also detected in noncancerous or cancerous parts of stomach and liver tumor tissues. However, it remains unclear why the mtDNA D-loop sequence is homoplasmic in each tumor cell line. The data indicate that mtDNA exhibits heterogeneity even in the noncancerous part and a slight decrease in heterogeneity during tumorigenesis and/or tumor progression. Homoplasmy of the mtDNA population in the tumor cell line would be acquired in the cloning process of establishing a cell line. Site-specific nucleotide substitutions might not be directly involved in the tumorigenesis process.     

A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Galpha and Galphao coupling to a human leukotriene B4 receptor

To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [3H]leukotriene B4 binding activity (Kd = 3.67 nm). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Galphai isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5'-(beta,gamma-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Galphai1beta1gamma2 (Kd = 0.17 nm). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and Galphai1 without Gbeta1gamma2 did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various Galpha subunits were combinatorially expressed in BV with BLT1 and Gbeta1gamma2. The BLT1 BV co-expressing GalphaoAbeta1gamma2 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galphai1beta1gamma2. Co-expression of other Galpha isoforms such as Galphas, Galpha11, Galpha14, Galpha16, Galpha12, or Galpha13 did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that Galphao as well as Galphai couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins.