FOXO3a is involved in the apoptosis of naked oocytes and oocytes of primordial follicles from neonatal rat ovaries.

Inhibition of the forkhead transcription factor, FOXO3a, can promote the transition from primordial to primary follicle and subsequent follicle development in mammalian ovaries. Stem cell factor (SCF) initiates anti-apoptotic signaling from its membrane receptor, c-kit, to Bcl-2 family members through PI3K/AKT in oocytes of primordial follicles. However, whether FOXO3a mediates the apoptosis of naked oocytes and oocytes of primordial follicles remains unknown. In the present study, oocytes from nests and primordial follicles from neonatal rat ovaries were cultured, and oocyte apoptosis was examined using the TUNEL technique. The pro-apoptotic action of FOXO3a and the potential signal transduction pathways were investigated using RT-PCR, Western blot, and immunocytochemistry. Culturing oocytes in the presence of SCF did not affect the level of total FOXO3a protein, but rapidly elevated the level of phosphorylated FOXO3a (indicating functional suppression). As phosphorylated FOXO3a increased, oocyte apoptosis was inhibited. The specific PI3K/Akt inhibitor, LY 294002, abolished the phosphorylation of FOXO3a and the anti-apoptotic action of SCF. SCF down-regulated the expression of p27KIP1 and pro-apoptotic factors such as Bim, Bad, and Bax, and this activity was reversed by LY 294002. SCF up-regulated the expression of MnSOD, which was also inhibited by LY 294002. However, SCF had no effect on Bcl-2 protein. These results suggest that FOXO3a is involved in oocyte apoptosis in the neonatal rat ovary, and the SCF-PI3K/Akt-FOXO3a signaling pathway mediates oocyte apoptosis and primordial follicle formation.

Genome-wide expression profiling of the response to terbinafine in Candida albicans using a cDNA microarray analysis.

BACKGROUND: Candida albicans is the most frequently seen opportunistic human fungal pathogen. Terbinafine is an allylamine antifungal agent that has been proven to have high clinical efficacy in the therapy of fungal infections, the mechanism of action of terbinafine involves the specific inhibition of fungal squalene epoxidase, resulting in ergosterol deficiency and accumulation of intracellular squalene. We used cDNA microarray analysis technology to monitor global expression profile changes of Candida albicans genes in response to terbinafine treatment, and we anticipated a panoramic view of the responses of Candida albicans cells to the representatives of allylamine antifungal agents at the molecular level in an effort to identify drug class-specific and mechanism-independent changes in gene expression. METHODS: Candida albicans strain ATCC 90028 was exposed to either medium alone or terbinafine at a concentration equivalent to the 1/2 minimal inhibitory concentrations (MICs, 4 mg/L) for 90 minutes. RNA was isolated and gene expression profiles were compared to identify the changes in the gene expression profile using a cDNA microarray analysis. Differential expression of 10 select genes detected by cDNA microarray analysis was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: A total of 222 genes were found to be responsive to terbinafine, including 121 up-regulated genes and 101 down-regulated genes. These included genes encoding membrane transport proteins belonging to the members of the ATP-binding cassette (ABC) or major facilitator superfamily (MFS; CDR1, AGP2, GAP6, PHO84, HOL3, FCY23, VCX1), genes involved in stress response and detoxification (CDR1, AGP2, HOL3), and gene involved in the ergosterol biosynthesis pathway (ERG12). The results of semi-quantitative RT-PCR were consistent with that of the cDNA microarray analysis. CONCLUSIONS: The up-regulation of the gene encoding the multidrug resistance efflux pump CDR1 may contribute to the terbinafine resistance in Candida albicans. However, the precise roles of other affected genes remain unclear, further studies of these genes and their respective products that play roles in the context of antifungal resistance are warranted.

[Effects of Klebsiella pneumoniae on the expression of cytokines by polymorphonuclear neutrophil granulocytes in mice].

OBJECTIVE: To observe the effect of Klebsiella pneumoniae (Kle.p) on the expression of cytokines in mouse polymorphonuclear neutrophil granulocytes (PMNs). METHODS: Mouse models of acute pneumonia were induced by intranasal inoculation of 30 microl PBS containing 1 x 10(7) Kle.p (heat-inactivated at 60 degrees C for 1.5 h). The mRNA and protein expressions of the cytokines in the isolated and purified PMNs were assayed by reverse transcriptional (RT)-PCR, Western blotting and enzyme- linked immunosorbent assay (ELISA), respectively. RESULTS: Kle.p infection induced the mRNA expressions of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the early stage, and TNF-alpha mRNA reached its peak at 6 h and IL-1beta mRNA at 12 h after infection, both of which decreased 24 h after infection. Western blot analysis showed that the protein expressions of TNF-alpha and IL-1beta were the highest at 6 h, and decreased obviously at 24 h after infection. ELISA showed that the secretion of TNF-alpha peaked at 6 h and decreased obviously at 24 h after infection, while the peak of IL-1beta production occurred at 12 h and maintained the high level till 24 h after the infection. CONCLUSION: TNF-alpha may participate in the immune defense of PMN against Kle.p infection mainly in the early stage of infection while IL-1beta mainly in the later stage.

[Cloning and expression of metallo beta-lactamase encoding gene found in a clinical isolate of Stenotrophomonas maltophilia].

OBJECTIVE: To learn Molecular characterization of metallo beta-lactamase (MBL) found in a clinical isolate of Stenotrophomonas maltophilia and confirm the role of MBL played in the antimicrobial-resistance of S. maltophilia by sequencing the encoding genes of the metallo beta-lactamase and construct the prokaryotic expression vector carrying the MBL gene and expressed in E. coli BL21. METHODS: The blaMBL gene was amplified by PCR and cloned into pMD18-T plasmid. The recombination was subcloned into pET32a+ plasmid and expressed in E. coli BL21. The susceptibility between expression vectors and strain 750 to antibiotics were compared. RESULTS: The 867 bp DNA fragment of MBL encoding gene was amplified from the strain 750 by PCR and sequenced. The gene was 99.31% homologous to blaS and blaL1 of MBL L1. After being transformed into the E. coli BL21 and induced with lmM IPTG, a recombinant protein of about 48 kDa was expressed in the pET32a+-blaMBL system. The susceptibility of pET32a+-blaMBL system and strain 750 showed MIC 12 mg/L and 128 mg/L to imipenem and MIC 2 mg/L and 2 mg/L to ceftazidime, respectively. CONCLUSION: The MBL produced by strain 750 was similar to the that in strain ULA511. The difference of MIC to imipenem between wild strain and E. coli BL21 transformant indicated that other unclear mechanism involving in imipenem resistance in the strain.

[Study on the relationship between genesis and development of cervical cancer and the infection of human papillomavirus type 16/18, human herpesvirus II and cytomegalovirus].

OBJECTIVE: To investigate the correlation between genesis and the development of cervical cancer and infection of human papillomavirus (HPV) type 16/18, human herpesvirus II (HSV- II) and cytomegalovirus(CMV). METHODS: Different viruses were determined by polymerase chain reaction in 156 specimens of uterine including cervix 43 cervical cancer specimens,47 cervical intraepithelial neoplasia (CIN) specimens, 56 cervicitis specimens and 10 normal cervix specimens. RESULTS: (1) Positive rates on different viruses: the positive rates of HSV- II, HPV16/18 and CMV were declining in the cervical cancer specimens, CIN specimens or CIN III specimens and CIN I - II specimens, with significant differences. (2)Positive rate and grading, staging and histogenesis of cervical cancer on different viruses as well as positive rates of HPV16/18 in II staging cervical cancer specimens were significantly higher than that in I staging cervical cancer specimens while positive rates of HPV16/18 and HSV- II in high differentiation of cervical cancer specimens were significantly higher than those with medium differentiation from cervical cancer specimens. Positive rates of CMV did not seem to correlate with positive rate of HSV- II and CMV was not correlated to grading, staging or histogenesis of cervical cancer. (3)Copies of infected virus, HSV-II and HPV16/18 showing cervical cancer>CIN> cervicitis while with CMV:cervical cancer>CIN. (4) There were mixed infections of different viruses as HPV16/18 + HSV- II > HPV16/18 + CMV seen in the study. CONCLUSION: HPV 16/18, HSV- II and CMV infection were closely related to the genesis of cervical cancer and quantity of viruses which might have played an important role in carcinogenesis of cervical lesions.

[Investigation on the antibiotic resistance and risky factors of nosocomial infections caused by Stenotrophomonas maltophilia].

OBJECTIVE: To investigate the antibiotic resistance and risky factors of nosocomial infections caused by Stenotrophomonas maltophilia, so as to help elucidate the difference of drug resistance between metallic beta-lactamase (MBL) producing and non-MBL producing strains. METHODS: Standard agar dilution method of NCCLS was employed in the isolation of 36 strains of Stenotrophomonas maltophilia from patients with nosocomial infection with respect to their in vitro antibiotic resistance to 18 kinds of antibiotics. MBL strains were identified by MBL-E test method. RESULTS: Stenotrophomonas maltophilia in our hospital was mainly identified in the lower respiratory tract (88.9%), in which 88.2% (30/34) of the patients had serious original diseases, 50% of whom had received Imipenem/cilastatin sodium treatment. Thirty-six strains of Stenotrophomonas maltophilia were susceptible to new types of fluoquinolone antibiotics, i.e. Sparfloxacin, levofloxacin, gatifloxacin and doxycycline, with inhibitory rate ranging 97.2%, 94.4%, 91.7% to 83.3%, respectively. They could also be inhibited by SMZ/TMP and Ticarcillin/Lavulanic acid with inhibitory rate of 63.9% and 58.3%, respectively. There were 16 strains out of 36 of MBL bacteria with complete resistance to Imipenem/cilastatin sodium, but with higher susceptibility to aztreonam than those non-MBL producing strains. CONCLUSION: The nosocomial infection in our hospital caused by Stenotrophomonas maltophilia seemed to be related with severe primary disease and the use of Imipenem/cilastatin sodium. The newly developed fluoroquinolones possessed powerful antibacterial potency on Stenotrophomonas maltophilia found in nosocomial infection.