Prevalence and antimicrobial-resistance phenotypes and genotypes of Escherichia coli isolated from raw milk samples from mastitis cases in four regions of China.

OBJECTIVES: The objective was to find the differences in the prevalence and resistance of Escherischia coli isolated from raw milk samples from mastitis cases in four regions of China. METHODS: A total of 750 bovine raw milk samples from mastitis cases were collected from four regions of China over two seasons. Antimicrobial resistance against 29 antimicrobial agents was determined, and 27 drug-resistant genes were tested. RESULTS: Eighty-three strains (11.1%) of E. coli were isolated and identified. No significant differences in the number of E. coli isolates were observed between the two sampling seasons in the same regions (P>0.05). However, a significant difference in E. coli prevalence was found among the four different regions (P<0.01). The isolates were most frequently resistant to penicillin (100%), acetylspiramycin (100%), lincomycin (98.8%), oxacillin (98.8%) and sulphamethoxazole (53%). All the E. coli strains were multiresistant to at least three antimicrobial classes, and the most frequent multidrug-resistance patterns for the isolates were resistant to three (36.1%) or four (39.8%) classes of drugs simultaneously. The blaTEM gene (n=69; 83.1%) was the most frequently detected resistance gene. The most frequent gene combinations were a four-gene pattern of blaCTX-M-sulII-blaTEM-sulI (n=13; 15.7%) and a three-gene pattern of blaCTX-M-aph (3)-II-blaTEM (n=11; 13.3%). CONCLUSIONS: This study indicated that there is a high incidence of E. coli with a great variation in resistance patterns and resistance genes; this is a matter of great concern for public and animal health in China.

The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis.

Mitochondrial topoisomerase IB (TOP1MT) is a nuclear-encoded topoisomerase, exclusively localized to mitochondria, which resolves topological stress generated during mtDNA replication and transcription. Here, we report that TOP1MT is overexpressed in cancer tissues and demonstrate that TOP1MT deficiency attenuates tumor growth in human and mouse models of colon and liver cancer. Due to their mitochondrial dysfunction, TOP1MT-KO cells become addicted to glycolysis, which limits synthetic building blocks and energy supply required for the proliferation of cancer cells in a nutrient-deprived tumor microenvironment. Mechanistically, we show that TOP1MT associates with mitoribosomal subunits, ensuring optimal mitochondrial translation and assembly of oxidative phosphorylation complexes that are critical for sustaining tumor growth. The TOP1MT genomic signature profile, based on Top1mt-KO liver cancers, is correlated with enhanced survival of hepatocellular carcinoma patients. Our results highlight the importance of TOP1MT for tumor development, providing a potential rationale to develop TOP1MT-targeted drugs as anticancer therapies.

Fine mapping and candidate gene analysis of Brtri1, a gene controlling trichome development in Chinese cabbage (Brassica rapa L. ssp pekinensis).

Trichomes are derived from the epidermis and constitute an ideal system for studying cell division in plants. Here, a Chinese cabbage doubled haploid (DH) line (FT) without trichomes was crossed with another DH line (PurDH-1) with trichomes to develop an F2 population for fine mapping of trichome control genes. Genetic analysis showed that the trichome phenotype was controlled by a single dominant gene, Brtri1. Using 1226 glabrous individuals in the F2 segregation population, Brtri1 was localized to a 16.84 kb region between markers Pur6-31 and Pur6-39 on chromosome A06. One of the four complete open reading frames within the mapping region, Bra025311, encodes a MYB transcription factor and is highly homologous to the trichome regulatory gene GL1 in Arabidopsis thaliana. It was thus regarded as a candidate gene for Brtri1. Comparative sequencing showed a 5-bp deletion in the third exon of Bra025311 in FT, resulting in a frame-shift mutation. No expression of Bra025311 was detected in FT. A co-dominant indel marker close to this mutation site was developed for marker-assisted selection in Chinese cabbage breeding.

Protection of dopamine neurons by vibration training and up-regulation of brain-derived neurotrophic factor in a MPTP mouse model of Parkinson's disease.

It is unknown whether the longer duration of vibration training (VT) has a beneficial effect on Parkinson's disease (PD). And also, the mechanisms underlying the reported sensorimotor-improvement in PD induced by short-duration of VT has not been determined. Here, we investigated the effects of longer duration (4 weeks) of low amplitude vibration (LAV) training on the numbers of dopaminergic neurons in the substantia nigra by immunostaining and the levels of dopamine (DA) and brain-derived neurotrophic factor (BDNF) in the striatum by HPLC and ELISA in the chronic MPTP lesion mouse. We demonstrated for the first time that the longer duration of VT could significantly increase the numbers of nigrostriatal DA neurons and the contents of striatal DA and BDNF in the MPTP mice. Our findings implied that longer duration of VT could protect dopaminergic neurons from the MPTP-induced damage probably by upregulating BDNF and also provided evidence for the beneficial effect of longer duration of VT on PD at the cellular and molecular level.

Functional significance of the Toll-like receptor 4 promoter gene polymorphisms in the Chinese Han population.

OBJECTIVE: Toll-like receptor 4 is an important signaling receptor for lipopolysaccharide in mammals, and the variation of the promoter may affect the activity of toll-like receptor 4 expression. Although 12 single nucleotide polymorphisms have been identified in the toll-like receptor 4 promoter, little is known about the functional significance of these single nucleotide polymorphisms. DESIGN: Genetic functional and association studies. SETTING: National Key Laboratory of Trauma and Departments of Traumatic Surgery in two teaching hospitals. SUBJECTS: Three hundred seventy-nine healthy volunteers and 303 patients with major trauma. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Five single nucleotide polymorphisms identified in the toll-like receptor 4 promoter in the Chinese Han population were selected. Three of them revealed a close relationship with transcription factor binding sites. Among the three single nucleotide polymorphisms, only the T-2242C polymorphism significantly increased transcriptional activities of the toll-like receptor 4 promoter, as shown by reporter gene assay. Results from flow cytometry and ex vivo responsiveness of peripheral blood leukocytes indicated that the T-2242C polymorphism was well-associated with increased expression of toll-like receptor 4 protein and production of tumor necrosis factor-alpha. The clinical relevance of these single nucleotide polymorphisms was then investigated in 303 patients with major trauma. The peripheral blood leukocytes of trauma patients with the variant C allele revealed greater capacity to produce tumor necrosis factor-alpha and interleukin-6 on the admission day. Furthermore, the toll-like receptor 4/2242 polymorphism was significantly associated with higher sepsis morbidity rates and multiple organ dysfunction scores in patients with major trauma. CONCLUSIONS: The toll-like receptor 4/2242 polymorphism is a functional variant and might be used as a relevant risk estimate for organ dysfunction and sepsis in trauma patients.

Supercritical carbon dioxide extract exhibits enhanced antioxidant and anti-inflammatory activities of Physalis peruviana.

Physalis peruviana L. (PP) is a medicinal herb widely used in folk medicine. In this study, supercritical carbon dioxide (SFE-CO2) method was employed to obtain three different PP extracts, namely SCEPP-0, SCEPP-4 and SCEPP-5. The total flavonoid and phenol concentrations, as well as antioxidant and anti-inflammatory activities of these extracts were analyzed and compared with aqueous and ethanolic PP extracts. Among all the extracts tested, SCEPP-5 demonstrated the highest total flavonoid (234.63+/-9.61 mg/g) and phenol (90.80+/-2.21 mg/g) contents. At concentrations 0.1-30 microg/ml, SCEPP-5 also demonstrated the strongest superoxide anion scavenging activity and xanthine oxidase inhibitory effect. At 30 microg/ml, SCEPP-5 significantly prevented lipopolysaccharide (LPS; 1 microg/ml)-induced cell cytotoxicity in murine macrophage (Raw 264.7) cells. At 10-50 microg/ml, it also significantly inhibited LPS-induced NO release and PGE2 formation in a dose-dependent pattern. SCEPP-5 at 30 microg/ml remarkably blocked the LPS induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. Taken together, these results suggest that SCEPP-5, an extract of SFE-CO2, displayed the strongest antioxidant and anti-inflammatory activities as compared to other extracts. Its protection against LPS-induced inflammation could be through the inhibition of iNOS and COX-2 expression.

Nonlinear adaptive control of interconnected systems using neural networks.

In this letter, we solve the problem of decentralized adaptive asymptotic tracking for a class of large scale systems with significant nonlinearities and uncertainties. Neural networks (NNs) are used as a control part to cancel the effect of the unknown nonlinearity. Semiglobal asymptotic stability results are obtained and the tracking error converges to zero.

Further results on adaptive control for a class of nonlinear systems using neural networks.

Zhang et al. presented an excellent neural-network (NN) controller for a class of nonlinear control designs. The singularity issue is completely avoided. Based on a modified Lyapunov function, their lemma illustrates the existence of an ideal control which is important in establishing the NN approximator. In this paper, we provide a Lyapunov function to realize an alternative ideal control which is more direct and simpler. The major contributions of this paper are divided into two parts. First, it proposes a control scheme which results in a smaller dimensionality of NN than that of Zhang et al. In this way, the proposed NN controller is easier to implement and more reliable for practical purposes. Second, by removing certain restrictions from the design reported by Zhang et al., we further develop a new NN controller, which can be applied to a wider class of systems.

A decentralized control of interconnected systems using neural networks.

We develop a decentralized neural-network (NN) controller for a class of large-scale nonlinear systems with the high-order interconnections. The controller is a mixed NN comprised of a conventional NN and a special NN. The conventional NN is used to approximate the unknown nonlinearities in the subsystem, while a special NN is used to counter the high-order interconnections. We prove that this NN structure can achieve a stable controller for the large-scale systems.

Riboflavin uptake in human trophoblast-derived BeWo cell monolayers: cellular translocation and regulatory mechanisms.

Riboflavin (vitamin B2) is essential for fetal development and must be acquired from maternal sources. The uptake mechanism of riboflavin and the major regulatory pathways involved were characterized in a model for the placental barrier, the human choriocarcinoma cell line, BeWo. Uptake of [3H]riboflavin was saturable (Kt = 1.32 +/- 0.68 nM, Jmax = 266.63 +/- 26.89 fmol/mg of protein/20 min), and was significantly reduced at low temperature and in the presence of metabolic inhibitors (azide, 2-deoxyglucose) or structural analogs. Ouabain, amiloride, sodium-free buffers, and medium with pH values ranging from 3 to 8 did not affect uptake of [3H]riboflavin. In contrast, substitution of chloride with other monovalent anions significantly inhibited its uptake. Induced differentiation of BeWo cells into syncytiotrophoblasts by forskolin or 8-bromo-cyclic adenosine monophosphate introduced a time-dependent decrease of riboflavin uptake. Preincubation with activators of cyclic nucleotide-dependent protein kinase pathways (3-isobutyl-1-methylxanthine and p-chlorophenylthio-cyclic guanosine monophosphate) and calmodulin antagonists (calmidazolium and W-13) resulted in a concentration-dependent reduction of [3H]riboflavin uptake, whereas specific modulators of protein kinase C pathways did not have significant effects. 3-Isobutyl-1-methylxanthine exerted its regulatory effect on riboflavin uptake via decreasing both Kt and Jmax of the riboflavin uptake process (Kt = 6.32 +/- 1.29 nM, Jmax = 135.57 +/- 10.42 fmol/mg of protein/20 min). In summary, we report the presence of high- affinity riboflavin transporter(s) on the microvillous membrane of BeWo cells that appears to be modulated by cellular cyclic nucleotide levels and calmodulin.

Involvement of a receptor-mediated component in cellular translocation of riboflavin.

This study addresses the transport mechanism of riboflavin (vitamin B(2)) across intestinal epithelium in the presence and absence of pharmacologically active compounds. A polarized transport process with a 6-fold higher basolateral (BL)-to-apical (AP) flux was observed in both a human intestinal cell model (Caco-2) and rat intestinal tissue. Riboflavin-specific translocation systems on both the AP and BL cell surfaces were saturable with affinity values close to most receptors (K(m): 9.72 +/- 0.85 and 4.06 +/- 0.03 nM, respectively). Pharmacological agents known to alter intracellular endocytic events were used to examine the potential involvement of receptor-mediated events. Nocodazole significantly inhibited AP uptake (58.4%), BL-to-AP riboflavin (56.7%) and fluorescein isothiocyanate-labeled transferrin (FITC-Tf) (31.8%) transport without affecting mannitol or cholic acid transport, whereas AP-to-BL riboflavin (252.8%) and FITC-Tf (145.1%) transport was increased. Brefeldin A significantly enhanced AP-to-BL riboflavin (37.1%) and bidirectional FITC-Tf transport (AP-to-BL: 13-fold; BL-to-AP: 5-fold). without affecting BL-to-AP riboflavin transport. Combined, these data suggest an essential role of microtubule-dependent movement and vesicular sorting component(s) in the bidirectional transport of riboflavin. Dissociation of riboflavin from the cell surface was pH-dependent with significantly higher substrate release at acidic pH, indicating the presence of riboflavin-specific cell surface receptors. In summary, our studies provide biochemical evidence of the involvement of a receptor-mediated mechanism in the cellular translocation of riboflavin.

Well-differentiated fetal adenocarcinoma of the lung.

We describe the case of a 43-year-old woman with a tumor shadow in the upper lobe of the left lung. The tumor was initially suspected to be a carcinoid tumor, following percutaneous needle biopsy. Subsequently, a left upper lobectomy was performed, and a well-differentiated fetal adenocarcinoma was diagnosed histologically. Unlike the biphasic epithelial and stromal features of pulmonary blastoma, it was composed solely of malignant glands of embryonal appearance.

Activation of nuclear factor kappaB in hepatitis C virus infection: implications for pathogenesis and hepatocarcinogenesis.

The hepatitis C virus (HCV) core protein is a multifunctional protein. It may bind to the death domain of tumor necrosis factor receptor 1 (TNFR1) and to the cytoplasmic tail of lymphotoxin-beta receptor, implying that it may be involved in the apoptosis and anti-apoptosis signaling pathways. In vitro studies have been inconclusive regarding its ability to inhibit or enhance TNF-alpha-induced apoptosis. To address this issue, electrophoretic mobility shift assay (EMSA) and immunohistochemical studies were used to show the activation of nuclear factor kappaB (NF-kappaB) in HCV-infected liver tissues and in HCV core-transfected cells. The activation of NF-kappaB was correlated with the apoptosis assays. The results showed that NF-kappaB activation could be shown in HCV-infected livers and HCV core-transfected cells. The data of EMSA correlated with those of immunohistochemical studies, which revealed a higher frequency of NF-kappaB nuclear staining in HCV-infected than in normal livers. NF-kappaB activation conferred resistance to TNF-alpha-induced apoptosis in HCV core-transfected cells. Inhibition of NF-kappaB activation by pyrrolidine dithiocarbamate sensitized them to TNF-alpha-induced apoptosis. These findings suggest that HCV infection may cause anti-apoptosis by activation of NF-kappaB and implicate a mechanism by which HCV may evade the host's immune surveillance leading to viral persistence and possibly to hepatocarcinogenesis.

Constitutive activation of nuclear factor kappaB in hepatocellular carcinoma.

BACKGROUND: Nuclear factor kappaB (NF-kappaB) is a transcription factor that plays important roles in cell proliferation and in immunity against viral infections. NF-kappaB is a dimer of Rel proteins that is sequestered in the cytoplasm as an inactive form through interaction with an inhibitory kappaB (IkappaB) protein. When IkappaB is degraded, the NF-kappaB dimer will enter the nucleus to activate the target genes. Chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection may activate NF-kappaB and, thus, may modulate cell apoptosis and may be associated with oncogenesis. The role of NF-kappaB in hepatocellular carcinoma (HCC) has not yet been explored. METHODS: Immunohistochemical staining to search for active nuclear RelA and nuclear IkappaBalpha proteins were done on formalin fixed liver tissues from 65 patients with HCC and from 9 normal control participants. Nuclear extracts of fresh-frozen tumor and nontumor liver tissues from 37 patients with HCC and from 7 normal controls were tested for NF-kappaB-DNA binding activity by electrophoretic mobility shift assay. The RelA and IkappaBalpha protein expressions were studied by Western blot analysis. RESULTS: Nuclear NF-kappaB stainings were significantly more abundant in HBV-infected or HCV-infected tumors as well as nontumor parts of HCC compared with normal controls. Nuclear NF-kappaB DNA binding activity and nuclear RelA protein expression were greater in tumor tissue compared with nontumor tissue, whereas cytosolic IkappaBalphs protein expression was generally greater in nontumor tissue compared with tumor tissue. CONCLUSIONS: Constitutive activation of NF-kappaB was found more frequently in tumor tissue compared with nontumor tissue. It is possible that NF-kappaB overexpression accompanied by dysregulation of IkappaBalpha may play a role in the hepatocarcinogenesis of HBV or HCV infection.

Development of a PCR assay for diagnosis of Pneumocystis carinii pneumonia based on amplification of the multicopy major surface glycoprotein gene family.

We have evaluated a PCR technique using primers based on Pneumocystis carinii major surface glycoprotein (MSG) genes, a multicopy gene family, for utility in detection of P. carinii in BAL and oropharyngeal samples obtained from immunosuppressed patients. These primers were able to detect P. carinii DNA in as little as 16 fg of genomic DNA. PCR using MSG primers detected P. carinii DNA in 7 smear-positive BAL samples (100% sensitivity), and found no P. carinii DNA in 12 smear-negative BAL samples (100% specificity). Mitochondrial ribosomal RNA (mrRNA) primers, commonly used in PCR studies of PCP, detected P. carinii in six of seven positive samples (85.7% sensitivity) and none of 12 were negative samples (100% specificity). Diagnosis of PCP by amplification of 81 oropharyngeal samples using MSG primers had a 50% sensitivity (4/8) and 96% specificity (70/73). PCR with mrRNA primers was 37.5% sensitive (3/8) and 100% specific (73/73). All three false-positive MSG results showed a very low intensity on Southern hybridization. PCR using MSG gene primers should prove valuable in the diagnosis of PCP.

Identification and characterization of novel variant major surface glycoprotein gene families in rat Pneumocystis carinii.

The major surface glycoprotein (MSG) is an abundant, immunodominant protein on the surface of the opportunistic pathogen Pneumocystis carinii. The current study identified two novel variant MSG (vMSG) gene families in rat P. carinii that are closely related to but distinct from MSG. These gene families encode proteins of approximately 90 kDa (v1MSG) and approximately 115 kDa (v2MSG). Compared with MSG, v1MSG is characterized by a deletion near the carboxyl terminus. The predicted v1MSG and v2MSG proteins are highly homologous to MSG at the carboxyl, but not the amino, terminus. Like MSG, they are cysteine-rich. Approximately 10% of the apparent molecular weight is due to N-linked glycosylation. Southern blotting studies demonstrated that, like MSG, v1MSG and v2MSG are the products of multicopy gene families. However, unlike MSG, each vMSG gene encodes a signal peptide, suggesting that the regulation of vMSG is different from that of MSG.

Histopathology and pathobiology of hepatotropic virus-induced liver injury.

The present report concerns current knowledge regarding immunopathogenesis that can be applied in the interpretation of histopathological changes in acute and chronic viral hepatitis. The histopathological features of viral hepatitis have not been changed and light microscopic examination remains essential for making a diagnosis and classification of chronic hepatitis and for the provision of objective parameters on grading and staging. However, new understanding and knowledge of viral pathogenesis, host immune responses, the biological behaviour of the causative viral agents and, in particular, viral interference in multiple hepatotropic viral infections must be taken into consideration in the interpretation of histopathological and immunopathological findings of liver tissues. This report also presents some histopathological analyses on multiple hepatotropic viral infections. It can be concluded that the diagnostic histological criteria for acute hepatitis remain applicable in such settings. However, the cause of acute flare up in chronic hepatitis could not be determined without clinical, virological and serological information. Routine histopathology cannot distinguish a new infection from an acute exacerbation due to a high level of viral replication or mutant virus. A repertoire of immunocytochemical stainings for viral antigens is helpful, but caution must be exercised in suggesting a specific viral aetiology due to the fact that suppression of pre-existing viral antigens can be pronounced when the new or concurrent infection is hepatitis C virus related.

T cell mechanisms in the immunopathogenesis of viral hepatitis B and C.

Considerable evidence suggests that immune mechanisms are involved in the pathogenesis of both hepatitis B and C. Both CD4+ and CD8+ T cell responses to viral antigens are important mechanisms that may be responsible for the hepatocyte damage in hepatitis B and C. CD4+ T cell proliferative responses to hepatitis B core antigen (HBcAg) in terms of stimulation index are correlated with hepatitis activity. These responses can be demonstrated in both adult and paediatric patients, and are more vigorous in patients with acute self-limited hepatitis B than in patients with chronic hepatitis B. Patients with hepatitis C also had a significant CD4+ T cell response to hepatitis C virus (HCV) antigens. These responses are also vigorous in acute hepatitis C with recovery than in those cases that evolve to chronic hepatitis C. In terms of human leucocyte antigen (HLA) class I-restricted, CD8+ cytotoxic T lymphocyte (CTL) response, antigenic peptides derived from HBcAg, hepatitis B surface antigen (HBsAg), and polymerase have been demonstrated as the targets for CTL recognition in hepatitis B patients. Multiple CTL epitopes within both HBsAg and HBcAg can be detected by sensitizing target cells with synthetic peptides. Similar to hepatitis B virus (HBV) infection, multispecific, HCV-specific CTL responses can coexist with an extensive quasispecies of viral variants. The mechanisms of viral persistence in both hepatitis B and C are not yet clarified.

Heat shock pretreatment prevents hydrogen peroxide injury of pulmonary endothelial cells and macrophages in culture.

The purpose of the present study was to determine whether heat shock pretreatment would protect pulmonary endothelial cells and alveolar macrophages against hydrogen peroxide (H2O2)-induced injury. The bovine pulmonary artery endothelial cells (BPAECs) heat-shocked (42 degrees C for 2 h) prior to exposure to H2O2 (1 mmol/L for 45 min) showed significant decrease in H2O2-mediated increment of release of lactate dehydrogenase and production of thiobarbituric acid-reactive substances, and obvious alleviation in H2O2-induced decrease in activities of catalase and superoxide dismutase. Heat-shocked (42 degrees C for 2 h) rat pulmonary alveolar macrophages (PAMs) also obtained acquired resistance to injury by subsequent exposure of 1, 2, or 3 mmol/L H2O2 for 45 min. Simultaneously with this acquired oxidative resistance, Northern blot analysis showed that heat-shocked BPAECs and PAMs, contained an increased level of mRNA coding for the inducible form of heat shock protein 70 (HSP70), and Western blot analysis indicated that there were increased expression of HSP70. Inhibition of protein synthesis by cycloheximide (25 micrograms/mL) and inhibition of RNA synthesis by actinomycin D (5 micrograms/mL) prevented the cytoprotection against H2O2. These results are consistent with the hypothesis that heat shock pretreatment would protect pulmonary endothelial cells and alveolar macrophages against H2O2-induced injury, and possibly that HSPs play a role in this cytoprotection.