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.

Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis.

The molecular and cellular mechanisms responsible for cytotoxic T lymphocyte (CTL)-induced immunopathology are not well defined. Using a model in which hepatitis B surface antigen (HBsAg)-specific CTL cause an acute necroinflammatory liver disease in HBsAg transgenic mice, we demonstrate that class I-restricted disease pathogenesis is an orderly, multistep process that involves direct as well as indirect consequences of CTL activation. It begins (step 1) almost immediately as a direct antigen-specific CTL-target cell interaction that triggers the HBsAg-positive hepatocyte to undergo programmed cell death (apoptosis). It progresses (step 2) within hours to a focal inflammatory response in which antigen-nonspecific lymphocytes and neutrophils amplify the local cytopathic effect of the CTL. The most destructive pathogenetic function of the CTL, however, is to secrete interferon gamma when they encounter antigen in vivo, thereby activating the intrahepatic macrophage and inducing a delayed-type hypersensitivity response (step 3) that destroys the liver and kills the mouse. We propose that the principles illustrated in this study are generally applicable to other models of class I-restricted, CTL-induced immunopathology, and we suggest that they contribute to the immunopathogenesis of viral hepatitis during hepatitis B virus infection in humans.

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.

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.

Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression.

Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three-dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.

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.

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.

Immunohistochemical localization of epidermal and Mallory body cytokeratin in undifferentiated epithelial tumors. Comparison with ultrastructural features.

Twenty-one anaplastic tumors were studied by light microscopy (LM), immunoperoxidase staining using anti-epidermal cytokeratin (ECK) and anti-Mallory body cytokeratin (MBCK) antibodies, and electron microscopy (EM), to determine whether an epithelial origin could be confirmed. The tumors were derived from lung, stomach, colon, breast, uterus, kidney, bladder, and mesothelium. By LM, the tumors consisted of either large and polygonal, spindle or small, round cells. With immunoperoxidase staining, 11 (52%) of the anaplastic tumors were positive for ECK, positivity being either absent or only weak in the main tumor mass, but marked in areas of infiltration and metastases. In contrast, all of the anaplastic tumors were positive for MBCK in the main tumor mass, infiltrating areas, and metastases. In the case of adenocarcinomas, staining was either web-like or diffuse throughout the cytoplasm with concentration occurring at the cell surface, whereas in mesotheliomas, the staining was either diffuse or showed focal perinuclear accentuation. Twelve of 13 anaplastic tumors examined by EM showed epithelial features (desmosomes, tonofilaments, lumina, and/or microvilli). As controls, 21 non-epithelial tumors (five melanomas, eight sarcomas, and eight lymphomas) showed no reactivity with either cytokeratin antibody. These studies show that the epithelial nature of undifferentiated and poorly differentiated tumors can be confirmed by immunohistochemistry using anti-cytokeratin antibodies.

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.

Structural and immunoreactive characteristics of hepatitis B core antigen.

The ultrastructural study of liver tissues from 38 patients with type B viral hepatitis consistently showed the presence of hepatitis B core antigen of 21-25 nm size in the liver cell nuclei and to a lesser extent in the cytoplasm. This finding and the demonstration of the tubular form of hepatitis B surface antigen in the proliferative degranulated endoplasmic reticulum constituted the etiologic criterion for the diagnosis of the disease. The double-shelled Dane-like particles were frequently found in association with the tubular form of the surface antigen. The core particles were found in the protoplasmic processes of hepatocytes and this correlated with the immunofluorescent microscopic findings that the antigen may be shed into circulation with the protoplasm. The core antigen was found to resist digestion by various enzymes such as protease, DNase, RNase, phospholipase C, lipase, lysozyme, diastase, neuraminidase and hyaluronidase, all of which did not destroy the immunoreactivity as demonstrated by immunoelectron and immunofluorescent microscopy. Similarly, sodium dodecyl sulfate, Tween 80 and mercaptoethanol also had no effect. The formalin-fixed paraffin-embedded liver tissue sections could be treated with protease to facilitate the immunofluorescent staining for the core antigen in tissue.

Ventricular preexcitation syndrome. Accessory left atrioventricular connection and rhabdomyomatous myocardial fibers.

A necropsy study of the heart of a 16-year-old girl with ventricular preexcitation syndrome and supraventricular tachycardia showed an accessory left atrioventricular connection partially within the mitral valve. Both the accessory pathway and the ventricular musculature showed multiple foci of cardiac muscle fibers with rhabdomyomatous features having the characteristic appearance of rhabdomyomatosis. The case is discussed in the context of a literature review of necropsy findings in cases of ventricular preexcitation.

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.

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.

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.

[Relation of HSP70 gene and spontaneously hypertension in rats].

The level of heat shock protein 70 messenger RNA was examined in aortic smooth muscle cells (ASMC) and hepatic tissue of Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The restriction fragment length polymorphism (RFLP) was also analysed in hepatic DNA. It was demonstrated that the basic level of heat shock protein 70 messenger RNA in ASMC and hepatic tissue of SHR were lower than that of the WKY rats. The extent of heat shock protein 70 messenger RNA accumulated in ASMC was higher in SHR than in WKY rats when the cells were heated by immersing the culture bottles in 42 degrees C water bath for 15 min and allowed to recover at 37 degrees C for 2 h. In contrast with WKY rats, the Bam HI restricted bepatic DNA showed that a fragment about 5.6 kb was lost in heat shock protein 70 gene from hepatic DNA of SHR. The results suggest that the ASMC of SHR is more sensitive to heat stress than that of WKY rats and the changes of heat shock protein gene sequence may be related to genetic hypertension.

[Transcription of HSP 70 gene induced by pressure overload in left ventricule of rats].

The changes of heat shock protein 70 (HSP70) messenger RNA was examined in rats after abdominal aortic partial ligation. Four hours after ligation, the systemic arterial pressure was increased and thereafter maintained at a high level. The ratio of left ventricule weight to body weight began to increase at the third day and increased by 59%, comparing with that of the sham-operated rats at 4 w. Accumulation of HSP 70 mRNA was observed at 4 h and maintained at high level at 1 d, 2 d, 1 w and then gradually disappeared there after. The above experimental results suggest that transcription of HSP 70 gene was induced at an early stage of cardiac hypertrophy caused by pressure overload.

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.