Hypoxia induces autophagy in PA-l ovarian teratoma cells and resistance to growth inhibition and apoptosis by chemotherapeutic agent cis-diamminedichloroplatinum.

Pathological hypoxia exists in solid tumors and it creates a microenvironment for tumor cells which has a critical and complicated implication for cancer. Hypoxia can also activate autophagy which plays a dual role in cancer. In this study the authors analyzed the effect of hypoxia, the autophagy inhibitor 3-methyladenine (3-MA), cis-diamminedichloroplatinum (CDDP, cisplatin), and any combination of them on PA-l cells (a human ovarian cancer cell line) with a series of assays, focusing on autophagy induction, cell growth inhibition, and cell death by CDDP. CDDP caused apoptosis in normoxic PA-l cells and autophagy upon hypoxia treatment decreased apoptosis induction in hypoxic cells by CDDP, which has implications in cancer chemotherapy resistance.

Evaluation of an objective method of image assessment for first-trimester nasal bone.

OBJECTIVE: To evaluate the degree of agreement in first-trimester nasal bone assessment in a group of sonographers before and after training, using a semi-quantitative scoring system. METHODS: Four sonographers who routinely perform first-trimester screening were first shown 46 images from both normal and trisomy 21 pregnancies. For each image, they were asked to score from 0 (disagree) to 3 (agree) on five different criteria that were deemed important in nasal-bone assessment, including image size, plane and visibility of nasal bone. A training program was then conducted, and a repeat exercise was carried out using the same 46 images. Finally, in a third exercise, images from 42 patients were presented, some having more than one image. The sonographers were required to give one overall nasal-bone score for each patient. In each exercise interobserver agreement was evaluated by intraclass correlation coefficient (ICC). RESULTS: Before training, the sonographers agreed reasonably well on the five proposed criteria (ICC, 0.752), with some disagreement on their perceived image quality. The training program further improved the agreement (ICC, 0.790), particularly on whether the nasal bone was the biggest and brightest echogenic component. Agreement was excellent when they were asked to give one overall score on the nasal bone based on multiple images from one patient (ICC, 0.929). CONCLUSION: The proposed scoring system can be used to improve consistency and reliability in first-trimester nasal-bone assessment.

A case of neuro-Behcet's disease presenting with lumbar spinal cord involvement.

STUDY DESIGN: Case report. OBJECTIVES: To report an unusual case of neuro-Behcet's disease (NBD) presenting with isolated myelitis that affected the lumbar cord. SETTING: Department of Neurology, West China Hospital, Chengdu, Sichuan, China. METHODS: A 19-year-old man presented with a 3-day history of paresthesia and weakness in both the legs. He had a 6-year history of recurrent oral and genital ulcers with erythema nodosum on his legs. Magnetic resonance imaging (MRI) revealed a T1-hypointense and T2-hyperintense lesion at the L1 level. Nodular enhancement of the lesion was achieved with gadolinium administration. Biopsy specimens from cutaneous lesions were consistent with an active perivasculitic process. RESULTS: Corticosteroid (pulsed methylprednisolone followed by oral prednisolone) therapy was administered immediately. Subsequently, the patient's clinical condition improved dramatically. The spinal cord lesion disappeared on the MRI performed 1 year later. Thus far, there has been no recurrence. CONCLUSION: NBD may involve the lumbar spinal cord. Prompt recognition of this disease and early vigorous steroid therapy are important to prevent further disability and recurrence.

Mapping quantitative trait loci for yield, yield components and morphological traits in an advanced backcross population between Oryza rufipogon and the Oryza sativa cultivar Jefferson.

An advanced backcross population between an accession of Oryza rufipogon (IRGC 105491) and the U.S. cultivar Jefferson (Oryza sativa ssp. japonica) was developed to identify quantitative trait loci (QTLs) for yield, yield components and morphological traits. The genetic linkage map generated for this population consisted of 153 SSR and RFLP markers with an average interval size of 10.3 cM. Thirteen traits were examined, nine of which were measured in multiple environments. Seventy-six QTLs above an experiment-wise significance threshold of P<0.01 (corresponding to an interval mapping LOD>3.6 or a composite interval mapping LOD>3.9) were identified. For the traits measured in multiple environments, 47% of the QTLs were detected in at least two environments. The O. rufipogon allele was favorable for 53% of the yield and yield component QTLs, including loci for yield, grains per panicle, panicle length, and grain weight. Morphological traits related to the domestication process and/or weedy characteristics, including plant height, shattering, tiller type and awns, were found clustered on chromosomes 1 and 4. Comparisons to previous studies involving wild x cultivated crosses revealed O. rufipogon alleles with stable effects in multiple genetic backgrounds and environments, several of which have not been detected in studies between Oryza sativa cultivars, indicating potentially novel alleles from O. rufipogon. Some O. rufipogon-derived QTLs, however, were in similar regions as previously reported QTLs from Oryza sativa cultivars, providing evidence for conservation of these QTLs across the Oryza genus. In addition, several QTLs for grain weight, plant height, and flowering time were localized to putative homeologous regions in maize where QTLs for these traits have been previously reported, supporting the hypothesis of functional conservation of QTLs across the grasses.

Francisella tularensis induces cytopathogenicity and apoptosis in murine macrophages via a mechanism that requires intracellular bacterial multiplication.

The murine macrophage-like cell line J774.A1 ingests and allows intracellular growth of Francisella tularensis. We demonstrate that, after 24 h of infection, a pronounced cytopathogenicity resulted and the J774 cells were undergoing apoptosis. Despite this host cell apoptosis, no decrease in bacterial numbers was observed. When internalization of bacteria was prevented or intracellularly located F. tularensis bacteria were eradicated within 12 h, the progression of host cell cytopathogenicity and apoptosis was prevented.

Cytocidal and apoptotic effects of the ClyA protein from Escherichia coli on primary and cultured monocytes and macrophages.

Cytolysin A (ClyA) is a newly discovered cytolytic protein of Escherichia coli K-12 that mediates a hemolytic phenotype. We show here that highly purified ClyA and ClyA-expressing E. coli were cytotoxic and apoptogenic to fresh as well as cultured human and murine monocytes/macrophages.

Molecular analysis of the cytolytic protein ClyA (SheA) from Escherichia coli.

Escherichia coli K-12 carries a gene for a protein denoted ClyA or SheA that can mediate a cytolytic phenotype. The ClyA protein is not expressed at detectable levels in most strains of E. coli, but overproduction suitable for purification was accomplished by cloning the structural gene in an hns mutant strain. Highly purified ClyA protein was cytotoxic to macrophage cells in culture and caused detachment and lysis of the mammalian cells. Results from osmotic protection assays were consistent with the suggestion that the protein formed pores with a diameter of up to 3 nm. Using Acholeplasma laidlawii cells and multilamellar liposomes, we studied the effect of ClyA on membranes with varying compositions and in the presence of different ions. ClyA induced cytolytic release of the fluorescent tracer from carboxyfluorescein-loaded liposomes, and the release was stimulated if cholesterol was present in the membranes whereas addition of calcium had no effect. Pretreatment of the ClyA protein with cholesterol inhibited the pore formation, suggesting that ClyA could bind to cholesterol. Efficient coprecipitation of ClyA with either cholesterol or 1,2,3-trioctadecanoylglycerol in aqueous solutions showed that ClyA directly interacted with the hydrophobic molecular aggregates. We tested the possible functional importance of selected ClyA protein regions by site-directed mutagenesis. Defined mutants of ClyA were obtained with alterations in postulated transmembrane structures in the central part and in a postulated membrane-targeting domain in the C-terminal part. Our results were consistent with the suggestion that particular amphiphilic segments are required for ClyA activity. We propose that these domains are necessary for ClyA to form pores.

An apoptotic response by J774 macrophage cells is common upon infection with diarrheagenic Escherichia coli.

Representative strains of the different diarrheagenic Escherichia coli virotypes were tested for their potential cytotoxicity in the J774 macrophage cell line. All the seven virotypes of E. coli were cytotoxic to J774 macrophages, and in most cases the bacteria induced an apoptotic response. With the exception of the enterotoxigenic E. coli (ETEC) strain, all the other six virotypes caused induction of apoptosis as evidenced by quantitative analysis of the characteristic DNA fragmentation at the individual cell level. These results suggest that apoptosis could be one of the mechanisms contributing to the diarrheal disease development.

Comparative Slow-Blasting in Rice Grown Under Upland and Flooded Blast Nursery Culture.

Evaluation of rice for resistance to rice blast disease caused by Pyricularia grisea usually is conducted in upland (nonflooded) disease nurseries, although all commercial U.S. rice is produced under flood irrigation. Upland rice is more susceptible to leaf blast than is flooded rice, and the magnitude of this differential susceptibility can vary among cultivars. This 2-year study was undertaken to determine (i) the relationship between rates of disease development (slow-blasting) in upland and flooded rice and (ii) the value to a rice breeding program of establishing a flooded blast nursery, a facility far more difficult to manage than an upland nursery. Among 200 rice lines compared for leaf blast susceptibility under upland and flooded cultures, only 7 were rated as slightly more resistant under upland culture and 136 rated more resistant under flooded culture. Disease ratings under upland and flooded cultures were highly correlated (R = 0.819). Among 14 preselected cultivars over 2 years, disease development curves under upland and flooded cultures were highly correlated (R = 0.990). The cultivars with intermediate susceptibility under upland culture appeared to benefit most from flooded culture. Upland culture provided more opportunities to assess slow-blasting than did flooded culture, since many lines that produced susceptible-type lesions in upland culture failed to do so under flooded culture. It was concluded that adequate information on comparative leaf blast resistance among rice lines was attainable from upland blast nurseries and that routine evaluation of rice breeding lines for blast resistance in flooded nurseries was not necessary.

Adherence patterns and DNA probe types of Escherichia coli isolated from diarrheal patients in China.

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P.R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).

Experimental infection of specific-pathogen-free mice with enterohemorrhagic Escherichia coli O157:H7.

By subcutaneous inoculation of 10(8) CFU of enterohemorrhagic E. coli O157:H7, specific-pathogen-free mice revealed most of the symptoms and histological changes observed in patients. The histological changes in intestine were mainly seen in the distal parts of small intestine and the cecum. Vacuolation of villi in the cecum was also observed. The histological changes in the kidneys of the infected mice were featured as the swollen epithelial cells of glomeruli and the marked thickening of glomerular capillaries with barely visible lumens. Unexpected findings in the bronchiole were characterized by sloughing of the epithelial cells of bronchiolar wall, leading to partial or complete obstruction of the lumens. Histological changes in the spleen, liver and lymphnodes were also observed. The bacteria were recovered from the feces, contents of small intestine, and samples taken from kidney, liver, heart, spleen, different parts of small intestine, cecum, and colon. By using peroxidase-antiperoxidase (PAP) assay with polyclonal antibodies against "O" antigen of E. coli O157:H7, it was observed that the samples taken from the brain, kidney, ileum, cecum, spleen, and liver gave positive reactions. Feces and contents of small intestine obtained from all of the infected animals were positive by occult blood test. These results show that the experimental infection of E. coli O157:H7 in this model is systemic in nature.