Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.     

用光谱技术鉴定植物色素突变体

1983年我国报道了从γ-射线处理的“矮杆齐”大麦中得到了一株黄绿色的突变体1832C[1]。本文用光谱技术对该突变体的光合色素成分进行了鉴定。1　材料和方法　　材料为六棱裸大麦“矮杆齐”和由该品种大麦诱变形成的黄绿色突变体1832C(Mb1832C),以及作为对照的缺失Chlb的突变体大麦Chlorina-f2[2]都于3月初播种于实验田中。　　每个样品取30g新鲜的叶片,先用自来水后用蒸馏水冲洗干净。把洗净的叶片摊放在干净的纱布上吸干表面水分,剪碎,加入100mL预冷的含有0.4mol/L山梨醇、0.1mol/LTris-HCl(pH7.6)的缓冲液,用组织捣碎机先慢速捣碎1…     

Suppression of oxidative damage by Saccharomyces cerevisiae ATX2, which encodes a manganese-trafficking protein that localizes to Golgi-like vesicles.

Oxygen toxicity in Saccharomyces cerevisiae lacking the copper/zinc superoxide dismutase (SOD1) can be suppressed by overexpression of the S. cerevisiae ATX2 gene. Multiple copies of ATX2 were found to reverse the aerobic auxotrophies of sod1(delta) mutants for lysine and methionine and also to enhance the resistance of these yeast strains to paraquat and atmospheric levels of oxygen. ATX2 encodes a novel 34.4-kDa polypeptide with a number of potential membrane-spanning domains. Our studies indicate that Atx2p localizes to the membrane of a vesicular compartment in yeast cells reminiscent of the Golgi apparatus. With indirect immunofluorescence microscopy, Atx2p exhibited a punctate pattern of staining typical of the Golgi apparatus, and upon subcellular fractionation, Atx2p colocalized with a biochemical marker for the yeast Golgi apparatus. We demonstrate here that this vesicle protein normally functions in the homeostasis of manganese ions and that this role in metal metabolism is necessary for the ATX1 suppression of SOD1 deficiency. First, overexpression of ATX2 caused cells to accumulate increased levels of manganese. Second, a deletion in ATX2 caused a decrease in the apparent available level of intracellular manganese and caused sod1(delta) mutants to become dependent upon exogenous manganese for aerobic growth. Third, ATX2 was incapable of suppressing oxidative damage in cells depleted of manganese ions or lacking the plasma membrane transporter for manganese. The effect of ATX2 overexpression on manganese accumulation and oxygen resistance is similar to what we have previously reported for mutations in PMR1, which encodes a manganese-trafficking protein that also resides in a vesicular compartment. Our studies are consistent with a model in which Atx2p and Pmr1p work in opposite directions to control manganese homeostasis.     

Toxicity to alveolar macrophages in rats following parenteral injection of mercuric chloride

Alveolar macrophages collected by pulmonary lavage from male Fisher-344 rats at intervals (24–72 h) after HgCl₂ injection (1–5 mg/kg, sc) were analyzed by several techniques. Within 24–72 h, the macrophages showed morphological signs of activation (hypertrophy and ruffled plasma membrane). Lipid peroxidation (increased malondialdehyde concentration) was not detected until 48 h. Dose- and time-related effects of HgCl₂ on malondialdehyde concentration and time-related effects of HgCl₂ on malondialdehyde concentration and mercury content of alveolar macrophages were observed 24–72 h postinjection. Diminished cell viability occurred only at 72 h after the highest dosage of HgCl₂. This study demonstrates that the alveolar macrophage was a cellular target for mercury toxicity following parenteral exposure to HgCl₂.     

RFLP mapping of QTLs for yield and related characters in rice (Oryza sativa L.)

Quantitative triat loci (QTLs) for yield and related traits in rice were mapped based on RFLP maps from two indica/indica F₂ populations, Tesanai 2/CB and Waiyin 2/CB. In Tesanai 2/CB, 14 intervals carrying QTLs for eight traits were detected, including 3 for grain weight per plant (GWT), 2 for number of panicles per plant (NP), 2 for number of grains per panicle (NG), 1 for total number of spikelets per panicle (TNS), 1 for spikelet fertility (SF), 3 for 1000-grain weight (TGWT), 1 for spikelet density (SD), and 1 for number of first branches per main panicle. The 3 QTLs for GWT were located on chromosomes 1, 2, and 4, with 1 in each chromosome. The additive effect of the single locus ranged from 2.0 g to 9.1 g. A major gene (np4) for NP was detected on chromosome 4 within the interval of RG143–RG214, about 4cM for RG143, and this locus explained 26.1% of the observed phenotypic variance for NP. The paternal allele of this locus was responsible for reduced panicles per plant (3 panicles per plant). In another population, Waiyin 2/CB, 12 intervals containing QTLs for six of the above-mentioned traits were detected, including 3 for GWT, 2 for each of NP, TNS, TGWT and SD, 1 for SF. Three QTLs for GWT were located on chromosome 1, 4, and 5, respectively. The additive effect of the single locus for GWT ranged from 6.7 g to 8.8 g, while the dominance effect was 1.7–11.5 g. QTL mapping in two populations with a common male parent is compared and discussed.     

Analysis of wild-type and mutant p21WAF-1 gene activities.

The p21WAF-1 gene is positively regulated by the wild-type p53 protein. p21WAF-1 has been shown to interact with several cyclin-dependent kinase complexes and block the activity of G1 cyclin-dependent kinases (cdks). Mutational analysis with the p21WAF-1 gene localized a site, at amino acid residues 21 and 24 in the amino terminus of the protein, for p21WAF-1 binding to cyclins D and E. This region of the protein is conserved (residues 21 to 26) in other p21WAF-1 family members, p27kip-1 and p57kip-2. The same p21WAF-121,24 mutant also fails to bind to cyclin D1-cdk 4 or cyclin E-cdk 2 complexes in vitro, suggesting that amino acid residues 21 and 24 are important for p21WAF-1-cdk-cyclin trimeric complex interactions. The p21WAF-1 wild-type protein will suppress tumor cell growth in culture while p21WAF-1 mutant proteins with defects in residues 21 and 24 fail to suppress tumor cell growth. The overexpression of cyclin D or E in these cells will partially overcome the growth suppression of wild-type p21WAF-1 protein in cells. These results provide evidence that p21WAF-1 acts through cyclin D1-cdk4 and cyclin E-cdk2 complexes in vivo to induce the growth suppression. The p21WAF-1 binding sites for cyclins (residues 21 to 26), cdk2 (residues 49 to 71), and proliferating-cell nuclear antigen (residues 124 to 164) have all been mapped to discrete sites on the protein.     

Physiological Elevations of Glucocorticoids Potentiate Glutamate Accumulation in the Hippocampus

Abstract: Glucocorticoids (GCs) are secreted during stress and can damage the hippocampus over the course of aging and impair the capacity of hippocampal neurons to survive excitotoxic insults. Using microdialysis, we have previously observed that GCs augment the extracellular accumulation of glutamate and aspartate in the hippocampus following kainic acid-induced seizures. In that study, adrenalectomized rats maintained on minimal GC concentrations were compared with those exposed to GCs elevated to near-pharmacological levels. We wished to gain insight into the physiological relevance of these observations. Thus, we have examined the effects of GCs over the normal physiological range on glutamate and aspartate profiles; this was done by implanting adrenalectomized rats with GC-secreting pellets, which produce stable and controllable circulating GC concentrations. We observe that incremental increases in GC concentrations cause incremental increases in glutamate accumulation before the kainic acid insult, as well as in the magnitude of the glutamate response to kainic acid. Elevating GC concentrations from the circadian trough to peak doubled cumulative glutamate accumulation, whereas a rise into the stress range caused a fourfold increase in accumulation. Similar, although smaller, effects also occurred with aspartate accumulation (as well as with taurine but not glutamine accumulation). These data show that the highly elevated GC concentrations that accompany neurological insults such as seizure or hypoxia-ischemia will greatly exacerbate the glutamate accumulation at that time. Furthermore, stress levels of GCs augmented glutamate accumulation even in the absence of an excitotoxic insult, perhaps explaining how sustained stress itself damages the hippocampus. Finally, even the moderately ?levated basal GC concentrations that typically occur in aged rats augmented glutamate accumulation, perhaps explaining how GCs damage the hippocampus over the course of normal aging.     

Development and Application of Cloned DNA Probes for Clavibacter xyli subsp. xyli, the Causal Agent of Sugarcane Ratoon Stunting

Eco RI restriction fragments of genomic DNA from Clavibacter xyli subsp. xyli (CXX) were ligated with plasmid pUC18 and cloned in Escherichia coli JM109. The cloned DNA inserts from recombinant plasmids were Eco RI-excised and labeled with non-radioactive digoxigenin and used as probes. Ten specific DNA probes, RSD3, 15, 30, 31, 32, 35, 37, 41, 71, and 73 were selected for disease detection and pathogen differentiation. In the specificity tests, all of the 10 CXX DNA, probes differentiated Clavibacter xyli from other bacteria specifically. Seven out of the 10 CXX probes crossreacted with C. x. subsp. cynodontis (CXC) very weakly under moderate stringency wash conditions of hybridization. In the sensitivity tests, all of the 10 DNA probes detected the homologous DNA of CXX from 0.19 to 0.75 ng. To detect various cell numbers of CXX, the DNA probes detected 10⁴ to 10⁵ cells effectively. In Southern hybridizations, distinctly different band patterns were shown when the probes hybridized with DNA from CXX and CXC. Among these probes, RSD3, 15, 30, 31, 35, 37, and 71, efficiently detected CXX present in the sap collected from symptomless sugarcane.