Molecular structure of Rarobacter faecitabidus protease I. A yeast-lytic serine protease having mannose-binding activity.

Rarobacter faecitabidus protease I (RPI) is a serine protease exhibiting lytic activity toward living yeast cells. RPI is similar to elastase in its substrate specificity and has a lectin-like affinity for mannose. The gene encoding RPI was cloned to elucidate its structure and function. And its nucleotide sequence revealed that it contains an open reading frame encoding a 525-amino acid protein. Homology comparison indicated that pre-pro-RPI consists of three domains: (1) an NH2-terminal prepro domain not found in the mature form of RPI, (2) a protease domain homologous to the trypsin family of serine proteases, and (3) a COOH-terminal domain homologous to the COOH-terminal part of Oerskovia xanthineolytica beta-1,3-glucanase and the NH2-terminal part of the ricin B chain, a lectin isolated from the part of the ricin B chain, a lectin isolated from the castor bean. The RPI gene and its mutant were subsequently expressed in Escherichia coli under its beta-galactosidase promoter to investigate the function of the COOH-terminal domain. The mutant RPI, whose COOH-terminal domain was truncated by site-directed mutagenesis, lost both its mannose-binding and yeast-lytic activity, although the protease activity was not affected. These findings suggest that the COOH-terminal domain actually participates in the mannose-binding activity and is required for yeast-lytic activity.     

D-ribose-5-phosphate isomerase from spinach: heterologous overexpression, purification, characterization, and site-directed mutagenesis of the recombinant enzyme

A cDNA encoding spinach chloroplastic ribose-5-phosphate isomerase (RPI) was cloned and overexpressed in Escherichia coli, and a purification scheme for the recombinant enzyme was developed. The purified recombinant RPI is a homodimer of 25-kDa subunits and shows kinetic properties similar to those of the homodimeric enzyme isolated from spinach leaves (A. C. Rutner, 1970, Biochemistry 9, 178-184). Phosphate, used as a buffer in previous studies, is a competitive inhibitor of RPI with a K(i) of 7.9 mM. D-Arabinose 5-phosphate is an effective inhibitor, while D-xylulose-5 phosphate is not, indicating that the configuration at carbon-3 contributes to substrate recognition. Although D-arabinose 5-phosphate binds to RPI, it is not isomerized, demonstrating that the configuration at carbon-2 is crucial for catalysis. Alignment of RPI sequences from diverse sources showed that only 11 charged amino acid residues of the 236-residue subunit are conserved. The possible function of four of these residues was examined by site-directed mutagenesis. D87A, K100A, and D90A mutants show greatly diminished k(cat) values (0. 0012, 0.074, and 0.38% of the wild type, respectively), while E91A retains substantial activity. Only insignificant or moderate changes in K(m) of D-ribose 5-phosphate are observed for D87A, K100A, and D90A, indicating a direct or indirect catalytic role of the targeted residues.     

Overexpression of RPI1, a novel inhibitor of the yeast Ras-cyclic AMP pathway, down-regulates normal but not mutationally activated ras function.

A high-copy-number plasmid genomic library was screened for genes that when overexpressed down-regulate Ras protein activity in Saccharomyces cerevisiae. We report on the structure and characterization of one such gene, RPI1, which potentially encodes a novel 46-kDa negative regulator of the Ras-cyclic AMP pathway. Three lines of evidence suggest that the RPI1 gene product operates upstream to negatively regulate the activity of normal but not mutationally activated Ras proteins: (i) overexpressed RPI1 lowers cyclic AMP levels in wild-type yeast cells but not in yeast cells carrying the RAS2Val-19 mutation, (ii) overexpressed RPI1 suppresses the heat shock sensitivity phenotype induced by overexpression of normal RAS2 but does not suppress the same phenotype induced by RAS2Val-19, and (iii) disruption of RPI1 results in a heat shock sensitivity phenotype which can be suppressed by mutations that lower normal Ras activity. Thus, RPI1 appears to encode an inhibitor of Ras activity that shares a common feature with Ras GTPase-activating proteins in that it fails to down-regulate activated RAS2Val-19 function. We present evidence that the down-regulatory effect of RPI1 requires the presence of one of the two Ras GTPase activators, IRA1 and IRA2.     

Microsequecing and cDNA cloning of the Calvin cycle/OPPP enzyme ribose-5-phosphate isomerase (EC 5.3.1.6) from spinach chloroplasts

Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.Abbreviations RPI ribose-5-phosphate isomerase - OPPP oxidative pentose phosphate pathway - CNBr cyanogen bromide - R5P ribose-5-phosphate - Ru5P ribulose-5-phosphate     

Ribose-5-phosphate isomerase deficiency: new inborn error in the pentose phosphate pathway associated with a slowly progressive leukoencephalopathy

The present article describes the first patient with a deficiency of ribose-5-phosphate isomerase (RPI) (Enzyme Commission number 5.3.1.6) who presented with leukoencephalopathy and peripheral neuropathy. Proton magnetic resonance spectroscopy of the brain revealed highly elevated levels of the polyols ribitol and D-arabitol, which were subsequently also found in high concentrations in body fluids. Deficient activity of RPI, one of the pentose-phosphate-pathway (PPP) enzymes, was demonstrated in fibroblasts. RPI gene-sequence analysis revealed a frameshift and a missense mutation. Recently, we described a patient with liver cirrhosis and abnormal polyol levels in body fluids, related to a deficiency of transaldolase, another enzyme in the PPP. RPI is the second known inborn error in the reversible phase of the PPP, confirming that defects in pentose and polyol metabolism constitute a new area of inborn metabolic disorders.     

Conformational changes in E. coli RNA polymerase during promoter recognition.

We analysed complexes formed during recognition of the lacUV5 promoter by E. coli RNA polymerase using formaldehyde as a DNA-protein and protein-protein cross-linking reagent. Most of the cross-linked complexes specific for the open complex (RPO) contain the beta' subunit of RNA polymerase cross-linked with promoter DNA in the regions: -50 to -49; -5 to -10; + 5 to +8 and +18 to +21. The protein-protein cross-linking pattern of contacting subunits is the same for the RNA polymerase in solution and in RPO: there are strong sigma-beta' and beta-beta' interactions. In contrast, only beta-beta' cross-links were detected in the closed (RPC) and intermediate (RPI) complexes. In presence of lac repressor before or after formation of the RPO cross-linking pattern is similar with that of RPI (RPC) complex.     

Studies on the genotoxicity of endosulfan in bacterial systems

Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage lambda induction assay in WP2s (lambda) and mutation induction in E. coli K12. The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535/pSK1002 cells. The differential sensitivity assay revealed that the recA 13 strain was the most sensitive. Endosulfan induced prophage lambda in E. coli and umu gene expression in S. typhimurium cells; however, the extent of the effects were low. Endosulfan also induced a dose-dependent increase in forward mutations in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance. Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage.     

The effect of organic pollution on the abundance and distribution of aquatic oligochaetes in an urban water basin, Taiwan

Aquatic oligochaetes are abundant in polluted areas and are, therefore, commonly used as bioindicators to study organic pollution in rivers and streams. In order to develop a species-level oligochaete biotic index to reflect the River Pollution Index (RPI) in the Taichung Water Basin in Taiwan, we conducted a systematic sampling scheme to collect aquatic oligochaetes from the sediment samples of watercourses in the Taichung Water Basin, Taiwan. We evaluated the relationships between aquatic oligochaetes and the sewage pollution using statistical methods. The distribution of aquatic oligochaetes in relation to environmental variables, such as water quality and sediment characteristics of the regional urban contaminated streams was expressed by Canonical Correspondence Analysis (CCA). We identified 17 species of aquatic oligochaetes (Annelida, Clitellata) including 3 species of Tubificidae, 13 species of Naididae, and 1 species of Enchytraeidae from the watercourses of an urban region in the Taichung Water Basin in Taiwan, during the summer and winter of 2005 and 2006. A positive correlation was found between the total abundance of aquatic oligochaetes and the RPI (r = 0.58, P < 0.05). However, only population density of the most abundant tubificid, Limnodrilus hoffmeisteri, increased with increasing RPI values and a significantly negative correlation was found between the population density of the naidid, Nais communis, and RPI values. The results of CCA indicated that certain naidids, such as Aulophorus furcatus and Allonais gwaliorensis also tolerated extremely polluted environments in upper stream or stony habitats, implying that tubificids should not be the sole representation of simple biotic indices but should also include pollution-tolerant naidids. We found that the community structure of aquatic oligochaetes was influenced by short-term variations in microhabitat rather than according to seasonal factors in our study region. The results proved that aquatic oligochaetes were sensitive enough to provide a supplement for the regional urban pollution assessment applications for biotic indicators at the species-level.     

大肠埃希菌泌尿系统感染患者临床分布及药物敏感性分析

摘要 目的：探讨大肠埃希菌引起泌尿系统感染患者的临床分布情况及其药物敏感性分析,为临床诊断及合理使用抗菌药物提供理论性依据。方法：选取2015年1月至2020年12月期间诊治的2052例大肠埃希菌泌尿系统感染患者为研究对象,使用MALDI-TOF MS全自动微生物鉴定质谱仪对细菌进行鉴定,应用VITEK 2 Compact全自动微生物鉴定及药敏分析仪进行药物敏感性分析。结果：2052例大肠埃希菌泌尿系统感染患者中泌尿外科患者占比最高,为17.50%,其次为肾病科患者,占12.87%。其中检出803株产ESBLs的大肠埃希菌,泌尿外科占比最高,为18.56%,其次是肾病科占13.95%,内分泌科占11.71%,重症医学科10.09%。2052株大肠埃希菌整体药敏分析结果显示,大肠埃希菌对美罗培南、亚胺培南、阿米卡星、哌拉西林/他唑巴坦、呋喃妥因、米诺环素、头孢西丁等药物敏感率高,均>90%,头孢唑啉、环丙沙星、左氧氟沙星、氨苄西林/舒巴坦、复方新诺明、氨苄西林敏感率低,均<50%,其中氨苄西林敏感率最低,只有28.17%。结论：大肠埃希菌泌尿系统感染主要发生在泌尿外科,产ESBLs大肠埃希菌整体检出率较高,临床应重视泌尿系统感染患者的尿细菌培养及药物敏感性分析,尽量避免仅凭经验性用药,根据药物敏感性结果合理选择抗菌药物。     

Deficiency in a cytosolic ribose-5-phosphate isomerase causes chloroplast dysfunction, late flowering and premature cell death in Arabidopsis

The oxidative pentose phosphate pathway (oxPPP) is part of central metabolism, consisting of two distinct phases: the oxidative phase and the non-oxidative phase. The non-oxidative phase of the oxPPP generates carbon skeletons for the synthesis of nucleotides, aromatic amino acids, phenylpropanoids and their derivatives, which are essential for plant growth and development. However, it is not well understood how the non-oxidative phase of the oxPPP contributes to plant growth and development. Here, we report the characterization of Arabidopsis T-DNA knockout mutants of the RPI2 gene (At2g01290), which encodes a cytosolic ribose-5-phosphate isomerase (RPI) that catalyzes the reversible interconversion of ribulose-5-phosphate and ribose-5-phosphate in the non-oxidative phase of the oxPPP. Although recombinant Arabidopsis RPI2 protein exhibits marked RPI enzymatic activity, knockout of the RPI2 gene does not significantly change the total RPI activity in the mutant plants. Interestingly, knockout of RPI2 interferes with chloroplast structure and decreases chloroplast photosynthetic capacity. The rpi2 mutants accumulate less starch in the leaves and flower significantly later than wild-type when grown under short-day conditions. Furthermore, the rpi2 mutants display premature cell death in the leaves when grown at an above-normal temperature (26°C). These results demonstrate that a deficiency in the non-oxidative phase of the cytosolic oxPPP has pleiotropic effects on plant growth and development and causes premature cell death.     

Effects of peroxide and catalase on near ultraviolet radiation sensitivity in Escherichia coli strains

The role of peroxide and catalase on NUV radiation sensitivity was examined in two repair competent E. coli strains, AB1157 and B/r. Exponential phase B/r is considerably more sensitive to NUV radiation than exponential phase AB1157. However, resistance to 5 mmol dm-3 H2O2 was induced in both AB1157 and B/r by pretreating growing cells with 30 mumol dm-3 H2O2. Pretreatment also induced resistance to broad-band NUV radiation in these strains. The addition of catalase to the post-irradiation plating medium increased survival to the same extent as that provided by pretreatment with 30 mumol dm-3 H2O2, in both strains. The NUV radiation sensitivity seen in B/r does not appear to be due to a deficiency in enzymes that scavenge H2O2, as a catalase deficient mutant, E. coli UM1, is more resistant to NUV radiation than B/r. Also, assays for H2O2 scavenging ability show little difference between AB1157 and B/r in this respect. Two hypotheses are put forward to account for the sensitivity of exponential phase B/r. Whilst it is apparent that peroxides and catalase do have a role in NUV radiation damage, it is clear that other factors also influence survival under certain conditions.     

Critical role of RPI1 in the stress tolerance of yeast during ethanolic fermentation

Stress tolerance of yeast Saccharomyces cerevisiae during ethanolic fermentation is poorly understood due to the lack of genetic screens and conventional plate assays for studying this phenotype. We screened a genomic expression library of yeast to identify gene(s) that, upon overexpression, would prolong the survival of yeast cells during fermentation, with the view to understand the stress response better and to use the identified gene(s) in strain improvement. The yeast RPI1 (Ras-cAMP pathway inhibitor 1) gene was identified in such a screen performed at 38 °C; introducing an additional copy of RPI1 with its native promoter helped the cells to retain their viability by over 50-fold better than the wild type (WT) parent strain, after 36 h of fermentation at 38 °C. Disruption of RPI1 resulted in a drastic reduction in viability during fermentation, but not during normal growth, further confirming the role of this gene in fermentation stress tolerance. This gene seems to improve viability by fortifying the yeast cell wall, because RPI1 overexpression strain is highly resistant to cell lytic enzyme zymolyase, compared with the WT strain. As the RPI1 overexpression strain substantially retains cell viability at the end of fermentation, the cells can be reused in the subsequent round of fermentation, which is likely to facilitate economical production of ethanol.     

A mutation in an Arabidopsis ribose 5-phosphate isomerase reduces cellulose synthesis and is rescued by exogenous uridine

The Arabidopsis radial swelling mutant rsw10 showed ballooning of root trichoblasts, a lower than wild-type level of cellulose and altered levels of some monosaccharides in non-cellulosic polysaccharides. Map-based cloning showed that the mutated gene (At1g71100) encodes a ribose 5-phosphate isomerase (RPI) and that the rsw10 mutation replaces a conserved glutamic acid residue with lysine. Although RPI is intimately involved with many biochemical pathways, media supplementation experiments suggest that the visible phenotype results from a defect in the production of pyrimidine-based sugar-nucleotide compounds, most likely uridine 5'-diphosphate-glucose, the presumed substrate of cellulose synthase. Two of three RPI sequences in the nuclear genome are cytoplasmic, while the third has a putative chloroplast transit sequence. The sequence encoding both cytoplasmic enzymes could complement the mutation when expressed behind the CaMV 35S promoter, while fusion of the RSW10 promoter region to the GUS reporter gene established that the gene is expressed in many aerial tissues as well as the roots. The prominence of the rsw10 phenotype in roots probably reflects RSW10 being the only cytosolic RPI in this tissue and the gene encoding the plastid RPI being relatively weakly expressed. We could not, however, detect a decrease in total RPI activity in root extracts. The rsw10 phenotype is prominent near the root tip where cells undergo division, endoreduplication and cell expansion and so are susceptible to a restriction in de novo pyrimidine production. The two cytosolic RPIs probably arose in an ancient duplication event, their present expression patterns representing subfunctionalization of the expression of the original ancestral gene.     

Spectral changes arising from the action of spinach chloroplast ribosephosphate isomerase on ribose 5-phosphate

Following addition of spinach chloroplast ribosephosphate isomerase (RPI) to ribose 5-phosphate (R5P) buffered with neutral phosphate, the sequential appearance of compounds absorbing at 280, 308.5, and 285 nm was observed. The 280-nm absorbing compound has previously been identified as ribulose 5-phosphate (Ru5P). The 308.5-nm absorbing compound has a high molar extinction coefficient and was a transient species, appearing only after production of Ru5P and preceding the accumulation of the 285-nm absorbing compound. The 285-nm absorbing compound has been identified as 4-hydroxy-5-methyl-3(2H)-furanone (I). Following addition of rabbit skeleton muscle RPI to R5P buffered with phosphate only the 280-nm absorbing compound, Ru5P, could be observed. Preparations of RPI from photosynthetic tissues have invariably led to the rapid accumulation of I whereas preparations of RPI from nonphotosynthetic tissues produce only Ru5P. In solutions of R5P buffered with citrate, the transient band at 308.5 nm was not observed although I accumulates in solution in the presence of spinach chloroplast RPI. Prior to formation of I a compound with a low molar extinction coefficient and wavelength of maximum absorption at approximately 310 nm can be detected. These results suggest that purified preparations of RPI from spinach chloroplasts and other photosynthetic tissues possess an enzymatic activity not present in crude extracts obtained from non-photosynthetic tissues. This activity is in addition to the aldol-ketol activity. The product of this heretofore unrecognized enzymic activity is postulated to be 3,4-epoxy-Ru5P and could be derived from Ru5P by removing the elements of water from the hydroxyl groups at C-3 and C-4.     

Mapping QTLs for root traits in a recombinant inbred population from two indica ecotypes in rice

Evaluation of root traits in rainfed lowland rice is very difficult. Molecular genetic markers could be used as an alternative strategy to phenotypic selection for the improvement of rice root traits. This research was undertaken to map QTLs associated with five root traits using RFLP and AFLP markers. Recombinant inbred lines (RILs) were developed from two indica parents, IR58821–23-B-1–2-1 and IR52561-UBN-1–1-2, that were adapted to rainfed lowland production systems. Using wax-petrolatum layers to simulate a hardpan in the soil, 166 RILs were evaluated for total root number (TRN), penetrated root number (PRN), root penetration index (RPI, the ratio of PRN to TRN), penetrated root thickness (PRT) and penetrated root length (PRL) under greenhouse conditions during the summer and the fall of 1997. A genetic linkage map of 2022 cM length was constructed comprising 303 AFLP and 96 RFLP markers with an average marker space of 5.0 cM. QTL analysis via interval mapping detected 28 QTLs for these five root traits, which were located on chromosomes 1, 2, 3, 4, 6, 7, 10 and 11. Individual QTLs accounted for between 6 and 27% of the phenotypic variation. Most of the favorable alleles were derived from the parent IR58821–23-B-1–2-1, which was phenotypically superior in root traits related to drought resistance. Three out of six QTLs for RPI were detected in both summer and fall experiments and they also were associated with PRN in both experiments. Out of eight QTLs for RPT, five were common in both seasons. Two genomic regions on chromosome 2 were associated with three root traits (PRN, PRT and RPI), whereas three genomic regions on chromosomes 2 and 3 were associated with two root traits (PRT and RPI). Two QTLs affecting RPI and two QTLs affecting PRT were also found in similar genomic regions in other rice populations. The consistent QTLs across genetic backgrounds and the common QTLs detected in both experiments should be good candidates for marker-assisted selection toward the incorporation of root traits in a drought resistance breeding program, especially for rainfed lowland rice. Received: 17 November 1999 / Accepted: 19 March 2000     

Comparison of dose distribution in IMRT and RapidArc technique in prostate radiotherapy

AimThe aim was to provide a dosimetric comparison between IMRT and RapidArc treatment plans with RPI index with simultaneous comparison of the treatment delivery time.BackgroundIMRT and RapidArc provide highly conformal dose distribution with good sparing of normal tissues. However, a complex spatial dosimetry of IMRT and RapidArc plans hampers the evaluation and comparison between plans calculated for the two modalities. RPI was used in this paper for treatment plan comparisons. The duration of the therapeutic session in RapidArc is reported to be shorter in comparison to therapeutic time of the other dynamic techniques. For this reasons, total treatment delivery time in both techniques was compared and discussed.Materials and methods15 patients with prostate carcinoma were randomly selected for the analysis. Two competitive treatment plans using respectively the IMRT and RapidArc techniques were computed for each patient in Eclipse planning system v. 8.6.15. RPIwin^® application was used for RPI calculations for each treatment plan.Additionally, total treatment time was compared between IMRT and RapidArc plans. Total treatment time was a sum of monitor units (MU) for each treated field.ResultsThe mean values of the RPI indices were insignificantly higher for IMRT plans in comparison to rotational therapy. Comparison of the mean numbers of monitor units confirmed that the use of rotational technique instead of conventional static field IMRT can significantly reduce the treatment time.ConclusionAnalysis presented in this paper, demonstrated that RapidArc can compete with the IMRT technique in the field of treatment plan dosimetry reducing the time required for dose delivery.     

Characterization of glycerol kinase and NAD-independent glycerol-3-phosphate dehydrogenase from Pediococcus pentosaceus N5p

The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157 : H7 in bovine faeces. To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested. These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean^® purification. Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g⁻¹ faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separation (IMS) to detect E. coli O157 : H7 in less than 8 h, but with a sensitivity of approximately 10³ cfu g⁻¹ faeces. These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers.     

Thymineless Death in Escherichia coli: Inactivation and Recovery

The effects of chloramphenicol (CAP) on the progress of thymineless death (TLD), nalidixic acid (NA) inactivation, ultraviolet (UV) irradiation, and mitomycin C (MC) inactivation were studied in Escherichia coli B, B(s-1), B(s-3), B(s-12), and B/r. This was done before, during, and after inactivation. During the progress of inactivation, it was found that at 10 to 20 mug of CAP per ml, up to 50% of the UV-sensitive bacteria survived TLD and about 10% survived NA. In E. coli B/r, at these concentrations of CAP, about 10 to 15% of the cells survived TLD and about 20 to 25% survived NA. Concentrations of CAP greater than 25 mug/ml actually increased the sensitivity of E. coli B, B(s-1), B(s-3), and B(s-12) to inactivation by either TLD or NA; at 150 mug of CAP per ml, the sensitivity of E. coli B/r to inactivation also increased. When E. coli B cells were incubated in CAP prior to inactivation, the longer the preincubation the longer onset of TLD was delayed; NA inactivation was also affected in that the rate of inactivation after CAP incubation was greatly decreased. Preincubation of E. coli B/r with CAP had much less effect on the progress of inactivation. After thymineless death, incubation in CAP plus thymine led to a rapid and almost complete recovery of E. coli B and B(s-12). Lesser recoveries were observed after inactivation due to UV, NA, or MC inactivation. E. coli B(s-1) and B/r did not recover viability after any mode of inactivation, and E. coli B(s-3) and B(s-12) recovered from UV to about 20% of the initial titer. It was suggested that protein synthesis, in particular proteins involved in deoxyribonucleic synthesis, was a determining factor in these inactivating and recovery events.