Effects of PIM3 in prognosis of colon cancer

Purpose PIM kinase is called proto-oncogene, but there are less research on PIM family in colon cancer. This study was designed to explore the prognosis of PIM3 in colon cancer. Methods In this study, we downloaded RNA-seq and clinical information of colon cancer from the Gene Expression Omnibus (GEO) database. Kaplan–Meier method was used for analyzing the impact of PIM3 on the survival of patients with colon cancer. Single-factor and multi-factor cox regression analysis were used for verifying the prognostic value of PIM3. Spearman correlation analysis was used for screening PIM3 related genes. Functional enrichment analysis was used for analyzing the biological functions and pathways in which PIM3 related genes may be involved. STRING online tools were used for building a co-expression network. Cytoscape was used for co-expression network visualization. Results Compared with the low expression group, the patients in the PIM3 high expression group lived longer time. Single-factor and multi-factor cox regression analysis indicated that PIM3 was an independent prognostic factor for colon cancer. Sixty-two PIM3 related genes were screened, and GO and KEGG enrichment analyses suggested that PIM3 related genes might be involved in the MAPK and WNT pathways. The co-expression network showed a strong correlation between PIM3 and MLKL, MYL5, PPP3R1 and other genes. Conclusions PIM3 is an independent prognostic factor of colon cancer and may be a target for the diagnosis and treatment of colon cancer (AU)

Effects of PIM3 in prognosis of colon cancer.

PURPOSE: PIM kinase is called proto-oncogene, but there are less research on PIM family in colon cancer. This study was designed to explore the prognosis of PIM3 in colon cancer. METHODS: In this study, we downloaded RNA-seq and clinical information of colon cancer from the Gene Expression Omnibus (GEO) database. Kaplan-Meier method was used for analyzing the impact of PIM3 on the survival of patients with colon cancer. Single-factor and multi-factor cox regression analysis were used for verifying the prognostic value of PIM3. Spearman correlation analysis was used for screening PIM3 related genes. Functional enrichment analysis was used for analyzing the biological functions and pathways in which PIM3 related genes may be involved. STRING online tools were used for building a co-expression network. Cytoscape was used for co-expression network visualization. RESULTS: Compared with the low expression group, the patients in the PIM3 high expression group lived longer time. Single-factor and multi-factor cox regression analysis indicated that PIM3 was an independent prognostic factor for colon cancer. Sixty-two PIM3 related genes were screened, and GO and KEGG enrichment analyses suggested that PIM3 related genes might be involved in the MAPK and WNT pathways. The co-expression network showed a strong correlation between PIM3 and MLKL, MYL5, PPP3R1 and other genes. CONCLUSIONS: PIM3 is an independent prognostic factor of colon cancer and may be a target for the diagnosis and treatment of colon cancer.

Research on the relationship between RAGE and its ligand HMGB1, and prognosis and pathogenesis of gastric cancer with diabetes mellitus.

OBJECTIVE: To investigate the relationship between the expression of receptor for advanced glycation end products (RAGE) and high-mobility group box-1 (HMGB1) and the clinical and pathological parameters and prognosis of the patients with gastric cancer (GC) with diabetes mellitus (DM). PATIENTS AND METHODS: 30 normal gastric mucosa, 30 tissues with GC, 90 tissues with GC and DM and their clinical data were collected. The expression levels of RAGE and HMGB1 were detected by immunohistochemistry. Kaplan-Meier survival curve was used to analyze the relationship between the expression levels of RAGE and HMGB1 and the 5-year survival rate. MTT and cell scratch assays were used to detect the effects of knockdown RAGE and HMGB1 on the proliferation and migration of BGC-823 cells. Real-Time PCR was used to detect the regulation of RAGE and HMGB1 on PTBP-1, and Spearman correlation analysis was performed to analyze the correlation between RAGE and HMGB1 and Polyprimidine tract protein (PTBP-1). RESULTS: Compared with the normal gastric mucosa group, the expression levels of RAGE and HMGB1 were significantly higher in the GC group, GC with DM group. The expression of RAGE and HMGB1 was related with lymph node metastasis, TNM staging, and tumor invasion (p<0.05). Age, TNM stage, tumor infiltration depth, the expression of RAGE and HMGB1 were related with prognosis of patients with GC and DM (p<0.05). Tumor infiltration depth, the expression of RAGE and HMGB1 could affect the 5-year survival rate of patients with GC and DM (p<0.05). CONCLUSIONS: Knockdown RAGE and HMGB1 increased the expression of PTBP-1, and RAGE and HMGB1 were negatively regulated with PTBP-1. RAGE and HMGB1 are independent risk factors for the prognosis of patients with GC with DM. RAGE and HMGB1 may regulate the expression of PTBP-1 and inhibit the glycolysis of cells, which may affect the cell proliferation and migration of GC.

[Epidemiological characteristics of COVID-19 in Wuchang district of Wuhan].

Objective: To analyzes epidemiological characteristics of COVID-19 and provide evidence for adjustment for COVID-19 prevention and control strategies. Methods: The data of COVID-19 cases in Wuchang district reported as of 19 March, 2020 were obtained from National Notifiable Disease Report System of Chinese Disease Prevention and Control Information System. The software's of Excel 2010, SPSSS 22.0, Arc GIS10.2 and Joinpoint regression program 4.8.0.0 were used for statistical analysis. Results: A total of 7547 COVID-19 cases had been reported as of 19 March, 2020 in Wuchang district, including 5 448 confirmed cases (72.19%), 2009 clinical diagnosed cases (26.62%) and 90 asymptomatic cases case (1.19%). The age of the cases was (56.65±16.25) years and age ranged from 2 days to 105 years among confirmed cases, 2634 were males (48.35%) and 2814 were females (51.65%), 2 492 were retirees (45.74%). A total of 545 health workers were infected with SARS-CoV-2 (7.22% of all cases) including 365 confirmed cases and 5 cases have died. A total of 430 cases of death were reported with case fatality rate of 7.89% (430/5 448), case fatality rate of males (10.9%, 266/2 634) was higher than that of females (5.82%, 164/2 814). The first phase of epidemic peak was from January 24 to January 26, the second phase of epidemic peak was from February 1 to February 5 and there was no one of new confirmed case in one day for the first time on March 18. The first four Streets with the highest incidence rates of confirmed cases were Huanghelou Street (1 043.77/100 000), Ziyang Street (627.97/100 000), Yangyuan Street (503.67/100 000) and Shuiguohu Street (486.02/100 000). Compared with females, aged ≤50 years and mild cases of clinical classification respectively, males (RR=0.690, 95%CI: 0.322-1.478), aged >50 years (RR=11.745, 95%CI: 6.878-20.058), severe cases (RR=2.317, 95%CI: 1.789-3.000) and critical cases of clinical classification (RR=10.794, 95%CI: 7.997-14.569), and gender time-dependent covariate (RR=1.392, 95%CI: 1.053-1.840) were major influencing factors of prognosis of COVID-19 confirmed cases. Conclusions: The gender, ages and occupation of distribution were wide among COVID-19 cases in Wuchang district. Males, aged >50 years, severe cases and critical cases of clinical classification were influencing factors of prognosis of COVID-19 confirmed cases. The standardized management of discharged cases, asymptomatic infected cases and close contact persons were main measures to reduce incidence rates of COVID-19 cases.

The safety and efficacy of endoscopic submucosal dissection for treating early oesophageal carcinoma: a meta-analysis.

INTRODUCTION: Oesophageal carcinoma is the sixth most lethal cancer in the world. At present, the choice of specific surgical methods is controversial. This study compares the safety and efficacy of endoscopic submucosal dissection and endoscopic mucosal resection in treating early oesophageal carcinoma. METHODS: We carried out a search of online databases including the Web of Science, PubMed, Embase and the Cochrane Library with no language restrictions. The inclusion criteria were patients with early oesophageal carcinoma who accepted the treatment of endoscopic submucosal dissection compared with endoscopic mucosal resection. FINDINGS: A total of 1,462 patients with 1,650 lesions from nine studies were included in the meta-analysis. When compared with the endoscopic mucosal resection group, the en bloc resection (endoscopic submucosal dissection 67.94% vs endoscopic mucosal resection 52.78%; odds ratio 19.79, p = 0.000) and complete resection (endoscopic submucosal dissection 75.57% vs endoscopic mucosal resection 59.47%; odds ratio 16.10, p = 0.000) rates were significantly higher in the endoscopic submucosal dissection group, while the local recurrence rate was significantly lower in the endoscopic submucosal dissection group (endoscopic submucosal dissection 0.08% vs endoscopic mucosal resection 2.66%; odds ratio 0.08, p = 0.000). The incidence of complications and procedural time were also tested.

Circular RNA hsa_circ_0010882 promotes the progression of gastric cancer via regulation of the PI3K/Akt/mTOR signaling pathway.

OBJECTIVE: Accumulating studies have reported that circular RNAs (circRNAs) can act as novel prognostic biomarkers in multiple malignant tumors. Here, we conducted a study to investigate the potential function and molecular mechanism of action of hsa_circ_0010882 in gastric cancer (GC). PATIENTS AND METHODS: The expression of hsa_circ_0010882 in the plasma of GC patients and in GC cell lines was verified by qRT-PCR. Its association with overall survival of GC patients was then analyzed by statistical analysis. Gain-of-function and loss-of-function assays were used to investigate the physiological function of hsa_circ_0010882 in GC cells in vitro in the context of proliferation, apoptosis, migration, and invasion. Moreover, the molecular mechanism of action of hsa_circ_0010882 was predicted using online databases and a literature review. A Western blot assay was used to detect the levels of proteins in the PI3K/Akt/mTOR signaling pathway. RESULTS: We found that hsa_circ_0010882 expression was significantly upregulated in the plasma of GC patients and GC cell lines. Increased expression of hsa_circ_0010882 was significantly correlated with tumor size and histological grade. In addition, GC patients with higher expression of hsa_circ_0010882 had significantly lower overall survival than patients with lower expression of hsa_circ_0010882. Multivariate analysis showed that hsa_circ_0010882 expression could be an independent prognostic factor for overall survival. The proliferation, migration, and invasiveness of GC cell lines were inhibited following hsa_circ_0010882 knock-down, while GC cellular apoptosis increased. Further, overexpression of hsa_circ_0010882 leads to increased proliferation, migration, and invasiveness of GC cell lines. While apoptosis was higher in the GC cell line group with low expressing hsa_circ_0010882 than the control group, no significant difference in apoptosis was detected between the hsa_circ_0010882 overexpressing and the control group. Finally, a mechanistic analysis demonstrated that the hsa_circ_0010882 was positively associated with PI3K/Akt/mTOR signaling pathway. CONCLUSIONS: Hsa_circ_0010882, as an oncogenic molecule, is highly expressed in the plasma of patients with GC and is associated with poor prognosis. It plays an important role in proliferation, migration, and invasive genotypes of GC cell lines via regulation of the PI3K/Akt/mTOR signaling pathway. Additionally, it might be a potential prognostic biomarker for GC patients.

[Influence of apatinib and VEGFR2-906T>C polymorphism on clinical outcomes of advanced non-small cell lung cancer patients].

Objective: To investigate the clinical outcomes of advanced non-small cell lung cancer (NSCLC) treated by apatinib regimens and the influence of VEGFR2-906T>C polymorphism. Methods: A total of 109 patients with advanced NSCLC who were treated by apatinib after three and more lines from March 2015 to December 2017 in the Department of Oncology of the First Affiliated Hospital of Zhengzhou University were included in this study. Overall response rates were evaluated after 2 cycles, then progression free survival (PFS) and overall survival (OS) were investigated, and safety data were recorded. Additionally, peripheral blood and the biopsy tissue specimens of some NSCLC patients were collected for the genotyping of genetic variation and VEGFR2 gene mRNA expression, respectively. The association between genotype and other characteristics and VEGFR2 gene mRNA expression were analyzed. The univariate analysis of genotypes and prognosis was carried out by Kaplan-Meier survival analysis, and multivariate analysis were adjusted by Cox regression analysis. Results: The treatment effect could be evaluated in all the 109 patients, among them, complete remission (CR) 0 case, partial remission (PR) 19 case, stable disease (SD) 58 case, progression disease (PD) 32 case. Overall response rate (ORR) was 17.43%, disease control rate (DCR) was 70.64%, median PFS was 4.35 months, median OS was 8.35 months. Of the polymorphisms analyzed, only -906T>C was of clinical significance. The prevalence of -906T>C in VEGFR2 among the study population were as follows: TT genotype 64 cases (58.72%), TC genotype 37 cases (33.94%), CC genotype 8 cases (7.34%), minor allele frequency of -906T>C was 0.24. The distribution of three genotypes was in accordance with Hardy-Weinberg Equilibrium (P=0.418). CC and TC genotype patients were merged in the comparison of clinical outcomes. The analysis of patients with different genotypes found that the ORR of CC/TC genotypes and TT genotypes were 13.33% and 20.31% (P=0.377), respectively. And the median PFS of patients with CC/TC genotype and TT genotype were 3.25 and 5.35 months, respectively, which was statistically significant (P=0.007). In terms of OS, the median OS of the two genotypes were 7.35 and 9.15 (P=0.014), respectively. Adjusted in multivariate Cox regression analysis of PFS, TC/CC genotypes were an independent factor for PFS (OR=1.83, P=0.015). The correlation between -906T>C and adverse reactions was not found in the safety analysis. Additionally, of the 69 biopsy tissue specimens, gene expression analysis was conducted. And the results show that the mRNA expression of VEGFR2 in cancer tissues of the patients with CC/TC genotypes were significantly higher than those of the TT genotype patients (P<0.001). Conclusions: Apatinib is safe and effective for patients with advanced non-small cell in multiline therapy. VEGFR2 -906T>C CC/TC genotype has a worse effect on apatinib multiline treatment in patients with advanced NSCLC.

Measurement of density fluctuation propagation direction via the far-forward collective scattering diagnostic based on polarimeter-interferometer.

Previously, the 17-channel three-wave polarimeter-interferometer system (POLARIS) on the J-TEXT tokamak has been implemented to measure far-forward collective scattering (FFCS) from electron density fluctuations. Recently, this system has been exploited to measure the propagation direction of density fluctuation. After considering the refraction of the laser probe beam passing through plasma, the ray tracing result shows that the detector of POLARIS may receive asymmetric far-forward scattering beams. Thus, the heterodyne detection of FFCS is available to identify the propagation direction of density fluctuation by resolving the asymmetric scattering spectrum. Experimentally, the transform of the heterodyne scattering spectrum from symmetry to asymmetry has been observed, while the refraction effect becomes strong demonstrating the capacity of measuring the propagation direction of fluctuation. Furthermore, by changing the plasma potential through the use of an applied positive electrode biasing, the reverse of frequency shift for the heterodyne scattering spectrum is identified, confirming the validity of direction discrimination of density fluctuation.

Effect of body mass index on overall survival of patients with allogeneic hematopoietic stem cell transplantation.

BACKGROUND/OBJECTIVES: The present work was performed to investigate the association between body mass index (BMI) before transplantation and the overall survival (OS) of patients with allogeneic hematopoietic stem cell transplantation (allo-HSCT). SUBJECTS/METHODS: Data from 310 adults who were diagnosed with acute leukemia and underwent allo-HSCT between March 2001 and December 2011 were analyzed. According to the suggested BMI categories for Asian population, patients with BMIs of ⩾23 and ⩾25 kg/m2 were identified as overweight and obese, respectively. Cox proportional hazards models was used to estimate hazard ratios (HRs) with 95% confidence intervals (CIs). RESULTS: The median follow-up time among the patients was 19.7 months (interquartile range=8.1-37.7). A total of 93 (34.8%) people died within the follow-up period. After adjusting for the potential confounders, normal-weight, overweight and obese patients showed significantly lower HRs than those of underweight patients, with a significant trend of OS improvement upon increasing BMI (P=0.019). Overweight and obese patients survived longer, with a significantly decreased HR by ~40% (HR=0.60; 95% CI: 0.38-0.95) compared with underweight and normal-weight patients. CONCLUSIONS: An increased OS was seen in allo-HSCT patients with BMI⩾23 kg/m2 compared to those with lower BMI. Further work are still needed to investigate of the effects of BMI or body composition on the survival of allo-HSCT patients.

Far-forward collective scattering measurements by FIR polarimeter-interferometer on J-TEXT tokamak.

The multi-channel three-wave polarimeter-interferometer system on J-TEXT tokamak has been exploited to measure far-forward collective scattering from electron density fluctuations. The diagnostic utilizes far infrared lasers operated at 432 µm with 17-channel vertical chords (3 cm chord spacing), covering the entire cross section of plasma. Scattering laser power is measured using a high-sensitivity Schottky planar diode mixer which can also detect polarimetric and interferometric phase simultaneously. The system provides a line-integrated measurement of density fluctuations with maximum measurable wave number: k⊥max ≤ 2 cm-1 and time response up to 350 kHz. Feasibility of the diagnostic has been tested, showing higher sensitivity to detect fluctuation than interferometric measurement. Capability of providing spatial-resolved information of fluctuation has also been demonstrated in preliminary experimental applications.

Significant impacts of CYP3A4*1G and CYP3A5*3 genetic polymorphisms on the pharmacokinetics of diltiazem and its main metabolites in Chinese adult kidney transplant patients.

WHAT IS KNOWN AND OBJECTIVE: The calcium channel blocker diltiazem has been used widely as a cyclosporine (CsA)/tacrolimus-sparing agent. However, considerable interpatient variability in diltiazem's CsA/tacrolimus-sparing effect has been observed in many clinical studies. This study was carried out to investigate the impacts of the CYP3A4*1G and CYP3A5*3 genetic polymorphisms on the trough concentration/dose ratios and pharmacokinetics of diltiazem and its main metabolites in Chinese adult renal transplant patients. METHODS: Two hundred and twenty-five Chinese renal transplant patients were genotyped for CYP3A4*1G and CYP3A5*3. The predose and post-dose plasma concentrations of diltiazem and its main metabolisms were determined by HPLC. The relationships between the genotypes and pharmacokinetics were investigated. RESULTS AND DISCUSSION: The dose-adjusted concentrations and pharmacokinetics of diltiazem and its main metabolites were significantly affected by CYP3A4 *1G and CYP3A5*3 alleles. Patients with a CYP3A4*1/*1 genotype were found to have a higher dose-adjusted trough concentration and AUC of diltiazem and its main metabolites compared with those with CYP3A4*1G*1G(P<0·05). The dose-adjusted trough levels and AUC of diltiazem and its main metabolites were significantly lower in CYP3A5*1*1 carriers than in CYP3A5*3 carriers (P < 0·05). WHAT IS NEW AND CONCLUSION: The CYP3A4*1G and CYP3A5*3 genetic polymorphisms are closely related to the trough concentration/dose ratios and pharmacokinetics of diltiazem and its main metabolites in Chinese adult renal transplant patients.

High resolution polarimeter-interferometer system for fast equilibrium dynamics and MHD instability studies on Joint-TEXT tokamak (invited).

A high-performance Faraday-effect polarimeter-interferometer system has been developed for the J-TEXT tokamak. This system has time response up to 1 µs, phase resolution < 0.1° and minimum spatial resolution ∼15 mm. High resolution permits investigation of fast equilibrium dynamics as well as magnetic and density perturbations associated with intrinsic Magneto-Hydro-Dynamic (MHD) instabilities and external coil-induced Resonant Magnetic Perturbations (RMP). The 3-wave technique, in which the line-integrated Faraday angle and electron density are measured simultaneously by three laser beams with specific polarizations and frequency offsets, is used. In order to achieve optimum resolution, three frequency-stabilized HCOOH lasers (694 GHz, >35 mW per cavity) and sensitive Planar Schottky Diode mixers are used, providing stable intermediate-frequency signals (0.5-3 MHz) with S/N > 50. The collinear R- and L-wave probe beams, which propagate through the plasma poloidal cross section (a = 0.25-0.27 m) vertically, are expanded using parabolic mirrors to cover the entire plasma column. Sources of systematic errors, e.g., stemming from mechanical vibration, beam non-collinearity, and beam polarization distortion are individually examined and minimized to ensure measurement accuracy. Simultaneous density and Faraday measurements have been successfully achieved for 14 chords. Based on measurements, temporal evolution of safety factor profile, current density profile, and electron density profile are resolved. Core magnetic and density perturbations associated with MHD tearing instabilities are clearly detected. Effects of non-axisymmetric 3D RMP in ohmically heated plasmas are directly observed by polarimetry for the first time.

Microvessel density of hepatocellular carcinoma: its relationship with prognosis.

PURPOSE: To elucidate the relationship between angiogenesis and prognosis after curative resection of hepatocellular carcinoma (HCC). METHODS: An immunohistochemical study using anti-CD34 monoclonal antibody was carried out on surgical specimens from 78 HCC patients who had undergone curative resection; microvessel density (MVD) was counted and the overall survival and disease-free survival were analyzed retrospectively. RESULTS: Blood vessels in the tumor were strongly stained by anti-CD34 antibody, but not those in the surrounding liver parenchyma. There were three types of tumor vessels: capillary-like (n = 59), sinusoid-like (n = 16) and mixed-type (n = 3). The median MVD count was 100 per field. The HCC were designated as hypovascular (n = 36) with an MVD count below 100, and hypervascular (n = 42) with an MVD count of 100 or more per field. The 5-year survival and disease-free survival rates were 49.7% and 42.8% respectively, and statistical analysis showed that the MVD level was not correlated with tumor size, capsule status, Edmondson's grade, alpha-fetoprotein level, associated cirrhosis, gamma-glutamyltransferase, and serum HBsAg status. The sinusoid-like tumor vessels appeared more frequently in the more differentiated tumors (P<0.05). No statistical difference in overall and disease-free survival between different MVD levels and microvessel types was found. Tumor size was the only predicting factor in the entire series. In patients with small HCC (< or =5 cm, n = 40), 5-year survival and disease-free survival rates were 58.9% and 52.7% respectively, higher than the values in large HCC (39.8% and 32.0% respectively, P<0.05). The MVD level was an independent predicting factor of disease-free survival, 5-year disease-free survival in the hypovascular group (74.6%) being better than that in the hypervascular group (34.7%, P<0.05). CONCLUSIONS: The MVD level was not related to tumor size, capsule statuo, Edmondson's grade, alpha-fetoprotein level, associated cirrhosis, gamma-glutamyltransferase and serum HBsAg status. In the entire series, tumor size was the only factor influencing survival after curative resection. However, in patients with small HCC, the MVD level was an independent factor of disease-free survival. The pathological and clinical implications of different types of tumor vessels in HCC remain to be studied.

The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli.

In Escherichia coli, stringently controlled genes are highly transcribed during rapid growth, but "turned off" under nutrient limiting conditions, a process called the stringent response. To understand how transcriptional initiation at these promoters is coordinately regulated, we analyzed the interactions between RNA polymerase (RNAP) (both wild type and mutants) and four stringently controlled promoters. Our results show that the interactions between RNAP and stringently controlled promoters are intrinsically unstable and can alternate between relatively stable and metastable states. The mutant RNAPs appear to specifically further weaken interactions with these promoters in vitro and behave like "stringent" RNAPs in the absence of the stringent response in vivo, constituting a novel class of mutant RNAPs. Consistently, these mutant RNAPs also activate the expression of other genes that normally require the response. We propose that the stability of initiation complexes is coupled to the transcription of stringently controlled promoters, and this unique feature coordinates the expression of genes positively and negatively regulated by the stringent response.

Multiple regions on the Escherichia coli heat shock transcription factor sigma32 determine core RNA polymerase binding specificity.

We have analyzed the core RNA polymerase (RNAP) binding activity of the purified products of nine defective alleles of the rpoH gene, which encodes sigma32 in Escherichia coli. All mutations studied here lie outside of the putative core RNAP binding regions 2.1 and 2.2. Based on the estimated K(s)s for the mutant sigma and core RNAP interaction determined by in vitro transcription and by glycerol gradient sedimentation, we have divided the mutants into three classes. The class III mutants showed greatly decreased affinity for core RNAP, whereas the class II mutants' effect on core RNAP interaction was only clearly seen in the presence of sigma70 competitor. The class I mutant behaved nearly identically to the wild type in core RNAP binding. Two point mutations in class III altered residues that were distant from one another. One was found in conserved region 4.2, and the other was in a region conserved only among heat shock sigma factors. These data suggest that there is more than one core RNAP binding region in sigma32 and that differences in contact sites probably exist among sigma factors.

RNA polymerase beta mutations have reduced sigma70 synthesis leading to a hyper-temperature-sensitive phenotype of a sigma70 mutant.

This work describes a mutational analysis of the interaction between the beta and sigma subunits of Escherichia coli RNA polymerase. The rpoD800 mutant has a temperature-sensitive growth phenotype because the mutant sigma70 polypeptide is not stable at a high temperature. Some rpoB mutations, including rpoB114, enhanced the temperature sensitivity of the rpoD800 mutant. We determined the mechanism by which the rpoB114 rpoD800 double mutant becomes hyper-temperature sensitive for growth. We found that the levels of the mutant sigma70 in the rpoB114 rpoD800 mutant were dramatically reduced compared to that in the rpoD800 mutant after temperature shift-up. The rate of synthesis of the sigma70 polypeptide was reduced in the rpoB114 rpoD800 double mutant compared to the rpoD800 mutant, whereas the half-life of the mutant sigma70 polypeptide after temperature shift-up was the same in both strains. We conclude that because of the reduction of expression of rpoD800 by rpoB114, in concert with the intrinsic instability of the mutant sigma70 polypeptide, the amount of holoenzyme containing sigma70 becomes limiting upon temperature shift-up. This results in the hyper-temperature sensitivity of the rpoB114 rpoD800 double mutant. Furthermore, the effect of rpoB114 on the expression of sigma70 is independent of the rpoD800 allele and is at the transcriptional level. In vitro transcription assays showed that the mutant RNA polymerase RpoB114 was defective in transcribing the two major promoters of the rpoD operon specifically. The effects of these rpoB mutations on gene expression are discussed.

Interaction of Gal repressor with inducer and operator: induction of gal transcription from repressor-bound DNA.

Gal repressor inhibits transcription from the gal promoter (P1) when it binds to the cognate operator (O(E)). The repression is relieved by the presence of the inducer D-galactose. Compared with its interaction with free repressor, D-galactose binds to the repressor-operator complex with 10-fold reduced affinity as determined by fluorescence enhancement measurements. Thermodynamic analysis and fluorescence anisotropy showed that the stability of the repressor-operator complex is reduced by only 7-fold by the presence of the inducer in the complex. The formation of the inducer-repressor-operator ternary complex has been confirmed by CD spectral analysis. Fluorescence spectroscopy and energy transfer experiments suggest that individual allosteric effects of the two ligands, inducer and operator, on Gal repressor are responsible for the slightly weakened stability of the ternary complex compared with the stability of the inducer-repressor and repressor-operator complexes. In vitro transcription results demonstrated full derepression of transcription of the P1 promoter under conditions in which the concentrations of the inducer-repressor binary complex are severalfold higher than the dissociation constant of the inducer-repressor-operator ternary complex into inducer-repressor and free DNA. These results strongly suggest that the inducer binding to the repressor-operator complex does not lead to dissociation of the repressor from the operator during transcription induction. Because Gal repressor inhibits transcription by modulating the alpha subunit of the P1-bound RNA polymerase, we conclude that the inducer binding to the operator-bound repressor only allosterically relieves the inhibitory effect of repressor on RNA polymerase without dissociating the repressor from DNA.

The non-inducible nature of super-repressors of the gal operon in Escherichia coli.

We isolated and characterized mutant repressors (GalR) of the gal operon in Escherichia coli. These repressors (super-repressors), called GalRs, have a non-inducible phenotype. Repression of the gal operon by super-repressors cannot be lifted by inducer. The mutant galR genes, galRs, have been cloned and the mutational changes determined. Two of them, galRuv7s and galR78s, were located in the proposed sugar binding domains of the repressor. The repressor from wild-type (galR+), as well as from mutant galRuv7s, was purified and characterized biochemically. The results showed that, like wild-type GalR+, GalRuv7s binds to DNA normally and represses transcription from the P1 promoter and stimulates that from the P2 promoter of the gal operon. Nevertheless, compared to GalR+, GalRuv7s is much less sensitive to the presence of the inducer, D-galactose. The affinity of D-galactose to GalRuv7s is 10 to 30-fold lower, as measured by the effect of the inducer on GalR tryptophan fluorescence; GalR complexes with DNA and on GalR repression of transcription. Our results suggest that the super-repressor phenotype of GalRuv7s is because of a defect in D-galactose binding rather than a defect in the ligand-induced allosteric change or increased affinity for the operator.

How a mutation in the gene encoding sigma 70 suppresses the defective heat shock response caused by a mutation in the gene encoding sigma 32.

In Escherichia coli, transcription of the heat shock genes is regulated by sigma 32, the alternative sigma factor directing RNA polymerase to heat shock promoters. sigma 32, encoded by rpoH (htpR), is normally present in limiting amounts in cells. Upon temperature upshift, the amount of sigma 32 transiently increases, resulting in the transient increase in transcription of the heat shock genes known as the heat shock response. Strains carrying the rpoH165 nonsense mutation and supC(Ts), a temperature-sensitive suppressor tRNA, do not exhibit a heat shock response. This defect is suppressed by rpoD800, a mutation in the gene encoding sigma 70. We have determined the mechanism of suppression. In contrast to wild-type strains, the level of sigma 32 and the level of transcription of heat shock genes remain relatively constant in an rpoH165 rpoD800 strain after a temperature upshift. Instead, the heat shock response in this strain results from an approximately fivefold decrease in the cellular transcription carried out by the RNA polymerase holoenzyme containing mutant RpoD800 sigma 70 coupled with an overall increase in the translational efficiency of all mRNA species.