[Predictive value of low-density lipoprotein cholesterol/remnant cholesterol coordination on cardiovascular and cerebrovascular events in elderly patients with hypertension].

Objective: To explore the predictive value of serum low-density lipoprotein cholesterol/residual cholesterol (LDL-C/RC) coordination on major adverse cardiovascular events (MACE) in elderly hypertensive patients. Methods: This is a prospective cohort study. Elderly hypertensive patients hospitalized in Beijing Anzhen Hospital from June 2018 to June 2020 were prospectively enrolled. According to the coordination of baseline LDL-C/RC, patients were divided into four groups: low LDL-C and low RC (LDL-C<2.6 mmol/L, and RC<0.62 mmol/L), low LDL-C and high RC (LDL-C<2.6 mmol/L, and RC≥0.62 mmol/L), high LDL-C and low RC (LDL-C≥2.6 mmol/L, and RC<0.62 mmol/L), and high LDL-C and high RC (LDL-C≥2.6 mmol/L, and RC≥0.62 mmol/L). Patients were followed up until June 2022. The primary outcome was MACE, including non-fatal acute coronary syndrome, non-fatal acute heart failure, non-fatal stroke and death. Kaplan-Meier survival analysis was used to evaluate MACE among the four groups, and Cox regression analysis was performed to evaluate the related factors of MACE. Results: A total of 847 hypertensive patients were enrolled. There were 453 males (53.5%), mean age was (72.4±8.8) years. There were 207, 162, 263, 215 patients in the low LDL-C and low RC, low LDL-C and high RC, high LDL-C and low RC and high LDL-C and high RC group, respectively. During a median follow-up of 37 months, 196 patients (23.1%) had MACE, including 77 cases (9.1%) of ACS, 56 cases (6.6%) of acute heart failure, 34 cases (4.0%)of non-fatal stroke, 29 cases(3.4%) of all-cause mortality, and 16 cases (1.9%) of cardiovascular death. Risk of MACE of the four groups was significantly different (log-rank P<0.001). Compared with low LDL-C and low RC groups, high LDL-C and high RC groups had the highest incidence of MACE (HR=2.237, 95%CI 1.328-3.783, P=0.004), followed by low LDL-C and high RC groups (HR=1.745, 95%CI 1.220-2.527, P=0.003) and high LDL-C and low RC groups (HR=1.393, 95%CI1.048-1.774, P=0.022). In addition, the risk of nonfatal ACS among the four groups was also statistically significant (P=0.037), while the risk of nonfatal acute heart failure, nonfatal stroke, all-cause mortality and cardiovascular death were similar (all P>0.05). Multivariate Cox regression analysis showed that age (HR=1.271), duration of hypertension (HR=1.339), diabetes (HR=1.415), hyperlipidemia (HR=1.348), serum creatinine (HR=1.263), N-terminal pro-B-type natriuretic peptide (HR=1.316), LDL-C (HR=1.205), RC (HR=1.302), low LDL-C and high RC (HR=1.745), high LDL-C and low RC (HR=1.393), high LDL-C and high RC (HR=2.237) were independently associated with the occurrence of MACE. Conclusion: The coordination of LDL-C/RC affects the risk of MACE in elderly hypertensive patients, and the risk of MACE is the highest in patients with high LDL-C and high RC.

[A prospective study on the safety and efficacy of excimer laser coronary angioplasty for the treatment of degenerated great saphenous vein graft].

Objective: To explore the safety and efficacy of excimer laser coronary angioplasty (ELCA) for the treatment of degenerated great saphenous vein graft (SVG). Methods: This is a single-center, prospective, single-arm study. Patients, who were admitted to the Geriatric Cardiovascular Center of Beijing Anzhen Hospital from January 2022 to June 2022, were consecutively enrolled. Inclusion criteria were recurrent chest pain after coronary artery bypass surgery (CABG), and coronary angiography confirmed that the SVG stenosis was more than 70% but not completely occluded, and interventional treatment for SVG lesions was planned. Before balloon dilation and stent placement, ELCA was used to pretreat the lesions. Optical coherence tomography (OCT) examination was performed and postoperative index of microcirculation resistance (IMR) were assessed after stent implantation. The technique success rate and operation success rate were calculated. The technique success was defined as the successful passage of the ELCA system through the lesion. Operation success was defined as the successful placement of a stent at the lesion. The primary evaluation index of the study was IMR immediately after PCI. Secondary evaluation indexes included thrombolysis in myocardial infarction (TIMI) flow grade, corrected TIMI frame count (cTFC), minimal stent area and stent expansion measured by OCT after PCI, and procedural complications (Ⅳa myocardial infarction, no reflow, perforation). Results: A total of 19 patients aged (66.0±5.6) years were enrolled, including 18 males (94.7%). The age of SVG was 8 (6, 11) years. The length of the lesions was greater than 20 mm, and they were all SVG body lesions. The median stenosis degree was 95% (80%, 99%), and the length of the implanted stent was (41.7±16.3)mm. The operation time was 119 (101, 166) minutes, and the cumulative dose was 2 089 (1 378, 3 011)mGy. The diameter of the laser catheter was 1.4 mm, the maximum energy was 60 mJ, and the maximum frequency was 40 Hz. The technique success and the operation success rate were both 100% (19/19). The IMR after stent implantation was 29.22±5.95. The TIMI flow grade of patients after ELCA and stent implantation was significantly improved (all P>0.05), and the TIMI flow grade of all patients after stent implantation was Grade Ⅲ. The cTFC decreased significantly after ELCA (33.2±7.8) and after stent placement (22.8±7.1) than preoperative level (49.7±13.0) (both P<0.001). The minimum stent area was (5.53±1.36)mm2, and the stent expansion rate was (90.0±4.3)%. Perforation, no reflow, type Ⅳa myocardial infarction and other complications were not observed. However, postoperative high-sensitivity troponin level was significantly increased ((67.937±33.839)ng/L vs. (5.316±3.105)ng/L, P<0.001). Conclusion: ELCA is safe and effective in the treatment of SVG lesions and could improve microcirculation and ensure full expansion of stent.

[A survey on clinical practice of tracheostomy decannulation in medical institutions at all levels across the country].

Objective: A questionnaire survey was conducted on the clinical practice of tracheostomy decannulation among medical staff in medical institutions at all levels across the country. Methods: The questionnaire was determined by literature review and expert consultation to investigate the clinical practice of tracheostomy decannulation among medical staff in comprehensive and rehabilitation hospitals of different levels across the country and the factors considered when deciding to decannulate. Statistical methods used χ² test and one-way ANOVA. Results: A total of 570 questionnaires were collected from all over the country, with 463 valid questionnaires. The survey results showed that the most important factors in clinical practice to determine the decannulation of the tracheostomy tube were upper airway patency, cough effectiveness, level of consciousness and oxygenation. Before decannulation, 220 (47.50%) would choose to change to metal cannula, and 384 (82.90%) would routinely occlude the tube. 294 (63.50%) thought that re-intubation within 24 hours after decannulation of the tracheostomy tube was failure of decannulation. The decannulation failure rate was mostly 2%-5%. Conclusions: Upper airway patency, cough effectiveness, level of consciousness and oxygenation were important factors when considering decannulation. Reintubation within 24 hours of decannulation was defined as failure by the majority of respondents.

[Clinical diagnosis and treatment of 27 cases of metanephric adenoma].

The clinical features, imaging features, treatment methods and pathological features of 27 patients with metanephric adenoma were analyzed. It was found that the clinical features and imaging features of metanephric adenoma were difficult to differentiate from renal malignantology. Pathology can be clearly diagnosed and some can be combined with malignant components. Nephron sparing surgery is the first choice, and the prognosis is good, but still need regular follow-up.

MicroRNA-95-3p promoted the development of prostatic cancer via regulating DKK3 and activating Wnt/ß-catenin pathway.

OBJECTIVE: Previous studies have shown that microRNA-95-3p (miR-95-3p) plays a crucial role in multiple human cancers except for prostatic cancer (PCa). Therefore, the function of miR-95-3p was investigated in PCa in the present work. PATIENTS AND METHODS: The expression of miR-95-3p was measured by Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) assay. Western blot assay was used to examine the protein expression of epithelial-mesenchymal transition (EMT) markers. In addition, the function of miR-95-3p was detected through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell assays. Dual Luciferase assay was applied to confirm the relationship between miR-95-3p and dickkopf-3 (DKK3). The tumor growth was observed through xenograft tumor formation assay. RESULTS: The upregulation of miR-95-3p was detected in PCa tissues and cell lines, which predicted poor prognosis of PCa patients. Moreover, miR-95-3p promoted cell proliferation, migration and invasion in PCa by targeting DKK3 and activating the Wnt/ß-catenin pathway. MiR-95-3p also promoted the tumor growth of PCa in vivo. Besides that, downregulation of DKK3 was identified in PCa and low DKK3 expression predicted poor prognosis of PCa patients. CONCLUSIONS: MiR-95-3p promoted the development of PCa via targeting DKK3 and activating the Wnt/ß-catenin pathway.

[Thyroglobulin measurement in fine-needle aspiration washout fluid for detection of differentiated thyroid cancer lymph nodes metastases].

Objective: To investigate the feasibiity of detecting lymph node metastasis in differentiated thyroid cancer patients by thyroglobin（Tg）measurement in fine-needle aspiration washout fluid. Method: Forty-seven differentiated thyroid cancer patients with cervical lymph node metastasis were included in this study. Fine-needle aspirations were performed on lymph nodes which were surgically resected for measurement of Tg in washout fluid. Diagnostic sensitivity, specificity and accuracy were evaluated according to three predetermined Tg threshold values: FNA-Tg/serum Tg＞1, 10 ng/ml and 100 ng/ml. Result: The meadian value of FNA-Tg in metastatic lymph node (n=46) was 5 177 ng/ml (1.79-408 300 ng/ml)，while lymph node without metastasis (n=11) showed a meadian level of 0.63 ng/ml (0.04-125.4 ng/ml). With the threshold of FNA-Tg level at FNA-Tg/serum Tg＞1, 10 ng/ml and 100 ng/ml respectively, the diagnostic sensitivity was 91.3%, 97.83% and 93.48% respectively,the specificity was 81.82%,63.64% and 90.90% respectively,and the accuracy was 89.47%,91.22% and 92.98% respectively. Conclusion: Thyroglobin measurement in fine-needle aspiration washout fluid is effective to diagnose lymph node metastasis in differentiated thyroid cancer.

[A multi-center study on the long-term mortality and related risk factors in patients with chronic heart failure receiving resynchronization therapy].

Objective: To analyze long-term mortality and patients characteristics of cardiac resynchronization therapy (CRT) for patients with chronic heart failure. Methods: In-patients with chronic heart failure who received CRT in the three medical centers(Bethune International Peace Hospital, General Hospital of Shenyang Military Command of Chinese People's Liberation Army, and 251 Hospital of People's Liberation Army)from March 2001 to June 2013 were included.Mortality and related causes, echocardiographic parameters were analyzed. Results: A total of 200 patients were treated with CRT therapy (154 males, mean age (60.57±11.75) years), 59 cases suffered from ischemic cardiomyopathy (ICM), patients were followed up from 0.5 to 12 years.The all-cause mortality rate was 25.50% (51/200), 20 out of 59 (33.90%) ICM patients died, as compared with 31 deaths out of 141 (21.98%) in non-ischemic cardiomyopath (NICM) patients.Thirty-six patients died due to cardiac death (70.59%), in which sudden death occurred in 21 patients (41.18%). Non-cardiac death occurred in 13 patients (25.49%), two patients died due to unknown reasons (3.92%). Incidence of cardiac death was significantly higher than non-cardiac death (P<0.01). The main cause for cardiac death was NICM (28/36, 77.78%), while the main cause of non-cardiac death was ICM (11/13, 84.62%, P<0.01). Patients died due to cardiac death were younger (P<0.01) and had larger left atrial end-diastolic diameter (LAEDD) and left ventricular end-diastolic diameter (LVEDD) (P<0.01), lower left ventricular ejection fraction (LVEF)(P<0.05), higher pulmonary artery pressure(P<0.05) compared to patients with non-cardiac death.One hundred and fifty-two cases received CRT-P and 48 cases received CRT-D, there were no significant differences in gender, the course of heart failure, serum creatinine levels, pre-operative and post-operative QRS duration and so on between the CRT-P and CRT-D groups(all P>0.05). Eleven out of the 48 cases with CRT-D died during the following-up (21.57%) , while 40 out of 152 cases with CRT-P died (78.43%) during the following-up(χ2=3.13, P<0.01). The proportional mortality rate in cause of death in patients with CRT-D was non-cardiac while in those with CRT-P was cardiac (χ2=2.66, P<0.01), sudden death rate was also significantly higher in CRT-P group than in CRT-D group (χ2=2.16, P<0.01). By using single- and multiple-factor Cox regression analysis, age, disease course, pre-operation QRS duration and NYHA classification were predictors of cardiac death(all P<0.05). Conclusions: The all-cause mortality of CRT is 25.50% in this patient cohort, mortality rate was higher in ICM patients than in NICM patients.Sudden cardiac death rate was the highest mortality reason.The mortality rate of patients with CRT-P was significantly higher than in patients with CRT-D.In comparison with patients of non-cardiac death, patients of cardiac death had larger left atrium and left ventricle and worse heart function before CRT implantation.

Analysis of genetic diversity and population structure in a tomato (Solanum lycopersicum L.) germplasm collection based on single nucleotide polymorphism markers.

Knowledge of genetic diversity is important to assist breeders in the selection of parental materials and in the design of breeding programs. In this study, we genotyped 348 inbred tomato lines, representing vintage and contemporary fresh-market varieties, by using 52 single nucleotide polymorphisms (SNPs); 45 of these were found to be polymorphic. The average minor allele frequency and unbiased expected heterozygosity were 0.315 and 0.356, respectively. Population structure analysis revealed that contemporary germplasm could be distinctly divided into six subpopulations representing three market classes and breeding programs (pink, green, and red). Vintage germplasm could be separated into at least two subpopulations, and more admixtures were found in vintage lines than in contemporary lines. These findings indicate that contemporary inbred lines are more diversified than vintage inbred lines. AMOVA of vintage and contemporary lines was performed. A significant difference was found (P < 0.01), which explained 17.4% of the total genetic variance. Subsequently, we constructed a core collection using 45 polymorphic SNP markers. The data showed that all alleles were captured by only 2% of lines, indicating that more alleles, as well as rare alleles, could enable more variation to be captured in the core collection. These data allow us to discard redundant inbred tomato lines and to select elite inbred lines, which will accelerate the breeding process.

[Salubrinal improves cardiac function in rats with heart failure post myocardial infarction through reducing endoplasmic reticulum stress-associated apoptosis].

OBJECTIVE: Endoplasmic reticulum (ER) stress plays an important role in ischemia-mediated cell death. The aim of the current study is to investigate the effects of salubrinal (Sal), a selective eIF2a dephosphorylation inhibitor, on heart failure rats and related mechanisms. METHODS: Heart failure was induced by coronary artery ligation (MI) in adult male Sprague-Dawley rats. To ensure comparable MI sizes post coronary artery ligation on various groups, echocardiography examination was performed before and 30 minutes after ligation in MI groups. Then rats were randomly assigned to 4 groups: Sham group (n=12), MI group (n=10), MI plus vehicle injections group (DMSO group, n=12) and MI plus Sal injection group (Sal group, n=12). Sal (1 mg/kg) or DMSO was injected via the tail vein daily for the first 3 days (starting at 30 minutes after ligation of the left coronary artery), followed by subcutaneous injections twice per week for 8 weeks. Cardiac function was assessed by echocardiography and cell apoptosis assessed by flow cytometric analysis after 8 weeks. Protein and mRNA levels of ER stress markers were evaluated by immunohistochemistry and real time RT-PCR respectively. RESULTS: Eight weeks later, LVEF was significantly higher, while LVESD and LVEDD values were significantly lower in Sal group compared to MI and DMSO groups (all P<0.05); LV/BW ratio was significantly higher in MI group than in Sham group ((2.30±0.40) mg/g vs.(1.78±0.31) mg/g, P<0.05), which was significantly reduced in Sal group ((1.88±0.25) mg/g), but not in DMSO group((2.25±0.36) mg/g, P<0.05 vs. MI). In addition, flow cytometric analysis showed that Sal treatment significantly reduced apoptosis but not necrosis in post MI. Immunohistochemistry and real time PCR analysis showed that the myocardial protein and mRNA expression of ER stress markers were significantly lower in Sal group than in MI group, myocardial caspase-12 expression was significantly upregulated in MI group and significantly reduced by Sal treatment. CONCLUSIONS: Our results suggest that reduction of ER stress and myocardial apoptosis through inhibition of eIF2α dephosphorylation may serve as the potential mechanisms for the improved cardiac function and attenuated cardiac remodeling post Sal treatment in this heart failure rat model.

Early enteral and parenteral nutrition on immune functions of neurocritically ill patients.

This study was designed to investigate the influence of early enteral and parenteral nutrition on immune functions of neurocritically ill patients. Patients who were admitted to the neurological intensive care unit (ICU) of The Second Affiliated Hospital of Zhengzhou University between May 2014 and January 2016 were selected. They had been hospitalized for more than one week and received enteral nutrition (EN) via nasogastric tube, with a gross energy of 25 kcal/(Kg • d). Patients were divided into EN group, EN + early PN (EPN) group and EN + supplemental PN (SPN) group according to the time of PN support. Differences in patients’ general information and changes in serum protein and immune indexes were compared between the three groups. On admission, patients’ Glasgow coma scale (GCS), age, immune functions and protein indexes had no obvious differences between the three groups. After nutritional support, serum protein level reduced in the EN group while prealbumin (PALB) and retinol binding protein (RBP) increased in the EN + EPN group and EN + SPN group after one week of admission to hospital, and the differences were statistically significant (p less than 0.05). Total protein (TP), albumin (ALB), PALB and transferrin (TRF) increased significantly in the EN + EPN group and EN + SPN group compared with the EN group (p < 0.05); before and after treatment, an increase was found in ALB in the EN + EPN group in comparison with EN + SPN group, with a notable difference (p < 0.05); C3, C4, immunoglobulin M (IgM) and immunoglobulin A (IgA) increased in the EN + SPN group after nutritional support compared with before treatment, and the difference was statistically significant (p < 0.05). Moreover, immunoglobulin G (IgG) and IgA in the EN + EPN group increased after nutritional support comparing to prior to nutritional support, and the difference was statistically significant (p < 0.05). After nutritional treatment, IgA and IgG increased markedly in the EN + EPN group, and there was a statistical significance between the groups (p < 0.05); the EN + EPN group and EN + SPN group exceeded the EN group in total lymphocyte count (TLC), and the difference had a statistical significance (p less than 0.05). These results demonstrate that neurocritically ill patients achieving the target energy can avoid malnutrition and immunodeficiency; serum protein decrease can cause malnutrition after one week of EN support; and enteral and parenteral nutrition can improve nutritional and immune indicators of neurocritically ill patients in the acute phase. In addition, EPN is more likely to improve malnutrition and immune functions of critical patients than SPN.

[The clinical analysis of extracorporeal membrane oxygenation for adult severe acute respiratory distress syndrome].

OBJECTIVE: To investigate the clinical experience and associated factors of extracorporeal membrane oxygenation(ECMO) for adult patients with severe acute respiratory distress syndrome (ARDS). METHODS: The clinical data of 58 adult patients with severe ARDS, which met the criteria for ECMO and treated in comprehensive intensive care unit of the Second Affiliated Hospital of Zhengzhou University from August 2012 to June 2015, were retrospectively collected and analyzed. The patients were divided into the ECMO group and the control group according to whether ECMO support was used. The ECMO group was further divided into the survival group and the non-survival group according to outcomes 3 months after the initiation of ECMO. The statistics software of SPSS 17.0 was used for data analysis. RESULTS: Of the 58 patients, 28 were treated with VV-ECMO. There were 22 males and 6 females, aged from 34 to 61 years, with a mean age of (49±8) years. Mean time of mechanical ventilation before ECMO therapy was(71±25) h. The main factors(the lowest PaO2/FiO2 and pH, the highest PEEP, PaCO2 and serum lactate level, the grade of APACHEⅡ, Murray and SOFA) between the ECMO group and the control group were not significantly different(all P>0.05). There were no significant differences in the factors before therapy(all P>0.05), except pH, heart rate (HR) and mean arterial pressure (MAP) between the ECMO survival group and the ECMO non-survival group. Compared with the factors at the same time point in the control group, those of the ECMO group were significantly improved after ECMO running 2 hours and 1 day (all P<0.05). Most factors of the ECMO group were also significantly improved after ECMO running 2 days (all P<0.05) except MAP; the MAP of the ECMO non-survival group being lower than the control group (t=2.433, P=0.019). After ECMO running 3 days, most factors of the ECMO survival group were better than those of the control group (all P<0.05) except MAP, but most factors between the ECMO non-survival group and the control group were not significantly different (all P>0.05), while the serum lactate level was higher (t=-3.156, P=0.003) and the MAP lower (t=3.578, P=0.001). Eighteen patients were successfully weaned from ECMO, and 15 patients survived to 3 months without severe disability. The survival rate between the ECMO group and the control group was not significantly different(53.57% and 43.33%, χ(2)=0.608, P=0.436). CONCLUSIONS: Our experience suggests that VV-ECMO may be an effective life support treatment for severe ARDS and should be used as early as possible when the ECMO criteria are met. Indexes such as age, time of mechanical ventilation before ECMO therapy, time between disease onset and ECMO therapy, HR, MAP, serum lactate level and serum procalcitonin level, may be the risk factors for prognosis.

Synaptic pattern formation during cellular recognition.

Cell-cell recognition often requires the formation of a highly organized pattern of receptor proteins (a synapse) in the intercellular junction. Recent experiments [e.g., Monks, C. R. F., Freiberg, B. A., Kupfer, H., Sciaky, N. & Kupfer, A. (1998) Nature (London) 395, 82-86; Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M. & Dustin, M. L. (1999) Science 285, 221-227; and Davis, D. M., Chiu, I., Fassett, M., Cohen, G. B., Mandelboim, O. & Strominger, J. L. (1999) Proc. Natl. Acad. Sci. USA 96, 15062-15067] vividly demonstrate a complex evolution of cell shape and spatial receptor-ligand patterns (several microns in size) in the intercellular junction during immunological synapse formation. The current view is that this dynamic rearrangement of proteins into organized supramolecular activation clusters is driven primarily by active cytoskeletal processes [e.g., Dustin, M. L. & Cooper, J. A. (2000) Nat. Immunol. 1, 23-29; and Wulfing, C. & Davis, M. M. (1998) Science 282, 2266-2269]. Here, aided by a quantitative analysis of the relevant physico-chemical processes, we demonstrate that the essential characteristics of synaptic patterns observed in living cells can result from spontaneous self-assembly processes. Active cellular interventions are superimposed on these self-organizing tendencies and may also serve to regulate the spontaneous processes. We find that the protein binding/dissociation characteristics, protein mobilities, and membrane constraints measured in the cellular environment are delicately balanced such that the length and time scales of spontaneously evolving patterns are in near-quantitative agreement with observations for synapse formation between T cells and supported membranes [Grakoui, A., Bromley, S. K., Sumen, C., Davis, M. M., Shaw, A. S., Allen, P. M. & Dustin, M. L. (1999) Science 285, 221-227]. The model we present provides a common way of analyzing immunological synapse formation in disparate systems (e.g., T cell/antigen-presenting cell junctions with different MHC-peptides, natural killer cells, etc.).

The proteome of Salmonella enterica serovar typhimurium: current progress on its determination and some applications.

Salmonella typhimurium (official designation Salmonella enterica serovar Typhimurium) is an enteric pathogen and a principal cause of gastroenteritis in humans. A comprehensive description of the proteins of Salmonella and their patterns of expression under different environmental conditions would greatly increase our understanding of the virulence of this organism at the molecular level and provide insights into many other aspects of Salmonella biology. While a variety of two-dimensional studies of Salmonella have been previously carried out to address specific questions, little systematic information is available at the protein level on the numbers of Salmonella polypeptides that have homologues in other organisms, their abundance, and the frequency of post-translational modifications. To test the feasibility of determining the proteome of Salmonella, the identities of 53 randomly sequenced cell envelope proteins have been determined by N-terminal sequencing of spots from two-dimensional gels. In addition to confirming the existence of previously hypothetical proteins predicted from genomic sequencing projects, we found that approximately 20% of the proteins had no matches in sequence databases. The results suggest that proteome analysis is an efficient way to identify novel proteins from prokaryotes and that the analysis provides a useful approach to the study of Salmonella virulence.

Proteome of Salmonella typhimurium SL1344: identification of novel abundant cell envelope proteins and assignment to a two-dimensional reference map.

Forty-nine cell envelope proteins of Salmonella typhimurium SL1344 have been identified by microsequencing and assigned to a two-dimensional reference map. Ten of the sequenced proteins appear to be novel. Several others closely match currently hypothetical proteins or proteins found in other bacteria but not previously reported in salmonellae.

Salmonella typhimurium responses to a bactericidal protein from human neutrophils.

Bactericidal/permeability-increasing protein [BPI] is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram-negative bacteria via the lipid A component of lipopolysaccharide. To obtain information about the responses of Salmonella typhimurium to cell-surface damage by BPI, two-dimensional gel electrophoresis and N-terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment. The majority of the affected proteins are involved in central metabolic processes. Upon addition of BPI, the beta-subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11-fold. Three of the latter were identified as lipoamide dehydrogenase, enoyl-acyl carrier protein reductase, and the heat-shock protein HtpG. Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes. A conserved direct-repeat motif is present in the regulatory regions of several BPI-inducible genes, including the bipA gene. Only one of the BPI-responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A. We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S. typhimurium even though they bind with high affinity to the same cell-surface component.

The region around residue 115 of human bactericidal/permeability-increasing protein is not involved in lipopolysaccharide binding or bactericidal activity. Chemical synthesis and expression of a gene coding for the active domain and characterization of recombinant proteins.

Bactericidal/permeability-increasing protein (BPI) is a potent antimicrobial agent produced by polymorphonuclear leucocytes that specifically interacts with and kills Gram-negative bacteria. An 825 bp gene determining the bactericidal N-terminal domain of human BPI was chemically synthesized and expressed as inclusion bodies in Escherichia coli. The recombinant polypeptide, BPI', was solubilized and conditions under which it folded to give the active protein were determined. Folding was critically dependent on the urea and salt concentrations as well as the pH. BPI' bound with high affinity to Salmonella typhimurium cells (apparent Kd = 36 nM), permeabilized their outer membranes to actinomycin D, specifically activated a synovial fluid phospholipase A2 and showed potent bactericidal activity. In contrast with the native protein, however, it could not be efficiently released from the cell surface by the addition of high concentrations of Mg2+ ions. Pre-incubation of the protein with lipopolysaccharide or trypsin prevented cytotoxicity. However, boiling BPI' immediately before its addition to cells did not block its bactericidal activity, suggesting that it may be able to function even when presented to cells in an unfolded form. A BPI' derivative, containing a 13-residue foreign antigenic determinant genetically inserted between Ala115 and Asp116, was also produced. The derivative was functional in the above assays and bound with high affinity to S. typhimurium (apparent Kd = 74 nM). These results imply that the region defined by these residues is not involved in the lipopolysaccharide-binding or bactericidal activities of BPI. The availability of functional, nonglycosylated recombinant derivatives of BPI should greatly aid detailed studies on its structure, interactions with lipopolysaccharide and mechanism of action.

Outer membrane permeability of Escherichia coli K12: isolation, cloning and mapping of suppressors of a defined antibiotic-hypersensitive mutant.

We have previously described defined mutants of the TraT protein, an outer membrane lipoprotein specified by F-like plasmids, which sensitize Escherichia coli and Salmonella typhimurium to antibiotics that are normally excluded from the cell. In this paper, the isolation, characterization and molecular cloning of suppressors of one such mutant (pDOC40) is reported. The suppressors, which were isolated by selection for vancomycin-resistant revertants, also restored resistance to several hydrophobic antibiotics although there were no detectable changes in lipopolysaccharides (LPS), phospholipids or outer membrane proteins. Three suppressor loci, provisionally designated sip, for suppression of increased permeability, were cloned in cosmids and mapped by a novel approach involving random sequencing of cloned DNA to identify flanking genes with known map positions. Our results indicate that the sipB locus is located in the 11 min region (485-510 kb) whereas sipC and sipD both map to 82 min (3850-3885 kb). Additionally, the previously sequenced nlpA gene was also mapped to the 82 min region. The cloned suppressor loci were specific for the permeability phenotype caused by the mutant R6-5 TraT protein and had no effect on the permeability phenotype caused by a related TraT mutant of S. typhimurium.

Characterization of the traT gene and mutants that increase outer membrane permeability from the Salmonella typhimurium virulence plasmid.

The nucleotide sequence of the traT gene present in the virulence-associated plasmid of Salmonella typhimurium was determined. The predicted TraT protein encoded by this gene was found to consist of 243 amino acids and to resemble the known TraT proteins of the plasmids of the F incompatibility group. Thus it contains a signal sequence of 20 amino acids, an amino-terminal lipid attachment site, and two strongly hydrophobic regions close to each other in the mature protein. A mutation leading to increased permeability of the outer membrane to hydrophobic agents, previously localized to the traT gene, was shown to change a glycine residue to arginine within one of these hydrophobic regions. The same principle was found to apply to TraT of R6-5: the introduction, by site-directed mutagenesis, of either positively or negatively charged amino acids or the helix-disrupting proline in the corresponding hydrophobic region led to increased hydrophobic permeability of the outer membrane.

[No significant change noted in sister chromatid exchanges, chromosomal aberrations and satellite associations in women taking oral contraceptive, methylnorethindrone compositae for a long term].

PIP: The lymphocytes in the peripheral blood taken from 10 women who were taking the oral contraceptive (OC) methylnorethindrone compositae and from 10 normal women who had never taken any contraceptives were cultured. Chromosomal aberrations, satellite associations, and sister chromatid exchanges were examined. No significant changes were noted. It is suggested that this kind of OC could not be a clastogen or mutagen-carcinogen. (author's modified)^ieng

[Sister chromatid exchanges in women taking oral contraceptive tablet norethindroni compositae for a long term].

PIP: The lymphocytes in the peripheral blood of 13 women who had been taking the oral contraceptive (OC) norethindrone and of 10 normal women who had not taken this drug were cultured. Sister chromatid exchange (SCE) frequencies were studied. The mean SCE frequency (-X +or- SE) for those women taking OCs was 9.06 +or- 0.33/metaphase, which was not significantly higher than the mean SCE score for 10 age-matched controls, 8.12 +or- 0.27 (P 0.05). These data indicate that this kind of OC is not a mutagen-carcinogen. (author's modified)^ieng