[Expression and prognostic value of programmed cell death ligand 1 in patients with locally advanced and non-EGFR-mutated non-small cell lung cancer receiving concurrent chemoradiotherapy].

Objective: To explore the expression of programmed cell death ligand 1 (PD-L1) in patients with locally advanced and non-EGFR-mutated non-small cell lung cancer (LA-NSCLC) undergoing concurrent chemoradiotherapy (cCRT) and its association with clinical outcome of patients. Methods: The basic clinical information of 19 patients with unresectable, non-EGFR mutated LA-NSCLC receiving radical cCRT in Cancer Hospital Chinese Academy of Medical Sciences from January 2016 to December 2017 was retrospectively analyzed. The rabbit monoclonal antibody SP263 was used for immunohistochemical analysis to detect the expression of PD-L1 in LA-NSCLC tissues and the tumor proportion score (TPS) equal to or greater than 1% was defined as PD-L1 positive. The associations between PD-L1 ≥1% and PD-L1 ≥25% with the clinical characteristics and clinical outcome of LA-NSCLC patients were evaluated respectively. Results: Among 19 LA-NSCLC patients, 13 had PD-L1 positive expression, and 4 had PD-L1 expression greater than or equal to 25%. No significant difference was observed between patients with PD-L1 positive and negative expressions regarding the distribution of age, smoking history, pathological classification, and TNM staging (P>0.05). A total of 15 patients could be evaluated for therapeutic effect, including 7 patients with partial response (PR), 7 patients with stable disease (SD), and 1 patient with progressive disease (PD). In the group with PD-L1 expression<1%, 3 patients were in objective response, and 4 patients were in disease control. In the group with PD-L1 expression ≥1%, 4 patients were in objective response, and 10 patients were in disease control. When the PD-L1 expression was less than 25%, 6 patients gained the objective response, and 11 patients gained the disease control. When the PD-L1 expression was greater than or equal to 25%, 1 patient gained the objective response, and 3 patients gained the disease control. The median overall survival (OS) was 35 (95%CI: 12.7-57.3) months for patients with PD-L1 ≥1% and 40 (95%CI: not reaching the end point) months for patients with PD-L1<1% (P=0.284). Patients with PD-L1 ≥25% had a median survival time of 12 (95%CI:0.0-34.5) months, and patients with PD-L1<25% had a median survival time of 40 (95%CI: 27.4-52.6) months (P=0.241). Conclusions: The prognosis of LA-NSCLC patients with PD-L1 positive and no-EGFR mutation receiving concurrent chemoradiation has a trend of poor prognosis. A larger sample size study is warranted to explore the prognostic value of PD-L1 expression in inoperable LA-NSCLC patients and to further explore the effect of immunotherapy on patients with different PD-L1 expression levels.

[The therapeutic experience of blunt pancreatic trauma].

Objective: To summarize the experience of treatment for blunt pancreatic trauma. Methods: The clinical data of 52 patients with blunt pancreatic trauma admitted to the Department of Pancreatic and Biliary Surgery of the First Affiliated Hospital of Harbin Medical University from January 2013 to June 2018 were analyzed retrospectively.There were 40 male and 12 female patients, aging from 12 to 112 years with a median age of 35.5 years.According to the organ injury scale by American Association for the Surgery of Trauma(AAST) for pancreatic injury severity, 15 cases were in grade Ⅰ(28.8%), 20 cases were in grade Ⅱ(38.5%), 10 cases were in grade Ⅲ(19.2%),5 cases were in grade Ⅳ(9.6%) and 2 cases were in grade Ⅴ(3.8%). Isolated blunt pancreatic trauma occurred in 11(21.2%) patients including 5 cases of grade Ⅰ,5 cases of grade Ⅱ and 1 case of grade Ⅲ, and associated injuries existed in 41 patients(78.8%). Results: Among 52 patients, 36 patients(69.2%) were transferred from other hospitals and 16(30.8%) patients were admitted through the emergency department. Finally, 49 patients(94.2%) were cured and 3 patients (5.8%) died.For the 15 cases of grade Ⅰ,9 patients were managed non-operatively, 5 cases underwent peritoneal lavage and drainage after surgery for the other injured abdominal organs, and 1 patient received percutaneous catheter drainage(PCD) with non-operative treatment. For the 20 cases of grade Ⅱ,4 cases only received non-operative treatment and 2 cases also received PCD. Besides, 2 cases underwent debridement and drainage for peripancreatic necrotic tissue and external drainage for pancreatic pseudocyst retrospectively after about 25 days of getting injured. As for patients who received exploratory laparotomy, 5 patients underwent suture repair associated with external drainage, and 7 patients were managed only with external drainage. For the 10 cases of grade Ⅲ,6 patients were cured through distal pancreatectomy and splenectomy with external drainage, while 2 patients underwent endoscopic retrograde cholangiopancreatography and ductal stenting, and the other 2 patients just received debridement and drainage for peripancreatic necrotic tissue.For the 5 cases of grade Ⅳ,2 patients underwent jejunostomy and abdominal cavity drainage, 1 patient had a pancreaticoduodenectomy with drainage,1 patient received suture repair of the pancreas and pancreaticojejunostomy, and 1 patient was managed with suture repair of the head of pancreas and external drainage.For the 2 patients of grade Ⅴ,1 patient received exploratory laparotomy and gauze compression packing hemostasis, and the other patient underwent pancreaticoduodenal repair, gastrointestinal anastomosis, duodenal exclusion surgery and external drainage. Conclusion: According to the AAST classifications, associated injuries, physiological status and intraoperative situation, it could be better to make a comprehensive judgment, achieve early diagnosis and take appropriate individualized treatment strategy, and to improve the overall therapeutic effect for blunt pancreatic trauma.

BRIEF-REPORT New set of microsatellites for Chinese tallow tree, Triadica sebifera.

Chinese tallow (Triadica sebifera) is an important crop and ornamental tree. After it was introduced into the USA, it gradually became a noxious invasive tree in south-eastern America since the middle of the 1900s. Because only six microsatellites were reported previously in T. sebifera, to better understand the genetic diversity and population dynamics of such species, we reported here 28 new microsatellite markers. For these 28 microsatellites, the number of alleles per locus ranged from 2-16. The expected heterozygosity and the expected heterozygosity corrected for sample size varied from 0.0796 to 0.9081 and from 0.0805 to 0.9176, respectively. These microsatellites will provide additional choice to investigate the genetic diversity and structure in T. sebifera.

A set of novel microsatellite markers developed for an economically important tree, Dracontomelon duperreanum, in China.

Dracontomelon duperreanum, the most representative species of the family Anacardiaceae, is an important multipurpose tree in China and Vietnam. However, no genetic diversity studies have been reported on this species. In this study, we identified 11 microsatellite markers for D. duperreanum by using the restriction-site-associated DNA sequencing (RAD-seq) method and examined their polymorphisms in 22 samples obtained from the South China Botanical Garden, South China. We could detect only two or three alleles for each microsatellite marker. The mean observed and expected heterozygosities were 0.41 and 0.39, respectively, which were lower than those reported for the species with similar life history forms. These relatively low genetic diversities in this common plant species are unexpected and might have resulted from its extensive cultivation. To our knowledge, this is the first report of microsatellite markers in the genus Dracontomelon. These microsatellite markers will be valuable for studying the genetic diversities and structures in D. duperreanum and other Dracontomelon species.

[Prognostic factors of postoperative delayed gastric emptying after pancreaticoduodenectomy: a predictive model].

Objective: To study the prognostic factors of delayed gastric emptying(DGE) after pancreaticoduodenectomy(PD) and construct a prognostic predictive model for clinical application. Methods: Clinic data of 401 consecutive patients who underwent PD between January 2012 and July 2016 in the First Affiliated Hospital of Harbin Medical University were retrospectively collected and analyzed. The patients were randomly selected to modeling group(n=299) and validation group(n=102) at a ratio of 3∶1. The data of modeling group were subjected to univariate and multivariate analysis for prognostic factors and to construct a prognostic predictive model of DGE after PD. The data of validation group were applied to test the prognostic predictive model. Results: DGE after PD occurred in 35 of 299 patients(11.7%) in the modeling group. The multivariate analysis of the modeling group showed that upper abdominal operation history(χ(2)=6.533, P=0.011), diabetes mellitus(χ(2)=17.872, P=0.000), preoperative hemoglobin <90 g/L(χ(2)=14.608, P=0.000) and pylorus-preserving pancreaticoduodenectomy(PPPD)(χ(2)=8.811, P=0.003) were associated with DGE after PD independently. A prognostic predictive model of DGE after PD was constructed based on these factors and successfully tested. The area under the receiver operating characteristic(ROC) curve was 0.761(95%CI: 0.666-0.856) of the modeling group and 0.750(95% CI: 0.577-0.923) of the validation group. Conclusions: Upper abdominal operation history, diabetes mellitus, preoperative hemoglobin<90 g/L and PPPD are associated with DGE after PD independently. The preoperative assessment of a patient's prognostic for DGE after PD is feasible. The model is a valid tool to take precautions against DGE after PD.

[Expression and intratumoral heterogeneity of LN-5γ2 in esophageal squamous cell carcinoma].

OBJECTIVE: The present study aimed to evaluate the expression and intratumoral heterogeneity of LN-5γ2 in esophageal squamous cell carcinoma (ESCC). METHODS: The expression of LN-5γ2 protein was examined in 135 ESCC cases by immunohistochemistry, and to analyze its relationship with the clinical relevance of patients. The protein expressions in different regions in the same tumor as well as different nests in the same region were compared. RESULTS: Moderate and high expression of LN-5γ2 protein was detected in 40.0% (54/135) of tumor tissues. Positive immunohistochemical staining was observed in 31.1% (23/74) of early stage (stages Ⅰ/Ⅱ) cases and 50.8% (31/61) of late stage (stage Ⅲ) cases, with a significant difference between these two groups (P=0.023). There was no statistical association of LN-5γ2 expression with age, sex of patients, PT sage, lymph node metastasis and degree of tumor differentiation (P>0.05). However, differential expression of LN-5γ2 protein was found at different sampling sites in the same tumor and the same sampling site in different carcinomas. CONCLUSION: High expression of LN-5γ2 is positively correlated with tumor clinical stages and there existed intratumoral heterogeneity of LN-5γ2 expression in ESCC tissues.

Analysis of genetic relationships and identification of lily cultivars based on inter-simple sequence repeat markers.

Inter-simple sequence repeat (ISSR) markers were used to discriminate 62 lily cultivars of 5 hybrid series. Eight ISSR primers generated 104 bands in total, which all showed 100% polymorphism, and an average of 13 bands were amplified by each primer. Two software packages, POPGENE 1.32 and NTSYSpc 2.1, were used to analyze the data matrix. Our results showed that the observed number of alleles (NA), effective number of alleles (NE), Nei's genetic diversity (H), and Shannon's information index (I) were 1.9630, 1.4179, 0.2606, and 0.4080, respectively. The highest genetic similarity (0.9601) was observed between the Oriental x Trumpet and Oriental lilies, which indicated that the two hybrids had a close genetic relationship. An unweighted pair-group method with arithmetic means dendrogram showed that the 62 lily cultivars clustered into two discrete groups. The first group included the Oriental and OT cultivars, while the Asiatic, LA, and Longiflorum lilies were placed in the second cluster. The distribution of individuals in the principal component analysis was consistent with the clustering of the dendrogram. Fingerprints of all lily cultivars built from 8 primers could be separated completely. This study confirmed the effect and efficiency of ISSR identification in lily cultivars.

Cloning and sequence analysis of the Blumea balsamifera DC farnesyl diphosphate synthase gene.

Blumea balsamifera DC is a member of the Compositae family and is frequently used as traditional Chinese medicine. Blumea balsamifera is rich in monoterpenes, which possess a variety of pharmacological activities, such as antioxidant, anti-bacteria, and anti-viral activities. Farnesyl diphosphate synthase (FPS) is a key enzyme in the biosynthetic pathway of terpenes, playing an important regulatory role in plant growth, such as resistance and secondary metabolism. Based on the conserved oligo amino acid residues of published FPS genes from other higher plant species, a cDNA sequence, designated BbFPS, was isolated from B. balsamifera DC using polymerase chain reaction. The clones were an average of 1.6 kb and contained an open reading frame that predicted a polypeptide of 342 amino acids with 89.07% identity to FPS from other plants. The deduced amino acid sequence was dominated by hydrophobic regions and contained 2 highly conserved DDxxD motifs that are essential for proper functioning of FPS. Phylogenetic analysis indicated that FPS grouped with other composite families. Prediction of secondary structure and subcellular localization suggested that alpha helices made up 70% of the amino acids of the sequence.

[Impact of Toxoplasma gondii human infections on blood lipid levels].

OBJECTIVE: To evaluate the impact of Toxoplasma gondii human infections on blood lipid levels. METHODS: A total of 1 000 healthy volunteers that were randomly sampled from a tertiary hospital during the period from December 2017 through December 2019 were enrolled, and assigned into the infection group and the control group according to the detection of serum anti-T. gondii antibody using enzyme-linked immunosorbent assay (ELISA). The serum levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglyceride (TG) were detected and compared between the two groups. RESULTS: The seroprevalence of anti-T. gondii antibody was 8.40% in the study subjects, and there were no significant differences between the infection and control groups in terms of gender (χ2 = 1.29, P > 0.05) or age (χ2 = 1.41, P > 0.05). The mean serum LDL (t = 3.89, P < 0.05) and TC levels (t = 3.81, P < 0.05) were significantly higher in the infection group than in the control group, while no significant differences were seen between the two groups in terms of mean serum TG (t = 0.97, P > 0.05) or HDL levels (t = 0.75, P > 0.05). In addition, the proportions of abnormal LDL (χ2 = 9.69, P < 0.01) and TC levels (χ2 = 10.39, P < 0.01) were significantly greater in the infection group than in the control group, while no significant differences were found in the proportion of abnormal TG (χ2 = 0.02, P > 0.05) or HDL level (χ2 = 0.11, P > 0.05) between the two groups. CONCLUSIONS: T. gondii human infections affect blood lipid levels, and the individuals sero-positive for anti-T. gondii antibody have higher mean serum LDL and TC, as well as higher proportions of abnormal TC and LDL than sero-negative individuals.

LncDBH-AS1 knockdown enhances proliferation of non-small cell lung cancer cells by activating the Wnt signaling pathway via the miR-155/AXIN1 axis.

OBJECTIVE: Dys-regulated long noncoding RNAs (lncRNAs) are involved in the cell growth of several malignancies and their aggressive phenotypes. LncRNA DBH-AS1 plays an important role in the advancement of various malignant tumors, but its contribution to non-small cell lung cancer (NSCLC) is still unexplored. This study intends to elucidate the role of the regulatory network of lncRNA DBH-AS1 in NSCLC progression. PATIENTS AND METHODS: The LncDBH-AS1 expression in 32 paired NSCLC patients' tissue samples and NSCLC cell lines were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The role of LncDBH-AS1 in NSCLC was investigated through cell counting kit-8 (CCK-8) assay and colony formation assay in vitro. Besides, the interaction between LncDBH-AS1 and miR-155 was also analyzed. RESULTS: The DBH-AS1 expression was significantly down-regulated in NSCLC cell lines and tissue samples. Decreased DBH-AS1 levels promoted the in vitro proliferation of the NSCLC cells. The mechanism was that DBH-AS1 regulated AXIN1 expression by sponging miR-155 in NSCLC cell lines. Importantly, LncDBH-AS1 might inhibit WNT/ß-CATENIN activation in NSCLC cells. CONCLUSIONS: The progression of NSCLC is facilitated by DBH-AS1 via miR-155 interaction and up-regulation of AXIN1 expression.

First Report of Pyricularia grisea Causing Gray Leaf Spot on Lily in Korea.

Lily is an economically important ornamental crop in Korea. In August 2008, severe leaf spot symptoms were observed on an oriental Lily 'Action' in a plant nursery in Daegu, Korea. Disease incidence was 20 to 30%. Initial symptoms were olive green-to-brown lesions on the leaf that developed into tan, elliptical, necrotic lesions. On severely infected leaves, lesions coalesced and killed the entire leaf blade. Infected leaves were surface disinfested with 70% ethanol for 30 s and 2% chlorox for 15 min before plating 1 cm2 sections onto potato dextrose agar. Hyphae appeared 5 days after inoculation and pure culture. Conidia were hyaline, transversely septate with one to three septa; most had two. Conidia were obpyriform and measured 29 to 46 µm long and 7 to 17 µm wide. Mycelia morphology and conidia production were consistent with that described previously for Pyricularia grisea (1). Koch's postulates were fulfilled by spraying five, healthy, vegetative-stage plants with 2 × 105 conidia per ml of sterile distilled water plus 0.05% Tween 20. As a control, five similar plants were sprayed with sterile water plus 0.05% Tween 20 only. Plants were placed inside plastic bags to maintain high relative humidity and incubated in a growth chamber at 25°C under fluorescent light for 14 h and at 20°C in darkness for 10 h. After 3 days, the plastic bags were removed and plants were maintained under the same conditions. Initial symptoms were observed 7 days after inoculation. Ten days after inoculation, disease symptoms on inoculated plants were similar to those previously described in the nursery. Control plants did not show any symptoms. Fungi isolated from these lesions had the same morphological characteristics as the ones isolated previously from plants in the nursery. To our knowledge, this is the first report of gray leaf spot on lily caused by P. grisea in Korea. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971.

Sec- and Tat-dependent translocation of beta-lactamases across the Escherichia coli inner membrane.

beta-Lactamases represent the major resistance mechanism of gram-negative bacteria against beta-lactam antibiotics. The amino acid sequences of these proteins vary widely, but all are located in the periplasm of bacteria. In this study, we investigated the translocation mechanism of representative beta-lactamases in an Escherichia coli model. N-terminal signal sequence analyses, antibiotic activity assay, and direct measurement of translocation of a green fluorescent protein (GFP) reporter fused to beta-lactamases revealed that most were exported via the Sec pathway. However, the Stenotrophomonas maltophilia L2 beta-lactamase was exported via the E. coli Tat translocase, while the S. maltophilia L1 beta-lactamase was Sec dependent. These results show the possible Tat-dependent translocation of beta-lactamases in the E. coli model system. In addition, the mutation of the cytoskeleton-encoding gene mreB, which may be involved in the spatial organization of penicillin-binding proteins, decreased the MIC of beta-lactams for beta-lactamase-producing E. coli. These findings provide new knowledge about beta-lactamase translocation, a putative new target for addressing beta-lactamase-mediated resistance.

Regulation of bacterial physiological processes by three types of protein phosphorylating systems.

A single type of protein-phosphorylating system, the ATP-dependent protein kinases, is employed in the regulation of a variety of cellular physiological processes in eukaryotes. By contrast, recent work with bacteria has revealed that three types of protein-phosphorylating systems are involved in regulation: (1) the classical protein kinases, (2) the newly discovered sensor-kinase/response-regulator systems, and (3) the multifaceted phosphoenolpyruvate-dependent phosphotransferase system. Physiological and mechanistic aspects of these three evolutionarily distinct systems are discussed.

Using the seasonal FGM(1,1) model to predict the air quality indicators in Xingtai and Handan.

The air pollution problem in Xingtai and Handan is the focus of public attention. The seasonal gray model with fractional order accumulation is proposed to predict the quarterly concentrations of PM2.5, PM10, NO2, and CO in Xingtai and Handan. The new model has higher forecasting performance and can describe the characteristics of seasonal fluctuation very well. The forecasting results indicated that except for the PM10 in Xingtai that will increase slowly, the other indicators in the two places will decrease. The changes of the air quality indicator concentration in different quarters are obvious, and in the same quarter tend to be stable. Except for CO and NO2 in some seasons, other indicators are in the state of exceeding the standard. The effect of air pollution control is not good. The governance needs to be further strengthened.

Knockdown of MiR-140-5 promotes osteogenesis of adipose-derived mesenchymal stem cells by targeting TLR4 and BMP2 and promoting fracture healing in the atrophic nonunion rat model.

OBJECTIVE: This study aims to assess the effect and mechanism of genetically modified adipose-derived mesenchymal stem cells (ASCs) with recombinant lentiviruses mediated knockdown of miR-140-5p in ASCs' osteogenesis in vitro and atrophic nonunion rat model. MATERIALS AND METHODS: This study included 36 male adult Sprague-Dawley (SD) rats weighing 400 g to 450 g from the experimental animal facility of our university. Approval was obtained from the University Animal Care Committee before the study. Rats' ASCs were prepared and genetically modified with lentivirus (Lv)-empty (NC) or Lv-miR-140-5p-TuD (inhibitors). After that, the expressions of RUNX2 and osteocalcin (OCN) were detected in the ASCs. To confirm the mechanisms of miR-140-5p in ASCs, we predicted the target genes by bioinformatics analysis and then the target genes were verified by luciferase reporting assay. The artificial atrophic nonunion was created in the rat's femoral bone. Animals were randomly divided into three groups according to the material implanted into bone defects space: AT scaffolds (AT group, n=12), AT scaffold with Lv-NC modified (AT+ASCs+Lv-NC group, n=12), AT scaffold with the Lv-miR-140-5p-TuD modified ASCs (AT+ASCs+Lv-miR-140-5p-TuD group, n=12). After four weeks, the rats were euthanized for the following radiographic examination, histologic study and biomechanical testing. RESULTS: MiR-140-5p was down-regulated during osteogenic differentiation of ASCs, and inhibition of MiR-140-5p promoted osteogenesis of ASCs in vitro. Inhibition of MiR-140-5p promoted osteogenesis of ASCs and enhanced fracture in the atrophic nonunion rat model: AT+ASCs+Lv-NC group, AT+ASCs+Lv-miR-140-5p-TuD group resulted in a better bone formation and higher BMD and BMC than AT group, while excellent bone formation and the highest BMD and BMC were observed in AT+ASCs+Lv-miR-140-5p-TuD group. Both AT+ASCs+Lv-NC group and AT+ASCs+Lv-miR-140-5p-TuD group presented more mature characteristics in the micro-architecture than AT group, whereas AT+ASCs+Lv-miR-140-5p-TuD group presented the highest BV/TV, Tb.Th and Tb.N as well as the lowest Tb.Sp. The peak load of the operated femur increased by 94.43% AT+ASCs+Lv-miR-140-5p-TuD group, 50.68% in AT+ASCs+Lv-NC group compared to the control AT group, respectively. The result of luciferase reporting assay showed that miR-140-5p could directly target TLR4 and BMP2. CONCLUSIONS: This study demonstrates that lentiviruses-mediated knockdown of miR-140-5p can significantly promote osteogenesis of ACSs by directly regulating its' target genes, TLR4 and BMP2, and that combined adipose scaffold with genetically modified ASCs can significantly enhance fracture-healing and bone formation in the atrophic nonunion rat model.

GACAT3 promoted proliferation of osteoarthritis synoviocytes by IL-6/STAT3 signaling pathway.

OBJECTIVE: Osteoarthritis (OA) is one of the most common chronic joint diseases, caused by lesions in articular cartilage and synovial membranes. Synovitis is a major characteristic of OA, due to the proliferation of synoviocytes. Long noncoding RNAs (lncRNAs) have implicated to play an important role in many different diseases. The aim of this study was to verify the role of lncRNA gastric cancer-associated transcript 3 (GACAT3) in osteoarthritis. MATERIALS AND METHODS: We utilized the qRT-PCR to detect the expression of lncRNA GACAT3 in osteoarthritis synoviocytes (OAS) and normal synoviocytes (NS). The cell proliferation in NS and OAS after transfection with lncRNA-NC or lncRNA-GACAT3 was detected. The cell cycle and apoptosis rate in NS and OAS were measured by the Flow cytometry analysis. Western blot was used to analyze the possible related mechanism that GACAT3 regulated the cells proliferation in osteoarthritis. RESULTS: We found that GACAT3 expression was significantly increased in OAS compared with NS. GACAT3 expression was decreased in OAS after transfection with siRNA and the cell proliferation in OAS after transfection with siRNA was significantly inhibited. The cell cycle was arrested in G0/G1 phase and the apoptosis rate was increased in OAS after transfection with siRNA. Moreover, GACAT3 could impact the proliferation of OAS by interleukin-6/signal transducer and activator of transcription-3 (IL-6/STAT3) signaling pathway. CONCLUSIONS: In this study, we found that lncRNA GACAT3 was closely related to the osteoarthritis. GACAT3 may be involved in the development and progression of osteoarthritis and become a potential target for treating.

Microbial hydrogenases: primary structure, classification, signatures and phylogeny.

Thirty sequenced microbial hydrogenases are classified into six classes according to sequence homologies, metal content and physiological function. The first class contains nine H2-uptake membrane-bound NiFe-hydrogenases from eight aerobic, facultative anaerobic and anaerobic bacteria. The second comprises four periplasmic and two membrane-bound H2-uptake NiFe(Se)-hydrogenases from sulphate-reducing bacteria. The third consists of four periplasmic Fe-hydrogenases from strict anaerobic bacteria. The fourth contains eight methyl-viologen- (MV), factor F420- (F420) or NAD-reducing soluble hydrogenases from methanobacteria and Alcaligenes eutrophusH16. The fifth is the H2-producing labile hydrogenase isoenzyme 3 of Escherichia coli. The sixth class contains two soluble tritium-exchange hydrogenases of cyanobacteria. The results of sequence comparison reveal that the 30 hydrogenases have evolved from at least three different ancestors. While those of class I, II, IV and V hydrogenases are homologous, i.e. sharing the same evolutionary origin, both class III and VI hydrogenases are neither related to each other nor to the other classes. Sequence comparison scores, hierarchical cluster structures and phylogenetic trees show that class II falls into two distinct clusters composed of NiFe- and NiFeSe-hydrogenases, respectively. These results also reveal that class IV comprises three distinct clusters: MV-reducing, F420-reducing and NAD-reducing hydrogenases. Specific signatures of the six classes of hydrogenases as well as some subclusters have been detected. Analyses of motif compositions indicate that all hydrogenases, except those of class VI, must contain some common motifs probably participating in the formation of hydrogen activation domains and electron transfer domains. The regions of hydrogen activation domains are highly conserved and can be divided into two categories. One corresponds to the 'nickel active center' of NiFe(Se)-hydrogenases. It consists of two possible specific nickel-binding motifs, RxCGxCxxxH and DPCxxCxxH, located at the N- and C-termini of so-called large subunits in the dimeric hydrogenases, respectively. The other is the H-cluster of the Fe-hydrogenases. It might comprise three motifs on the C-terminal half of the large subunits. However, the motifs corresponding to the putative electron transfer domains, as well as their polypeptides chains, are poorly or even not at all conserved. They are present essentially on the small subunits in NiFe-hydrogenases. Some of these motifs resemble the typical ferredoxin-like Fe-S cluster binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Evolution of permease diversity and energy-coupling mechanisms with special reference to the bacterial phosphotransferase system.

Different classes of apparently unrelated permeases couple different forms of energy to solute transport. While the energy coupling mechanisms utilized by the different permease classes are clearly distinct, it is proposed, based on structural comparisons, that many of these permeases possess transmembrane, hydrophobic domains which are evolutionarily related. Carriers may have arisen from transmembrane pore-forming proteins, and the protein constituents or domains which are specifically responsible for energy coupling may have had distinct origins. Thus, complex permeases may possess mosaic structures. This suggestion is substantiated by recent findings regarding the evolutionary origins of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). Mechanistic implications of this proposal are presented.

Structure and evolution of a multidomain multiphosphoryl transfer protein. Nucleotide sequence of the fruB(HI) gene in Rhodobacter capsulatus and comparisons with homologous genes from other organisms.

The gene order of the fructose (fru) operon and nucleotide sequence of the first gene (fruB(HI) of Rhodobacter capsulatus are reported, analyzed and compared with homologous genes from other bacteria, and the gene products are identified. Included within the region reported is a gene encoding a multiphosphoryl transfer protein (MTP) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). MTP consists of three moieties: a fructose-specific enzyme III (IIIfru)-like N-terminal moiety (residues 1 to 143) followed by an FPr(HPr)-like moiety (residues 157 to 245) and an enzyme I-like moiety (residues 273 to 827). The enzyme III-like moiety closely resembles the N-terminal 143 residues of the IIIfru-FPR fusion protein from Salmonella typhimurium (40.6% identity throughout its length) and the C-terminal 145 residues of the mannitol-specific enzyme II (IImtl) (37.8% identity throughout its length with the IIImtl moiety of IImtl). The FPr-like domain of MTP resembles the S. typhimurium FPr (42.4% identity) and the Escherichia coli or S. typhimurium HPr (38.8% identity). The enzyme I-like moiety resembles the E. coli enzyme I (38.9% identity). Predicted phosphorylation sites within the three functional units of MTP (His62 in the IIIfru-like moiety; His171 in the FPr-like moiety and His457 in the enzyme I-like moiety) were identified on the basis of sequence comparisons with the homologous proteins from enteric bacteria. The three functional domains of MTP are joined by two flexible "linkage" regions, rich in alanine, glycine and proline, which show 47% sequence identity with each other. They also exhibit a high degree of sequence identity with the linkage region of the mannose-specific enzyme III (IIIman) of the E. coli PTS as well as several other proteins of bacterial, eukaryotic and viral origin. At the RNA level, these linker regions formed hairpin structures with high (90%) G + C content. Analyses of the IIIfru-FPr fusion protein of S. typhimurium revealed that between the IIIfru and FPr moieties of this protein is a stretch of 142 amino acids that do not show homology to known PTS proteins. This region and the adjacent FPr-like region contain a sequence of 110 residues exhibiting 59% similarity to the receiver consensus motif defined by Kofoid and Parkinson. Because the Salmonella IIIfru-FPr fusion protein has been implicated in transcriptional regulation, this region of the Salmonella protein may prove to have regulatory significance.(ABSTRACT TRUNCATED AT 400 WORDS)