The prevalence of plasmid‐mediated quinolone resistance determinants among clinical isolates of ESBL or AmpC‐producing Escherichia coli from Chinese pediatric patients

Three kinds of PMQR determinants (qnr genes, aac(6’)‐Ib‐cr, and qepA) have been discovered and shown to be widely distributed among clinical isolates. To characterize the prevalence of PMQR determinants in ESBL or AmpC‐producing E. coli clinical isolates in Chinese children, a total of 292 ESBL or AmpC‐producing E. coli clinical isolates collected from five children's hospitals in China from 2005 to 2006 were screened for PMQR determinants by PCR. Twenty (6.8%) of the 292 isolates were positive for PMQR determinants. A total of 12 (4.1%) isolates were positive for qnr genes, comprising three positive for qnrA (1.0%), three for qnrB (1.0%), and six for qnrS (2.1%). Twenty‐four (8.2%) isolates were positive for aac(6’)‐Ib, of which 10 (3.4% of 292) had the –cr variant. There was no qepA gene detected in the isolates. Conjugation revealed that qnr, aac(6’)‐Ib‐cr, and ESBL‐encoding genes were transferred together.     

Involvement of osteopontin upregulation on mesangial cells growth and collagen synthesis induced by intermittent high glucose

Glucose fluctuations are strong predictor of diabetic vascular complications. We explored the effects of constant and intermittent high glucose on the proliferation and collagen synthesis of cultured rat mesangial cells. Furthermore, the possible involvement of osteopontin (OPN) was assessed. In rat mesangial cells cultured in 5, 25, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, β3 integrin receptor and β5 integrin receptor, the cell proliferation, collagen synthesis, and the expression of OPN and type IV collagen were assessed. In cultured mesangial cells, treatment with constant or intermittent high glucose significantly increased [³H]thymidine incorporation in a time‐dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum incorporation at 48 h. Treatment with constant high glucose for 48 h resulted in significant increases in [³H]thymidine incorporation, cell number, [³H]proline incorporation, mRNA, and protein levels of type IV collagen and OPN compared with mesangial cells treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose medium. In addition, neutralizing antibodies to either OPN or its receptor β3 integrin but not neutralizing antibodies to β5 integrin can effectively prevented proliferation and collagen synthesis of mesangial cells induced by constant or intermittent high glucose. Intermittent high glucose exacerbates mesangial cells growth and collagen synthesis by upregulation of OPN expression, indicating that glycemic variability have important pathological effects on the development of diabetic nephropathy, which is mediated by the stimulation of OPN expression and synthesis. J. Cell. Biochem. 109: 1210–1221, 2010. © 2010 Wiley‐Liss, Inc.     

Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China

A total of seventy-five symbiotic bacterial strains isolated from root nodules of wild Sophora alopecuroides grown in different regions of China's Loess Plateau were characterized. Based on the combined RFLP patterns, thirty-five genotypes were defined among the rhizobia and they were classified into nine genomic species, including Mesorhizobium alhagi and M. gobiense as the main groups, as well as Agrobacterium tumefaciens, M. amorphae, Phyllobacterium trifolii, Rhizobium giardinii, R. indigoferae, Sinorhizobium fredii and S. meliloti as the minor groups according to the 16S rRNA and recA gene analyses. Five and three lineages of nodA and nifH were found, respectively, in these strains, implying that the symbiotic genes of the S. alopecuroides rhizobia had different origins or had divergently evolved. Results of correspondence analysis showed that there was a correlation between rhizobial genotypes and the geographic origins. Possible lateral transfer of the recA and 16S rRNA genes between the P. trifolii and A. tumefaciens strains, and that of symbiotic genes (nodA, nifH) between different genera, was shown by discrepancies of the phylogenetic relationships of the four gene loci. These results revealed diverse rhizobia associated with wild S. alopecuroides grown in different regions of China's Loess Plateau, and demonstrated for the first time the existence of symbiotic A. tumefaciens strains in root nodules of S. alopecuroides.     

Partial Functional Rescue of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Infectivity by Replacement of F Protein with GP64 from Autographa californica Multicapsid Nucleopolyhedrovirus

Two distinct envelope fusion proteins (EFPs) (GP64 and F) have been identified in members of the Baculoviridae family of viruses. F proteins are found in group II nucleopolyhedroviruses (NPVs) of alphabaculoviruses and in beta- and deltabaculoviruses, while GP64 occurs only in group I NPVs of alphabaculoviruses. It was proposed that an ancestral baculovirus acquired the gp64 gene that conferred a selective advantage and allowed it to evolve into group I NPVs. The F protein is a functional analogue of GP64, as evidenced from the rescue of gp64-null Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV) by F proteins from group II NPVs or from betabaculoviruses. However, GP64 failed to rescue an F-null Spodoptera exigua MNPV (SeMNPV) (group II NPV). Here, we report the successful generation of an infectious gp64-rescued group II NPV of Helicoverpa armigera (vHaBacΔF-gp64). Viral growth curve assays and quantitative real-time PCR (Q-PCR), however, showed substantially decreased infectivity of vHaBacΔF-gp64 compared to the HaF rescue control virus vHaBacΔF-HaF. Electron microscopy further showed that most vHaBacΔF-gp64 budded viruses (BV) in the cell culture supernatant lacked envelope components and contained morphologically aberrant nucleocapsids, suggesting the improper BV envelopment or budding of vHaBacΔF-gp64. Bioassays using pseudotyped viruses with a reintroduced polyhedrin gene showed that GP64-pseudotyped Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) significantly delayed the mortality of infected H. armigera larvae.The envelope fusion protein (EFP) of budded viruses (BV) (30) of baculoviruses is critical for virus entry (attachment and fusion) and egress (assembly and budding) (7, 13, 21). Two types of BV EFPs have been identified in the Baculoviridae family of viruses. The F proteins are similar in structure, but they are very diverse in their amino acid sequences (20 to 40% identity). They are widespread within the baculovirus family (group II NPVs of the alphabaculoviruses and in beta- and deltabaculoviruses) (23) and are thought to be carried by ancestral members (26). In contrast, the baculovirus GP64 homologs are all closely related EFPs (>74% sequence identity) and found only in group I NPVs of the alphabaculoviruses (23). It has been suggested that a gp64 gene was acquired relatively recently by an ancestral virus of the group II NPV, thereby giving these viruses a selective advantage and obviating the need of the envelope fusion function of the F protein (23). A nonfusogenic F homolog (F-like protein), however, is maintained in the genome of group I NPVs, functioning as a virulence factor (9, 17, 24, 32).GP64 and F proteins play similar roles during the baculovirus infection processes, such as virus-cell receptor attachment, membrane fusion, and efficient budding. However, there are striking differences between the receptor usage of GP64 and F proteins as well. These two types of proteins are very different in structure, mode of action, and receptor exploitation. The crystal structure reveals that GP64 belongs to class III viral fusion proteins, with its fusion loop located in the internal region of the protein, and proteolytic cleavage is not required for activation of fusion activity (10). F proteins by contrast share common features of class I viral fusion proteins (12). The proteolytic cleavage of the F precursor (F₀) by a furin-like protease generates an N-terminal F₂ fragment and a C-teminal F₁ fragment. This cleavage is essential for exposing the N-terminal fusion peptide of F₁ and for activating F fusogenicity (8, 36). Although the nature of the baculovirus host cell receptors is still enigmatic, it has been reported that Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV)) and Orgyia pseudotsugata MNPV (OpMNPV), both using GP64 as their EFPs, exploit the same insect cell receptor, while Lymantria dispar MNPV (LdMNPV) with an F protein as the EFP utilizes a cell receptor different from that used by AcMNPV (7, 37). Additionally, in the case of SeMNPV, using competition assays, it was confirmed that the baculovirus F protein and GP64 recognized distinct receptors to gain entry into cultured insect cells (34).Pseudotyping viral nucleocapsid with heterologous EFPs to form pseudotype virions is a valuable approach to studying the structure, function, and specificity of heterologous EFPs. It has been a successful strategy to expand or alter viral host range, i.e., in gene delivery (3). For example, vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus and AcMNPV gp64-pseudotyped HIV-1 exhibit high virus titers and wider tropism (5, 14, 38); the gp64-pseudotyped human respiratory syncytial virus (HRSV) lacking its own glycoproteins is of high and stable infectivity (22); furthermore, pseudotyped lentiviruses with modified fusion proteins of GP64 with targeting peptides (i.e., hepatitis B virus PreS1 peptide, involved in viral attachment) or with the decay accelerating factor (DAF) facilitate the targeting to specific cell types or confer stability against serum inactivation, respectively (6, 19). For the Baculoviridae, a series of pseudotyping studies have investigated the functional analogy between GP64 and F proteins. F proteins of group II NPVs (SeMNPV, LdMNPV, and Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus [HearNPV]) can substitute for GP64 in gp64-null AcMNPV viruses (15, 16). Recent studies indicated that many granulovirus (GV) F proteins, but not F protein from Plutella xylostella GV (PxGV), can rescue a gp64-null AcMNPV (16, 39). These results demonstrated that baculovirus F proteins are functional analogues to GP64. Since it was postulated that GP64 was captured by a baculovirus during evolution (24), one would expect the functional incorporation of GP64 into the BV of an F-null group II NPV. However, the reverse substitution of a group II NPV (SeMNPV) F protein by GP64 failed to produce infectious progeny viruses (35).In this paper, we show that AcMNPV gp64 could be inserted into an F-null HearNPV genome and produce infectious progeny virus upon transfection of insect cells. The infectivity of the pseudotyped virus, however, was greatly impaired, and large amounts of morphologically defective BV were produced. Bioassay experiments indicated that the infectivity of GP64-pseudotyped F-null HearNPV for insect larvae was not reduced, but that the time to death was significantly delayed. These results demonstrate that GP64 alone can only partially complement HearNPV F protein function.     

Identification and Characterization of Bmi-1-responding Element within the Human p16 Promoter

Bmi-1, the first functionally identified polycomb gene family member, plays critical roles in cell cycle regulation, cell immortalization, and cell senescence. Bmi-1 is involved in the development and progression of carcinomas and is a potent target for cancer therapy. One important pathway regulated by Bmi-1 is that involving two cyclin-dependent kinase inhibitors, p16^Ink4a and p19^Arf, as Bmi-1 represses the INK4a locus on which they are encoded. A close correlation between the up-regulation of Bmi-1 and down-regulation of p16 has been demonstrated in various tumors; however, how Bmi-1 regulates p16 expression is not clear. In this study, we revealed that Bmi-1 regulates the expression of p16 by binding directly to the Bmi-1-responding element (BRE) within the p16 promoter. The BRE resided at bp −821 to −732 upstream of the p16 ATG codon. BRE alone was sufficient to allow Bmi-1-mediated regulation of the CMV promoter. Bmi-1 typically functions by forming a complex with Ring2; however, regulation of p16 was independent of Ring2. Chromatin immunoprecipitation sequencing of Bmi-1-precipitated chromatin DNA revealed that 1536 genes were targeted by Bmi-1, including genes involved in tissue-specific differentiation, cell cycle, and apoptosis. By analyzing the binding sequences of these genes, we found two highly conserved Bmi-1-binding motifs, which were required for Bmi-1-mediated p16 promoter regulation. Taken together, our results revealed the molecular mechanism of Bmi-1-mediated regulation of the p16 gene, thus providing further insights into the functions of Bmi-1 as well as a sensitive high-throughput platform with which to screen Bmi-1-targeted small molecules for cancer therapy.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

A Comprehensive Proteomic Analysis of the Type III Secretome of Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium belong to the family of attaching and effacing (A/E) bacterial pathogens. They intimately attach to host intestinal epithelial cells, trigger the effacement of intestinal microvilli, and cause diarrheal disease. Central to their pathogenesis is a type III secretion system (T3SS) encoded by a pathogenicity island called the locus of enterocyte effacement (LEE). The T3SS is used to inject both LEE- and non-LEE-encoded effector proteins into the host cell, where these effectors modulate host signaling pathways and immune responses. Identifying the effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Here we analyzed the type III secretome of C. rodentium using the highly sensitive and quantitative SILAC (stable isotope labeling with amino acids in cell culture)-based mass spectrometry. This approach not only confirmed nearly all known secreted proteins and effectors previously identified by conventional biochemical and proteomic techniques, but also identified several new secreted proteins. The T3SS-dependent secretion of these new proteins was validated, and five of them were translocated into cultured cells, representing new or additional effectors. Deletion mutants for genes encoding these effectors were generated in C. rodentium and tested in a murine infection model. This study comprehensively characterizes the type III secretome of C. rodentium, expands the repertoire of type III secreted proteins and effectors for the A/E pathogens, and demonstrates the simplicity and sensitivity of using SILAC-based quantitative proteomics as a tool for identifying substrates for protein secretion systems.