Germline mutations in TMEM127 confer susceptibility to pheochromocytoma

Pheochromocytomas, which are catecholamine-secreting tumors of neural crest origin, are frequently hereditary(1). However, the molecular basis of the majority of these tumors is unknown(2). We identified the transmembrane-encoding gene TMEM127 on chromosome 2q11 as a new pheochromocytoma susceptibility gene. In a cohort of 103 samples, we detected truncating germline TMEM127 mutations in approximately 30% of familial tumors and about 3% of sporadic-appearing pheochromocytomas without a known genetic cause. The wild-type allele was consistently deleted in tumor DNA, suggesting a classic mechanism of tumor suppressor gene inactivation. Pheochromocytomas with mutations in TMEM127 are transcriptionally related to tumors bearing NF1 mutations and, similarly, show hyperphosphorylation of mammalian target of rapamycin (mTOR) effector proteins. Accordingly, in vitro gain-of-function and loss-of-function analyses indicate that TMEM127 is a negative regulator of mTOR. TMEM127 dynamically associates with the endomembrane system and colocalizes with perinuclear (activated) mTOR, suggesting a subcompartmental-specific effect. Our studies identify TMEM127 as a tumor suppressor gene and validate the power of hereditary tumors to elucidate cancer pathogenesis.

Characterization of Leishmania (Leishmania) amazonensis promastigotes resistant to pentamidine

Pentamidine is a second-line agent used in the treatment of leishmaniasis and its mode of action and mechanism of resistance is not well understood. It was previously demonstrated that transfection of promastigotes and amastigotes with the ABC transporter PRP1 gene confers resistance to pentamidine. To further clarify this point, we generated Leishmania amazonensis mutants resistant to pentamidine. Our results indicated that this ABC transporter is not associated with pentamidine resistance in lines generated by drug pressure through amplification or overexpression mechanisms of PRP1 gene. (C) 2008 Elsevier Inc. All rights reserved.

Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

pido, a non-long terminal repeat retrotransposon of the chicken repeat 1 family from the genome of the Oriental blood fluke, Schistosoma japonicum

A newly described non-long terminal repeat (non-LTR) retrotransposon element was isolated from the genome of the Oriental schistosome, Schistosoma japonicum. At least 1000 partial copies of the element, which was named pido, were dispersed throughout the genome of S. japonicum. As is usual with non-LTR retrotransposons, it is expected that many pido elements will be 5'-truncated. A consensus sequence of 3564 bp of the truncated pido element was assembled from several genomic fragments that contained pido-hybridizing sequences. The sequence encoded part of the first open reading frame (ORF), the entire second ORF and, at its 3'-terminus, a tandemly repetitive, A-rich (TA(6)TA(5)TA(8)) tail, The ORF1 of pido encoded a nucleic acid binding protein and ORF2 encoded a retroviral-like polyprotein that included apurinic/apyrimidinic endonuclease (EN) and reverse transcriptase (RT) domains, in that order. Based on its sequence and structure, and phylogenetic analyses of both the RT and EN domains, pido belongs to the chicken repeat 1 (CR1)-like lineage of elements known from the chicken, turtle, puffer fish, mosquitoes and other taxa. pido shared equal similarity with CRI from chicken, an uncharacterized retrotransposon from Caenorhabditis elegans and SR1 (a non-LTR retrotransposon) from the related blood fluke Schistosoma mansoni; the level of similarity between pido and SR1 indicated that these two schistosome retrotransposons were related but not orthologous. The findings indicate that schistosomes have been colonized by at least two discrete CRI-like elements. Whereas pido did not appear to have a tight target site specificity, at least one copy of pido has inserted into the 3'-untranslated region of a protein-encoding gene (GeriBank AW736757) of as yet unknown identity. mRNA encoding the RT of pido was detected by reverse transcription-polymerase chain reaction in the egg, miracidium. and adult developmental stages of S. japonicum, indicating that the RT domain was transcribed and suggesting that pido was replicating actively and mobile within the S. japonicum genome. (C) 2002 Elsevier Science B.V. All rights reserved.

Molybdenum Induces the expression of a protein containing a new heterometallic Mo-Fe cluster in desulfoVibrio alaskensis

Biochemistry. 2009 Feb 10;48(5):873-82. doi: 10.1021/bi801773t.

Two Novel CMY-2-Type β-Lactamases Encountered in Clinical Escherichia Coli Isolates

BACKGROUND: Chromosomally encoded AmpC β-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal bla AmpC gene. Nowadays, these plasmid-mediated AmpC β-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of β-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type β-lactamase-encoding gene. FINDINGS: Both isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of bla CMY-46 and bla CMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-bla CMY-2-type -ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon. CONCLUSIONS: This study describes two new bla CMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level.

Antimicrobial resistance of Helicobacter pylori strains to five antibiotics, including levofloxacin, in Northwestern Turkey

INTRODUCTION: Antibiotic resistance is the main factor that affects the efficacy of current therapeutic regimens against Helicobacter pylori. This study aimed to determine the rates of resistance to efficacy clarithromycin, amoxicillin, tetracycline, levofloxacin and metronidazole among H. pylori strains isolated from Turkish patients with dyspepsia. METHODS: H. pylori was cultured from corpus and antrum biopsies that were collected from patients with dyspeptic symptoms, and the antimicrobial susceptibility of H. pylori was determined using the E-test (clarithromycin, amoxicillin, tetracycline, metronidazole and levofloxacin) according to the EUCAST breakpoints. Point mutations in the 23S rRNA gene of clarithromycin-resistant strains were investigated using real-time PCR. RESULTS: A total of 98 H. pylori strains were isolated, all of which were susceptible to amoxicillin and tetracycline. Of these strains, 36.7% (36/98) were resistant to clarithromycin, 35.5% (34/98) were resistant to metronidazole, and 29.5% (29/98) were resistant to levofloxacin. Multiple resistance was detected in 19.3% of the isolates. The A2143G and A2144G point mutations in the 23S rRNA-encoding gene were found in all 36 (100%) of the clarithromycin-resistant strains. Additionally, the levofloxacin MIC values increased to 32 mg/L in our H. pylori strains. Finally, among the clarithromycin-resistant strains, 27.2% were resistant to levofloxacin, and 45.4% were resistant to metronidazole. CONCLUSIONS: We conclude that treatment failure after clarithromycin- or levofloxacin-based triple therapy is not surprising and that metronidazole is not a reliable agent for the eradication of H. pylori infection in Turkey.

A minimal version of a Protein Tyrosine Phosphatase

Phosphatase and tensin homologue (PTEN) protein belongs to the family of protein tyrosine phos-phatase. Mutations on the phosphatase and tensin homologue (PTEN) protein are highly observed in diverse types of human tumors, being mostly identified on the phosphatase domain of the protein. Although PTEN is a modular protein composed by a phosphatase domain and a C2 domain for mem-brane anchoring, this work aimed at developing a minimal version of PTEN´s phosphatase domain. The minimal version (Small Domain) comprises a 28 residue peptide, with the PTEN 8-mer catalytic peptide accommodated between a α-helix and β-turn as observed in PTEN native structure. Firstly, a de novo prediction of the Small Domain´s secondary structure was carried out by molecular modeling tools. The stability of the predicted structures were then evaluated by Molecular Dynamics. Automated molecular docking of PTEN natural substrate PIP3, its analogue (Inositol) and a PTEN inhibitor (L-tar-tare) were performed with the modeled structure, and PTEN used as a positive control. The gene en-coding for Small Domain was designed and cloned into an expression vector at N-terminal of Green Fluorescence Protein (GFP) encoding gene. The fusion protein was then expressed in Escherichia coli cells. Different expression conditions have been explored for the production of the fusion protein to minimize the formation of inclusion bodies.

Dictyostelium discoideum as an expression host for the circumsporozoite protein of Plasmodium falciparum.

We have used the cellular slime mold, Dictyostelium discoideum (Dd), to express the Plasmodium falciparum circumsporozoite protein (CS), a potential component of a subunit vaccine against malaria. This was accomplished via an expression vector based on the discoidin I-encoding gene promoter, in which we linked a sequence coding for a Dd leader peptide to the almost complete CS coding region (pEDII-CS). CS production at both the mRNA and protein levels is induced by starving cells in a simple phosphate buffer. Variation in pH or cell density does not seem to influence CS synthesis. CS-producing cells can be grown either on their normal substrate, bacteria, or on a semi-synthetic media, without affecting CS accumulation level. The CS produced in Dd seems similar to the natural parasite protein as judged by its size and epitope recognition by a panel of monoclonal antibodies. We constructed a second expression vector in which the CS is under the control of a Dd ras promoter. CS accumulation can then be induced by external addition of cAMP. Such a tightly regulated promoter may allow expression of proteins potentially toxic to the cell. Thus, Dd could be a useful eukaryotic system to produce recombinant proteins, in particular from human or animal parasites like P. falciparum.

Mitochondrial targeting adaptation of the hominoid-specific glutamate dehydrogenase driven by positive Darwinian selection.

Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor approximately 18-25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.

Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector.

A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.

Genome-wide re-sequencing of multidrug-resistant Mycobacterium leprae Airaku-3.

Genotyping and molecular characterization of drug resistance mechanisms in Mycobacterium leprae enables disease transmission and drug resistance trends to be monitored. In the present study, we performed genome-wide analysis of Airaku-3, a multidrug-resistant strain with an unknown mechanism of resistance to rifampicin. We identified 12 unique non-synonymous single-nucleotide polymorphisms (SNPs) including two in the transporter-encoding ctpC and ctpI genes. In addition, two SNPs were found that improve the resolution of SNP-based genotyping, particularly for Venezuelan and South East Asian strains of M. leprae.

Molecular identification of Rickettsia felis in ticks and fleas from an endemic area for Brazilian Spotted Fever

Rickettsioses are arthropod-borne diseases caused by parasites from the Order Rickettsiales. The most prevalent rickettsial disease in Brazil is Brazilian Spotted Fever (BSF). This work intends the molecular detection of those agents in ectoparasites from an endemic area of BSF in the state of Espírito Santo. A total of 502 ectoparasites, among them Amblyomma cajennense, Amblyomma dubitatum (A. cooperi), Riphicephalus sanguineus, Anocentor nitens and Ctenocephalides felis, was collected from domestic animals and the environment and separated in 152 lots according to the origin. Rickettsia sp. was detected in pools of all collected species by amplification of 17kDa protein-encoding gene fragments. The products of PCR amplification of three samples were sequenced, and Rickettsia felis was identified in R. sanguineus and C. felis. These results confirm the presence of Rickettsia felis in areas previously known as endemic for BSF, disease caused by Rickettsia rickettsii. Moreover, they show the needing of further studies for deeper knowledge of R. felis-spotted fever epidemiology and differentiation of these diseases in Brazil.

Sequence analysis of coding DNA fragments of pfcrt and pfmdr-1 genes in Plasmodium falciparum isolates from Odisha, India

The global emergence and spread of malaria parasites resistant to antimalarial drugs is the major problem in malaria control. The genetic basis of the parasite's resistance to the antimalarial drug chloroquine (CQ) is well-documented, allowing for the analysis of field isolates of malaria parasites to address evolutionary questions concerning the origin and spread of CQ-resistance. Here, we present DNA sequence analyses of both the second exon of the Plasmodium falciparum CQ-resistance transporter (pfcrt) gene and the 5' end of the P. falciparum multidrug-resistance 1 (pfmdr-1) gene in 40 P. falciparum field isolates collected from eight different localities of Odisha, India. First, we genotyped the samples for the pfcrt K76T and pfmdr-1 N86Y mutations in these two genes, which are the mutations primarily implicated in CQ-resistance. We further analyzed amino acid changes in codons 72-76 of the pfcrt haplotypes. Interestingly, both the K76T and N86Y mutations were found to co-exist in 32 out of the total 40 isolates, which were of either the CVIET or SVMNT haplotype, while the remaining eight isolates were of the CVMNK haplotype. In total, eight nonsynonymous single nucleotide polymorphisms (SNPs) were observed, six in the pfcrt gene and two in the pfmdr-1 gene. One poorly studied SNP in the pfcrt gene (A97T) was found at a high frequency in many P. falciparum samples. Using population genetics to analyze these two gene fragments, we revealed comparatively higher nucleotide diversity in the pfcrt gene than in the pfmdr-1 gene. Furthermore, linkage disequilibrium was found to be tight between closely spaced SNPs of the pfcrt gene. Finally, both the pfcrt and the pfmdr-1 genes were found to evolve under the standard neutral model of molecular evolution.

Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.