Loop-mediated isothermal amplification as alternative to PCR for the diagnosis of extra-pulmonary tuberculosis.

BACKGROUND: The main challenge in combatting extra-pulmonary tuberculosis (EPTB) is the lack of a rapid, reliable and inexpensive diagnostic test for the detection of Mycobacterium tuberculosis. OBJECTIVE: To evaluate the diagnostic potential of an L-serine dehydratase gene (sdaA) loop-mediated isothermal amplification (LAMP) assay for the detection of M. tuberculosis in clinical specimens from presumptive EPTB patients. METHODS: An in-house sdaA LAMP assay was used to analyse clinical specimens (n = 315) for the diagnosis of EPTB compared with culture and the composite reference standard (CRS) comprising culture and polymerase chain reaction (PCR) using insertion sequence (IS) 6110 and mpb64 as target genes. RESULTS: The sdaA LAMP assay showed the highest sensitivity (93.3%) in comparison to culture; the sensitivity of IS6110 PCR, mpb64 and sdaA PCR assay was respectively 80%, 86.7% and 90%. In comparison to CRS, the LAMP assay had a sensitivity of 92.5% and a specificity of 99.2%, with a high positive (121.11) and a low negative likelihood ratio (0.08). CONCLUSION: Due to its speed, simplicity, sensitivity and specificity, the sdaA LAMP assay is a potential diagnostic test for the diagnosis of EPTB, particularly in resource-limited settings.

Platinum (IV) thiohydrazide, thiodiamine and thiohydrazone complexes: a spectral, antibacterial and cytotoxic study.

Some platinum (IV) complexes [Pt(L)2Cl2] [where, L=2-aminopyridine-N-thiohydrazide (L1), (2-aminopyridine-N-thio)-1,3-propanediamine (L2), benzaldehyde-2-aminopyridine-N-thiohydrazone (L3) and salicylaldehyde-2-aminopyrtidine-N-thiohydrazone (L4)] have been synthesized. The thiohydrazides, thiodiamine and thiohydrazones can exist as thione-thiol tautomer and coordinate as a bidentate N-S ligand. The ligands found to act in monobasic bidentate fashion. Analytical data reveals that metal to ligand stoichiometry is 1:2. The complexes have been characterized by elemental analysis, IR, mass, electronic and 1H NMR spectroscopic studies. In vitro antibacterial and cytotoxic study have also been carried out for some complexes.

Synthesis, characterization, antibacterial and cytotoxic study of platinum (IV) complexes.

Platinum (IV) complexes [Pt (L)2Cl2] [where, L= benzyl-N-thiohydrazide (L1), (benzyl-N-thio)-1,3-propanediamine (L2), benzaldehyde-benzyl-N-thiohydrazone (L3) and salicylaldehyde-benzyl-N-thiohydrazone (L4)] have been synthesized. The thiohydrazide, thiodiamine and thiohydrazones can exist as thione-thiol tautomer and coordinate as a bidentate N-S ligand. The ligands were found to act in monobasic bidentate fashion. Analytical data reveal that metal to ligand stoichiometry is 1:2. The complexes have been characterized by elemental analysis, IR, mass, electronic and 1H NMR spectroscopic studies. In vitro antibacterial and cytotoxic studies have been carried out for some complexes. Various kinetic and thermodynamic parameters like order of reaction (n), activation energy (Ea), apparent activation entropy (S#) and heat of reaction (DeltaH) have also been carried out for some complexes.

Mutation patterns in gyrA and parC genes of ciprofloxacin resistant isolates of Neisseria gonorrhoeae from India.

AIM: To analyse mutations in the gyrA and parC genes leading to possible increase in ciprofloxacin resistance (high MIC values for ciprofloxacin) in clinical isolates of Neisseria gonorrhoeae in Delhi, India. METHOD: MIC of ciprofloxacin for 63 clinical isolates of N gonorrhoeae were examined by the Etest method. Subsequently, gyrA and parC genes of these isolates were amplified and sequenced for possible mutations. RESULTS: Out of the 63 clinical isolates tested, only five (8%) isolates were found to be susceptible to ciprofloxacin (MIC <0.06 micro g/ml). DNA sequence analysis of the gyrA and the parC genes of all these isolates (n = 63) revealed that all isolates which were not susceptible to ciprofloxacin (n=58) had mutation(s) in gyrA and parC genes. 12 isolates (19%) exhibited high resistance with an MIC for ciprofloxacin of 32 micro g/ml. Two out of these 12 isolates (UD62 and UD63), harboured triple mutations (Ser-91 to Phe, Asp-95 to Asn and Val-120 to Leu) in the gyrA gene. The third mutation of Val-120 to Leu, lies downstream of the quinolone resistance determining region (QRDR) of the gyrA and has not been described before in gonococcus. In addition, both these isolates had a Phe-100 to Tyr substitution in the parC, a hitherto unknown mutation. CONCLUSIONS: Emergence of ciprofloxacin resistance with high levels of MIC values (up to 32 micro g/ml) in India is alarming. Double and triple mutations in gyrA alone or together in gyrA and parC could be responsible for such a high resistance.

Detection ofNeisseria Gonorrhoeae by polymerase chain reaction (PCR).

Three different sets of primers were designed using FASTA homology search and PRIMERSELECT for the specific detection ofNeisseria gonorrhoeae using polymerase chain reaction (PCR). These primers amplified the highly conserved regions of genes for Open Reading Frame (ORF), Outer Membrane Protein (OMP) and 23S rRNA sequences ofN. gonorrhoeae. Each of the PCR primer set was evaluated using the DNA samples isolated from eight different positive isolates ofN. gonorrhoeae cultured from urethral swabs of patients visiting Maulana Azad Medical College and Safdarjung Hospital. Amplification products were analyzed on agarose gel electrophoresis. Two sets of PCR primers, designated as Ngu1/Ngu2 and Ngu5/Ngu6, specific for ORF and OMP gene respectively, amplified four regions of the gene which may help to differentiate the various strains ofN. gonorrhoeae infecting indigenous population. In contrast, a single, specific PCR product of 650 bp was visualized on agarose gel with primers Ngu3/Ngu4, amplifying the 23S rRNA gene. Under optimum conditions, as low as 25ng of DNA isolated from eight different clinical strains ofN. gonorrhoeae could be detected by PCR using Ngu3/Ngu4 set of primers. Our results suggested that Ngu3/Ngu4 could serve as good primers for the specific, reproducible and sensitive diagnosis ofNeisseria gonorrhoeae from clinical samples.

Distinct subdomains of human TAFII130 are required for interactions with glutamine-rich transcriptional activators.

TFIID is a multiprotein complex consisting of the TATA box binding protein and multiple tightly associated proteins (TAFIIs) that are required for transcription by selected activators. We previously reported cloning and partial characterization of human TAFII130 (hTAFII130). The central domain of hTAFII130 contains four glutamine-rich regions, designated Q1 to Q4, that are involved in interactions with the transcriptional activator Sp1. Mutational analysis has revealed specific regions within the glutamine-rich (Q1 to Q4) central region of hTAFII130 that are required for interaction with distinct activation domains. We tested amino- and carboxyl-terminal deletions of hTAFII130 for interaction with Sp1 activation domains A and B (Sp1A and Sp1B) and the N-terminal activation domain of CREB (CREB-N) by using the yeast two-hybrid system. Our results indicate that Sp1B interacts almost exclusively with the Q1 region of hTAFII130. In contrast, Sp1A makes multiple contacts with Q1 to Q4 of hTAFII130, while CREB-N interacts primarily with the Q1-Q2 hTAFII130 subdomain. Consistent with these interaction studies, overexpression of the Q1-to-Q4 region in HeLa cells inhibits Sp1- but not VP16-mediated transcriptional activation. These findings indicate that the Q1-to-Q4 region of hTAFII130 is required for Sp1-mediated transcriptional enhancement in mammalian cells and that different activation domains target distinct subdomains of hTAFII130.

The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the alpha- and beta-globin gene loci in an erythroid cell line.

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human beta-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous beta- or alpha-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TAFII130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

Molecular cloning and analysis of two subunits of the human TFIID complex: hTAFII130 and hTAFII100.

Transcription factor TFIID is a multiprotein complex composed of the TATA box-binding protein (TBP) and multiple TBP-associated factors (TAFs). TFIID plays an essential role in mediating transcriptional activation by gene-specific activators. Numerous transcriptional activators have been characterized from mammalian cells; however, molecular analysis of the components of mammalian TFIID has been incomplete. Here we describe isolation of cDNAs encoding two TAF subunits of the human transcription factor TFIID. The first cDNA is predicted to encode the C-terminal 947 residues of the 130-kDa human TAF subunit, hTAFII130. The second cDNA encodes the C-terminal 801 residues of the 100-kDa subunit, hTAFII100. Recombinant TAFs expressed in human cells by transient transfections are capable of associating with the endogenous TAFs and TBP to form a TFIID complex in vivo. Protein binding experiments demonstrate that hTAFII130, like its Drosophila homolog dTAFII110, interacts with the glutamine-rich activation domains of the human transcription factor Sp1. Furthermore, hTAFII130 shows reduced binding to the Sp1 mutants with impaired ability to activate transcription, suggesting a role for hTAFII130 as a direct coactivator target for Sp1.

Biochemical characterization of Escherichia coli temperature-sensitive dnaB mutants dnaB8, dnaB252, dnaB70, dnaB43, and dnaB454.

By use of PCR, the dnaB genes from the classical temperature-sensitive dnaB mutants PC8 (dnaB8), RS162 (dnaB252), CR34/454 (dnaB454), HfrH165/70 (dnaB70), and CR34/43 (dnaB43) were isolated. The mutant genes were sequenced, and single amino acid changes were identified in all cases. The mutant DnaB proteins were overexpressed in BL21 (DE3) cells by using the T7 based pET-11c expression vector system. The purified proteins were compared in regard to activities in the general priming reaction of primer RNA synthesis (with primase and single-stranded DNA [ssDNA] as the template), ATPase activity, and helicase activity at permissive (30 degrees C) and nonpermissive (42 degrees C) temperatures. The DnaB252 mutation is at amino acid 299 (Gly to Asp), and in all in vitro assays the DnaB252 protein was as active as the wild-type DnaB protein at both 30 and 42 degrees C. This region of the DnaB protein is believed to be involved in interaction with the DnaC protein. The dnaB8, dnaB454, and dnaB43 mutations, although independently isolated in different laboratories, were all at the same site, changing amino acid 130 from Ala to Val. This mutation is in the hinge region of the DnaB protein domains and probably induces a temperature-sensitive conformational change. These mutants have negligible primer RNA synthesis, ATPase activity, and helicase activity at the nonpermissive temperature. DnaB70 has a mutation at amino acid 242 (Met to Ile), which is close to the proposed ATP binding site. At 30 degrees C this mutant protein has a low level of ATPase activity (approximately 25% of that of the wild type) which is not affected by high temperature. By using a gel shift method that relies upon ssDNA substrates containing the photoaffinity analog 5-(N-(p-azidobenzoyl)-3-aminoallyl)-dUMP, all mutant proteins were shown to bind to ssDNA at both 30 and 42 degrees C. Their lack of other activities at 42 degrees C, therefore, is not due to loss of binding to the ssDNA substrate.

Post-transcriptional regulation of S-adenosylmethionine synthetase from its stored mRNA in germinated wheat embryos.

About 2-3-fold stimulation of S-adenosylmethionine synthetase was witnessed in germinated wheat embryos (48 h). The enhancement of enzyme activity was significantly inhibited by cycloheximide and amino acid analogues. Simultaneous addition of corresponding amino acids alleviated the inhibitory effect of amino acid analogues. Conclusive proof for the de novo synthesis of S-adenosylmethionine synthetase was obtained by labelling this enzyme with [35SO4]2- in vivo. Thus de novo enzyme synthesis seemed necessary for the rise in activity of AdoMet synthetase in wheat embryos. Curiously, blocking of transcription with cordycepin failed to repress the de novo synthesis of AdoMet synthetase in germinated wheat embryos. We envisage the presence of stored mRNA for AdoMet synthetase in wheat embryos. Thus the regulation of this enzyme occurs at the post-transcriptional level. L-Methionine, which is one of the substrates of AdoMet synthetase, stimulated the enzyme activity (2-2.4-fold) over that observed in control germinated embryos. L-Methionine promotes increased de novo synthesis of AdoMet synthetase. Preincubation of enzyme fraction with L-Methionine failed to activate or stabilize the activity of AdoMet synthetase. Three isozymes of AdoMet synthetase were physically separated by DE-52 ion-exchange chromatography. One of the isozymes of AdoMet synthetase has been purified (1529-fold) to electrophoretic homogeneity by resorting to phenyl Sepharose and ATP Sepharose affinity chromatography. The purified enzyme catalyzed the synthesis of S-adenosylmethionine and also exhibited tripolyphosphatase activity. The reaction product of the purified enzyme was chemically and enzymatically characterized as S-adenosylmethionine. The molecular weight of the native enzyme is 174,000 and that of its subunit is 84,000 as determined on SDS-PAGE. Thus the native enzyme seems to be dimeric in nature.