Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria.

Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.

The human genome sequence: impact on health care.

The recent sequencing of the human genome, resulting from two independent global efforts, is poised to revolutionize all aspects of human health. This landmark achievement has also vindicated two differeint methodologies that can now be used to target other important large genomes. The human genome sequence has revealed several novel/surprising features notably the probable presence of a mere 30-35,000 genes. In depth comparisons have led to classification of protein families and identification of several orthologues and paralogues. Information regarding non-protein coding genes as well as regulatory regions has thrown up several new areas of research. Although still incomplete, the sequence is poised to become a boon to pharmaceutical companies with the promise of delivering several new drug targets. Several ethical concerns have also been raised and need to be addressed in earnest. This review discusses all these aspects and dwells on the possible impact of the human genome sequence on human health, medicine and also health care delivery system.

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans.

CaMDR1 encodes a major facilitator superfamily (MFS) protein in Candida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p in Sf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance in C. albicans, were independently expressed in a common hypersensitive host JG436 of Saccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.

Alterations in brain tumor DNA detected by a fingerprinting probe.

We used a novel DNA fingerprinting probe O-chi-1 (ref. 1) to detect differences in the hybridization pattern of brain tumor DNA and paired normal tissue of a given individual. Representatives of meningiomas (two), glioblastoma multeforme (three) and astrocytoma (one) were studied. Alterations, which included amplification as well as the loss of a normal band in tumor DNA, were observed in four of the six tumours. While the increased intensity of a band can be taken to imply increased copy number, the disappearance of bands could either be due to loss of DNA sequence or rearrangement resulting in different sized bands.

Effect of storage of blood samples on DNA yield, quality and fingerprinting: a forensic approach.

Various storage treatments on human blood samples have been described with respect to DNA yield, quality and fingerprinting. Blood samples were stored at room temperature, 4 degrees C, -20 degrees C and -70 degrees C for different duration varying from 1, 2, 4 and 8 weeks with or without anticoagulant/preservative (EDTA or Heparin). DNA was isolated from these stored samples, quantitated by spectrophotometry and subjected to DNA fingerprinting using a human alphoid satellite DNA sequence (TRF 5.6) and a 33 mer oligonucleotide (O-chi-1) as probes. The polymorphic DNA bands were apparent between 2 to 9 kb size range and the fingerprints were individual-specific. Our results suggest that higher amount of genomic DNA can be recovered from blood samples stored at temperatures 4 degrees C or below in the presence of EDTA or heparin.

Codon usage in the prototype baculovirus--Autographa californica nuclear polyhedrosis virus.

The overall codon usage profile of Autographa californica nuclear polyhedrosis virus (AcNPV) was analyzed, using UWGCG sequence analysis software package from the known protein coding gene sequences available in GenBank Release 72. The analysis revealed that although only 45% of the codon used by AcNPV have G/C at wobble base position, 15 out of 20 AcNPV codons over-utilized for their given amino acids has G/C at the wobble position indicating a possible selection of these codons. The differences in codon usage profile were studied using a parameter called D squared value, calculated with the aid of CORRESPOND program of UWGCG software package. While most of the codon usage profile of the individual genes was very similar to the overall AcNPV codon usage profile (D squared value less than 1.5), there were notable differences (D-squared value greater than 1.5). These genes were polh, p10, ub, sod, gp41, core, 25k, 39k, ie-n, etm, ets most of which, interestingly, belonged to late or very late class and were expressed relatively more efficiently. The two highly expressed genes of AcNPV, polh and the p10, differ from the overall AcNPV codon usage profile with respect to at least nine amino acids (Val, Ala, Ser, Lys, Ile, Thr, Leu, Phe, Arg). Our findings that the two highly expressed late genes polh and p10 utilize a codon usage profile different from the early genes have important implications.

Over-expression and characterization of recombinant beta subunit of the human chorionic gonadotropin hormone synthesized in insect cells infected with a genetically engineered baculovirus.

A recombinant baculovirus, vAc beta hCG, having a replacement of the viral polyhedrin gene with the cDNA encoding the beta subunit of hCG was used to express beta hCG, an extensively glycosylated hormone, in insect cells. Virus-infected cells, 72 hr pi, secreted approximately 8.02 micrograms beta hCG/2 x 10(6) cells/ml. The recombinant beta hCG purified from insect cells exhibited increased mobility on SDS-PAGE as compared to authentic urinary beta hCG, a reflection on differences in glycosylation between insect and mammalian systems. The insect derived beta hCG, however, was identical to the native hormonal peptide in terms of immunoreactivity and bioactivity on association with alpha-subunit, as evident by its binding to rat testicular receptors and induction of steroidogenesis in a mouse Leydig cell bioassay system. The implications of using the baculovirus system to study the importance of carbohydrates for biological activity are also discussed.

Construction, purification and characterization of a recombinant baculovirus containing the gene for alpha subunit of human chorionic gonadotropin.

A cDNA encoding the alpha subunit of human chorionic gonadotropin, a placental glycoprotein hormone, was cloned downstream to the viral polyhedrin gene promoter of Autographa california nuclear polyhedrosis virus and the recombinant transfer vector was used to co-transfect Spodoptera frugiperda cells growing in culture. Recombinant baculovirus carrying the alpha hCG gene was detected and isolated after dot hybridization using supernatant from co-transfected cells. Recombinant vAc alpha hCG having a replacement of the viral polyhedrin gene, which is hyper-transcribed very late in the infection cycle, with the alpha hCG cDNA was purified after a single round of plaque purification. Insect cell culture infected with vAc alpha hCG, secreted high levels of hCG which was biologically active.

Molecular cloning of human satellite DNA sequences and their use in detecting DNA polymorphism.

A approximately 400 bp HaeIII human genomic satellite DNA band was cloned into pUC18 to construct a partial library. A fragment of bacteriophage M13 containing a sequence homologous to the human minisatellite core was cloned in pUC18 and was used as a probe to isolate a approximately 350 bp human satellite clone (pTRF5.6) from the partial library. Other clones from this library showed a wide variation in terms of size and hybridization to the pTRF5.6 clone. Human DNA from different individuals was digested with restriction enzymes, Southern transferred and probed with TRF5.6. Individual-specific complex pattern of DNA bands was produced. TRF5.6, therefore, could be useful as a probe for detecting genetic polymorphism.