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Biomedical subjects

A Kohli

Publications and source records attributed to A Kohli.

At least 19 recordsLinked to original sources
PubMed2001

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Tagged Transcriptome Display (TTD) in indica rice using Ac transposition.

We have developed a population of transgenic indica rice lines containing the autonomous Activator transposon (Ac) from maize. A transposon excision assay using as the reporter the green fluorescent protein (GFP) gene driven by the ubiquitin promoter was used to monitor Ac excision in various tissues. Our results, based on Ac excision and re-insertion events in 289 independent rice transformants, provide an insight into transposon biology in this heterologous model cereal system. Twenty percent of the transformed calli displayed uniform GFP activity, indicating very early Ac excision, while later excision in another 40% of calli was revealed by mosaic GFP activity. Both phenotypes were confirmed by molecular analysis. Progeny analysis revealed active transposition, with some lines displaying transposition to unlinked positions. Amplification of the Ac copy number was observed in approximately 30% of the lines, thus tagging multiple sites. We developed a "transposon insertion display" procedure, involving the use of CpG methylation-sensitive enzymes and a macroarray-based approach with cDNA as a complex hybridization probe, to selectively detect transposon insertions in transcribed sequences. Sequencing of tagged sites identified by hybridization to leaf cDNA revealed that all the hybridizing tags were homologous to genes or ESTs present in databases. Extrapolation from these data suggests that 12% of all amplified tags display homology to genes or ESTs - five times more than would be expected on the basis of random transpositional insertion. This Tagged Transcriptome Display (TTD) technique, using a population of Ac insertion-tagged lines, therefore represents a convenient approach for the identification of tagged genes involved in specific processes, as revealed by their expression patterns.

Base Sequence↗
PubMed2001

Contrast sensitivity following focal laser photocoagulation in clinically significant macular oedema due to diabetic retinopathy.

PURPOSE: To evaluate the influence of focal aser photocoagulation on contrast sensitivity in diabetic patients with clinically significant macular oedema (CSMO). METHODS: A prospective non-comparative interventional study was performed on a group of patients with CSMO at Dr Rajendra Prasad Centre for Ophthalmic Sciences, New Delhi, a tertiary eye care centre. Thirteen diabetc patients (14 eyes) with CSMO and no history of prior photocoagulation were recruited for this study. Direct focal photocoagulation of all leaking microaneurysms was performed using an argon green laser (514 nm). A contact lens was used as a slit lamp delivery system. Evaluation of the best corrected Snellen visual acuity, contrast senstivity, slit lamp biomicroscopy, macular status on direct ophthalmoscopy and fluorescein angiography was carried out 1 month and 3 months after laser photocoagulation. RESULTS: Following direct focal laser photocoagulation, focal CSMO resolved completely in all but one eye, 4-8 weeks later, as seen on slit lamp biomicroscopy and/or fluorescein angiography. Post-treatment, visual acuity remained stationary in eight eyes, improved by one line in three eyes, by two lines in two eyes and by three lines in one eye. The mean +/- SD pretreatment and post-treatment decimal visual acuities were 0.49+/-0.30 and 0.59+/-0.28, respectively. The mean +/- SD pre-laser contrast sensitivity score was 121.3+/-83.6, which increased significantly to a mean +/- SD of 151.6+/-80.5 fo lowing direct focal photocoagulation. CONCLUSION: Focal argon laser photocoagulation in CSMO in diabetics helps in improving the contrast sensitivity and stabilizes the visual acuity. The changes in contrast sensitivity and visual acuity are independent of each other.

Contrast Sensitivity↗
PubMed2001

Pericardiectomy for constrictive pericarditis: is prolonged inotropic support a bad omen?

Sixty-seven patients who underwent pericardiectomy for constrictive pericarditis at JIPMER, Pondicherry between 1987 and 1998 were the subjects of the study. Pre-operatively 70% of cases were in the New York Heart Association classes III and IV categories with clinical signs suggestive of constriction ie, raised jugular venous pressure in 99%, pleural effusion in 77%, pedal oedema in 61% and ascites in 55% of the cases. Seventy-five per cent of the cases underwent pericardiectomy through a median sternotomy and the rest via left anterolateral thoracotomy. Low cardiac output was evidenced in 70% of cases postoperatively which was managed by early institution and prolonged use of inotropes. There was 9% mortality especially in the early part of the experience. Tuberculous pathology was confirmed histologically in 57% cases. Sixty-three per cent of cases are presenting in follow-up in New York Heart Association class I. Prolonged use of inotropes instituted early in postoperative period is recommended to prevent postoperative ventricular dysfunction with adrenaline being the preferred inotrope. It is concluded that postoperative New York Heart Association class and long term survival were not significantly influenced by pre-operative New York Heart Association class, operative approach or peri-operative low cardiac output syndrome requiring prolonged inotropic support.

Adolescent↗
PubMed2000

Alternative silencing effects involve distinct types of non-spreading cytosine methylation at a three-gene, single-copy transgenic locus in rice.

We investigated transgene silencing in a line of rice plants that carries a single-copy 6.6-kb transgenic locus comprising three heterologous transgenes: bar, hpt and gusA. We identified at least three distinct types of silencing effects associated with different methylation patterns, including a novel form of transcriptional silencing involving methylation of cytosine residues only at non-conventional acceptor sites in the coding region. Silencing arose de novo in individual R1, R2 and R3 plants despite the stability of the transgenic locus, although the basic structure of the locus, transgene dosage and position effects remained constant within the line. We found that different silencing effects could occur concurrently in adjacent heterologous transgenes in the same plant, with no evidence for spreading of silenced states or methylation patterns from one transgene to another.

Base Sequence↗
PubMed2000

Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns.

Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.

Acetyltransferases↗
PubMed2000

The effect of intravenous verapamil on cerebral hemodynamics in a migraine patient with hemiplegia.

OBJECTIVE: To describe the use of intravenous verapamil in a migraine patient with hemiplegia to reverse the symptomatology and hemodynamics of the middle cerebral artery as determined by transcranial Doppler. CASE SUMMARY: A 31-year-old white woman was admitted with an acute exacerbation of migraine with hemiplegia. A transcranial Doppler showed an increased flow velocity through the middle cerebral artery consistent with a migrainous process. The patient was treated with verapamil 5 mg iv and the hemiplegia gradually resolved. A transcranial Doppler indicated that the flow velocity through the middle cerebral artery was decreased after verapamil administration, indicating reversal of the vasospasm. DISCUSSION: Transcranial Doppler has not been previously used to determine the effect of intravenous verapamil on the migrainous process. Intravenous verapamil reversed the altered hemodynamics of the middle cerebral artery as determined by transcranial Doppler. This finding correlated with the gradual resolution of hemiplegia. Whether both subjective and objective findings in this patient can be attributed to the reversal of the cerebral artery hemodynamics is not known. CONCLUSIONS: Intravenous verapamil appears to reverse the vasospasm that may be associated with a migrainous process. Whether this effect is solely responsible for clinical improvement is not known. Verapamil may be a consideration for the treatment of intractable migraine, especially when there is evidence of spasm of the major cerebral arteries.

Adult↗
PubMed1999

Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination.

The characterization of plasmid-genomic DNA junctions following plant transformation has established links between DNA double-strand break repair (DSBR), illegitimate recombination and plasmid DNA integration. The limited information on plasmid-plasmid junctions in plants comes from the dicot species tobacco and Arabidopsis. We analyzed 12 representative transgenic rice lines, carrying a range of transforming plasmid rearrangements, which predominantly reflected microhomology mediated illegitimate recombination involving short complementary patches at the recombining ends. Direct end-ligation, in the absence of homology between the recombining molecules, occurred only rarely. Filler DNA was found at some of the junctions. Short, purine-rich tracts were present, either at the junction site or in the immediate flanking regions. Putative DNA topoisomerase I binding sites were clustered around the junctions. Although different regions of the transforming plasmid were involved in plasmid-plasmid recombination, we showed that a 19 bp palindromic sequence, including the TATA box of the CaMV 35S promoter, acted as a recombination hotspot. The purine-rich half of the palindromic sequence was specifically involved at the recombination junctions. This recombination hotspot is located within the 'highly recombinogenic' region of the full-length CaMV RNA that has been shown to promote viral recombination in dicot plants. Clustering of plasmid recombination events in this highly recombinogenic region, even in the absence of viral enzymes and other cis-acting elements proves that the plant cellular machinery alone is sufficient to recognize and act on these viral sequences. Our data also show the similarity between mechanisms underlying junction formation in dicot and monocot plants transformed using different procedures.

Base Sequence↗
PubMed1998

Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots.

Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a two-phase integration mechanism. In the "preintegration" phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium, the qualitative and/or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of Agrobacterium vir genes in the integration process.

DNA, Plant↗
PubMed1998

Identification of polymorphic mutant alleles of CaMDR1, a major facilitator of Candida albicans which confers multidrug resistance, and its in vitro transcriptional activation.

CaMDR1 (Candida albicans Multi Drug Resistance) encodes a major facilitator whose expression in Saccharomyces cerevisiae confers resistance to several unrelated drugs. We describe here the identification and molecular characterization of seven mutant alleles of CaMDR1 (CaMDR1-1 to 1-7). The complete sequencing of CaMDR1 alleles revealed several in-frame point mutations leading to a change in amino-acid residues where insertion/replacement of an aspartate residue in a serine-asparagine-aspartate-rich domain was most noteworthy. Interestingly, these alleles showed a distinct drug resistance profile. The expression of CaMDR1, or of its alleles, in C. albicans cells was enhanced by benomyl, methotrexate and several other unrelated drugs, and was more pronounced in at least one of the azole-resistant clinical isolates.

Alleles↗
PubMed1998

Study of head injury victims in fatal road traffic accidents in Delhi.

The study revealed 31% were the victims of Head injury injury in this part of Delhi in vehicular accidents. Although majority of the fatalities were on the spot quite a number of such victims survived for a varied period. A multipronged approach including the preventive and curative measures for this man-made calamity is the need of the hour. Amongst various preventive measure are wearing helmet be made compulsory for both the driver and pillion riders of two-wheelers, development of a safe traffic sense amongst the road users. Traffic Police should be honest, exemplary punishment for the reckless driver, decongestion of Delhi roads etc. The curative measures are facilities of CT scanners and neurosurgeons in all the major hospitals, provision of first-aid in the PCR (Police control room) vans, to institute immediate treatment to the victim without waiting for the medico-legal formalities, opening of more fully equipped Accident and trauma centres. It is high time the concerned authorities gave some serious thought towards this avoidable epidemic on Delhi roads.

Accidents, Traffic↗
PubMed1997

MRI demonstration of Wallerian degeneration in various intracranial lesions and its clinical implications.

Dynamic signal intensity changes of Wallerian degeneration (WD) are well documented in cases of stroke. These changes have been staged I-IV, depending on time-specific signal intensity changes in corticospinal tract with magnetic resonance imaging (MRI). We performed both prospective and retrospective evaluation of various intracranial lesions to look for evidence of WD and to assess its prognostic implications. Eighteen patients of acute stroke were studied prospectively. Their functional disability was evaluated by using a modified Barthel index of activity of daily living (ADL) at presentation, at 1 month and at 4 months, and was correlated with presence or absence of WD on MRI. 10/18 patients showed signal intensity changes of WD on MRI and their mean ADL score changed from 9.1 at 1 month to 11.4 at 4 months duration suggestive of moderate to severe disability after 4 months of stroke. The rest of the eight patients, where MRI did not reveal signal intensity changes of WD, the mean ADL score improved from 10.37 at 1 month to 17.5 at 4 months, suggesting significant improvement in their clinical disability. 520 patients were studied retrospectively, out of whom 31 showed signal intensity changes of WD in various intracranial lesions, i.e. infarcts (14/220), intracranial haematoma (4/147), arterio-venous malformation (1/20), tumour (6/98), multiple sclerosis (5/20) and encephalitis (1/15). Presence of WD in these intracranial lesions correlated well with persistent clinical disability. This observation has immense prognostic value, particularly in relapsing and remitting disease like multiple sclerosis. We conclude that WD can be seen secondary to any CNS insult with MRI and its presence correlates well with persistent functional disability. It thus has prognostic value.

Adult↗
PubMed1997

MRI of the brain in patients with miliary pulmonary tuberculosis without symptoms or signs of central nervous system involvement.

MRI was performed on patients with miliary pulmonary tuberculosis to look for brain involvement and to study the features sequentially, during treatment. We studied seven patients with typical radiographic tuberculosis, and no symptoms or signs of central nervous system involvement. Conventional spin-echo (SE) imaging, including contrast enhanced images, was performed in all cases. All patients showed brain involvement: four patients showed lesions mainly less than 3 mm in diameter, better seen on contrast-enhanced images. These patients showed oedema around the lesions after 2 months of treatment, with subsequent regression on follow-up. The remaining three patients had multiple lesions, 3 mm or more in diameter, which showed a gradual decrease on follow-up. We conclude that the brain may commonly be involved in miliary pulmonary tuberculosis. The response to treatment depends on the stage of the granuloma and shows a definite pattern of healing on follow-up.

Adolescent↗
PubMed1997

Anterior cerebral artery territory infarction in neurocysticercosis: evaluation by MR angiography and in vivo proton MR spectroscopy.

Vascular complications of neurocysticercosis are common, but frequently not recognized. Mostly these are in the form of an endarteritis involving the smaller basal vessels due to basal exudates. Large vessel territory infarction has been clearly documented in not more than 10 cases. All these cases had either a chronic meningeal state or close proximity of cysts to the vessel wall explaining the vasculitis. We report a case that developed anterior as well as segmental middle cerebral artery territory infarction in the acute encephalitic state of neurocysticercosis. MR angiography showed constriction in the proximal segment of the right anterior cerebral artery. There was no biochemical or imaging evidence of a meningeal reaction. In vivo MR spectroscopy over the infarction showed absent N-acetyl-aspartate, low creatine and high lactate. This is the first case showing a large vessel territory infarction in the encephalitic state of neurocysticercosis, with no meningeal reaction. Focal arteritis due to an adjacent brain parenchymal reaction could be a possible mechanism for the vasculitis. MR spectroscopy may have a potential role in assessing tissue viability and therapeutic modalities in such infective vasculitis.

Albendazole↗