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Biomedical subjects

P Kumar

Publications and source records attributed to P Kumar.

At least 793 records · Page 44Linked to original sources
PubMedDate not supplied

A rapid method for the construction of oligonucleotide arrays.

A simple method has been devised to construct oligonucleotide array on a variety of surfaces using commonly available reagents and chemistry with good efficiency and accuracy. The method involves the generation of hydroxyl functionalities on glass, polypropylene, polyethylene, and commonly used surfaces for construction of oligonucleotide arrays followed by their activation with trifluoroethanesulfonyl chloride (tresyl chloride). The activated surface in the subsequent reaction is used to covalently immobilize oligonucleotides in regioselective fashion to create an oligonucleotide array. The surface bound tresyl sulfonate esters allow the immobilization of oligonucleotides specifically via their 3'- or 5'-end having mercaptohexyl- or aminohexyl functionalities. The constructed oligonucleotide arrays were successfully used to analyze oligonucleotides by hybridization technique.

Hydroxyl Radical↗
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N-(3-Trifluoroethanesulfonyloxypropyl)anthraquinone- 2-carboxamide: a new heterobifunctional reagent for immobilization of biomolecules on a variety of polymer surfaces.

A new heterobifunctional reagent, N-(3-trifluoroethanesulfonyloxypropyl)anthraquinone-2-carboxamide (NTPAC) has been developed, useful for making bioconjugates and immobilization of biomolecules, viz., oligonucleotides, peptides, proteins, etc., on a variety of carbon-containing solid surfaces. Its trifluoroethanesulfonate ester group reacts with aminoalkyl or mercaptoalkyl functions present in biomolecules, and the anthraquinone structure reacts with a variety of carbon-containing polymers under ultraviolet irradiation (365 nm). The reagent has been used in two ways. First, the reagent, NTPAC, was first brought in contact with the above said supports and exposed to long wavelength ultraviolet light (365 nm), thereby generating active trifluoroethanesulfonate ester functions on the support, which subsequently react with appropriate mercaptoalkyl- or aminoalkyl-containing biomolecules to fix them on the supports. In another route, the proposed reagent was allowed to react first with proteins or 5'-aminoalkyl- or mercaptoalkyl-modified oligonucleotides to form the appropriate biomolecule-anthraquinone conjugate, which was then brought in contact with a variety of carbon-containing polymers, viz., modified controlled pore glass (CPG), modified glass microslides, cross-linked polystyrene, nylon, cross-linked polysaccharides, polypropylene (PP), polyethylene (PE), etc., and exposed to long wavelength ultraviolet light (365 nm), resulting in immobilization of the conjugates on the support. Both of the routes work satisfactorily and we could successfully immobilize a number of enzymes and modified oligonucleotides on a variety of supports.

Alkanesulfonates↗
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Oligonucleotide microarrays: immobilization of phosphorylated oligonucleotides on epoxylated surface.

A facile and efficient method for direct immobilization of phosphorylated oligonucleotides on an epoxy-activated glass surface is described. The new immobilization strategy has been analyzed for its performance in DNA microarray under both microwave and thermal conditions. It reflects high immobilization efficiency ( approximately 23%), and signal-to-noise ratio ( approximately 98) and resulted in high hybridization efficiency ( approximately 36%) in comparison to those obtained with standard methods, viz., NTMTA ( approximately 9.76%) and epoxide-amine ( approximately 9.82%). The probes immobilized through the new strategy were found to be heat-stable, since the performance of microarray decreased by only approximately 7% after subjecting it to 20 PCR-like heat cycles, suggesting that the chemistry could be used in integrated PCR/microarray devices. The immobilization of probes following the proposed chemistry resulted in spots of superior quality in terms of spot morphology, spot homogeneity, and signal reproducibility. The constructed microarrays have been successfully used for the discrimination of nucleotide mismatches. In conclusion, these features make the new immobilization strategy ideal for facile, efficient, and cost-effective manufacturing of DNA microarrays.

Epoxy Compounds↗
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N-(Iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED): a new heterobifunctional reagent for the preparation of biochips.

Design and synthesis of a new heterobifunctional reagent, N-(iodoacetyl)-N'-(anthraquinon-2-oyl)-ethylenediamine (IAED), have been described for the preparation of oligonucleotide-based biochips. The performance of the featured reagent is probed by the immobilization of thiolated and thiophosphorylated oligonucleotides on modified glass microslides via two routes (routes A and B). The immobilization procedure was accelerated by performing a chemical reaction between thiolated oligomers and the iodoacetyl moiety of the reagent under microwaves (MW), where it is completed in just 10 min. The quality of the constructed oligonucleotide microarrays was tested by performing a hybridization assay with a complementary target and subsequently used for the detection of base mismatches. The immobilized probes were found to be thermally stable.

Microchip Analytical Procedures↗
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Relation of HTLV-4 to simian and human immunodeficiency-associated viruses.

Human immunodeficiency virus type 1 (HIV-1) is the aetiologic agent of AIDS (acquired immune deficiency syndrome) in most countries and probably originated in Central Africa like the AIDS epidemic itself. Evidence for a second major group of human immunodeficiency-associated retroviruses came from a report that West African human populations like wild-caught African green monkeys had serum antibodies that reacted more strongly with a simian immunodeficiency virus (STLV-3Mac) (ref.6) than with HIV-1. Novel T-lymphotropic retroviruses were reported to have been isolated from healthy Senegalese West Africans (HTLV-4) (ref. 4) and from African green monkeys (STLV-3AGM) (ref. 7), and a different retrovirus (HIV-2) was identified in other West African AIDS patients. Genomic analysis of HIV-2 clearly distinguished it from STLV-3 (ref. 9), but restriction enzyme site-mapping of three different HTLV-4 isolates and six different STLV-3AGM isolates showed them to be essentially indistinguishable. In this report we clone, restriction map, and partially sequence three isolates of HTLV-4 (PK82, PK289, PK190) (ref. 4). We find that these viruses differ in nucleotide sequence from each other and from three isolates of STLV-3AGM (K78, K6W, K1) (ref. 7) by 1% or less. We also report the isolation of a T-lymphotropic retrovirus from the peripheral blood of a healthy Senegalese woman which hybridizes preferentially to HIV-2 specific DNA probes. We conclude that HTLV-4 (ref. 4) and STLV-3AGM (ref. 7) are not independent virus isolates and that HIV-2 is present in Senegal as it is in other West African countries.

Base Sequence↗
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Identifying at risk infants following neonatal extracorporeal membrane oxygenation.

OBJECTIVE: To identify infants at risk of death and abnormal neurodevelopmental outcome following extracorporeal membrane oxygenation (ECMO) in the neonatal period. METHODS: The medical records of 82 neonates treated with ECMO were reviewed to evaluate risk of death. All survivors were followed by neurologic examinations and tested using the Bayley Scales of Infant Development or McCarthy Scale of Children's Abilities, and risk for abnormal neurodevelopmental outcome was assessed. RESULTS: The overall survival was 91% (75 of 82). The mean gestational age and birth weight of nonsurvivors were lower than those of survivors (37 +/- 1 weeks vs 40 +/- 0 weeks; 2734 +/- 230 vs 3325 +/- 69 gm, p < 0.05). Infants who were lost to follow-up (16%) did not differ from those with follow-up in demographic variables or clinical indicators of illness severity. Thirty-five of 63 infants (56%) with follow-up had normal neurodevelopmental outcome. Risk of abnormal outcome was higher in infants requiring assisted ventilation for > or = 15 days (relative risk [RR] 5.5; 95% confidence interval [CI] 2.0 to 14.8), supplemental oxygenation for > or = 22 days (RR 3.1; 95% CI 1.3 to 7.6), and black race (RR 8.9; 95% CI 1.3 to 62.9). None of the neuroimaging studies accurately predicted the neurodevelopmental outcome of these infants. CONCLUSION: We conclude that ECMO in critically ill infants is associated with good survival. The need for prolonged respiratory support may help in identifying infants at risk for abnormal neurodevelopmental outcome.

Cause of Death↗
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Variability of milk fat globule membrane protein gene between cattle and riverine buffalo.

A study on butyrophilin (BTN) gene was conducted to detect variability at nucleotide level between cattle and buffalo. Hae III PCR-RFLP was carried out in crossbred cattle and it revealed polymorphism at this locus. Three genotypes namely, AA, BB and AB and two alleles were observed with frequencies 0.78, 0.17, 0.04 and 0.87, 0.13, respectively. The sequences of different cattle, buffalo and sheep breeds have been reported in the EMBL gene bank with accession numbers: AY491468 to AY491475. The nucleotides, which have been substituted from allele A to B, were found to be C to G (71st nucleotide), C to T (86th nucleotide), A to T (217th nucleotide), G to A (258th nucleotide), A to C (371st nucleotide) and C to T (377th nucleotide). The nucleotide substitution at 71st, 86th and 377th position of the fragment were expected to be a silent mutation where as nucleotide changes at 217th, 258th and 371st positions were expected to be substituted by lysine with arginine, valine with isoleucine and leucine with proline in allele B. The differences of nucleotides and amino acids between cattle, buffalo and sheep breeds have been revealed and on the basis of nucleotide as well as protein variability the phylogenetic diagram have been developed indicating closeness between cattle and buffalo.

Animals↗
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Microwave assisted high yielding preparation of N-protected 2'-deoxyribonucleosides useful for oligonucleotide synthesis.

A rapid and high yielding method for the synthesis of precursors of synthons for DNA synthesis, N-protected 2'-deoxyribonucleosides is described, which occur under mild conditions using microwave irradiation. The desired material, N-protected nucleosides, was obtained in 93-96% yield in few minutes. The final products were then characterized by 1H-NMR and MALDI-TOF and compared with the standard samples. The method is amenable to small to moderate scale of synthesis.

Deoxycytidine↗
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Brown larva: an allele of the green larva mutation in the malaria mosquito, Anopheles stephensi.

We isolated a new larval color mutant, brown larva (b), from the Bangalore, India strain of Anopheles stephensi Liston. The gene b is an autosomal recessive with uniform expression and complete penetrance. We conducted extensive crosses to establish allelism between brown larva (b) and green larva (g) reported previously in An. stephensi from our laboratory. The wild-type is dominant to green larva, which, in turn, is dominant to brown larva. These larval color mutants belong to an allelic series.

Alleles↗
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Gene activation and protein expression following ischaemic stroke: strategies towards neuroprotection.

Current understanding of the patho-physiological events that follow acute ischaemic stroke suggests that treatment regimens could be improved by manipulation of gene transcription and protein activation, especially in the penumbra region adjacent to the infarct. An immediate reduction in excitotoxicity in response to hypoxia, as well as the subsequent inflammatory response, and beneficial control of reperfusion via collateral revascularization near the ischaemic border, together with greater control over apoptotic cell death, could improve neuronal survival and ultimately patient recovery. Highly significant differences in gene activation between animal models for stroke by middle cerebral artery occlusion, and stroke in patients, may explain why current treatment strategies based on animal models of stroke often fail. We have highlighted the complexities of cellular regulation and demonstrated a requirement for detailed studies examining cell specific protective mechanisms after stroke in humans.

Animals↗
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Urinary retention and the role of indwelling catheterization following total knee arthroplasty.

INTRODUCTION: We aimed to investigate the rate of urinary retention after knee arthroplasty, the various factors involved in predicting those at risk for retention and to assess the impact of retention and catheterization on joint sepsis. MATERIALS AND METHODS: A retrospective review was conducted of all available case notes of patients undergoing total knee arthroplasty in a consecutive 2-year period (2000-2002). Adequate data was available for 142 patients. RESULTS: 142 patients underwent total knee arthroplasty. 19 patients were catheterized preoperatively for monitoring urine output. 123 patients were not catheterized. Urinary retention occurred in 19.7% (28/142). The mean day of catheterization for retention was 0.66. The mean duration of catheterization in patients developing retention was 3.58 days and was 3 days in the patients catheterized pre- or perioperatively. Deep joint sepsis occurred in 2.1% (3/142)--only one had been catheterized and that was preoperatively. No case of infection had urinary retention or had a symptomatic urinary tract infection. The only factors predicting those at significant risk of retention following knee arthroplasty was a past medical history of urinary retention (p = 0.049) and postoperative morphine requirement (p = 0.035). No patients required urological surgical intervention at mean follow up of 1.97 years. CONCLUSIONS: This study supports the use of indwelling urinary catheterization for patients developing urinary retention after total knee arthroplasty.

Aged↗
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Samarium-153 ethylenediamine tetramethylene phosphonate therapy for bone pain palliation in skeletal metastases.

BACKGROUND: Systemic therapy with radionuclides may be used for the treatment of patients with painful skeletal metastases owing to its efficacy, low cost and low toxicity. Imported radionuclides for pain palliation, like Strontium-89 are expensive; particularly for developing countries. In the Indian scenario, Samarium-153 (Sm-153) is produced in our own reactors and as a result, it is readily available and economical. AIM: We undertook this study to determine the efficacy and toxicity of single-dose Sm-153 ethylenediamine tetramethylene phosphonate as a palliative treatment for painful skeletal metastases. MATERIALS AND METHODS: Eightysix patients with painful skeletal metastases from various primaries, were treated with Sm-153 EDTMP at a dose of 37 MBq/kg. The effects were evaluated according to change in visual analogue pain score, analgesic consumption, Karnofsky performance score, mobility score and blood count tests, conducted regularly for 16 weeks. STATISTICS: Repeated measures analysis. RESULTS: The overall response rates were 73%, while complete response was seen in 12.4%. Reduction in analgesic consumption with improvement in Karnofsky performance score and mobility score, was seen in all responders. Response rates were 80.3 and 80.5% in breast and prostate cancer, respectively. One case, each of Wilms tumor, ovarian cancer, germ cell tumor testis, multiple myeloma, primitive neuroectodermal tumor and oesophageal cancer, did not respond to therapy. No serious side-effects were noted, except for fall in white blood cell, platelet and haemoglobin counts, which gradually returned to normal levels by six-eight weeks. CONCLUSION: Sm-153 EDTMP provided effective palliation in 73% patients with painful bone metastases: the major toxicity was temporary myelosuppression.

Adolescent↗
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Balloon dilatation in achalasia cardia.

Treatment of achalasia cardia by pneumatic dilatation is an established method. However, data on results of pneumatic dilatation in Indian patients with achalasia cardia are scarce. We report our experience with treatment of achalasia cardia by pneumatic dilatation in 38 patients. The diagnosis of achalasia cardia was based on clinical examination, barium swallow and upper gastrointestinal (GI) endoscopy. Twenty nine (76.3%) patients improved with first dilatation, 8 (21%) patients had to undergo second dilatation and 1 (2.6%) required a third. Most of the patients remained asymptomatic during a follow-up of 6 months to 6 years. Immediate and late complications occurred in 5 (13%) and 2 (5.2%) patients, respectively. Thus, pneumatic dilatation is a simple, safe and effective method for treating Indian patients with achalasia cardia.

Adolescent↗
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Symphysis fundal height curve--a simple method for foetal growth assessment.

In this prospective study, symphisis fundal height (SFH) was measured in centimeters at different weeks of gestation from 20th week onwards in 100 healthy women with uncomplicated pregnancies. A curve was plotted based on the mean SFH measurements with standard deviation. Readings were also arranged on the basis of 10th, 50th and 90th percentiles. Percentile curve was similar to the curve based on mean with standard deviation. The rate of growth was 1 cm per week between 20-32 weeks. Thereafter, there was a slight fall in the rate of growth. SFH measurement is a simple method of foetal growth assessment which can be utilized even by paramedical workers to screen for small for gestational age babies. It is better to have a standard curve derived from the population as there is regional variation.

Adult↗
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Mycoplasma pneumoniae and community-acquired pneumonia.

BACKGROUND: Community-acquired pneumonia is an important cause of mortality and hospitalization in all age groups. In temperate climates, Mycoplasma pneumoniae is a common respiratory pathogen causing pneumonia. Information on human Mycoplasma infection in India is scarce. METHODS: We aimed to determine the frequency of Mycoplasma pneumoniae infection among patients with community-acquired pneumonia in a prospective cross-sectional study. The assessment included clinical and radiological evaluation followed by microbiological evaluation for the specific pathogen. Microbiological investigations included aerobic and anaerobic blood culture, anti-Mycoplasma IgM antibody detection by gelatin particle agglutination test and ELISA, culture of respiratory tract secretions for Mycoplasma pneumoniae and other organisms, and detection of specific Mycoplasma pneumoniae antigen by indirect immunofluorescence. RESULTS: Sixty-two patients (42 men and 20 women; mean age 41.7 years) with community-acquired pneumonia were investigated prospectively. They included 42 immunocompetent and 20 immunocompromised patients. Six patients had definitive evidence of Mycoplasma pneumoniae infection and an additional 16 patients had a probable diagnosis. In all, 22 (35.5%) patients with pneumonia had Mycoplasma pneumoniae infection. Of these, 12 patients belonged to the immunocompromised group and 10 to the immunocompetent group. Patients with Mycoplasma pneumoniae infection also had secondary bacterial infection as evidenced by organisms isolated from blood in 50% and from respiratory tract secretions in 68%. CONCLUSION: Community-acquired pneumonia has a polymicrobial aetiology, of which the prevalence of Mycoplasma pneumoniae is 35%. The study has two implications: (i) Mycoplasma pneumoniae infection is frequently associated with secondary bacterial infection; and (ii) initial empirical antibiotic therapy for community-acquired pneumonia in India must include antibiotics with activity against Mycoplasma pneumoniae.

Adolescent↗