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Biomedical subjects

D Subramanian

Publications and source records attributed to D Subramanian.

18 recordsLinked to original sources
PubMed2003

Rapid spread of penicillin-resistant Streptococcus pneumoniae among high-risk hospital inpatients and the role of molecular typing in outbreak confirmation.

This study describes an outbreak of penicillin-resistant Streptococcus pneumoniae among patients on an ear, nose and throat (ENT) ward. Over a period of 10 days, S. pneumoniae [penicillin minimum inhibitory concentration (MIC) 0.75] was isolated from a total of seven patients with symptoms and signs of lower respiratory tract infection. Standard source isolation was implemented and the ward was closed to admissions and discharges. Screening of nasopharyngeal secretions was undertaken on all patients and staff contacts. Three patients (of eight possible contacts) and none of the staff (47 screened) were identified as colonized with the same strain. Nasal mupirocin and oral rifampicin were administered to carriers. Confirmation of the outbreak was achieved rapidly using a contemporary molecular typing method (pulsed-field gel electrophoresis) and timely introduction of infection control measures successfully contained the outbreak. Implications for pneumococcal vaccination are discussed.

Carrier State↗
PubMed2001

Mechanism of topology simplification by type II DNA topoisomerases.

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

Computer Simulation↗
PubMed2000

Analysis of the binding of p53 to DNAs containing mismatched and bulged bases.

The tumor suppressor protein p53 modulates cellular response to DNA damage by a variety of mechanisms that may include direct recognition of some forms of primary DNA damage. Linear 49-base pair duplex DNAs were constructed containing all possible single-base mismatches as well as a 3-cytosine bulge. Filter binding and gel retardation assays revealed that the affinity of p53 for a number of these lesions was equal to or greater than that of the human mismatch repair complex, hMSH2-hMSH6, under the same binding conditions. However, other mismatches including G/T, which is bound strongly by hMSH2-hMSH6, were poorly recognized by p53. The general order of affinity of p53 was greatest for a 3-cytosine bulge followed by A/G and C/C mismatches, then C/T and G/T mismatches, and finally all the other mismatches.

Base Pair Mismatch↗
PubMed2000

Statistical methods for the objective design of screening procedures for macromolecular crystallization.

The crystallization of a new macromolecule is still very much a trial-and-error process. As is well known, it requires the search of a large parameter space of experimental settings to find the relatively few idiosyncratic conditions that lead to diffraction-quality crystals. Crystallographers have developed a variety of screens to help identify initial crystallization conditions, including those based on systematic grids, incomplete factorial and sparse-matrix approaches. These are somewhat subjectively formulated based on accumulated data from past crystallization experiments. Ideally, one would prefer as objective a procedure as possible; however, that requires objective methods that incorporate a broad source of crystallization data. The Biological Macromolecular Crystallization Database (BMCD), a repository of all published crystallization conditions, is an obvious source of this data. This database has been augmented with a hierarchical classification of the macromolecules contained in the BMCD as well as extensive data on the additives used with them. A statistical analysis of the augmented BMCD shows the existence of significant correlations between families of macromolecules and the experimental conditions under which they crystallize. This in turn leads to a Bayesian technique for determining the probability of success of a set of experimental conditions based on the data in the BMCD as well as facts about a macromolecule known prior to crystallization. This has been incorporated into software that enables users to rank experimental conditions for new macromolecules generated by a dense partial factorial design. Finally, an additional advantage of the software described here is that it also facilitates the accumulation of the data required for improving the accuracy of estimation of the probabilities of success - knowledge of the conditions which lead to failure of crystallization.

Crystallization↗
PubMed1999

hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA.

Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch. Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular. We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches.

Adenosine Triphosphatases↗
PubMed1998

Ultraviolet-induced DNA damage stimulates topoisomerase I-DNA complex formation in vivo: possible relationship with DNA repair.

An antibody-based method was used to examine genomic DNA cleavage by endogenous topoisomerases in living cells. The method quantifies cleavable (covalent) complex formation in vivo after exposure to topoisomerase poisons, as reported previously (D. Subramanian et al., Cancer Res., 55: 2097-2103, 1995). Unexpectedly, exposing cells to UVB irradiation stimulated endogenous topoisomerase I-DNA covalent complex formation by as much as 8-fold, even in the absence of drugs that stabilize the cleavable complex. Covalent complexes are not a result of nonspecific UV protein-DNA cross-linking; rather, they result from the enzymatic activity of topoisomerase I on genomic DNA. Because the action of topoisomerase II on genomic DNA was not affected by UVB exposure, the observation appears to be specific for type I. Topoisomerase I is rapidly mobilized onto the genome (within 12 min after UVB exposure); however, topoisomerase I polypeptide levels did not show a corresponding increase, suggesting that preexisting enzyme is being recruited to sites of DNA damage. Complexes persist up to 5 h post-UV exposure (concurrent with the period of active DNA repair), and their formation is independent of S phase. These findings can be partially explained by the fact that in vitro topoisomerase I activity on UV-damaged DNA tends to favor formation of cleavage complexes; thus, a higher yield of covalent complexes are detected at or near cyclopyrimidine dimer lesions. Because repair-deficient cells are additionally compromised in their ability to recruit topoisomerase I, a direct role for the enzyme in DNA excision repair process in vivo is proposed that may be related to the activity of the xeroderma pigmentosum complementation group D helicase. Finally, these results collectively demonstrate that topoisomerase I is a repair-proficient topoisomerase in vivo.

DNA↗
PubMed1997

The involvement of topoisomerase I in the induction of DNA-protein crosslinks and DNA single-strand breaks in cells of ultraviolet-irradiated human and frog cell lines.

Exposure of GM 4390 human skin fibroblasts and ICR 2A frog cells to 10 kJ m(-2) of ultraviolet B (UVB) radiation resulted in the formation of DNA-protein crosslinks (DPCs) and DNA single-strand breaks (SSBs). However, upon incubation, there were rapid increases in the yields of both DPCs and SSBs. An enhancement in these DNA alterations was detected within 12 min after irradiation and their levels continued to rise by 5-8-fold within 15 h after exposure to UV radiation. Using an antibody-based assay that measures covalent complex formation between topoisomerase (topo) I and genomic DNA, it was found that topo I is one of the proteins involved in these DPCs induced by UV radiation. The levels and rate of increase of topo I-DNA covalent complexes were similar to the UV-radiation-dependent formation of DPCs and SSBs. A UV-radiation-sensitive mutant frog cell line, DRP 153, was also examined and was found to be deficient in this induction of DPCs and SSBs by UV radiation. When these cells were transfected with the human SUVCC3 gene, the resulting transformant displayed kinetics for the induction of DPCs and SSBs similar to the human and parental frog cells. However, human topo I was not defected in the transformed frog cells, indicating that SUVCC3 does not encode topo I. It is likely that SUVCC3 encodes an associated enzymatic activity which permits normal stimulation of topo I-DNA covalent complexes in UV-irradiated cells.

Animals↗
PubMed1995

Analysis of topoisomerase I/DNA complexes in patients administered topotecan.

As part of a Phase II clinical trial of topotecan, DNA breakage in vivo was measured by detecting covalent topoisomerase/DNA intermediates in peripheral blood. The ICE (in vivo complex of enzyme) bioassay was used to assess topotecan activity in the peripheral blood of patients before, during, and after infusion therapy. The results can be summarized as: (a) ICE bioassay is a specific, antibody-based assay for topoisomerase I-mediated DNA damage. Topoisomerase I/DNA complex formation can be monitored unambiguously in the absence of topotecan to establish a basal level of endogenous enzyme action on DNA; (b) infusion of topotecan significantly stimulated formation of covalent enzyme/DNA complexes. Complexes were detected within 5 min postinfusion and increased over the course of a 30-min treatment; (c) after termination of infusion, complex formation decreased by 3-4-fold within 30 min, showing that cleavage complexes quickly reseal after drug withdrawal; and (d) formation of complexes varied widely between patients. The ICE bioassay can evaluate the effects of topoisomerase I inhibitors on target tissues; thus, it may valuable in predicting response to these drugs.

Antineoplastic Agents↗
PubMed1995

Liposomal encapsulation increases the activity of the topoisomerase I inhibitor topotecan.

Topotecan, a topoisomerase I poison and water-soluble derivative of camptothecin, has shown promise in treating solid tumors; however, the drug is unstable under physiological conditions and converts to an inactive form within 30 minutes. Encapsulating topotecan in liposomes (LIP-TPT) minimizes inactivation. The efficacy of LIP-TPT was examined with a novel in vivo bioassay called ICE for In vivo Complexes of Enzyme. This bioassay uses antibodies to probe DNA for the presence of topoisomerase I covalent complexes and thereby allows direct quantification of topoisomerase I driven DNA adducts in living cells. We report that LIP-TPT was three- to fourfold more effective than free TPT in stabilizing covalent topoisomerase I-DNA intermediates inside tumor cells. These findings reveal that liposomal wrapping permitted effective delivery of camptothecin derivatives to active enzyme in the nucleus of the cell.

Antineoplastic Agents↗
PubMed1994

Association between the p170 form of human topoisomerase II and progeny viral DNA in cells infected with herpes simplex virus type 1.

Endogenous host topoisomerase II acts upon herpes simplex virus type 1 (HSV-1) DNA in infected cells (S.N. Ebert, S.S. Shtrom, and M.T. Muller, J. Virol. 56:4059-4066, 1990), and cleavage is directed exclusively at progeny viral DNA while parental DNA is resistant. To evaluate the possibility that HSV-1 induces topoisomerase II activity which could account for the preferential cleavage of progeny viral DNA, we assessed topoisomerase II cleavage activity on cellular and viral DNA substrates before and after the initiation of viral DNA replication. We show that cleavage of a host gene in mock-infected cells was similar to that observed in HSV-1-infected cells, regardless of whether viral DNA replication had occurred. In addition, quantitative measurements revealed comparable amounts of topoisomerase II activity in infected and mock-infected cells; thus, HSV-1 neither induces nor encodes its own type II topoisomerase and cleavages in vivo are due to a preexisting host topoisomerase. Human cells contain two isozymes of topoisomerase II (p170 and p180), encoded by separate genes. Through the use of isozyme-specific antibodies, we demonstrate that only p170 was found to be cross-linked to HSV-1 DNA even though both forms were present at nearly constant levels in HSV-1-infected cells. Immunofluorescence revealed that by 6 h postinfection, p170 becomes redistributed and localized to sites of active viral DNA synthesis. The data suggest that p170 gains preferential access to replicated viral DNA molecules, which explains why topoisomerase II activity is concentrated on progeny DNA.

Amino Acid Sequence↗
PubMed1994

Induction of rules for biological macromolecule crystallization.

X-ray crystallography is the method of choice for determining the 3-D structure of large macromolecules at a high enough resolution. The rate limiting step in structure determination is the crystallization itself. It takes anywhere between a few weeks to several years to obtain macromolecular crystals that yield good diffraction patterns. The theory of forces that promote and maintain crystal growth is preliminary, and crystallographers systematically search a large parameter space of experimental settings to grow good crystals. There is a wealth of experimental data on crystal growth most of which is in paper laboratory notebooks. Some of the data has been gathered in electronic form, e.g., the Biological Macromolecular Crystallization Database (BMCD) which is a repository of successful experimental conditions for growing over 800 different macromolecules (Gilliland 1987). Crystallographers are in need of computational tools to gather and analyze past data to design new crystal growth trails. We are building the Crystallographer's Assistant (CA) to help crystallographers record and maintain experimental context in electronic form, offer suggestions on experimental conditions that are likely to be successful, and provide explanations for failed experiments. As an initial step in this project, we have applied RL, an inductive learning program, to the BMCD. In this paper we report initial experiments and findings in applying RL to the BMCD. From the point of view of crystallography, we have discovered possibly significant new empirical relationships in crystal growth. From the point of view of machine learning, our work suggests refinements of existing methods for incorporating detailed domain knowledge into inductive analysis techniques.

Animals↗
PubMed1994

Diagnosing obstructive lung disease. Why is differentiating COPD from asthma important?

Chronic obstructive pulmonary disease (COPD) and asthma are the main causes of obstructive lung disease, and mortality rates of both diseases are on the increase. Smoking is the major risk factor for COPD. Airway hyperreactivity and reversibility of airflow obstruction, once considered hallmarks of asthma, may also be seen in COPD. Asthma is increasingly being recognized as a chronic inflammatory airway disease. COPD and asthma can be diagnosed through careful history taking, physical examination, spirometry, and other ancillary tests. More widespread use of peak expiratory flow rates should aid in diagnosis and monitoring.

Algorithms↗
PubMed1993

Primary pulmonary lymphoma. Diagnosis by immunoglobulin gene rearrangement study using a novel polymerase chain reaction technique.

A 63-yr-old-woman presented with pulmonary infiltrates and macroglobinemia. Open lung biopsy revealed an interstitial plasma cell infiltrate. Immunocytochemical staining to determine clonality of the infiltrate was negative for both kappa and lambda light chains and therefore was not helpful. Polymerase chain reaction (PCR) amplification of the CDR-III region of the immunoglobulin heavy chain gene revealed that the plasma cells were either monoclonal or biclonal, thus suggesting the diagnosis of a plasmacytoid pulmonary lymphoma. The advantages and limitations of the PCR technique over the traditional method of detecting gene rearrangements, i.e., restriction-digestion of DNA and Southern blotting, are discussed. To our knowledge this patient represents the first reported application of the PCR technique for detecting gene rearrangements to determine the clonality of a lymphoid infiltrate in the lung. This technique can also be applied to determine the clonality of lymphocytes obtained by bronchoalveolar lavage or needle biopsy, or from a pleural effusion.

Biopsy↗
PubMed1992

Imported malaria in pregnancy: report of four cases and review of management.

Malaria is a common infection worldwide. Increased travel by pregnant women makes it likely that physicians in the United States will see cases of malaria in this population. We observed four cases of malaria during pregnancy over an 8-month period at a general hospital in the United States. These cases illustrate the association between pregnancy and severe malaria in the mother and congenital infection in the newborn. We also noted delays in diagnosis because malaria was mistaken for other common illnesses. Therapy was complicated by concerns about the safety of antimalarial agents for the fetus and newborn as well as drug resistance. While chloroquine is safe for use in pregnancy, drug resistance is now common, especially when the etiologic organism is Plasmodium falciparum. There are concerns about the safety of administering other antimalarial agents during pregnancy (e.g., mefloquine). Concerns about the safety and availability of these agents limit options for prophylaxis.

Adult↗
PubMed1992

Case report: severe symptomatic hyponatremia associated with lisinopril therapy.

A 63-year-old white female who was being treated for hypertension with lisinopril presented with seizures, altered mental status, and a serum sodium of 101 mEq/L. Serum sodium prior to initiation of lisinopril therapy was 137 mEq/L. The hyponatremia was corrected and did not recur after lisinopril was stopped. The hyponatremia may have been a result of polydipsia and inappropriate antidiuresis secondary to ACE-inhibitor therapy.

Angiotensin-Converting Enzyme Inhibitors↗