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D M Dwyer

Publications and source records attributed to D M Dwyer.

At least 55 records · Page 3Linked to original sources
PubMed1995

Identification of differentially expressed Leishmania donovani genes using arbitrarily primed polymerase chain reactions.

Arbitrarily primed polymerase chain reactions (AP-PCR) were used to amplify polymorphic DNA fragments from the genomes of a variety of geographic isolates of Leishmania donovani (Ld). From the latter, five polymorphic DNA fragments were cloned and sequence analysis identified 15 unique clones. Northern blot analysis showed that 13 of the 15 clones hybridized to transcribed RNAs isolated from Ld. Eight of these 13 AP-PCR clones specifically hybridized to Ld RNAs that were differentially expressed in promastigote and 'amastigote' cells. Comparative Northern analysis of four differentially expressed AP-PCR clones indicated that two clones, LdS-14-14 and LdI-9-7, were expressed in Ld and several other Leishmania species. However, RNAs corresponding to two other AP-PCR clones, LdE-6-1 and LdI-9-5, were detected only in members of the Ld complex, and not in L. major (Lm) or L. tropica (Lt). Comparative Southern blot analysis of the LdS-14-14 locus revealed numerous restriction-fragment length polymorphisms (RFLP) distinguishing Lm and Lt from the Ld isolates and L. infantum. However, the LdS-14-14 loci were mapped to similar-sized chromosomes observed among all Old World Leishmania species tested, indicating that localized nucleotide divergence, not chromosomal rearrangement, was responsible for altered Southern blot patterns. These results demonstrate that AP-PCR is a very useful method for identifying expressed gene sequences in organisms of relatively low-complexity genomes. Interestingly, the majority of these sequences identified in this study correspond to differentially expressed genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Relapsing invasive group B streptococcal infection in adults.

OBJECTIVE: To study recurrent group B streptococcal infection in adults. DESIGN: Patients with more than one reported group B streptococcal infection were identified through active surveillance for this infection. Sterile-site group B streptococcal isolates were evaluated for serotype and molecular subtyping using restriction endonuclease analysis of chromosomal DNA (REAC). SETTING: All acute-care hospitals in Maryland. PATIENTS: Nonpregnant residents of Maryland 18 years of age or older. RESULTS: 22 adults had at least two group B streptococcal episodes that were separated by 2 to 95 weeks (mean, 24 weeks). Of 395 patients with invasive group B streptococcal infection who survived the first episode and were followed for at least 1 year, 17 (4.3% [95% CI, 2.6% to 6.9%]) had more than one episode. Several patients were found to have endocarditis or osteomyelitis during the second episode. Group B streptococcal isolates from both episodes were obtained from 18 of 22 patients. Of the 18 isolate pairs, 13 (72% [CI, 46% to 90%]) had identical REAC patterns; the probability that at least 13 matches would be found by chance alone was less than 0.000001. Among patients with recurrent infection caused by the same strain, the interval between episodes was shorter (mean, 14 weeks) than that among patients with recurrent infection caused by another strain (mean, 43 weeks; P = 0.05). CONCLUSIONS: Recurrent group B streptococcal infection is common among adults and in most cases appears to be caused by relapse. The optimal management of adults with a first episode of group B streptococcal infection needs to be further defined to minimize the likelihood of recurrent disease.

Adolescent↗
PubMed1995

Crithidia luciliae: effect of purine starvation on S-adenosyl-L-methionine uptake and protein methylation.

The utilization of S-adenosyl-L-[methyl-3H]methionine ([3H-methyl]AdoMet) by Crithidia luciliae was assessed under nutrient-replete and purine-starvation conditions. Uptake experiments with intact cells demonstrated that the radiolabel from this molecule was accumulated by purine-starved organisms at a rate approximately 10-fold greater than that observed in those cultivated in nutrient-replete medium. Purine-starved cells also incorporated the radiolabel into trichloroacetic acid insoluble material at an approximately 10-fold faster rate than nutrient-replete cells. No differences, however, were observed in the intracellular levels of AdoMet and its metabolites between organisms cultivated under the two conditions. Results of comparative labeling studies with [3H-methyl]AdoMet, S-adenosyl-L-[carboxyl-14C]methionine, L-[methyl-3H]methionine and L-[35S]methionine in the presence and absence of cycloheximide demonstrated that the incorporation of label from [3H-methyl]AdoMet was due to transmethylation and was independent of protein synthesis. Further, approximately 15 methylated protein bands were identified by SDS-PAGE analysis. Lysates from both purine-starved and nutrient-replete organisms demonstrated similar levels of activity of three protein methyltransferases (PMI, II, III). The differences observed in [3H-methyl]AdoMet utilization between purine-starved and nutrient-replete C. luciliae may reflect the enhanced purine transport capacity which results from purine starvation.

Adenosine↗
PubMed1995

Identification of intra- and interspecific Leishmania genetic polymorphisms by arbitrary primed polymerase chain reactions and use of polymorphic DNA to identify differentially regulated genes.

Arbitrary primed polymerase chain reactions (AP-PCR) were used to amplify different polymorphic genomic DNA fragments from various Old World Leishmania species. Using four 10-mer AP primers, geographic isolates of L. donovani and various Old World species of Leishmania could be readily distinguished from one another by the pattern of amplified DNA products. Our studies confirmed two important characteristics of AP-PCR: its abilities to amplify a consistent pattern of DNA fragments from the genomes of different isolates of a single species and to identify genetic polymorphisms between the species isolates. We selected three polymorphic DNA fragments that differentiate L. donovani geographic isolates for further analysis. Sequence analysis of the clones derived from these three polymorphic fragments revealed eight unique sequences. Six of eight unique clones hybridized to distinct RNAs upon Northern-blot analysis. Three of these six clones hybridized to RNAs expressed differentially in in vitro grown L. donovani pro- and "amastigotes." One of the differentially expressed clones, LdE-6-1, exhibited restriction length polymorphisms that distinguished L. donovani from L. tropica and L. major. Comparative Northern blotting revealed that LdE-6-1 was differentially expressed in some members of the L. donovani species complex but not in L. major or L. tropica. These results demonstrate that AP-PCR can be used to generate products reflecting particular genes in organisms with low-complexity genomes.

Animals↗
PubMed1995

Isolation and characterization of the gene encoding the surface membrane 3'-nucleotidase/nuclease of Leishmania donovani.

Leishmania donovani and related trypanosomatid protozoa possess an externally oriented surface membrane enzyme capable of hydrolyzing both 3'-nucleotides and nucleic acids. By virtue of these activities, this 3'-nucleotidase/nuclease (3'-NT/Nu), previously shown to be analogous to fungal and plant class-I single-strand-specific nucleases, is thought to play a critical role in the salvage of purines, essential for the survival of these organisms. The 43-kDa 3'-NT/Nu was purified from L. donovani promastigotes and trypsin treated. Four of the released tryptic peptide fragments yielded amino-acid sequence information (Pept-1 to Pept-4) which provided the basis for the preparation of oligonucleotide primers used for PCR amplification of an approx. 300-bp DNA fragment. This fragment was cloned, sequenced and used to probe a genomic L. donovani cosmid library. Nucleotide sequence analysis of a 4.5-kb SmaI fragment, isolated from a cosmid clone, revealed an open reading frame (ORF) of 1434 nt encoding a 477-amino-acid protein. Pept-1 to Pept-4 were mapped onto the ORF-deduced protein sequence. Peptides corresponding to Pept-1 to Pept-4 were synthesized and used to immunize rabbits. The resulting anti-peptide antibodies recognized the 43-kDa protein on Western blots and immunoprecipitated the native 3'-nucleotidase activity from L. donovani membrane extracts. Further, the ORF-deduced protein shared significant sequence identity with the S1 and P1 fungal nucleases of Aspergillus oryzae and Penicillium citrinum, respectively. Cumulatively, these results demonstrated that the ORF corresponded to a gene for the L. donovani 3'-nucleotidase/nuclease. In Northern blots a nucleotide probe specific for the 3'-NT/Nu gene hybridized to a single 2.5-kb messenger RNA. Results of Southern blot analyses were consistent with the 3'-NT/Nu being encoded by a single copy gene. These data constitute the first report of the gene for this unique trypanosomatid surface membrane enzyme.

Amino Acid Sequence↗
PubMed1994

Immunization coverage in a population-based sample of Maryland children.

OBJECTIVE: To describe the vaccination coverage of a population-based sample of Maryland children. DESIGN: Historical cohort study using stratified cluster sampling. Strata were formed from five state regions and public and private schools within each region. SETTING: Random sample of elementary schools in Maryland. SUBJECTS: Immunization records were obtained for a randomly selected cohort of 1938 first-grade children in Maryland. OUTCOME MEASURES: Age of completion of recommended childhood vaccines and proportion of vaccines given simultaneously. RESULTS: By first grade, 95.2% of the sample of Maryland children were documented to have received at least four doses of the diphtheria and tetanus toxoids and pertussis (DTP) vaccine, three doses of oral poliovirus (OPV) vaccine, and one dose of measles-mumps-rubella (MMR) vaccine. However, only 54.8% of children surveyed had achieved this level of immunization by 24 months of age. For children whose immunizations were up to date on their first birthday (78.7% of children surveyed), vaccine providers failed to administer one dose of DTP, OPV, and MMR vaccines to 30% of these children during the second year of life. Only 16.2% (n = 313) of children sampled received an MMR vaccination simultaneously with any dose of DTP or OPV vaccine. CONCLUSIONS: This study shows that while immunization levels are very high at school entry, there are serious delays in vaccine administration before school entry. These delays are observed regardless of whether children are in public or private schools or attend school in an urban, suburban, or rural area. Physicians and health care providers could play an important role in age-appropriate vaccination by administering vaccines simultaneously.

Child↗
PubMed1994

Immunization coverage and its relationship to preventive health care visits among inner-city children in Baltimore.

OBJECTIVE: To provide empirical data on immunization coverage and the receipt of preventive health care to inform policy makers' efforts to improve childhood immunization. DESIGN AND METHODS: We surveyed a random sample drawn from a birth cohort of 557 2-year-old children living in the inner-city of Baltimore. Complete information on all their preventive health care visits and immunization status was obtained from medical record audits of their health care providers. MAIN OUTCOME MEASURES: Age-appropriate immunizations and preventive health care visits. RESULTS: By 3 months of age, nearly 80% made an age-appropriate preventive health visit, but by 7 months of age, less than 40% had a preventive visit that was age-appropriate. In the second year of life, 75% made a preventive health visit between their 12- and 17-month birthdays. The corresponding age-appropriate immunization levels were 71% for DTP1, 39% for DTP3, and 53% for measles-mumps-rubella vaccine. Infants who received their DTP1 on-time were twice as likely to be up-to-date by 24 months of age. CONCLUSIONS: Our analyses focus attention on the performance of the primary health care system, especially during the first 6 months of life. Many young infants are underimmunized despite having age-appropriate preventive visits, health insurance coverage through Medicaid, and providers who receive free vaccine from public agencies. Measles vaccination coverage could be improved by initiating measles-mumps-rubella vaccine vaccination, routinely, at 12 months among high risk populations.

Baltimore↗
PubMed1993

Leishmania: immunochemical comparison of the secretory (extracellular) acid phosphatases from various species.

Promastigotes of all Leishmania species, except most Leishmania major stocks, synthesize and secrete extracellular soluble acid phosphatases [SAcP(s)] which are capable of dephosphorylating a wide range of substrates and, consequently, of modifying the parasite environment. To assess their antigenic/structural relatedness, the SAcP(s) from various species were compared immunochemically using both a monospecific rabbit antiserum and several monoclonal antibodies made against the purified promastigote SAcP of a cloned line of Leishmania donovani donovani. Results obtained from antibody-bridged enzyme activity assays demonstrated that these reagents quantitatively immunoprecipitated the enzymatic activities of SAcP(s) from 18 different W.H.O. reference stocks of Leishmania, including two L. major strains. Those results showed, for the first time, that all SAcP(s) possessed some common cross-reactive antigenic epitopes. Immunoprecipitates obtained from [35S]methionine, metabolically labeled promastigote culture supernatants of the various species, were analyzed by SDS-PAGE/fluorography. These analyses showed that marked differences existed among the SAcP(s) both in their relative mobilities and the number of constituent bands resolved. Tunicamycin treatment resulted in a significant reduction in the apparent molecular weights of SAcP(s) from three different isolates, confirming the presence of N-linked carbohydrate side chains in these enzymes. In addition, the SAcP released by L. donovani intracellular amastigotes was also immunoprecipitated with the monospecific rabbit antisera from the culture supernatant of infected macrophages. Cumulatively, these data demonstrate that despite individual species variations, the SAcP(s) from all Leishmania tested have retained certain common antigenic/structural epitopes and functional protein domains. Such conservation among SAcP(s) suggests that this enzyme must play an essential role in the survival of promastigotes and amastigotes of all Leishmania species.

Acid Phosphatase↗
PubMed1993

Cloning and characterization of differentially expressed genes from in vitro-grown 'amastigotes' of Leishmania donovani.

Leishmanial parasites routinely undergo cyclic differentiation from promastigotes to amastigotes during their life cycle. This process involves both morphological and macromolecular changes. To study such changes, we used a axenic culture system which permits the continuous generation and cycling of Leishmania donovani from promastigotes to 'amastigotes' in vitro. cDNA libraries were constructed from poly(A)+ RNA isolated from both the pro- and amastigote forms. Using differential cDNA hybridization techniques, 3 unique cDNAs clones (P17, A41 and A45) were isolated from the amastigote library. To assess whether these clones were differentially expressed by the pro-or 'amastigotes' forms, they were hybridized to RNA isolated from each of these parasite forms in Northern and slot-blots. Results of these analyses showed that 'amastigotes' had approx. 2-fold higher levels of the A41 and A45 RNAs compared to the promastigotes. Conversely, promastigotes showed approx. 2-fold higher levels of the P17 RNA than 'amastigotes'. Nucleotide sequence analysis and comparison with those in Gene bank, revealed that the 3 cDNAs represent unique leishmanial genes. Comparison of the deduced amino acid sequences revealed that P17 open reading frame (ORF) had significant similarity with a soybean ribosomal protein S11; A41 ORF with a Bacillus subtilis spore germination gene (gerC) and A45 ORF with yeast stress-inducible protein (STI1). It is of interest to note that, of the 3 cDNAs identified, the A45-encoded protein was recognized by sera from patients with clinically active visceral leishmaniasis and was encoded by a single copy gene.

Amino Acid Sequence↗
PubMed1993

Chronic obstructive pulmonary disease mortality. A role for altitude.

A map of US COPD mortality rates by state suggested that the relative hypoxia of increased altitude may be independently associated with COPD mortality. This was investigated using linear regression analysis of 1986 state-specific data on COPD mortality rates, history of cigarette consumption, and altitude. County seat altitudes and county populations were used to calculate the median altitude of state residents. We found independent significant associations between COPD and both smoking and altitude. State COPD mortality rose by 1/10(5) for every 5.4 increase in mean packs consumed per capita per year or for each 95-m increase in resident altitude. There was no association between altitude and smoking. If increased altitude does contribute to COPD mortality, persons with this disease may benefit from down-migration.

Altitude↗
PubMed1993

Gastroenteritis outbreak in a Maryland nursing home.

From December 25, 1990, to January 12, 1991, an outbreak of acute viral gastroenteritis occurred among 132 residents and 102 employees of a Maryland nursing home. Illness typically lasted 24 hours and was characterized by diarrhea, vomiting, and fever. The attack rate was 46.2% (61/132) for residents and 42.2% (43/102) for employees. No differences in attack rates were observed by station in the facility. The risk of having an early case (before the peak of the outbreak on January 2, 1991) was 3.5 times greater for employees with patient contact than for employees without patient contact. Analyses of temporal and geographic clustering of cases suggest that person-to-person transmission was an important transmission mode. Although an etiologic agent was not identified, the short duration of illness, high rates of vomiting and diarrhea, and high attack rate are consistent with Norwalk-like viral infection.

Adult↗
PubMed1993

Characteristics of epidemic hepatitis A in Baltimore City: implications for control measures.

In 1988, a hepatitis A epidemic began in Baltimore, Maryland. A total of 607 cases were reported to the Baltimore City Health Department between the period November 1, 1988, and December 31, 1989. The health department conducted case investigations to identify factors important to the development of control measures. Immunoglobulin was given to appropriate contacts, and an educational campaign was initiated. Of the 607 reported cases, 63% were white and 57% were male. Cases were geographically clustered within 5 of Baltimore's 23 zip codes. Among the 242 adults interviewed, 43% self-reported drug use and 44% were unemployed. Twenty-one percent of the adults and 56% of the children reported contact with either a suspected or confirmed case of hepatitis. Despite control efforts, the epidemic continued through 1990. Barriers to implementing traditional control measures resulted in continuing transmission of hepatitis A in the community.

Adolescent↗
PubMed1992

Purification and properties of 3'-nucleotidase of Leishmania donovani.

A surface membrane 3'-nucleotidase from Leishmania donovani promastigotes has been purified to SDS/PAGE homogeneity. The enzyme has apparent subunit molecular mass of 38 kDa, pI 5.8 and a broad pH optimum, 5.5-7.5. EDTA partially inhibited the enzyme activity, which was fully restored by Co2+; Mg2+, Ca2+ or Mn2+ had no effect on the activity. ZnCl2 or dithiothreitol at 1 mM was inhibitory at pH 7.5, but was without effect at pH 5.5, whereas at both pH values 5 mM of either compound inhibited the enzyme. The substrate-specificity of the purified enzyme is restricted to ribonucleoside 3'-phosphates. 3'-AMP and 3'-IMP are the best substrates, whereas ADP, ATP, 2'-deoxyadenosine 3'-phosphate and 5'-AMP are competitive inhibitors of the enzyme. The enzyme showed low latency in intact-cell preparations. The kinetic properties and the surface membrane localization of the enzyme suggest its implication in the formation of nucleosides from 3'-nucleotides of the parasite's host.

Adenine Nucleotides↗
PubMed1992

Nursing home outbreak of influenza A (H3N2): evaluation of vaccine efficacy and influenza case definitions.

OBJECTIVES: Describe an outbreak of influenza A (H3N2); provide an analysis of vaccine efficacy; measure the sensitivity, specificity, and positive predictive value of 3 clinical case definitions of influenza. SETTING: A nursing home in Washington County, Maryland. The outbreak involved 52 residents (attack rate = 47.7%) and at least 10 of 140 employees (minimum attack rate = 7.1%). RESULTS: Twenty-five residents exhibited a 4-fold or greater increase in titer to influenza A/Sichuan/2/87. Vaccine efficacy was measured at -7.1%, suggesting that the influenza vaccine in 1988/1989 did not offer optimal protection against influenza A infection for the institutionalized elderly. CONCLUSIONS: The outbreak was a clear indicator of the need for rapid diagnosis. With the use of rapid diagnostic tests, influenza A could have been detected in time to use amantadine.

Aged↗
PubMed1992

Changing clonal patterns of Salmonella enteritidis in Maryland: evaluation of strains isolated between 1985 and 1990.

We examined isolates from 203 sporadic and outbreak-associated Salmonella enteritidis cases occurring in Maryland between 1985 and 1990. Plasmid profiles were determined for all isolates; 52 isolates were phage typed. Ten plasmid profiles were identified. A single profile (consisting of a single ca. 55-kb plasmid) emerged as the predominant profile in Maryland during the study period. This profile (which was closely associated with phage type 8) accounted for 86% of a group of isolates from sporadic cases in 1988 and 1989, compared with 43% of the 1985 isolates. Strains with this profile were identified in four of nine outbreaks, including one of three outbreaks in which eggs were implicated as a vehicle. While plasmid profiles and phage typing appear to provide complementary means of identifying specific strains of S. enteritidis, the emergence of what appears to be a single predominant clone has reduced the discriminant ability of both typing systems. The factors be a single predominant clone has reduced the discriminant ability of both typing systems. The factors contributing to the emergence of this one clone are still not well understood.

Bacteriophage Typing↗