PubMed Health⌕ Search

Biomedical subjects

D J Kelly

Publications and source records attributed to D J Kelly.

At least 91 records · Page 5Linked to original sources
PubMed1995

Identification of carboxylation enzymes and characterization of a novel four-subunit pyruvate:flavodoxin oxidoreductase from Helicobacter pylori.

The enzyme activities responsible for carboxylation reactions in cell extracts of the gastric pathogen Helicobacter pylori have been studied by H14CO3- fixation and spectrophotometric assays. Acetyl coenzyme A carboxylase (EC 6.4.1.2) and malic enzyme (EC 1.1.1.40) activities were detected, whereas pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.3.1) and phosphoenolpyruvate carboxykinase (EC 4.1.1.49) activities were absent. However, a pyruvate-dependent, ATP-independent, and avidin-insensitive H14CO3- fixation activity, which was shown to be due to the isotope exchange reaction of pyruvate:flavodoxin oxidoreductase (EC 1.2.7.1), was present. The purified enzyme is composed of four subunits of 47, 36, 24, and 14 kDa. N-terminal sequence analysis showed that this enzyme is related to a recently recognized group of four-subunit pyruvate:ferredoxin oxidoreductases previously known only from hyperthermophiles. This enzyme from H. pylori was found to mediate the reduction of a number of artificial electron acceptors in addition to a flavodoxin isolated from H. pylori extracts, which is likely to be the in vivo electron acceptor. Indirect evidence that the enzyme is capable of in vitro reduction of the anti-H. pylori drug metronidazole was also obtained.

Acetyl-CoA Carboxylase↗
PubMed1995

Rickettsia tsutsugamushi infection in cell culture: antibiotic susceptibility determined by flow cytometry.

Recent unpublished reports from northern Thailand of severe and sometimes fatal cases of scrub typhus, despite appropriate antibiotic therapy, suggest that resistance may occur. Current antibiotic susceptibility methods that use direct microscopic counts of Giemsa-stained cells or mouse protection assays are slow, labor-intensive, and expensive. We explored the use of flow cytometry to measure rickettsial infection in vitro in L-929 cells treated with and without doxycycline, ciprofloxacin, erythromycin, and chloramphenicol. It was possible to detect the rickettsiae down to a level of 83% of the cells infected, mean of 37 rickettsiae per cell, and 40% of cells with too many rickettsiae to count. This level of sensitivity was sufficient to determine the inhibitory effect of all four drugs at standard screening concentrations. At lower concentrations of doxycycline, flow cytometry detected inhibition of rickettsial growth at a concentration of 6.25 x 10(-2) micrograms/ml but not at 6.25 x 10(-3) micrograms/ml, suggesting that the minimum inhibitory concentration is somewhere between these two values. The data from this study show that flow cytometry permits the rapid screening of numerous rickettsial isolates for their susceptibility to a variety of antibiotics, but that visual counts of infected cells provide a more precise indication of rickettsial growth.

Animals↗
PubMed1995

Effectiveness of a dot-blot immunoassay of anti-Rickettsia tsutsugamushi antibodies for serologic analysis of scrub typhus.

We compared a commercially available dot-blot immunoassay system with the indirect immunofluorescence assay (IFA) in tests of known negative and known positive sera from scrub typhus cases. Using a panel of 100 sera from patients with various rickettsial and nonrickettsial infections, we observed that the IFA was 99% specific and the dipstick assay was 98% specific. In tests of 91 sera (30 negative and 61 positive for scrub typhus antibodies) from a study of febrile patients in Malaysia, using the standard of an IFA titer < 1:64 as negative, an IFA titer > 1:128 as positive, and an IFA titer = 1:64 as either positive or negative (supported by clinical records), dipsticks were 83% specific and 90% sensitive. The quantitative correlation of the dipsticks to IFA titers was confirmed by significant differences in geometric means of inverse IFA titers corresponding to the number of positive dipstick spots (no dots = 8.5, one dot = 43.3, two dots = 206.7, and three dots = 676.9). The assay would enable physicians and public health workers who deal with patients to quickly diagnose and appropriately treat most cases of the disease, especially in areas of high prevalence where the proportion of false-positive results to true-positive results would be low.

Antibodies, Bacterial↗
PubMed1994

An improved procedure and new vectors for transposon Tn5 mutagenesis of the phototrophic bacterium Rhodospirillum rubrum.

A detailed examination of vectors and procedures used for Tn5 mutagenesis of the phototrophic purple non-sulfur bacterium Rhodospirillum rubrum has been performed. The mobilizable Tn5 suicide vectors currently available show a frequency of Tn5 mutagenesis for R. rubrum of approx. 10(-7)-10(-8), approx. 100-1000-fold lower than observed for the related bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides. Using the blue-to-red reversion of a blue-green mutant, R. rubrum ST6, containing a single Tn5 lesion in one of the early genes for carotenoid biosynthesis, we have shown that the frequency of precise excision of a chromosomally inserted Tn5 element, to restore the wild-type phenotype in the absence of selection, is 10(-6). We have constructed three new suicide vectors for Tn5 mutagenesis, where the transposase encoded by the IS50R element was placed in the same (pSUPEG11, pSUPEG21) or in the opposing (pSUPEG22) orientation from the weak promoter of the RK2-derived tetR gene. With the vector pSUPEG11, the frequency of Tn5 mutagenesis was increased to 10(-5), approx. 100-fold higher than observed previously.

Conjugation, Genetic↗
PubMed1994

Gram-positive cell wall structure of the A3 gamma type in heliobacteria.

The amino acid composition and structure of the peptidoglycan from Heliobacillus mobilis was determined by one- and two-dimensional thin-layer chromatography of completely and partially hydrolysed cell wall preparations. The structure was found to be of the A3 gamma type, with L,L-diaminopimelate in position 3, D-alanine in position 4 and a glycine interpeptide bridge, as found in certain groups of Gram-positive bacteria including Clostridium perfringens and Nocardioides simplex. The presence of a Gram-positive type of cell wall in heliobacteria is consistent with their phylogenetic relationship to the 'low G + C' Gram-positive bacteria, as previously demonstrated by 16S rRNA sequencing.

Amino Acid Sequence↗
PubMed1994

Renin processing and secretion in adrenal and retina of transgenic (mREN-2)27 rats.

Two extrarenal tissue sources of renin were studied using quantitative assays and immunocytochemical methods during 12 hours following binephrectomy (BNx) in anesthetized hypertensive homozygous Ren-2 transgenic (TG) rats maintained off hypotensive drugs for three weeks. Compared to normal rats, circulating active renin was depressed 50% in conscious TG rats and prorenin was 5- to 10-fold higher. Post-BNx, arterial active and prorenin increased progressively to 10-fold, at which time adrenal venous outputs were 0.1 and 20 mGU/min, respectively. The ratio of active to prorenin (3.1 +/- 0.6%) remained unchanged with increasing plasma levels. Thus, either intrinsic enzyme activity of the transgenic prorenin contributed a constant proportion to the measured active renin, or processing to mature renin was coupled to prorenin synthesis and secretion in extrarenal tissues. In the TG rat eye, renin protein labeling was localized throughout retinal Müller cells with prosequence more obvious posteriorly, consistent with directional processing. Immunogold studies are in progress. In adrenal following BNx, labeling for renin and prosequence increased uniformaly in all zones of the cortex and in scattered medullary chromaffin cells. In cortex, both renin and prosequence were strongly located in intramitochondrial dense bodies. In chromaffin cells, renin labeling was present in both cytoplasmic vesicles and electron-dense granules, while prosequence was predominantly in cytoplasmic vesicles, consistent with processing of prorenin prior to storage in chromaffin granules.

Adrenal Glands↗
PubMed1994

Preferences for verb interpretation in children with specific language impairment.

This study examined initial preferences for verb interpretation by children with specific language impairment (SLI), MLU-matched children, and age-matched children. Each child watched motion and change-of-state activity scenes on videotape and was then asked to point to the scene that depicted a novel verb, thereby indicating a preferred interpretation. The children were also asked to label the same activity scenes on a second tape. The findings indicated that the 5-year-old age-matched children exhibited a significant verb interpretation preference for the change-of-state scenes, whereas the children with specific language impairment and their 3-year-old MLU-matched peers did not have an interpretation preference for either the motion or change-of-state scenes. The children's labeling of the activity scenes yielded findings that further supported group differences on the two semantic verb categories. The findings suggest that children's initial verb interpretation biases vary relative to age and language proficiency.

Age Factors↗
PubMed1994

Identification of Rickettsia conorii infection by polymerase chain reaction in a soldier returning from Somalia.

A soldier developed characteristic manifestations of boutonneuse fever shortly after leaving Somalia. Rickettsial DNA was detected in a biopsy sample of the tache noire by a polymerase chain reaction (PCR) in which primers derived from the 190-kD antigen gene of Rickettsia rickettsii were used. The source of this DNA was identified as Rickettsia conorii by restriction fragment length polymorphism (RFLP) analysis of the PCR product. R. conorii was also isolated from the skin biopsy specimen. The patient did not develop a significant increase in specific antibodies, as assessed by indirect fluorescent antibody testing, until several weeks after the onset of symptoms. This case demonstrates that the PCR/RFLP technique can be used for the direct identification of rickettsiae from clinical specimens. To our knowledge, this is the first confirmed case of R. conorii infection in Somalia.

Adult↗
PubMed1994

Rickettsia tsutsugamushi in chiggers (Acari: Trombiculidae) associated with rodents in central Thailand.

Chiggers were collected from rodents trapped at two military bases located 10 km apart in central Thailand. One site was swampy and nearly treeless and the other site was well-drained and partially wooded. Although 13 species of chiggers were collected, only three species were found to be positive for Rickettsia tsutsugamushi (Hayashi) Ogata using a direct fluorescent antibody test: Blankaartia acuscutellaris Walch (7.3% infected), Leptotrombidium delinese Walch (3.1% infected), and an undescribed species of Ascoschoengastia near A. indica Hirst (1.2% infected). This was the first record of R. tsutsugamushi in B. acuscutellaris. The Ascoschoengastia species occurred with equal frequency at the two study sites, L. deliense occurred more frequently at the well-drained site, and B. acuscutellaris occurred more frequently at the swampy site. Results suggest that there are important foci of scrub typhus in central Thailand and that B. acuscutellaris may be a vector in this area.

Animals↗
PubMed1994

Detection and characterization of Rickettsia tsutsugamushi (Rickettsiales: Rickettsiaceae) in infected Leptotrombidium (Leptotrombidium) fletcheri chiggers (Acari: Trombiculidae) with the polymerase chain reaction.

We developed a method for detecting and characterizing the DNA of Rickettsia tsutsugamushi in chiggers (larval trombiculid mites) by polymerase chain reaction (PCR). Three procedures for extracting DNA from frozen chiggers were compared by evaluating the yield of PCR amplicand obtained with nine oligonucleotide primer pairs derived from the rickettsial 22 kD, 47 kD, groESL, 56 kD, and 110 kD antigen genes. Although extracts and primer pairs differed in amplification efficiency, R. tsutsugamushi DNA was successfully detected in extracts of colonized infected Leptotrombidium (Leptotrombidium) fletcheri (Wormersley & Heaslip) chiggers and in uninfected chigger extracts seeded with known amounts of Karp-strain rickettsiae. The 22 kD gene restriction fragment length polymorphisms (RFLP) observed in PCR amplicands from five rickettsial isolates obtained from the infected chigger colony over a 26-yr period were identical to those of PCR amplicands derived directly from infected chiggers taken from the same colony. This suggests that stable transmission of R. tsutsugamushi occurs in mites (62 generations), and isolates encompass the full genetic heterogeneity found in the chigger. PCR/RFLP analysis is an important new tool for investigating the complex epidemiology of scrub typhus rickettsiae in their mite vectors.

Animals↗
PubMed1994

The bacterial degradation of benzoic acid and benzenoid compounds under anaerobic conditions: unifying trends and new perspectives.

Simple homocyclic aromatic compounds are extremely abundant in the environment and are derived largely from lignin. Such compounds may enter anaerobic environments and several groups of bacteria, exhibiting diverse energy-yielding mechanisms, have evolved the capacity to overcome the thermodynamic stability of the benzene nucleus and degrade aromatic compounds under these conditions. Over the last few years considerable advances have been made in our understanding of the biochemical strategies underlying the bacterial degradation of aromatic compounds in anoxic environments. The study of the biochemistry, and more recently the molecular genetics of the photosynthetic bacterium Rhodopseudomonas palustris and several strains of denitrifying pseudomonads, has provided the greatest insight into the mechanism and regulation of aromatic degradation under anaerobic conditions. Research has centred around the anaerobic degradation of benzoic acid. This involves the initial activation to form benzoyl-Coenzyme A, reduction of the aromatic nucleus--a reaction that has only recently been demonstrated in vitro--and the subsequent degradation of the alicyclic intermediates. Recently, much information regarding the exact nature of these intermediates has been obtained. Also through recent studies, it has become increasingly clear that benzoyl-CoA is a central metabolic intermediate during the anaerobic degradation of structurally diverse aromatic compounds. The initial metabolism of these compounds involves the formation of a carboxyl group on the aromatic nucleus (if necessary) and the synthesis of the respective Coenzyme A thioester; this results in the direct formation of benzoyl-Coenzyme A rather than benzoate. In many cases of anaerobic aromatic degradation studied in batch culture, aromatic intermediates are transiently excreted into the medium. It is argued that the study of this phenomenon may facilitate the understanding of the regulation and kinetics of the aromatic degradative pathways.

Acyl Coenzyme A↗
PubMed1994

First cases of spotted fever group rickettsiosis in Thailand.

The first three cases of spotted fever group rickettsiosis from Thailand are reported. The patients presented with fever, headache, lymphadenopathy, and petechial maculopapular rash. One patient also had an eschar and overt evidence of confusion. An indirect fluorescent antibody test, an indirect immunoperoxidase test, and an enzyme-linked immunosorbent assay demonstrated a broad, strong reactions of the sera of the patients with spotted fever group rickettsia antigens of many species, but not with antigens of typhus or scrub typhus rickettsiae. All three patients responded to treatment with a single dose of doxycycline.

Adult↗
PubMed1994

Prevalence of antibodies to rickettsiae in the human population of suburban Bangkok.

Following a report of three cases of scrub typhus in suburban Bangkok, we performed a serosurvey in the patients' communities. Both IgG and IgM antibodies were measured in an indirect immunoperoxidase assay, using separate spots of antigen from Rickettsia tsutsugamushi (scrub typhus), R. typhi (murine typhus), and TT-118 spotted fever group rickettsiae. Of 215 people donating blood, antibody levels indicative of most recent exposure to R. tsutsugamushi were most prevalent (21%), followed by R. typhi (8%), and TT-118 (4%). Seroprevalence suggesting most recent exposure to R. tsutsugamushi varied by location (range 13-31%), gender (26% of females and 13% of males), age (61-80-year-old individuals were the highest, with a prevalence of 38%), and contact with orchards and orchid farms (29% of those with extensive contact, 38% of those with occasional contact, and 10% of those with no contact). These patterns indicated that exposure to R. tsutsugamushi was related to occupation and behavior, as has been observed in areas of rural transmission. Expansion of metropolitan Bangkok has created a situation in which people employed in agriculture live with people employed in the city. As a result, a serosurvey in suburban Bangkok reveals evidence of murine typhus, which is usually transmitted in urban areas, as well as scrub and tick typhus, which are usually transmitted in rural areas.

Adolescent↗
PubMed1994

Pathology of Rickettsia tsutsugamushi infection in Bandicota savilei, a natural host in Thailand.

Following rodent surveys in a rice-growing area of central Thailand where we found Bandicota savilei, B. indica, and Rattus rattus infected with Rickettsia tsutsugamushi, we performed a study of pathogenesis of R. tsutsugamushi in laboratory-reared B. savilei. Eight animals were injected with saline and 19 animals were injected with 4.0 x 10(6) mouse 50% lethal dose units of a strain of R. tsutsugamushi isolated from a human in central Thailand. Animals were evaluated at intervals for IgG and IgM antibodies to R. tsutsugamushi by an indirect immunoperoxidase assay, the presence of the pathogen in liver and spleen by murine inoculation, and the pathology of representative tissues by gross and microscopic examination. The infected animals began to show internal evidence of mild illness 7-14 days after inoculation, and exhibited no changes in behavior. Total white blood cell counts decreased on day seven (including lymphocytes and polymorphonuclear leukocytes), followed by an almost equal increase on day 14. Gross pathology noted at necropsy was limited to slight liver and spleen enlargement accompanied by low numbers of abscesses and fibrinous tags present in the abdominal cavity. In addition to the gross morphologic changes, histopathologic lesions noted were all mild, consisting of vasculitis of the lung, activation of the mononuclear phagocyte system, abdominal mesothelial cell hyperplasia, and peritonitis. Rickettsiae were isolated from liver and spleen on days 0, 7, and 14, but not thereafter. Specific antibody response was first observed on day 14, peaked on day 21, and it decreased to levels observed in uninfected animals between days 120 and 180.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Sequence analysis and interposon mutagenesis of a sensor-kinase (DctS) and response-regulator (DctR) controlling synthesis of the high-affinity C4-dicarboxylate transport system in Rhodobacter capsulatus.

A two-component sensor-regulator system has been identified in the purple photosynthetic bacterium Rhodobacter capsulatus, which controls the expression of high-affinity C4-dicarboxylate transport activity in these cells. Nucleotide sequencing has revealed the existence of two genes, dctS and dctR, which together form an operon linked to, but divergently transcribed from, the previously identified dctP gene, which encodes the periplasmic binding protein of the transport system. The DctS protein is predicted to be a membrane-bound sensor-kinase with two potential membrane-spanning sequences in the N-terminal region. DctR was found to have sequence similarity throughout its entire length with proteins in the FixJ subfamily of response-regulators, especially to FixJ itself (42% identical residues). Insertional inactivation of the dctS and dctR genes resulted in the inability of the resulting mutants to grow on or transport malate, succinate or fumarate under aerobic conditions in the dark, and such mutants did not express the DctP protein. The mutants were complemented in trans by plasmids containing intact copies of the dctS and dctR genes.

Amino Acid Sequence↗
PubMed1992

Perturbation of the equilibrium between open and closed conformations of the periplasmic C4-dicarboxylate binding protein from Rhodobacter capsulatus.

A kinetic and thermodynamic analysis has been carried out on the conformational transitions of the periplasmic C4-dicarboxylate binding protein (DctP) from the photosynthetic bacterium Rhodobacter capsulatus. This protein is distinct from other periplasmic binding proteins characterized to date in that the transition between the putative closed-unliganded (BP1) and open-unliganded (BP2) conformations is slow compared to the rate of ligand binding [Walmsley, A. R., Shaw, J. G., & Kelly, D. J. (1992) J. Biol. Chem. 276, 8064-8072]. Using stopped-flow fluorescence techniques, we have probed the conformational dynamics of the closed to open transition of DctP in the absence and presence of ligand. Both the forward rate constant for the BP1 to BP2 interconversion (k1) and the fumarate dissociation rate constant (k-3) were found to increase in a biphasic manner between pH 5 and pH 11. The data were fitted to a two-pKa function which gave pKa values of 10.3 and 5.4 for the BP1 to BP2 interconversion and 8.9 and 4.5 for the closed-liganded (BP3L) to open-liganded (BP2L) transition. An increase in ionic strength at constant pH resulted in a hyperbolic increase in both k1 and k-3 to maximal rates that were similar in each case to the values obtained in pH variation experiments. Measurement of the temperature dependencies of k1 and k-3 also gave similar activation energies. Gibbs free energy, enthalpy, and entropy changes were determined for the open to closed transitions of DctP in both the presence and absence of ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Carrier Proteins↗
PubMed1992

The mechanism of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus.

The kinetics of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus were investigated by exploiting the changes in the intrinsic fluorescence of the protein upon binding ligands. Steady state measurements have shown that L-malate, succinate, and fumarate are all bound with sub-micromolar Kd values, whereas D-malate is bound 2 orders of magnitude more weakly. Stopped-flow studies have revealed that the binding process involves at least three steps. In the absence of ligand, the protein is in equilibrium between an essentially nonbinding form, BP1, and the binding form, BP2. Ligands bind to the BP2 form, shifting the equilibrium toward the BP2-L conformation, and also inducing a further isomerization of the protein, to the BP3-L form. The kinetic properties of the four different conformational states of the DctP protein identified in this study would be consistent with their identification as the closed-conformation, the open-conformation, an open-liganded conformation, and a closed-liganded conformation. The latter three states have been identified by x-ray crystallographic studies of binding proteins, but no kinetic or structural data have been presented previously to support the possibility of a closed but unliganded conformation.

Bacterial Proteins↗