PubMed Health⌕ Search

Biomedical subjects

D J Kelly

Publications and source records attributed to D J Kelly.

At least 73 records · Page 4Linked to original sources
PubMed1998

Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays.

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States.

Amino Acid Sequence↗
PubMed1998

Topological analysis of DcuA, an anaerobic C4-dicarboxylate transporter of Escherichia coli.

Escherichia coli possesses three independent anaerobic C4-dicarboxylate transport systems encoded by the dcuA, dcuB, and dcuC genes. The dcuA and dcuB genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity. A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S. Six, S. C. Andrews, G. Unden, and J. R. Guest, J. Bacteriol. 176:6470-6478, 1994). These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a beta-lactamase protein lacking the N-terminal signal peptide. The resulting topological model differs from those previously predicted. It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm and the predicted orientation is consistent with the "positive-inside rule." Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region. The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB. Overproduction of DcuA to 15% of inner-membrane protein was obtained with the lacUV5-promoter-based plasmid, pYZ4.

Amino Acid Sequence↗
PubMed1998

Transcriptional regulation and organization of the dcuA and dcuB genes, encoding homologous anaerobic C4-dicarboxylate transporters in Escherichia coli.

The dcuA and dcuB genes of Escherichia coli encode homologous proteins that appear to function as independent and mutually redundant C4-dicarboxylate transporters during anaerobiosis. The dcuA gene is 117 bp downstream of, and has the same polarity as, the aspartase gene (aspA), while dcuB is 77 bp upstream of, and has the same polarity as, the anaerobic fumarase gene (fumB). To learn more about the respective roles of the dcu genes, the environmental and regulatory factors influencing their expression were investigated by generating and analyzing single-copy dcuA- and dcuB-lacZ transcriptional fusions. The results show that dcuA is constitutively expressed whereas dcuB expression is highly regulated. The dcuB gene is strongly activated anaerobically by FNR, repressed in the presence of nitrate by NarL, and subject to cyclic AMP receptor protein (CRP)-mediated catabolite repression. In addition, dcuB is strongly induced by C4-dicarboxylates, suggesting that dcuB is under the control of an uncharacterized C4-dicarboxylate-responsive gene regulator. Northern blotting confirmed that dcuA (and aspA) is expressed under both aerobic and anaerobic conditions and that dcuB (and fumB) is induced anaerobically. Major monocistronic transcripts were identified for aspA and dcuA, as well as a minor species possibly corresponding to an aspA-dcuA cotranscript. Five major transcripts were observed for dcuB and fumB: monocistronic transcripts for both fumB and dcuB; a dcuB-fumB cotranscript; and two transcripts, possibly corresponding to dcuB-fumB and fumB mRNA degradation products. Primer extension analysis revealed independent promoters for aspA, dcuA, and dcuB, but surprisingly no primer extension product could be detected for fumB. The expression of dcuB is entirely consistent with a primary role for DcuB in mediating C4-dicarboxylate transport during anaerobic fumarate respiration. The precise physiological purpose of DcuA remains unclear.

Amino Acid Sequence↗
PubMed1998

Helicobacter pylori porCDAB and oorDABC genes encode distinct pyruvate:flavodoxin and 2-oxoglutarate:acceptor oxidoreductases which mediate electron transport to NADP.

Helicobacter pylori, a major cause of human gastric disease, is a microaerophilic bacterium that contains neither pyruvate nor 2-oxoglutarate dehydrogenase activity. Previous studies (N. J. Hughes, P. A. Chalk, C. L. Clayton, and D. J. Kelly, J. Bacteriol. 177:3953-3959, 1995) have indicated that the major routes for the generation of acetyl coenzyme A (acetyl-CoA) and succinyl-CoA are via pyruvate:flavodoxin oxidoreductase (POR) and 2-oxoglutarate:acceptor oxidoreductase (OOR), respectively. The purified POR is a heterotetrameric protein, with subunits of 48 (PorA), 36 (PorB), 24 (PorC), and 14 (PorD) kDa. In this study OOR has been purified, and it is similarly composed of polypeptides of 43 (OorA), 33 (OorB), 24 (OorC), and 10 (OorD) kDa. Both POR and OOR are oxygen labile and are likely to be major contributors to the microaerophilic phenotype of H. pylori. Unlike POR, OOR was unable to use a previously identified flavodoxin (FldA) as an electron acceptor. Although the purified enzymes were unable to reduce NAD(P), electrons from both pyruvate and 2-oxoglutarate could reduce NADP in cell extracts, consistent with a role for these oxidoreductases in the provision of NADPH as a respiratory electron donor. The H. pylori por, oor, and fldA genes were cloned and sequenced. The deduced por gene products showed significant sequence similarity to archaeal four-subunit 2-oxoacid:acceptor oxidoreductases. However, the amino acid sequences of OorA and -B were more closely related to that of the two-subunit POR of the aerobic halophile Halobacterium halobium. Both porD and oorD encode integral ferredoxin-like subunits. POR and OOR are probably essential enzymes in H. pylori, as insertion inactivation of porB and oorA appeared to be lethal.

Amino Acid Sequence↗
PubMed1998

Laboratory evaluation of a dot-blot enzyme immunoassay for serologic confirmation of illness due to Rickettsia conorii.

Of the 169 United States Army soldiers who deployed on a field training exercise to a remote area of Botswana for two weeks in January 1992, more than 30% developed a febrile illness within five days of their return. A diagnosis of South African tick typhus was suggested by soldiers' exposure to ticks, as well as the presence of eschars and vesicles at the site of tick bites, and tender regional lymphadenopathies. This high attack rate, experienced during such a short exposure period, emphasizes the hazard of illness due to Rickettsia conorii to persons visiting endemic areas. A rapid, diagnostic, semiquantitative enzyme immunoassay (DS) for detection of IgG and IgM antibodies to R. conorii was performed on 209 acute and convalescent sera from soldiers in the outbreak and on 75 control sera. For the acute sera from soldiers meeting the probable case definition of having both regional lymphadenopathy and tick bite eschar, as judged by an IgG indirect fluorescent antibody (IFA) test, the resulting sensitivity and specificity of the DS test were 100% and 48%, respectively. In the analysis of the acute sera, the DS test identified as reactive more of the probable cases (62%) than either the IgG (16%) or IgM (55%) IFAs. This simple and rapid diagnostic test could be useful in establishing a preliminary diagnosis of R. conorii rickettsiosis in remote settings when immediate confirmation by IFA is impossible.

Antibodies, Bacterial↗
PubMed1998

Vascular endothelial growth factor and its receptors in control and diabetic rat eyes.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and permeability-inducing factor that has been implicated in the pathogenesis of diabetic retinopathy. In the present study, the localization and magnitude of VEGF, VEGF receptor-1 (VEGFR-1), and VEGF receptor-2 (VEGFR-2) gene expression were examined in the eye of streptozotocin-induced diabetic rats using quantitative in situ hybridization. VEGF protein was also examined by immunohistochemistry. Abundant VEGF mRNA and protein were present in the retinae of control rats. In the retinae of diabetic rats, VEGF gene expression was increased compared with control animals (p = 0.001). The increase in VEGF mRNA was noted in the ganglion cell layer and inner nuclear layer but not in the pigment epithelium of the retina. VEGF was also detected in blood vessels, ciliary body, and lens epithelium in both control and diabetic rats. The distributions of VEGFR-1 and VEGFR-2 were similar in both control and diabetic rats. VEGFR-1 mRNA was present beneath the inner limiting membrane and in the ganglion cell layer, inner nuclear layer, outer plexiform layer, and outer limiting membrane of the retina; it was also detected in blood vessels, the ciliary body, and the cornea. The magnitude and distribution of ocular VEGFR-1 mRNA were not affected by experimental diabetes. Expression of VEGFR-2 mRNA was noted in the inner nuclear layer and pigment epithelium of the retina and in blood vessels. An increase in VEGFR-2 mRNA in the diabetic retina was restricted to the inner nuclear layer. The presence of VEGF and its receptors in the control retina suggests a physiologic role for VEGF within the eye. The changes in retinal expression of VEGF and VEGFR-2 in association with diabetes suggest a role for this pathway in diabetic retinopathy.

Animals↗
PubMed1997

TRAP transporters: a new family of periplasmic solute transport systems encoded by the dctPQM genes of Rhodobacter capsulatus and by homologs in diverse gram-negative bacteria.

The dct locus of Rhodobacter capsulatus encodes a high-affinity transport system for the C4-dicarboxylates malate, succinate, and fumarate. The nucleotide sequence of the region downstream of the previously sequenced dctP gene (encoding a periplasmic C4-dicarboxylate-binding protein) was determined. Two open reading frames (ORFs) of 681 bp (dctQ) and 1,320 bp (dctM) were identified as additional dct genes by insertional mutagenesis and complementation studies. DctQ (24,763 Da) and DctM (46,827 Da) had hydropathic profiles consistent with the presence of 4 and 12 potential transmembrane segments, respectively, and were localized in the cytoplasmic membrane fraction after heterologous expression of the dctQM ORFs in Escherichia coli. DctP, DctQ, and DctM were found to be unrelated to known transport proteins in the ABC (ATP-binding cassette) superfamily but were shown to be homologous with the products of previously unidentified ORFs in a number of gram-negative bacteria, including Bordetella pertussis, E. coli, Salmonella typhimurium, Haemophilus influenzae, and Synechocystis sp. strain PCC6803. An additional ORF (rypA) downstream of dctM encodes a protein with sequence similarity to eukaryotic protein-tyrosine phosphatases, but interposon mutagenesis of this ORF did not result in a Dct- phenotype. Complementation of a Rhizobium meliloti dctABD deletion mutant by heterologous expression of the dctPQM genes from R. capsulatus demonstrated that no additional structural genes were required to form a functional transport system. Transport via the Dct system was vanadate insensitive, and in uncoupler titrations with intact cells, the decrease in the rate of succinate transport correlated closely with the fall in membrane potential but not with the cellular ATP concentration, implying that the proton motive force, rather than ATP hydrolysis, drives uptake. It is concluded that the R. capsulatus Dct system is a new type of periplasmic secondary transporter and that similar, hitherto-unrecognized systems are widespread in gram-negative bacteria. The name TRAP (for tripartite ATP-independent periplasmic) transporters is proposed for this new group.

Adenosine Triphosphate↗
PubMed1997

Amylin stimulates proximal tubular sodium transport and cell proliferation in the rat kidney.

In autoradiographic studies in anesthetized rats, 125I-labeled amylin binding was associated with proximal convoluted tubules but not distal tubules, interstitium, or glomeruli in the renal cortex. Split-drop micropuncture experiments showed that perfusion of the peritubular capillaries with amylin (10(-9) M) stimulated proximal tubular fluid absorption by 28%. This effect was inhibited by luminal addition of ethylisopropylamiloride, indicating mediation by a brush-border Na+/H+ exchanger. Intravenous infusion of an amylin binding antagonist, AC-187, reduced proximal fluid reabsorption (22%) in anesthetized rats, indicating a role for endogenous amylin in salt homeostasis. In primary cultures of rat proximal tubule cells, amylin (10(-7) M) stimulated proliferation with a potency equal to epidermal growth factor. Peptide antagonists (AC-187, AC-413, and AC-512) of the amylin binding sites in the renal cortex blocked the mitogenic action of amylin. We conclude that amylin acts on renal proximal tubules to promote sodium and water reabsorption and cell proliferation. These novel actions may have implications for the development of hypertension for example in non-insulin-dependent diabetes mellitus and obesity in which hyperamylinemia has been observed.

Absorption↗
PubMed1997

Evaluation of an enzyme-linked immunosorbent assay in Thai scrub typhus patients.

We report the development of an improved enzyme-linked immunosorbent assay (ELISA) to detect Orientia (formerly Rickettsia) tsutsugamushi antibody in human sera. Results were compared with a standard test, the indirect immunoperoxidase assay (IIP). Control serum samples were collected from 96 American soldiers and 198 Royal Thai Army soldiers with no recent history of clinical illness. Sera were examined from 79 febrile, Thai scrub typhus patients presenting at Chiang Rai (76) and Bangkraui Nontaburi (3) Provincial hospitals (cases confirmed by elevated IIP IgG levels > or = 1:1,600, IgM levels > or = 1:400, or presence of an eschar). The mean + 2 SD, used for the upper limit of normal reactions in the IgG ELISA, was 0.10 for U.S. soldiers and 0.42 for Thai soldiers. Using the 0.10 cutoff value, 29% of the asymptomatic Thai soldiers would be designated as antibody positive. Variability of IgG ELISA values was greater in the Thai soldier group than in American soldiers, possibly reflecting previous exposure to O. tsutsugamushi. In the Thai patients, there was a significant correlation between IIP titers and single serum dilution (1:100) ELISA values (IgG, r = 0.75, n = 104; P < 0.0005; IgM, r = 0.70, n = 75; P < 0.0005) and between IIP titers and ELISA titers (IgG, r = 0.87, n = 103; P < 0.0005; IgM, r = 0.76, n = 75; P < 0.0005). The single serum dilution ELISA was as effective as the titration in determining presence of specific antibodies. The O. tsutsugamushi ELISA is a rapid and objective test amenable to accurately testing the large numbers of sera often obtained in seroepidemiologic investigations.

Animals↗
PubMed1997

Short report: surveillance of rickettsial infections in Indonesian military personnel during peace keeping operations in Cambodia.

Indonesian peacekeepers in Cambodia provided a unique study population to estimate the threat of rickettsial exposure to Rickettsia typhi (murine typhus), Orientia tsutsugamushi, (scrub typhus), and R. conorii (spotted fever) for the region. Prescreening prevalence measure showed a large proportion (36%) of soldiers with antibodies to R. typhi. Predeployment prevalence for antibodies to O. tsutsugamushi was 8%, with no evidence of background R. conorii infections. Actual seroconversions of R. typhi (3) and O. tsutsugamushi (1), attributed to exposure(s) in Cambodia, translated into annualized incidence rates of 24 and 8 per 1,000 per year, respectively. Surveillance of rickettsial infections and/or disease is particularly warranted in Cambodia with recent recognition of drug-resistant scrub typhus in neighboring Thailand.

Antibodies, Bacterial↗
PubMed1997

Seroepidemiologic evidence for murine and scrub typhus in Malang, Indonesia.

Indonesian military personnel stationed in Malang, East Java were among troops deployed to central Cambodia as part of the United Nations' Transition Authority Cambodia peace-keeping operation in 1992. Predeployment blood samples obtained from a cohort of Indonesian soldiers indicated a high prevalence of antibodies to antigens of Rickettsia typhi or Orientia (formerly Rickettsia) tsutsugamushi, the etiologic agents for murine and scrub typhus, respectively. To evaluate the potential risk of these rickettsial diseases in the Malang area, a subsequent seroepidemiologic survey was conducted. This study involved civilian personnel residing within one of three Malang kelurahans (neighborhoods) representing urban, suburban, and rural communities. The heads-of-households from 197 homes completed a detailed epidemiologic survey. In addition, blood samples were collected from 464 individuals residing within the households surveyed. Examination of civilian blood samples disclosed that 34.7% and 1.3% of the study participants were seroreactive to R. typhi and O. tsutsugamushi, respectively. These results were similar to those obtained earlier from the military samples. In addition, assessment of 78 blood samples obtained from peridomestic rodents trapped from within or near the households surveyed showed that 28 were reactive to R. typhi antigens and four were reactive to O. tsutsugamushi antigens. These data indicate that military and civilian personnel living in the Malang area of East Java are at risk of infection with rickettsiae that are antigenically indistinguishable from those that cause murine and scrub typhus.

Adolescent↗
PubMed1996

Adrenaline cells of the rat adrenal cortex and medulla contain renin and prorenin.

The distribution and content of renin in Sprague-Dawley (SD) and transgenic (mREN-2)27 rats (TG) were compared to further define the cellular basis and function of the adrenal renin-angiotensin system. Antibody binding (to rat and mouse renin protein and prosequence) was visualised in serial paraffin sections using an avidin-biotin peroxidase technique. Chromaffin and adrenaline cells were identified by tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase immunoreactivity, respectively. In SD zona glomerulosa (ZG), renin and its prosequence localised to small steroid cells while in homozygous (receiving lisinopril) and heterozygous (untreated) TG, steroid cells labelled in all cortical zones. In addition, throughout the cortex of each strain, large polyhedral adrenaline chromaffin cells occurring singly or in small groups and occasionally in rays labelled for renin and prosequence. Similar large adrenaline cells immunolabelled for all antisera in medulla while other cells were only TH-positive. Total adrenal renin content was 53 times higher in heterozygous transgenics than SD rats and was mainly (74%) prorenin. In SD, 37% of cortical renin was prorenin but in adrenal medulla only active renin was detected. Thus, from present and previous work both renin and prorenin occur not only in mitochondrial dense bodies of the ZG, but also in secretory granules of adrenaline chromaffin cells in both cortex and medulla implying in situ synthesis and paracrine functions.

Adrenal Cortex↗
PubMed1995

Measuring children's lexical diversity: differentiating typical and impaired language learners.

This study evaluated the extent to which measures of lexical diversity (type-token ratio and number of different words produced) differentiated children with specific language impairment (SLI) from children whose language skills were following typical developmental expectations. Analysis of 50- and 100-utterance samples revealed that children with SLI did not significantly differ from their age- and language-equivalent peers on type-token ratio; however, children with SLI did use significantly fewer different words than their age-equivalent peers in these samples. When samples of 100 and 200 tokens were considered, the children with SLI also used significantly fewer different words than their age-equivalent counterparts. Overall, the findings of this study discourage the use of type-token ratio, as traditionally calculated, in many clinical and research activities related to children with SLI. In contrast, the number of different words produced provides a more sensitive and informative estimate of lexical diversity.

Child Language↗
PubMed1995

Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography.

Pyruvate carboxylase (PC) was purified to homogeneity from an overexpressing strain of the purple photosynthetic bacterium Rhodobacter capsulatus using a rapid dye-ligand affinity chromatography procedure, in which dye-bound enzyme was specifically eluted with a low concentration of acetyl-CoA, an allosteric activator of the enzyme. The enzyme purified by this method was obtained in 75% yield with a specific activity of 40 U (mg protein)-1. In contrast, affinity chromatography on a monomeric avidin column, commonly used in the purification of biotin-containing carboxylases, resulted in a yield of < 40%, with a specific activity of 10 U (mg protein)-1. The enzyme purified by the dye-linked procedure had a subunit molecular mass of 140,000 Da and was absolutely dependent on acetyl-CoA for activity. Acetyl-CoA was also effective in protecting the enzyme from thermal denaturation. The enzyme was inhibited by 2-oxoglutarate and, to a lesser extent, L-aspartate, with sigmoidal kinetics with respect to acetyl-CoA concentration. The amino acid composition, pH optimum and kinetic constants for pyruvate, ATP and bicarbonate were determined. An N-terminal sequence of 26 residues was obtained, which was homologous to the N-terminal regions of several eukaryotic PCs, propionyl-CoA carboxylases and acetyl-CoA carboxylase.

Acetyl Coenzyme A↗
PubMed1995

Comparative evaluation of a commercial enzyme immunoassay for the detection of human antibody to Rickettsia typhi.

A commercial enzyme immunoassay kit called the Dip-S-Ticks (DS) for the detection of total immunoglobulin (Ig) G and IgM human antibodies to Rickettsia typhi was evaluated. In tests with 340 serum samples from patients with diagnosed cases of rickettsial diseases, patients suffering from other febrile illnesses, and normal subjects, the DS compared favorably with the standard indirect fluorescent-antibody (IFA) test. At IFA cutoff titers of > or = 1.64 and > or = 1:128, the DS showed sensitivities of 88.2 and 91.4% and specificities of 91.8 and 87.7%, respectively. The DS test correlated significantly with both the IFA IgG (r = 0.84, P < 0.0005) and IgM (r = 0.63, P < 0.0005) titers. Only 80% of IgG and 82% of IgM IFA readings determined by two technicians were within one dilution, while the DS was more reliable, with 100% within one dot. The rapidity, reliability, and simplicity of the DS suggest that it is a suitable test for use in clinical laboratories unable to perform the IFA test.

Antibodies, Bacterial↗