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Biomedical subjects

B E Dunn

Publications and source records attributed to B E Dunn.

At least 19 recordsLinked to original sources
PubMed1995

Antimicrobial activity of calprotectin isolated from human empyema fluid supernatants.

Abscess and empyema fluid supernatants have zinc-reversible antimicrobial activity that is thought to be due to calprotectin, a calcium- and zinc-binding protein present within neutrophil cytoplasm. The present studies were undertaken to determine if calprotectin isolated from human empyema fluid supernatants demonstrated similar antimicrobial activity to that of the original specimens. The characteristics of the calprotectin complex on SDS-PAGE and Western blotting with specific antisera were similar in neutrophil lysates and in empyema fluid supernatants. Ion-exchange and size-exclusion chromatography were used to obtain highly purified preparations of calprotectin from empyema fluids, and these preparations demonstrated zinc-reversible anti-Candida albicans activity which was similar to that observed in the original specimens. These findings suggest that calprotectin is responsible for most of the growth-inhibitory activity of empyema fluid supernatants against this organism.

Antifungal Agents↗
PubMed1994

Correlation between serological and mucosal inflammatory responses to Helicobacter pylori.

In 82 patients who underwent gastroduodenoscopy, acute and chronic gastric mucosal inflammation was scored for severity, and systemic humoral immune responses to Helicobacter pylori antigens were assessed by enzyme-linked immunosorbent assays. On the basis of culture, gastric histology, and serologic evaluation, 33 patients were classified as H. pylori infected and 36 were classified as uninfected. Thirteen patients had negative cultures and stains but were seropositive and were analyzed separately from the other two groups. Specific serum immunoglobulin G (IgG) subclass responses to H. pylori whole-cell antigens and specific IgG responses to the 54-kDa heat shock protein homolog (Hp54K) and vacuolating cytotoxin were significantly greater in infected than in uninfected patients as were specific IgA responses to whole-cell antigens and cytotoxin (P < 0.001). Among the H. pylori-infected persons, serum IgG responses to Hp54K and to the vacuolating cytotoxin were correlated with acute mucosal inflammatory scores. In contrast, serum IgA responses to whole-cell sonicate and to vacuolating cytotoxin were inversely related to chronic inflammatory scores. By multivariant regression analysis, only specific serum IgG responses to Hp54K correlated with severity of inflammation (both acute and chronic; P < 0.001); these responses may be markers of inflammation or these antibodies could play a direct role in the pathogenesis of H. pylori-induced inflammation.

Antibodies, Bacterial↗
PubMed1992

Molecular cloning and nucleotide sequence analysis of the gene encoding the immunoreactive Brucella abortus Hsp60 protein, BA60K.

A recombinant 60 kDa Brucella abortus protein expressed in Escherichia coli was recognized in immunoblots by sera from mice experimentally infected with B. abortus and a dog experimentally infected with B. canis. Sera from humans and dogs with naturally acquired brucellosis also recognized this protein, which was designated BA60K. The gene encoding BA60K was localized within an 18 kb B. abortus genomic fragment and its direction of transcription determined by subcloning and maxicell analysis of selected restriction fragments. The nucleotide sequence of 1800 bases encompassing the predicted gene location was determined, revealing an open reading frame encoding a protein of 546 amino acids (predicted relative molecular mass of 57515). Solid phase micro-sequencing of BA60K eluted from two-dimensional polyacrylamide gels confirmed the predicted amino acid sequence. Comparison of the predicted amino acid sequence of BA60K with a protein sequence database revealed that BA60K shares 67.9% identity with the GroEL protein of E. coli, a member of the Hsp60 family of chaperonins. The immunodominant Hsp60 homologs from Legionella pneumophila, Chlamydia trachomatis and Mycobacterium tuberculosis were also found to share greater than 59% amino acid sequence identity with the BA60K protein. The identification of BA60K as a member of the Hsp60 family of chaperonins supports its role in stimulating a prominent host immune response during the course of Brucella infections. It also indicates that BA60K is an important candidate for studies aimed at identifying the antigens responsible for eliciting the protective immune response to brucellosis.

Amino Acid Sequence↗
PubMed1992

Identification and purification of a cpn60 heat shock protein homolog from Helicobacter pylori.

Helicobacter pylori is associated with gastritis and peptic ulcer disease in humans. We have identified a homolog of the chaperonin cpn60 family of heat shock proteins in H. pylori, referred to as Hp54K. Hp54K, purified from water-extractable H. pylori proteins, migrated as a single band at 54 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its native molecular mass was 740 kDa; thus, Hp54K apparently comprises a 14-mer. The N-terminal 33 residues of Hp54K exhibited 60.6, 57.6, 54.5, 54.5, 51.5, and 51.5% identity with corresponding sequences in the following cpn60 homologs: HtpB (Legionella pneumophila), P1 (human mitochondria), GroEL (Escherichia coli), BA60K (Brucella abortus), HypB (Chlamydia trachomatis), and the 65-kDa immunodominant protein of Mycobacterium bovis BCG, respectively. Hp54K was the only protein recognized in whole-cell preparations of H. pylori by immunoblotting using monospecific antisera against cpn60 homologs from L. pneumophila, E. coli, C. trachomatis, and M. bovis BCG. Antiserum against Hp54K recognized proteins with molecular masses of 50 to 60 kDa in a large number of gram-negative bacteria, consistent with the known highly conserved nature of cpn60 proteins. Hp54K is a major protein and is immunogenic in humans infected with H. pylori. Thus, Hp54K shares many similarities with known cpn60 homologs. On the basis of the proposed role of other cpn60 proteins in induction of chronic inflammation, immune cross-reactivity between Hp54K and gastric tissue may provide an important link between H. pylori infection and gastritis.

Amino Acid Sequence↗
PubMed1992

Identification of Ehrlichia chaffeensis morulae in cerebrospinal fluid mononuclear cells.

We report a case of ehrlichiosis in a 72-year-old man who developed extreme lethargy, acute renal failure requiring hemodialysis, and respiratory insufficiency requiring intubation. Lumbar puncture performed on the second day of hospitalization revealed significant cellular pleocytosis. Ehrlichia morulae were tentatively identified in mononuclear cells in routinely processed Wright-stained cytospin preparations of cerebrospinal fluid (CSF). Identification was confirmed by a specific immunocytochemical staining procedure. Subsequent identification specifically as Ehrlichia chaffeensis morulae was established by polymerase chain reaction analysis, which revealed E. chaffeensis-specific DNA in CSF, bone marrow, and blood samples; by indirect fluorescent-antibody analysis, the patient developed an antibody titer of 32,768 against E. chaffeensis antigen. The patient responded to intravenous therapy with doxycycline and dexamethasone. Subsequently, neurologic, hematologic, renal, and pulmonary status had returned to baseline at follow-up 12 weeks after admission. To our knowledge, this is the first identification of E. chaffeensis morulae in CSF cells in an infected patient.

Aged↗
PubMed1991

Purification and characterization of Helicobacter mustelae urease.

Helicobacter mustelae is a urease-rich bacterium associated with gastritis in ferrets. The ureases of H. mustelae and Helicobacter pylori, a bacterium implicated in human gastritis, share many characteristics. Helicobacter sp. ureases appear to be unique among bacterial enzymes in exhibiting submillimolar Km values and in being composed of two subunits.

Campylobacter↗
PubMed1990

Purification and characterization of urease from Helicobacter pylori.

Urease was purified 112-fold to homogeneity from the microaerophilic human gastric bacterium, Helicobacter pylori. The urease isolation procedure included a water extraction step, size exclusion chromatography, and anion exchange chromatography. The purified enzyme exhibited a Km of 0.3 +/- 0.1 mM and a Vmax of 1,100 +/- 200 mumols of urea hydrolyzed/min/mg of protein at 22 degrees C in 31 mM Tris-HCl, pH 8.0. The isoelectric point was 5.99 +/- 0.03. Molecular mass estimated for the native enzyme was 380,000 +/- 30,000 daltons, whereas subunit values of 62,000 +/- 2,000 and 30,000 +/- 1,000 were determined. The partial amino-terminal sequence (17 residues) of the large subunit of H. pylori urease (Mr = 62,000) was 76% homologous with an internal sequence of the homohexameric jack bean urease subunit (Mr = 90,770; Takashima, K., Suga, T., and Mamiya, G. (1988) Eur. J. Biochem. 175, 151-165) and was 65% homologous with amino-terminal sequences of the large subunits of heteropolymeric ureases from Proteus mirabilis (Mr = 73,000) and from Klebsiella aerogenes (Mr = 72,000; Mobley, H. L. T., and Hausinger, R. P. (1989) Microbiol. Rev. 53, 85-108). The amino-terminal sequence (20 residues) of the small subunit of H. pylori urease (Mr = 30,000) was 65 and 60% homologous with the amino-terminal sequences of the subunit of jack bean urease and with the Mr = 11,000 subunit of P. mirabilis urease (Jones, B. D., and Mobley, H. L. T. (1989) J. Bacteriol. 171, 6414-6422), respectively. Thus, the urease of H. pylori shows similarities to ureases found in plants and other bacteria. When used as antigens in an enzyme-linked immunosorbent assay, neither purified urease nor an Mr = 54,000 protein that co-purified with urease by size exclusion chromatography was as effective as crude preparations of H. pylori proteins at distinguishing sera from persons known either to be infected with H. pylori or not.

Amino Acid Sequence↗
PubMed1990

Seroprevalence of Helicobacter pylori infections in Thailand.

Serologic studies in developed countries indicate that Helicobacter (formerly Campylobacter) pylori infection is uncommon until the third decade of life and achieves a peak prevalence of 50% in the seventh decade. In developing countries the epidemiology of H. pylori has not well been described. A sensitive and specific serologic assay for H. pylori infection was validated in Thai patients also studied by culture and histologic examination of biopsy specimens. The prevalence of H. pylori antibodies in persons from a rural Thai community began early (17.5% of children 5-9 years old), increased to 55% during the third decade of life, and peaked (75%) in the 30- to 49-year age group. At a Bangkok orphanage where enteric infections are hyperendemic, 74% of children 1-4 years old were seropositive. This study shows that the prevalence of H. pylori infection in Thailand is higher than in industrialized countries. The high infection rate at the orphanage suggests that person-to-person transmission of H. pylori may be occurring.

Antibodies, Bacterial↗
PubMed1990

Lactoferrin and transferrin damage of the gram-negative outer membrane is modulated by Ca2+ and Mg2+.

Lactoferrin and transferrin have antimicrobial activity against selected Gram-negative bacteria, but the mechanism of action has not been defined. We studied the ability of lactoferrin and transferrin to damage the Gram-negative outer membrane. Lipopolysaccharide release by the proteins could be blocked by concurrent addition of Ca2+ and Mg2+. Addition of Ca2+ also blocked the ability of lactoferrin to increase the susceptibility of Escherichia coli to rifampicin. Transferrin, but not lactoferrin, increased susceptibility of Gram-negative bacteria to deoxycholate, with reversal of sensitivity occurring with exposure to Ca2+ or Mg2+. In transmission electron microscopy studies polymyxin B caused finger-like membrane projections, but no morphological alterations were seen in cells exposed to EDTA, lactoferrin or transferrin. These data provide further evidence that lactoferrin and transferrin act as membrane-active agents with the effects modulated by Ca2+ and Mg2+.

Calcium↗
PubMed1990

Cognitive correlates of headache intensity and duration.

Cognitive processes and cognitive styles of persistent headache sufferers were examined in relation to indices of intensity and duration of head pain. One hundred and eight persistent headache patients, referred for cognitive-behavioral management of their disorder, completed questionnaires designed to assess: distressing thoughts and feelings experienced during headache attacks, evaluation of the experienced pain, perceived influence of stress and worry on headaches, and capacity to verbally express emotions. The results showed that the intensity of head pain correlated with measures of headache-related distress and with the proportion of headache-related distress to situation-related distress. Duration of head pain was also associated with the proportion of headache-related distress to situation-related distress, and additionally with a tendency to deny the influence of stress and worry on headaches and with difficulty in expressing emotions. These findings were taken as support of our previously stated hypothesis that head pain of increased severity is associated with a cognitive shift whereby the patient's primary concern moves from situational and interpersonal distress to distress associated with the disorder itself. In addition, these findings point to affective distress and lack of emotional expressiveness as correlating differentially with the headache dimensions of intensity and duration.

Cognition↗
PubMed1989

Two-dimensional gel electrophoresis and immunoblotting of Campylobacter pylori proteins.

Whole-cell, outer-membrane protein, flagellum-associated antigens and partially purified urease of Campylobacter pylori were analyzed by two-dimensional gel electrophoresis. C. pylori strains were readily distinguished from strains of Campylobacter jejuni, C. coli, and C. fetus by absence of major outer membrane proteins with Mrs of 41,000 to 45,000. C. pylori strains also lacked the acidic surface-array proteins at Mr 100,000 to 149,000 identified previously in serum-resistant strains of C. fetus. Surface labeling of intact C. pylori cells with 125I revealed two common major proteins, which we have designated protein 2 (pI 5.6 to 5.8, Mr 66,000) and protein 3 (pI 5.2 to 5.5, Mr 63,000). Proteins 2 and 3 were also the major components (subunits) observed in partially purified urease. Partially purified preparations of flagella consistently contained proteins 2 and 3. Thus, urease appears to be associated with both outer membranes and flagella of C. pylori. C. pylori strains also possessed an antigen at Mr 59,000 which was cross-reactive with antiserum against flagella of C. jejuni. However, the antigen did not appear to be associated with flagella per se in C. pylori. Protein 2 was unique to C. pylori among the Campylobacter species studied. It was not recognized by antibody against whole cells of C. jejuni or C. fetus or flagella of C. jejuni. Protein 3 was cross-reactive with antiserum against whole cells of C. jejuni and C. fetus, as were several other major protein antigens. Because protein 2 is a major outer membrane protein that is apparently unique to C. pylori, development of monospecific antibodies against this antigen may be useful for the identification of C. pylori in tissues, and purified antigen may be useful for serologic tests for specific diagnosis of C. pylori infections.

Animals↗
PubMed1988

Purification of Escherichia coli alkaline phosphatase on an ion-exchange high-performance liquid chromatographic column using carboxymethyl dextrans.

Carboxymethyl dextrans (CM-Ds) were used on an HPLC ion-exchange column to obtain significantly enriched alkaline phosphatase (EC 3.1.3.1) from a sample of Escherichia coli periplasmic space proteins without significant loss of enzymatic activity. The ability of CM-Ds to separate alkaline phosphatase even when the column was 80-85% saturated with protein demonstrates the potential for high column capacity using CM-Ds. In addition, the fractions containing alkaline phosphatase and CM-Ds were reapplied to the same ion-exchange column under different buffer conditions and purified to homogeneity by salt gradient elution chromatography, thus demonstrating the compatibility of CM-Ds with the latter chromatographic method. The two-step chromatographic procedure yielded enzyme of purity comparable to that of electrophoretically purified E. coli alkaline phosphatase obtained commercially. These studies demonstrate that HPLC displacement chromatography is a mild procedure which allows rapid, quantitative purification of an enzyme. Scaling up with larger columns should allow purification of enzymes of a commercial basis.

Alkaline Phosphatase↗
PubMed1987

Two-dimensional gel electrophoresis and immunoblotting of Campylobacter outer membrane proteins.

We characterized outer membrane proteins (OMPs) from selected Campylobacter jejuni, C. coli, and C. fetus strains by two-dimensional gel electrophoresis (2DGE), using isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and by immunoblotting with immune rabbit serum. The flagellar band with a molecular mass of 63 kilodaltons (kDa) demonstrated previously by one-dimensional SDS-PAGE was shown by 2DGE to consist of one or several charge trains, depending upon the species, strain, and type of preparation studied; each of the individual peptides was found to be antigenic by immunoblotting. In contrast, in all of the strains studied, the major OMP (43 to 44 kDa) of C.jejuni and C. coli consisted of a single isomeric form which was weakly immunogenic. Several minor proteins (29 to 31 kDa) were found to be strongly immunogenic by immunoblotting. C. fetus strains possessed two major OMPs of 45 to 47 kDa, each of which consisted of either a single isomer or a major isomer comprising at least 90% of the major OMP. Serum-resistant strains of C. fetus possessed an acid-labile 100-kDa glycoprotein (pI, 4.1) which was markedly diminished or absent in serum-sensitive strains. These 2DGE analyses provide information that is useful in taxonomic and epidemiologic studies and for the purification of surface antigens for the development of campylobacter vaccines and may also facilitate the identification of specific virulence factors.

Bacterial Outer Membrane Proteins↗
PubMed1987

Endocytosis in the embryonic chick chorionic epithelium.

The chorionic epithelium of the developing chick embryo consists of two major cell types, villus cavity (VC) and sinus-covering (SC) cells. Ultrastructurally, VC cells contain apical microvilli with numerous underlying vesicles, whereas SC cells possess thin cytoplasmic processes (containing occasional vesicles) overlying adjacent blood sinuses. This study was performed to determine whether vesicles observed in VC and SC cells are endocytic in nature and whether endocytosis is limited to either VC or SC cells. Exposure of air space chorionic epithelium in ovo to microperoxidase (MRP-11) resulted in endocytic uptake of marker by the thin cytoplasmic processes of 70% of SC cells. Vesicles containing MRP-11 reaction product were also observed in approximately 20% of VC cells. Exposure of the chorionic epithelium of shell-less cultured embryos to horseradish peroxidase (HRP) resulted in endocytic uptake of HRP in approximately 80% of SC cells and in 33% of VC cells. Thus, VC and SC cells demonstrate endocytic activity both in ovo and in shell-less culture. In ovo, endocytosis may be involved in the recovery of water and/or ions from the eggshell or shell membranes.

Animals↗
PubMed1987

Effect of calcium supplementation on growth of shell-less cultured chick embryos.

Chick embryos grown in shell-less culture are calcium-deficient by 9 days and retarded in growth by 13 days of incubation (Dunn and Boone, Poult. Sci., 56:662-672, 1977). To determine whether addition of exogenous calcium might stimulate growth and/or survival of cultured embryos, calcium supplementation was attempted. Calcium supplementation between days 11 and 17 resulted in significant increases in both total embryo and serum calcium. Addition of shell pieces oriented with shell membranes onto the chorioallantoic membrane (CAM) resulted in significant stimulation of calcium transport by the CAM. However, growth of supplemented embryos was not increased to the same degree as were embryonic serum and total calcium levels. It is concluded that at least one factor other than calcium deficiency is responsible for retarded growth of shell-less cultured embryos.

Animals↗
PubMed1986

Calcium regulation in the embryonic chick. II. Ultrastructure of the parathyroid glands in shell-less and in ovo embryos.

The ultrastructure of the parathyroid glands was studied in chick embryos developing normally in ovo or in shell-less culture (after removal of the eggshell). Shell-less chick embryos are significantly hypocalcemic relative to their in ovo counterparts. At 12 days of incubation, the parathyroid glands of shell-less embryos contain more lipid and show evidence of increased protein synthetic activity relative to those grown in ovo (more rough endoplasmic reticulum, presence of some dense secretory granules). The glands from in ovo embryos do not contain secretory granules at this age. At 15 days of incubation, the in ovo glands have developed signs of protein synthetic activity similar to those of the 12-day shell-less embryos. However, the parathyroids of the 15-day shell-less embryos appear strikingly more active than at 12 days, containing stacks of concentric RER membranes and increased numbers of secretory granules. By 18 days of incubation, the ultrastructure of the glands of the two groups is indistinguishable, both appearing to be more active than the 15-day shell-less group. Thus, protein synthetic activity of the parathyroid glands, as detected by ultrastructural alterations of the chief cells, normally appears to be initiated during the latter part of embryogenesis (by approximately 15 days incubation) and its onset can be stimulated at least 3 days prematurely by hypocalcemia.

Animals↗
PubMed1986

Embryonic chick allantois: functional isolation and development of sodium transport.

By removing the shell membranes from the chorioallantoic membrane, the chorion is damaged, as visualized by electron microscopy, and rendered permeable, as evidenced by penetration of horseradish peroxidase and increased inhibition of the allantoic Na+-K+ pump by ouabain applied on the chorionic side. The short-circuit current (SCC) of this functionally isolated allantoic epithelium is augmented by nystatin, a channel-forming ionophore, when applied to the mucosal surface. Electrical parameters were determined for three age groups between 12 and 19 days of incubation. The SCC approximately doubled from the youngest (12-13 days) to the oldest (18-19 days) groups, whereas the transepithelial resistance (Re) of 700-900 omega X cm2 remained the same. Amiloride, an inhibitor of apical Na+ uptake, inhibited 98-100% of the SCC at 10(-4) M in both 15-16 and 18-19 day epithelia. In the 12- to 13-day preparation 20-25% of the SCC was insensitive to 10(-3) M amiloride. The Ki's for amiloride were similar in all preparations, at about 5 X 10(-7) M. Determination of the Hill coefficients for inhibition revealed a lower value (0.75 +/- 0.03) for the 12-13 day preparation compared with the two older preparations with coefficients not significantly different from unity. Replacing Na+ in the bathing solutions abolished the SCC of 18-19 day epithelia, whereas about 15% of the SCC remained at 12-13 days. Thus, during development, the SCC of the allantoic epithelium increases in magnitude and becomes increasingly (to 100%) amiloride-sensitive and Na+-dependent.

Allantois↗