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D A Dean

Publications and source records attributed to D A Dean.

At least 55 records · Page 3Linked to original sources
PubMed1994

Identification of Schistosoma mansoni antigens recognized by spleen cells of C57B1/6 mice immunized with ultraviolet-irradiated cercariae.

Spleen cells of C57B1/6 mice immunized twice with Schistosoma mansoni cercariae attenuated by ultraviolet irradiation proliferated and produced interleukin-(II)-2 and/or II-4 in response to both soluble schistosomular and adult worm antigens of 72-68, 60-62, 50, 45, 29.5 and 28 kDa. All of these bands, except the 45 kDa, were also recognized by serum antibodies in Western blotting.

Animals↗
PubMed1994

Identification of Schistosoma mansoni antigens recognized by T cells of C57BL/6 mice immunized with gamma-irradiated cercariae.

Immunization of C57Bl/6 mice with gamma-irradiated Schistosoma mansoni cercariae induces highly significant protection against subsequent challenge with unattenuated living cercariae. The antigens that evoke T cell-mediated immunity in this model are vaccine antigen candidates since T lymphocytes mediate antibody-independent immune responses and control the production of most antibodies. To identify these antigens, spleen cells of mice immunized twice with gamma-irradiated cercariae were tested for proliferative responsiveness and production of interleukins (Il) and interferon-gamma (IFN-gamma) following incubation with separated soluble schistosomular (SSA) and adult worm (SAWA) antigens in T cell western assays. Data obtained at 3, 4, 5, and 6 wk post-secondary immunization in 2 experiments indicated that SSA and SAWA bands of 62-60, 50, and 45 kDa reproducibly elicited T cell proliferation and production of Il-2, Il-4, and IFN-gamma by spleen cells from immunized, but not unimmunized, mice. Bands of 72-68, 29.5, and 28 kDa elicited proliferation and production of Il-2 and Il-4, but not IFN-gamma. Bands of 35-33 and 24 kDa induced either proliferation or Il production at some intervals.

Animals↗
PubMed1993

Identification of a DNA binding domain in simian virus 40 capsid proteins Vp2 and Vp3.

We have identified both biochemically and genetically a protein domain within the simian virus 40 virion protein Vp3, and within Vp2 since its carboxyl two-thirds are identical to the full-length Vp3, that binds DNA in a sequence nonspecific manner. Both the Vp2 and Vp3 (Vp2/3) components of SV40 and mutant SV40(202T) bound either SV40 or pBR322 DNA equally well. Wild type and mutant Vp2/3 proteins, expressed as fusion proteins with glutathione S-transferase (GST), were tested for their ability to bind DNA. GST-Vp3 bound DNA at physiological salt concentrations with an apparent Kd of 2.5 x 10(-8) M and also bound RNA with 4-fold higher affinity. Over 90% of the nucleic acid binding, and all of the activity, was lost upon removal of the carboxyl-terminal 13 and 35 residues, respectively. The DNA binding domain was shown to be distinct and separable from the Vp2/3 nuclear transport signal since mutations within the nuclear transport signal that reduce or abolish nuclear localization of Vp2/3 had no effect on the DNA binding activity of mutant Vp2/3 fusion proteins. The carboxyl-terminal 40 residues of Vp2/3 in the form of a beta-galactosidase fusion protein, F6, are sufficient for DNA binding and may cause compaction of the DNA. The significance of this DNA binding and possible compaction are discussed in relation to the assembly of virion particles.

Animals↗
PubMed1992

Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli.

Active transport of maltose in Escherichia coli requires the presence of both maltose-binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants in malF or malG were isolated that are able to grow on maltose in the complete absence of MBP. When the wild-type malE+ allele, coding for MBP, was introduced into these MBP-independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal- strains, Mal+ secondary mutants that contained suppressor mutations in malE were isolated. In this study, we examined the interaction of wild-type and mutant MBPs with wild-type and mutant FGK2 complexes by using right-side-out membrane vesicles. The vesicles from a MBP-independent mutant (malG511) transported maltose in the absence of MBP, with Km and Vmax values similar to those found in intact cells. However, addition of wild-type MBP to these mutant vesicles produced unexpected responses. Although malE+ malG511 cells could not utilize maltose, wild-type MBP at low concentrations stimulated the maltose uptake by malG511 vesicles. At higher concentrations of the wild-type MBP and maltose, however, maltose transport into malG511 vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype of malE+ malG511 cells, which were found to be capable of transporting maltose from a low external concentration (1 microM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2 complex, containing MalG511, had a much higher apparent affinity towards the wild-type MBP than did the wild-type FGK2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1992

Combined microautoradiographic and histopathologic analysis of the fate of challenge Schistosoma mansoni schistosomula in mice immunized with irradiated cercariae.

Combined microautoradiographic and histopathologic methods were used to locate and examine schistosomula of Schistosoma mansoni in the lungs of irradiated cercaria-immunized mice 21 days after percutaneous challenge infection with 75Se-labeled cercariae. Of 75 schistosomula examined in serial sections, 53% were located in the pulmonary microvasculature, 23% in alveolar spaces, 3% with one end in a vessel and the other in an alveolar space, and the locations of 21% were not identified. Inflammatory reactions of variable intensity were observed around schistosomula in both vascular and alveolar sites, although the most intense category of reactions was associated almost entirely with alveolar larvae. All autoradiographic foci contained recognizable schistosomula. Although the concentration of reduced silver grains precluded cyto-structural analysis, observations on schistosomular contour and shape provided no evidence of larval damage. Our findings suggest that immune elimination of schistosomula in mice immunized with irradiated cercariae is partly or largely effected by a process of alveolar extrusion of viable parasites during their lung migration.

Animals↗
PubMed1992

Evidence that both normal and immune elimination of Schistosoma mansoni take place at the lung stage of migration prior to parasite death.

The number and distribution of autoradiographic foci observed in this and previous studies following percutaneous infection with 75Se-labeled Schistosoma mansoni cercariae indicate that the lungs are the principal site of worm elimination in both normal mice and mice immunized with irradiated cercariae. It was observed in the present study, however, that the intensities of the autoradiographic foci produced in the lungs during both the normal (early) and immune (late) phases of elimination were identical to those of foci produced in the livers of the same mice by larvae shown to be alive. In contrast, foci produced in the lungs by heat-killed, intravenously injected, lung schistosomula became smaller and fainter with time, disappearing completely between seven and 10 days after injection in normal mice and between four and six days in immunized mice. These results indicate that although the targets of both normal and immune elimination do not proceed beyond the lung stage of migration, they do not die in the lungs. A possible explanation for this paradoxical situation, for which there is some experimental evidence, is that unsuccessful migrators leave the blood stream, enter alveoli, pass up the trachea, and are eventually digested in the gastrointestinal tract or eliminated from the body intact.

Animals↗
PubMed1992

The role of IgG antibodies from irradiated cercaria-immunized rabbits in the passive transfer of immunity to Schistosoma mansoni-infected mice.

Antibodies of the IgG subclass isolated from the sera of rabbits immunized with cercariae subjected to 50 kilorads of gamma irradiation passively provided partial immunity against Schistosoma mansoni challenge in C57B1/6J mice. These mice exhibited reductions in adult worm burdens of 43-61% compared with recipients of normal rabbit antibodies. Passively transferred IgG antibodies were most effective when given 4-7-days postchallenge; they were less effective when given just before challenge, and were totally ineffective when given 15 days postchallenge. It was also shown that the Fc portion of the IgG molecule was important for passive transfer of immunity. Finally, we observed that although some antibodies from irradiated cercaria-immunized rabbits recognized keyhole limpet hemocyanin (KLH), these KLH cross-reacting antibodies were not necessary for successful passive transfer of immunity. Antibodies from a KLH-immunized rabbit also failed to passively protect mice.

Animals↗
PubMed1991

Modification of the coding sequence enhances plant expression of insect control protein genes.

Increased expression of the insect control protein genes of Bacillus thuringiensis in plants has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. The expression of the cryIA(b) gene was compared to partially modified (3% nucleotide difference) and to fully modified (21% nucleotide difference) cryIA(b) and cryIA(c) genes in tobacco and tomato. The modified genes increased the frequency of plants that produced the proteins at quantities sufficient to control insects and dramatically increased the levels of these proteins. Among the most highly expressing transformed plants for each gene, the plants with the partially modified cryIA(b) gene had a 10-fold higher level of insect control protein and plants with the fully modified cryIA(b) had a 100-fold higher level of CryIA(b) protein compared with the wild-type gene. Similar results were obtained with the fully modified cryIA(c) gene in plants. Specific sequences of the partially modified cryIA(b) gene were analyzed for their ability to affect cryIA(b) gene expression in tobacco. The DNA sequence of a single region was identified as important to the improvement of plant expression of the cryIA(b) gene. The increased levels of cryIA(b) mRNA were not directly proportional to the increased levels of CryIA(b) protein in plants transformed with the modified cryIA(b) genes, indicating that the nucleotide sequence of these genes had an effect in improving their translational efficiency in plants.

Bacillus thuringiensis↗
PubMed1990

Regulation of the maltose transport system of Escherichia coli by the glucose-specific enzyme III of the phosphoenolpyruvate-sugar phosphotransferase system. Characterization of inducer exclusion-resistant mutants and reconstitution of inducer exclusion in proteoliposomes.

Maltose transport in Escherichia coli is regulated at the protein level by the glucose-specific enzyme III (IIIglc) of the phosphoenolpyruvate-sugar phosphotransferase system, by a mechanism known as inducer exclusion. We have isolated and characterized four mutants in the maltose transport system, all of which are in malK, which are resistant to inducer exclusion. The mutations in three of these mutants fall within the COOH-terminal domain of MalK and suggest the first reported function for this domain. Two of these are in a region which shows sequence similarity to lacY and melB, both of which are also regulated by IIIglc, and thus may define a IIIglc-binding domain. We have also reconstituted inducer exclusion in proteoliposomes made from membranes overexpressing the maltose permease. Maltose transport is inhibited by 50-60% when IIIglc is included in the intravesicular space. The inhibition is due to a decrease in the Vmax of transport by a factor of 2. IIIglc does not affect the coupling of ATP hydrolysis to maltose transport, since the ratio of ATP hydrolyzed/maltose transported remained constant in the presence and absence of IIIglc. Finally, the Ki for IIIglc was 40 microM, roughly the same as the in vivo concentration of IIIglc.

Amino Acid Sequence↗
PubMed1989

Resistance of Drosophila to cadmium: biochemical factors in resistant and sensitive strains.

With regard to cadmium toxicity, Drosophila strains v;bw and Austin represent extremes in resistance and sensitivity, respectively. Both strains produced metallothionein (MT) in response to Cd ions in their diet. Austin produced more metallothionein than v;bw at Cd ion levels below 0.2 mM, when both strains were allowed lifetime development on Cd2+-containing media. When the rate of MT appearance was measured for 4 days in young adults the results showed no clear trend with time within a strain or between strains. The plot of LC50 vs. MT levels for identical developmental conditions revealed that for v;bw small increases in MT corresponded to large increases in resistance whereas for the sensitive Austin even large increases in MT had comparatively little effect on increasing LC50. Results given here suggest that differences in total MT content do not explain the genetically demonstrable difference in Cd2+-resistance between v;bw and Austin. However, since two MT genes are identified in Drosophila, differences in resistance could be reflective of greater relative amounts of one "more important" MT in the resistant fly.

Animals↗
PubMed1989

Maltose transport in membrane vesicles of Escherichia coli is linked to ATP hydrolysis.

We examined the energy requirement for maltose transport in right-side-out membrane vesicles derived from Escherichia coli. When membrane vesicles were made from strains producing tethered maltose-binding proteins by dilution of spheroplasts into phosphate buffer, those from an F0F1 ATPase-containing (unc+) strain transported maltose in the presence of an exogenous electron donor, such as ascorbate/phenazine methosulfate, at a rate of 1-5 nmol/min per mg of protein, whereas those from an isogenic unc- strain failed to transport maltose. Transport in vesicles obtained from the latter strain could be restored in the presence of electron donors if the vesicles were made to contain NAD+ and either ATP or an ATP-regenerating system. ATP hydrolysis was apparently required for transport, since nonhydrolyzable ATP analogues did not sustain transport. Maltose transport significantly increased ATP hydrolysis in ATP-containing vesicles from unc- cells. Finally, ATP-containing vesicles from unc- strains producing normal maltose-binding proteins could accumulate maltose in the absence of electron donors. These results provide convincing evidence that it is the hydrolysis of ATP that drives maltose transport, and probably also other periplasmic-binding-protein-dependent transport systems.

Adenosine Triphosphate↗
PubMed1989

Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process.

ATP-Binding Cassette Transporters↗
PubMed1987

Nucleotide sequence analysis of alpha-amylase and thiol protease genes that are hormonally regulated in barley aleurone cells.

We have determined the nucleotide sequences of Amy32b, a type A alpha-amylase gene, and of the gene for aleurain, a thiol protease closely related to mammalian cathepsin H. Both are expressed in barley aleurone cells under control of the plant hormones gibberellic acid and abscisic acid, but only aleurain is expressed at high levels in other barley tissues. Sequence analysis indicates that the 5' end of the aleurain gene, comprising 3 exons and 2 introns, may have become associated with the remainder of the gene, encoding the protease domain of the protein, by some sort of recombination event. This 5' domain of the gene is very G + C-rich and is flanked by inverted repetitive sequences. We found two different groups of homologous sequence elements. The first group consists of four blocks of sequences conserved in the same spatial arrangement in both genes; these are arranged at similar intervals upstream from the Amy32b TATA box and from a TATA box present in intron 3 of aleurain, outside of the 5' domain and upstream from the protease domain. A part of two of these conserved sequences is similar to the core sequence of certain enhancer elements characterized from mammalian cells. The second group of homologous elements is present in the upstream region of both genes. We speculate that these conserved sets of sequences may have some role in either the tissue specificity of expression of the genes or in some part of the hormonal regulation imposed on them.

Abscisic Acid↗
PubMed1987

Schistosoma mansoni: mechanism of attrition and routes of migration from lungs to hepatic portal system in the laboratory mouse.

The number of schistosomula in the axillary lymph nodes of mice was determined by compressed tissue autoradiography at 13 intervals from 0.5 to 28 days after exposure of abdominal skin to 75Se-labeled cercariae of S. mansoni. Significant accumulations were observed between days 3 and 6 and peaked on day 4 at which time 9.4 +/- 1.1% of the schistosomula present in the whole body were found in the axillary lymph nodes. The total number and distribution of schistosomula in all tissues of mice were likewise determined at 12 intervals from 3 to 24 days following exposure. The frequent appearance of small numbers of schistosomula in trachea and esophagus suggested that normal attrition resulted at least in part from physical expulsion of schistosomula from the body by way of the tracheobronchial tree and gastrointestinal tract. The distribution of schistosomula observed in heart chambers, caudal vena cava, hepatic portal vein, aorta, intestinal wall, thoracic cavity rinses, and diaphragm supported all 3 standing hypotheses regarding route of migration from lungs to hepatic portal system, i.e., that schistosomula migrate via (1) the pulmonary artery, right heart, caudal vena cava, and hepatic veins, (2) the pulmonary vein, left heart, aorta, and cranial mesenteric artery, and (3) the thoracic cavity and diaphragm.

Animals↗
PubMed1987

Intraspecific cross-protection in mice immunized with irradiated Schistosoma mansoni cercariae.

Several laboratory-maintained strains of Schistosoma mansoni were tested for their relative immunogenicity or susceptibility to anti-schistosome immunity in irradiated cercaria-immunized mice. A total of 11 strains and substrains were used; 7 were of Puerto Rican origin, 3 from Brazil, and 1 from Egypt. Mice were immunized by percutaneous exposure to 50-krad-irradiated cercariae. Immunity was assessed following challenge with cercariae of the homologous or a heterologous strain. The results showed that the choice of either the challenge or immunizing strains was not critical in the development of significant levels of protection. Extensive degrees of cross-protection developed in all intrastrain combinations tested.

Animals↗
PubMed1986

Identification of Schistosoma mansoni glycoproteins recognized by protective antibodies from mice immunized with irradiated cercariae.

The humoral immune responses of mice patently infected with Schistosoma mansoni and of mice vaccinated with radiation-attenuated cercariae were compared by radioimmunoassays and one- and two-dimensional polyacrylamide gel analyses of radioimmunoprecipitates. The binding observed with antibodies of mice vaccinated twice with radiation-attenuated cercariae over a period of 7 to 11 wk was less than 50% of the binding observed with antibodies of mice patently infected for 20 wk, but three to four times greater than that obtained with antibodies of mice infected for 6 wk, irrespective of whether the test antigen extracts were derived from schistosomula or adult worms. Sera of vaccinated mice precipitated a restricted number of predominantly high m.w. glycoproteins of both schistosomula and adult worms metabolically labeled with [35S] methionine. Each of the glycoproteins of 36 hr in vitro-cultured schistosomula that was precipitated by the sera of vaccinated mice was also precipitated by sera of infected mice. In contrast, sera of vaccinated mice uniquely precipitated a 38,000 m.w. glycoprotein of schistosomula cultured for 5 days and a 94,000 m.w. glycoprotein of adult male worms. Although radiation-attenuated larvae do not reach the adult stage, mice vaccinated with these still elicit a strong immune response against egg glycoproteins. In particular, an egg glycoprotein of 85,000 to 70,000 and isoelectric point of 4.8 showed an enhanced reactivity with sera of vaccinated mice in comparison with infected mice. These results show that the antibody response in mice vaccinated with radiation-attenuated larvae differs qualitatively and quantitatively from that of infected mice.

Acute Disease↗
PubMed1986

Passive transfer with serum and IgG antibodies of irradiated cercaria-induced resistance against Schistosoma mansoni in mice.

The role of humoral immunity to Schistosoma mansoni infection in C57BL/6J mice was examined by employing a passive transfer system. Sera from highly resistant mice that had been exposed to two or three immunizations with 50-kilorad-gamma-irradiated cercariae were tested for their ability to transfer protection against S. mansoni challenge. All five batches of serum tested were observed to have protective activity. Immune serum recipients exhibited statistically significant reductions in challenge worm burdens of 20 to 50% compared with recipients of normal serum or no serum. The most consistent level of resistance was obtained when immune serum was administered several days post-challenge, i.e., at a time coincident with schistosomulum residence in the lungs. Furthermore, it was shown that the protective activity in immune serum was associated with factors that bind to staphylococcal protein A and that are precipitated by 50% ammonium sulfate; thus it appears that the protective factors in immune serum are IgG antibodies.

Animals↗