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Biomedical subjects

B A Webb

Publications and source records attributed to B A Webb.

35 records · Page 2Linked to original sources
PubMed1996

Expression of polydnavirus genes under polydnavirus promoter regulation in insect larvae infected with baculovirus recombinants.

We have evaluated the use of baculoviruses to deliver Campoletis sonorensis polydnavirus (CsPDV) genomic DNA into lepidopteran larvae to facilitate the identification of functional CsPDV genes. Genomic fragments consisting of regulatory (promoter) and coding sequences for two CsPDV genes (VHv1.1 and WHv1.6) were used to generate CsPDV-baculovirus recombinants and evaluate the expression of genes under the regulation of the CsPDV promoters. Northern blot and primer extension studies established that CsPDV genes were expressed under the control of their own promoters in these CsPDV-baculovirus recombinants. Transcripts were detected as early as 4 h post-infection indicating that temporal activity of CsPDV promoters was retained. The VHv1.1 gene product as expressed from CsPDV-baculovirus recombinants was identical in size and in functional properties to that produced in CsPDV-infected insects. CsPDV-baculovirus recombinants may be useful for the screening and characterization of polydnavirus genes with functional activities that can only be evaluated in insect larvae.

Animals↗
PubMed1994

Evidence for an early immunosuppressive role for related Campoletis sonorensis venom and ovarian proteins in Heliothis virescens.

Shared epitopes among venom, ovarian, and viral proteins may indicate that related proteins have similar functional roles during parasitization of Heliothis virescens by Campoletis sonorensis. Venom and ovarian proteins are introduced directly into the hemolymph during parasitization where they may target hemocytes or other components of the immune system. Polydnavirus expression has been detected in hemocytes, fat body, and other tissues but has not been detected earlier than 4 h after parasitization. Therefore, effects on hemocytes at times earlier than 4 h may not be caused by polydnavirus proteins synthesized in the parasitized insect. Visualization of hemocyte F-actin with fluorescently labeled phallicidin indicated that a dramatic alteration of plasmatocyte and granulocyte cytoskeletons occurred within 1.5 h after parasitization. The predominant non-viral proteins in the ovary introduced during parasitization were immunologically related to venom and viral envelope proteins. These ovarian proteins persist in the hemolymph. Antisera to the ovarian proteins bound to granulocytes and to plasmatocytes to a lesser degree, suggesting that ovarian proteins may be involved in early suppression of the host's immune response after parasitization.

Animals↗
PubMed1994

Polydnavirus infection inhibits synthesis of an insect plasma protein, arylphorin.

The wasp Campoletis sonorensis injects a segmented, double-stranded DNA polydnavirus (CsPDV) along with its egg during parasitization of Heliothis virescens larvae. After parasitization, CsPDV protects the wasp egg and larva by selectively disabling the host's cellular immune response. Other host physiological systems including growth and development are affected to the apparent benefit of the parasite. To begin the characterization of the biochemical effects and mode of action of CsPDV on host growth, the titre of a developmentally regulated insect storage protein, arylphorin, was studied. Parasitized or virus-infected insects had substantially less circulating arylphorin than control insects. Fat bodies from parasitized larvae also synthesized less arylphorin in vitro. However, Northern blots of total RNA from parasitized and non-parasitized, control insects showed that the arylphorin transcript level was unaffected by parasitization suggesting a biochemical block at the translational level. In vitro translation followed by immunoprecipitation of arylphorin indicated that the mRNA was present and translatable at equal levels in both parasitized and control insects. Injection of purified virus elicited the response observed in naturally parasitized larvae, demonstrating that the effect on arylphorin synthesis is mediated, either directly or indirectly, by polydnavirus gene product(s).

Animals↗
PubMed1994

Apparent functional role for a cysteine-rich polydnavirus protein in suppression of the insect cellular immune response.

Polydnaviruses suppress the cellular immune response and inhibit growth and development in their lepidopteran host, allowing survival of their endoparasitic hymenopteran host. Characterization of genes disrupting insect physiological systems is a major objective in the study of polydnaviruses. Recently, a cysteine-rich gene family encoding a motif composed of invariable cysteine residues flanking hypervariable intercysteine amino acids was described (S.D. Dib-Hajj, B.A. Webb, and M.D. Summers, Proc. Natl. Acad. Sci. USA 90:3765-3769, 1993). They noted similarities to the positive selection pressure for mutations within the vertebrate major histocompatibility complex (MHC) class II genes and speculated that this class of polydnavirus genes may target and disrupt the insect immune system. To study the functional activity of this family of predicted cysteine-rich proteins, the VHv1.1 gene product was produced from bacterial and baculovirus expression systems. Polyclonal antiserum produced from the bacterial fusion protein reacted with a 30-kDa protein from hemocytes, cell-free plasma, and fat body of parasitized larvae. Immunofluorescence analysis of hemocytes from parasitized insects detected the 30-kDa protein bound to granulocytes and plasmacytes. To assay the functional activity of the 30-kDa VHv1.1 protein, a recombinant baculovirus was constructed allowing in vivo expression of the 30-kDa polydnavirus protein from infected insects. Expression of the VHv1.1 protein from the baculovirus system reduced the encapsulation response to washed wasp eggs relative to controls. The experimental evidence demonstrates that Campoletis sonorensis polydnavirus-infected cells secrete VHv1.1 into the hemolymph, where it binds to hemocytes and is associated with the inhibition of the cellular immune response.

Animals↗
PubMed1993

Structure and evolutionary implications of a "cysteine-rich" Campoletis sonorensis polydnavirus gene family.

For successful parasitization, the female Campoletis sonorensis endoparasitic wasp injects a polydnavirus into its host, Heliothis virescens, during oviposition. Viral gene expression induces immunosuppression and alters development of the host. We report here that three abundantly expressed genes, VHv1.1, WHv1.0, and WHv1.6, describes a polydnavirus "cysteine-rich" gene family which may be important in inducing these host manifestations. These genes have a similar primary gene structure and their proteins contain cysteine motifs characteristic of snail ion-channel ligands, the omega-conotoxins. Like the omega-conotoxins, the intercysteine amino acid residues are hypervariable with only three identical amino acids in all motifs. The conservation of this domain in the three viral genes may reflect an important functional role for these viral proteins in the parasitization of H. virescens. The three genes also contain introns similar in sequence at comparable positions in their 5' untranslated leaders and coding sequences. VHv1.1 contains two cysteine motifs, and each motif is interrupted by an intron at the same position as in the cysteine motifs of WHv1.0 and WHv1.6. Intron 2 sequences of WHv1.0 and WHv1.6 are 92% identical, while the immediately flanking exon sequences encoding the cysteine motifs are only 76% identical. This provides an example of nuclear pre-mRNA introns which are more conserved than flanking exons among members of a gene family.

Amino Acid Sequence↗
PubMed1992

Stimulation of polydnavirus replication by 20-hydroxyecdysone.

During oviposition the endoparasitic wasp Campoletis sonorensis, introduces a polydnavirus into parasitized insects where viral gene expression is required for endoparasite survival. The polydnavirus is integrated into wasp chromosomal DNA and replicates only in the ovary. Ecdysteroids regulate the developmental expression of many insect genes and may regulate polydnavirus replication. Direct verification of viral replication was performed by dot blot hybridization and by amplifying DNA sequences containing the viral integration site; this 'junction' fragment cannot be amplified from integrated virus. Thoracic ligations and in vitro ecdysteroid treatments of wasp ovaries support the hypothesis that polydnavirus DNA replication is regulated by ecdysteroid during parasite development.

Animals↗
PubMed1990

Developmental expression, synthesis, and secretion of insecticyanin by the epidermis of the tobacco hornworm, Manduca sexta.

Insecticyanin was found to be synthesized in several isoelectric forms and stored in the pigment granules in the epidermis. Both major epidermal forms (INS-a, pl 5.5; INS-b, pl 5.7) were found in the cuticle, but only the most basic form, INS-b, was present in the hemolymph. In vitro the epidermis synthesized and secreted both forms into both the cuticle and the medium. Isolation of two cDNA clones for insecticyanin followed by hybridization to epidermal mRNA showed the presence of only one 1.1 kb mRNA, but transcription of the longer cDNA yielded a RNA which produced INS-a but no INS-b. Insecticyanin mRNA was present during the intermolt feeding stages of the 4th and 5th instars and absent during the larval molt and after the onset of metamorphosis. Exposure of either day 2 4th-instar or day 1 5th-instar larval epidermis to 20-hydroxyecdysone (20HE) in vitro caused a dose-dependent decline in this mRNA that was not prevented by simultaneous exposure to JH. When synthesis resumes just before ecdysis, INS-b appears before INS-a; then on the final day of feeding, synthesis of INS-a ceases before that of INS-b. Culture experiments showed that exposure of day 15th epidermis to a small pulse of 20HE followed by an ecdysteroid-free period was best to mimic the in vivo situation, indicating that the small rise of ecdysteroid in the absence of JH on day-2 was responsible for this differential cessation.

Animals↗
PubMed1990

Venom and viral expression products of the endoparasitic wasp Campoletis sonorensis share epitopes and related sequences.

Endoparasitic wasps of lepidopteran insects must induce changes in host immunity and development to survive. Depending on the species, this may require wasp venom proteins and/or a polydnavirus. We describe an immunological and genetic relationship between the Campoletis sonorensis polydnavirus and the wasp's venom gland. Monoclonal antibodies raised against venom glands recognized epitopes conserved on several polydnavirus proteins and on multiple wasp oviduct and venom proteins. The viral envelope proteins had molecular masses of 16, 20, 45, and 50 kDa, while a complex of at least five immunoreactive venom-gland and soluble oviduct proteins ranged in size from 24 to 36 kDa. Since the conserved epitopes were present on the viral envelope, neutralization assays were performed. Monoclonal antibodies added to purified virus blocked the normal viral inhibition of host growth and development. To determine whether venom mRNA and viral genes were also related, venom-related cDNA clones were isolated from the wasp oviduct with a venom-gland cDNA probe. Venom-related viral clones were then identified and selected from a viral genomic library and from a parasitized Heliothis virescens cDNA library. Venom-related mRNAs were expressed in the venom gland, the oviduct, and the parasitized host. We propose that the immunological relationship between venom and viral proteins, and the hybridization of venom and viral genes, may reflect an evolutionary relationship in which venom gene homologs were incorporated into the viral genome, thereby allowing viral expression of venom-related genes and enhancing parasite survival.

Animals↗
PubMed1988

Iron binding proteins and their roles in the tobacco hornworm, Manduca sexta (L.).

Manduca sexta larvae accumulate large amounts of iron during their larval feeding period. When 59Fe was fed to 5th instar larvae, it was evenly distributed among the hemolymph, gut and carcass until the cessation of feeding. By pupation 95% of the labelled iron was found in the fat body. In the adult a significant portion of this iron was found in flight muscle. Studies of the hemolymph disclosed two iron-containing proteins. The first was composed of a single polypeptide chain of 80 kD, containing one atom of iron. This protein bound ionic iron in vitro and was able to transfer this iron to ferritin when incubated with fat body in vitro. Therefore, it appeared to serve a transport function. The second protein had a molecular weight of 490 kD with subunits of 24 and 26 kD and contained 220 micrograms of iron/mg protein. Its chemical and ultrastructural characteristics were those of ferritin. These studies demonstrate the presence of both a transport protein and a unique circulating ferritin in Manduca sexta, the latter serving a storage function during development and possibly also a transport function.

Animals↗
PubMed1988

Synthesis of two storage proteins during larval development of the tobacco hornworm, Manduca sexta.

Studies of synthesis and accumulation of the two storage proteins arylphorin and female-specific protein (FSP) during the final two larval instars of the tobacco hornworm showed both stage and temporal specificity. Arylphorin was present in both stages, but its synthesis ceased during the molt, during starvation, and at the wandering stage, and then resumed about 24 hr after the onset of feeding. During the larval molt about 25% of injected iodinated arylphorin was incorporated into the newly forming fifth instar cuticle. The cessation of arylphorin synthesis was mimicked by exposure of the fat body to 1 microgram/ml 20-hydroxyecdysone (20HE) in complete Grace's medium or to dilutions of Grace's medium greater than 50%. Lower concentrations of 20HE were ineffective, indicating that the cessation of synthesis in vivo was likely due to a combination of lack of excess nutrients and the hormonal milieu. The female-specific protein was not synthesized until the final larval instar, appearing first in females on Day 2 and later in males at the time of wandering, with synthesis continuing throughout the prepupal period. In vitro studies showed that this protein was synthesized as a 620-kDa protein, and then during secretion a 730-kDa immunoreactive form also appeared. Synthesis of FSP was inhibited by exposure of Day 2 fat body to 1 microgram/ml 20HE for 24 hr. Ligation followed by 20HE infusion showed that the disappearance of FSP from the hemolymph during the prepupal period was controlled by the rising ecdysteroid titer.

Adipose Tissue↗
PubMed1988

Regulation of expression of arylphorin and female-specific protein mRNAs in the tobacco hornworm, Manduca sexta.

Two non-cross-hybridizing cDNA clones were isolated from a lambda gt11 cDNA library prepared from Day 2 fifth instar female fat body of Manduca sexta and shown by hybrid selection to code respectively for the two storage proteins arylphorin and female-specific protein (FSP). Analysis of the developmental expression of arylphorin showed its presence during the feeding phases of the penultimate (fourth) and final (fifth) larval instars and its absence during the molt. Abdominal ligation of larvae followed by infusion of Grace's medium showed that this amino acid-rich medium was able to maintain arylphorin expression in fourth instar larvae, but not continued high expression in fifth instar larvae. This nutrient medium however was sufficient to allow initiation of expression in newly ecdysed fifth larval abdomens. Infusion of 5 micrograms 20-hydroxyecdysone (20HE) caused a significant reduction of arylphorin RNA in ligated fourth larval abdomens, whereas 50 micrograms was required in Day 2 fifth larval abdomens to suppress this RNA. Thus, both the lack of incoming nutrients and the rising titer of ecdysteroid contribute to the loss of arylphorin mRNA at the molts and at wandering. By contrast, FSP mRNA was first detected in females on Day 2 of the fifth instar, but not in males until wandering, and then was present throughout the prepupal period. In females allatectomy caused the precocious appearance of FSP mRNA which was prevented by application of 10 micrograms methoprene, a juvenile hormone analog. Expression of FSP mRNA in males however appeared to be independent of hormonal milieu.

Adipose Tissue↗
PubMed1988

Identification of factors limiting the accurate measurement of plasma D-penicillamine in rheumatoid arthritis patients.

Free and total reduced concentrations of D-penicillamine have been measured in the plasma of rheumatoid arthritis patients by HPLC and electro-chemical detection. A reverse-phase ion-pair separation in conjunction with a dual porous graphite electrode satisfied the requirements of robustness, sensitivity, selectivity and suitable retention time. Plasma levels measured between 1.5 and 3 h after an oral dose, were less than 0.3 to 57.6 mumol/L and 0.6 to 85.0 mumol/L (n = 26) for free and total reduced drug concentrations, respectively. Sources of error in the accurate measurement of peak plasma D-penicillamine levels were identified as oxidative loss and alteration in the free to protein-bound ratio in the period following sample collection.

Arthritis, Rheumatoid↗
PubMed1983

A potential systematic error in using lysivane as inhibitor in the measurement of amniotic fluid acetylcholinesterase by the Ellman method.

The measurement of low levels of cholinesterase or acetylcholinesterase by the Ellman method requires correction for a non-enzymatic increase in absorption at 412 millimicron that is due both to non-enzymatic hydrolysis of the acetylthiocholine substrate and to modification of the colour reagent. The rate of increase in absorption is dependent on temperature and pH. Addition of an acidic solution of lysivane to the assay solution for selective measurement of amniotic fluid acetylcholinesterase gives rise to a shift in pH; the use of methanol is suggested as an easier method of dissolving the inhibitor and does not affect the pH of the assay, obviating any need to redetermine the background absorption. There is, however, no improvement in ability of the method to predict pregnancies associated with neural-tube defects.

Acetylcholinesterase↗
PubMedDate not supplied

Interaction of a wasp ovarian protein and polydnavirus in host immune suppression.

During parasitization of Heliothis virescens, Campoletis sonorensis deposits an egg along with venom, polydnavirus particles (CsPDV), and ovarian proteins (OPs). Oviposited eggs are not encapsulated, while washed eggs are encapsulated rapidly by H. virescens. Early protection from encapsulation is afforded by a group of 29-36 kD OP glycoforms. These glycoforms are endocytosed by host hemocytes within 30 min post-parasitization (pp) and disrupt hemocyte spreading behavior and egg encapsulation through at least 24 h p.i. Purified CsPDV does not protect eggs from encapsulation early, but disrupts hemocyte spreading and egg encapsulation from 24 h through at least 5 days p.i. Functional activity of CsPDV appears to be correlated with time-dependent accumulation of virus-specific proteins in parasitized insects. We propose that the fast-acting 29-36 kD OP protects Campoletis eggs from encapsulation until accumulation of CsPDV proteins which sustain immunosuppression.

Animals↗