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P Willadsen

Publications and source records attributed to P Willadsen.

At least 19 recordsLinked to original sources
PubMed1999

Peritrophic matrix proteins.

The peritrophic matrix (or peritrophic membrane) lines the gut of most insects at one or more stages of the life cycle. It has important roles in the facilitation of the digestive processes in the gut and the protection of the insect from invasion by microorganisms and parasites. The traditional view of the peritrophic matrix as a relatively insert sieve, composed largely of proteins and glycosaminoglycans embedded in a chitinous matrix, is under revision as more is learned about the molecular characteristics of the peritrophic matrix proteins. This review summarizes emerging knowledge of the main protein constituents of the peritrophic matrix. The availability of the first sequences of integral peritrophic matrix proteins has coincided with the explosion of information in sequence databases. It is therefore possible to examine common structural themes in this family of proteins as well as in proteins of unknown location and function from a variety of other insects, nematodes and viruses. The review concludes with speculation about the biological functions of the proteins in this matrix.

Animals↗
PubMed1998

Vaccination of cattle against the Boophilus microplus using a mucin-like membrane glycoprotein.

An antigen, BMA7, which induced partial immunity against tick infestation has been isolated from Boophilus microplus using two different protein fractionation protocols, accompanied by vaccination and parasite challenge trails. The antigen is a 63 kDa glycoprotein isolated from semi-engorged adult female ticks. Though significant, the induced immunity is less striking than that previously reported for antigen Bm86 from the same parasite. However, co-vaccination with Bm86 and BMA7 can enhance immunity over that seen with a commercial vaccine based on Bm86 alone. Limited peptide sequence information shows significant variation in the BMA7 protein occurs. The antigen has approximately 36 kDa of glycosylation, in both N-linked and O-linked oligosaccharides. There is evidence that both polypeptide and oligosaccharide are antigenic, but the chemical nature of the protective antigenic sites is not clear. There is little or no immunological response to the antigen during natural infestation with parasites, suggesting the antigen is 'concealed' and protective immunity dependent on artificial vaccination. The antigen has some similarities with the vertebrate mucins. It is widely distributed in tick tissues and membrane bound but its function is currently unknown.

Amino Acid Sequence↗
PubMed1997

Novel vaccines for ectoparasites.

Novel vaccines against ectoparasites have the potential to be cost-effective new technology for pest control that avoids some of the real and perceived problems with insecticide and acaricide usage. Nevertheless, their development is in its infancy. A vaccine against the cattle tick Boophilus microplus, the world's first vaccine against an ectoparasite, is in field use in Australia. Considerable effort had gone into the development of a vaccine against the sheep blowfly Lucilia cuprina, while other vaccines are at an earlier stage of development. The identification of critical antigens and their production as effective recombinant proteins remains the greatest hurdle. Characteristics of the few known antigens and the mode of action of the protective immune response are discussed. Development of further vaccines will depend on recognition of likely antigenic targets. The efficacy of such vaccines will depend on the characteristics of the target species, in particular its digestive biology and the way in which the novel vaccine impacts on the parasite population.

Animals↗
PubMed1996

Cloning and characterisation of angiotensin-converting enzyme from the dipteran species, Haematobia irritans exigua, and its expression in the maturing male reproductive system.

The angiotensin-converting enzymes (ACE) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. A carboxydipeptidase similar to mammalian ACE has now been identified in the adult stage of the haematophagous fly, Haematobia irritans exigua (buffalo fly), a close relative of the horn fly of North America. The enzyme was purified by lectin-affinity chromatography and ion-exchange chromatography and migrated as a doublet of 70 kDa upon reducing SDS/PAGE. Unlike mammalian ACE, the fly carboxydipeptidase (HieACE) is not membrane bound. The amino acid sequence of an internal peptide from HieACE and a conserved amino acid region present in all mammalian ACE were used to design degenerate oligonucleotide primers suitable for PCR. A DNA fragment amplified from adult buffalo fly cDNA was used to identify a cDNA clone that encoded the enzyme. The cDNA sequence encodes a carboxydipeptidase with 41-42% amino acid identity to the mammalian testicular ACE. The active-site regions of mammalian ACE are conserved in the deduced amino acid sequence of HieACE. Enzymatically, HieACE is very similar to its mammalian counterparts, with comparable Km and V(max) values for the synthetic substrate, benzoylglycylglycylglycine, and similar patterns of inhibition by EDTA, ACE inhibitor peptide and captopril. HieACE also specifically activates angiotensin I to angiotensin II and degrades other mammalian ACE substrates such as bradykinin, substance P and cholecystokinin-8. In the adult fly, HieACE is expressed in the compound ganglion and in the posterior region of the midgut. Similar to the mammalian system, expression of this enzyme is induced in the maturing male reproductive system, which suggests conservation of ACE function in these species.

Amino Acid Sequence↗
PubMed1996

Endocytosis by digest cells of the cattle tick Boophilus microplus: regulation by protein kinase C.

Endocytosis of fluorescently-labeled bovine serum albumin by digest cells of the gut of the cattle tick Boophilus microplus is inhibited by approx 60% in the presence of the tumour promoter 12-O-tetradecanoylphorbol 13-acetate. The results are consistent with a role for protein kinase C in regulating the uptake of blood meal by digest cells. Protein kinase C activity has been measured in the digest cell and the amount of enzyme has also been determined using a phorbol ester binding assay. The presence of a small number of specific protein kinase C substrates in the plasma membrane of the digest cell has been demonstrated. Preliminary experiments indicate that one of these substrates, a protein of approximately 30 kDa, is an integral membrane protein, part of which is exposed on the extracellular surface of the digest cell.

Amino Acid Sequence↗
PubMed1996

Comparative vaccination of cattle against Boophilus microplus with recombinant antigen Bm86 alone or in combination with recombinant Bm91.

Cattle were vaccinated either with a single recombinant tick antigen, Bm86 or with a combination of two recombinant antigens, Bm86 and Bm91 from the tick Boophilus microplus. In three experiments, the responses of cattle to subsequent challenge with the tick were assessed. The addition of the Bm91 antigen enhanced the efficacy of the vaccination over that with Bm86 alone to a statistically significant degree. Moreover, co-vaccination with two antigens did not impair the response of cattle to the Bm86 antigen. Finally, responses of individual cattle to the two antigens were independent. All of these results may be relevant to the increase in efficacy expected from a dual antigen vaccine.

Animals↗
PubMed1995

Carboxydipeptidase from Boophilus microplus: a "concealed" antigen with similarity to angiotensin-converting enzyme.

A protein, Bm91, which was first identified as a protective vaccine antigen from the tick Boophilus microplus, has regions of very strong amino acid sequence similarity to mammalian carboxydipeptidases or angiotensin converting enzymes (ACE; E.C. 3.4.15.1). This protein is now shown to share many biochemical and enzymatic properties with mammalian carboxydipeptidases. It is enzymatically active in a conventional assay for ACE using hippuryl-Gly-Gly as substrate. The hydrolysis of the C-terminal nonapeptide of the insulin B chain proceeds by sequential removal of carboxy-terminal dipeptides. The similarities extend to the dependence of activity on pH and added salt. Bm91 is inhibited by two well-characterized inhibitors of the mammalian enzymes, the drug Captopril and a nonapeptide, and the inhibition occurs in similar concentration ranges to those effective with the mammalian enzymes. However, the natural substrates of the tick enzyme are unknown. Angiotensin I itself is a poor substrate and the enzyme's natural substrates are likely to be one or more of the pharmacologically active peptides occurring in insects and arthropods.

Amino Acid Sequence↗
PubMed1995

Commercialisation of a recombinant vaccine against Boophilus microplus.

Increasingly, there is need for methods to control cattle tick (Boophilus microplus) infestations by the use of non-chemical technology. This need is brought about by a mixture of market forces and the failure or inadequacy of existing technology. A recombinant vaccine has now been developed against the tick. This vaccine relies on the uptake with the blood meal of antibody directed against a critical protein in the tick gut. The isolation of the vaccine antigen, Bm86, and its production as a recombinant protein is briefly described. The vaccine has been tested in the field, has been taken through the full registration process and is now in commercial use in Australia. A related development has occurred in Cuba. The potential for improvement of the current vaccine and for the development of similar vaccines against other haematophagous parasites is discussed.

Administration, Oral↗
PubMed1994

A protective "concealed" antigen from Boophilus microplus. Purification, localization, and possible function.

A membrane protein that can be used successfully to vaccinate cattle against the tick Boophilus microplus has been purified and characterized. The mature protein, which is referred to as Bm91, has an apparent m.w. of 86,000, an isoelectric point of 4.8 to 5.2, and is glycosylated, with an affinity for lentil lectin. Bm91 is of relatively low abundance, with approximately 300 to 400 micrograms being recovered from 1 kg of semiengorged adult female ticks. The protein is located largely in the salivary gland and gut of these ticks. Partial amino acid sequence data for the protein show striking similarities to that of mammalian angiotensin-converting enzyme, suggesting that the Ag may have an enzymatic function. The protein seems not to be recognized by sera from cattle with extensive exposure to ticks under natural conditions. The immunity induced by vaccination, therefore, represents another example of vaccination against a hematophagous parasite with "concealed" Ags.

Amino Acid Sequence↗
PubMed1994

Prostaglandin E2 production by the cattle tick (Boophilus microplus) into feeding sites and its effect on the response of bovine mononuclear cells to mitogen.

Prostaglandin E2 (PGE2) secretion by the cattle tick Boophilus microplus into feeding sites was quantified. It was detected by the in vitro tube feeding experiment and it was determined that a semi-engorged female tick could produce and transmit 1.8 ng PGE2 into the feeding site. Using the in vitro membrane feeding system, newly molted adult ticks were also shown to secrete 0.04-0.15 ng PGE2 into the feeding site; however, female ticks produced more PGE2 than male ticks. The immune suppressive effect of PGE2 in the saliva of B. microplus on the bovine mononuclear cells (MNC) was also examined. PGE2 in the saliva was suspected of being a major component that inhibited the blastogenic response of MNC to a T-cell mitogen phytohemagglutinin. As bovine MNC are sensitive to low level concentration of PGE2, the PGE2 transmitted into feeding sites was suspected to be sufficient to produce physiological effects on the bovine host.

Animals↗
PubMed1994

Comparison of prostaglandin E2 (PGE2) in salivary gland of Boophilus microplus, Haemaphysalis longicornis and Ixodes holocyclus, and quantification of PGE2 in saliva, hemolymph, ovary and gut of B. microplus.

The amount of prostaglandin E2 (PGE2) in salivary gland of semi-engorged adult female Boophilus microplus, Haemaphysalis longicornis and Ixodes holocyclus were 374.3 pg, 427.0 pg and 825.0 pg per one tick, respectively. It was thought that the PGE2 production is a common phenomenon among ticks. Then PGE2 concentrations in saliva and hemolymph, salivary gland, ovary and gut of fully-engorged adult female B. microplus were compared. The PGE2 concentration in saliva induced by pilocarpine was 40.3 ng/ml and hemolymph was 19.1 ng/ml. Salivary gland, ovary and gut from a tick contains 35.5 pg., 27.0 pg and 2.5 ng of PGE2, respectively.

Animals↗
PubMed1994

Histamine receptors on bovine peripheral blood lymphocytes.

Histamine receptors on bovine peripheral blood lymphocytes (PBL) were detected by three different methods: a rosetting technique, binding to histamine-bearing Sepharose beads and immunofluorescence staining. The rosetting technique used histamine-rabbit serum albumin (H-RSA) conjugated to bovine red blood cells to detect histamine receptors and this showed that 10.8% of bovine PBL were positive. A method using H-RSA conjugate coupled Sepharose beads also detected histamine receptor bearing PBL but was not quantitative. The indirect immunofluorescence method, by which the subpopulation of histamine receptor bearing lymphocytes can be easily double stained to concurrently identify the B cell marker, revealed that PBL, the B cell and T cell fraction of bovine PBL contained 18.4, 52.8 and 9.3% histamine receptor bearing cells, respectively. This method was found to be more stable and more easily quantifiable than the other two methods. Blocking tests using the histamine H1 receptor antagonist diphenhydramine and the histamine H2 receptor antagonist cimetidine suggested that bovine PBL have both H1 and H2 receptors on their surfaces. Addition of histamine into cultures of PBL at the concentration range 10(-6) to 10(-3) M suppressed the response of PBL to the mitogen phytohemagglutinin. The histamine induced suppression of mitogenesis could be reduced partially by the H2 receptor antagonist cimetidine, but not by the H1 antagonist diphenhydramine. It is possible that histamine induced suppression of PBL mitogenesis was mediated by H2 receptors on T cells.

Animals↗
PubMed1993

Effects of cattle tick (Boophilus microplus) infestation on the bovine immune system.

The immunosuppressive effect of experimental Boophilus microplus infestation on bovine peripheral blood lymphocytes (PBL) and on host antibody production to a protein antigen (ovalbumin) was examined. Boophilus microplus infestation caused a marginal decrease in the percentage of T lymphocytes in PBL, which was observed in both lightly (5000 larvae) and heavily (40,000 larvae) infested cattle, and began at the second infestation and continued until the end of the fourth infestation. The percentage of B lymphocytes in heavily tick-infested cattle was less than that in non-infested control cattle after the fourth infestation. The response of PBL from tick-infested cattle to phytohemagglutinin (PHA) was always less than that of tick-free cattle after the second infestation. No noteworthy differences were detected between the three stages of tick infestation, that is, 1 week before the peak of adult engorgement, the middle of the peak and 1 week after all ticks had dropped. Boophilus microplus saliva (100 microliters ml-1) suppressed 47% of the response of bovine PBL to PHA in vitro. This suppressive effect of saliva may contribute to the lower responsiveness of PBL from tick-infested cattle. Antibody production by tick-infested cattle was examined during the third and fourth heavy tick infestation. Tick-infested cattle showed a diminished response against ovalbumin after the second immunization. The immunosuppressive effects of tick infestation may play an important role in tick survival or in the transmission of tick-borne diseases in the field.

Animals↗
PubMed1993

The nucleotidase of Boophilus microplus and its relationship to enzymes from the rat and Escherichia coli.

Boophilus microplus contains a nucleotidase-like enzyme which is able to hydrolyze a range of nucleoside 5'-mono-, di- and triphosphates to the nucleoside. Its relationship to several other nucleotide hydrolyzing enzymes has been explored. Limited peptide sequencing shows similarities to both mammalian nucleotidases and the Escherichia coli uridine diphosphate sugar hydrolase. The tick enzyme also hydrolyzes UDP-glucose, though by a mechanism different to that of the bacterial enzyme. On the other hand, it resembles the mammalian nucleotidases in that there is evidence that it is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor.

5'-Nucleotidase↗
PubMed1992

Successful vaccination against Boophilus microplus and Babesia bovis using recombinant antigens.

Current methods for the control of the cattle tick Boophilus microplus and the agent of bovine babesiosis, Babesia bovis are unsatisfactory. Effective immunological control of both parasites would have great advantages. However, naturally acquired immunity to the tick is generally unable to prevent serious production losses. A vaccine against the tick, based on a novel form of immunization, is being developed. A protective antigen has been isolated from the tick, characterized and produced as an effective, recombinant protein. A vaccine incorporating this antigen is currently undergoing field trials. In the Australian situation, improved tick control will probably increase endemic instability with respect to B. bovis. Fortunately, a trivalent, recombinant B. bovis vaccine has also been developed. This too is now undergoing pre-registration field trials.

Animals↗
PubMed1991

Vaccination with 'concealed' antigens: myth or reality?

Cattle infested with the tick Boophilus microplus produce antibodies to intrinsic membrane glycoproteins of the tick, as well as to Bm86, a well characterized antigen from the tick gut. Several factors explain how cattle could produce antibody to such antigens, which one would expect to be 'concealed' from the host's immune system, during natural infestation. It has been shown that the carbohydrate determinants on many tick glycoproteins are cross-reactive immunologically and that the reaction of bovine antibodies with intrinsic membrane glycoprotein is at least partially blocked by low molecular weight carbohydrate. Further, antisera from cattle exposed to ticks react with a glycosylated, native Bm86 but not with a non-glycosylated, recombinant Bm86. Thus the reaction of concealed antigens with antibodies produced as a result of tick infestation appears to be due to a relatively non-specific reaction with carbohydrate determinants on tick glycoprotein. Evidence is also presented that antibodies directed against carbohydrate determinants of Bm86 are not protective. Care must therefore be exercised in interpreting the results of antibody reaction with glycoproteins in such complex organisms.

Animals↗
PubMed1990

Assay of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum.

It has been proposed that Clostridium aminobutyricum contains an enzyme catalyzing an unusual reaction: the dehydration of 4-hydroxybutyryl-CoA to vinylacetyl-CoA. 4-Hydroxy-[3-3H]butyric acid has been prepared which allows the activity of this enzyme to be assayed in the presence of acetyl-CoA under anaerobic conditions by the release of tritiated water. Initial characterization of the enzyme from C. aminobutyricum has shown it to be largely membrane or particle bound in the crude lysates. It can be solubilized in detergent. It is inactivated by oxygen, but stable under anaerobic conditions. Only 49 +/- 2% of the label is removed after enzyme-catalyzed equilibration with water. This stereospecific release is consistent with the formation of vinylacetyl-CoA and excludes a vitamin B12 coenzyme-dependent rearrangement to 3-hydroxybutyryl-CoA followed by dehydration to crotonyl-CoA.

Clostridium↗