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Biomedical subjects

R L Tellam

Publications and source records attributed to R L Tellam.

At least 37 records · Page 2Linked to original sources
PubMed1996

The major excretory/secretory protease from Lucilia cuprina larvae is also a gut digestive protease.

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsia (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.

Animals↗
PubMed1996

Growth of Lucilia cuprina larvae following treatment of sheep divergently selected for fleece rot and fly strike with monoclonal antibodies to T lymphocyte subsets and interferon gamma.

Intensive lymphocytic infiltration of the underlying dermis occurs during cutaneous myiasis caused by larvae of the blow fly, Lucilia cuprina. To determine the effect of this infiltrate on larval growth, monoclonal antibodies (mAb) to CD4, CD8 or WC1 lymphocyte subset determinants were injected intravenously before and during experimental infection of sheep with larvae. The effect of intravenous injection of mAb to ovine interferon (IFN) gamma was also examined. The experiments were performed in 18-month-old maiden ewes with genetic resistance or susceptibility to the disease complex, bacterial dermatitis/cutaneous myiasis. mAbs induced profound depletion of CD8+ and WC1+ subpopulations from blood and skin at sites of larval growth. mAb to CD4+ gave only a moderate reduction in lymphocytes from blood or skin. mAb treatments did not modify larval growth or survival at 20 or 50 h after infection. Larval growth rates did not differ between resistant and susceptible genotypes. No evidence was found for a role of T lymphocyte subpopulations or the cytokine IFN, in modifying larval growth during the first 50 h of infection. It seems unlikely that T lymphocyte-dependent immunological effector mechanisms contribute to the lower prevalence of fly strike seen in the resistant genotype in the field.

Animals↗
PubMed1994

Vaccination of sheep with purified serine proteases from the secretory and excretory material of Lucilia cuprina larvae.

Sheep were vaccinated with two purified serine proteases, LCT25a and LCT25b, isolated from the secretory and excretory material from first instar larvae of Lucilia cuprina. The immunization produced a strong antibody response to LCT25b and a weaker response to LCT25a as measured by ELISA. However, neither protease induced an ovine immune response which affected the development of first instar larvae growing on sera derived from these sheep. Further, direct in vivo bioassays of larval growth on the backs of vaccinated sheep also indicated a lack of induction of an immune response which prevented establishment of the larvae. Sera from unvaccinated sheep which had previous experience of blowfly strike, in general, strongly recognised the serine protease LCT25b. It was concluded from all of these results that serine proteases from the secretory and excretory material of L. cuprina are unlikely to be effective antigens in a vaccine designed to protect sheep from blowfly strike.

Animals↗
PubMed1994

Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA.

Two chymotrypsin-like proteases were purified from the secretory and excretory material of first-instar larvae of Lucilia cuprina. The hydrolysis of N-succinyl-L-phenylalanine-nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor-Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease (Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis-causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.

Amino Acid Sequence↗
PubMed1993

Lucilia cuprina: inhibition of larval growth induced by immunization of host sheep with extracts of larval peritrophic membrane.

A culture system has been established to produce gram amounts of peritrophic membrane from larvae of the sheep blowfly, Lucilia cuprina. Peritrophic membrane obtained from this culture has been used to immunize sheep. The immunization produced an immune response which resulted in the average weight of larvae on immunized sheep being only 50% of that of larvae grown on control sheep (P < 0.05). Fractionation of the components of the peritrophic membrane followed by immunization trials showed that the protective antigen fraction comprised material that could only be solubilized by harsh agents such as 4 M-urea. Even after solubilization by 4 M-urea, the protective antigens were able to produce a protective immune response which reduced growth of larvae on immunized sheep to 55% of larvae grown on control sheep (P < 0.05). This immune response which reduced growth of the larvae did not cause gross morphological damage to the larvae.

Animals↗
PubMed1993

'Concealed' antigens: expanding the range of immunological targets.

For many parasites, the interaction between the immunogenicity of the parasite and the immunological response of the host is a dynamic equilibrium that allows both to survive, albeit often with severe consequences for the host. Vaccines, if intended as a means of parasite control, are unlikely to be generally successful if they do no more than mimic an immunological equilibrium that would be reached after natural exposure to the parasites. The situation must be tipped in favour of the host. It has been difficult to find ways around this impasse. One approach has been receiving practical attention over recent years, an approach that Peter Willadsen, Craig Eisemann and Ross Tellam have called vaccination against 'concealed' antigens.

Journal Article↗
PubMed1993

Native and baculovirus-expressed forms of the immuno-protective protein BM86 from Boophilus microplus are anchored to the cell membrane by a glycosyl-phosphatidyl inositol linkage.

A glycoprotein (BM86) from the gut cells of the cattle tick Boophilus microplus, when used to vaccinate cattle, has been shown to protect cattle from tick infestation. A recombinant BM86 protein is the principal component of a novel tick vaccine currently under development. The nature of the anchorage of BM86 to tick gut epithelial cells has been investigated using BM86 from B. microplus and recombinant BM86 proteins expressed in insect cells using the baculovirus expression system. BM86 from B. microplus and a full length recombinant BM86 are shown to be anchored to the extracellular surface of tick gut epithelial cells and baculovirus-infected insect cells, respectively by a glycosyl-phosphatidyl inositol membrane anchor. A recombinant BM86 truncated by the removal of a hydrophobic region coding for thirty amino acids at the carboxy-terminal end was secreted from baculovirus-infected Sf9 cells. This secreted form of recombinant BM86 showed strong protective activity against ticks in cattle vaccinated with this protein.

Animals↗
PubMed1991

The binding of terbium ions to gelsolin reveals two classes of metal ion binding sites.

Spectroscopically active terbium ions have been used to probe the Ca2+ ion-binding sites on human plasma gelsolin. The luminescence of Tb3+ ions bound to gelsolin is markedly enhanced when excited indirectly at 295 nm due to Förster type dipole-dipole energy transfer from neighboring tryptophan residues. Titration of this luminescence with increasing concentrations of Tb3+ ions was saturable although the shape of this titration curve was complex indicating the involvement of multiple classes of sites. Luminescence lifetime measurements (obtained by indirect excitation at 295 nm) demonstrate the presence of two classes of sites characterized by a major lifetime of 1.0-1.1 ms and a minor lifetime of 0.7-0.8 ms. However, while the amplitude of the minor lifetime showed a hyperbolic dependence on the Tb3+ ion concentration, the amplitude of the major lifetime showed a strongly sigmoidal dependence. Different classes of Tb3+ ion binding sites can also be distinguished by the different Ca2+ ion concentrations needed to displace Tb3+ ions from these sites on gelsolin. It is proposed that the occupancy of one class of Tb3+ ion binding sites on gelsolin causes a conformational change in gelsolin which then allows a second class of cryptic Tb3+ ion binding sites to be expressed. The implications of these results in terms of the binding of Ca2+ ions to gelsolin and the regulation of the activities of gelsolin by calcium are discussed.

Binding Sites↗
PubMed1991

Dextran sulfate induces changes in the free intracellular calcium ion concentration of a subpopulation of immature thymocytes.

Previous studies have shown that certain sulfated polysaccharides, such as dextran sulfate (DxS), can induce a rapid and sustained increase in the free intracellular calcium ion concentration ([Ca2+]i) of thymocytes, whereas they cannot induce a change in the [Ca2+]i of peripheral lymphocytes. This study examined this phenomenon in more detail and clearly demonstrated that only immature thymocytes can respond to DxS. Cortisone-resistant thymocytes failed to exhibit an increase in [Ca2+]i when exposed to DxS. Furthermore, analysis of [Ca2+]i changes at the single cell level using a flow cytometer demonstrated that only a subpopulation of immature thymocytes responded to DxS. Additional flow cytometry studies revealed that DxS and Con-A induce a [Ca2+]i response in different subpopulations of thymocytes, approximately 30% of thymocytes responding to Con-A and 15-20% to DxS. Thus, responsiveness to Con-A and DxS, in terms of increases in [Ca2+]i, may represent a novel marker for two different thymocyte subpopulations. Such metabolic markers could be used to complement the conventional serological methods currently employed to define stages in thymocyte differentiation.

Animals↗
PubMed1990

Microfilament organization correlates with increased cellular content of gelsolin.

The relative amounts of intracellular gelsolin were determined in a number of human somatic cell hybrids and parental cell lines which greatly differ in their microfilament organizations. In contrast to the disruptive effect of gelsolin on actin filament formation in vitro, there is a correlation between the degree of microfilament organization and the amount of gelsolin within these cell lines.

Actin Cytoskeleton↗
PubMed1989

Immunologic control of a parasitic arthropod. Identification of a protective antigen from Boophilus microplus.

Cattle can be vaccinated against the tick Boophilus microplus by inducing an immunologic reaction against Ag in the tick gut. The uptake of antibody during feeding leads to severe damage to the parasite. One of the responsible tick gut Ag has now been purified and characterized: the Bm86 Ag. It is a membrane-bound glycoprotein present in very low abundance in extracts of partially engorged adult female ticks. It has an apparent m.w. of 89,000, an isoelectric point of 5.1 to 5.6 and an affinity for wheat germ lectin. Microgram amounts of this Ag are able to induce effective protection in cattle against the parasite, as shown by the decreased survival of ticks on vaccinated cattle and a reduction in engorgement weights and egg laying capacity of the survivors. Antisera to the Ag react with the surface of digest cells in the tick gut. As a result of the reaction with antibody, the endocytotic activity of these cells, which is a critical step in bloodmeal digestion in this tick, is strongly and rapidly inhibited. A number of peptides from this Ag, produced by digestion of the reduced and alkylated protein with endoproteinase lys-C, have been sequenced. One peptide has significant amino acid sequence homology with the epidermal growth factor precursor and a second peptide has homology with a putative protective antigen from Plasmodium falciparum.

Amino Acid Sequence↗
PubMed1987

The effect of sulfated polysaccharides on the free intracellular calcium ion concentration of lymphocytes.

Recent studies have demonstrated that murine lymphocytes express specific cell-surface receptors for a range of sulfated polysaccharides. In order to determine whether polysaccharide binding induces transmembrane signaling, the effects of sulfated polysaccharides on the free intracellular calcium ion concentration [( Ca2+]i) of mouse thymocytes and spleen cells were determined. Cells were loaded with Indo-I, a fluorescent indicator of calcium ion concentration. The validity and limitations in the use of this indicator in the determination of [Ca2+]i are documented. Dextran sulfate (Mn = 500,000), iota-carrageenan, lambda-carrageenan and kappa-carrageenan all cause relatively large changes in the [Ca2+]i of thymocytes (change in [Ca2+]i greater than 50 nM). Of these, dextran sulfate (Mn = 500,000) always had the greatest effect on [Ca2+]i. Smaller responses were obtained with heparin and dextran sulfate (Mn = 5000), while no response was obtained with chondroitin 4-sulfate, chondroitin 6-sulfate, pentosan sulfate or fucoidin. This response pattern (with the exception of fucoidin and pentosan sulfate) corresponds with the expression of thymocyte receptors for these polysaccharides. The increase in [Ca2+]i caused by the sulfated polysaccharides requires extracellular Ca2+ ions however, it is unlikely that voltage-dependent ion channels are involved in these responses. In contrast to thymocytes, although spleen cells express receptors for sulfated polysaccharides, they were unresponsive to all of the sulfated polysaccharides tested, suggesting a basic difference between thymocytes and peripheral T and B lymphocytes in their response to the binding of sulfated polysaccharides.

Animals↗
PubMed1987

Lipids from Plasmodium vinckei-infected erythrocytes and their susceptibility to oxidative damage.

The constituent lipids of plasma and red blood cells (RBC) from mice late in infection with the malarial parasite Plasmodium vinckei were analyzed and compared with those obtained from uninfected animals. On a dry weight basis, the total extractable lipids of RBC increased threefold during infection, while those of the plasma did not change significantly. In general, changes in individual plasma lipid constituents paralleled those found in RBC of infected mice but were of smaller magnitude. While the increase in the total lipids of parasitized RBC was largely attributable to an increase of more than fourfold in total phospholipids, a significant increase in neutral lipids was also observed. Phosphatidylcholine and phosphatidylethanolamine were the major phospholipids present within RBC, and their total and relative concentrations increased as a result of the infection. A parallel increase occurred in the ratio of unsaturated to saturated fatty acids in the parasitized RBC phospholipids. Infection was also associated with decreases in the relative amount of cholesterol present in RBC and in the ratio of cholesterol to phospholipid. Consistent with this, the fluorescence polarization of 1[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene within parasitized RBC plasma membranes was decreased in comparison with its value in noninfected RBC, indicating that malarial infection decreases the "order" of membrane lipids. These modifications, in conjunction with the increased levels of vitamin E and malonyldialdehyde reported elsewhere, are important determinants of the susceptibility of the different membrane compartments within infected RBC to peroxidative damage.

Animals↗
PubMed1986

Gelsolin inhibits nucleotide exchange from actin.

The effects of platelet gelsolin on the state and exchangeability of the nucleotide bound to skeletal muscle actin monomer have been investigated. In the presence of Ca2+, a stable ternary complex consisting of two actins and one gelsolin is formed. Removal of Ca2+ from this species results in the formation of a highly stable binary gelsolin-actin complex. The interaction of gelsolin with actin monomer has no effect on the virtually negligible [less than 0.01 mol of Pi X h-1 X (mol of actin)-1] intrinsic ATPase activity of actin monomer (in the absence of Mg2+). A single molecule of ATP is bound to the binary complex while two molecules of ATP are bound to the actins within the ternary complex. The ATP within the binary complex is nonexchangeable, and only one of the two ATP molecules in the ternary complex is exchangeable. In the latter case the rate constant for this nucleotide exchange is decreased compared to that for free actin monomer. These results demonstrate the nonequivalence of actin monomers within the ternary complex. The involvement of these oligomeric complexes of gelsolin and actin in the expression of the activity(ies) of gelsolin is discussed.

Actins↗
PubMed1986

Increased actin nucleating activity in tumorigenic cells.

The kinetics of actin polymerization has been used to quantitate the relative levels of actin nucleating activity in extracts from a number of related tumorigenic and non-tumorigenic cells. The level of nucleating activity was significantly elevated in the tumorigenic compared with the non-tumorigenic cell extracts whether the results were expressed on the basis of per protein (2-3 fold increase) or per total endogenous cellular actin (3-4 fold increase). It is concluded that this activity is probably due to an actin filament capping/severing regulatory protein(s) and that this protein(s) may be, at least partially, responsible for the microfilament disruption observed in transformed cells.

Actin Cytoskeleton↗