Investigation of the uptake of drugs by individual cells using a scanning proton microprobe (SPM).

In this paper we demonstrate the use of micro-PIXE (proton induced X-ray emission) for measuring the quantitative uptake of anti-AIDS drugs, containing metal atoms, by individual Vero cells (African green monkey kidney cell line). Hetero-polytungstates, which are assessed to present an activity against the HIV virus, were studied using Vero cells. It was found that unlike other techniques, SPM offers both the sensitivity and the spatial resolution to carry out these programs of investigations. The use of elemental analysis in single cells of cultured cell lines has shown to have distinct advantages over peripheral blood lymphocytes.

Systemic immunization of sheep with surface antigens from Haemonchus contortus larvae.

Sheep immunized systemically with a surface extract from third-stage H. contortus larvae showed high serum antibody reactivity against surface antigens and whole, viable larvae. After a first infection, no significant difference was found between the mean egg counts of the vaccinates and controls although most vaccinated sheep seemed to show an increased susceptibility to infection. The local abomasal response was stimulated by giving both vaccinated and control sheep a large, abbreviated infection cured after 11 days by drenching. Thereafter, a second challenge infection was given. This immunization regime resulted in seven of the nine vaccinated sheep showing clear protection against the second challenge infection.

Expression and secretion in Aspergillus nidulans and Aspergillus niger of a cell surface glycoprotein from the cattle tick, Boophilus microplus, by using the fungal amdS promoter system.

A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.

Chromatography and generation of specific antisera to synthetic peptides from a protective Boophilus microplus antigen.

Four oligopeptides corresponding to predicted antigenic regions of the protective Bm86 glycoprotein of the cattle tick Boophilus microplus were synthesized and purified. Three were conjugated to carrier proteins and antisera raised in rabbits and cows. All elicited antipeptide antibodies that recognized Bm86 and recombinant derived products in Western blots; however, only one produced antiserum capable of recognizing native Bm86 in an indirect immunofluorescence assay. Ticks fed in vitro on this antiserum showed no obvious gut damage.

Human immunoglobulin variable region genes: a new VH sequence used to detect polymorphism.

A human heavy chain variable region subgroup III pseudogene (HV3.3) was isolated, characterized, and sequenced. When HV3.3 was hybridized to Southern blots of human DNA, two potentially informative polymorphic bands, resulting from 2.7 kb Hind III (HH2.7) and 7.3 kb Eco RI (HE7.3) fragments, were detected with frequencies of 0.553 and 0.606, respectively. These polymorphic bands showed Mendelian segregation in families and appeared to be in tight linkage disequilibrium with each other (chi 2(1) = 24.91, P less than 0.001). Evidence from sibling-pair data indicated linkage of the Hind III polymorphic band to constant region allotypic and restriction fragment length polymorphism markers. Bands representing alternative forms of the polymorphic restriction sites were not detected for either HH2.7 or HE7.3. This indicates either that the alternative fragments comigrate with homologous fragments resulting from conserved restriction sites, or that the polymorphism is due to a gene duplication or deletion. No band segregating with HH2.7 was found in separate digests using eight other enzymes. Although this indicates that a major deletion or unlikely, it does not exclude the possibility of a gene deletion or duplication affecting the intergenic region(s) of one or more homologous genes. Whatever the precise explanation, these findings support the hypothesis that there is polymorphic variation of VH gene repertoires in man.

Human immunoglobulin variable region genes: V kappa polymorphisms suggest variation in V-gene repertoires.

The present study characterizes four potentially informative polymorphic bands of 5.2, 2.3, 1.9, and 1.2 kb, detected by Southern blot hybridization of Eco RI digests of human DNA using HK101/80 (an immunoglobulin V kappa I probe). These restriction fragment length polymorphisms (RFLP) show Mendelian segregation and they are linked to each other and to Km(1), the allotypic marker on the kappa constant region. There is strong linkage disequilibrium between the 2.3 and 1.2 kb polymorphisms. A 0.7 kb Pst I polymorphic band and a 2.9 kb Sac I polymorphic band were also identified; the latter may reflect a region of tandem repeats in the V kappa region. No bands representing the alternative forms of any of the polymorphic restriction sites were identified. This implies either that genes are missing from the V kappa repertoire or that such bands are hidden because of comigration of fragments due to conservation of restriction sites. Evidence for comigration of fragments was obtained from independent V kappa clones and suggests that dark bands on Southern blots of Pst I digests must often represent several superimposed genes which have conserved restriction sites. The demonstration of RFLP within the V kappa region provides circumstantial evidence for polymorphic variation in the repertoire of V kappa structural genes. The RFLP reported here should be useful as genetic markers in future studies on the immune response and disease susceptibility in man.

Integumental chitin synthase activity in cell-free extracts of larvae of the Australian sheep blowfly, Lucilia cuprina, and two other species of diptera.

Chitin synthase activity has been demonstrated in crude homogenates of larval integuments from L. cuprina and in similar preparations from Musca domestica and Calliphora erythrocephala. This is the first report of an insect integumental chitin synthase. This activity brings about the incorporation of radioactivity from UDP-N-acetyl-[14C]glucosamine into an ethanol- and alkali-insoluble form. A major part of this labelled product has been characterized as chitin by its insolubility in alkali, resistance to degradation by proteases and its susceptibility to digestion by chitinase and HCl. Most of the radioactivity solubilized during digestion by chitinase co-migrates with N-acetylglucosamine, glucosamine and chitobiose during paper chromatography. Some radioactivity also becomes incorporated into non-chitin products in this system. There is substantial evidence that incorporation is not brought about by whole epidermal cells or by microbial contamination in the homogenates. The extent of incorporation obtained with the homogenates is limited by the presence of degradative enzymes which rapidly break down the substrate (UDP-N-acetylglucosamine). The incorporation was partially inhibited (50-70%) by both polyoxin-D (apparent Ki 0.04 microM) and diflubenzuron (apparent Ki 5-8 microM). This is the first report of a cell-free chitin-synthesizing system derived from insect tissue which is sensitive to inhibition by diflubenzuron.

Larvicidal activity of inhibitors of DOPA decarboxylase on the Australian sheep blowfly, Lucilia cuprina.

Inhibitors of DOPA decarboxylase, the key enzyme in the formation of the sclerotizing agent (N-acetyl dopamine) of the blowfly cuticle, have been tested for larvicidal activity against L. cuprina. A significant level of DOPA decarboxylase activity has been shown to be present throughout larval life in this species. Four potent in vitro inhibitors of L. cuprina larval DOPA decarboxylase (carbidopa, benserazide, methyl tyrosine and methyl DOPA) have been shown to be effective larvicides when fed to first- or second-instar larvae. However, no correlation is seen between the apparent Ki and LD50 values for these compounds. Treated larvae are observed to die at the next moult but death can be averted by the addition of N-acetyl dopamine to the food. Thus the toxic effects of the DOPA decarboxylase inhibitors appear to result from an inhibition of the formation of the sclerotizing agent in the cuticles of treated larvae.