Biomedical subjects

M F Lavin

Publications and source records attributed to M F Lavin.

At least 127 records · Page 7Linked to original sources

PubMed1992

Early detection of bovine leukemia virus by using an enzyme-linked assay for polymerase chain reaction-amplified proviral DNA in experimentally infected cattle.

Bovine leukemia virus is the causative agent of bovine leukosis and has been described in many countries throughout the world. We describe here a sensitive and readily applicable assay for the detection of bovine leukemia proviral DNA. Detection relies on initial amplification of proviral DNA by using polymerase chain reaction (PCR) followed by an enzyme-linked assay (PCR-ELA). Amplification is carried out by using one biotinylated primer and a second primer containing the GCN4 protein binding site. DNA is detected by a colorimetric assay after it is coupled to GCN4-coated plates and subsequently incubated with horseradish-streptavidin peroxidase and the appropriate substrate to produce a chromogenic reaction. It was possible to detect proviral DNA for all of eight bovine leukemia virus-infected calves by 2 weeks postinfection. Use of the more conventional agar gel immunodiffusion assay failed to reveal the presence of the virus in any of the animals up to 4 weeks postinfection. The PCR-ELA detected as little as 0.1 to 0.2 ng of amplified DNA per well, which compares very favorably with ethidium bromide staining of gels, by which 1 to 2 ng per lane was detected. This method lends itself to mass screening, is carried out in a similar way to an enzyme-linked immunosorbent assay, and does not require gel electrophoresis or the use of radioactive gene probes.

Animals↗

PubMed1991

Identification of cis-regulatory sequences in ascidian ribosomal DNA using a rapid filter-binding assay.

Using 32P-labelled random primed ribosomal DNA (rDNA) from the ascidian, Herdmania momus, multiple and large-scale filter-binding assays were performed to identify cis-regulatory sequences interacting with H. momus oocyte germinal vesicle protein. A vacublot apparatus was used to isolate DNA-protein complexes, providing a means of filtering multiple binding reactions simultaneously and for isolating sufficient amounts of bound DNA for further investigations. DNA bound to the filter was used to identify unknown cis-elements in the rDNA by Southern-blot analysis. The trapped rDNA hybridized specifically to the intergenic spacer, a region which contains cis-regulatory sequences that interact with rDNA transcription factors in several other species. Gel shift analysis of intergenic spacer fragments and native Southwestern blots confirmed that cis-elements were localized in the rDNA intergenic spacer. In principle, this method allows for the rapid identification of cis-regulatory sequences within any large, cloned DNA fragment which interact with nuclear extract.

Animals↗

PubMed1991

Isolation of a cDNA clone, encoding a human beta-galactoside binding protein, overexpressed during glucocorticoid-induced cell death.

In this report we describe the isolation and characterization of a cDNA clone overexpressed tenfold during the induction of apoptosis in the glucocorticoid-sensitive human leukaemia cell line CEM C7. This clone was shown by DNA sequence analysis to represent the human HL14 gene, encoding a beta-galactoside binding protein, the mouse homologue of which has recently been reported to act as a cell growth inhibitory factor.

Amino Acid Sequence↗

PubMed1991

Use of DNA-protein interaction to isolate specific genomic DNA sequences.

We describe a simple and rapid method for the isolation of specific genomic DNA sequences recognized by DNA-binding proteins. This procedure consists of four steps: (1) restriction enzyme digestion and size fractionation of genomic DNA; (2) DNA--protein binding using the gel mobility-shift assay; (3) ligation of isolated DNA fragments followed by transformation of Escherichia coli; and (4) screening of recombinant clones for inserts containing specific DNA--protein binding sequences. We have used this protocol to isolate human DNA sequences, 100-200 bp in size, that are recognized by both partially purified and affinity purified proteins. Unlike other procedures designed to identify genomic target sequences, the method described does not require polymerase chain reaction or successive immunoprecipitations.

Ataxia Telangiectasia↗

PubMed1991

T cell-T cell killing is induced by specific epitopes: evidence for an apoptotic mechanism.

Epstein-Barr virus-specific cytotoxic T lymphocyte clones were shown to be an effective target for their own lysis when incubated in the presence of their specific epitopes but not in the presence of irrelevant epitopes. The mode of cell killing appeared to be by apoptosis and was prevented by previously described inhibitors of the process. Degranulation, as measured by serine esterase activity, was involved in this form of T cell-T cell killing. This is the first report of T cell-T cell killing by apoptosis and is only observed in the presence of a specific epitope. This result may be of significance in the use of peptide-based vaccines.

Amino Acid Sequence↗

PubMed1991

Integration of bovine leukaemia virus at all stages of enzootic bovine leukosis.

Integration of bovine leukaemia virus DNA was investigated at all stages of infection in cattle. We report here the detection of integrated proviral DNA in the majority of antibody positive animals without lymphocytosis. In all but one case virus was integrated at a number of different sites. Hybridization analysis failed to detect proviral sequences in animals shown to be BLV antibody-negative by the Agar Gel Immunodiffusion assay. The pattern of integration in leukocytes from animals with persistent lymphocytosis was similar to that in sero-positive animals without lymphocytosis in that multiple sites of integration were evident. As reported by others only one or a few sites of integration were detected in tumours from enzootic bovine leukosis animals. Tumours from different sites in individual animals were either monoclonal or oligoclonal.

Animals↗

PubMed1991

Early detection of bovine leukosis virus DNA in infected sheep using the polymerase chain reaction.

The early diagnosis of bovine leukosis virus (BLV) infection, the aetiological agent in enzootic bovine leukosis, is important for the implementation of control measures. BLV infection is currently assessed by the detection of circulating antibodies against the viral envelope protein, gp51. However, this approach has shortcomings in the time taken to detect anti-BLV antibodies (three to four weeks after infection), and in the failure to detect antibodies in some animals. Clearly a technique such as the polymerase chain reaction (PCR), which directly detects the presence of viral DNA, has advantages over methods designed to measure host antibodies. The use of PCR for the detection of proviral DNA in an affected DNA sample with as little as 10(-5) micrograms of host DNA using agarose gel electrophoresis followed by ethidium bromide staining is described here. It was possible to improve the sensitivity of this assay by using hybridisation analysis with a BLV gene probe. PCR used in combination with hybridisation analysis will provide a sensitive diagnostic assay to detect BLV when antibody tests give weakly positive or equivocal results.

Animals↗

PubMed1991

Demonstration of the induction of apoptosis (programmed cell death) by tetrandrine, a novel anti-inflammatory agent.

Tetrandrine, a bisbenzylisoquinoline alkaloid, was found to cause death of malignant lymphoid and myeloid cells but not of Epstein-Barr virus-transformed lymphoblastoid cells. The death took the form of apoptosis (programmed cell death), the nature of the process being confirmed by DNA gel electrophoresis and electron microscopy. The induction of apoptosis by tetrandrine was much more rapid in CEM-C7 cells (4 h) than in the same cells treated with glucocorticoids (40 h), and did not require de novo protein synthesis. These results suggest that the anti-inflammatory and immunosuppressive properties of tetrandrine are mediated by novel mechanisms worthy of further investigation. They also indicate that tetrandrine may have value as an anti-neoplastic agent.

Alkaloids↗

PubMed1991

DNA fingerprints of sheep using an M13 probe.

The bacteriophage M13 DNA was used to detect hypervariable minisatellites in several families of Booroola sheep as well as Merino and Suffolk sheep. Digestion of sheep DNA gave rise to three to eight fragments with different restriction enzymes demonstrating considerable polymorphism between the different breeds. The length of informative DNA fragments varied in size from 6 to 20kb. The DNA fingerprints generated were individual specific and allowed for differentiation between closely related animals. The pattern obtained with sheep DNA was different from that observed with humans and other vertebrates in the proportion of high molecular weight DNA fragments present. Pedigree analysis of DNA patterns of dams and their offspring for several sets of twins and triplets showed a clear distinction between individuals and failed to reveal the presence of monozygosity.

Animals↗

PubMed1991

The nucleotide sequence of the genes, fanE and fanF of Escherichia coli K99 fimbriae.

K99 fimbriae of enterotoxigenic Escherichia coli consist of eight different subunits. A major subunit called fimbrillin forms fimbrial structure and a minor subunit called adhesin localizes at the tip of fimbriae and recognizes host receptor ganglioside. Within this eight gene cluster, fanE and fanF have not yet been sequenced. In this study, fanE and fanF genes were sequenced by analyzing several DNA fragments produced by endonuclease or exonuclease digestion. The fanE gene encoded 227 amino acids containing 20 amino acids of signal peptide starting from GTG (valine) and showed a homology to fanA-fanB. The fanF gene encoded 271 amino acids containing 20 amino acids of signal peptide starting from ATG (methionine) and showed homologies to the fanD gene, fimbrillin gene of F41, adhesin gene of P fimbriae (papG) and adhesin gene of Type 1 fimbriae (fimH). E and F subunits had fifteen and fourteen hydrophobic domains, respectively, which periodically appeared possibly forming a hydrophobic region.

Amino Acid Sequence↗

PubMed1991

Molecular diversity of marine glues: polyphenolic proteins from five mussel species.

Adhesive polyphenolic proteins have been purified and characterized from the feet of five marine mussels (Brachidontes exustus, Modiolus modiolus squamosus, Mytella guyanensis, Septifer bifurcatus, and Trichomya hirsuta). All five proteins contain high levels of 3,4-dihydroxyphenylalanine (DOPA), lysine, glycine, and serine or threonine. All but B. exustus also contain high levels (> or = 10%) of proline or 4-hydroxyproline. The polyphenolic proteins of all the mussels have repeated sequences of the motif X1-Y*-X2-Y*-X3-K, where Y* denotes tyrosine or DOPA. In two species (S. bifurcatus and B. exustus), X2 represents 3 amino acids (frequently glycine) and X3 is absent. M. guyanensis is similar except that X2 is reduced to 2 amino acids. In T. hirsuta and M. m. squamosus, however, X2 is absent and X3 occurs as alanine or hydroxyproline. All proteins share approximately equimolar proportions of tyrosyl- and lysyl-derived residues. Although all of the mussels examined thus far are adhesively opportunistic with respect to substratum type, a rigidly invariant sequence does not appear to be necessary for achieving this.

Amino Acid Sequence↗

PubMed1991

The response of ataxia-telangiectasia lymphoblastoid cells to neutron irradiation.

The response of control and ataxia-telangiectasia (A-T) cells to increasing doses of high-linear-energy-transfer (LET) ionizing radiation (neutrons) was compared. Ataxia-telangiectasia cells were markedly more sensitive to neutron irradiation than were control cells. The D0 value for the two A-T cell lines was 0.4 Gy while the value for controls was approximately 1.4 Gy. Fast neutrons were considerably more effective than gamma rays in inducing cell death in both cell types, but the sensitivity factor remained approximately the same as with gamma rays. A minimal depression of DNA synthesis was observed in ataxia-telangiectasia cells after neutron irradiation, similar to that reported previously after gamma irradiation. The extent of inhibition was not significantly greater in control cells, contrary to that seen with gamma rays. In time-course experiments a significant difference in degree of inhibition of DNA synthesis was observed between the cell types. Low doses of fast neutrons induced a G2-phase delay in both cell types, but the degree and extent of this delay was greater in ataxia-telangiectasia cells as observed previously with low-LET radiation.

Ataxia Telangiectasia↗

PubMed1990

rRNA genes from the lower chordate Herdmania momus: structural similarity with higher eukaryotes.

Ascidians, primitive chordates that have retained features of the likely progenitors to all vertebrates, are a useful model to study the evolutionary relationship of chordates to other animals. We have selected the well characterized ribosomal RNA (rRNA) genes to investigate this relationship, and we describe here the cloning and characterization of an entire ribosomal DNA (rDNA) tandem repeat unit from a lower chordate, the ascidian Herdmania momus. rDNA copy number and considerable sequence differences were observed between two H. momus populations. Comparison of rDNA primary sequence and rRNA secondary structures from H. momus with those from other well characterized organisms, demonstrated that the ascidians are more closely related to other chordates than invertebrates. The rDNA tandem repeat makes up a larger percentage (7%) of the genome of this animal than in other higher eukaryotes. The total length of the spacer and transcribed region in H. momus rDNA is small compared to most higher eukaryotes, being less than 8 kb, and the intergenic spacer region consists of smaller internal repeats. Comparative analysis of rDNA sequences has allowed the construction of secondary structures for the 18S, 5.8S and 26S rRNAs.

Animals↗

PubMed1990

Expression of bovine leukaemia virus envelope gene by recombinant vaccinia viruses.

Recombinant vaccinia viruses (VV) containing the envelope gene of bovine leukaemia virus (BLV) were constructed. Three virus constructs were designed: VV-BLV1 which contained the open reading frame for envelope glycoprotein gp51 alone, under control of VVP7.5 promoter; VV-BLV2 and VV-BLV3 contained the entire gene (gp51 + gp30) coding sequence downstream of VP7.5 and the fowlpox virus early/late promoter (PFE/L) respectively. All three VV recombinants expressed envelope glycoproteins as determined by the agar gel diffusion assay. By immunofluorescence techniques it was shown that while VV-BLV2 and VV-BLV3 expressed envelope glycoprotein on the surface of virus-infected cells, VV-BLV1 failed to do so. Rabbits inoculated with VV-BLV1 failed to show an anti envelope glycoprotein antibody response, however, significant levels of antibodies against envelope glycoprotein were detected in sera from rabbits inoculated with VV-BLV2 and VV-BLV3.

Animals↗

PubMed1990

Bovine leukaemia proviral DNA detection in cattle using the polymerase chain reaction.

Bovine leukaemia virus (BLV) is the causative agent in enzootic bovine leukosis a disease occurring worldwide. This virus is normally detected by the agar gel immunodiffusion or ELISA assays which rely on the appearance of antibodies to a major surface protein of the virus, gp51, present in the serum of infected cattle. We have used the polymerase chain reaction, which depends on the amplification of specific DNA sequences as a sensitive assay for the detection of BLV. It was possible to detect proviral DNA in 100 pg of tumour DNA from an infected host using agarose gel electrophoresis followed by ethidium bromide staining. The sensitivity of the assay was increased by two log orders when hybridization analysis, using a BLV proviral DNA probe, was used in combination with amplification of the DNA. Proviral DNA was detected in both lymphocytic and tumour DNA and at all stages of infection in cattle.

Animals↗

PubMed1990

Structure-activity relationships of the lissoclinamides: cytotoxic cyclic peptides from the ascidian Lissoclinum patella.

Two new lissoclinamides (lissoclinamides 7 and 8) have been isolated from the aplousobranch ascidian Lissoclinum patella. These lissoclinamides are cyclic heptapeptides with the same structural features as lissoclinamides 4 and 5 reported earlier, containing an oxazoline ring, one proline, one valine, two phenylalanine residues, and thiazole and/or thiazoline rings. All four peptides have the same sequence of amino acids around the ring and differ from one another only in their stereochemistry or the number of thiazole and thiazoline rings. The cytotoxicities of the compounds were tested with human fibroblast and bladder carcinoma cell lines and normal lymphocytes. Slight changes in structure resulted in marked differences in the cytotoxicities of these compounds. The most potent is lissoclinamide 7, containing two thiazoline rings, which rivals didemnin B in cytotoxicity in vitro.

Animals↗

PubMed1990

Molecular cloning and sequencing of an Australian isolate of proviral bovine leukaemia virus DNA: comparison with other isolates.

The molecular cloning and characterization of an EcoRI fragment, 8.26 kb in size, of an Australian isolate of bovine leukaemia virus (pBLV-A1) is described. This fragment includes most of the proviral genome as well as 340 bp of flanking bovine DNA sequence at the 5' end. Approximately 790 bp, including the 3' long terminal repeat, was missing from this clone. At the level of restriction enzyme mapping, this isolate could be distinguished from American, Belgian and Japanese isolates. DNA sequencing of the entire clone demonstrated some variation at the amino acid level between pBLV-A1 and the Japanese and Belgian isolates, particularly in the gag gene. In that gene there were 59 amino acid changes compared to the Japanese isolate and 24 compared to the Belgian isolate. The greater number in the case of the Japanese isolate was due to both single nucleotide changes and frameshift in a single region of the gene. This study also demonstrates that there are large tracts of amino acid sequence, particularly within the env and pol genes, that are highly conserved in different isolates. Some of these conserved sequences exist in regions containing epitopes important in virus infectivity.

Amino Acid Sequence↗

PubMed1990

Serum immunoglobulin concentrations in cattle naturally infected with bovine leukemia virus.

In order to elucidate whether natural infection of BLV in cattle might induce humoral immunological responses, changes in IgG1, IgG2, and IgM concentrations in the sera of infected cattle were determined. Twelve BLV-infected cattle were used. Cattle of different breeds were classified serologically and haematologically into BLV + PL+, BLV + PL- and BLV-free groups. Ig concentrations in the sera of the three groups were quantitated using a commercial single radial immunodiffusion assay. The findings were compared to those of BLV-free cattle. The serum IgM concentrations were significantly lower in cattle with PL (P less than 0.001) than in BVL + PL- and BLV-free cattle. The IgM concentrations tended to be lower in BLV+ PL- than those of BVL-free cattle. There were no significant differences in IgG1 and IgG2 serum concentrations between the three experimental cattle groups. IgG1 was the predominant subtype in the sera of all cattle groups.

Animals↗