Search PubMed⌕ Search

Biomedical subjects

M Carrington

Publications and source records attributed to M Carrington.

At least 109 records · Page 6Linked to original sources
PubMed1996

Localization of the recombination points in a family with two DR/DP recombinations.

In a family with a maternal DR/GLO recombination, cellular DP typing showed it to be located between DR and DP. RFLP studies done during the 9th international histocompatibility workshop gave anomalous segregation patterns of DPA and DPB bands that could be interpreted as being due to a second, paternal DR/DP recombination. This assumption was confirmed later by PCR-SSO typing. A more precise mapping has been done by new markers showing the maternal recombination to be within the TAP2 locus and the paternal recombination to be between DQB1 and DQB3. This supports earlier suggestions of a hot spot of recombination in the TAP region. The recombinations involve parental haplotypes that presently show DR/DP linkage disequilibrium in the French population and it is proposed that DR/DP recombinations occur randomly while B/DR recombinations preferentially occur on haplotypes without strong linkage disequilibrium. Existing DR/DP linkage disequilibria in a given population will thus be broken down with time. The mixed lymphocyte culture response towards an isolated DP difference was tested in this and another DR/DP recombinant family. It showed that an alloresponse towards DP may be highly variable and this suggests that it might be important to define the rules for the strength of this reaction and the possible implications for allotransplantation.

Female↗
PubMed1996

A distinct cyclin A is expressed in germ cells in the mouse.

In this paper, the existence of two A-type cyclins in the mouse is demonstrated. In the adult mouse, the expression of cyclin A1, which has greatest sequence identity with Xenopus cyclin A1, is restricted to germ cells. In contrast cyclin A2, which has greatest sequence identity with human cyclin A and Xenopus cyclin A2, is expressed in all tissues analysed. In order to explore the function of cyclin A1 in germ cells, its expression during the meiotic cell cycle and its associated kinase subunits have been characterised in the testis. The levels of cyclin A1 mRNA rise dramatically in late pachytene spermatocytes and become undetectable soon after completion of the meiotic divisions; thus its expression is cell cycle regulated. In lysates of germ cells from adult testes, cyclin A1 is present in p13suc1 precipitates, and cyclin A1 immunoprecipitates possess histone H1 kinase activity. Three kinase partners of cyclin A1 were identified: p34cdc2, a polypeptide of 39 x 10(3) M(r) that is related to p33cdk2 and, in lesser quantities, p33cdk2. Cyclin A1 was also detected in oocytes; in metaphase I and metaphase II oocytes, a proportion of the cyclin A1 colocalises with the spindle, possibly suggestive of a functional interaction. These data indicate that mammalian germ cells contain cyclin A1-dependent kinases that either act as a substitute for, or in addition to, the cyclin A2-dependent kinases characterised in somatic tissues.

Amino Acid Sequence↗
PubMed1996

Identification of the C-terminal region of 70 kDa heat shock protein from Leishmania (Viannia) braziliensis as a target for the humoral immune response.

A Leishmania (Viannia) braziliensis (Lb) promastigote cDNA library in lambda gt11 was screened with patients' sera with the aim of identifying antigens specifically related to mucocutaneous leishmaniasis (MCL). One of the clones isolated, 133P, consistently reacted with MCL sera; it was sequenced and found to encode the C-terminal three-quarters of a protein belonging to the highly conserved Hsp70 family. Since Hsp70 proteins from different species tend to be less conserved through the C-terminal end, it was predicted that this region would be more antigenic and was likely to bear the discriminatory epitopes. In order to test this hypothesis, the N- and C-terminal halves of the polypeptide encoded by 133P, 133P-N and 133P-C, respectively, were expressed in Escherichia coli. Immunoblotting analysis demonstrated that 133P-C reacted more strongly with a pool of MCL sera than 133P-N, and both recombinant proteins reacted faintly with pools of cutaneous (CL) and visceral (VL) leishmaniasis sera. These results confirmed the predicted epitope location in the C-terminal region. The 133P-C fragment was also expressed as a fusion protein with glutathione-S-transferase (GST-133P-C), affinity-purified with glutathione-agarose and tested by ELISA with individual sera. From 46 Lb-infected patients, 41 sera (89%) were positive, no cross-reactivity was observed with healthy, Trypanosoma cruzior L. amazonensis-infected individuals. Despite a relatively high cross-reactivity with VL sera, the enhanced humoral response of MCL as compared with CL patients might be interesting for studies on disease aggravation.

Amino Acid Sequence↗
PubMed1995

Susceptibility to Reiter's syndrome is associated with alleles of TAP genes.

OBJECTIVE: Although HLA-B27 is strongly associated with susceptibility to Reiter's syndrome (RS), recent data suggest that an additional modifying or susceptibility gene(s) acts in concert with HLA-B27 to contribute to disease pathogenesis. The recently described TAP genes (transporters associated with antigen processing) are potential candidates because they are polymorphic and their function is to transport antigenic peptides to be loaded in HLA class I molecules. METHODS: TAP1 and TAP2 alleles were determined for 34 patients with RS (28 HLA-B27 positive, 6 HLA-B27 negative), and their frequencies were compared with those observed for 52 HLA-B27 positive and 80 random disease-free control subjects. RESULTS: The allele frequency of TAP1C was greater in patients with RS (8 of 62, 13%) than in random controls (5 of 160, 3%) (P = 0.009). The frequency of TAP2A was greater in RS patients (51 of 66, 77%) than in random controls (88 of 160, 55%) (P = 0.002); likewise, the frequency was greater in HLA-B27 positive RS patients (41 of 54, 76%) than in HLA-B27 positive disease-free controls (49 of 94, 52%) (P = 0.004). Furthermore, the TAP2A allele was present in all RS patients (100%), whereas TAP2A was present in 79% (63 of 80) of the random controls (P = 0.003). CONCLUSION: The association observed between TAP alleles and RS is independent of the presence of HLA-B27, and despite the physical proximity of TAP and HLA class II genes, linkage disequilibrium does not account for the observed associations between TAP and RS. Thus, TAP genes are genetically separated but functionally linked to class I genes, and both contribute to susceptibility to RS.

ATP-Binding Cassette Transporters↗
PubMed1995

Recombination rates across the HLA complex: use of microsatellites as a rapid screen for recombinant chromosomes.

Meiotic recombination does not appear to occur randomly across chromosomes, but rather seems to be restricted to specific regions. A striking example of this phenomenon is illustrated by the HLA class II region. No recombination within the 100 kb encompassing the DRB1-DQA1-DQB1 loci has been reported, whereas the random association of TAP1 with TAP2 alleles suggests the presence of a hotspot for recombination within the 15 kb separating the closest variant sites of these two loci. Recombination rates between loci may provide clues to the functional properties of haplotypes. Absence of recombination may suggest the necessity to keep alleles of certain genes in phase and, alternatively, high recombination rates may suggest selective pressure to diversify haplotypes within the population. To address this issue, recombination rates across the HLA complex were determined using the 59 Centre d'Etude Polymorphisme Humain (CEPH) pedigrees. The allele frequencies of four microsatellite markers which map at sites ranging from the telomeric to centromeric ends of the complex were determined and the markers were used as a rapid means for identification of recombinant chromosomes. Typing these as well as other polymorphic loci within the HLA class I, II and III regions allowed assignment of the segments where recombination occurred. Recombination rates within the class II region (defined here as DRB1 to DPB1) and class III region (defined here as HLA-B to DRB1) regions were 0.74% and 0.94%, respectively, both of which are within an expected range given the standard of 1% recombination rate per megabase of DNA per meiosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1995

Molecular mapping of a recombination hotspot located in the second intron of the human TAP2 locus.

Recombination across the HLA class II region is not randomly distributed, as indicated by both strong linkage disequilibrium within the 100 kb encompassing the DRB1-DQA1-DQB1 loci and complete equilibrium between TAP1 and TAP2, the closest variant sites of which are < 15 kb. In an attempt to explain these observations, 39 novel polymorphic markers in a region encompassing the TAP, LMP, and DOB genes were used to delineate the site of crossover in 11 class II recombinant chromosomes. SSCP demonstrated that two recombination events occurred within an 850-bp interval in the second intron of TAP2, which separates the variant sites of TAP1 and TAP2. These data indicate the presence of a recombination hotspot, the first to be identified from the analysis of familial transmission in the human major histocompatibility complex. The region of crossover was cloned and sequenced from one of the recombinants, further defining the crossover site to a 138-bp segment nested within the 850-bp region. This represents the most precisely defined region of recombination in the human genome.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Discordant patterns of linkage disequilibrium of the peptide-transporter loci within the HLA class II region.

Disequilibrium between genetic markers is expected to decline monotonically with recombinational map distance. We present evidence from the HLA class II region that seems to violate this principle. Pairwise disequilibrium values were calculated from six loci ranging in physical separation from 15 kb to 550 kb. The histocompatibility loci DRB1, DQA1, and DQB1, located on the distal end of the class II region, behave as a single evolutionary unit within which extremely high linkage disequilibrium exists. Lower but still significant levels of disequilibrium are present between these loci and DPB1, located at the proximal edge of the HLA complex. The peptide-transporter loci TAP1 and TAP2, located in the intervening region, reveal no disequilibrium with each other and low or negligible disequilibrium with the flanking loci. The action of two genetic process is required to account for this phenomenon: a recombinational hotspot operating between TAP1 and TAP2, to eliminate disequilibrium between these loci, and at the same time selection operating on particular combinations of alleles across the DR-DP region, to create disequilibrium in the favored haplotypes. The forces producing the patterns of disequilibrium observed here have implications for the mapping of train loci and disease genes: markers of TAP1, for example, would give a false impression as to the influence of DPB1 on a trait known to be associated with DQB1.

Alleles↗
PubMed1994

Cystic fibrosis-related diabetes is associated with HLA DQB1 alleles encoding Asp-57- molecules.

The incidence of insulin-dependent diabetes in individuals with cystic fibrosis is nearly 100 times greater than in the general population. In the latter group, strong associations with specific HLA DQA1 and DQB1 alleles have been observed. To determine if a similar distribution of alleles occurs in cystic fibrosis patients with diabetes, a cohort of these individuals was typed for DQA1 and DQB1 alleles. HLA DQB1*0201 (Asp57-) was more frequent in diabetics compared to controls (40.4 vs 28%), while the frequency of alleles encoding Asp57+ molecules was lower in diabetics relative to both the cystic fibrosis-only controls (P = 0.025) and the general population (P = 0.008). The presence of at least one protective DQA1-DQB1 heterodimer (i.e., Arg52- and Asp57+, respectively) in cis or trans was significantly lower in the diabetics than in either of the control groups. Thus, the HLA alleles known to be associated with insulin-dependent diabetes mellitus in the general population are also found in diabetics with cystic fibrosis.

Adolescent↗
PubMed1994

Polymorphism of SPAG-1, a candidate antigen for inclusion in a sub-unit vaccine against Theileria annulata.

SPAG-1, a Theileria annulata sporozoite surface antigen, is a vaccine candidate. Data is presented, based on the clonal segregation of SPAG-1 associated RFLPs, showing that this antigen is encoded by a single copy gene. We have cloned and sequenced a full-length genomic copy of the SPAG-1 gene and a comparison of this with a previously published SPAG-1 cDNA sequence demonstrates a high degree of polymorphism. We infer that these sequences represent two distinct allelic SPAG-1 variants. The deduced polypeptides show an overall identity of 92% with the most variable stretch (60% identity) occurring towards the middle of the molecule. The N and C termini are more conserved with identities of 92% and 97% respectively. The elastin receptor ligand, VGVAPG, present 3 times in the protein sequence derived from the cDNA is not found in that deduced from the genomic copy. Evidence for 2 further SPAG-1 alleles was obtained from PCR based sequences using macroschizont clones containing different SPAG-1 associated RFLPs. In summary we have shown the existence of at least 4 highly polymorphic SPAG-1 alleles. The implications of such polymorphism between and within distinct geographical isolates for the development of a SPAG-1 based subunit vaccine is discussed.

Alleles↗
PubMed1994

The role of GPI-PLC in Trypanosoma brucei.

The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG). However the evidence that it is involved in VSG metabolism in the living trypanosome is circumstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotonic lysis. Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease. To try to determine the role of the GPI-PLC, a homozygous null mutant T. brucei has been constructed. The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes. The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype. The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be.

Animals↗
PubMed1994

Selective labelling of proteins synthesized by intracellular parasites using ricin and host cells lacking mitochondrial DNA.

Studies focused on the synthesis of developmentally regulated proteins by intracellular parasites have been limited due to the lack of a simple method for selectively labelling proteins produced by the parasite. A method has now been developed in which ricin, the toxin, is employed to selectively inhibit host cell protein synthesis while protein synthesis by the intracellular parasite is unaffected. Ricin is composed of two subunits, one of which binds to cell surface receptors containing terminal galactose residues while the other subunit enters the cell, inactivates ribosomes and, as a consequence, cytoplasmic protein synthesis. Due to the loss of the receptor-binding subunit, ricin cannot permeate the host cell mitochondria or the intracellular parasite, and therefore protein synthesis within these compartments continues uninterrupted. This system was explored using Eimeria tenella- and Toxoplasma gondii-infected avian rho0 cells. This host cell type was selected because it lacks mitochondrial DNA and supports the intracellular development of E. tenella sporozoites through first-generation merogony. Host mitochondrial proteins are not synthesized when labelling in the presence of ricin because these cells lack mitochondrial DNA. Therefore, those proteins which are radiolabelled with 35S methionine in ricin-treated infected monolayers are exclusively those of the intracellular parasite. Alternatively cells with intact mitochondria can be utilized, and in this case the host mitochondrial protein synthesis can be inhibited by chloramphenicol.

Animals↗
PubMed1994

Expression of Mycobacterium leprae 18-kDa antigen in yeast in a GPI-anchored form.

The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form.

Bacterial Proteins↗
PubMed1993

A structural motif in the variant surface glycoproteins of Trypanosoma brucei.

The variable domain of the trypanosome variant surface glycoprotein (VSG) ILTat 1.24 has been shown by X-ray crystallography to resemble closely the structures of VSG MITat 1.2, despite their low sequence similarity. Specific structural features of these VSGs, including substitution of carbohydrate for an alpha-helix, can be found in other VSG sequences. Thus antigenic variation in trypanosomes is accomplished by sequence variation, not gross structural alteration; the extensive sequence differences among VSGs may be required for another reason, such as the avoidance of recognition by helper T cells. Additionally, VSG sequences are found to define families, within a VSG superfamily, which have evolved in the trypanosome genome.

Amino Acid Sequence↗