Biomedical subjects

A F Markham

Publications and source records attributed to A F Markham.

At least 19 recordsLinked to original sources

PubMed2002

Expression of survivin, a novel inhibitor of apoptosis and cell cycle regulatory protein, in pancreatic adenocarcinoma.

Survivin is unique for its expression in human malignancies but not in normal adult cells. It has been implicated in sensitisation to chemotherapy and as a prognostic marker in several common cancers. Immunohistochemistry for Survivin, P53 and BCL-2 expression as well as cell proliferative index (Ki-67) and apoptosis index (TUNEL) was conducted on 52 pancreatic and 12 ampullary adenocarcinomas. Survivin was detected in the cytoplasm of carcinoma cells in 46 (88%) of pancreatic tumours. P53 and BCL-2 were detected in 54% and 12% of pancreatic tumours, respectively. Proliferative index was 26.2+/-10.5% and apoptosis index was 1.38+/-0.69%. Prevalence of Survivin expression was significantly higher in P53-positive than in P53-negative cases (P=0.05) but was not associated with BCL-2 expression. Incrementally higher weighted scores of Survivin expression were associated with increased proliferative index (P=0.001). Furthermore, there was linear correlation between increased proliferative index and higher apoptosis index (P<0.001). Surprisingly, higher scores of Survivin expression were associated with increased apoptosis index (P=0.007). Survival characteristics were not influenced by Survivin, P53 or BCL-2 expression, apoptosis index or proliferative index. Ampullary carcinoma showed Survivin expression in 83% of cases. However, unlike pancreatic carcinoma, there was no correlation between Survivin and P53 expression or proliferative index. In conclusion, Survivin is expressed in the majority of pancreatic adenocarcinomas and correlates with both cellular proliferation and apoptosis. Molecular manipulation of Survivin expression may enhance chemotherapy and radiation therapy for pancreatic cancer.

Adenocarcinoma↗

PubMed2002

BCL10 in malignant lymphomas--an evaluation using fluorescence in situ hybridization.

BCL10 is a tumour suppressor gene originally cloned from a t(1;14)(p22;q32) breakpoint in a case of mucosa-associated lymphoid tissue (MALT) lymphoma. Translocations involving this gene, though uncommon, are sometimes encountered in MALT lymphomas. This gene is thought to play an important role in the development of malignant lymphomas. Fluorescence in situ hybridization (FISH) was therefore undertaken on 22 cases of malignant lymphoma of varying histology to establish the incidence of rearrangements involving the BCL10 gene. Initially, one case with a novel t(1;2)(p22;p12) translocation involving the BCL10 gene was identified, in a marginal zone lymphoma of the MALT type, and was reported elsewhere. Seven other cases were subsequently identified with abnormalities in the 1p region, including a translocation with a breakpoint in the 1p22 region in a case of lymphoblastic lymphoma. However, none of these involved the BCL10 gene. Mutation analysis of BCL10 was then performed on 57 cases of malignant lymphoma, including 17 MALT lymphomas, by single-strand conformational polymorphism (SSCP) analysis of tumour DNA. Tissue was obtained for mutation analysis for 12 of the 22 cases analysed by FISH. Selected cases with SSCP band shifts were further studied by direct sequencing. Polymorphisms were identified in eight cases, but no mutations of pathogenic significance were identified. Further RT-PCR and mutation analysis was performed on cDNAs from 12 cases (four MALT, seven diffuse large B-cell lymphoma, one Hodgkin's disease) in which DNA analysis had already been completed. This included the MALT lymphoma with the t(1;2)(p22;p12) rearrangement. Again, no mutations were identified in the coding sequence. This study confirms that rearrangements of the BCL10 gene are uncommon in lymphoma (1/22) and may be limited tothe MALT subtype of non-Hodgkin's lymphomas. It was also found that breakpoints or rearrangements in the 1p22 region do not necessarily involve the BCL10 gene. Moreover, the absence of mutations at both the DNA (0/60) and the mRNA (0/12) level indicates that this gene is not frequently inactivated by mutation, in those tumours in which it is not involved in translocations. Our findings suggest that the BCL10 gene is unlikely to have a frequent or key role in general lymphomagenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2002

A family study and the natural history of prenatally detected unilateral multicystic dysplastic kidney.

PURPOSE: We document the inheritance pattern of multicystic dysplastic kidney in 3 affected families and screen first-degree relatives of a cohort of children with prenatally detected multicystic dysplastic kidney for renal anomalies. The study also afforded an opportunity to document the natural history of prenatally detected multicystic dysplastic kidney. MATERIALS AND METHODS: We identified 3 families during clinical treatment of children with prenatally detected multicystic dysplastic kidneys. Other members of these families were evaluated with renal ultrasonography. For the family screening study index cases were identified from a fetal uropathy database. A total of 94 first-degree relatives (52 parents, 35 full siblings and 7 half siblings) of 29 children with prenatally detected multicystic dysplastic kidneys were studied with urinary tract ultrasonography, blood pressure measurement, urinalysis and plasma biochemistry. RESULTS: Two families had affected sibling pairs, 1 of which also had a half sibling with vesicoureteral reflux. The third family included 3 individuals with multicystic dysplastic kidney and 1 with renal agenesis thought to have resulted from involution of multicystic dysplastic kidney. This family is consistent with autosomal dominant inheritance with variable expressivity and reduced penetrance. In the screening study ultrasonography did not demonstrate significant renal anomalies in any of the 94 first-degree relatives of the multicystic dysplastic kidney index cases. Followup assessment of prenatally detected multicystic dysplastic kidneys in index cases demonstrated total involution in 52% at a median age of 6.5 years with no multicystic dysplastic kidney related morbidity. CONCLUSIONS: Multicystic dysplastic kidney can be familial but is most commonly a sporadic anomaly. Formal screening of relatives is not recommended. Followup data on a cohort of children with prenatally detected multicystic dysplastic kidney add further support to conservative management.

Female↗

PubMed2001

Genetic events during the transformation of a tamoxifen-sensitive human breast cancer cell line into a drug-resistant clone.

Tamoxifen resistance is a serious clinical problem commonly encountered in the management of patients with breast cancer. The mechanisms leading to its development are unclear. Tamoxifen acts via multiple pathways and has diverse effects. Hence transformation from a tamoxifen-sensitive to a resistant phenotype could involve multiple genetic events. Knowledge of the genetic pathways leading to resistance may facilitate the development of novel therapeutic strategies. In this study, a variation of conventional comparative genomic hybridization (CGH) has been employed to detect genetic alterations associated with tamoxifen resistance. MCF-7, a tamoxifen-sensitive human breast cancer cells line, and its tamoxifen-resistant clone, CL-9 were used. Both cell lines showed extensive areas of concordance but consistent differences were seen with the acquisition of tamoxifen resistance. These differences included the amplification of 2p16.3 approximately p23.2, 2q21 approximately q34, 3p12.3 approximately p14.1, 3p22 approximately p26, 3q, 12q13.2 approximately q22, 13q12 approximately q14, 17q21.3 approximately q23, 20q11.2 approximately q13.1 and 21q11.2 approximately q21 as well as the deletion of 6p21.1, 6p23 approximately p25, 7q11.1 approximately q31, 7q35 approximately q36, 11p15, 11q24, 13q33, 17p, 18q12 approximately q21.1, 19p, 19q13.3, 22q13.1 approximately q13.2. These findings were supported by conventional cytogenetics and chromosome painting. The regions identified by CGH potentially harbor genes that could be important in the development of tamoxifen resistance.

Antineoplastic Agents, Hormonal↗

PubMed2001

Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13).

Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive degeneration of the peripheral retina leading to night blindness and loss of visual fields. With an incidence of approximately 1 in 4000, RP can be inherited in X-linked, autosomal dominant or autosomal recessive modes. The RP13 locus for autosomal dominant RP (adRP) was placed on chromosome 17p13.3 by linkage mapping in a large South African adRP family. Using a positional cloning and candidate gene strategy, we have identified seven different missense mutations in the splicing factor gene PRPC8 in adRP families. Three of the mutations cosegregate within three RP13 linked families including the original large South African pedigree, and four additional mutations have been identified in other unrelated adRP families. The seven mutations are clustered within a 14 codon stretch within the last exon of this large 7 kb transcript. The altered amino acid residues at the C-terminus exhibit a high degree of conservation across species as diverse as humans, Arabidopsis and trypanosome, suggesting that some functional significance is associated with this part of the protein. These mutations in this ubiquitous and highly conserved splicing factor offer compelling evidence for a novel pathway to retinal degeneration.

Animals↗

PubMed2001

Spectrum, frequency and penetrance of OPA1 mutations in dominant optic atrophy.

Dominant optic atrophy (DOA) is the commonest form of inherited optic neuropathy. Although heterogeneous, a major locus has been mapped to chromosome 3q28 and the gene responsible, OPA1, was recently identified. We therefore screened a panel of 35 DOA patients for mutations in OPA1. This revealed 14 novel mutations and a further three known mutations, which together accounted for 20 of the 35 families (57%) included in this study. This more than doubles the number of OPA1 mutations reported in the literature, bringing the total to 25. These are predominantly null mutations generating truncated proteins, strongly suggesting that the mechanism underlying DOA is haploinsufficiency. The mutations are largely family-specific, although a common 4 bp deletion in exon 27 (eight different families) and missense mutations in exons 8 (two families) and 9 (two families) have been identified. Haplotype analysis of individuals with the exon 27 2708del(TTAG) mutation suggests that this is a mutation hotspot and not an ancient mutation, thus excluding a major founder effect at the OPA1 locus. The mutation screening in this study also identified a number of asymptomatic individuals with OPA1 mutations. A re-calculation of the penetrance of this disorder within two of our families indicates figures as low as 43 and 62% associated with the 2708del(TTAG) mutation. If haploinsufficiency is the mechanism underlying DOA it is unlikely that this figure will be mutation-specific, indicating that the penetrance in DOA is much lower than the 98% reported previously. To investigate whether Leber's hereditary optic neuropathy (LHON) could be caused by mutations in OPA1 we also screened a panel of 28 LHON patients who tested negatively for the three major LHON mutations. No mutations were identified in any LHON patients, indicating that DOA and LHON are genetically distinct.

Alternative Splicing↗

PubMed2001

Characterisation and expression of Sox9 in the Leopard gecko, Eublepharis macularius.

Since the discovery of the sex-determining gene, Sry, a number of genes have been identified which are involved in sex determination and gonadogenesis in mammals. Although Sry is known to be the testis-determining factor in mammals, this is not the case in non-mammalian vertebrates. Sox9 is another gene that has been shown to have a male-specific role in sex determination, but, unlike Sry, Sox9 has been shown to be involved in sex determination in mammals, birds, and reptiles. This is the first gene to be described that has a conserved role in sex determination in species with either chromosomal or environmental sex-determining mechanisms. Many reptiles do not have sex chromosomes but exhibit temperature-dependent sex determination (TSD). Sox9 has been shown to be expressed in both turtle and alligator during gonadogenesis. To determine if Sox9 also has a role in a gecko species with TSD, we studied gonadal expression of Sox9 during embryonic development of the Leopard gecko (Eublepharis macularius). Gecko Sox9 was found to be highly conserved at the nucleotide level when compared to other vertebrate species including human, chick, alligator, and turtle. Sox9 was found to be expressed in embryos incubated at the male-producing temperature (32.5 degrees C) as well as in embryos incubated at the female-producing temperatures (26 and 34 degrees C), Northern blot analysis showed that Sox9 was expressed at both temperatures from morphological stages 31 to 37. mRNA in situ hybridisation on isolated urogenital systems showed expression at both female- and male-producing temperatures up to stage 36. After this stage, no expression was seen in the female gonads but expression remained in the male. These data provide further evidence that Sox9 is an essential component of a testis-determining pathway that is conserved in species with differing sex-determining mechanisms.

Alligators and Crocodiles↗

PubMed2001

Features of the parkin/ariadne-like ubiquitin ligase, HHARI, that regulate its interaction with the ubiquitin-conjugating enzyme, Ubch7.

We recently reported the identification of a RING finger-containing protein, HHARI (human homologue of Drosophila ariadne), which binds to the human ubiquitin-conjugating enzyme UbcH7 in vitro. We now demonstrate that HHARI interacts and co-localizes with UbcH7 in mammalian cells, particularly in the perinuclear region. We have further defined a minimal interaction region of HHARI comprising residues 186-254, identified individual amino acid residues essential for the interaction, and determined that the distance between the RING1 finger and IBR (in between RING fingers) domains is critical to maintaining binding. We have also established that the RING1 finger of HHARI cannot be substituted for by the highly homologous RING finger domains of either of the ubiquitin-protein ligase components c-CBL or Parkin, despite their similarity in structure and their independent capabilities to bind UbcH7. Furthermore, mutation of the RING1 finger domain of HHARI from a RING-HC to a RING-H2 type abolishes interaction with UbcH7. These studies demonstrate that very subtle changes to the domains that regulate recognition between highly conserved components of the ubiquitin pathway can dramatically affect their ability to interact.

Amino Acid Sequence↗

PubMed2001

Expression of the Sonic Hedgehog receptor "PATCHED" in basal cell carcinomas and odontogenic keratocysts.

Basal cell carcinoma (BCC) is a common invasive skin lesion in Caucasians. Odontogenic keratocysts (OKs) are developmental, non-inflammatory oral cysts. They can be sporadic and/or multiple and are locally destructive. Basal cell naevus syndrome (BCNS) comprises both multiple BCCs and multiple OKs, in addition to several other systemic manifestations. The genetic defect underlying this autosomal dominant syndrome is a germ line mutation in the Sonic Hedgehog receptor PATCHED (PTCH) gene. For this study, a rabbit anti-peptide PTCH antiserum was produced. Immunohistochemistry procedures were performed using PTCH antibody and commercially produced GLI-1 antibody (downstream member in the hedgehog pathway) to stain 11 BCNS-OKs, eight sporadic OKs, two BCNS-BCCs, and six sporadic BCCs. Most of these lesions had been previously screened for PTCH mutation. Most BCCs (n=7) demonstrated moderate staining, with the heaviest staining in the outer palisading cell layer, except a BCNS-BCC which had mutation proximal to the sequence used for production of immunogenic peptide; this demonstrated only weak staining. Although moderate to heavy staining with PTCH antibody was demonstrated in the epithelium of both types of OK (n=19), a quite different pattern of staining of the basal cell layer was observed in the two patient groups. In BCNS, OK staining was heaviest in basal epithelial layers. In contrast, staining in non-BCNS odontogenic keratocysts was exclusively located in the superficial epithelial layers. Up-regulation of PTCH and GLI-1 protein was demonstrated in both BCCs and OKs. The pattern of PTCH expression matched the PTCH transcript pattern previously reported in BCCs and appeared sufficiently characteristic in OKs to allow differentiation between syndromic and non-syndromic cysts.

Basal Cell Nevus Syndrome↗

PubMed2001

Immunohistochemical detection of the anti-apoptosis protein, survivin, predicts survival after curative resection of stage II colorectal carcinomas.

BACKGROUND: This study examined the role of Survivin protein, a novel inhibitor of apoptosis, in determining prognosis after curative resection of stage II colorectal carcinomas. METHODS: Expression of Survivin, P53, and BCL-2 was evaluated immunohistochemically in stage II colorectal carcinomas from 49 patients who were followed for up to 9 years after operation. The Cox proportional hazards regression model was used to examine the predictive value of several covariates. RESULTS: The patients comprised 33 men and 16 women with a median age of 71 years. There were 32 colonic and 17 rectal cancers comprising 40 T3 and nine T4 primary tumors. Survivin was expressed in 30 (61.2%), P53 in 30 (61.2%), and BCL-2 in 21 (42.9%) tumors. Expression of Survivin was independent of P53 or BCL-2 expression and histopathological characteristics of the tumor. The 5-year survival rate of patients with Survivin-positive tumors was significantly lower than that of patients with Survivin-negative tumors (52.5% vs. 94.1%, respectively; P = .01). On multivariate analysis, expression of Survivin (Hazard Ratio [HR] = 9; P = .03), and rectal origin of cancer (HR = 3; P = .05) were the only factors which independently predicted an increased risk of death from recurrent cancer. CONCLUSION: Survivin expression within the tumor can identify patients with stage II colorectal carcinoma who are at increased risk of death from recurrent disease and might particularly benefit from adjuvant therapy.

Aged↗

PubMed2001

Application of differential display to immunological research.

The majority of immunological processes are mediated by cell-to-cell contact or receptor-ligand interactions that transmit intracellular signals and affect the regulation of transcription in the nucleus. As a consequence, precursor cells develop into their respective lineages and cells differentiate further during an immune response. In order to study changes in normal cells or even cells that have been isolated from diseased tissue, a number of approaches have been developed. One such method, differential display (DDRT-PCR), is a versatile technique for the analysis of gene expression that is based on RT-PCR and denaturing polyacrylamide gel electrophoresis. This technique is applicable to multiple samples of clonal or purified cell populations as well as to complex tissues and can be used to provide mRNA fingerprints. However, the main purpose of DDRT-PCR is to isolate differentially regulated genes in biological systems. The method is carried out without prior hypothesis as to which genes should be examined and so increases the possibility of identifying completely novel and unexpected changes in transcription. A major drawback has been the isolation of false positive clones and the need to confirm the results of analysis by another method. This makes DDRT-PCR labour intensive. A number of strategies have been recommended to reduce these problems, including reverse-northern analysis as a confirmatory step for screening putative differentials. In order to reduce the number of gel fingerprints that would be required to cover all the mRNAs in a cell, several focused approaches have been suggested. These include targeted differential display for the isolation of multigene families that have conserved protein domains or gene signatures and subtractive differential display whereby one population is subtracted from the other prior to screening. The purpose of this review is to provide some guidance to the immunologist who might wish to apply DDRT-PCR in their research. A number of examples where DDRT-PCR has been used successfully in immunological research are included.

Animals↗

PubMed2001

Mutation in the gene encoding ferritin light polypeptide causes dominant adult-onset basal ganglia disease.

We describe here a previously unknown, dominantly inherited, late-onset basal ganglia disease, variably presenting with extrapyramidal features similar to those of Huntington's disease (HD) or parkinsonism. We mapped the disorder, by linkage analysis, to 19q13.3, which contains the gene for ferritin light polypeptide (FTL). We found an adenine insertion at position 460-461 that is predicted to alter carboxy-terminal residues of the gene product. Brain histochemistry disclosed abnormal aggregates of ferritin and iron. Low serum ferritin levels also characterized patients. Ferritin, the main iron storage protein, is composed of 24 subunits of two types (heavy, H and light, L) which form a soluble, hollow sphere. Brain iron deposition increases normally with age, especially in the basal ganglia, and is a suspected causative factor in several neurodegenerative diseases in which it correlates with visible pathology, possibly by its involvement in toxic free-radical reactions. We found the same mutation in five apparently unrelated subjects with similar extrapyramidal symptoms. An abnormality in ferritin strongly indicates a primary function for iron in the pathogenesis of this new disease, for which we propose the name 'neuroferritinopathy'.

Adult↗

PubMed2001

Maternally inherited hearing impairment in a family with the mitochondrial DNA A7445G mutation.

Despite the increasing number of reports of families with hearing impairment and mitochondrial DNA (mtDNA) mutations, the frequency of these mutations as causes of non-syndromic sensorineural hearing impairment (NSSHI) remains unknown. Mutations such as A1555G, A7445G and 7472insC have been found in several unrelated families implying they are more frequent than initially thought. We describe a family with NSSHI due to the presence of the homoplasmic mtDNA A7445G mutation in the tRNASer(UCN) gene. This is the fourth such family described with this mutation, all of different genetic backgrounds. Our study also demonstrates the difficulties sometimes encountered in establishing mitochondrial inheritance of hearing impairment in some families.

DNA↗

PubMed2001

In vivo episomal maintenance of a herpesvirus saimiri-based gene delivery vector.

Herpesvirus saimiri (HVS) has several properties that make it amenable to development as a gene delivery vector. HVS offers the potential to incorporate large amounts of heterologous DNA and infect a broad range of human cell lines. Upon infection the viral genome can persist by virtue of episomal maintenance and stably maintains heterologous gene expression. Here we report an evaluation of the in vivo properties of HVS, with a view to its development as a gene delivery system. We demonstrate for the first time, the long-term persistence of the HVS genome in tumour xenografts generated from HVS-infected human carcinoma cell lines. The HVS-based vector remained latent in the xenograft without spreading to other organs. Moreover, the long-term in vivo maintenance of the HVS genome, as a nonintegrated circular episome, provided efficient sustained expression of a heterologous transgene. These in vivo results suggest that HVS-based vectors have potential for gene therapy applications.

Animals↗

PubMed2001

Lack of inducible nitric oxide synthase promotes intestinal tumorigenesis in the Apc(Min/+) mouse.

BACKGROUND & AIMS: The role of the inducible isoform of nitric oxide synthase (Nos2 or iNOS) in intestinal tumorigenesis is unclear. Conflicting data also exist regarding the ability of Nos2 to modulate expression and/or activity of cyclooxygenase 2 (Cox-2), which promotes intestinal tumorigenesis. Therefore, we determined the effect of a null Nos2 genotype on intestinal tumorigenesis and Cox-2 expression/activity in the Apc(Min/+) mouse model of familial adenomatous polyposis. METHODS: Apc(Min/+)Nos2(-/-) mice were generated by successive crosses between C57BL/6-Apc(Min/+) and C57BL/6-Nos2(tm1Lau) mice. Adenoma characteristics of age-matched Apc(Min/+)Nos2(+/+) and Apc(Min/+)Nos2(-/-) mice were compared. The level and cellular localization of Nos2 messenger RNA (mRNA) expression in Apc(Min/+)Nos2(+/+) mouse intestine was determined. Cox-2 expression and activity were measured in both intestinal tissue and bone marrow-derived macrophages in vitro. RESULTS: Apc(Min/+)Nos2(-/-) mice developed significantly more intestinal adenomas than Apc(Min/+)Nos2(+/+) littermates. Epithelial cell Nos2 mRNA expression was decreased in adenomas compared with histologically normal Apc(Min/+)Nos2(+/+) intestine. There was no significant difference in Cox-2 expression or activity in either intestine or bone marrow-derived macrophages from Apc(Min/+)Nos2(+/+) and Apc(Min/+)Nos2(-/-) animals. CONCLUSIONS: Nos2 plays an antineoplastic role in the Apc(Min/+) mouse model of familial adenomatous polyposis. Nos2 does not modulate Cox-2 expression or activity in the Apc(Min/+) mouse.

Adenomatous Polyposis Coli Protein↗

PubMed2001

Genomic structures of the human angiopoietins show polymorphism in angiopoietin-2.

The family of human angiopoietins comprises factors with important roles in vascular development and angiogenesis. All angiopoietins bind with similar affinity to the endothelial cell-specific receptor, Tie2. The mechanism by which they contribute to angiogenesis is thought to involve regulation of endothelial cell interactions with supporting perivascular cells. In this study the genomic structures of all three human angiopoietins were characterised by direct sequencing of human genomic DNA from the appropriate P1 artificial chromosome (PAC) clones. The exact positions at the intron/exon boundaries and the lengths of all eight introns were determined. As would be expected from the homology of these three proteins, the positions of the introns in the three genes are highly conserved. The putative RNA transcription start site for each angiopoietin gene was also determined. Intron-specific primers were used to amplify each exon of angiopoietin-1 and angiopoietin-2 from individual genomic DNAs. Although no polymorphism has been detected in the coding region of angiopoietin-1, three independent polymorphisms have been identified for angiopoietin-2.

Angiopoietin-1↗

PubMed2001

Rheumatoid arthritis synovial T cells regulate transcription of several genes associated with antigen-induced anergy.

Rheumatoid arthritis (RA) is a chronic, inflammatory synovitis whose pathogenesis may involve autoimmune mechanisms. Anergy is a state of T-cell nonresponsiveness characterized by downregulated IL-2 production. Paradoxically, RA T cells are hyporesponsive and proliferate poorly to antigens and mitogens, thus sharing some characteristics with anergic T cells. We analyzed the molecular basis of anergy in cloned human CD4+ T cells using differential display RT-PCR and subsequently examined the levels of differentially expressed transcripts in RA and, as control, reactive arthritis (ReA) synovium. Several transcriptional events were common to anergic T cells and RA synovium. These included downregulation of CALMODULIN:, which is critical to T-cell activation, and of cellular apoptosis susceptibility protein, which may mediate resistance to apoptosis in RA. Transcription of CALMODULIN: in RA synovium was less than 1% of that in ReA and was lower in RA synovial fluid mononuclear cells than in paired PBMCs. Following anti-TNF-alpha therapy in vivo, RA PBMC CALMODULIN: transcripts increased five- to tenfold. Pharmacological calmodulin blockade in vitro impaired antigen-specific proliferation. These data provide a link between reduced CALMODULIN: transcription and impaired T-cell responsiveness in RA. The identification of transcriptional changes common to anergic and RA synovial T cells should help interpret some of the characteristic RA cellular defects.

Antigens↗

PubMed2000

Promoter analysis of the human ubiquitin-conjugating enzyme gene family UBE2L1-4, including UBE2L3 which encodes UbcH7.

UbcH7 is a ubiquitin-conjugating enzyme mediating c-fos degradation, transcription factor NF-kappaB maturation, human papilloma virus-mediated p53 and Myc protein degradation, in vitro. Previously, we characterised a highly dispersed gene family, UBE2L1-UBE2L4, whose members could potentially encode different isoforms of the UbcH7 protein. UBE2L3, located at chromosome 22q11.2, is the only identified family member with introns and encodes a polypeptide sequence identical to that of UbcH7. Promoter characterisation of UBE2L1, UBE2L3 and UBE2L4 5'-upstream regions was performed to establish which are transcribed under normal physiological conditions and after heat shock. Promoter activity was observed only with the UBE2L3 construct, the minimal promoter lying within a region 100 bp upstream of the transcriptional start site. No evidence for the presence of UBE2L1 or UBE2L4 transcripts was observed in human or murine tissues and cell lines. These data strongly suggest that UBE2L1 and UBE2L4 are likely to encode pseudogenes. Sequencing revealed that the UBE2L3 promoter contained no TATA or CCAAT boxes. Protein:DNA interaction studies confirmed the presence of binding sites for the transcription factors AP2 and Sp1 in the UBE2L3 minimal promoter. Deletion of these binding sites indicated that these factors are crucial for transcription of this gene.

Animals↗