FANCD2 protein is expressed in proliferating cells of human tissues that are cancer-prone in Fanconi anaemia

Fanconi anaemia (FA) is an inherited form of progressive pancytopenia associated with developmental defects, chromosomal instability, and cancer predisposition. At least seven distinct FA proteins function in concert to protect the genome, a key step being the activation of FANCD2 by mono-ubiquitination. This paper reports an immunohistochemical analysis of FANCD2 expression in normal human tissue. The highest expression was observed in maturing spermatocytes and fetal oocytes (consistent with a role for FANCD2 in meiosis) and in germinal centre cells of the spleen, tonsil, and lymph nodes (consistent with a role in proliferation). FANCD2 expression was also seen in tissues predisposed to cancer development in FA patients: haematopoietic cells, especially in the fetus, and squamous cell epithelia, particularly in the head and neck region and uterine cervix. FANCD2 expression was also occasionally seen in the breast and Fallopian tube epithelium, the respiratory epithelium of the trachea, and the exocrine cells of the pancreas, indicating that these tissues may also be cancer-prone in FA. FANCD2 expression is frequently expressed in proliferating cells as demonstrated by Ki-67 immunofluorescence double staining, consistent with a function of FANCD2 in DNA replication.     

Novel protein-truncating mutations in the ASPM gene in families with autosomal recessive primary microcephaly

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder that causes reduction in brain size. Individuals affected with the disorder show a small but architecturally normal cerebral cortex and are associated with mental retardation of mild-to severe form. MCPH is genetically heterogeneous with six loci, and four genes have been identified so far. Homozygous mutations in the ASPM gene, located at MCPH5 locus on chromosome 1q31, are the most common cause of MCPH particularly in the Pakistani population. In the present study, we have ascertained ten Pakistani and one Kashmiri family with primary microcephaly. We screened for potential mutations of the ASPM gene in seven consanguineous families (six Pakistani and one Kashmiri) linked to MCPH5 locus. Two previously reported (8508delGA, W1326X) and four novel sequence variants (Y1712X, I1717X, Y3353X, R3244X) were detected and all were predicted to be protein truncating. The degree of mental retardation in the affected individuals of the seven families varied from mild to moderate, and was not dependent on the location of mutations in the ASPM gene.     

ASPM mutations identified in patients with primary microcephaly and seizures

Background: Human autosomal recessive primary microcephaly (MCPH) is a heterogeneous disorder with at least six genetic loci (MCPH1–6), with MCPH5, caused by ASPM mutation, being the most common. Despite the high prevalence of epilepsy in microcephaly patients, microcephaly with frequent seizures has been excluded from the ascertainment of MCPH. Here, we report a pedigree with multiple affected individuals with microcephaly and seizures. Objective: To identify the gene responsible for microcephaly and seizures in this pedigree. Methods: Clinical assessments of three patients and brain MRIs of two patients were obtained. Genome-wide linkage screen with 10 k SNP microarray, fine mapping with microsatellite markers, and mutational analysis of the genomic DNA were performed on the pedigree. Results: We found that the family was linked to the MCPH5 locus on chromosome 1q31.2–q32.1. We screened ASPM and identified a previously unreported nonsense mutation that introduced a premature stop codon in exon 18 of the ASPM gene. Conclusions: We thus expand the clinical spectrum of ASPM mutations by showing that they can occur in patients with seizures and that the history of seizures alone should not necessarily preclude the diagnosis of primary microcephaly.     

The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein

Adrenoleukodystrophy is a severe genetic demye-llnating diseaseassociated with an impairment of ß-oxidation of verylong chain fatty acids (VLCFA) In peroxisomes. Earlier studieshad suggested that a deficiency in VLCFA CoA synthetase wasthe primary defect. A candidate adrenoleukodystrophy gene hasrecently been cloned and was found unexpectedly to encode aputative ATP-binding cassette transporter. We have raised monoclonalantibodies against this protein, that detect a 75kDa band. Thisprotein was absent in several patients with adrenoleukodystrophy.Immunofluorescence and Immunoelectron microscopy showed thatthe adrenoleukodystrophy protein (ALDP) is associated with theperoxisomal membrane. Distinct Immunofluorescence patterns wereobserved in cell lines from patients with Zellweger syndrome(a peroxisomal biogenesis disorder) belonging to different complementationgroups.     

The acute phase protein haptoglobin is locally expressed in arthritic and oncological tissues

Haptoglobin is an acute phase protein known to be highly expressed in the liver. Recently, we showed increased local arterial haptoglobin expression after flow-induced arterial remodelling and found that haptoglobin is involved in cell migration and arterial restructuring probably through accumulation of a temporary gelatin matrix. Since cell migration and matrix turnover are important features in the pathology of arthritis and cancer, we hypothesized that haptoglobin is also locally expressed in arthritic and oncological tissues. In this study, we investigated local haptoglobin expression in arthritic rats (n = 12) using semi-quantitative PCR and Western blotting, and we studied haptoglobin mRNA localization in human kidney tumours (n = 3) using in situ hybridization. The arthritic rats demonstrated an increase of haptoglobin mRNA (2.5-fold, P < 0.001) and protein (2.6-fold, P < 0.001) in the arthritic Achilles tendon. Haptoglobin protein was also increased in the arthritic ankle (2.6-fold, P < 0.001) but not in the non-arthritic knee. In human kidney tumours, tumour and stromal cells produced haptoglobin mRNA. This study shows that the liver protein haptoglobin is, in addition to the artery, also expressed in arthritic and oncological tissues that are recognized for enhanced cell migration and matrix turnover.     

The MUC6 secretory mucin gene is expressed in a wide variety of epithelial tissues

Secretory mucins play an important role in the cytoprotection of epithelial surfaces and are used as tumour markers in a variety of cancers. The MUC6 secretory mucin was originally isolated from a gastric cDNA library. The aim was to determine the specific type and location of MUC6 mucin gene expression in a wide range of human adult and fetal epithelial tissues. In situ hybridization, RNA analysis, and immunohistochemistry were used to quantify and localize mucin gene expression. The data obtained show that MUC6 is highly expressed in gastric mucosa, duodenal Brunner's glands, gall bladder, seminal vesicle, pancreatic centroacinar cells and ducts, and periductal glands of the common bile duct; focal expression is seen in basal endometrial and endocervical glands. MUC6 epitopes were also highly expressed in 7/10 pancreatic cancers and 7/10 cholangiocarcinomas and focally expressed in 4/10 endocervical adenocarcinomas. Expression of MUC6 occurs early in fetal development and was observed in Brunner's glands and pancreatic ducts at 18-19 weeks and in gastric glands at 20 weeks' gestation. The tissue distribution of the MUC6 secretory mucin indicates that it may function to protect epithelial tissues from a wide range of substances. Expression of MUC6 is frequently preserved in pancreatic and bile duct adenocarcinomas, but it is only sparsely expressed in endocervical carcinomas.     

The Mycobacterium tuberculosis complex-restricted gene cfp32 encodes an expressed protein that is detectable in tuberculosis patients and is positively correlated with pulmonary interleukin-10

Human tuberculosis (TB) is caused by the bacillus Mycobacterium tuberculosis, a subspecies of the M. tuberculosis complex (MTC) of mycobacteria. Postgenomic dissection of the M. tuberculosis proteome is ongoing and critical to furthering our understanding of factors mediating M. tuberculosis pathobiology. Towards this end, a 32-kDa putative glyoxalase in the culture filtrate (CF) of growing M. tuberculosis (originally annotated as Rv0577 and hereafter designated CFP32) was identified, cloned, and characterized. The cfp32 gene is MTC restricted, and the gene product is expressed ex vivo as determined by the respective Southern and Western blot testing of an assortment of mycobacteria. Moreover, the cfp32 gene sequence is conserved within the MTC, as no polymorphisms were found in the tested cfp32 PCR products upon sequence analysis. Western blotting of M. tuberculosis subcellular fractions localized CFP32 predominantly to the CF and cytosolic compartments. Data to support the in vivo expression of CFP32 were provided by the serum recognition of recombinant CFP32 in 32% of TB patients by enzyme-linked immunosorbent assay (ELISA) as well as the direct detection of CFP32 by ELISA in the induced sputum samples from 56% of pulmonary TB patients. Of greatest interest was the observation that, per sample, sputum CFP32 levels (a potential indicator of increasing bacterial burden) correlated with levels of expression in sputum of interleukin-10 (an immunosuppressive cytokine and a putative contributing factor to disease progression) but not levels of gamma interferon (a key cytokine in the protective immune response in TB), as measured by ELISA. Combined, these data suggest that CFP32 serves a necessary biological function(s) in tubercle bacilli and may contribute to the M. tuberculosis pathogenic mechanism. Overall, CFP32 is an attractive target for drug and vaccine design as well as new diagnostic strategies.     

The HTLV receptor is a widely expressed protein

The receptor for human T-cell leukemia virus type 1 (HTLV-1) was found to be expressed on a broad range of cell lines derived from multiple species. Receptor expression was assessed using human immunodeficiency virus type 1 particles, pseudotyped with the HTLV-1 envelope glycoprotein, and expressing luciferase under the control of an SV40 enhancer and promoter. Infection by pseudotyped virus was blocked with neutralizing antibodies to HTLV-1, and infection was dependent on the presence of the cleavage and fusogenic sequences in the envelope protein precursor. Trypsin treatment of susceptible target lymphocytes reduced entry. Entry was partially resistant to ammonium chloride.     

Compound heterozygous ASPM mutations associated with microcephaly and simplified cortical gyration in a consanguineous Algerian family

Homozygous mutations in the ASPM gene are a major cause of autosomal recessive primary microcephaly (MCPH). Here we report on a consanguineous Algerian family in which three out of five children presented with severe microcephaly, simplified cortical gyration, mild to severe mental retardation and low to low-normal birth weight. Given the parental consanguinity with the unaffected parents being third cousins once removed, the most probable pattern of inheritance was autosomal recessive. Linkage and mutational analyses identified compound heterozygous truncating mutations within the ASPM gene segregating with MCPH (c.2389C > T [p.Arg797X] and c.7781_7782delAG [p.Gln2594fsX6]). These results highlight some of the pitfalls of genetic analysis in consanguineous families. They also suggest that low birth weight may be a feature of MCPH, a finding that needs confirmation, and confirm that ASPM mutations are associated with simplified cortical gyration.     

The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling

Talpid3 is a classical chicken mutant with abnormal limb patterning and malformations in other regions of the embryo known to depend on Hedgehog signaling. We combined the ease of manipulating chicken embryos with emerging knowledge of the chicken genome to reveal directly the basis of defective Hedgehog signal transduction in talpid3 embryos and to identify the talpid3 gene. We show in several regions of the embryo that the talpid3 phenotype is completely ligand independent and demonstrate for the first time that talpid3 is absolutely required for the function of both Gli repressor and activator in the intracellular Hedgehog pathway. We map the talpid3 locus to chromosome 5 and find a frameshift mutation in a KIAA0586 ortholog (ENSGALG00000012025), a gene not previously attributed with any known function. We show a direct causal link between KIAA0586 and the mutant phenotype by rescue experiments. KIAA0586 encodes a novel protein, apparently specific to vertebrates, that localizes to the cytoplasm. We show that Gli3 processing is abnormal in talpid3 mutant cells but that Gli3 can still translocate to the nucleus. These results suggest that the talpid3 protein operates in the cytoplasm to regulate the activity of both Gli repressor and activator proteins.     

The mitotic spindle is required for loading of the DASH complex onto the kinetochore.

A role for the mitotic spindle in the maturation of the kinetochore has not been defined previously. Here we describe the isolation of a novel and conserved essential gene, ASK1, from Saccharomyces cerevisiae involved in this process. ask1 mutants display either G(2)/M arrest or segregation of DNA masses without the separation of sister chromatids, resulting in massive nondisjunction and broken spindles. Ask1 localizes along mitotic spindles and to kinetochores, and cross-links to centromeric DNA. Microtubules are required for Ask1 binding to kinetochores, and are partially required to maintain its association. We found Ask1 is part of a multisubunit complex, DASH, that contains approximately 10 components, including several proteins essential for mitosis including Dam1, Duo1, Spc34, Spc19, and Hsk1. The Ipl1 kinase controls the phosphorylation of Dam1 in the DASH complex and may regulate its function. We propose that DASH is a microtubule-binding complex that is transferred to the kinetochore prior to mitosis, thereby defining a new step in kinetochore maturation.     

The MAL63 gene of Saccharomyces encodes a cysteine-zinc finger protein

Summary Inducible maltose fermentation by tSaccharomyces carlesbergensis requires the product of the MAL63 gene of the MAL6 locus. It has been suggested that this gene product is an activator protein controlling the expression of the structural genes encoding the maltose fermentative enzymes perhaps by binding to DNA sequences upstream of these genes. We report the sequence of the MAL63 gene. A single open reading frame is seen capable of encoding a protein of 470 amino acid residues. The deduced sequence of this protein indicates that it is a cysteine-zinc forger protein supporting the hypothesis that the MAL63 gene product is a DNA binding protein.     

Identification of beta-subunit of GTP-binding regulatory protein in mitotic spindle.

BACKGROUND: The heterotrimeric GTP-binding regulatory protein (G protein) is important in membrane signal transduction. Since the function of the beta-subunit of G protein (G beta) in tumor cells is not well-documented, identification of G beta in tumor cells was performed. EXPERIMENTAL DESIGN: Immunolocalization, Western blotting, and immunoprecipitation of G beta in mammalian tumor and normal cells were investigated using rabbit antisera against amino and carboxyl terminal peptide fragments of G beta. A human nasopharyngeal carcinoma cell line (NPC-TW039) was used as the cell model because of its short doubling time. RESULTS: Anti-G beta immunoreactivity was found to be associated with the plasma membrane and the mitotic spindle throughout the mitotic phase of cell replication. Colcemid pretreatment resulted in random distribution of the anti-G beta reaction product in the mitotic cells. The same phenomenon was also seen in various other tumor and normal cell lines. When solubilized membranous, cytosolic, and mitotic spindle fractions were analyzed by Western blotting, G beta (35 +/- 1 kDa) was found to be associated with the plasma membrane and mitotic spindle fractions. Immunoprecipitation of isolated mitotic spindles with anti-G beta further confirmed these results. CONCLUSIONS: These findings indicate that G beta is closely associated with the mitotic spindle as well as the plasma membrane and may be important in regulation of cell mitosis in addition to transmembrane signal transduction.     

ES2, a gene deleted in DiGeorge syndrome, encodes a nuclear protein and is expressed during early mouse development, where it shares an expression domain with a Goosecoid-like gene

ES2 is a gene deleted in DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS) which has homologs in species as distant as Caenorhabditis elegans and Drosophila . The function of ES2 is unknown, and the predicted protein sequence does not contain motifs which suggest a particular role in the developmental defects present in DGS and VCFS. Here we show that the mouse homolog, Es2 , is transcribed in two forms resulting from the use of alternative polyadenylation signals. Structural analysis programs predict that the Es2 -encoded peptide has a coiled-coil domain, and transfection experiments with an Es2 -green fluorescent protein (GFP) fusion construct show that the peptide is recruited into the nucleus. Es2 is highly expressed during mouse embryogenesis from E7 onwards. In situ hybridization with an RNA probe revealed that the gene is widely expressed; however, relatively higher expression was detected in the nervous system, with a particularly high area of expression in a sub-region of the pons. The Es2 expression domain in the pons is shared with a Goosecoid-like gene ( Gscl) which is located upstream of Es2 , and raises the possibility that the two genes share regulatory elements and/or interact in this region of the developing brain. This finding suggests that different genes in the deleted region may be functionally related and might explain the occurrence of the characteristic phenotype in patients with non-overlapping genetic lesions.