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Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation. A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1-7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined. MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from -3 to+458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1alpha. HNF4alpha induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1alpha while HNF4alpha induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression. This study emphasizes the complex regulation of MRP2 with HNF1alpha and HNF4alpha playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes.

Biliary excretion is the rate-limiting step in transfer of bilirubin, other organic anions, and xenobiotics across the liver. Multidrug resistance-associated protein 2 (Mrp2, Abcc2) is the major transporter for conjugated endo- and xenobiotic-conjugated compounds into bile. Hormones regulate bilirubin and xenobiotic secretion into bile, which have dimorphic differences. Therefore, we examined the possible role of sex steroids and growth hormone in the regulation of Mrp2. In ∼8-wk-old rats, mRNA, transcriptional activity, and hepatic content of Mrp2 were selectively increased fourfold ( P < 0.001) in females compared with males. In males, estrogens increased and testosterone decreased Mrp2 mRNA and protein, whereas no significant effect was measured in females, suggesting either a direct effect on the liver or an alteration in growth hormone secretory pattern. After hypophysectomy, Mrp2 mRNA was markedly reduced and the effects of estrogens and testosterone on Mrp2 were prevented, supporting the role of pituitary hormones in controlling Mrp2 expression. Mrp2 increased following growth hormone infusion in males. Mrp2 mRNA was decreased in growth hormone-deficient “Little” mice. Growth hormone infusions in hypophysectomized rats partially restored Mrp2 levels, whereas thyroxine addition returned Mrp2 mRNA and protein to basal levels. Morphology as well as biochemical measurements demonstrated that Mrp2 was localized to the bile canaliculus in equal density in both genders, whereas hormone replacements increased Mrp2 in hypophysectomized animals. In cultured hepatocytes, thyroxine did not have an effect, but growth hormone alone and combined with thyroxine increased Mrp2 mRNA levels. In conclusion, Mrp2 levels are regulated by the combination of thyroxine and different growth hormone secretory patterns.

The semaphorins comprise a family of secreted and membrane bound proteins that influence development of the nervous system as well as non-neural organs. H.SemaIV was originally isolated from a homozygously deleted region involving a subset of small cell lung cancers, a neuroendocrine derived neoplasm. To investigate H.SemaIV expression, specific polyclonal antibodies directed against a unique polypeptide (amino acids 758-773) were developed and their specificity confirmed. In cell lines, H.SemaIV staining was observed in cytoplasmic granules. In the normal adult human brain, we noted three general characteristics of H.SemaIV expression. H.SemaIV was strongly present in specific nuclei or in neuronal regions arranged in defined subnuclear structures. It was also present in neurons but not glial cells or ependymocytes. Lastly, H.SemaIV was not present in cell bodies, but rather in fibers and nerve terminals. Interestingly, an altered pattern of staining was detected in brains of three patients with Alzheimer's disease.

X-linked recessive myotubular myopathy (XLMTM) is a very severe congenital muscular disease characterised by an impaired maturation of muscle fibres, and caused by defects in the MTM1 gene. This gene defines a new family of putative tyrosine phosphatases conserved through evolution. We have determined intronic flanking sequences for all the 15 exons to facilitate the detection of mutations in patients and genetic counselling. We characterised a new polymorphic marker in the immediate vicinity of the gene, which might prove useful for linkage analysis. Sequencing of the TATA-less predicted promoter provides the basis for transcriptional regulatory studies.

X-linked recessive myotubular myopathy (XLMTM) is a severe neonatal neuro-muscular disease characterized by muscle weakness, hypotonia, and respiratory problems. The locus for the XLMTM gene (MTM1) has previously been mapped to Xq28 between the markers DXS304 and DXS497 by linkage analyses and by determining the breakpoints of deletion patients. We report linkage analysis data or 20 XLMTM families who were tested using the DNA markers DXS1113, DXS304, DXS455, DXS1684, DXS305 and DXS52 and present two families showing recombination between MTM1 and either DXS304, DXS334 or DXS305. We found each of the families to be informative for at least three markers. Based on these findings we excluded 30 women from being carriers, the carrier status of 17 obligate carrier mothers could be confirmed and eight mothers and sisters were identified as to be at high risk of carrying a MTM1 mutation. By combining recently published data with the results of our recombinant families, we suggest that the MTM1 locus maps between DXS334 and DXS497 narrowing the region of interest from 600 kb to an estimated500 kb interval. This additional refinement in the localization of MTM1 means a further step towards the isolation of the gene in the near future, and allows more reliable and efficient carrier detection and prenatal diagnosis.

X-linked recessive myotubular myopathy (MTM1) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. We have restricted the candidate region to 280 kb and characterized two candidate genes using positional cloning strategies. The presence of frameshift or missense mutations (of which two are new mutations) in seven patients proved that one of these genes is indeed implicated in MTM1. The protein encoded by the MTM1 gene is highly conserved in yeast, which is surprising for a muscle specific disease. The protein contains the consensus sequence for the active site of tyrosine phosphatases, a wide class of proteins involved in signal transduction. At least three other genes, one located within 100 kb distal from the MTM1 gene, encode proteins with very high sequence similarities and define, together with the MTM1 gene, a new family of putative tyrosine phosphatases in man.

X-linked myotubular myopathy (MTM1) is a severe congenital myopathy characterized by hypotonia, muscle weakness, and associated respiratory insufficiency. Perinatal death is common. The disease locus was shown to be linked to polymorphic markers in Xq28 and we have recently refined the MTM1 locus to a physical region of less than one megabase (Mb) at proximal Xq28. Two new microsatellite markers were developed and assigned in the MTM1 candidate region. We applied them and other DNA markers for prenatal diagnosis in two families. In one case, an affected fetus was predicted and a recombination event was observed with two more distal markers in the region. The second fetus was born unaffected as predicted. The new DNA markers and the precise location of the MTM1 gene provide an improvement for early prenatal diagnosis of the disease. We present suggestions for different combinations of linked and flanking DNA markers for maximal informativeness and accuracy.

Small mutations in the POU domain gene POU3F4 were recently shown to cause X-linked deafness type 3 (DFN3) in nine unrelated males. The POU3F4 gene was found to be located outside four of five deletions associated with DFN3. Two of these deletions were situated more than 400 kb proximal to POU3F4. Employing PCR analysis of sequence tagged sites from this region we initially identified novel deletions in two DFN3 patients. To investigate this chromosomal segment in more detail, we extended a previously established 850 kb cosmid contig in the centromeric direction to a total size of 1500 kb. Cosmids from this contig were hybridized to DNA of 11 unrelated males with DFN3. In two patients, we identified deletions encompassing the POU3F4 gene and variably sized segments of Xq21.1. In six of the nine remaining patients which lacked mutations in the POU3F4 gene, smaller deletions were identified which, with one exception, overlap in a 8 kb segment 900 kb proximal to the POU3F4 gene. In one patient, we identified several small deletions in the vicinity of the 8 kb DNA segment. Together, deletions account for 56% (13/23) of all known DFN3 mutations, most (10/13) of which do not encompass the POU3F4 gene. The combined molecular data suggest that the deletion hot spot region in Xq21.1 contains another DFN3 gene or, alternatively, a sequence element involved in transcriptional regulation of POU3F4.

Background Although melanoma can elicit robust tumor antigen-specific immune responses, advanced melanoma is associated with immune tolerance. We have previously described several mechanisms of melanoma-induced immunosuppression, including the skewing of the immune response towards a Th2 cytokine profile and the induction of regulatory T cells. Since dendritic cells (DCs) are potentially important players that can direct other cells of the immune system towards a cytotoxic, humoral, or regulatory phenotype, we hypothesized that melanoma-produced factors directly affect the maturation and function of DCs, influencing the nature and magnitude of the resulting immune response. Materials and Methods To test this hypothesis, immature myeloid-derived DCs (mdDCs) were derived with cytokines from CD14+ peripheral blood mononuclear cells (PBMCs) and exposed to 20% melanoma-conditioned media (MCM). After 2 d, the expression of maturation markers and the function of these mdDCs, measured by cytokine production, the amount of endocytosis, expression of the inhibitory molecule indoleamine 2,3-dioxygenase (IDO), and the ability to stimulate T cells were determined. Results We found that incubation with MCM did not inhibit the expression of maturation markers or IDO, the production of cytokines, the amount of antigen uptake, or the ability to induce T cell proliferation in mixed-lymphocyte reactions by mdDC. Conclusions These results suggest that the immunosuppressive effects of melanoma-produced factors are independent of directly measurable changes in mdDC function or maturation in vitro .

Objective Cerebral cavernous malformations (CCMs) are focal dysmorphic blood vessel anomalies predisposing individuals to hemorrhagic stroke and epilepsy. CCMs are sporadic or inherited as autosomal dominant disease with three known genes. The hypothesis that genetic heterogeneity would account for the remarkable variability in CCM manifestations was tested. Methods CCM cases were prospectively enrolled. Germline CCM1 gene mutations were sought in 89 CCM samples. Associations with clinical manifestations and lesion characteristics were made among 41 symptomatic familial cases, including one cohort of 26 cases with CCM1 mutations and a second cohort of 15 cases without identifiable CCM1 mutations. The 15 cases were screened for CCM2 and CCM3 mutations. Results CCM1 mutations were found in 34 out of 50 subjects with familial disease and in none of 39 sporadic CCM cases. CCM2 and CCM3 mutations were found in three out of 10 families screened without CCM1 mutations. Clinical manifestations in 22 Hispanic-American cases with identical CCM1 mutations were highly variable. Fewer CCM1 patients experienced hemorrhage than others with familial disease (P = 0.0139 for all cases and P = 0.0442 for symptomatic cases). Adjusting for sex and age improved the logistic regression model, suggesting decreased numbers of patients with hemorrhage in CCM1 familial disease (P = 0.003 for all cases and P = 0.014 for symptomatic cases). Hemorrhage differences were not related to size or number of lesions. Conclusion Factors in addition to CCM1 germline mutation contribute to CCM clinical manifestations. However, this evidence suggests that familial cases with CCM1 mutations may have less severe clinical manifestations than other familial cases.

Cerebrovascular malformations (CVMs) are lesions with an abnormal vessel phenotype that predisposes patients to hemorrhagic strokes, seizures, focal neurological deficits, and other clinical manifestations (4, 22). They include arteriovenous malformations (AVMs) and cerebral cavernous malformations (CCMs) and have distinct clinicopathological radiological profiles (14, 30). The AVMs are tangled complexes of tortuous vessels representing fistulous connections between arteries and veins, and they lack an intervening capillary bed. They reveal preserved features of mature vessel wall phenotype altered by high flow and hemodynamic stress, including arterial, nidal, and venous aneurysms (5, 6, 28). The CCMs are characterized by caverns filled with blood or thrombus and are lined with a single layer of endothelial cells. These low-flow lesions are associated with brittle vasculature and repetitive oozing (5, 6). The CCMs lack inter-endothelial cell tight junctions and mature vessel wall angio-architecture (8, 37). Little is known about the mechanisms of genesis or the progression of these lesions. Several proteins are abnormally expressed in AVMs and CCMs. Our group and others have demonstrated by performing immunohistochemical analysis that vascular endothelial growth factor (VEGF) and the VEGF receptors VEGF-r1 (flt1) and VEGF-r2 (flk1) are overexpressed in both AVMs and CCMs compared with normal brain vessels (21, 35). Fibronectin is expressed to a greater extent in CCMs than in AVMs, consistent with the proliferative immature vessel phenotype (21). Laminin and smoothelin, both of which reflect mature vessel wall phenotype, are underexpressed in CCMs as compared with AVMs (21, 34). Distinct genes have been identified that predispose individuals to familial manifestations of these lesions, which is related to transforming growth factor-β receptor binding proteins (1, 13, 18, 19) and the Krev Interaction Trapped 1 (krit1) signaling pathway (9, 17, 23, 26, 32) for AVMs and CCMs, respectively. We hypothesize that different groups of genes are involved in the pathogenesis of AVMs and CCMs and that other genes are nonspecifically associated with both lesion types. In the experiments described in this article, we applied gene microarray analysis to correlate alterations in ribonucleic acid (RNA) transcription in AVMs and CCMs to the previously published abnormal protein expression in these lesions. In addition, we found numerous other genes differentially expressed in one or both lesion types, which has not been reported previously.

We have identified a novel human gene that is entirely deleted in two boys with abnormal genital development and myotubular myopathy (MTM1). The gene, F18, is located in proximal Xq28, approximately 80 kb centromeric to the recently isolated MTM1 gene. Northern analysis of mRNA showed a ubiquitous pattern and suggested high levels of expression in skeletal muscle, brain, and heart. A transcript of 4.6 kb was detected in a range of tissues, and additional alternate forms of 3.8 and 2.6 kb were present in placenta and pancreas, respectively. The gene extends over 100 kb and is composed of at least seven exons, of which two are noncoding. Sequence analysis of a 4.6-kb cDNA contig revealed two overlapping open reading frames (ORFs) that encode putative proteins of 701 and 424 amino acids, respectively. Two alternative spliced transcripts affecting the large open reading frame were identified that, together with the Northern blot results, suggest that distinct proteins are derived from the gene. No significant homology to other known proteins was detected, but segments of the first ORF encode polyglutamine tracts and proline-rich domains, which are frequently observed in DNA-binding proteins. The F18 gene is a strong candidate for being implicated in the intersexual genitalia present in the two MTM1-deleted patients. The gene also serves as a candidate for other disorders that map to proximal Xq28.

We have recently refined the localization of the myotubular myopathy (MTM1) gene to a 430-kb region between DXS304 and DXS1345 in proximal Xq28. We report two new polymorphic microsatellite markers, DXS8377 and DXS7423, that were physically mapped within the critical interval. A recombination event in a family segregating for MTM1 placed the disease gene telomeric to the trinucleotide polymorphism DXS8377. Together with the recent mapping of two microdeletions associated with MTM1, the recombination refines the critical region to 280 kb. A second recombination event was observed distal to the tetranucleotide repeat DXS7423. This recombination has occurred in the off-spring of a female with a more than 67% probability of being a carrier and very likely restricts the MTM1 gene to a 130-kb region. This physical refinement is significant for positional cloning of the disease gene. The highly polymorphic markers and the precise localization of the MTM1 gene will facilitate genetic diagnosis of the disorder.

We have recently described a female patient with myotubular myopathy (MTM1) and an interstitial deletion at Xq28. Characterisation of the deletion allowed us to position the MTM1 gene to a 600 kb region between DXS304 and DXS497. In order to further restrict the region we screened for deletions in a set of 38 patients. We found two overlapping deletions in boys that in addition to MTM1 showed an unexpected abnormal genital development. As the latter phenotype is not found in the other non-deleted MTM1 patients, our observations are best explained by a contiguous gene syndrome. The deletions define a 430 kb region that contains the MTM1 gene and most likely a gene implicated in male sexual development. A high resolution physical map of this region is presented.

CPPs is characterized by hyperproliferation of keratinocytes (KC) and T-cell (Tc)/DCs activation. IL-32, which induces TNF and activates NFkB and p38 MAPK, is involved in early inflammation. We investigated the role of IL-32 in the pathogenesis of CPPs by IHC, ECL and qRT-PCR analysis. Biopsy tissues were obtained from CPPs patients with active disease (N=20) and healthy human controls (N=15). A mouse Mab against human IL-32 and mouse IgG1 isotype antibody were used. IHC staining of IL-32 was performed on deparaffinized sections of formalin-fixed tissues by an indirect imunoperoxidase method (EnVision, Dako). IL-32 was expressed in basal KC with cytoplasmic granular staining. IL-32 was also expressed in DCs with strong nuclear and cytoplasmic staining in dermis. IL-32 was weakly expressed in the nucleus of KC in healthy human skin. Induction of IL-32 was detected in human KC lysates after stimulation with TNF and IL-1 or cell contact with activated Tc, but not in KC supernatants, indicating IL-32 is not secreted from KC in vitro. ECL/qRT-PCR results showed IL-32 and other CPPs-relevant cytokines (IL-18, IL-20, 22 and 23) were significantly (P

During development, growth cones can be guided at a distance by diffusible factors, which are attractants and/or repellents. The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic, motor, and forebrain axons. We found that in rat embryos, the olfactory epithelium releases a diffusible factor that repels olfactory bulb axons. In addition, Sema A and Sema IV, but not Sema III, Sema E, or Sema H, are able to orientin vitrothe growth of olfactory bulb axons; Sema IV has a strong repulsive action, whereas Sema A appears to attract those axons. The expression patterns ofsema Aandsema IVin the developing olfactory system confirm that they may play a cooperative role in the formation of the lateral olfactory tract. This also represents a further evidence for a chemoattractive function of secreted semaphorins.