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Objective: To conduct the genotype-phenotype correlation in a family in which several individuals share clinical and electrophysiologic features of paramyotonia congenita (PC). Background: PC, hyperkalemic periodic paralysis (HyperPP), and potassium-aggravated myotonias form the group of hereditary sodium channelopathies. Each of these disorders is associated with different point mutations in SCN4A , the gene encoding the α-subunit of the adult human skeletal muscle sodium channel. However, in HyperPP families, evidence of a causative gene different from SCN4A has been found. Methods: We conducted direct clinical examination, electrophysiologic (EMG/electroneurographic) and cardiologic studies, as well as laboratory screening in several affected and nonaffected members of the family. We performed the genotype-phenotype correlation by microsatellite linkage and cDNA-mutation analyses of the SCN4A gene. Results: Affected members in this family showed clinical and electrophysiologic features typical of PC. The disease phenotype segregated with the chromosomal region that includes the SCN4A gene. Analysis of the entire cDNA sequence of the SCN4A gene in the index case disclosed a G3826A transition, which results in the Val1276Ile substitution. However, PCR–single-stranded confirmation polymorphism and direct sequencing analysis of the segment coding for Val-1276 on genomic DNA confirmed the G3826A transition in the index case but was negative in 11 affected members of the family; however, neither mutations nor aberrant splicings causative of the PC phenotype in this family were found on SCN4A . Conclusion: The existence of a second gene different from SCN4A that can give rise to a clinical PC phenotype can be speculated upon.

Double-stranded DNA fragments were selected from a random pool by repeated cycles of estrogen receptor-specific immunoprecipitation in the presence of a nuclear extract and PCR amplification (cyclic amplification and selection of target, CAST, for multiple elements). Fragments were cloned and sequence analysis indicated the 5-nucleotide word TTGGC was the most recurrent sequence unrelated to the known estrogen responsive element. Screening a HeLa cell expression library with a probe designed with multiple repeats of this sequence resulted in the identification of a 1700-aa protein showing a complete homology with the product of the human retinoblastoma-interacting zinc-finger gene RIZ. In transfection experiments, RIZ protein was able to bestow estrogen inducibility to a promoter containing an incomplete estrogen responsive element and a TTGGC motif. RIZ protein present in MCF-7 cell nuclear extract retarded the TTGGC-containing probe in an EMSA. Estrogen receptor was co-immunoprecipitated from MCF-7 cell extract by antibodies to RIZ protein and vice versa, thus indicating an existing interaction between these two proteins.

Among the heterogeneous group of autosomal recessive limb-girdle muscular dystrophies (AR LGMDs), the sarcoglycanopathies (LGMD2C-2F) represent a subgroup characterised by defects in the gamma, alpha, beta, and delta sarcoglycan genes, respectively. Genotype-phenotype correlations in these forms of AR LGMD are important to enhance our understanding of protein function. Regarding LGMD2F, only two homozygous frameshift mutations have been reported to date in patients with a severe phenotype. In the present report, through screening 23 unrelated AR LGMD patients, we identified three subjects with LGMD2F, two with a previously reported frameshift mutation and the other homozygous for a new missense mutation in the delta sarcoglycan gene. Interestingly, this new mutation is also associated with a severe clinical course. In addition, our results suggest that this form of severe AR LGMD is not very rare in our population.

hGH-1 gene deletions are detected by simultaneous PCR amplification along the two homologous DNA sequences flanking the hGH-1 gene on both sides and are differentiated by SmaI restriction enzyme digestion. We have observed that among the SmaI digested PCR products from normal homozygous subjects, from those heterozygous for the 7.6 kb deletion and from those heterozygous for a 6.7 kb deletion, along with the expected fragments there is an unexpected 1470 bp fragment. This fragment arises from the co-amplification of a third homologous sequence located downstream from the hGH-1 gene and it confuses differentiation between normal homozygous and heterozygous for 7.6 kb subjects from the 6.7 kb heterozygous subjects. To overcome this problem we have improved PCR conditions using a different reverse primer. These changes avoid the interaction of the primers with the third homologous sequence located downstream from the hGH-1 gene and prevent the appearance of this additional band that complicates the interpretation of the results. We conclude that the new reverse primer sequence avoids the amplification of the downstream hGH-1 gene sequence and the production of the 1474 bp band after SmaI endonuclease enzyme digestion and makes it possible to differentiate homozygous normal subjects and those who are heterozygous for a 7.6 kb deletion from those who are heterozygous for a 6.7 kb deletion.

A 104-kD protein was coimmunoprecipitated with the estrogen receptor from the flowtrough of a phosphocellulose chromatography of MCF-7 cell nuclear extract. mAbs to this protein identified several cDNA clones coding for the human 104-kD major vault protein. Vaults are large ribonucleoprotein particles of unknown function present in all eukaryotic cells. They have a complex morphology, including several small molecules of RNA, but a single protein species, the major vault protein, accounts for >70% of their mass. Their shape is reminiscent of the nucleopore central plug, but no proteins of known function have been described to interact with them. Western blot analysis of vaults purified on sucrose gradient showed the presence of estrogen receptor co-migrating with the vault peak. The AER317 antibody to estrogen receptor coimmunoprecipitated the major vault protein and the vault RNA also in the 20,000 g supernatant fraction. Reconstitution experiments of estrogen receptor fragments with the major vault protein mapped the site of the interaction between amino acids 241 and 280 of human estrogen receptor, where the nuclear localization signal sequences are located. Estradiol treatment of cells increased the amount of major vault protein present in the nuclear extract and coimmunoprecipitated with estrogen receptor, whereas the anti-estrogen ICI182,780 had no effect. The hormone-dependent interaction of vaults with estrogen receptor was reproducible in vitro and was prevented by sodium molybdate. Antibodies to progesterone and glucocorticoid receptors were able to coimmunoprecipitate the major vault protein. The association of nuclear receptors with vaults could be related to their intracellular traffic.

We found a novel dystrophin-associated protein (DAP) exhibiting almost the same mobility as γ-sarcoglycan on SDS-PAGE. This novel DAP with basic charge is separated from γ-sarcoglycan by 2-dimensional PAGE or de- N -glycosylation followed by SDS-PAGE. This DAP is most likely the rabbit homologue of “δ-sarcoglycan”, the γ-sarcoglycan-like protein identified previously [Nigro et al. (1996) Hum. Mol. Genet. 5, 1179–1186], since an internal amino acid sequence from the DAP matched the predicted amino acid sequence of “human δ-sarcoglycan” within the limits of species difference and this DAP was recognized by anti-“δ-sarcoglycan” antibody. The DAP was found to be contained in the sarcoglycan fraction which was prepared by treatment of the dystrophin-DAP complex with n -octyl β- d -glucoside and crosslinked with β- and/or γ-sarcoglycan by a chemical crosslinker, dithiobis(succinimidyl propionate). Therefore, we concluded that the DAP is the fourth component of the sarcoglycan complex. © 1997 Federation of European Biochemical Societies.

Analysis of the exon-intron organization of the human dystrophin gene has been hampered by its enormous size. By using a YAC-based exon mapping approach and long PCR, we have succeeded in defining the size of the gene and its organization. Our results, compared with data on the distribution of deletion breakpoints by intron, elucidate the topography of the intragenic deletion-prone regions. Within the central high-frequency deletion region, the small, 6.6-kb, intron 49 shows a much higher density of deletion breakpoints than intron 44, which was previously believed to coincide with the most mutable zone of the gene. On the other hand, in the proximal part of the gene, deletion breakpoints do not preferentially occur in a few introns, but are spread over a large DNA segment containing introns 2 to 42.

The BIO14.6 hamster is a widely used model for autosomal recessive cardiomyopathy. These animals die prematurely from progressive myocardial necrosis and heart failure. The primary genetic defect leading to the cardiomyopathy is still unknown. Recently, a genetic linkage map localized the cardiomyopathy locus on hamster chromosome 9qa2.1-b1, excluding several candidate genes. We now demonstrate that the cardiomyopathy results from a mutation in the delta-sarcoglycan gene that maps to the disease locus. This mutation was completely coincident with the disease in backcross and F2 pedigrees. This constitutes the first animal model identified for human sarcoglycan disorders.

We have raised an anti-emerin polyclonal antibody against a fusion protein encompassing most of the hydrophilic portion of emerin. Using this antibody, we have analyzed emerin expression in Emery-Dreifuss muscular dystrophy (EDMD) patients and controls, by immunocytochemistry, in skeletal muscle and skin, and by immunoblot, in peripheral blood mononuclear cells and lymphoblasts. Emerin was localized on the surfaces of nuclei in control skeletal muscle and skin but was absent or reduced in patient skeletal muscle, was absent from the skin of patients, and was expressed only in a few nuclei in a patient's mother. Immunoblot of peripheral blood cells from EDMD patients showed absence of the emerin band, altered-size emerin, or a protein of normal molecular mass but slightly reduced quantity. The diagnosis of X-linked EDMD is normally confirmed by genetic analysis of the STA gene coding for emerin. We propose immunocytochemical evaluation of emerin expression in skin biopsies as a sensitive and more convenient tool for diagnosing X-linked EDMD and, in particular, for distinguishing it from the autosomal dominant form. This technique may be applied to suspected EDMD patients, especially sporadic cases or those with incomplete clinical phenotype, and also suspected carriers. Immunoblot of peripheral blood cells is also useful, but it may not unequivocally identify carriers and some patients.

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of inherited neuromuscular disorders characterized by proximal muscular weakness of the pelvic and shoulder girdles and a variable progression with symptoms, ranging from very severe to mild1,2. One autosomal dominant (LGMD1 A, at chromosome 5q22.3–31.3) (ref. 3) and five autosomal recessive (AR) loci responsible for this phenotype have been identified: LGMD2A at 15q (ref. 4); LGMD2B at 2p (ref. 5), LGMD2C at 13q (ref. 6), LGMD2D at 17q (ref. 7) and LGMD2E at 4q (refs 8, 9). In the muscle membrane, dystrophin associates with several proteins and glycoproteins organized in two main subcomplexes: the dystroglycan (DG) and sarcoglycan (SG) complexes9, 10–14. The genes for LGMD2C, LGMD2D and LGMD2E code for proteins of the SG complex7, 8, 15–17. We recently mapped a sixth AR form of LGMD, LGMD2F, to chromosome 5q33–3418 in two Brazilian families. In the same chromosomal interval we also mapped the δSG gene, encoding a novel 35-kD component of the sarcoglycan (SG) complex19. We now show that a homozygous mutation in the δSG gene (a single nucleotide deletion that alters its reading frame) is the cause of LGMD2F.

Objective. —To characterize the presence and behavior of the dystrophinopathic myocardial damage in female carriers of a gene defect at the Xp21 locus of the X chromosome that causes Duchenne and Becker muscular dystrophies (DMD and BMD). Design. —Cohort study from April 1, 1985, to April 30, 1995, with cardiologic follow-up performed yearly for a minimum of 3 to a maximum of 10 years. Setting. —Counseling center for genetic muscular disorders. Patients. —A total of 197 women and girls aged 5 to 60 years ascertained to be carriers of the DMD (n=152) or BMD (n=45) gene. Main Outcome Measures. —Cardiac status at yearly examinations as determined by 12-lead electrocardiogram (ECG), 24-hour ambulatory ECG, M-mode and 2-dimensional echocardiography, and carotid pulse tracing. Myocardial scintigram was performed on each individual at least twice during the study. Immunohistochemical analysis of dystrophin from myocardium and/or skeletal muscle biopsy was performed in 12 carriers. Results. —Preclinical or clinically evident myocardial involvement was found in 166 cases (84.3%), without significant differences in percentage and behavior between DMD and BMD carriers. Its occurrence increased significantly with age, from 54.5% (18 cases) in carriers aged between 5 and 16 years to 90.2% (148 cases) in carriers older than 16 years. Dystrophin anomalies were detected at the membrane level of the myocardial fibers in all endomyocardial biopsy specimens. Conclusions. —Genetic anomalies can be considered the primary cause of myocardial damage in carriers of dystrophinopathic myopathies; myocardial damage shows the same behavior already described in DMD and BMD patients and progresses from preclinical to dilated cardiomyopathy, passing through stages of myocardial hypertrophy or dysrhythmias. ( JAMA . 1996;275:1335-1338)

Mutations in any of the genes encoding the alpha, beta or gamma-sarcoglycan components of dystrophin-associated glycoproteins result in both sporadic and familial cases of either limb-girdle muscular dystrophy or severe childhood autosomal recessive muscular dystrophy. The collective name 'sarcoglycanopathies' has been proposed for these forms. We report the identification of a fourth member of the human sarcoglycan family. We named this novel cDNA delta-sarcoglycan. Its mRNA expression is abundant in striated and smooth muscles, with a main 8 kb transcript, encoding a predicted basic transmembrane glycoprotein of 290 amino acids. Antibodies specifically raised against this protein recognized a single band at 35 kDa on western blots of human and mouse muscle. Immunohistochemical staining revealed a unique sarcolemmal localization. FISH, radiation hybrid and YAC mapping concordantly linked the delta-sarcoglycan gene to 5q33, close to D5S487 and D5S1439. The gene spans at least 100 kb and is composed of eight exons. The identification of a novel sarcoglycan component modifies the current model of the dystrophin-glycoprotein complex.

Next generation sequencing (NGS) is having a tremendous impact on our knowledge of different aspects of biology. It can be also very powerful to study patients with heterogeneous genetic conditions, like muscular dystrophies. First, to identify new genes using “exome resequencing”. Second, to diagnose mutations in all the known causative genes, when used as targeted approach. Third to obtain a knowledge of the impact of mutations on mRNA expression and splicing in diseased muscle. We used NGS to identify new genes by whole exome sequencing. We sequenced the whole exome of four family members with LGMD1F separated by up to eleven meioses and identified a single shared novel heterozygous frame-shift variant. This causes a nonstop change in the Transportin 3 (TNPO3) gene that encodes a member of the importin-β super-family. To reach the second task, we first recruited 160 familial cases of nonspecific limb-girdle muscular dystrophies with apparent autosomal inheritance. All DNA samples were first enriched for 486,480 bp, covering 2447 exons of 98 genes by using the Haloplex technology with the use of barcodes. We the performed pooled NGS of all samples and identified a number of mutations, then verified by Sanger sequencing. Cases were also studied by the Agilent MotorChip CGH array version 3.0 to identify deletions or duplications. Finally, in selected cases, we performed the RNA-Seq starting from a muscle biopsy sample. We converted mRNA to cDNA and purified it by a customized SureSelect Target Enrichment System, focused on the same 98 mRNAs. The probes had a 4× coverage with a total target of 1.41 Mb of sequences/sample. These cDNAs were sequenced using barcodes trying to obtain an average sequencing coverage of at least 100×. Our results confirm that there is a very high genetic heterogeneity in muscular dystrophies and that NGS-based DNA and RNA testing are ready for diagnostic use.

""Critical functional properties are embedded in the non-coding portion of the human genome. Recent successful studies have shown that variations in distant-acting gene enhancer sequences can contribute to disease. In fact, various disorders, such as thalassaemias, preaxial polydactyly or susceptibility to Hirschsprung’s disease, may be the result of rearrangements of enhancer elements. We have analyzed the distribution of enhancer loci in the genome and compared their localization to that of previously described copy-number variations (CNVs). These data suggest a negative selection of copy number variable enhancers. To identify CNVs covering enhancer elements, we have developed a simple and cost-effective test. Here we describe the gene selection, design strategy and experimental validation of a customized oligonucleotide Array-Based Comparative Genomic Hybridization (aCGH), designated Enhancer Chip. It has been designed to investigate CNVs, allowing the analysis of all the genome with a 300 Kb resolution and specific disease regions (telomeres, centromeres and selected disease loci) at a tenfold higher resolution. Moreover, this is the first aCGH able to test over 1,250 enhancers, in order to investigate their potential pathogenic role. Validation experiments have demonstrated that Enhancer Chip efficiently detects duplications and deletions covering enhancer loci, demonstrating that it is a powerful instrument to detect and characterize copy number variable enhancers.""

DNA from tumor tissue and peripheral blood lymphocytes of primary breast cancer patients was screened for the presence of p53 mutations. In DNA from one tumor we found that the histidine codon 193 (CAT) was somatically converted to arginine (CGT). This amino acid residue is highly conserved in many species, thus suggesting that such mutation plays an important role in the loss of wt-p53 function.

Receptors are expressed by eukaryotic cells, either as intracellular or cell surface proteins. They allow signalling molecules to be detected which influence cells to proliferate or differentiate. Receptors activate a cascade of events that ultimately involves the transcriptional responses of a definite set of genes (Kerr et al., 1992). Whilst cell surface receptors transmit an external signal via the production of intracellular second messengers, nuclear receptors provide a direct link between extracellular signals and the genetic apparatus functioning as hormone-dependent transcription factors (Beato, 1991). Steroid, thyroid, retinoid and related hormones influence cell fate during embryonic development and modulate organ function in the adult. The main requirement of a hormone response pathway is to be highly specific and flexible, allowing different responses under different conditions (Laudet and Stehelin, 1992).

A new panel of monoclonal antibodies to the calf uterus estrogen receptor was prepared. Thirteen antibodies were characterized for their isotype and for the affinity for the antigen. These antibodies recognize the human receptor and can be used in Western blot analysis. The location of the epitopes was mapped on the antigen structure using synthetic fragments of estrogen receptor, and it was possible to group the antibodies in five groups. Many antibodies were useful for the purification of estrogen receptor from tissue extracts by immunoaffinity chromatography. The reciprocal inhibition of the antibodies for the antigen binding was measured with an immunoadsorption assay. This was maximal and symmetrical for antibody pairs within the same group, but was incomplete and, in some instances, asymmetrical between pairs of antibodies from different groups. One antibody was able to inhibit the estrogen receptor-DNA interaction, whereas two others were unable to recognize the receptor-DNA complexes. This new panel of antibodies is a useful addition to the existing tools for studying structure and function of the estrogen receptor.

Objective: Evaluation of hepatitis C virus (HCV) by reverse transcription-polymerase chain reaction (RT-PCR) in peripheral nerve tissues from HCV infected patients with peripheral neuropathy. METHODS: RT-PCR was performed on homogenates of nerve biopsies from 17 consecutive HCV-positive patients with peripheral neuropathy, with or without mixed cryoglobulinemia, hospitalised from 1996 to 2000. Sural nerve specimens were frozen in iso-pentane pre-cooled in liquid nitrogen and stored at −80°C until use. RNA was extracted from ten 7-μm thick cryostatic sections or from a nerve trunk specimen of about 3 mm length, collected from each biopsy. Three different protocols of RNA extraction were tested (1–3). Complementary DNAs (cDNAs) were obtained without or with RNasin (Promega, Madison, WI) addition in the reaction mixture to inhibit residual RNase activity. Two sets of commercially available PCR primers for the outer and the nested reaction were used. PCR products were analysed by agarose gel electrophoresis and ethidium bromide staining. Serum samples and liver specimens from proven HCV positive patients served as positive controls, whereas sera from healthy subjects were negative controls. RESULTS: Sufficient amount of RNA could be obtained either by cryostatic sections or by in toto nerve specimens. Extraction by Trizol (Gibco-BRL) allowed the best concentration and purity of RNA as assessed by biophotometry. The presence of RNasin didn't improve the cDNA synthesis. The resulting amplification product of the nested PCR was 187 bp long. We have always observed this product in our positive controls and never in the negative. Six samples from patients either with or without cryoglobulinemia resulted positive; 7 were negative. Four samples gave variable results. CONCLUSIONS: While 40% of the nerves in our series were undoubtedly HCV positive, the cause(s) of negative and variable results in the remaining samples is likely more complex than variations in the detection protocols and deserve further investigations. REFERENCES: 1) Chomczynski P, Sacchi N (1987). Anal Biochem 162:156. 2) Marquardt O et al. (1996). Med Microbiol Lett 5:55. 3) Chomczynski P (1993). Bio/Techniques 15:532.

A combination of multiplex PCR with the single strand conformation polymorphism (SSCP) technique was employed to screen for point mutations in the human dystrophin gene. Co-amplification of 11 exons from genomic DNA of Duchenne and Becker muscular dystrophy (DMD/BMD) patients with no deletion or duplication was performed and the samples subjected to multiple SSCP analysis. We report the case of a nonsense mutation in a Duchenne patient identified by this approach. The mutation introduces a termination codon within exon 8 of the dystrophin gene. It is predicted to cause a very premature translational termination accounting for the severe phenotype observed. The patient inherited this mutation from his mother. In addition the analysis revealed 5 polymorphisms useful for internal control.

An in vitro transcription system was developed to investigate the mechanisms of gene regulation by the estrogen receptor (ER). ER purified from calf uterus was highly active in enhancing RNA transcription from a template DNA containing estrogen response elements (EREs) upstream from a minimal promoter. Under the conditions employed, no addition of tissue specific factors was required and both estrogen or antiestrogens were ineffective. The stimulation of transcription correlated with the copy number of EREs in the template. The addition of competitor ERE oligonucleotides specifically inhibited the ER-induced transcription. We suggest that the ER may be involved in the formation of the stable initiation complex.

Background The BIO14.6 hamster is an excellent animal model for inherited cardiomyopathy, because of its lethal and well-documented course, due to a spontaneous deletion of delta-sarcoglycan gene promoter and first exon. The muscle disease is progressive and average lifespan is 11 months, because heart slowly dilates towards heart failure. Methodology/Principal Findings Based on the ability of adeno-associated viral (AAV) vectors to transduce heart together with skeletal muscle following systemic administration, we delivered human delta-sarcoglycan cDNA into male BIO14.6 hamsters by testing different ages of injection, routes of administration and AAV serotypes. Body-wide restoration of delta-SG expression was associated with functional reconstitution of the sarcoglycan complex and with significant lowering of centralized nuclei and fibrosis in skeletal muscle. Motor ability and cardiac functions were completely rescued. However, BIO14.6 hamsters having less than 70% of fibers recovering sarcoglycan developed cardiomyopathy, even if the total rescued protein was normal. When we used serotype 2/8 in combination with serotype 2/1, lifespan was extended up to 22 months with sustained heart function improvement. Conclusions/Significance Our data support multiple systemic administrations of AAV as a general therapeutic strategy for clinical trials in cardiomyopathies and muscle disorders.

The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity.

We studied the expression of 38 human homeobox genes belonging to the four HOX complex loci in embryonal carcinoma (EC) cells induced to differentiate by culturing them in a medium containing retinoic acid (RA). Genes located at the 3′ end of each one of the four HOX loci are activated by RA in a sequential order colinear with their 3′ to 5′ arrangement in the cluster: 3′ HOX genes respond early to the drug while upstream genes respond progressively later. Among the genes located at the 5′ end of HOX loci RNase protection analysis reveals that one HOX3 gene and four HOX4 genes are weakly expressed in EC stem cells and downregulated upon treatment with 10−5 M RA. While activation of early responding genes does not require continuous protein synthesis, the observed timing and polarity of gene activation is disrupted in the absence of protein synthesis.

We studied the expression of 33 human homeobox genes belonging to four complex HOX loci in embryonal carcinoma NT2/D1 cells. These cells can be induced to differentiate by culturing them in media containing retinoic acid. Northern blot analysis reveals that no expression of these genes was detectable in NT2/D1 stem cells, whereas 22 HOX genes are well expressed in NT2/D1 cells treated with 10 microM retinoic acid for 14 days. The 11 HOX genes the expression of which remained undetectable in NT2/D1 cells after this treatment are located at the 5' end of their loci: four in HOX1, five in HOX3 and two in HOX4. The boundary between induced and silent genes roughly corresponds to the HOX genes constituting the homology group 5, related to the Abdominal-B homeotic gene of Drosophila. All nine identified HOX2 genes are well expressed in fully induced NT2/D1 cells and none of them maps 5' genes of this homology group. We conclude that HOX genes are differentially activated by retinoic acid in these cells according to their physical location within the four chromosomal loci.

It has been proposed that the estrogen receptor bears proteolytic activity responsible for its own transformation. This activity was inhibited by aprotinin. Incubation of transformed ER with aprotinin modified the proteolytic digestion of the hormone binding subunit by proteinase K. The smallest hormone-binding fragment of the ER, obtained by tryptic digestion, was still able to bind to aprotinin. These results suggest that aprotinin interacts with ER and the hormone-binding domain of ER is endowed with a specific aprotinin-binding site.

The existence of putative metal binding sites on the estradiol receptor (ER) molecule from calf uterus was evaluated by immobilizing various divalent metals to iminodiacetate-Sepharose. ER from both crude and highly purified preparations binds to metal-containing adsorbents complexed with Zn(II), Ni(II), Co(II), and Cu(II), but not to those complexed with Fe(II) and Cd(II). Elution of ER was obtained by chelating agents or by imidazole, thus indicating that histidine residues on the ER molecule are involved in the interaction with the metal. Analysis of affinity-labeled ER by [3H]tamoxifen aziridine after elution from a column of Zn(II)-charged iminodiacetate-Sepharose showed that ER fragments obtained by extensive trypsinization were also bound. Zn(II) and the same other metals able to bind ER, when immobilized on resins, inhibit the binding of estradiol to the receptor at micromolar concentrations. This inhibition is noncompetitive and can be reversed by EDTA. The inhibition of the hormone binding was still present after trypsin treatment of the cytosol, and it was abolished by preincubation with the hormone. Micromolar concentrations of these metals were able to block those chemical-physical changes occurring during the process of ER transformation in vitro. Furthermore, if added to pretransformed ER-hormone complex, they strongly inhibited the binding of the complex to isolated nuclei. The presence of metal binding sites that modulate the ER activity in the hormone binding domain of ER is therefore speculated. Since progesterone receptor showed the same pattern of binding and elution from metal-containing adsorbents, the presence of metal binding regulatory sites could be a property of all steroid receptors.

A new member of the dystrobrevin gene family was identified using a bioinformatics approach. Sequence analysis indicates that this gene, named DTN-B, is highly homologous to the rabbit A0, the previously described dystrobrevin (DTN), Torpedo 87 kDa and to the C-terminus of dystrophin. The coiled-coil domain, shown to be the site of interaction between dystrobrevins and dystrophin, is highly conserved. Immunostaining studies indicate that DTN-B and DTN expression is absent in affected muscle fibers from DMD patients and carriers.

BackgroundGenetic testing of patients with inherited kidney diseases has emerged as a tool of clinical utility by improving the patients’ diagnosis, prognosis, surveillance and therapy.MethodsThe present study applied a Next Generation Sequencing (NGS)-based panel, named NephroPlex, testing 115 genes causing renal diseases, to 119 individuals, including 107 probands and 12 relatives. Thirty-five (poly)cystic and 72 non (poly)cystic individuals were enrolled. The latter subgroup of patients included Bardet-Biedl syndrome (BBS) patients, as major components.ResultsDisease-causing mutations were identified in 51.5 and 40% of polycystic and non-polycystic individuals, respectively. Autosomal dominant polycystic kidney disease (ADPKD) patients with truncatingPKD1variants showed a trend towards a greater slope of the age-estimated glomerular filtration rate (eGFR) regression line than patients with (i) missense variants, (ii) anyPKD2mutations and (iii) no detected mutations, according to previous findings. The analysis of BBS individuals showed a similar frequency ofBBS4,9,10and12mutations. Of note, allBBS4-mutated patients harbored the novel c.332+1G>GTT variant, which was absent in public databases, however, in our internal database, an additional heterozygote carrier was found. AllBBS4-mutated individuals originated from the same geographical area encompassing the coastal provinces of Naples.DiscussionIn conclusion, these findings indicate the potential for a genetic panel to provide useful information at both clinical and epidemiological levels.Graphic abstract

Micromolar concentrations of the proteinase inhibitor, aprotinin, produced a dose-dependent inhibition in the binding capacity of the estrogen receptor from calf uterus. Aprotinin inhibition was greater at 28 degrees C than at 4 degrees C and only occurred when conditions allowed the receptor transformation. When aprotinin was tested in the presence of transformation inhibitors, its effect was no longer seen. The binding capacity of the highly purified estrogen-binding subunit was similarly inhibited.

Experimental conditions affecting the partitioning of the estrogen receptor were studied. Homogenization of rat uteri at 25 degrees C resulted in a particulate partitioning of the estrogen receptor. The use of frozen tissue (-70 degrees C) or pre-exposure of the tissue to 0 degrees C prior to 25 degrees C homogenization, homogenization at 0 degrees C and tissue dilution all induced soluble partitioning of the receptor. The estrogen receptor found in the particulate fraction was mostly associated with the nuclei, even in the absence of hormone. The interaction between estradiol and the estrogen receptor induced modification in the receptor's charge and size that promoted its cold-insensitive association with the nuclei of target cells. These modifications were studied in a cell-free in vitro system and were reversibly blocked by molybdate. Similar changes occurred in vivo when estradiol interacted with the receptor in the nuclei of target cells.