RESUMO
Congenital generalized hypertrichosis (CGH) is a rare, fully penetrant X-linked dominant trait previously described in a single, multigenerational Mexican family. CGH is a visually striking phenotype characterized by excessive facial and upper torso hair in males and by less severe asymmetric hairiness in females. We have found significant evidence for linkage with several markers from the long arm of the X chromosome. Recombinant chromosomes place the CGH gene within a 22 cM interval between DXS425 and DXS1227 in Xq24-Xq27.1. The localization of a gene for CGH represents the first step towards the isolation of genes involved in hair growth pattern, particularly those involved in restriction of areas in humans.
Assuntos
Hipertricose/genética , Cromossomo X , Criança , Mapeamento Cromossômico , Feminino , Genes Dominantes , Ligação Genética , Genótipo , Humanos , Masculino , Linhagem , Polimorfismo de Fragmento de RestriçãoRESUMO
Titanium dioxide, or titania, is perhaps the most well-known and widely studied photocatalytic material, with myriad applications, due to a high degree of tunability achievable through the incorporation of dopants and control of phase composition and particle size. Many of the applications of titanium dioxide require particular forms, such as gels, coatings, or thin films, making the development of hybrid solution processable nanoparticles increasingly attractive. Here we report a simple solvothermal route to highly dispersible anatase phase titanium dioxide hybrid nanoparticles from amorphous titania. Solvothermal treatment of the amorphous titania in trifluoroacetic acid leads to the formation of anatase phase nanoparticles with a high degree of size control and near complete surface functionalisation. This renders the particles highly dispersible in simple organic solvents such as acetone. Dopant ions may be readily incorporated into the amorphous precursor by co-precipitation, with no adverse effect on subsequent crystallisation and surface modification.
RESUMO
Synthetic oligonucleotides containing GC-rich triplet sequences were used in a scanning strategy to identify unstable genetic sequences at the myotonic dystrophy (DM) locus. A highly polymorphic GCT repeat was identified and found to be unstable, with an increased number of repeats occurring in DM patients. In the case of severe congenital DM, the paternal triplet allele was inherited unaltered while the maternal, DM-associated allele was unstable. These studies suggest that the mutational mechanism leading to DM is triplet amplification, similar to that occurring in the fragile X syndrome. The triplet repeat sequence is within a gene (to be referred to as myotonin-protein kinase), which has a sequence similar to protein kinases.
Assuntos
Distrofia Miotônica/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 19 , Clonagem Molecular , DNA/química , Humanos , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Polimorfismo GenéticoRESUMO
Here we report a new low temperature dry ice carbonation approach for the synthesis of carbonate-based nano- and micro-particulate materials, which enables the preparation of monodispersed calcium carbonate nanoparticles and microspheres with very high purity phases.
RESUMO
A new method for the large-scale hydrothermal production and activation of ZIF-8 is presented in this communication. Activated ZIF-8 has been produced, at lab-scale and pilot-scale, at a rate of 27 g h(-1) and 810 g h(-1) respectively with the activated material showing a surface area of 1800 m(2) g(-1).
RESUMO
Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Células COS , Ativação Enzimática , Humanos , Miotonina Proteína QuinaseRESUMO
The mutation responsible for myotonic dystrophy (DM) is an unstable expansion of the CTG repeat within the myotonin protein kinase gene. To examine whether the parental origin of the expanded repeat influences the repeat size in offspring, we studied 51 father-child and 59 mother-child pairs with DM. Small expansions in fathers resulted in larger size expansions in their offspring, while large paternal expansions resulted in less size change in their offspring. However, there was no correlation between maternal size expansion and size increase in offspring for either congenital or noncongenital DM. These data suggest that the sex of the affected parent influences the unstable expansion of the repeat in DM offspring. While some evidence suggests that DNA methylation status cannot explain this observation, the mechanism for differential maternal/paternal transmission expansion is currently unknown.
Assuntos
Distrofia Miotônica/congênito , Distrofia Miotônica/genética , Pais , Sequências Repetitivas de Ácido Nucleico , Caracteres Sexuais , Adulto , Sequência de Bases , Feminino , Humanos , Recém-Nascido , Masculino , Dados de Sequência MolecularRESUMO
We studied the expansion of the GCT repeats within the myotonic dystrophy protein kinase gene in nine myotonic dystrophy (DM) kindreds. Southern blot and polymerase chain reaction analyses of the repeat region demonstrated the expansion in all 62 patients with the diagnosis of DM. Among 43 DM parent-child pairs, age of onset in the child was earlier than in the parent in 36 pairs, in the same decade as the parent in five, and undetermined in two. The clinical anticipation observed in the 36 pairs accompanied an increase in the fragment size in 32, a decrease in two, and no apparent change in two pairs. In the remaining pairs without documented clinical anticipation, the fragment size increased in four, decreased in two, and was apparently unchanged in one. Overall, the size of expansion showed an inverse correlation with the age of onset (p < 0.001). In all seven pairs in which the fragment did not increase in size, the affected parent was male. Two congenital DM children born to affected mothers had expanded DNA greater than 4.5 kb. The differences between parent and child in age of onset significantly correlated with the differences in the expansion size among father-child pairs (p < 0.001) but not mother-child pairs (p > 0.5). Our data suggest that the expansion of the GCT repeats plays an important role in anticipation although other factors, including the sex of the affected parent, may have significant effects on molecular mechanisms of anticipation.
Assuntos
DNA , Distrofia Miotônica/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , Haplótipos , Humanos , Masculino , Distrofia Miotônica/diagnóstico , Distrofia Miotônica/fisiopatologia , LinhagemRESUMO
The molecular revolution that is transforming the entire biomedical field has had far-reaching impact in its application to inherited human muscle disease. The gene for Duchenne muscular dystrophy was one of the first cloned without knowledge of the defective protein product. This success was based upon the availability of key chromosomal aberrations that provided molecular landmarks for the disease locus. Subsequent discoveries regarding the mode of expression for this gene, the structure and localization of its protein product dystrophin, and molecular diagnosis of affected and carrier individuals constitute a paradigm for investigation of human genetics. Finding the gene for myotonic muscular dystrophy is requiring the brute force approach of cloning several million bases of DNA, identifying expressed sequences, and characterizing candidate genes. The gene that causes hypertrophic cardiomyopathy has been found serendipitously to be one of the genetic markers on chromosome 14, the beta myosin heavy chain.
Assuntos
Doenças Musculares/genética , Humanos , Doenças Musculares/terapia , Distrofias Musculares/genética , Distrofias Musculares/terapiaRESUMO
We have developed a procedure for determining the rates of mitotic recombination of an interrupted duplication created by integration of transforming plasmid sequences at the benA, beta-tubulin, locus of Aspergillus nidulans. Transformation of a strain carrying a benomyl-resistant benA allele with plasmid AIpGM4, which carries the wild-type benA allele and the pyr4 (orotidine-5'-phosphate decarboxylase) gene of Neurospora crassa, creates an interrupted duplication with plasmid sequences flanked by two benA alleles, one wild type and one benomyl resistant. Such transformants will not grow in the presence of high levels of benomyl. Mitotic recombination causes the loss of the wild-type benA allele or conversion of the wild-type to the mutant allele resulting in nuclei carrying only the benomyl-resistant allele. Conidia containing such nuclei can be selected on media with high benomyl allowing easy quantitation of mitotic recombination. We found that the rate of recombination giving rise to benomyl-resistant conidia was 4.6 x 10(-4). Reciprocal recombination leading to benomyl-resistant conidia lacking plasmid sequences occurred at a rate of 2.0 x 10(-4) and gene conversion leading to benomyl-resistant conidia occurred at a rate of 2.6 x 10(-4). We selected for reciprocal recombination leading to loss of pyr4 sequences on 5-fluoro-orotic acid and used this selection for two-step gene replacement of a mutant benA allele with the wild-type allele.
Assuntos
Aspergillus nidulans/genética , Genes Fúngicos , Alelos , Benomilo/farmacologia , Resistência Microbiana a Medicamentos/genética , Conversão Gênica , Mitose , Plasmídeos , Recombinação Genética , Tubulina (Proteína)/genéticaRESUMO
The predicted protein kinase activity of the cloned gene product of the human myotonic dystrophy locus has been experimentally verified. Affinity-purified recombinant DM protein kinase became phosphorylated itself and transphosphorylated histone H1. These activities were not present in the bacterial host cells and were exhibited by DMPK and DMPKH, recombinant proteins which contain the protein kinase domain but exhibit distinct sizes, 43 and 66 kDa, respectively. DMPKH was further purified by velocity sedimentation on sucrose gradients; both activities migrated with the recombinant protein at 41 S, consistent with discrete multimeric particles. Phosphoamino acid analysis showed that threonine (predominantly) and serine were phosphorylated in both DMPKH and histone H1. Although PKA and PKC are the known types of protein kinase with closest sequence homology to the DM protein kinase domain, purified DMPKH was inhibited by 4 mM but not 0.04-0.4 mM H7 and H8, which inhibit PKA and PKC with Ki's of 0.4-15 microM. Specific inhibitors of other classes of multifunctional serine/threonine protein kinases such as casein kinases I (CKI-7) and II (heparin) and calcium/calmodulin-dependent protein kinase II (KN-62) did not inhibit DMPKH. DMPKH did not phosphorylate membrane-associated phosphoproteins such as acetylcholine receptor or spectrin which are known to be substrates for PKA, PKC, and CKI and -II, respectively. These experimental results suggest that the active center of the recombinant human myotonic dystrophy protein kinase may have properties distinct from the well-studied classes of serine/threonine protein kinases, in contrast to predictions based upon primary structure alone.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Bases , Clonagem Molecular , Primers do DNA/química , Escherichia coli , Humanos , Dados de Sequência Molecular , Peso Molecular , Miotonina Proteína Quinase , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Myotonic dystrophy (DM) is an autosomal dominant disorder which affects skeletal muscle, heart, eye lens, brain, and endocrine functions. The disease-causing mutations are expansions of the triplet repeat CTG in the 3' untranslated region of a locus which encodes a serine/threonine protein kinase that represents a new family of protein kinases. A monoclonal antibody to a recombinant DM protein kinase (mAb DM-1) reacts specifically with the 64 kDa isoform of DM protein kinase in type I fibers in skeletal muscle, the fiber type which characteristically atrophies in the disease. Within type I fibers of normal muscle the isoform may be localized with mAb DM-1 to the triad region. In the DM disease state, the enzyme is redistributed to the pathologically characteristic peripheral sarcoplasmic masses. In markedly affected human distal myotonic muscle, the levels of the 64 kDa DM kinase isoform are elevated relative to slow skeletal myosin heavy chain. These results suggest that, consistent with the dominant clinical phenotype, the localization and accumulation of the 64 kDa isoform are altered in the heterozygous disease state.
Assuntos
Isoenzimas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Miotônica/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Resinas Acrílicas/metabolismo , Adulto , Animais , Sítios de Ligação , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/patologia , Distrofia Miotônica/patologia , Miotonina Proteína Quinase , Fosforilação , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/metabolismo , Sarcolema/metabolismoRESUMO
cDNA surveying is a straightforward approach for identifying sequences in genomic clones expressed in specific tissues. It has been applied to a subchromosomal region of human chromosome 19 (19q13.2-q13.4), a region that contains several known expressed sequences including the locus for myotonic dystrophy (DM). Genomic clones were selected from this region by probing a human placental cosmid library with a chromosome 19q-specific minisatellite sequence, or human genomic clones were isolated from a cosmid library constructed from a human chromosome 19q13.2-q13.3 hamster hybrid cell line using human repetitive DNA as probe. Pooled cDNAs synthesized from RNA of specific tissues characteristically affected in DM were depleted in repetitive sequences and used as hybridization probes against gridded cosmid arrays. DNA from the cDNA-positive cosmid clones was transferred to nylon filters and reprobed with cDNAs to identify restriction fragments that were expressed in these tissues. Hybridizing restriction fragments were subcloned, sequenced, and demonstrated to be nonrepetitive. Primer pairs complementary to subcloned sequences were constructed and used for PCR amplification of cDNA synthesized from RNA of tissues affected in myotonic dystrophy. PCR products were sequenced to verify the identity of expressed genomic DNA and its corresponding cDNA.
Assuntos
Cromossomos Humanos Par 19 , DNA/genética , Animais , Sequência de Bases , Southern Blotting , Cosmídeos , DNA/análise , DNA Satélite/genética , Expressão Gênica , Técnicas Genéticas , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Distrofia Miotônica/genética , Reação em Cadeia da PolimeraseRESUMO
Myotonic dystrophy (DM) is an autosomal dominant trait closely associated with CGT repeat expansions in the same locus on human chromosome 19q13.3. The expansions occur in the 3'untranslated region of a transcription unit encoding a serine-threonine kinase (DM kinase) of a new class based upon structure and function. Lens cataracts are a prominent finding in myotonic dystrophy. DM kinase was shown to be expressed in human and bovine lenses at the RNA level and in human lenses at the protein level. Sequencing of PCR products of RNA extracted from normal human lenses demonstrated an exact match to published genomic and cDNA 3' UTR sequences. Northern blots of bovine lens RNA showed that the transcript is similar in size to the transcript detected in other tissues that are affected in myotonic dystrophy. A polyclonal antibody (DM-2) was produced against recombinant DM protein kinase in rabbits. Development of Western blots with DM-2 showed a single reactive band of 67 kDa. Immunofluorescent studies of formalin-fixed human lens sections detected the DM kinase in the perinuclear cytoplasm of normal human lens epithelial cells and more diffusely in superficial subcapsular cortical fibers. In contrast, the same antibody labeled the nucleus most prominently in a single DM lens.
Assuntos
Cristalino/enzimologia , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Anticorpos , Sequência de Bases , Northern Blotting , Bovinos , Mapeamento Cromossômico , Cromossomos Humanos Par 19 , Primers do DNA , Epitélio/enzimologia , Imunofluorescência , Expressão Gênica , Humanos , Dados de Sequência Molecular , Distrofia Miotônica/enzimologia , Distrofia Miotônica/genética , Miotonina Proteína Quinase , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Coelhos , Proteínas Recombinantes/análise , Transcrição Gênica , Repetições de TrinucleotídeosRESUMO
The clinical manifestation of myotonic dystrophy (DM) is correlated to the extent of expansion of an unstable [CTG]n DNA motif. Recent studies have demonstrated that this trinucleotide motif forms part of the last, 3' untranslated exon of a gene which potentially encodes multiple protein isoforms of a serine/threonine protein kinase (myotonic dystrophy protein kinase, DM-PK). We report here on the development of antisera against synthetic DM-PK peptide antigens and their use in biochemical and histochemical studies. Immunoreactive DM-kinase protein of 53 kD is present at low levels in skeletal and cardiac muscle extracts of DM patients and normal controls. Immunohistochemical staining revealed that DM-PK is localised prominently at sites of neuromuscular and myotendinous junctions (NMJs and MTJs) of human and rodent skeletal muscles. Furthermore, very low levels of immunoreactive DM-PK protein are present in the sarcoplasm of predominantly type I fibres in various muscles. Strikingly, presence of the protein can also be demonstrated for NMJs of muscular tissues of adult and congenital cases of DM, with no gross changes in structural organisation. Our findings provide a basis for further characterisation of the role of the kinase in protein assembly processes or signal mediation at synaptic sites and ultimately for the understanding of the complex pathophysiology of DM.