RESUMEN
Genetic causes of skeletal disorders are manifold and affect, among others, enzymes of bone and connective tissue synthesis pathways. We present a twelve-year-old boy with a mild skeletal dysplasia, hypermobility of joints and axial malalignment of lower limbs and feet. Exome sequencing revealed a biallelic loss of function mutation in CSGALNACT1, which encodes chondroitin sulfate N-acetylgalactosaminyltransferase 1 and plays a major role in the chondroitin sulfate chain biosynthesis and therefore in the synthesis of glycosaminoglycans. Recently, the first case of a pediatric patient with a mild skeletal dysplasia due to a compound heterozygous large intragenic deletion and a damaging missense variant in CSGALNACT1 was reported. We here identify a second case and the first juvenile patient with a homozygous frameshift variant in CSGALNACT1 which corroborates its role in mild and non-progressive skeletal dysplasia with joint laxity.
Asunto(s)
Alelos , Mutación/genética , N-Acetilgalactosaminiltransferasas/genética , Osteocondrodisplasias/enzimología , Osteocondrodisplasias/genética , Estatura , Peso Corporal , Niño , Humanos , Masculino , Osteocondrodisplasias/diagnóstico por imagenRESUMEN
Most bacterial membranes contain one or two type I signal peptidases (SPases) for the removal of signal peptides from export proteins. For Streptomyces lividans, four different type I SPases (denoted SipW, SipX, SipY, and SipZ) were previously described. In this communication, we report the experimental determination of the membrane topology of these SPases. A protease protection assay of SPase tendamistat fusions confirmed the presence of the N- as well as the C-terminal transmembrane anchor for SipY. SipX and SipZ have a predicted topology similar to that of SipY. These three S. lividans SPases are currently the only known prokaryotic-type type I SPases of gram-positive bacteria with a C-terminal transmembrane anchor, thereby establishing a new subclass of type I SPases. In contrast, S. lividans SipW contains only the N-terminal transmembrane segment, similar to most type I SPases of gram-positive bacteria. Functional analysis showed that the C-terminal transmembrane anchor of SipY is important to enhance the processing activity, both in vitro as well as in vivo. Moreover, for the S. lividans SPases, a relation seems to exist between the presence or absence of the C-terminal anchor and the relative contributions to the total SPase processing activity in the cell. SipY and SipZ, two SPases with a C-terminal anchor, were shown to be of major importance to the cell. Accordingly, for SipW, missing the C-terminal anchor, a minor role in preprotein processing was found.
Asunto(s)
Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Streptomyces/enzimología , Secuencia de Bases , Membrana Celular/enzimología , Cartilla de ADN , Bacterias Grampositivas/enzimología , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Plásmidos , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/genética , Streptomyces/genéticaRESUMEN
Traditional as well as biotechnological processing of coal leads to complex mixtures of products. Besides chemical and physical characterization, which provides the information for product application, there is a need for bioassays to monitor properties that are probably toxic, mutagenic or cancerogenic. Investigations carried out focused on the selection, adaptation and validation of bioassays for the sensitive estimation of toxic effects. Organisms like bacteria, Daphnia magna and Scenedesmus subspicatus, representing different complexities in the biosphere, were selected as test systems for ecotoxicological and mutagenicity studies. The results obtained indicate that bioassays are, in principle, suitable tools for characterization and evaluation of coal-derived substances and bioconversion products. Using coal products, coal-relevant model compounds and bioconversion products, data for risk assessment are presented.
Asunto(s)
Bioensayo/métodos , Carbón Mineral , Sustancias Húmicas/toxicidad , Hidrocarburos Aromáticos/toxicidad , Animales , Biodegradación Ambiental , Chlorophyta/crecimiento & desarrollo , Daphnia/crecimiento & desarrollo , Ecosistema , Monitoreo del Ambiente , Pruebas de Mutagenicidad , Vibrio/crecimiento & desarrolloRESUMEN
We report here on the isolation and identification of a gene coding for a novel subtilisin inhibitor (VSI) isolated from Streptomyces venezuelae CBS762.70. The vsi gene was isolated on a 5-kb chromosomal PvuII fragment as identified by DNA sequencing and inhibitor activity testing of the gene product. Primer extension studies revealed that the mRNA transcriptional start point was situated at -37 and -36 relatively to the ATG start codon assuming the presence of solely one promoter. Vsi promoter strength was about double of those of ermE-P1a and aph-P1, as tested with the mRNA production of the aphII gene preceded by the respective promoters. Translation of the vsi coding sequence revealed a 28 amino acids long signal peptide. The mature VSI protein consists of 118 amino acids of which 87% was verified by N-terminal amino acid sequence analysis. Compared with the already known Streptomyces proteinase inhibitors, VSI shows a relatively high amino acid identity in the conserved domains. Nevertheless, only a maximum amino acid identity of 56.1% was noticed and some highly conserved residues were substituted in VSI. As a consequence, VSI could be classified within a separate group of Streptomyces subtilisin inhibitors.
Asunto(s)
Proteínas Bacterianas/genética , Streptomyces/genética , Subtilisinas/antagonistas & inhibidores , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cartilla de ADN , ADN Bacteriano/aislamiento & purificación , Genes Bacterianos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Streptomyces/metabolismo , Subtilisinas/metabolismoRESUMEN
With the aim of investigating determining factors for secretion of heterologous proteins by streptomycetes, we analysed the effect of charge variation in the Streptomyces venezuelae ATCC15068 alpha-amylase signal peptide on expression and secretion of mouse tumour necrosis factor alpha (mTNF) by Streptomyces lividans. To this end, the mTNF cDNA was fused to the wild-type alpha-amylase (aml) signal sequence and the fusion gene was expressed under the control of the S. venezuelae CBS762.70 subtilisin inhibitor gene (vsi) promoter, which has been shown to be very effective in initiating transcription. In addition, the number of positive charges in the N region of the alpha-amylase signal peptide was altered by in vitro mutagenesis. Secreted and intracellular mTNF levels were determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and biological activity measurements. This revealed moderate amounts of secreted mTNF compared to the levels obtained in previous experiments using the vsi promoter in combination with the Vsi signal peptide. Levels of secreted mTNF could be increased sevenfold by introducing one extra positive charge in the N region of the signal peptide.
Asunto(s)
Proteínas Recombinantes/metabolismo , Streptomyces/genética , Factor de Necrosis Tumoral alfa/metabolismo , alfa-Amilasas/química , Animales , Vectores Genéticos , Immunoblotting , Ratones , Mutagénesis Insercional/genética , Señales de Clasificación de Proteína/genética , Streptomyces/enzimología , Streptomyces/crecimiento & desarrollo , Transcripción Genética , alfa-Amilasas/genéticaRESUMEN
Type I signal peptidases (SPases) are a widespread family of enzymes which remove signal peptides from proteins translocated across cellular membranes. Here, we report the first isolation of a gene coding for type I signal peptidase of Streptomyces, denoted Sip(Sli). The sip(sli) gene specifies a protein of 291 amino acids. Thus Sip(Sli) is much larger (approximately 100 amino acids) than other known SPases of Gram-positive bacteria and resembles SPases of Gram-negative bacteria, showing the highest degree of similarity to an SPase of the cyanobacterium Phormidium laminosum. Sip(Sli) contains conserved serine and lysine residues, which are believed to be required for the catalytic activity. Similar to other known SPases from Gram-positive bacteria, Sip(Sli) seems to have only one N-terminal transmembrane anchor. In addition, Sip(Sli) seems to contain a second transmembrane anchor at the C-terminus, which is an unusual feature for type I signal peptidases.
Asunto(s)
Genes Bacterianos , Proteínas de la Membrana , Serina Endopeptidasas/genética , Streptomyces/genética , Secuencia de Aminoácidos , Cartilla de ADN , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Biblioteca de Genes , Datos de Secuencia Molecular , Plásmidos/genética , Reacción en Cadena de la Polimerasa/métodos , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Streptomyces/enzimologíaRESUMEN
In order to evaluate the expression and secretion signals of the highly secreted subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (VSI) for the production of heterologous proteins by Streptomyces lividans, mouse tumor necrosis factor alpha (mTNF) was chosen as a model protein. The mTNF cDNA was fused to the vsi signal sequence. The analysis of secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and biological activity measurements revealed an efficient translocation of mTNF. Up to 300 mg of secreted biologically active mTNF per liter could be obtained in shaken-flask cultures. By analyzing the effects of mutations in the N region of the VSI signal peptide on secretion, we found that decreasing the +3 charge of the wild-type protein to +2 resulted in a 3- to 10-fold increase in secretion.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Streptomyces/genética , Streptomyces/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Clonación Molecular , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Ratones , Señales de Clasificación de Proteína , ARN Mensajero/análisis , Recombinación Genética , Transducción de Señal , Translocación Genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Ordered mesostructured porous silicas that are also macroscopically structured were created by control of the interface on two different length scales simultaneously. Micellar arrays controlled the nanometer-scale assembly, and at the static boundary between an aqueous phase and an organic phase, control was achieved on the micrometer to centimeter scale. Acid-prepared mesostructures of silica were made with the p6, Pm3n, and the P63/mmc structures in the form of porous fibers 50 to 1000 micrometers in length, hollow spheres with diameters of 1 to 100 micrometers, and thin sheets up to 10 centimeters in diameter and about 10 to 500 micrometers in thickness. These results might have implications for technical applications, such as slow drug-release systems or membranes, and in biomineralization, where many processes are also interface-controlled.
RESUMEN
Bicyclams are a novel class of antiviral compounds which act as potent and selective inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) and HIV-2. They block an early step in the viral life cycle following adsorption to the CD4 receptor and preceding reverse transcription. To identify the molecular target of these compounds, we genetically analyzed variants of the HIV-1 molecular clone NL4-3, which developed resistance against two structurally related bicyclams, JM2763 and the more potent SID791. The resistant strains were obtained after long-term passaging in MT-4 cells in the presence of progressively increasing compound concentrations. Recombinants between selected genes of the resistant strains and the parental NL4-3 provirus were generated by adapting the marker rescue technique to MT-4 cells. The bicyclam-resistant phenotype was rescued by transferring the envelope gp120 gene of bicyclam-resistant virus into the NL4-3 parental genetic background. In the gp120 genes of the resistant strains, we identified several mutations leading to amino acid substitutions in the V3 loop. Furthermore, two substitutions of highly conserved amino acids in close proximity to the disulfide bridges of the V3 and V4 loops were found in both SID791- and JM2763-resistant strains. Additional mutations in regions encoding V3, C4, V5, and C5 were present in SID791-resistant viruses. Recombination experiments with overlapping parts of the envelope gene indicated that most, if not all, of the mutations were necessary to develop the fully SID791 resistant phenotype. The mutations in the C-terminal part of gp120 downstream of the V3 loop sequence conferred partial resistance to JM2763 but did not significantly decrease susceptibility to SID791. The genetic data and the biological properties of the resistant viruses point to inhibition of entry and fusion as the mode of action of the HIV-inhibitory bicyclams. A possible mechanism of binding of bicyclams to gp120 leading to inhibition of unfolding of gp120 and its shedding from the gp41 fusion domain is discussed.
Asunto(s)
Antivirales/farmacología , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Secuencia de Aminoácidos , Antivirales/química , Secuencia de Bases , Bencilaminas , Sitios de Unión , Línea Celular , Ciclamas , Farmacorresistencia Microbiana/genética , Genes env , Proteína gp120 de Envoltorio del VIH/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/aislamiento & purificación , Compuestos Heterocíclicos/química , Humanos , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/efectos de los fármacos , Fragmentos de Péptidos/genética , Relación Estructura-ActividadRESUMEN
2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The Km-value of 1 microM for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with 18O2 and H2(18)O indicated the incorporation of 18O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).
Asunto(s)
Burkholderia cepacia/metabolismo , Dioxigenasas , Oxigenasas/metabolismo , Éteres Fenílicos/metabolismo , Biodegradación Ambiental , Burkholderia cepacia/enzimología , Oxigenasas/química , Oxigenasas/aislamiento & purificación , Fenol , Fenoles/metabolismo , Pironas/metabolismoRESUMEN
The Premenstrual Assessment Form (PAF) is a new self report procedure designed to measure changes in mood, behavior, and physical condition during the premenstrual period. It reflects the great variability of premenstrual syndromes as opposed to the common practice of viewing these changes as a single entity. In comparison to commonly used procedures, the PAF 1) contains a broader variety and more specific descriptions of positive as well as negative changes; 2) provides Unipolar Summary Scales and Bipolar Continua which are sensitive measures for indexing levels of severity on various types of change; and 3) provides specific criteria for Typological Categories descriptive of different syndromes of change, especially those of mood and behavior. The paper describes the development of the PAF and the three scoring systems and illustrates the sensitivity of the individual items and scoring systems in reflecting the great diversity of change manifested during the premenstrual period.
Asunto(s)
Síndrome Premenstrual/diagnóstico , Adulto , Emociones/fisiología , Femenino , Humanos , Menstruación , Persona de Mediana Edad , Síndrome Premenstrual/fisiopatología , Síndrome Premenstrual/psicología , SíndromeRESUMEN
The differential relationship between specific subtypes of premenstrual changes and specific subtypes of mental disorder was studied. Premenstrual changes were evaluated with the Premenstrual Assessment Form, which provides specific criteria for the classification of the various subtypes of premenstrual change. The Research Diagnostic Criteria were used to make lifetime diagnoses of mental disorder. Differential relationships were found between subtypes of premenstrual change and subtypes of mental disorder. The results suggest that premenstrual changes characterized by a depressive syndrome may represent a mild or subclinical manifestation of affective disorder.