RESUMEN
PURPOSE: We determined whether genetic variation at 3 loci coding for putative crystallization inhibitors is linked to calcium urolithiasis. MATERIALS AND METHODS: We studied a cohort of 64 French-Canadian sibships including multiple recurrent calcium stone formers, comprising 154 independent pairs of siblings with at least 1 stone episode. Physical and meiotic mapping of the genes coding for osteopontin and uromodulin (Tamm-Horsfall protein) as well as the osteocalcin related gene (ORG or putative nephrocalcin) was performed and microsatellite markers were identified. We used nonparametric linkage analysis in the whole affected sib pair cohort as well as in affected pairs without hypercalciuria, that is concordant for 24-hour urine calcium excretion in the first quartile (mean plus or minus standard deviation 0.053 +/- 0.020 mmol./kg. or 3.4 +/- 1.3 mmol. daily), and in the first and second quartiles (mean 0.064 +/- 0.027 mmol./kg. or 4.9 +/- 2.1 mmol. daily, respectively). RESULTS: Lod scores were less than 0.3 for all 3 loci using these affection statuses. Further analysis enabled the exclusion of uromodulin at (relative risk or lambda = 3.9 and 2.5), osteopontin (lambda = 2.7 and 1.6) and ORG (lambda = 5.5 and 3.7) for affected sib pairs concordant for urine calcium excretion in the lowest and 2 lowest quartiles, respectively. CONCLUSIONS: Loci encoding 3 crystallization inhibitors are unlikely to be major genes involved in calcium stone formation in our population.
Asunto(s)
Oxalato de Calcio/metabolismo , Glicoproteínas/genética , Mucoproteínas/genética , Sialoglicoproteínas/genética , Cálculos Urinarios/genética , Cálculos Urinarios/metabolismo , Mapeo Cromosómico , Cristalización , Variación Genética , Humanos , Osteopontina , UromodulinaRESUMEN
BACKGROUND: A family history increases the risk of kidney stone passage independent of dietary risk factors. However, the metabolic basis for familial aggregation of urolithiasis is unknown. METHODS: We evaluated metabolic risk factors in families with at least two sibs with a history of calcium stones. Sibs underwent outpatient evaluations simultaneously, including 24-hour urine collection and oral calcium loading. Phenotypes were compared between affected and unaffected sibs from the same sibship. RESULTS: Eighty-three sibships comprising 388 sibs (212 affected sibs, 114 males and 98 females, and 176 unaffected sibs, 68 males and 108 females) from 71 families were analyzed. Daily urine calcium excretion was higher in affected compared with unaffected sibs (0.64 +/- 0.33 vs. 0.50 +/- 0.22 mmol Ca(2+)/mmol creatinine, respectively, P < 10(-5)). This corresponded to absolute values of 7.4 +/- 3.9 and 5.1 +/- 2.3 mmol Ca(2+)/day, respectively, for affected and unaffected males, and 5.4 +/- 2.6 and 4.2 +/- 1.9 mmol Ca(2+)/day, respectively, for affected and unaffected female sibs. When analyzed by tertile of onset age of stone passage, the differences in urine calcium were only significant in the first two tertiles (with onset age of stone passage <35 years). The fasting urine Ca(2+)/creatinine ratio was significantly higher in stone formers compared with control sibs (0.46 +/- 0.27 vs. 0.40 +/- 0.27, P = 0.04), as was the postcalcium load Ca(2+)/creatinine ratio (0.57 +/- 0.46 vs. 0.43 +/- 0.41, respectively, P = 0.02). Body mass index was marginally significantly higher in stone forming sibs (P = 0.04). Other urine phenotypes, including oxalate, phosphate, magnesium, citrate, urate, sodium, ammonium, and volume, were not associated with stone passage. CONCLUSION: Increased urine calcium excretion is the only phenotype associated with a kidney stone formation in these French-Canadian families.
Asunto(s)
Calcio/orina , Núcleo Familiar , Cálculos Urinarios/genética , Adolescente , Adulto , Anciano , Calcio/sangre , Calcio/metabolismo , Canadá , Familia , Francia/etnología , Humanos , Magnesio/sangre , Persona de Mediana Edad , Fenotipo , Fosfatos/sangre , Encuestas y Cuestionarios , Cálculos Urinarios/sangre , Cálculos Urinarios/orinaRESUMEN
Association between the ER and mitochondria has long been observed, and the formation of close contacts between ER and mitochondria is necessary for the ER-mediated sequestration of cytosolic calcium by mitochondria. Autocrine motility factor receptor (AMF-R) is a marker for a smooth subdomain of the ER, shown here by confocal microscopy to be distinct from, yet closely associated with the calnexin- or calreticulin-labeled ER. By EM, smooth ER AMF-R tubules exhibit direct interactions with mitochondria, identifying them as a mitochondria-associated smooth ER subdomain. In digitonin-permeabilized MDCK cells, the addition of rat liver cytosol stimulates the dissociation of smooth ER and mitochondria under conditions of low calcium. Using BAPTA chelators of various affinities and CaEGTA buffers of defined free Ca(2+) concentrations and quantitative confocal microscopy, we show that free calcium concentrations <100 nM favor dissociation, whereas those >1 microM favor close association between these two organelles. Therefore, we describe a cellular mechanism that facilitates the close association of this smooth ER subdomain and mitochondria when cytosolic free calcium rises above physiological levels.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico Liso/metabolismo , Mitocondrias/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Biomarcadores , Calcio/análisis , Línea Celular , Quelantes/farmacología , Citosol/metabolismo , Citosol/ultraestructura , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Retículo Endoplásmico Liso/química , Retículo Endoplásmico Liso/ultraestructura , Técnica del Anticuerpo Fluorescente , Riñón/citología , Hígado/metabolismo , Microscopía Electrónica , Mitocondrias/ultraestructura , Ratas , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/análisis , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: Calcium urolithiasis is in part genetically determined and associated with idiopathic hypercalciuria. METHODS: We have used a candidate gene approach to determine whether the calcium-sensing receptor (CaR) gene is linked to idiopathic hypercalciuria and calcium urolithiasis in a cohort of French Canadian sibships with multiple affected members (64 sibships from 55 pedigrees yielding 359 affected sibling pairs with > or =1 stone episode). RESULTS: Using nonparametric linkage analysis with various intragenic and flanking markers, we showed that the CaR gene could be excluded as a major gene for hypercalciuric stone formation. We excluded the CaR (lod score <-2) at lambdas values of 1.5, 1.68, and 2.6 for sib pairs concordant for at least one stone passage, at least two stone passages, and at least one stone passage and calciuria above the 75th percentile, respectively. Quantitative trait linkage analyses did not suggest that the CaR gene was linked to biochemical markers of idiopathic hypercalciuria. CONCLUSIONS: This study shows that genetic variants of the CaR gene are not associated with idiopathic hypercalciuria and calcium nephrolithiasis in this population of French Canadians.
Asunto(s)
Calcio/orina , Cromosomas Humanos Par 3 , Ligamiento Genético , Cálculos Renales/genética , Cálculos Renales/orina , Receptores de Superficie Celular/genética , Mapeo Cromosómico , Salud de la Familia , Humanos , Núcleo Familiar , Fenotipo , Carácter Cuantitativo Heredable , Receptores Sensibles al CalcioRESUMEN
Transfection of Mv1Lu mink lung type II alveolar cells with beta1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the beta1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella.
Asunto(s)
Autofagia/fisiología , Orgánulos/fisiología , Animales , Línea Celular , Lisosomas/ultraestructura , Visón , N-Acetilglucosaminiltransferasas/genética , Orgánulos/ultraestructura , Transfección , Vacuolas/ultraestructuraRESUMEN
Calcium is the principal crystalline constituent in up to 80% of kidney stones. Epidemiologic studies have suggested that genetic predisposition plays a major role in the etiology of this condition. This study evaluates by a candidate-gene approach whether the vitamin D receptor (VDR) locus on chromosome 12q12-14 is implicated in idiopathic hypercalciuria and calcium nephrolithiasis in a cohort of 47 French Canadian pedigrees. These comprised 54 sibships with a total of 303 pairs of siblings concordant for > or =1 stone episode. Evidence is provided for linkage to nephrolithiasis with microsatellite marker D12S339 (near the VDR locus, P = 0.01), as well as with flanking markers (D12S1663: P = 0.03 and D12S368: P = 0.01). Inclusion of unaffected sibs in the analyses also supported evidence for linkage. Quantitative trait linkage analysis of urinary calcium excretion yielded linkage to some, but not all, markers. This appears to be the first study to suggest linkage for idiopathic calcium stone formation.
Asunto(s)
Mapeo Cromosómico , Predisposición Genética a la Enfermedad/genética , Cálculos Renales/genética , Receptores de Calcitriol/genética , Adulto , Secuencia de Bases/genética , Exones/genética , Femenino , Ligamiento Genético/genética , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Polimorfismo Genético/genética , Polimorfismo Conformacional Retorcido-Simple , Carácter Cuantitativo HeredableRESUMEN
Calcium urolithiasis is often associated with increased intestinal absorption and urine excretion of calcium, and has been suggested to result from increased vitamin D production. The role of the enzyme 1 alpha-hydroxylase, the rate-limiting step in active vitamin D production, was evaluated in 36 families, including 28 sibships with at least a pair of affected sibs, using qualitative and quantitative trait linkage analyses. Sibs with a verified calcium urolithiasis passage (n = 117) had higher 24-h calciuria (P = 0.03), oxaluria (P = 0.02), fasting and postcalcium loading urine calcium/creatinine (Ca/cr) ratios (P = 0.008 and P = 0.002, respectively), and serum 1,25(OH)2 vitamin D levels (P = 0.02) compared with nonstone-forming sibs (n = 120). Markers from a 9-centiMorgan interval encompassing the VDD1 locus on chromosome 12q13-14 (putative 1 alpha-hydroxylase) were analyzed in 28 sibships (146 sib pairs) of single and recurrent stone formers and in 14 sibships (65 sib pairs) with recurrent-only (> or = 3 episodes) stone-forming sibs. Two-point and multipoint analyses did not reveal excess in alleles shared among affected sibs at the VDD1 locus. Linkage of stone formation to the VDD1 locus could be excluded, respectively, with a lambda d of 2.0 (single and recurrent stone formers) and 3.25 (recurrent stone formers). Quantitative trait analyses revealed no evidence for linkage to 24-h calciuria and oxaluria, serum 1,25(OH)2 vitamin D levels, and Ca/cr ratios. This study shows absence of linkage of the putative 1 alpha-hydroxylase locus to calcium stone formation or to quantitative traits associated with idiopathic hypercalciuria. In addition, there is coaggregation of calciuric and oxaluric phenotypes with stone formation.
Asunto(s)
25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , Calcio/orina , Cálculos Renales/enzimología , Cálculos Renales/genética , Población Blanca/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Adulto , Canadá , Salud de la Familia , Femenino , Francia/etnología , Ligamiento Genético , Marcadores Genéticos/genética , Humanos , Masculino , Persona de Mediana Edad , Núcleo Familiar , Linaje , Fenotipo , Vitamina D/sangreRESUMEN
A sulfathiazole-resistant dihydropteroate synthase (DHPS) present in two different laboratory strains of Escherichia coli repeatedly selected for sulfathiazole resistance was mapped to folP by P1 transduction. The folP mutation in each of the strains was shown to be identical by nucleotide sequence analysis. A single C-->T transition resulted in a Pro-->Ser substitution at amino acid position 64. Replacement of the mutant folP alleles with wild-type folP significantly reduced the level of resistance to sulfathiazole but did not abolish it, indicating the presence of an additional mutation(s) that contributes to sulfathiazole resistance in the two strains. Transfer of the mutant folP allele to a wild-type background resulted in a strain with only a low level of resistance to sulfathiazole, suggesting that the presence of the resistant DHPS was not in itself sufficient to account for the overall sulfathiazole resistance in these strains of E. coli. Additional characterization of an amplified secondary resistance determinant, sur, present in one of the strains, identified it as the previously identified bicyclomycin resistance determinant bcr, a member of a family of membrane-bound multidrug resistance antiporters. An additional mutation contributing to sulfathiazole resistance, sux, has also been identified and has been shown to affect the histidine response to adenine sensitivity displayed by these purU strains.
Asunto(s)
Antibacterianos/farmacología , Dihidropteroato Sintasa/genética , Escherichia coli/enzimología , Sulfatiazoles/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Farmacorresistencia Microbiana/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Mutación , Fenotipo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Autocrine motility factor receptor (AMF-R) is localized to an intracellular microtubule-associated membranous organelle, the AMF-R tubule. In well-spread untransformed MDCK epithelial cells, the microtubules originate from a broad perinuclear region and AMF-R tubules extend throughout the cytoplasm of the cells. In Moloney sarcoma virus (mos)-transformed MDCK (MSV-MDCK) cells, microtubules accumulate around the centrosome, forming a microtubule domain rich in stabilized detyrosinated microtubules. AMF-R tubules are quantitatively associated with this pericentriolar microtubule domain and the rough endoplasmic reticulum and lysosomes also co-distribute with the pericentriolar mass of microtubules. The Golgi apparatus is closely associated with the microtubule organizing center (MTOC) within the juxtanuclear mass of AMF-R tubules, and no co-localization of AMF-R tubules with the Golgi marker beta-COP could be detected by confocal microscopy. After nocodazole treatment and washout, microtubule nucleation occurs exclusively at the centrosome of MSV-MDCK cells, and only after microtubule extension to the cell periphery does the microtubule cytoskeleton reorganize to generate the pericentriolar microtubule domain after 30-60 min. AMF-R tubules dispersed by nocodazole treatment concentrate in the pericentriolar region in parallel with the reorganization of the microtubule cytoskeleton. MSV transformation of epithelial MDCK cells results in the stabilization of a pericentriolar microtubule domain responsible for the concentration and polarized distribution of AMF-R tubules.
Asunto(s)
Microtúbulos/metabolismo , Receptores de Citocinas/análisis , Tubulina (Proteína)/análisis , Animales , Antígenos/análisis , Antígenos CD/análisis , Proteínas de Unión al Calcio/análisis , Calnexina , Línea Celular Transformada/química , Núcleo Celular/ultraestructura , Centriolos/ultraestructura , Proteína Coatómero , Perros , Epitelio/química , Epitelio/ultraestructura , Técnica del Anticuerpo Fluorescente Indirecta , Aparato de Golgi/ultraestructura , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/análisis , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/análisis , Microtúbulos/efectos de los fármacos , Virus del Sarcoma Murino de Moloney , Nocodazol/farmacología , Receptores del Factor Autocrino de Motilidad , Ubiquitina-Proteína LigasasRESUMEN
This paper describes a morphologically unusual feature occurring in lymph nodes of some aged euthymic animals but mostly athymic animals. It initially consists of small alveole-like excrescences of the cortical wall of the subcapsular sinus. With dilatation, an excrescence becomes an ectasia which expands into the cortex. Observations suggest that ectasias enlarge under the influence of an increased pressure of the afferent lymph of a node. Such condition conceivably results from a greater lymph formation due to inflammation of the drained tissue site, combined with an impairment to the flow of lymph from the subcapsular sinus into medullary sinuses. A probable relation of ectasia formation to immunodeficiency is discussed. This formation results in the atrophy of the affected lymphoid cell populations of a node which likely contributes to aggravate the deficiency of the immune system.
Asunto(s)
Ganglios Linfáticos/patología , Timo/fisiología , Envejecimiento/inmunología , Animales , Animales Recién Nacidos , Atrofia/patología , Dilatación Patológica/inmunología , Inmunidad/fisiología , Ganglios Linfáticos/citología , Ratas , Ratas Desnudas , Ratas Sprague-DawleyRESUMEN
Genetic analysis of the tetA(C) gene of pBR322 indicates the importance of two-cytoplasmic loops in the TetA(C) protein (P. McNicholas, I. Chopra, and D. M. Rothstein, J. Bacteriol. 174:7926-7933, 1992). In this study, we characterized second-site suppressor mutations that suggest a functional interaction between these two cytoplasmic regions of the protein.
Asunto(s)
Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , Plásmidos/genética , Secuencia de Aminoácidos , Antiportadores/genética , Proteínas Bacterianas/genética , Codón/genética , Citoplasma , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Genes Bacterianos/genética , Datos de Secuencia Molecular , Mutación Puntual , Supresión Genética , Tetraciclina/farmacología , Resistencia a la Tetraciclina/genéticaRESUMEN
Escherichia coli JRG582 is an ampD ampE deletion derivative of strain HfrH and accordingly it is derepressed for expression of the cloned inducible beta-lactamase gene of Citrobacter freundii, carried on plasmid pNU305. Following chemical mutagenesis of JRG582(pNU305) a cefotaxime sensitive mutant was isolated, CS51(pNU305), which produced low levels of beta-lactamase due to a mutation in the host chromosome. Two recombinant plasmids containing genomic DNA from E. coli HfrH, namely pUB5608 and pUB5611, were isolated as a consequence of their ability to restore the beta-lactam resistant phenotype to CS51(pNU305). This ability was due to direct transcriptional activation of the beta-lactamase gene, ampC, rather than complementation of the CS51 mutation. Transposon mutagenesis and subcloning showed that restoration of ampicillin resistance to CS51(pNU305) was the function of a single gene, which maps at 60.3 min on the E. coli chromosome. The gene encodes a 33 kDa protein with significant homology to members of the LysR family of bacterial activator proteins, in particular the AmpR protein from C. freundii. Homology is especially strong over the N-terminal region which includes the helix-turn-helix DNA-binding motif. This gene was shown to complement the gcvA1 mutation at 60.3 min on the E. coli chromosome, and the DNA sequence agrees exactly with the published sequence of gcvA which encodes the transcriptional activator of the inducible glycine cleavage enzyme system. It is suggested that GcvA can activate transcription of ampC by binding to the AmpR binding region upstream of ampC so as to mimic the activated state of AmpR and hence provides an example of cross-talk between DNA-binding proteins of different inducible enzyme systems.
Asunto(s)
Proteínas Bacterianas/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Genes Reguladores/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cefotaxima/farmacología , Citrobacter freundii/enzimología , Citrobacter freundii/genética , Clonación Molecular , Farmacorresistencia Microbiana/genética , Escherichia coli/efectos de los fármacos , Prueba de Complementación Genética , Imitación Molecular , Datos de Secuencia Molecular , Mutagénesis Insercional , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The endothelium of the high endothelial venules (HEVs) of lymph nodes is normally considered to inhibit an association with neutrophils. The present paper shows that for a few weeks after birth, however, neutrophils are commonly associated with the walls of HEVs, the extent depending on the site of the lymph node. Overall, neutrophils increase in numbers in rat nodes from birth until about day 11, and vanish progressively thereafter. Moreover, neutrophils are more abundant in the nodes of standard neonates than in the nodes of pathogen-free neonates raised in an aseptic milieu. It is concluded that the postnatal recruitment of neutrophils by nodal HEVs relates to the then prevailing state of immaturity of the immune system. An explanation is proposed as to why neonatal HEVs of nodes recruit neutrophils and not only lymphocytes, as is the case later in ontogeny.
Asunto(s)
Endotelio Vascular/citología , Ganglios Linfáticos/citología , Neutrófilos/citología , Análisis de Varianza , Animales , Animales Recién Nacidos , Endotelio Vascular/inmunología , Femenino , Inmunocompetencia , Ganglios Linfáticos/inmunología , Masculino , Neutrófilos/inmunología , Embarazo , Ratas , Ratas Sprague-Dawley , Organismos Libres de Patógenos EspecíficosRESUMEN
Alaska Department of Environmental Conservation took indoor and ambient air samples during the winter of 1992-93 in Fairbanks. The samples showed a significant increase in the level of benzene in the air between December and February. The highest levels occurred in indoor air and exceeded workplace standards in garages. These findings were corroborated by NIOSH and OSHA personal monitoring studies done at the same time in Fairbanks and by blood samples taken by Centers for Disease Control and Prevention from workers exposed to gasoline in December and February. The blood samples showed a 300 percent increase in the amount of benzene in worker's blood after MTBE was taken out of gasoline. The most likely cause of this air pollution is gasoline evaporation and exhaust emissions. The benzene content of Alaska gasoline is 5 percent which is the highest in the nation. In view of these findings the Environmental Protection Agency is funding a new study of indoor air organic compounds including benzene in Anchorage, Alaska.
Asunto(s)
Contaminación del Aire/análisis , Benceno/análisis , Exposición a Riesgos Ambientales , Monitoreo del Ambiente , Estaciones del Año , Contaminación del Aire Interior/análisis , Alaska , HumanosRESUMEN
The tetA(B) gene from transposon Tn10 fails to mediate resistance to the novel tetracycline analog 9-(dimethylglycylamido)minocycline (DMG-Mino) (P. E. Sum, V. J. Lee, R. T. Testa, J. J. Hlavka, G. A. Ellestad, J. D. Bloom, Y. Gluzman, and F. P. Tally, J. Med. Chem. 37:184-188, 1994; R. T. Testa, P. Petersen, N. V. Jacobus, P. E. Sum, V. J. Lee, and F. P. Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). Mutations in either of two codons of tetA(B) that resulted in increased resistance to DMG-Mino also caused diminished resistance to tetracycline, identifying amino acid residues critical for the recognition of tetracycline.
Asunto(s)
Staphylococcus aureus/metabolismo , Resistencia a la Tetraciclina/genética , Tetraciclina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico Activo , Membranas/metabolismo , Minociclina/farmacología , Datos de Secuencia Molecular , Plásmidos , Estructura Secundaria de Proteína , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genéticaRESUMEN
The corrected DNA sequence of the tet(K) structural gene, encoding the tetracycline efflux protein from Staphylococcus aureus, is reported. The Tet(K) protein is homologous to related tetracycline efflux proteins throughout the protein, including at the C-terminal end. Hydropathy plotting now clearly indicates that the Tet(K) protein contains 14 transmembrane alpha-helices.
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Proteínas de la Membrana/genética , Estructura Secundaria de Proteína , Factores R , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Resistencia a la Tetraciclina/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
The tet(K) gene, encoding the tetracycline efflux protein from Staphylococcus aureus, mediates the transport of potassium in an Escherichia coli mutant defective in potassium uptake. Deletion mapping indicates that the first third of the tet(K) gene is sufficient to mediate potassium transport.
Asunto(s)
Proteínas Bacterianas/metabolismo , Genes Bacterianos/fisiología , Potasio/metabolismo , Staphylococcus aureus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Transporte Biológico/genética , Escherichia coli , Eliminación de Gen , Genes Bacterianos/genética , Datos de Secuencia Molecular , Staphylococcus aureus/genética , Resistencia a la Tetraciclina/genéticaRESUMEN
The tetK gene, which encodes a tetracycline efflux pump from Staphylococcus aureus, was expressed in Escherichia coli by using an inducible, low-level expression system. The tetK gene, as well as the tetA(B) gene from the transposon Tn10 and the tetA(C) gene from plasmid pBR322, was subjected to the regulatory control of the lac repressor, and resistance to tetracycline was measured as a function of the isopropyl-beta-D-thiogalactopyranoside concentration. The maximum resistance of the E. coli strain containing the tetK construct was comparable to the maximum resistance of the strain containing the tetA(C) construct but was less than the resistance of the strain containing the tetA(B) construct. Overexpression of the tetK, tetA(B), or tetA(C) genes was toxic. When expression was regulated so that resistance to tetracycline was comparable, then the TetA(B) and TetA(C) proteins conferred very similar levels of resistance to a variety of tetracycline derivatives. In contrast, the TetK protein was less capable of conferring resistance to the tetracycline derivatives minocycline, 6-deoxy-6-demethyltetracycline, and doxycycline. The implications for the recognition of various tetracycline substituents by the TetK protein are discussed.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Staphylococcus aureus/genética , Resistencia a la Tetraciclina/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Datos de Secuencia Molecular , Mutación , Plásmidos/genética , Proteínas Represoras , Especificidad por Sustrato , Tetraciclina/farmacologíaRESUMEN
The expression of gamma-aminobutyric acid was studied in sensory neurons and peripheral target tissues of the chick dorsal root ganglia by combining immunocytochemistry and electron microscopy. In the chick embryos, the first immunoreaction was observed at embryonic day 12 in 1.4% of ganglion cell bodies. The intensity of immunostaining gradually increased during development and the percentage of immunostained neurons reached an average of 7.3% after hatching. These immunostained cell bodies could be identified as sensory neurons belonging either to some large neurons of the A1 subclass or to a few small neurons of the B1 subclass. The other neuronal cell bodies, corresponding to the A2 and B2 subclasses, as well as the satellite and glial cells were apparently devoid of any gamma-aminobutyric acid immunostaining. Among the peripheral tissues innervated by the primary sensory neurons, the nerve endings of Achilles' tendon and the paravertebral autonomic ganglia appeared devoid of immunoreactivity. In contrast, immunoreactivity was found within nerve endings located in some neuromuscular spindles of the skeletal muscles and within some Herbst's corpuscles in the subcutaneous tissue of the skin. Thus, the present results provide evidence that gamma-aminobutyric acid may be expressed by neuronal cell bodies belonging to two subclasses of primary sensory neurons and could be a putative neurotransmitter involved in the peripheral sensory innervation of, at least in part, skin and skeletal muscles.
Asunto(s)
Ganglios Espinales/citología , Neuronas Aferentes/ultraestructura , Ácido gamma-Aminobutírico/análisis , Animales , Embrión de Pollo , Pollos , Ganglios Espinales/química , Ganglios Espinales/embriología , Ganglios Espinales/crecimiento & desarrollo , Microscopía Inmunoelectrónica , Músculos/inervación , Neuronas Aferentes/química , Piel/inervaciónRESUMEN
The differentiation of brown adipocyte precursor cells was studied in interscapular brown adipose tissue of adult mice by electron microscopy. Different stages of cell differentiation were characterized in situ. Previous autoradiographic studies suggested that interstitial cells represent the precursor cells of fully differentiated brown adipocytes. The present observations provide morphological evidence for a progressive differentiation of interstitial stem cells into mature brown adipocytes. Four typical stages of development were identified: (1) interstitial cells, (2) protoadipocytes, (3) preadipocytes, and (4) mature brown adipocytes. Interstitial stem cells were small spindle shaped cells, situated between brown adipocytes and characterized by a high nuclear-cytoplasmic ratio, the scarcity of organelles, and the absence of lipid inclusions. Protoadipocytes resembled interstitial cells except that they contained a few tiny lipid droplets in their cytoplasm. Preadipocytes had a larger cytoplasm enclosing many mitochondria and lipid droplets; the smooth endoplasmic reticulum was well developed surrounding the lipid droplets, and was closely associated with the mitochondria. Preadipocytes had the typical structure of growing cells, developing long cytoplasmic processes between and around blood capillaries. Mature brown adipocytes represented the final stage of differentiation. Almost all their cellular volume was occupied by lipid droplets and numerous mitochondria with very dense cristae. Brown adipocytes were also characterized by a tight association with blood capillaries, as expected from metabolically active cells requiring oxygen and substrates. These observations provide direct ultrastructural evidence for a progressive differentiation of interstitial cells into brown adipocytes with a continuum of intermediate cellular types.