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61.
Christophe Sola Anne Devallois Khye Seng Goh Eric Legrand Nalin Rastogi 《Current microbiology》1996,33(6):352-358
A genetic fingerprinting analysis of Caribbean isolates of M. avium complex (MAC) from AIDS patients by a Southern blotting technique after Pstl digestion with nonradioactive DNA probes coding for single-copy sequences DT1 and DT6 was performed. In parallel, a selective
amplification of a 187-bp fragment within the DT6 sequence with AV6/AV7 primers for Mycobacterium avium and of a 666-bp fragment within the DT1 sequence of M. intracellulare with the IN38/IN41 primers was also performed, and the molecular speciation with these two methods was compared with results
obtained with DNA probes of the Accuprobe system. 66 strains investigated comprised 31 international reference isolates of
MAC belonging to serovars 1–28 and 42–44, and 35 clinical isolates including 24 strains from Caribbean AIDS patients. 91.43%
of the clinical isolates tested gave concordant data with the DT1/DT6 Southern hybridization and PCR as compared with 74.28%
for PCR and Accuprobe, and 71.43% for Accuprobe and Southern hybridization. Our results corroborated previous findings showing
that the DT1 probe was specific for M. intracellulare, whereas the DT6 probe was specific for M. avium (reference serovars 2 and 3 probed positive both with DT1 and DT6 probes). Contrary to DT1 probe, which did not reveal sufficient
polymorphism to discriminate between MAC isolates, DT6 probe showed an interesting polymorphism giving four distinct clusters.
Three clusters corresponded to profiles previously reported for reference and/or clinical isolates; however, a fourth cluster
was discovered in five Caribbean isolates from four AIDS patients that did not correspond to previously published genetic
patterns. When probed with the insertion sequence IS1245, this cluster retained its homogeneity.
Received: 9 May 1996 / Accepted: 10 June 1996 相似文献
62.
Cell walls were prepared fromMycobacterium leprae (separated and purified from experimentally infected armadillo),M. tuberculosis, M. smegmatis, andMicrococcus lysodeikticus. The purity of the above wall preparations was confirmed after negative staining and shadow-casting and subsequent observation under the electron microscope. As judged from the electron microscopic observations, the bacteria were of different fragility in the following increasing order:M. tuberculosis, M. smegmatis, M. leprae, andMicro. lysodeikticus. The cell walls were hydrolyzed with 6N HCl, and the amino acids were identified by thin-layer chromatography compared with the authentic standards. With the same purification procedures, it was not possible to obtain satisfactorily pure peptidoglycan fromM. leprae. In leprosy bacilli,meso-DAP was found to be present, ín the walls; however, contamination by nonwall amino acids did not allow the confirmation of previous results, a finding that suggests that glycine completely replaced L-alanine inM. leprae cell walls. 相似文献
63.
D R Thakker W Levin H Yagi H J Yeh D E Ryan P E Thomas A H Conney D M Jerina 《The Journal of biological chemistry》1986,261(12):5404-5413
Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols. 相似文献
64.
S. A. Forbes A. A. J. Pannett J. H. D. Bassett B. Harding C. Wooding R. V. Thakker R. Butler D. Ogilvie R. Anand P. Gaudray G. Weber C. Larsson C. X. Zhang A. Calender J. W. M. Höppener C. J. M. Lips K. Kas 《Human genetics》1997,100(3-4):481-485
The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localised to 11q13 by combined tumour deletion mapping
and recombination studies, and a 0.5-Mb region, flanked by PYGM and D11S449, has been defined. In the course of constructing
a contig, we have identified the location of the gene encoding the B56β subunit of protein phosphatase 2A (PP2A), which is
involved in cell signal transduction pathways and thus represents a candidate gene for MEN1. We have searched for mutations
in the PP2A-B56β coding region, together with the 5′ and 3′ untranslated regions in six MEN1 patients. DNA sequence abnormalities
were not identified and thus the PP2A-B56β gene is excluded as the candidate gene for MEN1. However, our precise localisation
of PP2A-B56β to this region of 11q13 may help in elucidating the basis for other disease genes mapping to this gene-rich region.
Received: 17 April 1997 / Accepted: 22 April 1997 相似文献
65.
D R Thakker C Boehlert W Levin D E Ryan P E Thomas H Yagi D M Jerina 《Archives of biochemistry and biophysics》1991,288(1):54-63
Metabolism of 3H-labeled (+)-(S,S)- and (-)-(R,R)-1,2-dihydrodiols of triphenylene by rat liver microsomes and 11 purified isozymes of cytochrome P450 in a reconstituted monooxygenase system has been examined. Although both enantiomers were metabolized at comparable rates, the distribution of metabolites between phenolic dihydrodiols and bay-region, 1,2-diol 3,4-epoxide diastereomers varied substantially with the different systems. Treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) caused a slight reduction or less than a twofold increase, respectively, in the rate of total metabolism (per nanomole of cytochrome P450) of the enantiomeric dihydrodiols compared to microsomes from control rats. Among the 11 isozymes of cytochrome P450 tested, only cytochromes P450c (P450IA1) and P450d (P450IA2) had significant catalytic activity. With either enantiomer of triphenylene 1,2-dihydrodiol, both purified cytochrome P450c (P450IA1) and liver microsomes from MC-treated rats formed diol epoxides and phenolic dihydrodiols in approximately equal amounts. Purifed cytochrome P450d (P450IA2), however, formed bay-region diol epoxides and phenolic dihydrodiols in an 80:20 ratio. Interestingly, liver microsomes from control or PB-treated rats produced only diol epoxides and little or no phenolic dihydrodiols. The diol epoxide diastereomers differ in that the epoxide oxygen is either cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 1-hydroxyl group. With either purified cytochromes P450 (isozymes c or d) or liver microsomes from MC-treated rats, diol epoxide-2 is favored over diol epoxide-1 by at least 4:1 when the (-)-enantiomer is the substrate, while diol epoxide-1 is favored by at least 5:1 when the (+)- enantiomer is the substrate. In contrast, with liver microsomes from control or PB-treated rats, formation of diol epoxide-1 relative to diol epoxide-2 was favored by at least 2:1 regardless of the substrate enantiomer metabolized. This is the first instance where the ratio of diol epoxide-1/diol epoxide-2 metabolites is independent of the dihydrodiol enantiomer metabolized. Experiments with antibodies indicate that a large percentage of the metabolism by microsomes from control and PB-treated rats is catalyzed by cytochrome P450p (P450IIIA1), resulting in the altered stereoselectivity of these microsomes compared to that of the liver microsomes from MC-treated rats. 相似文献
66.
Earlier studies using affinity labeling reagents have suggested the existence of two nucleophilic groups at the active site of catechol-O-methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6). Both nucleophilic residues are critical for catalytic activity. In an effort to elucidate the nature of these residues and to further characterize the relationship between the chemical structure and the catalytic function of this enzyme, inactivation studies using N-ethylmaleimide were undertaken. Inactivation of the enzyme by N-ethylmaleimide under pseudo first-order conditions exhibited a non-linear relationship between the log of the fraction of enzyme activity remaining and preincubation time. Kinetic analysis of this inactivation process suggested the modification by N-ethylmaleimide of two residues at the active site of the enzyme, both crucial for catalytic activity. Detailed analysis of the inactivation process including substrate protection studies, pH profiles of inactivation, and incorporation studies using N-ethyl[2,3-14C2]maleimide provided additional evidence to support this conclusion. 相似文献
67.
Norma Candia Beatriz Lopez Thierry Zozio Marcela Carrivale Chyntia Diaz Graciela Russomando Nilda J de Romero Juan C Jara Lucia Barrera Nalin Rastogi Viviana Ritacco 《BMC microbiology》2007,7(1):1-10
Background
Francisella tularensis is a gram negative, facultative intracellular bacterium that is the etiological agent of tularemia. F. novicida is closely related to F. tularensis but has low virulence for humans while being highly virulent in mice. IglA is a 21 kDa protein encoded by a gene that is part of an iglABCD operon located on the Francisella pathogenicity island (FPI).Results
Bioinformatics analysis of the FPI suggests that IglA and IglB are components of a newly described type VI secretion system. In this study, we showed that IglA regulation is controlled by the global regulators MglA and MglB. During intracellular growth IglA production reaches a maximum at about 10 hours post infection. Biochemical fractionation showed that IglA is a soluble cytoplasmic protein and immunoprecipitation experiments demonstrate that it interacts with the downstream-encoded IglB. When the iglB gene was disrupted IglA could not be detected in cell extracts of F. novicida, although IglC could be detected. We further demonstrated that IglA is needed for intracellular growth of F. novicida. A non-polar iglA deletion mutant was defective for growth in mouse macrophage-like cells, and in cis complementation largely restored the wild type macrophage growth phenotype.Conclusion
The results of this study demonstrate that IglA and IglB are interacting cytoplasmic proteins that are required for intramacrophage growth. The significance of the interaction may be to secrete effector molecules that affect host cell processes. 相似文献68.
69.
NL Subasinghe E Khalil JM Travins F Ali SK Ballentine HR Hufnagel W Pan K Leonard RF Bone RM Soll CS Crysler N Ninan J Kirkpatrick MX Kolpak KA Diloreto SH Eisennagel ND Huebert CJ Molloy BE Tomczuk MD Gaul 《Bioorganic & medicinal chemistry letters》2012,22(16):5303-5307
Complement C1s protease inhibitors have potential utility in the treatment of diseases associated with activation of the classical complement pathway such as humorally mediated graft rejection, ischemia-reperfusion injury (IRI), vascular leak syndrome, and acute respiratory distress syndrome (ARDS). The utility of biphenylsulfonyl-thiophene-carboxamidine small-molecule C1s inhibitors are limited by their poor in vivo pharmacokinetic properties. Pegylation of a potent analog has provided compounds with good potency and good in vivo pharmacokinetic properties. 相似文献
70.
Alicia De Maria Yanrong Shi Nalin M. Kumar Steven Bassnett 《The Journal of biological chemistry》2009,284(20):13542-13550
In animal models, the dysregulated activity of calcium-activated proteases,
calpains, contributes directly to cataract formation. However, the
physiological role of calpains in the healthy lens is not well defined. In
this study, we examined the expression pattern of calpains in the mouse lens.
Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7
were expressed in lens fiber cells. Using controlled lysis, depth-dependent
expression profiles for each calpain were obtained. These indicated that,
unlike calpain 1, 2, and 7, which were most abundant in cells near the lens
surface, calpain 3 expression was strongest in the deep cortical region of the
lens. We detected calpain activities in vitro and showed that
calpains were active in vivo by microinjecting fluorogenic calpain
substrates into cortical fiber cells. To identify endogenous calpain
substrates, membrane/cytoskeleton preparations were treated with recombinant
calpain, and cleaved products were identified by two-dimensional difference
electrophoresis/mass spectrometry. Among the calpain substrates identified by
this approach was αII-spectrin. An antibody that specifically recognized
calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved
in vivo, late in fiber cell differentiation, at or about the time
that lens organelles are degraded. The generation of the calpain-specific
spectrin cleavage product was not observed in lens tissue from calpain 3-null
mice, indicating that calpain 3 is uniquely activated during lens fiber
differentiation. Our data suggest a role for calpains in the remodeling of the
membrane cytoskeleton that occurs with fiber cell maturation.Calpains comprise a family of cysteine proteases named for the calcium
dependence of the founder members of the family, the ubiquitously expressed
enzymes, calpain 1 (μ-calpain) and calpain 2 (m-calpain). The calpain
family includes more than a dozen members with sequence relatedness to the
catalytic subunits of calpain 1 and 2. Calpains have a modular domain
architecture. By convention, the family is subdivided into classical and
nonclassical calpains, according to the presence or absence, respectively, of
a calcium-binding penta-EF-hand module in domain IV of the protein
(1). Classical calpains include
calpain 1, 2, 3, 8, 9, and 11. Nonclassical calpains include calpain 5, 6, 7,
10, 12, 13, and 14.Transgenic and gene knock-out approaches in mice have demonstrated an
essential role for calpains during embryonic development. Knock-out of the
small regulatory subunit (Capn4) results in embryonic lethality
(2,
3). Similarly, inactivation of
the Capn2 gene blocks development between the morula and blastocyst
stage (4). In humans, mutations
in CAPN3 underlie limb-girdle muscular dystrophy-2A, and
polymorphisms in CAPN10 may predispose to type 2 diabetes mellitus
(5,
6).Even under conditions of calcium overload, where calpains are presumably
activated maximally, only a subset (<5%) of cellular proteins are
hydrolyzed (7). Calpains
typically cleave their substrates at a limited number of sites to generate
large polypeptide fragments that, in many cases, retain bioactivity. Thus,
under physiological conditions, calpains probably participate in the
regulation of protein function rather than in non-specific protein
degradation.More than 100 proteins have been shown to serve as calpain substrates
in vitro, including cytoskeletal proteins
(8), signal transduction
molecules (9), ion channels
(10), and receptors
(11). In vivo,
calpains are believed to function in myoblast fusion
(12), long term potentiation
(13), and cellular mobility
(14). Unregulated calpain
activity, secondary to intracellular calcium overload, is associated with
several pathological conditions, including Alzheimer disease
(15), animal models of
cataract (16), myocardial
(17), and cerebral ischemia
(18).In addition to their domain structure, calpains are often classified
according to their tissue expression patterns. Calpain 1, 2, and 10 are widely
expressed in mammalian tissues, but other members of the calpain family show
tissue-specific expression patterns. Calpain 8, for example, is a
stomach-specific calpain (19),
whereas expression of calpain 9 is restricted to tissues of the digestive
tract (20). The expression of
calpain 3 was originally thought to be limited to skeletal muscle
(21), but splice variants of
calpain 3 have since been detected in a range of tissues. At least 12 isoforms
of calpain 3 have been described in rodents
(22), of which several are
expressed in the mammalian eye, including Lp82 (lens), Cn94 (cornea), and Rt88
(retina) (23).Calpains have been studied intensively in the ocular lens because of their
suspected involvement in lens opacification (cataract). Calpain-mediated
proteolysis of lens crystallin proteins causes increased light scatter
(24). Unregulated activation
of calpains is observed in rodent cataract models
(25), where calpain-mediated
degradation of crystallin proteins
(26) and cytoskeletal elements
(27) is commonly observed.
Calpain inhibitors are effective in delaying or preventing cataract in
vitro (28,
29) and in vivo
(30,
31).It is likely, however, that calpains have important physiological roles in
the lens beyond their involvement in tissue pathology. Terminal
differentiation of lens fiber cells involves a series of profound
morphological and biochemical transformations. For example, differentiating
lens fiber cells undergo an enormous (>100-fold) increase in cell length,
accompanied by extensive remodeling of the plasma membrane system
(32). Early in the
differentiation process, fusion pores are established between cells, as
neighboring fibers are incorporated into the lens syncytium
(33). A later stage of fiber
cell differentiation involves the dissolution of all intracellular organelles,
a process that is thought to eliminate light-scattering particles from the
light path and contribute to the transparency of the tissue
(34). Any or all of these
phenomena might require the developmentally regulated activation of calpains.
This is consistent with our previous observation that in calpain 3 knock-out
mice the transition zone is altered, suggesting a change in the
differentiation program
(35).In the current study, therefore, we examined the depth-dependent expression
pattern and activity of calpains in the mouse lens. Fluorogenic substrates
were microinjected into the intact lens to visualize calpain activity
directly, and proteomic approaches were used to identify endogenous calpain
substrates. The cleavage pattern of one of these, αII-spectrin, was
examined in detail. Immunocytochemical and immunoblot analysis with wild type
and calpain 3-null lenses indicated that αII-spectrin is a specific
calpain 3 substrate in maturing lens fiber cells. Together, the data suggest
that calpains are activated relatively late in fiber cell differentiation and
may contribute to the remodeling of the membrane cytoskeleton that accompanies
fiber cell maturation. 相似文献