RESUMO
Neurodevelopmental disorder with microcephaly, hypotonia, and variable brain anomalies (NMIHBA) (OMIM #617481) is an autosomal recessive disease characterized by progressive microcephaly, plagiocephaly, hypotonia, spastic quadriparesis, global developmental delay, intellectual disability, optic features and abnormal brain magnetic resonance imaging (MRI). NMIHBA was recently reported to be caused by PRUNE1 mutations. Eight mutations have been reported in 13 unrelated families. Here, we report 3 PRUNE1 mutations in 1 Caucasian and 3 Japanese families. One recurrent missense mutation (p.Asp106Asn) was previously reported in Turkish and Italian families, while the other 2 mutations (p.Leu18Serfs*8 and p.Cys180*) are novel. We also show that mutant PRUNE1 mRNA can be subject to nonsense-mediated mRNA decay. The patients presented in this study showed atypical NMIHBA phenotypes with no progressive microcephaly. Furthermore, one Caucasian case had significant macrocephaly; therefore, patients with PRUNE1 mutations can exhibit a broad and heterogeneous spectrum of phenotypes.
Assuntos
Encéfalo/anormalidades , Microcefalia/genética , Hipotonia Muscular/genética , Monoéster Fosfórico Hidrolases/genética , Encéfalo/diagnóstico por imagem , Criança , Feminino , Humanos , Itália , Imageamento por Ressonância Magnética , Masculino , Mutação de Sentido Incorreto , Linhagem , RNA Mensageiro/genética , TurquiaAssuntos
Neuropatia Hereditária Motora e Sensorial/genética , Atrofia Muscular Espinal/genética , Simportadores/genética , Feminino , Neuropatia Hereditária Motora e Sensorial/fisiopatologia , Humanos , Japão , Mutação com Perda de Função/genética , Masculino , Pessoa de Meia-Idade , Atrofia Muscular Espinal/fisiopatologia , Linhagem , País de GalesAssuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Quinazolinas/administração & dosagem , Quinazolinas/sangue , Administração Metronômica , Adulto , Idoso , Esquema de Medicação , Feminino , Humanos , Lapatinib , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/sangueRESUMO
BACKGROUND AND PURPOSE: Mutations in the valosin-containing protein (VCP) gene are known to cause inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and familial amyotrophic lateral sclerosis (ALS). Despite an increasing number of clinical reports, only one Asian family with IBMPFD has been described. METHODS: To characterize patients with VCP mutations, we screened a total of 152 unrelated Asian families who were suspected to have rimmed vacuolar myopathy. RESULTS: We identified VCP mutations in seven patients from six unrelated Asian families. Five different missense mutations were found, including a novel p.Ala439Pro substitution. All patients had adult-onset progressive muscle wasting with variable involvement of axial, proximal, and distal muscles. Two of seven patients were suggested to have mild brain involvement including cerebellar ataxia, and only one showed radiological findings indicating a change in bone. Findings from skeletal muscle indicated mixed neurogenic and myogenic changes, fibers with rimmed vacuoles, and the presence of cytoplasmic and nuclear inclusions. These inclusions were immunopositive for VCP, ubiquitin, transactivation response DNA-binding protein 43, and also histone deacetylase 6 (HDAC6), of which function is regulated by VCP. Evidence of early nuclear and mitochondrial damage was also characteristic. CONCLUSIONS: Valosin-containing protein mutations are not rare in Asian patients, and gene analysis should be considered for patients with adult-onset rimmed vacuolar myopathy with neurogenic changes. A wide variety of central and peripheral nervous system symptoms coupled with rare bone abnormalities may complicate diagnosis.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Miopatias Distais/genética , Miopatias Distais/patologia , Músculo Esquelético/patologia , Mutação , Miosite de Corpos de Inclusão/genética , Miosite de Corpos de Inclusão/patologia , Adulto , Sequência de Aminoácidos , Povo Asiático , Sequência de Bases , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Dados de Sequência Molecular , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Linhagem , Proteína com ValosinaRESUMO
RNA fractions extracted from the spleens of immunized animals prepared against L1210 leukemia cells can transfer allospecific cell-mediated immunity both in vitro and in vivo. Both preculture of nonsensitized lymphocytes prior to the treatment with immune RNA and additional culture of lymphocytes after 1-hr treatment with immune RNA enhance the growth-inhibitory activity of immune RNA-treated lymphocytes. Preculture for 5 hr and additional culture for 24 hr are sufficient for maximal enhancement of the growth-inhibitory activity. There is no significant difference in inhibition between lymphocytes treated with immune RNA for 1 hr and 24 hr. The growth inhibition by lymphocytes is augmented proportionally to the increase in the dose of immune RNA in vitro but not in vivo. In mice given i.p. injections of immune RNA-treated syngeneic spleen cells (before or after inoculation with L1210 cells), a significant prolongation of the mean survival time [27.0 +/- 5.4 (S.D.) days, 0.05 < p < 0.1] was not achieved when compared with control mice (21.4 +/- 1.6 days). Evidence is presented that suppressor cells in the spleens of L1210-bearing mice are involved in preventing effector cell function in vivo. The present study revealed that 8 X 10(3) and 5 X 10(5) suppressor cells were sufficient to inhibit completely the activity of immune RNA-treated effector cells in vitro and in vivo, respectively. Suppressive activity was abolished by treatment with anti-Thy 1.2 serum and complement and partially abolished with irradiation (1500 R). The suppressor cells belonged to a radiation-sensitive T-cell population. The limited activity of immune RNA in vivo is probably due to the destruction of immune RNA by RNase present in the host plasma and tissues and the acquisition of suppressor cells which inhibits nonadherent peritoneal exudate cell activity in the animal.
Assuntos
Imunidade Celular , Leucemia L1210/imunologia , RNA/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Imunização Passiva , Imunoterapia , Leucemia L1210/terapia , Camundongos , Linfócitos T Reguladores/imunologiaRESUMO
The role of inhibin in gonadal function and reproduction has been confirmed by the measurement of plasma inhibin levels, but there has been no clinical data available on activin because of the lack of a good assay method. We measured plasma free activin levels during the normal menstrual cycle using a newly developed competitive protein binding assay with follistatin as the binding protein. Plasma inhibin levels were measured simultaneously using an alpha-subunit N-terminal fragment RIA with recombinant inhibin as the reference standard. Four normal women, aged 23-29 years, were investigated by obtaining plasma at 3-day intervals. Plasma inhibin levels showed some variation during the follicular phase, but a parallel rise in inhibin and progesterone was observed during the luteal phase. These findings confirmed those of previous studies. In contrast, plasma free activin levels did not show any substantial changes during the menstrual cycle. This study suggests that activin has no endocrine role in modulating the pituitary-gonadal axis during the normal menstrual cycle, while changes of inhibin reflect cyclic gonadal function and indicate an endocrine role for this protein in modulating gonadal activity.
Assuntos
Inibinas/sangue , Ciclo Menstrual/sangue , Ativinas , Adulto , Feminino , Hormônios Esteroides Gonadais/sangue , Humanos , Concentração Osmolar , Radioimunoensaio , Valores de ReferênciaRESUMO
The erythrocytes of various vertebrates, such as mice, rabbits, sheep, chickens, bullfrogs, and toads are lysed by normal snake sera. However, snake erythrocytes were not lysed by serum from different snake species. Putative natural antibody seems with different specificities to comprise heterogeneous antibodies. Thus, absorption of snake serum with mouse erythrocytes, for example, abrogated hemolytic activity for mice but not for rabbit or sheep erythrocytes. We observed no significant intraspecies individual differences in serum hemolytic titer, but interspecies differences were obvious. Immunization of snakes with sheep erythrocytes caused no further elevation of hemolytic activity, though high titer antibody was produced in response to certain bacterial antigens. Even the sera of newly-hatched snakes showed hemolytic activity at modestly high levels. No seasonal change in hemolytic activity was observed.
Assuntos
Anticorpos , Hemólise , Absorção , Aglutininas , Envelhecimento , Animais , Antígenos de Bactérias , Anuros , Galinhas , Imunização , Camundongos , Coelhos , Rana catesbeiana , Estações do Ano , Ovinos , SerpentesRESUMO
Vibrio anguillarum strains were isolated from chloramphenicol-resistant bacteria in diseased fish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418-encoded chloramphenicol acetyltransferase (CAT) [EC 2.3.1.99] was isolated and purified to homogeneity using affinity chromatography on immobilized p-amino-chloramphenicol or ATP. The general CAT could be adsorbed by a matrix with a chloramphenicol base ligand (Zaidenzaig, Y. & Shaw, W.V. (1976) FEBS Lett. 62,266-271), but the Rms418-encoded CAT was not bound under these conditions. The specific activity of the enzyme, when measured by the spectrophotometric assay, was 71.4 units/mg protein at 37 degrees C. The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol was shown to be approximately 22,000. The molecular weight of the native enzyme, as determined by gel filtration, was approximately 69,000, and the optimal pH was 7.8. The Km values for chloramphenicol and CoASAc were 34.5 and 150 microM, respectively. Enzyme activity was inhibited by HgCl2, p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and ethylendiaminotetraacetic acid (EDTA). The half life at 53 degrees C was approximately 100 min.
Assuntos
Cloranfenicol O-Acetiltransferase/genética , Escherichia coli/genética , Fatores R , Vibrio/genética , Cátions Bivalentes , Cloranfenicol O-Acetiltransferase/isolamento & purificação , Cloranfenicol O-Acetiltransferase/metabolismo , Cloromercurobenzoatos/farmacologia , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Cromatografia em Gel , Ácido Ditionitrobenzoico/farmacologia , Resistência Microbiana a Medicamentos/genética , Ácido Edético/farmacologia , Estabilidade Enzimática , Escherichia coli/enzimologia , Peso Molecular , Vibrio/enzimologia , Ácido p-CloromercurobenzoicoRESUMO
The vanadate (Vi)-mediated photocleavage reaction was used to study the interaction between the regulatory segment and the catalytic site of smooth muscle myosin light chain kinase (MLCK). When MLCK was irradiated with long-wave UV (366 nm) in the presence of ADP and Vi, kinase activity was substantially decreased, and the MLCK polypeptide of 130 kDa was cleaved into several smaller fragments with apparent molecular masses of 100, 70, 60, 32, and 28 kDa. Inhibition of kinase activity and photocleavage were both competitively antagonized by the addition of ATP. Inconsistency between the observed maximum levels of UV-induced inhibition of MLCK-mediated phosphorylation (80%) and photocleavage (15-20%) suggested that the photocleavage reaction proceeds as a two-step process. Monoclonal antibodies recognizing the C-terminus of MLCK labeled the 60- and 28-kDa fragments, indicating that MLCK was cleaved at two sites, at 28 and 60 kDa from the C-terminus, within what are believed to be the autoinhibitory region and the catalytic site, respectively. Moreover, Ca2+-calmodulin (Ca2+-CaM) protected against cleavage at the site at 28 kDa from the C-terminus. Analysis of the amino acid composition of the fragment revealed that the cleavage site at 28 kDa from C-terminus occurred at Lys 799 +/- 3 amino acid residues, which is in a region where the CaM-binding and pseudosubstrate regions overlap. These results suggest that the three-dimensional structure of MLCK brings the regulatory segment into direct contact with the ATP-binding site. Moreover, the binding of Ca2+-CaM displaces the regulatory segment away from the catalytic site.
Assuntos
Difosfato de Adenosina/farmacologia , Quinase de Cadeia Leve de Miosina/metabolismo , Vanadatos/farmacologia , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Calmodulina/metabolismo , Bovinos , Galinhas , Hidrólise , Cinética , Dados de Sequência Molecular , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/química , Fosforilação , Fotoquímica , Turquia , Raios UltravioletaRESUMO
An extended-spectrum beta-lactamase, the gene for which is located on plasmid pMS350 in Pseudomonas aeruginosa strains, hydrolyzes carbapenems and other extended-spectrum beta-lactam antibiotics. We cloned the pMS350 beta-lactamase gene in an Escherichia coli K-12 strain using the vector plasmid pHSG398, and subcloned it into pMS360, a plasmid with a wide host-range. This resulted in the formation of the recombinant plasmid, pMS363, containing a 4.1-kb DNA insert that includes the extended-spectrum beta-lactamase gene. Plasmid pMS363 was introduced into the P. aeruginosa PAO strain or into six species of Enterobacteriaceae, and the specific activities of the beta-lactamase and MICs of various beta-lactam antibiotics were estimated. The cloned gene was capable of expression in these strains and caused resistance to carbapenem, penem and other beta-lactam antibiotics, with the exception of aztreonam.
Assuntos
Enterobacteriaceae/genética , Pseudomonas aeruginosa/genética , beta-Lactamases/genética , Clonagem Molecular , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , PlasmídeosAssuntos
Antibacterianos/metabolismo , Resistência Microbiana a Medicamentos , Escherichia coli/enzimologia , Glicosídeos/metabolismo , Canamicina/metabolismo , Fosfatos/metabolismo , Trifosfato de Adenosina/metabolismo , Fosfatase Alcalina , Cromatografia por Troca Iônica , Sulfato de Di-Hidroestreptomicina/metabolismo , Escherichia coli/efeitos dos fármacos , Glucosamina/metabolismo , Hexoses/metabolismo , Concentração de Íons de Hidrogênio , Canamicina/farmacologia , Neomicina/farmacologia , Paromomicina/farmacologiaRESUMO
The clinical findings and prognosis in 15 patients with primary Ki-1 anaplastic large cell lymphoma (ALCL) were analyzed and compared with those of patients with T cell and B cell lymphoma and Hodgkin's disease. Clinical data revealed lymphadenopathy in 13 patients (87%) and skin lesions in eight (53%). Other organic involvements were hepatomegaly in two patients (13%), splenomegaly in five (33%), and bone marrow involvement in three (20%). The rate of skin involvement was significantly higher than that in B cell lymphoma and Hodgkin's disease. In laboratory findings the gamma-globulin concentration was significantly higher than that in T cell lymphoma, and the erythrocyte sedimentation rate (ESR) was significantly higher than that in B cell lymphoma. Complete remission was achieved in 11 patients (73%) and the five-year relapse-free survival was 27%. The overall survival was 4.0-69.8 months (mean 30.6 months). The mean survival was compatible with that of T cell lymphoma and was significantly shorter than that in Hodgkin's disease. Ki-1 ALCL can be distinguished from other lymphomas clinically as well as pathologically. Because Ki-1 ALCL is chemosensitive and the prognosis is as poor as that of T cell lymphoma, aggressive chemotherapy should be employed for the treatment of this disease.
Assuntos
Doença de Hodgkin/patologia , Linfoma de Células B/patologia , Linfoma Anaplásico de Células Grandes/patologia , Linfoma de Células T/patologia , HumanosRESUMO
There have been several reports on the use of extracorporeal shock waves in the treatment of pseudarthrosis, calcifying tendinitis, and tendinopathies of the elbow. However, the pathomechanism of pain relief has not been clarified. To investigate the analgesic properties of shock wave application, we analyzed whether it produces morphologic changes in cutaneous nerve fibres. In normal rat skin, the epidermis is heavily innervated by nerve fibres immunoreactive for protein gene product (PGP) 9.5 and by some fibres immunoreactive for calcitonin gene-related peptide (CGRP). There was nearly complete degeneration of epidermal nerve fibres in the shock wave-treated skin, as indicated by the loss of immunoreactivity for PGP 9.5 or CGRP. Reinnervation of the epidermis occurred 2 weeks after treatment. These data show that relief of pain after shock wave application to the skin results from rapid degeneration of the intracutaneous nerve fibres.
Assuntos
Ondas de Choque de Alta Energia , Fibras Nervosas/efeitos da radiação , Pele/efeitos da radiação , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Epiderme/inervação , Epiderme/efeitos da radiação , Membro Posterior , Imuno-Histoquímica , Masculino , Degeneração Neural , Fibras Nervosas/metabolismo , Medição da Dor , Ratos , Ratos Sprague-Dawley , Pele/inervação , Tioléster Hidrolases/metabolismo , Ubiquitina TiolesteraseRESUMO
A radioimmunoassay (RIA) for growth hormone-releasing hormone (GHRH) using a polyclonal antibody against synthetic GHRH(1-29)-Gly4-Cys-NH2 has been developed. The antiserum (RBM105) showed full cross-reactivity with GHRH-(1-44)NH2, GHRH-(1-40)OH, GHRH-(1-37)OH and GHRH-(3-44)NH2, and probably recognized the region of Ala4 to Lys12 of GHRH. Since the sensitivity of the GHRH RIA was 1.5 pg/tube, the lowest detectable plasma level was 5 ng/l when an extract of 0.3 ml of plasma per tube was used. On gelfiltration chromatography, the GHRH immunoreactivity of normal plasma was eluted in the same position as synthetic GHRH. The plasma GHRH concentration in healthy subjects was 20.5 +/- 6.5 ng/l (mean +/- SD), and in patients with hypothalamic disorders was 17.4 +/- 2.0 ng/l. In contrast, the plasma GHRH level in hemodialysis-dependent, chronic renal failure (CRF-HD) patients (38.7 +/- 13.1 ng/l) was significantly higher than normal. The acromegalic patients were 24.3 +/- 11.9 ng/l, except for one patient with ectopic GHRH syndrome (990 ng/l): his plasma GHRH level reached 7,100 ng/l during operation, and then decreased logarithmically to 70 ng/l after 6 h. Somatostatin at concentrations of 10 and 1,000 nmol/l significantly suppressed (GHRH release) from primary culture cells of the GHRH-producing tumor from 17.3 +/- 0.92 ng/2 x 10(5) cells to 9.98 +/- 3.61 and 4.32 +/- 1.01 ng/2 x 10(5) cells, respectively after 48 h. These data indicate that this GHRH RIA is useful for determining the plasma GHRH concentration in normal and diseased states and also for in vitro studies of GHRH release.
Assuntos
Acromegalia/sangue , Hormônio Liberador de Hormônio do Crescimento/sangue , Hormônio Liberador de Hormônio do Crescimento/imunologia , Neoplasias Hipotalâmicas/sangue , Falência Renal Crônica/sangue , Fragmentos de Peptídeos/imunologia , Adulto , Anticorpos , Cromatografia em Gel/métodos , Reações Cruzadas , Hormônio do Crescimento/sangue , Hormônio Liberador de Hormônio do Crescimento/isolamento & purificação , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Humanos , Cinética , Microquímica , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/cirurgia , Radioimunoensaio/métodos , Valores de Referência , Somatostatina/farmacologia , Células Tumorais CultivadasRESUMO
Table 5 summarizes the activity of the newer quinolones against various bacteria including intracellular bacteria and the other microorganisms. In this table, the overall MIC ranges of NFLX, ENX, OFLX, and CPFX, for susceptible isolates of each bacteria are schematically presented. The newer quinolones are considered to have sufficient activity against gram-negative enteric bacteria, N. gonorrhoeae, and H. influenzae and Legionella spp. Since OFLX and CPFX show only moderate activity against staphylococci, streptococci, and P. aeruginosa, improvement is expected. As shown in Table 3, TFLX and the recent investigational quinolones such as SPFX and KB-5246 show higher and promising activity against gram-positive bacteria. However, further studies are needed with longer periods to indicate whether these newer agents will be able to stop the increase of quinolone-resistant staphylococci. Furthermore, the activity of the newer quinolones against obligate anaerobes such as clostridia and bacteroides are considered to be insufficient for clinical use. Whether it will be possible to synthesize quinolones with anti-anaerobic activity sufficiently for clinical treatment is uncertain. Although Mycobacterium tuberculosis is susceptible to the newer quinolones, other mycobacteria are somewhat less susceptible to this class of agents. In addition, the newer quinolones have adequate activity against mycoplasma, chlamydia and rickettsia. From a microbiological viewpoint the prospects for the newer quinolones would be primarily to find agents that have higher anti-staphylococcal and anti-streptococcal activity. Secondly, agents possessing superior activity against obligate anaerobes such as Bacteroides spp. and Clostridium spp. are expected to synthesize. Furthermore, it may be possible to synthesize compounds that are sufficiently active for clinical use against atypical mycoplasma, chlamydia, and rickettsia.
Assuntos
Anti-Infecciosos/farmacologia , 4-Quinolonas , Bactérias/efeitos dos fármacos , Testes de Sensibilidade MicrobianaRESUMO
Thyrsiferyl 23-acetate (TF-23A) has been shown to potently and specifically inhibit PP2A. TF-23A also induced a rapid cell death in various leukemic T- and B-cell lines. The TF-23A induced cell death with a typical apoptotic process. TF-23A and its several analogous compounds showed apoptosis-inducing activity. However, only TF-23A out of these compounds showed an inhibitory activity for PP2A. These results suggest that a portion of TF-23A involved in induction of apoptosis is different from that involved in the PP2A inhibition. Then, the effects of tautomycin and its derivatives on PP1 and PP2A and their apoptosis-inducing activity were examined. The C22-C26 moiety was essential for inhibition of protein phosphatase activity, whereas the C1-C18 moiety was essential for induction of apoptosis. Therefore, different moieties of tautomycin are involved in protein phosphatase inhibition and induction of apoptosis. From these results, it was concluded that the biological effects of phosphatase inhibitors are not necessarily induced by the inhibition of PP1 and PP2A but through other different molecular mechanisms which remain to be elucidated.
Assuntos
Apoptose , Inibidores Enzimáticos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Piranos/metabolismo , Compostos de Espiro , Animais , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Piranos/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Microbial kinetics of Escherichia coli NIHJ JC-2 and E. coli B/r were investigated in the presence of beta-lactam antibiotics. To maintain a constant drug concentration during the experiment, a novel technique, using a dialysis membrane tube containing the drug solution, was successfully employed. The drug-affected generation curves of E. coli exhibited common features. After the addition of drug, an apparent lag period was noted, followed by a first-order decrease of the sensitive organisms and, 6 h later, by a regrowth of resistant organisms, depending on the antibiotic concentration used. The relationship between the apparent generation rate constant, kapp, and the antibiotic concentration was found to be nonlinear. This phenomenon is consistent with a saturable receptor site model for the drug action. A good linear free energy relationship was observed between the microbial kinetic parameter, kmax, and the alkaline degradation rate constants, kOH, of the cephalosporins studied.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Técnicas Bacteriológicas , Divisão Celular , Meios de Cultura , Diálise , Resistência Microbiana a Medicamentos , Cinética , Relação Estrutura-Atividade , beta-LactamasRESUMO
The generation curves of Escherichia coli B/r and E. coli NIHJ JC-2 in the presence of several beta-lactam antibiotics were studied from the kinetic point of view. Apparent first-order regrowth of resistant organisms was observed approximately 6 h after addition of these antibiotics. The time courses of apparent viable counts could be interpreted in terms of the sum of the viable counts of sensitive and resistant organisms. To clarify the nature of the regrowth, experiments involving a second addition of antibiotic, single colonization by subculture, and synchronous cell culture were carried out. Several possible explanations for the results are discussed, including beta-lactamase production, selection in terms of membrane permeability, and mutation to acquire drug resistance. A selection process or a modification of membrane permeability caused by contact with the drug seems to be the most probable reason for the regrowth of the organisms.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/crescimento & desenvolvimento , Ampicilina/farmacologia , Técnicas Bacteriológicas , Divisão Celular/efeitos dos fármacos , Meios de Cultura , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Cinética , Testes de Sensibilidade Microbiana , Modelos Biológicos , Resistência às Penicilinas , Álcool Feniletílico/farmacologia , beta-Lactamases/metabolismoRESUMO
A plasmid, Rms425, mediating resistance to streptomycin(Sm) and mercury(Hg) was isolated from Pseudomonas cepacia GN11131 of clinical origin. Rms425 was transferred to Pseudomonas cepacia, Pseudomonas aeruginosa and Escherichia coli strains by transformation with purified DNA and by conjugation between isogenic strains of P. aeruginosa or of E. coli by mixing on membrane filters. The molecular weight of Rms425 was estimated to be about 32 megadaltons by electron microscopy and it was classified as incompatibility group P. Examining the incorporation of [gamma-32P] or [8-14C] from isotope-labelled ATP into Sm using the cell-free extracts from P. cepacia or E. coli strains harboring Rms425, we found that the Sm resistance conferred by Rms425 was due to the phosphorylation of the drug.
Assuntos
Pseudomonas/genética , Fatores R , Estreptomicina/farmacologia , Trifosfato de Adenosina/metabolismo , Meios de Cultura , DNA Bacteriano/isolamento & purificação , Pseudomonas/efeitos dos fármacos , Pseudomonas/ultraestrutura , Transformação Bacteriana/efeitos dos fármacosRESUMO
The cephalosporin beta-lactamase (cephalosporinase) was purified from a strain of Proteus morganii which showed resistance to cephalosporins. The optimal pH was about 8.5, and the optimal temperature was 40 degrees C. The isoelectric point was 8.7 and the molecular weight was estimated to be about 41,000 from sodium dodecyl sulfate-acrylamide gel electrophoresis. The enzyme activity was inhibited by cloxacillin, ampicillin, carbenicillin, cefuroxime, cefotaxime, ceftizoxime (FK749), cefmenoxime (SCE-1365), cefoxitin, cefmetazole, YM09330 and moxalactam (6059-S), but not by clavulanic acid or CP-45899. The beta-lactamase also hydrolyzed cephaloridine, cefazolin, cephalothin, cephalexin, cefotiam, cefamandole and benzylpenicillin. These results suggest the possibility that the properties of beta-lactamases may be characterized by measuring the kinetic parameters of the enzyme toward newly-introduced beta-lactam antibiotics and beta-lactamase inhibitors.