RESUMO
OBJECTIVES: To evaluate the efficacy of a birth ball exercise programme conducted by physiotherapists on pain relief, psychological care, and facilitation of the labour process at a labour ward in a regional hospital. DESIGN: Case series with before-after comparisons. SETTING: Kwong Wah Hospital, Hong Kong. PARTICIPANTS: Chinese women admitted to the labour ward for spontaneous vaginal delivery between April and August 2012 were recruited. Physiotherapists taught birth ball exercises in groups or individually for 30 minutes. Labour pain intensity, back pain intensity, frequency of labour pain, stress and anxiety levels, and subjective pressure level over the lower abdomen were captured before and after birth ball exercises. Most of the parameters were measured using self-reported visual analogue scales. After the exercise session, physiotherapists measured the women's satisfaction level. Midwives recorded pethidine usage. RESULTS: A total of 203 pregnant women participated in this programme; 181 were in the latent phase group, whereas 22 were categorised into the no-labour-pain group. In both groups, there were statistically and clinically significant differences in back pain level, stress and anxiety levels, as well as pressure level over the lower abdomen before and after the exercise (P<0.05). In the latent phase group, significant decreases in labour pain and frequency of labour pain were demonstrated. Mean satisfaction scores were high, with visual analogue scale scores higher than 8.2 in both groups. Pethidine usage showed a further decreasing trend (6.4%) compared with the past 2 years. CONCLUSION: Birth ball exercise could be an alternative means of relieving back pain and labour pain in the labour ward, and could decrease pethidine consumption in labouring women.
Assuntos
Terapia por Exercício/métodos , Dor do Parto/terapia , Trabalho de Parto , Satisfação do Paciente , Adulto , Analgésicos Opioides/administração & dosagem , Ansiedade/etiologia , Ansiedade/terapia , Parto Obstétrico , Feminino , Hong Kong , Humanos , Meperidina/administração & dosagem , Medição da Dor , Gravidez , Estresse Psicológico/etiologia , Estresse Psicológico/terapiaRESUMO
PURPOSE: To assess a protected mobilisation programme (dynamic treatment) for proximal phalangeal fracture of the hand, irrespective of the geometry. METHODS: Clinical and radiological results of 32 consecutive patients with proximal phalangeal fracture of the hand treated from January 2001 to February 2007 were evaluated. Our supervised rehabilitation programme was strictly followed to gain full range of movement of the proximal interphalangeal joint and to prevent the development of an extension lag contracture. Patients were followed up for a mean period of 15 (range, 13-16) months. Results were evaluated using the Belsky classification. RESULTS: The results were excellent in 72% of the patients, good in 22%, and poor in 6%. Some patients defaulted follow-up, which made long-term assessment difficult. The poor results may have been related to patient non-compliance or default from rehabilitation. Many good results upgraded to excellent following further rehabilitation. CONCLUSION: Skeletal stability, not rigidity, is necessary for functional movements of the hand. Proximal phalangeal fractures can be effectively treated by closed methods, using the stabilising effect of soft tissues (zancolli complex-metacarpophalangeal retention apparatus) and external devices (metacarpophalangeal block splint), thus enabling bone healing and movement recovery at the same time.
Assuntos
Traumatismos dos Dedos/fisiopatologia , Falanges dos Dedos da Mão/lesões , Fixação de Fratura/métodos , Fraturas Ósseas/terapia , Adolescente , Adulto , Idoso , Criança , Feminino , Traumatismos dos Dedos/diagnóstico por imagem , Articulações dos Dedos/diagnóstico por imagem , Articulações dos Dedos/fisiopatologia , Falanges dos Dedos da Mão/diagnóstico por imagem , Seguimentos , Fraturas Ósseas/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade , Radiografia , Amplitude de Movimento Articular , Estudos Retrospectivos , Fatores de Tempo , Resultado do TratamentoRESUMO
Occupational risk factors of carpal tunnel syndrome (CTS) are popular current research targets, with main emphasis put on wrist posture and dynamics. In this study, we do not intend to pinpoint individual occupations, but aim to identify high risk wrist postures and actions which may occur across various occupations. It is hoped that prevention can thus be instituted in a general population by directing at the particular causative wrist actions rather than exclusively targeting isolated occupations. We performed a case-control study with 166 cases and 111 controls recruited from different hospitals in Hong Kong in 2004. All cases and controls completed the survey on their general health condition, smoking status, wrist posture and motion as well as psychosocial status at the time of diagnosis of CTS. Frequent flexion OR = 4.436 (95% CI: 1.833-10.734), frequent extension OR = 2.691 (95% CI: 1.106-6.547) of the wrist were found to be associated with CTS. Frequent sustained forceful motion of the wrist OR = 2.588 (95% CI: 1.144-5.851) was also found to be associated with CTS. Neutral wrist position and repetitive wrist motion were not associated with CTS. Adjustment was made for age, sex, BMI, smoking and psychosocial stress. Our study confirms that frequent flexion, extension and sustained force of the wrist increase the risk of developing CTS.
Assuntos
Síndrome do Túnel Carpal/epidemiologia , Punho/fisiopatologia , Adolescente , Adulto , Fenômenos Biomecânicos , Síndrome do Túnel Carpal/fisiopatologia , Síndrome do Túnel Carpal/psicologia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Fatores de Risco , Fatores Sexuais , Fumar/epidemiologia , Estresse PsicológicoRESUMO
The gamma polypeptide of brain G-proteins is carboxyl methylated when the purified beta gamma subunit complex is reconstituted with S-adenosyl-[3H-methyl]-L-methionine and a methyltransferase present in detergent-stripped brain membranes. By chromatographic analysis of the 3H-amino acid generated by exhaustive proteolysis and performic acid oxidation of the 3H-methylated beta gamma complex, we show that this modification occurs on the alpha-carboxyl group of a C-terminal cysteine residue. Our result suggests that brain G-protein may undergo multiple covalent modification steps, including proteolytic removal of the three terminal amino acids from the predicted common C-terminal Cys-Xaa-Xaa-Xaa sequence, and the methyl esterification of the resulting terminal cysteine residue. This modification is likely to be associated with lipidation at the sulfhydryl group of the same cysteine, which would explain the tight membrane binding property of the brain beta gamma complex.
Assuntos
Química Encefálica , Ácidos Carboxílicos/análise , Cisteína/análise , Ésteres/análise , Proteínas de Ligação ao GTP/isolamento & purificação , Animais , Bovinos , Membrana Celular/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Hidrólise , Lipídeos de Membrana/fisiologia , Metilação , Ligação Proteica , S-AdenosilmetioninaRESUMO
In the inherited retinal degeneration of rd mice, cyclic GMP accumulates in affected rod photoreceptors prior to their degeneration. A deficiency in the activity of the visual cell phosphodiesterase apparently results in the accumulation of cyclic GMP. The cyclic GMP phosphodiesterase (PDE) of normal mouse photoreceptors is a heteromeric protein complex of about 170 kDa, consisting of the alpha beta catalytic unit and the gamma inhibitory unit. The isolated complex has low enzyme activity but it can be activated by incubation with histone. Affinity-purified polyclonal antibodies against the PDE complex of bovine rod outer segments were prepared and used to identify in retinas of both normal and rd mice PDE-immunoreactive polypeptides which comigrated on SDS-polyacrylamide gels with the large subunits (88 kDa) of the normal PDE complex. During development of normal retinas, the 88 kDa immunoreactive component of the PDE complex were detected by day 7, with immunoreactivity increasing throughout the second postnatal week. In rd retinas, the 88 kDa immunoreactivity increased after 9 postnatal days, decreased during rod photoreceptor degeneration, and was undetectable in mature rd retinas. Under nondenaturing conditions, the PDE-immunoreactive polypeptide of rd retinas sedimented on sucrose gradients with a sedimentation coefficient of 5.6S and an apparent molecular mass of about 105 kDa; no associated histone-activated PDE activity was detected. These findings show that PDE-immunoreactive polypeptides are synthesized in immature rd photoreceptors and that the PDE-immunoreactive polypeptides fail to form a PDE complex which is comparable to that of normal photoreceptors.
Assuntos
Camundongos Mutantes/imunologia , Peptídeos/imunologia , Diester Fosfórico Hidrolases/imunologia , Células Fotorreceptoras/imunologia , Retina/crescimento & desenvolvimento , Degeneração Retiniana/genética , Animais , Especificidade de Anticorpos , Bovinos , Centrifugação com Gradiente de Concentração , Técnicas Imunológicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes/genética , Células Fotorreceptoras/crescimento & desenvolvimento , Coelhos , Valores de Referência , Retina/imunologia , Degeneração Retiniana/imunologia , Degeneração Retiniana/fisiopatologiaRESUMO
PURPOSE: To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors. METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors. RESULTS: Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification. CONCLUSIONS: These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.
Assuntos
Receptores de Fatores de Crescimento/metabolismo , Malha Trabecular/metabolismo , Adolescente , Idoso , Idoso de 80 Anos ou mais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Pré-Escolar , Primers do DNA/química , Glaucoma/metabolismo , Glaucoma/patologia , Substâncias de Crescimento/farmacologia , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento/genética , Malha Trabecular/citologia , Malha Trabecular/efeitos dos fármacosRESUMO
BACKGROUND: Continuous epidural analgesia is a popular technique provided for labor pain relief. However, the possible association between epidural analgesia and increased rate of cesarean delivery is always a concern. The purpose of this study was to explore if epidural analgesia would affect the rate of cesarean delivery in healthy term parturient women. METHODS: A retrospective review of obstetric records of 822 parturients was done to compute the proportions and odds ratio of cesarean delivery between parturients receiving epidural analgesia for labor pain relief and those not receiving. RESULTS: Results indicated that the overall proportion of cesarean delivery was 11.1% in the epidural group as against 16.2% in the control group and the odds ratio was 0.65 (95% confidence interval: 0.37 to 1.08). The difference between groups was not statistically significant. However, for nulliparous women alone, the proportions of cesarean delivery were 11.6% and 25.1% in exposure group and control group respectively. The odds ratio was 0.39 (95% CI: 0.20 to 0.71). This difference was statistically significant. CONCLUSIONS: The findings may support the conclusion that continuous epidural analgesia for pain relief during labor would not increase the rate of cesarean delivery. In contrast, it may reduce this risk in nulliparas.
Assuntos
Analgesia Epidural , Analgesia Obstétrica , Cesárea , Adulto , Feminino , Humanos , Gravidez , Estudos RetrospectivosRESUMO
BACKGROUND: Administration of general anesthesia necessitates certain amounts of manipulation or instrumentation within the mouth that may cause some oral tissue injuries. The purpose of this study was to determine the incidence of oral tissue trauma after receiving general anesthesia. Some possible risk factors were explored. METHODS: Four hundred and four adult patients receiving surgical procedures under general anesthesia facilitated by endotracheal intubation were included in this prospective survey. In each patient detailed oral and dental conditions were evaluated and recorded by an oral surgeon before and after operation. The extent of oral tissue trauma were computed and the type and character of the injury were recorded. RESULTS: Twenty-eight patients (6.9%) were found to sustain various degrees of oral trauma that included soft tissue laceration, tooth fracture, prosthesis damage, tooth avulsion, ecchymosis and numbness of tongue. Preexistence of missing tooth, crown, and residual root were some possible risk factors that might predispose to complication. CONCLUSIONS: Oral tissue trauma as it seems to be a common anesthetic complication that should be considered as a recognized hazard of general anesthesia. Understanding and recognizing oral anatomical condition and pathological change before anesthesia may help the anesthesiologists in preventing oral and dental complications and thus to avoid legal suits.
Assuntos
Anestesia Geral/efeitos adversos , Intubação Intratraqueal/efeitos adversos , Boca/lesões , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Ferimentos e Lesões/epidemiologiaRESUMO
BACKGROUND: Postoperative wound pain is a remarkable distress and may precipitate major complication. We attempted to develop a reliable and effective technique for postoperative analgesia that is safe and simple enough to be provided in an ordinary ward of a rural hospital. MATERIALS AND METHODS: A retrospective study of continuous epidural infusion of morphine 7.5 mg in 60 mL diluted lidocaine for postoperative pain control in 156 patients in the past year was undertaken. Among the patients, one hundred and twelve patients had undergone. Cesarean Section, and 44 patients had uterine or adnexal operations. All patients received epidural anesthesia. RESULTS: Effective analgesia was achieved in all cases throughout the postoperative period. Pain relief was long lasting and there were only 5 patients (3.2%) who required some additional narcotics. The mean interval between completion of the operation and passage of flatus was 33.2 h. Only 48 patients (30.8%) complained of skin itching, 39 patients (25.0%) reported nausea and vomiting and 62 cases (39.7%) experienced constipation. Urinary retention after Foley catheter removal was noted in 9 cases (5.8%). No major complication pertaining to the technique was encountered. CONCLUSIONS: We concluded that continuous epidural infusion of morphine and diluted lidocaine is an effective, simple and safe technique for postoperative pain control in obstetric and gynecologic operation and it can be administered routinely even in a rural hospital.
Assuntos
Analgesia Epidural , Lidocaína/administração & dosagem , Morfina/administração & dosagem , Dor Pós-Operatória/tratamento farmacológico , Adolescente , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
OBJECTIVE: To examine the efficacy of a strength-focused mutual support group for reducing stress and enhancing psychological well-being of the caretakers of children with cerebral palsy. METHODS: This pre- and post-intervention outcome study, conducted by Department of Orthopaedics and Traumatology, Duchess of Kent Children's Hospital, recruited 12 primary caretakers of children with cerebral palsy in Hong Kong. A strength-focused support group manual was developed to help such caretakers to identify and cultivate the character strengths of their children and enhance their own positive emotions. Participants were asked to complete a full set of questionnaires at 3 time-points: before and immediately after the intervention (consisting of 4 weekly sessions), and after the 1-month follow-up booster session. Two additional questionnaires were administered before each session to check mood. Parenting stress, anxiety, depression, social support, hope, and other psychological well-being measures were also assessed. RESULTS: Half of the caretakers (n = 6) who had attended the full intervention programme were included in the data analysis. Participants exhibited a significantly lower level of parental stress and higher hope level both after the 4 intervention sessions and at the booster session. Their perceived social support was significantly increased when the group was ongoing but not after it ended. CONCLUSION: This group intervention programme could effectively help caretakers reduce their parenting stress and enhance their hopefulness. Launching a similar programme with more sessions and regular follow-up sessions might help maintain positive effects and establish a social support network.
Assuntos
Cuidadores/psicologia , Paralisia Cerebral/terapia , Grupos de Autoajuda/estatística & dados numéricos , Adaptação Psicológica , Adulto , Paralisia Cerebral/psicologia , Emoções , Feminino , Seguimentos , Hong Kong , Humanos , Masculino , Pessoa de Meia-Idade , Pais/psicologia , Satisfação Pessoal , Avaliação de Programas e Projetos de Saúde/métodos , Avaliação de Programas e Projetos de Saúde/estatística & dados numéricos , Apoio Social , Estresse Psicológico/psicologia , Estresse Psicológico/terapia , Inquéritos e QuestionáriosAssuntos
Lipossomos , Fosfatidilcolinas , Fosfatidiletanolaminas , Pigmentos da Retina , Rodopsina , Animais , Membrana Celular/ultraestrutura , Escuridão , Radioisótopos do Iodo , Marcação por Isótopo/métodos , Lactoperoxidase , Pigmentos da Retina/metabolismo , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/ultraestruturaAssuntos
3',5'-GMP Cíclico Fosfodiesterases/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Células Fotorreceptoras/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Adenilil Ciclases/metabolismo , Animais , Ativação Enzimática , Proteínas de Ligação ao GTP , Luz , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Métodos , Peso Molecular , Receptores de Superfície Celular/metabolismo , Transducina , Visão OcularAssuntos
Alquil e Aril Transferases , Cisteína/análogos & derivados , Diterpenos/síntese química , Proteínas de Ligação ao GTP/metabolismo , Prenilação de Proteína , Sequência de Aminoácidos , Animais , Cisteína/síntese química , Cisteína/metabolismo , Diterpenos/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Farnesiltranstransferase , Proteínas de Ligação ao GTP/isolamento & purificação , Substâncias Macromoleculares , Ácido Mevalônico/metabolismo , Dados de Sequência Molecular , Peso Molecular , Técnica de Diluição de Radioisótopos , Ratos , Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Transferases/metabolismo , TrítioAssuntos
Mãos/microbiologia , Infecções por Mycobacterium não Tuberculosas/complicações , Tenossinovite/microbiologia , Idoso , Antibacterianos/administração & dosagem , Sedimentação Sanguínea , Proteína C-Reativa/análise , Claritromicina/administração & dosagem , Histiócitos/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/patologia , Ofloxacino/administração & dosagem , Tenossinovite/patologiaRESUMO
Transducin, a guanine nucleotide regulatory protein found in the bovine retinal rod outer segment, mediates the signal coupling between rhodopsin and a cyclic GMP phosphodiesterase. Previous studies have demonstrated that photolyzed rhodopsin catalyzed the exchange of GTP for GDP bound to transducin. The transducin-GTP complex, in turn, activates the phosphodiesterase. Purified transducin (T) has been resolved by omega-amino octylagarose chromatography into two functional subunits: T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). The guanine nucleotide binding site is on the T alpha subunit. Neither the T alpha nor the T beta gamma subunit showed significant GTPase activity, Gpp(NH)p-GDP exchange, and ability to bind to rhodopsin when assayed in the presence of reconstituted membranes containing photolyzed rhodopsin. However, all the above activities were restored if the two subunits were recombined. Analysis of the reconstituted GTPase activity as a function of the T alpha subunit concentration revealed a linear relationship. On the other hand, GTPase activity rapidly saturated at T beta gamma concentration much lower than the T alpha concentration, indicating that the two subunits were dissociated during GTP hydrolysis. These findings strongly suggest that the T beta gamma subunit is an activator of the GTPase activity. Its mode of action is to enable the T alpha subunit to interact with rhodopsin effectively.
Assuntos
Proteínas de Membrana/análise , Células Fotorreceptoras/análise , Segmento Externo da Célula Bastonete/análise , Animais , Bovinos , GTP Fosfo-Hidrolases/metabolismo , Guanilil Imidodifosfato/metabolismo , Imidoésteres/metabolismo , Substâncias Macromoleculares , Peso Molecular , TransducinaRESUMO
A panel of monoclonal antibodies has been developed against the T alpha, T beta and T gamma subunits of bovine transducin. Two anti-T alpha antibodies from this panel (TF15 and TF16) and a third one (4A) against frog T alpha (Witt, P. L., Hamm, H. E., and Bownds, M. D. (1984) J. Gen. Physiol. 84, 251-263) were characterized. Each of these monoclonal antibodies recognizes a different region of T alpha and has a specific effect on the function of transducin. The binding of TF15 is reversibly enhanced by treating T alpha with either 1 M guanidinium chloride or, to a smaller extent, by the removal of bound guanine nucleotide. Its epitope is located in a 12-kDa tryptic fragment containing the binding site for the guanine moiety of GTP. Taken together, these results support previous observations that the conformation of T alpha is modulated by the occupancy of the guanine nucleotide binding site. In contrast to TF15, TF16 recognizes only the native form of T alpha. Its epitope resides within the central portion of the T alpha molecule. While T alpha-bound TF16 does not inhibit either pertussis toxin-catalyzed ADP-ribosylation, rhodopsin binding, or transducin subunit interaction, it blocks both the light-activated uptake of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and the GTP-dependent elution of transducin from photolyzed rhodopsin. These effects are unlikely to be caused by the occupation of the guanine nucleotide binding site by TF16 because this antibody quantitatively precipitates T alpha-GTP gamma S. We propose that bound TF16 locks T alpha in a conformation that prevents the entrance of guanine nucleotide and favors T beta gamma association. In contrast to TF16, the epitope of 4A was mapped to the amino-terminal region of T alpha. This monoclonal antibody blocks pertussis toxin-catalyzed ADP-ribosylation, GTP gamma S uptake, and T alpha-T beta gamma association. Moreover, the binding site for 4A becomes inaccessible when transducin binds to photolyzed rhodopsin. These results suggest that the inhibitory effects of 4A are due to a simultaneous steric blockage of both the interaction of T alpha with T beta gamma and their binding to photolyzed rhodopsin. The results obtained from these studies are correlated with the structure and function of T alpha.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/metabolismo , Células Fotorreceptoras/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Animais , Anticorpos Monoclonais , Complexo Antígeno-Anticorpo , Ligação Competitiva , Bovinos , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Cinética , Substâncias Macromoleculares , Proteínas de Membrana/imunologia , Tionucleotídeos/metabolismo , TransducinaRESUMO
We have purified to homogeneity a 23-kDa protein from bovine brain membranes using [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding as an assay. GTP gamma S binding to the purified protein is inhibited by GDP, GTP, and GTP analogs but not by cGMP, GMP, or adenine nucleotides, consistent with the nucleotide-binding behavior of members of the family of GTP-binding regulatory proteins. On addition of the methyl donor S-adenosyl-L-methionine and a methyltransferase present in bovine brain membranes, the purified 23-kDa G-protein is carboxyl methylated. When subjected to limited tryptic proteolysis, the 23-kDa protein is converted to a 22-kDa major fragment with concomitant release of a carboxyl methylated protein fragment of 1 kDa. Furthermore, when the cleaved protein is reconstituted with stripped bovine brain membranes, the small carboxyl-methylated fragment but not the 22-kDa major fragment is found to reassociate with the membranes. These results indicate that the site of carboxyl methylation and the region responsible for membrane anchoring, most likely, are localized to a small region at the carboxyl terminus. It is attractive to speculate that carboxyl methylation and membrane anchoring are interrelated processes and play key roles in the function of this small G-protein.
Assuntos
Proteínas de Ligação ao GTP/isolamento & purificação , Animais , Ligação Competitiva , Encéfalo/metabolismo , Bovinos , Membrana Celular/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Proteínas de Ligação ao GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/farmacologia , Cinética , Metilação , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Ligação Proteica , Ribonucleotídeos/farmacologia , Tionucleotídeos/metabolismo , Tionucleotídeos/farmacologia , TripsinaRESUMO
The GTP-induced dissociation of T alpha from T beta gamma initiates the release of transducin from photolyzed rhodopsin and the subsequent activation of the cGMP phosphodiesterase. In this study, site-specific proteolysis and immunoprecipitation were used to map the domain of T alpha that interacts with T beta gamma. We found that Staphylococcus aureus V8 protease rapidly removes a small fragment from T alpha under native conditions, resulting in the formation of a single 38-kDa polypeptide (T alpha'). Under the same conditions, T beta gamma remains intact. A 4.5-fold decrease in the rate of T alpha cleavage by S. aureus protease was observed in the presence of T beta gamma, suggesting T beta gamma binding blocks the protease-sensitive site on T alpha. Amino acid sequence analysis indicated that T alpha' is derived from the cleavage of T alpha at Glu-21. The ability of T alpha' to interact with and activate the retinal phosphodiesterase is not diminished. However, T alpha' is unable to participate in T beta gamma-dependent activities such as the light-stimulated binding of guanine nucleotides, binding to photoexcited rhodopsin, and ADP-ribosylation catalyzed by pertussis toxin. Moreover, the anti-T alpha monoclonal antibody TF16 was able to precipitate T beta gamma in the presence of T alpha, but not with either T alpha' or T alpha-guanosine 5'-O-(3-thiotriphosphate). We conclude that the amino-terminal region of T alpha participates in T beta gamma interaction and discuss our results with respect to the known structure and function of transducin.
Assuntos
Proteínas de Membrana/análise , Células Fotorreceptoras/análise , Segmento Externo da Célula Bastonete/análise , Adenosina Difosfato Ribose/metabolismo , Animais , Anticorpos Monoclonais , Sítios de Ligação , Bovinos , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/metabolismo , Substâncias Macromoleculares , Metaloendopeptidases , Toxina Pertussis , Tionucleotídeos/farmacologia , Transducina , Fatores de Virulência de Bordetella/farmacologiaRESUMO
We have shown by intravitreal injection of [3H]mevalonolactone that a 65 kDa protein in rat photoreceptors is posttranslationally modified by farnesylation. We further identified this 65 kDa prenylated protein as rhodopsin kinase based on its affinity for photolyzed rhodopsin and its ability to autophosphorylate in the presence of [gamma-32P]ATP. The farnesylation of rhodopsin kinase may be important for correctly targeting this enzyme to the photoreceptor outer segments, allowing it to phosphorylate photolyzed rhodopsin efficiently.