Characterization of the heme and pyridoxal phosphate cofactors of human cystathionine beta-synthase reveals nonequivalent active sites
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Journal Article↗
Biomedical subjects
M West
Publications and source records attributed to M West.
Characterization of the heme and pyridoxal phosphate cofactors of human cystathionine beta-synthase reveals nonequivalent active sites.
Cystathionine beta-synthase is an unusual enzyme that requires the cofactors heme and pyridoxal phosphate (PLP) to catalyze the condensation of homocysteine and serine to generate cystathionine. This transsulfuration reaction represents one of two major cellular routes for detoxification of homocysteine, which is a risk factor for atherosclerosis. While the beta-replacement reaction catalyzed by this enzyme suggests a role for the pyridoxal phosphate, the role of the heme is uncertain. In this study we have examined the effect of changing one of the ligands to the heme on the activity of the enzyme. Binding of carbon monooxide results in the displacement of a thiolate ligand to the ferrous heme, and is accompanied by complete loss of cystathionine beta-synthase activity. Furthermore, inhibition by CO is competitive with respect to homocysteine, providing the first indication that the homocysteine binding site is in the proximity of heme. Binding of both CO and cyanide to ferrous cystathionine beta-synthase occurs in two distinct isotherms and indicates that the hemes are nonequivalent. We have employed fluorescence spectroscopy to characterize the bound PLP and its interaction with serine. PLP bound to cystathionine beta-synthase is weakly fluorescent and exists as a mixture of the protonated and unprotonated tautomers. Reaction with hydroxylamine releases the oxime and greatly enhances the associated fluorescence. Binding of serine is accompanied by a shift to the unprotonated tautomer of the external aldimine as well as the appearance of a new fluorescent species at approximately 400 nm that could be due to the aminoacrylate or to a gemdiamine intermediate. These data provide the first characterization of the PLP bound to cystathionine beta-synthase. Treatment of cystathionine beta-synthase with hydroxylamine releases two PLPs after 1 day and results in complete loss of activity. Incubation for an additional 3-4 days results in the release of two more PLPs. These data lead us to revise the PLP stoichiometry to 4 per tetramer, and to the conclusion that the heme and PLP sites in cystathionine beta-synthase are nonequivalent.
Binding Sites↗
New methods of time series analysis of non-stationary EEG data: eigenstructure decompositions of time varying autoregressions.
OBJECTIVE: Those who analyze EEG data require quantitative techniques that can be validly applied to time series exhibiting ranges of non-stationary behavior. Our objective is to introduce a new analysis technique based on formal non-stationary time series models. This novel method provides a decomposition of the time series into a set of 'latent' components with time-varying frequency content. The identification of these components can lead to practical insights and quantitative comparisons of changes in frequency structure over time in EEG time series. METHODS: The technique begins with the development of time-varying autoregressive models of the EEG time series. Such models have been previously used in EEG analysis but we extend their utility by the introduction of eigenstructure decomposition methods. We review the basis and implementation of this method and report on the analysis of two channel EEG data recorded during 3 generalized tonic-clonic seizures induced in an individual as part of a course of electroconvulsive therapy for major depression. RESULTS: This technique identified EEG patterns consistent with prior reports. In addition, it quantified a decrease in dominant frequency content over the seizures and suggested for the first time that this decrease is continuous across the end of the seizures. The analysis also suggested that the seizure EEG may be best modeled by the combination of multiple processes, whereas post-ictally there appears to be one dominant process. There was also preliminary evidence that these features may differ as a function of ECT therapeutic effectiveness. CONCLUSIONS: Eigenanalysis of time-varying autoregressive models has promise for improving the analysis of EEG time series.
Brain↗
Clostridium difficile colitis after kidney and kidney-pancreas transplantation.
OBJECTIVE: To determine the timing and risk factors involved in the development of Clostridium difficile (CD) colitis in kidney and kidney-pancreas transplant recipients. BACKGROUND DATA: The incidence of CD colitis after kidney and kidney-pancreas transplantation has not been studied in detail. The question of whether the immunosuppressed transplant recipient is more prone to CD colitis and its complications (i.e., megacolon, perforations) and the risk factors involved have not been determined. METHODS: We retrospectively reviewed our experience in kidney and kidney-pancreas recipients who received transplants between January 1, 1985 and December 31, 1994. We divided these recipients into three groups: pediatric kidney recipients, adult kidney recipients, and kidney-pancreas recipients. For each group, we assessed the timing of infection, primary disease, colitis treatment, and any concurrent complications or risk factors. RESULTS: Of 1932 transplants, 159 recipients developed post-transplant CD colitis. 132 charts were available for review. Forty-three pediatric kidney recipients developed CD colitis. Their mean age was 3.2 yr; 74% (n = 37) of them developed their colitis during their initial hospital stay, with the mean timing of infection being 33 d. Forty-one (95%) had undergone intra-abdominal placement of the graft, with renal artery anastomoses to the aorta. Fifty adult kidney recipients developed CD colitis. Thirteen (26%) developed colitis during their initial hospital stay, with the mean timing of infection (for all adult kidney recipients) being 15 months. Thirty-nine kidney-pancreas recipients developed CD colitis. Mean timing of infection was 6 months. The overall incidence of CD colitis was 8%, with 16% in the pediatric kidney group, 15.5% in the kidney-pancreas group, and 3.5% in the adult kidney group. The difference in mean timing of infection was significant between the three groups (p < 0.001 for pediatric versus adult kidney recipients, p = 0.002 for pediatric kidney versus kidney-pancreas recipients, and p = 0.2846 for adult kidney versus kidney-pancreas recipients). CONCLUSION: The incidence of CD colitis is increased in pediatric kidney and kidney-pancreas recipients. Young recipient age ( < 5 yr), female gender, treatment of rejection with monoclonal antibodies, antibiotic use, and intra-abdominal graft placement have been shown to increase the incidence of this disease. Further studies concerning prevention in the high-risk groups are needed.
Adolescent↗
Portrayal of lobotomy in the popular press: 1935-1960.
This study analyzed the content of popular press articles on lobotomy between the years 1935 and 1960. Both a qualitative and quantitative analysis provided evidence that the press initially used uncritical and sensational reporting styles, with the content of articles on lobotomy becoming increasingly negative through time. The initial positive bias occurred despite opposing views in the medical community, which provided a basis for more balanced coverage. These findings support the theory that biased reporting in popular press articles may have been a factor influencing the quick and widespread adoption of lobotomy as a psychiatric treatment.
Brain↗
Abuse and violence in intimate adult relationships: new perspectives from attachment theory.
Previous attachment writing about violence between intimate adult partners has implicated the role of insecure attachment. This paper proposes that insecure attachment is not sufficient to explain intimate partner violence. Instead, it is suggested that recent evidence leads to the conclusion that such violent behavior is best explained in terms of attachment disorganization, a more profound form of relational insecurity. We discuss the contributions of this perspective to intervention.
Adult↗
Assessing attachment: convergent validity of the adult attachment interview and the parental bonding instrument.
OBJECTIVE: The aim of this study was to determine whether or not the Parental Bonding Instrument (PBI) can provide information about parent-child attachment that is comparable to information obtained from the Adult Attachment Interview (AAI), a more complex measure of attachment. METHOD: One hundred and thirty emotionally and/or behaviourally disturbed adolescents (73 male, 57 female; ages 13-19 years, x = 15.3 +/- 1.47 years) participating in a study of attachment and suicidality completed the PBI and the AAI. Data from these measures were compared within participants. RESULTS: Maternal care and overprotection on the PBI differed significantly by AAI attachment classification (F3,122 = 2.79, p = 0.012), with autonomous participants showing the most optimal and unresolved participants the least optimal PBI results. Maternal love and maternal involvement/role reversal on the AAI were significant predictors of maternal care and maternal overprotection, respectively, on the PBI (R2 = 0.15; R2 = 0.16). These predictions improved when AAI scales measuring idealisation and involving anger towards the mother were included in the regression analyses (R2 = 0.35; R2 = 0.20). Autonomous participants on AAI showed the highest scale correlations across instruments. CONCLUSIONS: Attachment information obtained from the PBI and the AAI is comparable in participants with optimal attachment histories, but not in participants showing idealisation or anger towards their mothers. Caution is, therefore, advisable when using the PBI to obtain attachment information in clinical samples where suboptimal attachment histories are likely.
Adolescent↗
Epstein-Barr virus nuclear antigen 3C interacts with histone deacetylase to repress transcription.
EBNA3C can specifically repress the expression of reporter plasmids containing EBV Cp latency-associated promoter elements. Cp is normally the main promoter for EBNA mRNA initiation, so it appears that EBNA3C contributes to a negative autoregulatory control loop. By mutational analysis it was previously established that this repression is consistent with EBNA3C being targeted to Cp by binding the cellular sequence-specific DNA-binding protein CBF1 (also known as recombination signal-binding protein [RBP]-Jkappa. Further analysis suggested that in vivo a corepressor interacts with EBNA3C in this DNA binding complex. Results presented here are all consistent with a component of such a corepressor exhibiting histone deacetylase activity. The drug trichostatin A, which specifically inhibits histone deacetylases, relieved two- to threefold the repression of Cp induced by EBNA3C in two different cell types. Moreover, repression of pTK-CAT-Cp4x by EBNA3C was specifically enhanced by cotransfection of an expression plasmid for human histone deacetylase-1 (HDAC1). Consistent with these functional assays, in vitro-translated HDAC1 bound to a glutathione S-transferase (GST) fusion protein including full-length EBNA3C, and in the reciprocal experiment EBNA3C bound to a GST fusion with the N terminus of HDAC1. Coimmunoprecipitations also revealed an EBNA3C-HDAC1 interaction in vivo, and GST-EBNA3C bound functional histone deacetylase enzyme activity from HeLa cell nuclear extracts. The region of EBNA3C involved in the interaction with HDAC1 appears to correspond to the region which is necessary for binding to CBF1/RBP-Jkappa. A direct physical interaction between EBNA3C and HDAC1 was demonstrated with recombinant proteins purified from bacterial cells, and we therefore conclude that HDAC1 and CBF1/RBP-Jkappa bind to the same or adjacent regions of EBNA3C. These data suggest that recruitment of histone deacetylase activity makes a significant contribution to the repression of transcription from Cp because EBNA3C bridges an interaction between CBF1/RBP-Jkappa and HDAC1.
Animals↗
A comparison of image reject rates when using film, hard copy computed radiography and soft copy images on picture archiving and communication systems (PACS) workstations.
A comparison has been made of the reject rates of plain images for three separate periods when film, computed radiography (CR) and PACS systems were in operation throughout the Hammersmith Hospital, London. There was a statistically significant reduction in the overall percentage reject rate across all examinations from 9.9% to 8.1% when the hospital changed from using a conventional film based system to a CR system. There was a further reduction in the reject rate to 7.3% when the hospital moved to a hospital-wide PACS system, but this change was not statistically significant. Using estimations of the total number of images used, the percentage reject rates were 6.6% for film, 5.5% for CR and 5.5% for PACS. Thus, if the radiation dose for each image is unchanged, and the same types of images are used for the examination of each body area, a move from conventional film imaging to phosphor plate imaging provides the potential to reduce the patient population dose.
Computer Systems↗
Anxious attachment and severity of depressive symptomatology in women.
The present research evaluated a conceptual model that links anxious attachment to depressive symptomatology in women. Four hundred and twenty women completed the following measures: Socio-demographic questionnaire, the Centre for Epidemiological Studies Depression Scale (CES-D), the Reciprocal Attachment Questionnaire, the Social Support Questionnaire, the Rosenberg Self-Esteem Scale and the Global Assessment of Recent Stress Scale. Results indicate that feared loss of the attachment figure, intense proximity seeking and lack of use of the attachment figure were predictive of depressive symptomatology. We also found that lower levels of self-esteem and higher levels of recent stress were predictive of depressive symptomatology. The implications of these findings for the understanding of symptoms of depression in women is discussed.
Adult↗
Developmental vs. social personality models of adult attachment and mental ill health.
Both the developmental and social personality approaches to the study of adult attachment are concerned with understanding those factors that describe an individual's quality of relational adaptation and risk for mental ill health. This paper examines the theoretical and methodological assumptions of these alternative models and how these assumptions have markedly different implications for addressing clinical issues. It is suggested that recent evidence necessarily leads to the conclusion that mental and relational difficulties, such as partner violence and victimization, borderline personality, dissociation, suicidal behaviour and other clinical symptomology thought to be related to experiences of severe relationship distress, are best explained in terms of attachment disorganization rather than as normative forms of attachment insecurity or fearful avoidant adult romantic attachment.
Adaptation, Psychological↗
The Medicaid Home and Community-Based Waiver and supported employment.
The Medicaid Home and Community-Based (HCB) Waiver is the primary funding source for long-term care for individuals with developmental disabilities. Findings from a national survey of 48 state coordinators regarding waiver-funded supported employment services are presented. Very low utilization for this service was found, primarily due to limits on eligibility. However, respondents in 18 states reported large numbers of waiver participants receiving employment services under other service categories. Eleven states had waiting lists for waiver-funded supported employment, totalling three times those actually receiving services. Low reimbursement rates to provider agencies and disinterest of providers in using waiver funds were significant barriers to expansion of service capacity.
Centers for Medicare and Medicaid Services, U.S.↗
Hilar somatostatin-mRNA containing neurons are preserved after perforant path kindling in the rat.
The loss of neurons in the dentate gyrus have been evaluated in rats in which kindling had been induced through stimulation of the hippocampal perforant path. The total number of granule cells, hilar neurons and the total number of neurons expressing the message for somatostatin (SS-mRNA) were estimated with stereological methods in five rats sacrificed 24 h after the last seizure and compared with estimates made in five control animals. The mean total number of granule cells was 981,000 in control rats and 943,000 in kindled rats, a non-significant difference at the 5% level of significance (P = 0.78, Student's t-test). The mean total number of hilar neurons was 52,100 compared with a mean of 58,520 in the control group, the difference being statistically significant (P = 0.04). The mean total number of SSergic hilar neurons was 16,630 compared with a mean of 15,430 in the control rats (non-significant, P = 0.12). These findings indicate that hilar neurons are lost after kindling but that these neurons are not somatostatinergic.
Animals↗
Carcinogens preferentially bind at methylated CpG in the p53 mutational hot spots.
The major mutational hot spots in human cancers occur at CpG sequences in the p53 gene. It is generally presumed that the majority of mutations at these sites result from the endogenous deamination of methylated cytosine. Using a UvrABC incision method, we have found that cytosine methylation greatly enhances guanine alkylation at all CpG sites in the p53 gene by a variety of carcinogens, including benzo(a)pyrene diol epoxide, benzo(g)chrysene diol epoxide, aflatoxin B1 8,9-epoxide, and N-acetoxy-2-acetylaminofluorene. These findings suggest that mutational hot spots at methylated CpG sequences in the p53 gene may be a consequence of preferential carcinogen binding at these sites.
Acetoxyacetylaminofluorene↗
A novel role for Rab5-GDI in ligand sequestration into clathrin-coated pits.
BACKGROUND: Clathrin-coated pits are formed at the plasma membrane by the assembly of the coat components, namely clathrin and adaptors from the cytosol. Little is known about the regulation and mechanism of this assembly process. RESULTS: We have used an in vitro assay for clathrin-coated pit assembly to identify a novel component required for the invagination of newly formed coated pits. We have purified this cytosolic component and shown it to be a complex of Rab5 and GDI (guanine-nucleotide dissociation inhibitor), that was previously demonstrated to be involved in downstream processing of endocytic vesicles. Using a combination of quantitative electron microscopy and in vitro endocytosis assays, we have demonstrated that although coat proteins and ATP are sufficient to increase the number of new coated pits at the cell surface in permeabilised cells, the Rab5-GDI complex is required for ligand sequestration into clathrin-coated pits. CONCLUSIONS: We have identified Rab5 as a critical cytosolic component required for clathrin-coated pit function. Given the well-established role of Rab5 in the fusion of endocytic vesicles with endosomes, our results suggest that recruitment of essential components of the targeting and fusion machinery is coupled to the formation of functional transport vesicles.
Amino Acid Sequence↗
Surgical complications after conversion from bladder to enteric drainage in pancreaticoduodenal transplantation.
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Anastomosis, Roux-en-Y↗
Mycophenolate mofetil and tacrolimus for induction and maintenance therapy after pancreas transplantation.
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Adult↗
A comparison of two measures to assess adult attachment.
The purpose of this study was to determine the relationship between two different approaches to the assessment of adult attachment. The approaches were C. Hazan and P. R. Shaver's (1987) categorization of three attachment styles and the dimensional system of the Reciprocal Attachment Questionnaire (RAQ; M. West & A. Sheldon-Keller, 1992). Participants were 196 (72 men and 124 women) respondents to a community survey. Multiple regression results showed that the three attachment groups differed significantly on three dimensions of the RAQ: feared loss of the attachment figure, perceived availability of the attachment figure, and use of the attachment figure. In addition, men and women differed significantly on use of the attachment figure, independent of their attachment style. The results indicate that the two approaches to the assessment of adult attachment are interrelated in meaningful ways.
Adult↗