Resolution of transducin subunits by chromatography on blue sepharose

The retinal guanine nucleotide-binding protein, transducin (TD), was subjected to chromatography on Blue Sepharose (BLS). A simple two-step protocol was developed, allowing the resolution of the alpha-subunit and the beta gamma-complex of the protein extracted from bovine retina by the use of a poorly hydrolysable GTP analogue. If TD was applied to BLS in a divalent cation-containing buffer, the beta gamma-complex did not bind to the resin, whereas the alpha-subunit was retained; elution of the latter was achieved by removing the divalent cation from the buffer. Binding of the alpha-subunit to BLS was not affected by nucleotides or by ADP ribosylation catalysed by bacterial toxins. However, adsorption of the alpha-subunit by BLS or by a strong cation exchanger (Mono S) depended strictly on divalent cations. In contrast to previous reports, the data suggest the formation of a complex between a sulphonyl residue of Cibacron Blue, a divalent metal ion, and the alpha-subunit as the relevant binding mechanism causing adsorption of the alpha-subunit to BLS

Identification and characterization of the 35-kDa beta subunit of guanine-nucleotide-binding proteins by an antiserum raised against transducin

Antisera were raised against the retinal guanine-nucleotide-binding protein (N-protein), transducin, purified from bovine rod outer segments. Sera obtained after repeated injections of antigen recognized all transducin subunits (alpha, beta and gamma). One antiserum, tested for cross-reactivity with non-retinal N-proteins, was found to cross-react with the beta subunits of the ubiquitously occurring N-proteins, Ns and Ni, but not with their respective alpha and gamma subunits. The antiserum also cross-reacted with the beta subunit of the recently identified N-protein, No, which has been found in high abundance in the central nervous system. These data support the similarity of the beta subunits of the N-proteins identified so far. Purification of N-proteins from porcine cerebral cortex without the use of activating ligands yielded fractions containing the isolated alpha subunit of No, free beta gamma complex, Ni, No and fractions containing both N-proteins in various proportions. The purity of the preparations was at least 80% as judged by Coomassie-blue-stained SDS gels. No pure Ns was obtained. Use of the transducin antibody during the course of the purification revealed that the beta subunits coeluted from a gel filtration column largely with the alpha subunits of Ni and No but were hardly detectable in fractions that were able to reconstitute Ns activity into membranes of an Ns-deficient cell line (S49 cyc- lymphoma cells). This indicates that in the central nervous system the concentrations of Ni and No are of magnitudes higher than that of Ns. Two-dimensional gel electrophoresis of N-proteins, purified from porcine cerebral cortex, resulted in the resolution of two major peptides in the 35-kDa region, which differed in their pI values and were identified as beta subunits by the use of the antiserum. Identical results were achieved using crude cholate extracts from membranes of the same tissue instead of purified proteins. The occurrence of different beta subunits may be explained by posttranslational N-protein modification

Measuring frailty in Dutch community-dwelling older people: reference values of the Tilburg Frailty Indicator (TFI)

Objectives The objectives of this study were to provide reference values of the Tilburg Frailty Indicator (TFI) for community-dwelling older people by age, sex, marital status, ethnicity, education, income, and residence, and examine the effects of these seven socio-demographic variables on frailty. Methods 47,768 individuals aged 65 years and older living in the Netherlands completed a health questionnaire (58.5% response rate), including the TFI. The TFI is a self-report questionnaire for measuring frailty, developed from an integral approach of frailty, including physical, psychological, and social domains. Results Reference values were provided for men and women separately, as a function of age. We found associations of all socio-demographic variables with frailty, also after controlling for the effects of age. These associations held for both sexes and for big cities as wells as more rural areas. For instance, the effect of age was large for total and physical frailty, women were more frail than men, and some very large average frailty differences between the ethnic groups were found, with autochthon people having the lowest frailty score. Conclusions In conclusion, this study offers reference values of the TFI by socio-demographic characteristics and explains frailty using these characteristics. This information will support researchers, policymakers and health care professionals in interpreting scores of the TFI, which may guide their efforts to reduce frailty and its adverse outcomes

Measuring frailty in Dutch community-dwelling older people: reference values of the Tilburg Frailty Indicator (TFI)

Objectives The objectives of this study were to provide reference values of the Tilburg Frailty Indicator (TFI) for community-dwelling older people by age, sex, marital status, ethnicity, education, income, and residence, and examine the effects of these seven socio-demographic variables on frailty. Methods 47,768 individuals aged 65 years and older living in the Netherlands completed a health questionnaire (58.5% response rate), including the TFI. The TFI is a self-report questionnaire for measuring frailty, developed from an integral approach of frailty, including physical, psychological, and social domains. Results Reference values were provided for men and women separately, as a function of age. We found associations of all socio-demographic variables with frailty, also after controlling for the effects of age. These associations held for both sexes and for big cities as wells as more rural areas. For instance, the effect of age was large for total and physical frailty, women were more frail than men, and some very large average frailty differences between the ethnic groups were found, with autochthon people having the lowest frailty score. Conclusions In conclusion, this study offers reference values of the TFI by socio-demographic characteristics and explains frailty using these characteristics. This information will support researchers, policymakers and health care professionals in interpreting scores of the TFI, which may guide their efforts to reduce frailty and its adverse outcomes

Methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms resulting in suboptimal oocyte maturation: a discussion of folate status, neural tube defects, schizophrenia, and vasculopathy.

Contains fulltext : 69270.pdf (publisher's version ) (Open Access)ABSTRACT: Several conditions apparent at birth, e.g., neural tube defects (NTDs) and cardiac anomalies, are associated with polymorphisms in folate-related genes, such as the 677C --> T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene. Similar associations have been established for several constitutional chronic diseases in adulthood, such as schizophrenia, cardiovascular diseases, dementia, and even neoplasias in different organ systems. This spectrum of developmental anomalies and constitutional diseases may be linked to high-risk conceptions related to preovulatory overripeness ovopathy (PrOO). Some developmental anomalies, such as NTDs, are to a large extent prevented by supplementation of folic acid before conception, but supplementation does not seem to prevent cardiovascular disease or cognitive decline. These diverging results can be elucidated by introduction of the PrOO concept, as MTHFR polymorphisms and inherent low folate levels induce both non-optimal maturation of the oocyte and unsuccessful DNA methylation and demethylation, i.e. epigenetic mutations. The PrOO concept is testable and predicts in a random population the following: (1) female carriers of specific genetic MTHFR variants exhibit more ovulatory disturbances and inherent subfecundity traits, (2) descendents from a carrier mother, when compared with those from a wild-type mother, are more frequently conceived in PrOO high-risk conditions and, thus, (3) disadvantaged in life expectancy. If so, some MTHFR polymorphisms represent a novel, genetically determined, PrOO high-risk conception category comparable to those which are environmentally and behaviorly influenced. These high-risk conditions may cause developmental anomalies and defective epigenetic reprogramming in progeny. The interaction between genetic and environmental factors is a plausible mechanism of multifactorial inheritance