Search PubMed⌕ Search

Biomedical subjects

J R Savage

Publications and source records attributed to J R Savage.

At least 19 recordsLinked to original sources
PubMed2001

Modulation of the G protein regulator phosducin by Ca2+/calmodulin-dependent protein kinase II phosphorylation and 14-3-3 protein binding.

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a Gbetagamma-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. Dephosphorylated Pd binds Gbetagamma with high affinity and inhibits the interaction of Gbetagamma with Galpha or other effectors, whereas phosphorylated Pd does not. These results have led to the hypothesis that Pd down-regulates the light response. Consequently, it is important to understand the mechanisms of regulation of Pd phosphorylation. We have previously shown that phosphorylation of Pd by cAMP-dependent protein kinase moderately inhibits its association with Gbetagamma. In this study, we report that Pd was rapidly phosphorylated by Ca(2+)/calmodulin-dependent kinase II, resulting in 100-fold greater inhibition of Gbetagamma binding than cAMP-dependent protein kinase phosphorylation. Furthermore, Pd phosphorylation by Ca(2+)/calmodulin-dependent kinase II at Ser-54 and Ser-73 led to binding of the phosphoserine-binding protein 14-3-3. Importantly, in vivo decreases in Ca(2+) concentration blocked the interaction of Pd with 14-3-3, indicating that Ca(2+) controls the phosphorylation state of Ser-54 and Ser-73 in vivo. These results are consistent with a role for Pd in Ca(2+)-dependent light adaptation processes in photoreceptor cells and also suggest other possible physiological functions.

14-3-3 Proteins↗
PubMed2001

Collaborative exercise on the use of FISH chromosome painting for retrospective biodosimetry of Mayak nuclear-industrial personnel.

PURPOSE: To investigate within the framework of a multilaboratory study the suitability of FISH chromosome painting to measure so-called stable translocations in peripheral lymphocytes of Mayak nuclear-industrial workers (from the Southern Urals) and their use for retrospective biodosimetry. MATERIALS AND METHODS: Chromosime analyses were carried out from 69 workers who had received protracted occupational radiation exposures (0.012-6.065 Gy) up to approximately 40 years before blood sampling. Twenty-one unexposed people living in the same area were controls. A multicolour FISH-painting protocol with the target chromosomes 1, 4 and 8 simultaneously with a pancentromeric probe was used to score potentially transmissible chromosome-type aberrations (reciprocal translocations 2B and related 'one-way' patterns I-III according to the S&S classification). RESULTS: Individual biodosimetry estimates were obtained in terms of these potentially long-term surviving aberration types based on the linear component of a low dose-rate gamma-ray calibration curve produced using identical staining and scoring protocols. For comparison, the workers personal and total background doses were converted to red bone marrow doses. The estimated doses were mainly lower than would be predicted by the calibration curve, particularly at accumulated higher dose levels. CONCLUSIONS: Owing to the limited life-time of circulating T-lymphocytes, the long-term persistence of translocations in vivo requires the assumption of a clonal repopulation of these naturally senescing cells from the haemopoietic stem cell compartments. Obviously such a replacement cannot be fully achieved, leading to a temporal decline even of the yield of transmissible aberrations types. Assuming further a highly selective capacity of stem cells against any type of chromosomal damage and the fact that one must rely on partial genome findings, the potential of FISH chromosome painting for retrospective dose reconstruction is probably limited to a decade or so after high-level protracted radiation exposure.

Adult↗
PubMed2000

Functional roles of the two domains of phosducin and phosducin-like protein.

Phosducin and phosducin-like protein regulate G protein signaling pathways by binding the betagamma subunit complex (Gbetagamma) and blocking Gbetagamma association with Galpha subunits, effector enzymes, or membranes. Both proteins are composed of two structurally independent domains, each constituting approximately half of the molecule. We investigated the functional roles of the two domains of phosducin and phosducin-like protein in binding retinal G(t)betagamma. Kinetic measurements using surface plasmon resonance showed that: 1) phosducin bound G(t)betagamma with a 2. 5-fold greater affinity than phosducin-like protein; 2) phosphorylation of phosducin decreased its affinity by 3-fold, principally as a result of a decrease in k(1); and 3) most of the free energy of binding comes from the N-terminal domain with a lesser contribution from the C-terminal domain. In assays measuring the association of G(t)betagamma with G(t)alpha and light-activated rhodopsin, both N-terminal domains inhibited binding while neither of the C-terminal domains had any effect. In assays measuring membrane binding of G(t)betagamma, both the N- and C-terminal domains inhibited membrane association, but much less effectively than the full-length proteins. This inhibition could only be described by models that included a change in G(t)betagamma to a conformation that did not bind the membrane. These models yielded a free energy change of +1.5 +/- 0.25 kcal/mol for the transition from the G(t)alpha-binding to the Pd-binding conformation of G(t)betagamma.

Animals↗
PubMed2000

Underprediction of visibly complex chromosome aberrations by a recombinational-repair ('one-hit') model.

PURPOSE: Published low-LET FISH data were used to test two models of chromosome aberration production based on breakage-and-reunion or recombinational repair. MATERIALS AND METHODS: Randomness of DNA double strand break induction and misrejoining is analyzed comprehensively and adopted as a working hypothesis. Proximity effects are approximated by using interaction sites. Model results are calculated using CAS (chromosome aberration simulator) Monte Carlo computer software with two adjustable parameters. CAS can emulate the specifics of any experimental painting protocol, allowing very detailed tests of the models. RESULTS: To reasonable approximation, breakage-and-reunion model predictions are consistent with low-LET FISH results, including two large, elaborate, one-paint data sets. An explicitly specified version of the recombinational-repair model severely underpredicts the frequency of the visibly complex aberration patterns most commonly observed with one-paint FISH, and is inconsistent with some observed multi-paint patterns. When high-dose effects (distortion and saturation) are taken into account quantitatively, a dose-response relation for apparently simple interchanges slightly favours the breakage-and-reunion model over the recombinational-repair model, despite being approximately linear over the dose range 2-6 Gy. CONCLUSIONS: The random breakage-and-reunion model gives comprehensive baseline predictions that are sufficiently accurate for the organization of experimental results. The data speak against complex aberrations being formed by the random recombinational repair pathway discussed here.

Chromosome Aberrations↗
PubMed1999

The immunolocalization and divergent roles of phosducin and phosducin-like protein in the retina.

PURPOSE: These investigations were undertaken to compare and contrast the roles of phosducin and phosducin-like protein in the retina. METHODS: Phosducin and phosducin-like protein were compared in an in vitro assay measuring their inhibition of transducin binding to light-activated rhodopsin. The two proteins were localized within the retina by immunoblot analyses and immunocytochemistry using affinity-purified antibodies with high specificity for each of the two homologs. The sensitivity of phosducin-like protein to phosphorylation was probed using in vitro protein kinase reactions. RESULTS: Phosducin and phosducin-like protein were found to have similar, though not identical, transducin inhibiting activity in vitro. These two proteins were found to be localized dissimilarly within the retina, with spatial overlap limited to the inner segments of the photoreceptors. Phosducin is found exclusively in photoreceptor cells, including the synaptic and nuclear layers, while phosducin-like protein is found throughout the inner retinal layers, most abundantly in the bipolar cells of the inner nuclear layer. Phosducin-like protein is not efficiently phosphorylated by the protein kinases tested, indicating that its regulation differs from that of phosducin. CONCLUSIONS: It appears that phosducin and phosducin-like protein play distinct roles in the retina. While phosducin is likely to be important in feedback regulation of the visual signal, such as in light adaptation, phosducin-like protein probably has little if any function in the phototransduction cascade. Phosducin-like protein may have a role in regulating the processing of visual signals by the neural cells of the inner retina.

Animals↗
PubMed1999

A molecular mechanism for the phosphorylation-dependent regulation of heterotrimeric G proteins by phosducin.

Visual signal transduction is a nearly noise-free process that is exquisitely well regulated over a wide dynamic range of light intensity. A key component in dark/light adaptation is phosducin, a phosphorylatable protein that modulates the amount of transducin heterotrimer (Gt alpha beta gamma) available through sequestration of the beta gamma subunits (Gt beta gamma). The structure of the phosphophosducin/Gt beta gamma complex combined with mutational and biophysical analysis provides a stereochemical mechanism for the regulation of the phosducin-Gt beta gamma interaction. Phosphorylation of serine 73 causes an order-to-disorder transition of a 20-residue stretch, including the phosphorylation site, by disrupting a helix-capping motif. This transition disrupts phosducin's interface with Gt beta gamma, leading to the release of unencumbered Gt beta gamma, which reassociates with the membrane and Gt alpha to form a signaling-competent Gt alpha beta gamma heterotrimer.

Animals↗
PubMed1999

Interphase chromosome domain re-organization following irradiation.

PURPOSE: To investigate chromosome domain movements during interphase in stationary cell nuclei. MATERIALS AND METHODS: Contact-inhibited primary human fibroblasts were irradiated with 4.0 Gy X-rays, BrdU was added, and air-dried cell preparations made at intervals up to 48 h. Chromosome 4 domain signals (1, 2 and >2) in BrdU negative nuclei (almost exclusively G0/G1) were counted using FISH. A similar experiment was performed using unstimulated human lymphocytes. RESULTS: A very significant rise in nuclei with >2 signals was found within 1 h after radiation. The frequencies observed were in very good agreement with those expected for simple and complex interchanges involving chromosome 4, scored at metaphase in these materials. CONCLUSIONS: The observations constitute evidence for significant domain movement and re-organization within a short time of radiation exposure in G0/G1 interphase nuclei, presumably induced by the formation of inter-domain exchanges. Such re-organization must be a very complicated and delicate topological problem for relaxed chromatin, and must have an important bearing on the interpretation of mechanistic premature chromosome condensation experiments performed whilst it is in operation.

Cell Line↗
PubMed1999

Clustering of radiation-produced breaks along chromosomes: modelling the effects on chromosome aberrations.

PURPOSE: For high-LET radiations, and perhaps even for hard X-rays, DNA double-strand breaks (dsb) are clustered nonrandomly along chromosomes; disproportionately, many inter-dsb segments are less than a few Mbp (10(6) base pairs). The implications of such dsb clustering for chromosome aberrations are analysed. METHODS: Chromosome segments between different dsb within one dsb cluster are assumed too small to detect in the aberration assay. Enumeration or Monte-Carlo computer simulations are used to compute the relative frequencies of many observable aberration patterns: apparently simple or visibly complex. The theoretical predictions are compared with X-ray data for human fibroblasts, involving painted chromosomes 1, 2, 4, 5, 7 or 13. RESULTS AND CONCLUSIONS: Surprisingly, cryptic dsb multiplicity does not affect the frequency ratios predicted for aberration patterns by a random breakage-and-rejoining model. The model is generally consistent with current data on many different types of aberrations, whether or not dsb usually occur in cryptic clusters. For a Revell-type exchange model, however, the predictions do depend on clustering configurations; they gradually approach the predictions of the breakage-and-rejoining model as average cluster multiplicity increases. The model is consistent with the data, for example with the ratio of visibly complex to apparently simple aberrations, only if there is considerable dsb clustering even at low-LET, with approximately 1.5 or more reactive dsb per cluster on average.

Chromosome Aberrations↗
PubMed1999

X-ray-induced simple, pseudosimple and complex exchanges involving two distinctly painted chromosomes.

PURPOSE: To detect simple, pseudosimple and complex chromosome exchanges in X-ray-induced aberrations involving two distinctly painted chromosomes. Each visibly complex two-paint exchange was analysed to determine the number of breaks and chromosomes necessary to derive the pattern. In addition, the number of associated paint junctions was scored to assess the frequency of non-reciprocal exchanges. MATERIALS AND METHODS: Metaphase spreads were prepared from a human primary fibroblast cell line irradiated with 2, 4 and 6 Gy 250kV X-rays. FISH-painting was performed with distinctly labelled probes for chromosomes 1 and 2, and a pancentromeric probe. RESULTS: From a total of 78 two-paint exchanges observed, 35 were apparently simple, with no additional counterstain chromatin, and 43 were visibly complex with two-colour painting, of which 23 contained at least one pseudosimple exchange. A detailed analysis of the number of two-paint colour junctions showed that at least 50% of the visibly complex exchange patterns involved non-reciprocal exchanges. The simple and complex exchange dose-response curves were considered to be linear and curvilinear respectively. CONCLUSION: The frequency of non-reciprocal rejoining events within complex exchanges is consistent with an interaction model based on the free exchange of multiple break-ends. In addition, the simple and complex exchanges have distinct dose-response curves, in agreement with previous data for single-painted exchanges corrected for pseudosimples.

Chromosome Breakage↗
PubMed1998

An investigation of LET 'finger-prints' in Tradescantia.

Chromosome-type aberration scores, obtained in the Tradescantia microspore system following exposure to radiation, have been extracted from all significant papers since 1940, and augmented with unpublished results from this laboratory. The data include aberrations produced by X-, gamma-rays and neutrons, and cover an enormous range of qualities, doses, dose-rates and ambient gas conditions. Two proposed LET 'finger-print' ratios were examined: Dicentrics/Centric-rings (D/R or F-ratio) and Total deletions/Dicentrics+Centric-rings. There was no significant effect of LET on either of these. Further, D/R was independent of dose, whereas Deletions/D+R showed, as expected, a positive dose effect. In a very small subset of experiments, sizes of interstitial deletions had been recorded, and a significant reduction in modal size was observed for neutrons compared to X-rays. Even if this observation is confirmed by future work, it will not provide a usable 'finger-print' for long-term studies since, being acentric, deletions are rapidly eliminated from a dividing cell population.

Chromosome Aberrations↗
PubMed1998

A brief survey of aberration origin theories.

The salient points of three currently debated theories for chromosomal aberration origins (the Classic Breakage-and-Reunion theory, the Exchange theory, and the Molecular theory) are outlined, and some comments are made on each in the light of recent research.

Chromatids↗
PubMed1998

The naevoid basal-cell carcinoma syndrome (Gorlin syndrome) is a chromosomal instability syndrome.

The Gorlin syndrome, or naevoid basal-cell carcinoma syndrome (NBCS) is an autosomal dominant cancer prone disease (at risk of multiple basal cell carcinomas, and other malignant or benign proliferations). We have previously reported data from peripheral blood lymphocytes of patients with this condition, showing a significant level of spontaneous chromatid and chromosome rearrangements and an overall lengthening of the cell cycle. In this paper, we confirm this disease to be a chromosome instability syndrome from studies on fibroblasts of 5 patients. Spontaneous chromosomal rearrangements, an increased frequency of sister chromatid exchanges and a slowing of the cell cycle were found, compared to age-matched control material. There was also an increased sensitivity to aberration production by mechlorethamine in patient fibroblasts. The chromosome instability we found was not restricted to a given cell lineage, but appears to be part of the general condition of this syndrome. The recently discovered gene responsible for Gorlin syndrome, PTC (or PTCH), encodes a transmembrane protein with yet poorly known functions. However, the demonstration of Gorlin syndrome as a chromosome instability syndrome suggests that this protein has a role in DNA maintenance, repair and/or replication.

Basal Cell Nevus Syndrome↗
PubMed1998

Effectiveness of 0.28 keV carbon K ultrasoft X-rays at producing simple and complex chromosome exchanges in human fibroblasts in vitro detected using FISH.

PURPOSE: To study the effects of carbon K ultrasoft X-rays, which produce a single photoelectron with a track length of < 7 nm, on the production of structural chromosome-type changes. MATERIALS AND METHODS: Untransformed human fibroblasts (HF12) were irradiated in G1 phase. Aberrations were analysed using fluorescence in situ hybridization using multi-coloured chromosome specific DNA probes for chromosomes 1 and 2 and an alpha-satellite pan-centromeric probe. RESULTS: CK X-rays have a high efficiency per unit absorbed dose for producing simple and complex exchanges. Mean absorbed doses of 0.33-1.31 Gy produce simple exchanges with a predominantly linear dose dependency, and visibly complex exchanges increased by more than the power 2 of the dose, with no evidence of a linear component. The proportion of exchanges that are visibly complex ranged from 9% to 46%. CONCLUSIONS: The linear response for simple exchanges provides further support to the hypothesis that damaged DNA may be able to interact with undamaged DNA. The high proportion of complex exchanges may be due to the increased efficiency of double-strand break induction and to the high density of tracks per unit absorbed dose targeting pre-existing sites, some of which may be close to the incident nuclear membrane.

Cell Line↗
PubMed1997

Chromosomal aberrations induced by defined DNA double-strand breaks: the origin of achromatic lesions.

The mechanisms of formation of chromosomal aberrations are poorly understood, despite the common use of aberrations as a measure of the genetic effects of physical and chemical agents. We have used restriction endonucleases to introduce defined DNA double-strand breaks into mammalian cells, and measured chromosomal aberration formation relative to the activity of the endonuclease. The endonucleases AluI and Sau3AI remain active for a relatively short time under simulated cellular conditions and induce achromatic lesions ('gaps') in chromatids only within the first hour or two following treatment. In contrast, the endonuclease MboI (an isoschizomer of Sau3AI) is active for an extremely long time and continues to produce chromatid gaps during the whole 12 hr sampling period. This observation strongly suggests that the aberrations classified as gaps are a manifestation of unrejoined DNA double-strand breaks. The formation of gaps may relate to the opportunities for repair of DNA breaks in relation to cell-cycle position. It is more difficult to relate the formation of structural chromatid aberrations to the endonuclease activity, although at relatively low concentrations all 3 endonucleases gave similar levels of structural aberrations.

Animals↗
PubMed1997

Investigating the nature of chromatid breaks produced by restriction endonucleases.

It is a basic assumption of the breakage-and-reunion theory that the majority of open chromatid breaks seen at metaphase are the residue of unrejoined primary breaks that have neither restituted nor rejoined illegitimately to form exchange aberrations. If Chinese hamster chromosomes with BrdU sister-chromatid differentiation are irradiated, and chromatid aberrations scored from G2 cells, some 15-20% of open breaks show a colour-jump at the point of discontinuity, indicating a two-lesion intrachange origin. Since we see complete forms of several intrachanges whose incomplete forms will also look like breaks, but devoid of a colour-jump, it appears that a substantial proportion of observed breaks are intrachange derived. Experiments to date show that the colour-jump proportion is constant, irrespective of radiation dose, radiation quality, BrdU concentration and hamster cell origin. It is the same for the very low "spontaneous' breaks found in control samples. Restriction endonucleases (RE) can be introduced into cells by various poration methods, and are highly efficient at producing all types of aberrations. This is taken as strong evidence that DNA dsb are significant lesions triggering aberrations. One might anticipate, therefore, that observed breaks will be predominantly unrejoined dsb, and the proportion of colour-jump break correspondingly low. We tested this supposition using three RE; Alu 1, a blunt-end cutter, Sau3A 1, a cohesive-end cutter, both with a short life-time in vivo, and Mbo 1, an isoschizomer of Sau3A 1, which has a long cutting life-time in vivo. Although there were differences in absolute yields of breaks, and of relative frequencies of aberration types recovered, the proportion of colour-jump breaks was as high as that in a parallel X-ray experiment, and fell well within the range encountered in all our previous experiments. It is difficult to reconcile this universal constancy of colour-jump breaks with the expectations of breakage-and-reunion theory, where the occurrence of two-break events must be a treatment variable. Rather, our results suggest that most open breaks are secondary, resulting from a regular intrachange processing mechanism.

Animals↗