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M P Richards

Publications and source records attributed to M P Richards.

At least 19 recordsLinked to original sources
PubMed1995

Comparison of different techniques for the analysis of metallothionein isoforms by capillary electrophoresis.

We have investigated free-solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC) separations of metallothionein (MT) isoforms conducted in uncoated and surface-modified fused-silica capillaries. At alkaline pH, FSCE rapidly resolves isoforms belonging to the MT-1 and MT-2 charge classes. At acidic pH, additional resolution of MT isoforms is achieved. The use of high-ionic-strength (0.5 M) phosphate buffers can result in high peak efficiencies and increased resolution for some MT isoforms. Interior capillary surface coatings such as polyamine and linear polyacrylamide polymers permit separation of MT isoforms with enhanced resolution through their effects on electroosmotic flow (EOF) and protein-wall interactions. Improvements in MT isoform resolution can also be achieved by MECC using 100 mM borate buffer pH 8.4 containing 75 mM SDS. Deproteinization of tissue cytosol samples with acetonitrile (60-80%) or perchloric acid (7%) produces extracts that can be subjected to direct analysis of MT by FSCE or MECC. We conclude that optimal separation of MT isoforms by capillary electrophoresis (CE) can be achieved with the appropriate combination of different capillaries, buffers and sample preparation techniques.

Animals↗
PubMed1995

Analysis of metallothionein isoforms by capillary electrophoresis: optimisation of protein separation conditions using micellar electrokinetic capillary chromatography.

Capillary electrophoresis (CE) techniques have been successfully applied to the separation of metallothionein (MT) isoforms and have proved to be rapid, practical and economical. Study of a variety of different electrolytes and capillaries has shown that electrolyte buffer composition and capillary wall surface modifications can have considerable influence on isoform separation and resolution. Ionic surfactants such as sodium dodecyl sulphate (SDS) form micelles at elevated concentrations and the partitioning of molecules between the hydrophobic micelle phase and the aqueous phase and their resulting migration in an electric field is the basis of the technique known as micellar electrokinetic capillary chromatography (MECC). In the present work, we have used sheep and rabbit MT to optimise MECC conditions for analysis of MT isoforms. Capillaries of 57 cm gave much better separations than shorter columns although analysis times were increased to about 12 min. Changing the buffer and SDS concentration or the pH affected the selectivity of isoform separation and up to 5 isoforms in sheep MT and 6 in rabbit MT were completely or partially resolved. Comparing different diameter capillaries we conclude that 25 microns I.D. columns give better separations than 50 or 75 microns I.D. columns although sensitivity is reduced by a factor of about 3 and 5, respectively. Using our MECC conditions, columns coated with C1 or C18 hydrophobic material were not found to be useful in improving MT separation or resolution although further evaluation of these columns is in progress.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The use of solid phase concentrators for on-line preconcentration of metallothionein prior to isoform separation by capillary zone electrophoresis.

UV absorbance is a convenient detection method for monitoring a wide variety of different components separated by capillary electrophoresis (CE). However, a significant disadvantage of UV detection is its low sensitivity and this is a major factor limiting the application of CE as an analytical technique. In order to improve sensitivity, several methods of on-line sample preconcentration by electrofocusing, including "stacking" and isotachophoresis, have been investigated and the potential of on-line solid phase preconcentrators for this purpose has also been demonstrated. We have developed methods for the separation of isoforms of metallothionein (MT), a low M(r) metal-binding protein which functions as a detoxification system and cellular buffer for heavy metals, primarily in liver and kidney. While a variety of modifications to the capillary surface and electrolyte chemistry have been used to resolve many MT isoforms in liver samples from a variety of species, there are few available alternatives to UV detection and thus sensitivity limits the application of these methods to samples with low MT levels, such as urine or plasma. In this study we have investigated the use of an inexpensive and easy to assemble C18 concentrator which was fitted to the capillary inlet to extend the detection limits for MT isoform analysis. Samples were pressure loaded onto the concentrator for up to 5 min, eluted using 33% acetonitrile and then subjected to electrophoresis in borate or phosphate buffer. The purified isoforms MT-1 and MT-2 from rabbit liver were best resolved using an acidic acetonitrile eluent and a phosphate buffer electrolyte at pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Application of a polyamine-coated capillary to the separation of metallothionein isoforms by capillary zone electrophoresis.

In this study a fused-silica capillary treated internally with a polyamine coating which reverses electroosmotic flow in the direction of the anode was evaluated for its ability to resolve metallothionein (MT) isoforms. Analysis of different MTs purified from liver and kidney tissue revealed the following numbers of putative isoform peaks resolved: rabbit (3-6); horse (3-5); rat (2-3), chicken (1); human MT-1 (5-6); sheep (4-5) and pig (4-5). The greater degree of MT isoform heterogeneity detected in this study using the polyamine-coated capillary suggested a higher resolving capacity for capillary zone electrophoresis conducted with this capillary compared to an uncoated one. Using the single isoform of chicken MT (cMT) as a reference standard, relative standard deviations of 2.53, 1.85 and 2.21% for peak migration time, area and height, respectively, were observed for eight consecutive runs. A standard curve for cMT established linearity (r2 = 0.99) for integrated peak area over three log units of cMT concentration with a lower limit of detection estimated to be < or = 5 micrograms/ml. Acetonitrile extracts of chick liver tissue homogenates were successfully analyzed for the presence of MT isoforms from both control and zinc-injected animals. Based on our initial evaluation, capillary zone electrophoresis using the polyamine-coated capillary appears to be a very useful analytical method for the separation and quantification of individual MT isoforms.

Animals↗
PubMed1994

Calcium homeostasis in in ovo and ex ovo turkey embryos.

Calcium homeostasis of in ovo (normal) and ex ovo (shell-less) turkey embryos was investigated at 15, 18, and 21 days of incubation. Hypocalcemia and an elevation in circulating 1,25(OH)2D3 in ex ovo embryos were observed by 15 days. Calcium and phosphorus concentrations in femora and tibiae in ex ovo embryos were significantly lower compared to their normal counterparts. These results suggest that shell calcium mobilization is required prior to 15 days of incubation for maintaining serum calcium and supporting bone mineralization. Furthermore, the elevation of 1,25(OH)2D3 is indicative of a functional calcium homeostatic mechanism responding to the absence of the primary calcium source (eggshell) during the second half of turkey embryonic development.

Animals↗
PubMed1991

Mineral metabolism in the developing turkey embryo--I. The effects of developmental age and shell-less culture on trace element contents of selected tissues.

1. Turkey embryos were incubated in ovo or in long-term shell-less culture (ex ovo) for 14, 18, 22 or 26 days. The embryos incubated ex ovo exhibited a progressive decline in the rate of growth and were hypocalcemic and hypoproteinemic compared to their in ovo counterparts from day 18 to day 26 of incubation. 2. The ratio of the concentrations of alpha-fetoprotein and albumin (AFP/A) in serum was determined for both groups of embryos. The AFP/A ratio may be useful as a biochemical index to stage avian embryonic development. Using this index it was concluded that ex ovo embryos exhibited a progressive developmental retardation compared to in ovo embryos. 3. Significant differences were observed in serum trace element concentrations for embryos incubated in ovo vs ex ovo. Most notably, serum copper concentration was significantly lower in ex ovo embryos on days 18 and 22 of incubation and significantly higher on day 26 of incubation compared to serum from embryos incubated in ovo. 4. Livers from embryos incubated ex ovo exhibited significant differences trace element levels compared to those incubated in ovo. By day 26 of incubation the concentration and total amount of zinc and iron were markedly elevated, whereas copper was greatly reduced in the livers of embryos incubated ex ovo compared to the corresponding in ovo levels. 5. Hearts from embryos incubated ex ovo contained less zinc and copper and more iron by day 26 of incubation than those from embryos incubated in ovo.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Mineral metabolism in the developing turkey embryo--II. The role of the yolk sac.

1. Turkey embryos were incubated in ovo or in long-term shell-less culture (ex ovo) for 14, 18, 22 or 26 days, at which time the concentrations of zinc, copper, iron, manganese and calcium in yolk and yolk sac membrane were determined. 2. Yolk manganese and calcium concentrations increased during incubation in ovo while the concentrations of zinc, copper and iron declined. The concentrations of zinc, copper and iron in yolk from ex ovo embryos did not decline. Yolk calcium concentration increased during incubation ex ovo, although to a much lesser extent than that observed in ovo. 3. The concentration of zinc, copper and iron declined in yolk sac tissue during incubation in ovo whereas no decline was observed for yolk sac tissue from ex ovo embryos. Yolk sac calcium and manganese concentrations increased during incubation in ovo and ex ovo, although the increase in calcium concentration for ex ovo yolk sac was much smaller than that observed in ovo. 4. A peak corresponding to metallothionein (MT) which bound both zinc and copper was isolated from yolk sac cytosol on day 14 of incubation in ovo using gel-permeation column chromatography. 5. Further fractionation of the MT peak by anion exchange chromatography revealed three metal-binding peaks designated MT-1, MT-2a and MT-2b. The majority of the zinc was bound to MT-2a and MT-2b whereas most of the copper was bound to a single peak (MT-2b). 6. The concentrations of zinc and copper in yolk sac cytosol reached a maximum on day 14 of incubation in ovo and declined through to day 28 (hatching).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Night waking in infants: effects of providing advice and support for parents.

A randomized controlled study was conducted with three groups of mothers with night waking infants less than 18 mths. Groups received a specially written advice booklet and support visits, the booklet only or no intervention. A fourth group of non-night waking infants acted as a comparison group. Mothers' estimates of waking frequency and infant sleep diaries and maternal well being measures were used to assess change. No significant differences in terms of outcome measures were found between three waking groups. Mothers of night wakers showed higher levels of distress than those in the sleeping group. Parents of wakers were positive about both the support they had received and the booklet.

Community Health Nursing↗
PubMed1989

Characterization of the metal composition of metallothionein isoforms using reversed-phase high-performance liquid chromatography with atomic absorption spectrophotometric detection.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to separate metallothionein (MT) isoforms and on-line atomic absorption spectrophotometric (AAS) detection was used to quantitatively determine their metal content. With this coupled system (HPLC-AAS), it was possible to determine the zinc, cadmium and copper content of individual horse kidney MT isoforms. When rabbit liver MT and the purified isoforms (MT-1 and MT-2) were subjected to RP-HPLC and the zinc-containing peaks of the MT sample to MT-1 or MT-2. HPLC-AAS was used to identify zinc-induced MT in heat-treated cytosol from turkey hen liver, thereby demonstrating its application to the analysis of crude tissue extracts. A standard curve was established using turkey liver MT for the quantitative determination of the zinc content of MT isoforms. There was excellent linear correlation between the micrograms of zinc bound to MT injected onto the column (ranging from 0.34 to 3.43 micrograms of MT-bond zinc) and the integrated peak area of the atomic absorbance for zinc. Using this standard curve, it was possible to quantitate the amount of MT-bound zinc in cytosol extracts of cultured turkey embryo hepatocytes exposed to varying levels of supplemental zinc in the culture medium.

Animals↗
PubMed1989

Influence of egg production on zinc, copper and iron metabolism in the turkey hen (Meleagris gallopavo).

1. Serum concentrations of zinc, copper, iron, calcium and protein increased significantly with the onset of egg production in turkey hens. 2. Hepatic concentrations of zinc, copper and iron declined significantly with the onset of egg production, whereas the total hepatic content of zinc and copper increased significantly. 3. When plasma from laying and nonlaying hens was subjected to gel-permeation column chromatography, a high molecular weight protein fraction was detected in laying but not in nonlaying plasma which bound significant amounts of zinc, copper and iron and which coeluted with purified vitellogenin. 4. Vitellogenin purified from the plasma of laying hens is a metalloprotein which upon analysis was found to contain 0.54, 0.09 and 0.72 micrograms of zinc, copper and iron, respectively, per mg of protein.

Animals↗
PubMed1989

Reflections on the withdrawal of treatment from newborn infants.

The paper reviews the principal arguments that have been used to justify or otherwise the withdrawal of treatment for severely damaged neonates. The present confused legal situation is outlined and also the various considerations that have been put forward as relevant to the process of making decisions. It is argued that these decisions are not technical but are moral and social ones taken in the light of professional and technical expertise. Therefore parents should be involved in them as they have to live with the consequences of what is decided. The legal system does not seem very helpful in resolving present difficulties. At best, it forms a framework within which we can evolve appropriate decision-making procedures.

Decision Making↗
PubMed1989

Recent developments in trace element metabolism and function: role of metallothionein in copper and zinc metabolism.

Metallothionein is a copper- and zinc-binding protein present in most, if not all, tissues of higher eukaryotic species of animals. This review focuses on what is currently known about the characteristics, detection, occurrence, synthesis and turnover of metallothionein, specifically in livestock animals. The function of metallothionein in copper and zinc metabolism, though widely acknowledged, remains controversial. Current evidence in support of a role in the detoxification as well as in the homeostasis of copper and zinc is presented, along with a proposed model summarizing the involvement of metallothionein in basic cellular metabolism of copper and zinc. Also discussed are potential implications of metallothionein in animal agriculture.

Amino Acid Sequence↗
PubMed1989

A diabetic-like condition of turkey embryos maintained in shell-less culture.

Serum insulin concentration and pancreatic insulin content were determined for turkey embryos incubated in ovo and in long-term shell-less culture (ex ovo). Insulin was undetectable (less than 10 pg) in serum from 87% of the ex ovo embryos compared with their in ovo counterparts. This was evident at all incubation ages, although insulin was detectable in more of the ex ovo embryos on Day 24. Insulin increased in the embryos incubated in ovo from 122 (Day 15) to levels exceeding 2000 pg/ml at hatching. Total pancreatic insulin content was greater in the cultured embryos on Days 15, 17, and 22 compared with their in ovo counterparts. Serum glucose was significantly greater (P less than 0.05) in the ex ovo embryos at all ages. In response to an infusion of L-arginine, serum insulin increased from 566 to 1256 pg/ml in the in ovo embryos, whereas no change was evident in the ex ovo embryos (233 vs 257 pg/ml). When embryos incubated in ovo were injected with insulin, a significant (P less than 0.05) reduction of serum glucose was observed at 60 min after injection. Serum glucose concentrations remained elevated in the embryos incubated ex ovo despite the insulin injection. Liver glucose 6-phosphatase activity, assessed on Days 15 and 22 of incubation, was found to be significantly (P less than 0.05) lower in the ex ovo embryos. Turkey embryos incubated in shell-less culture exhibited chronic hyperglycemia in concert with extremely low circulating levels of insulin. The pancreatic beta cells of these embryos were not responsive to arginine or elevated glucose. Taken together these findings suggest the occurrence of a diabetic-like condition in the ex ovo embryos. This defect in insulin secretion may, in part, be responsible for some of the developmental abnormalities characteristic of the turkey embryo cultured ex ovo.

Animals↗
PubMed1988

AIDS in Britain.

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Acquired Immunodeficiency Syndrome↗