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Biomedical subjects

A H Kingston-Smith

Publications and source records attributed to A H Kingston-Smith.

8 recordsLinked to original sources
PubMed2005

Evidence in support of a role for plant-mediated proteolysis in the rumens of grazing animals.

The present work aimed to differentiate between proteolytic activities of plants and micro-organisms during the incubation of grass in cattle rumens. Freshly cut ryegrass was placed in bags of varying permeability and incubated for 16 h in the rumens of dairy cows that had previously grazed a ryegrass sward, supplemented with 4 kg dairy concentrate daily. Woven polyester bags (50 microm pore size) permitted direct access of the micro-organisms and rumen fluid enzymes to the plant material. The polythene was impermeable even to small molecules such as NH(3). Dialysis tubing excluded micro-organisms and rumen enzymes/metabolites larger than 10 kDa. DM loss was 46.3 % in polyester, 36.2 % in polythene and 38.1 % in dialysis treatments. It is possible that the DM loss within polythene bags occurred due to a solubilisation of plant constituents (e.g. water-soluble carbohydrates) rather than microbial attachment/degradation processes. The final protein content of the herbage residues was not significantly different between treatments. Regardless of bag permeability, over 97 % of the initial protein content was lost during incubations in situ. Electrophoretic separation showed that Rubisco was extensively degraded in herbage residues whereas the membrane-associated, light-harvesting protein remained relatively undegraded. Protease activity was detected in herbage residues and bathing liquids after all incubation in situ treatments. Although rumen fluid contains proteases (possibly of plant and microbial origin), our results suggest that, owing to cell compartmentation, their activity against the proteins of intact plant cells is limited, supporting the view that plant proteases are involved in the degradation of proteins in freshly ingested herbage.

Animal Nutritional Physiological Phenomena↗
PubMed2003

Proteolysis and cell death in clover leaves is induced by grazing.

Programmed plant cell death is a widespread phenomenon resulting in the formation of xylem vessels, dissected leaf forms, and aerenchyma. We demonstrate here that some characteristics of programmed cell death can also be observed during the cellular response to biotic and abiotic stress when plant tissue is ingested by grazing ruminants. Furthermore, the onset and progression of plant cell death processes may influence the proteolytic rate in the rumen. This is important because rapid proteolysis of plant proteins in ruminants is a major cause of the inefficient conversion of plant to animal protein resulting in the release of environmental N pollutants. Although rumen proteolysis is widely believed to be mediated by proteases from rumen microorganisms, proteolysis and cell death occurred concurrently in clover leaves incubated in vitro under rumenlike conditions (maintained anaerobically at 39 degrees C) but in the absence of a rumen microbial population. Under rumenlike conditions, both red and white clover cells showed progressive loss of DNA, but this was only associated with fragmentation in white clover. Cell death was indicated by increased ionic leakage and the appearance of terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labelled nuclei. Foliar protein decreased to 50% of the initial values after 3 h incubation in white clover and after 4 h in red clover, while no decrease was observed in ambient (25 degrees C, aerobic) incubations. In white clover, decreased foliar protein coincided with an increased number of protease isoforms.

Aerobiosis↗
PubMed2000

Overexpression of Mn-superoxide dismutase in maize leaves leads to increased monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities.

The effect of increased Mn-superoxide dismutase (SOD) on antioxidant enzymes and metabolites was studied using transformed maize, TG1+ and TG2+. The progeny of the backcross of each of the primary transformants with the parental line generated two populations denoted M6884 and M6885. These were grown at optimal (25 degrees C) and sub-optimal (18, 14 and 10 degrees C) temperatures to assess the impact of elevated SOD activity on cold tolerance and the antioxidant defences in maize. The plants of the M6885 population had similar foliar SOD activities to the untransformed maize plants. Within the segregating M6884 population 50% of the plants had elevated SOD activity (up to four times the activity of the untransformed controls) and 50% of the plants contained the product of the transgene. In untransformed plants grown at 25 degrees C and 18 degrees C, SOD activity was not detectable in mesophyll extracts. Similarly, increased foliar SOD activity in the M6884 transformed maize did not lead to detectable mesophyll SOD activity. Increased foliar KCN-insensitive SOD activities were accompanied by enhancement of monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities; enzymes which are localized exclusively in the leaf mesophyll tissues. Increased foliar SOD activity had no effect on the hydrogen peroxide, glutathione or ascorbate contents of the leaves. This suggests that increased recycling of reduced ascorbate was required to compensate for enhanced hydrogen peroxide production in transformed plants.

Antioxidants↗
PubMed2000

Bundle sheath proteins are more sensitive to oxidative damage than those of the mesophyll in maize leaves exposed to paraquat or low temperatures.

In maize leaves growth at low temperatures causes decreases in maximum catalytic activities of photosynthetic enzymes and reduced amounts of proteins, rather than effects on regulation or co-ordination of the photosynthetic processes. To test the hypothesis that differential localization of antioxidants between the different types of photosynthetic cell in maize leaves is a major determinant of the extreme sensitivity of maize leaves to chilling damage, oxidative damage to proteins, induced by incubation of maize leaves with paraquat, has been measured and compared with the effects incurred by growth at low temperatures. While the increase in protein carbonyl groups caused by paraquat treatment was much greater than that caused by low temperature growth conditions, most carbonyl groups were detected on bundle sheath proteins in both stress conditions. With one or two exceptions proteins located in the mesophyll tissues were free of protein carbonyl groups in both situations. Paraquat treatment caused a complete loss of the psaA gene products, modified the photosystem II reaction centre polypeptide, D1, and increased the number of peptides arising from breakdown of ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco). In contrast, growth at 15 degrees C increased the abundance (but not number) of Rubisco breakdown products and decreased that of the psaB gene product while the psaA gene product and PEP carboxylase were largely unaffected. Since bundle sheath proteins are more susceptible to oxidative damage than those located in the mesophyll cells, strategies for achieving a more balanced system of antioxidant defence may be effective in improving chilling tolerance in maize.

Adaptation, Physiological↗
PubMed1999

Ascorbate metabolism in potato leaves supplied with exogenous ascorbate.

Photosynthesis and leaf ascorbate were measured in potato (Solanum tuberosum L.) plants grown in low light and then transferred to high light. Total foliar ascorbate content in low light-grown plants was 4.72+/-0.42 micromol mg(-1)chl. Over 80% of the ascorbate pool was found in the reduced form irrespective of position on the stem. No statistically-significant light-dependent effects were observed. Leaf discs supplied with [14C]-ascorbate in the dark showed significant ascorbate uptake such that after a 16h incubation over half of the total ascorbate pool in the discs was labelled [14C]-ascorbate. No ascorbate efflux from the leaves occurred during the period of [14C]-ascorbate uptake. The total amount of ascorbate did not increase, however, implying modified ascorbate turnover. The turnover of the [14C]-ascorbate in the leaves occurred at similar rates in both light and darkness. Little degradation of labelled ascorbate was observed, suggesting that uptake of exogenous ascorbate leads to inhibition of de novo ascorbate biosynthesis in potato leaf discs.

Ascorbic Acid↗
PubMed1999

Evidence of a role for plant proteases in the degradation of herbage proteins in the rumen of grazing cattle.

Protein breakdown in the rumen is generally regarded as a two-stage process in which proteases produced by rumen microorganisms cleave plant protein into peptides and amino acids. However, many of the fiber-degrading cellulolytic species in the rumen are not in fact proteolytic, and the proteolytic activity of the entire rumen microbial population is only moderate when compared to the gastric and pancreatic secretions in the abomasum. Moreover, plant cell walls remain largely intact after initial chewing (particularly in cattle), presenting a physical barrier that must be breached prior to their effective colonization. The present study considers the hypothesis that the plant enzymes are at least partly responsible for herbage protein degradation in grazing ruminants. Ryegrass, red clover, white clover, and bird's-foot trefoil were incubated in the presence and absence of rumen microorganisms. The production of volatile fatty acids indicated the level of microbial activity, whereas the relative disappearance of the large subunit of ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco LSU) indicated proteolytic activity. In all incubations, the relative abundance of the Rubisco LSU decreased as the incubation progressed. When rumen microorganisms were absent, low molecular weight peptides (below 20 kDa) accumulated as the incubation progressed. This accumulation was not observed in the presence of rumen microorganisms. Therefore we suggest that the intrinsic plant proteases contribute to the initial stages of proteolysis of grazed herbage.

Animal Feed↗
PubMed1992

Purification and properties of a phosphatase in French bean (Phaseolus vulgaris L.) leaves that hydrolyses 2'-carboxy-D-arabinitol 1-phosphate.

An enzyme that releases P(i) from 2-carboxy-D-arabinitol 1-phosphate, a naturally occurring tightly binding inhibitor of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), was purified from leaves of French bean seedlings. It was a monomeric protein of M(r) about 56,000. Catalytic activity was stimulated by increased concentrations of inorganic salts to a maximum at an ionic strength above 0.2. NADPH and D-fructose 1,6-bisphosphate increased the activity of the enzyme in both the presence and absence of 0.2 M-KCl. The pure enzyme did not require dithiothreitol for activity. The pH optimum was 7, the Km for 2-carboxy-D-arabinitol 1-phosphate was 0.43 mM and the specific activity 6.8 mumol/min per mg of protein. The enzyme had little or no activity against phosphate ester intermediates of photosynthetic metabolism and glycolysis but hydrolysed the 1,5-bisphosphates of 2'-carboxy-D-ribitol and 2'-carboxy-D-arabinitol more rapidly than 2'-carboxy-D-arabinitol 1-phosphate.

Dithiothreitol↗