Insights into the mechanism and regulation of pyruvate carboxylase by characterisation of a biotin-deficient mutant of the Bacillus thermodenitrificans enzyme

Pyruvate carboxylase is a biotin-dependent enzyme in which the biotin is carboxylated by a putative carboxyphosphate intermediate that is formed in a reaction between ATP and bicarbonate. The resultant carboxybiotin then transfers its carboxyl group to pyruvate to form oxaloacetate. In the Bacillus thermodenitrificans enzyme the biotin is covalently attached to K1112. A mutant form of the enzyme (K1112A) has been prepared which is not biotinylated. This mutant did not catalyse the complete reaction, but did catalyse ATP-cleavage and the carboxylation of free biotin. Oxaloacetate decarboxylation was not catalysed, even in the presence of free biotin, suggesting that only the biotin carboxylation domain of the enzyme is accessible to free biotin. This mutant allowed the study of ATP-cleavage both coupled and not coupled to biotin carboxylation. Kinetic analyses of these reactions indicate that the major effect of the enzyme activator, acetyl CoA, is to promote the carboxylation of biotin. Acetyl CoA reduces the K(m)s for both MgATP and biotin. In addition, pH profiles of the ATP-cleavage reaction in the presence and absence of free biotin revealed the involvement of several ionisable residues in both ATP-cleavage and biotin carboxylation. K1112A also catalyses the phosphorylation of ADP from carbamoyl phosphate. Stopped-flow studies using the fluorescent ATP analogue, formycin A-5'-triphosphate, in which nucleotide binding to the holoenzyme was compared to K1112A indicated that the presence of biotin enhanced binding. Attempts to trap the putative carboxyphosphate intermediate in K1112A using diazomethane were unsuccessful.     

Potential risk of biologic pollution associated to the introduction of <i>Pinus radiata</i> in grassland areas

Afforestation is a recommended practice to mitigate global warming. However, their implementation may generate undesirable impacts, mostly if exotic species are used. Plantations of Pinus radiata D Don in Ventania (Bs. As., Argentina) soils showed notorious increments of extractable P (Pe), which could affect the dynamic of this element as well as the degree of phosphorus saturation (GSP_Bray). The objectives of this study were: i) to quantify the GSP_Bray in Mollisols afforested with P. radiata comparing the results with those coming from adjacent, natural grassland areas (base line); ii) to evaluate the potential environmental risk induced by afforestation through the identification of a change point (PC) in the GSP_Bray indicative of a phosphate leaching increment. Treatments included mature stands of P. radiata (TB) and adjacent areas with natural grassland vegetation (TP). Samples were taken at 0-15; 15-30 and 30-45 cm soil depth, and texture, pH, total organic carbon (COT), Pe, soluble reactive phosphorus (PSR), phosphorus sorption index (ISP) and GSP_Bray were determined. The results showed a significant acidification in TB and an increase in the COT stock, indicating an additional atmospheric CO₂ sequestration by the trees. The Pe and PSR values were notoriously higher in TB, and they were reflected in a significant increment in the GSP_Bray with respect to TP. The detection of a significant PC in the GSP_Bray-PSR regression indicates higher chances of phosphate leaching in the forest stands, which could reach water courses, lakes and artificial reservoirs promoting their eutrophication. Because of the potential environmental pollution risk of biologic origin derived from the afforestation with P. radiata in Mollisols areas, their inclusion in clean development practices must be reconsidered.     

Regulation of root respiration in two species of Plantago that differ in relative growth rate: the effect of short- and long-term changes in temperature

This study investigates the effect of short‐ and long‐term changesin temperature on the regulation of root respiratory O₂ uptakeby substrate supply, adenylate restriction and/or the capacityof the respiratory system. The species investigated were the lowland Plantagolanceolata L. and alpine Plantago euryphylla Briggs, Carolin& Pulley, which are inherently fast‐ and slow‐growing, respectively. Theplants were grown hydroponically in a controlled environment (constant23 °C). The effect of long‐term exposure to lowtemperature on regulation of respiration was also assessed in P.lanceolata using plants transferred to 15/10 °C(day/night) for 7 d. Exogenous glucose and uncoupler (CCCP)were used to assess the extent to which respiration rates were limitedby substrate supply and adenylates. The results suggest that adenylatesand/or substrate supply exert the greatest control overrespiration at moderate temperatures (e.g. 15–30 °C)in both species. At low temperatures (5–15 °C),CCCP and glucose had little effect on respiration, suggesting thatrespiration was limited by enzyme capacity alone. The Q₁₀ (proportionalincrease of respiration per 10 °C) of respirationwas increased following the addition of CCCP and/or exogenousglucose. The degree of stimulation by CCCP was considerably lowerin P. euryphylla than P. lanceolata. This suggeststhat respiration rates operate much closer to the maximum capacity in P.euryphylla than P. lanceolata. When P. lanceolata wastransferred to 15 °C for 7 d, respirationacclimated to the lower growth temperature (as demonstrated by an increasein respiration rates measured at 25 °C). In addition,the Q₁₀ was higher, and the stimulatory effectof exogenous glucose and CCCP lower, in the cold‐acclimated rootsin comparison with their warm‐grown counterparts. Acclimation of P.lanceolata to different day/night‐time temperatureregimes was also investigated. The low night‐time temperature wasfound to be the most important factor influencing acclimation. The Q₁₀ valueswere also higher in plants exposed to the lowest night‐time temperature.The results demonstrate that short‐ and long‐term changes in temperaturealter the importance of substrate supply, adenylates and capacityof respiratory enzymes in regulating respiratory flux.     

Schistosoma ovuncatum n. sp. (Digenea: Schistosomatidae) from northwest Thailand and the historical biogeography of Southeast Asian Schistosoma Weinland, 1858

Schistosoma sinensium Bao, 1958 was first isolated from an unidentified snail in Sichuan Province, PR China. This species was apparently rediscovered in Chiang Mai Province, northwest Thailand (Baidikul et al., 1984); the definitive host was the rat Rattus rattus and the intermediate host was the snail Tricula bollingi. In this paper S. sinensium is rediscovered in Sichuan Province and compared with worms recovered from experimentally infected mice, which had been exposed to cercariae shed by T. bollingi from Chiang Mai. Evidence is presented suggesting that the schistosome collected by Baidikul was not S. sinensium and that a new species is involved. The new species, named Schistosoma ovuncatum (etymology: ovum (egg) + uncatus (hooked)), is described and compared with related taxa. All previous papers on the Thai schistosome have used worms recovered from field-collected rodents only; this is the first account in which the life-cycle has been completed in the laboratory, using cercariae shed by T. bollingi, and the resulting worms described. S. ovuncatum differs from S. sinensium in terms of size and shape of body and egg, number of testes, size of ovary, length of vitellarium, intermediate host and biogeographical distribution. The relationships of the two taxa and their position with respect to the Schistosoma indicum- and S. japonicum-groups are discussed. The implications of the findings for the evolution of human schistosomiasis in the region are also commented upon.     

Patterns in the benthic communities on the shelf of the sub-Antarctic Prince Edward Islands

Summary Benthic communities at the sub-Antarctic Prince Edward Islands were sampled qualitatively with an epi-benthic sled at 57 stations over a five year period. Additional information on these communities was obtained from replicate underwater photographs. In total, 546 macrofaunal species were found. Despite the extreme isolation and geological youth of the islands, there seems to have been a diverse colonisation. A cluster-analysis based on species abundance aggregated most stations into 8 groups. Each of these groups had a unique depth and substratum combination. Thourella variabilis (Octocoralea), and Cabarea darwinii (Bryozoa) characterised deep rocky assemblages and Serpula vermicularis, Lanice conchilega (Polychaeta) and Magellania kerguelenensis (Brachiopoda) characterised soft substrata. Indicator species separating each of the 8 clusters are given. Trends in percentage cover and densities of major taxa with respect to depth and substratum, as shown by photographs, are described. Deep, rocky substrata were dominated by Porifera, Bryozoa and Cnidaria, while soft-substrata were dominated by Polychaeta, Bivalvia and Brachiopoda. Errant forms consisted predominantly of Echinodermata, with Crustacea being important in shallower habitats. Filter-feeders dominated the benthos of the islands in all habitats.     

SOMAP: a novel interactive approach to multiple protein sequences alignment

A novel interactive method for generating multiple protein sequencealignments is described. The program has no internal limit tothe number or length of sequences it can handle and is designedfor use with DEC VAX processors running the VMS operating system.The approach used is essentially one of manual sequence manipulation,aided by built-in symbolic displays of identities and similarities,and strict and ‘fuzzy’ (ambiguous) pattern-matchingfacilities. Additional flexibility is provided by means of aninterface to a publicly available automatic alignment systemand to a comprehensive sequence analysis package. Received on August 28, 1990; accepted on November 20, 1990