Cloning of the amphibolic Calvin cycle/OPPP enzyme d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects

Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T₇ promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E d-ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - PRK phosphoribulokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - FBP fructose-1,6-bisphophatase - FBP fructose 1,6-bisphosphate - G6PDH glucose-6-phosphate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - OPPP oxidative pentose phosphate pathway - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphophate aldolase - IPTG isopropyl -d-thiogalactoside - GST glutathione S-tranferase - PBS phosphate-buffered saline - TPI triosephosphate isomerase     

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: Functional and structural considerations

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca²⁺ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca²⁺ binding site are thereby changed, permitting vectorial dissociation of bound Ca²⁺ against a concentration gradient. A long range linkage between phosphorylation and Ca²⁺ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.     

Formulation and Evaluation of Taste Masked Oral Reconstitutable Suspension of Primaquine Phosphate

The purpose of this research was to mask the intensely bitter taste of primaquine phosphate (PRM) and to formulate suspension powder (cachets) of the taste masked drug. Taste masking was done using beta-cyclodextrin. To characterize and formulate taste masked cachets of PRM, the 1:25 M physical mixture was selected based on bitterness score. Phase solubility studies, fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and X-ray powder diffraction (XRPD) were performed to identify the physicochemical interaction between drug and carrier, hence its effect on dissolution. Cachets were evaluated for angle of repose, sedimentation characterization and pH. In vitro drug release studies for physical mixture and kneaded system were performed at pH, 1.2 and 6.8. Bitterness score was evaluated using gustatory sensation test. Phase solubility studies showed weak interaction between PRM and CD. The FTIR, DSC and XRPD studies indicated inclusion complexation in physical mixture and kneaded system. In addition, kneaded system and physical mixture exhibited better drug release at pH 1.2 and negligible effect at pH 6.8. Cachets prepared using physical mixture, (DS24), showed complete bitter taste masking and easy redispersibility. Taste evaluation of cachets in human volunteers rated tasteless with a score of 0 to DS24 and 3 to DS25. Thus, results conclusively demonstrated successful taste masking and formulation of cachets with taste masked drug.     

Dual regulation of the cardiac L-type calcium channel in L6 cells by protein kinase C

The role of protein kinase C (PKC) in the regulation of cardiac L-type Ca²⁺ channel activity (LCC) was investigated in L6 rat neonatal myoblasts. Depolarization of fura-2 loaded cells with 140 mM KCl activated a Ba²⁺ influx pathway that was blocked by nifedipine and stimulated by (−) Bay K 8644. At least two splice variants of the α_1C subunit of the cardiac LCC were identified by PCR; the α_1S subunit of the skeletal muscle LCC was not detected. Peptides that specifically inhibit translocation of the novel, Ca²⁺-independent δ and PKC isozymes reduced Ba²⁺ influx by 27% and 19%, respectively, whereas a corresponding peptide directed against translocation of classical PKC α had no effect. Ingenol 3,20-dibenzoate, an agent reported to selectively activate novel PKCs, increased Ba²⁺ uptake by 31% while ethanol, a PKC agonist, enhanced uptake by 38%. In contrast, selective activation of classical PKCs with thymeleatoxin or an agonist peptide reduced Ba²⁺ influx by 23–33%. Ba²⁺ influx was reduced by 30–40% when cells were treated with either a PKC inhibitor (Gö 6983, bisindolylmaleimide) or the PKC activator phorbol-12-myristate-13-acetate. We propose that novel, Ca²⁺-insensitive PKC(s) enhance cardiac Ca²⁺ channel activity in L6 cells under basal conditions while activation of the classical, Ca²⁺-sensitive PKC(s) inhibits channel activity. These findings provide the first evidence that different PKC isozymes exert class-specific opposing effects on cardiac L-type Ca²⁺ channel activity in L6 myoblasts.     

Caries clinical trial of a remineralising toothpaste in radiation patients

Objectives: The purpose of this study was to determine the efficacy and safety of a specially formulated remineralising toothpaste in controlling caries in a high‐risk population: head and neck radiation patients. Design: The study compared the performance of the remineralising toothpaste with a conventional fluoride dentifrice using double‐blind randomisation. Materials and methods: Test products: The products compared contained equivalent quantities of fluoride (1100 p.p.m.). The dual‐phase remineralising toothpaste, Enamelon^®, also delivered soluble calcium and phosphate ions, essential components of teeth, from separate phases. Both groups had all caries restored at baseline and used a fluoride rinse daily. Subjects: Fifty‐seven subjects who received radiation to the head and neck causing saliva hypofunction, entered the study, while 44 completed the 10–12 month visit. Measurements: Examinations included coronal and root caries using the Pitts Diagnostic Criteria, salivary flow rate, plaque and gingival indices and microbiological counts over a 1‐year period. Results: The average net increment per year for root caries per subject was 0.04 (±.052) in subjects completing the study using the remineralising toothpaste and 1.65 (±0.51) for root caries in subjects completing the study using the conventional fluoride dentifrice. The difference was statistically significant (p = 0.03), suggesting lower net root surface increment/year for the remineralising toothpaste relative to the conventional toothpaste. No significant differences were noted on coronal surfaces. Conclusion: The results indicate that the remineralising toothpaste provides a significant benefit in preventing and remineralising root caries in high‐risk patients.     

Methylene substitution at the alpha-beta bridging position within the phosphate chain of dUDP profoundly perturbs ligand accommodation into the dUTPase active site

dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species-specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP-analogous compounds will be required as a second step to invoke species-specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, alpha,beta-methylene-dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of alpha,beta-imino-dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:alpha,beta-methylene-dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the "trans" alpha-phosphate conformation of alpha,beta-methylene-dUDP differs from the catalytically competent "gauche" alpha-phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the alpha-phosphorus by more than 3 A. The three-dimensional structures determined in this work show that the binding of the methylene analogue with the alpha-phosphorus in the "gauche" conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of alpha,beta-methylene-dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while alpha,beta-methylene-dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein.     

Structural Basis for Catalysis of a Tetrameric Class IIa Fructose 1,6-Bisphosphate Aldolase from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), currently infects one-third of the world's population in its latent form. The emergence of multidrug-resistant and extensive drug-resistant strains has highlighted the need for new pharmacological targets within M. tuberculosis. The class IIa fructose 1,6-bisphosphate aldolase (FBA) enzyme from M. tuberculosis (MtFBA) has been proposed as one such target since it is upregulated in latent TB. Since the structure of MtFBA has not been determined and there is little information available on its reaction mechanism, we sought to determine the X-ray structure of MtFBA in complex with its substrates. By lowering the pH of the enzyme in the crystalline state, we were able to determine a series of high-resolution X-ray structures of MtFBA bound to dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and fructose 1,6-bisphosphate at 1.5, 2.1, and 1.3 Å, respectively. Through these structures, it was discovered that MtFBA belongs to a novel tetrameric class of type IIa FBAs. The molecular details at the interface of the tetramer revealed important information for better predictability of the quaternary structures among the FBAs based on their primary sequences. These X-ray structures also provide interesting and new details on the reaction mechanism of class II FBAs. Substrates and products were observed in geometries poised for catalysis; in addition, unexpectedly, the hydroxyl-enolate intermediate of dihydroxyacetone phosphate was also captured and resolved structurally. These concise new details offer a better understanding of the reaction mechanisms for FBAs in general and provide a structural basis for inhibitor design efforts aimed at this class of enzymes.     

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+‐associated opening of plasma membrane anion channels

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe‐associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN‐SENSING 2 (FLS2) and EF‐Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode‐impaled mesophyll cells and root hairs of Col‐0 Arabidopsis plants revealed rapid, dose‐dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion‐selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species‐independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2‐associated receptor‐like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1‐dependent, calcium‐associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.     

大鼠脑片中细胞内游离钙动态变化的研究

目的：探索大鼠急性脑片中电刺激诱发的细胞内钙的动态变化规律。方法：采用表面灌流的急性脑片模型,结合电生理和激光共聚焦技术,利用细胞内钙荧光探针进行细胞内游离钙标记,观察电刺激诱发的脑片中神经细胞内游离钙的变化情况。结果：急性脑片组织中,钙标记染料的神经细胞内钙探针荧光强度,电刺激后出现显著增强,且具有波样特征,而Suramin明显抑制此反应,表现为钙探针荧光强度下降和钙反应时间出现延迟,两组之间差异具有统计学意义（P〈0．05）.结论：刺激诱发的大鼠急性脑片中瞬时动态钙信号变化具有一定的时空发生特征,且这种钙信号的时空变化过程可能与嘌呤能信号的作用有关。     

Bismuth citrate in the quantification of inorganic phosphate and its utility in the determination of membrane-bound phosphatases

This paper describes a rapid and sensitive method to determine inorganic phosphate, even in the presence of labile organic phosphate compounds and large quantities of proteins. The method eliminates the use of sodium arsenite, a highly toxic compound, substituting bismuth citrate for it to stabilize the phosphomolybdic acid complex formed during the interaction of inorganic phosphate and molybdate reduced by ascorbic acid. This method has also been adapted to microplates and has been used to determine the activities of Na/K ATPase and alkaline phosphatase of intestinal basolateral and luminal plasma membranes.