Cloning and Functional Analysis of an Allene Oxide Synthase in Physcomitrella patens

Jasmonic acid (JA) is a plant hormone that plays important roles in a large number of processes in stress adaptation and development in flowering plants. A search of genome database indicated the existence of allene oxide synthase (AOS), an enzyme of JA biosynthesis, in Physcomitrella patens, a model plant among mosses. In this study, the presence of JA was detected in P. patens. The recombinant AOS of P. patens, which was overexpressed in Escherichia coli, showed AOS activity. These data suggest that the octadecanoid pathway also exists in P. patens.     

A tailed primers protocol to identify the association of eNOS gene variable number of tandem repeats polymorphism with ischemic stroke in Chinese Han population by capillary electrophoresis

Endothelial nitric oxide synthase (eNOS) plays an important role in mediating endothelium-dependent vasodilatation and antithrombotic action and is thus involved in the development of ischemic stroke (IS). Controversial results regarding the association of eNOS gene variable number of tandem repeats (VNTR) polymorphism with IS have been reported by conventional PCR-polyacrylamide gel electrophoresis methods. We aimed to identify any common association of eNOS gene VNTR polymorphism with IS in Chinese Han population by capillary electrophoresis (CE). The VNTR polymorphism of 27 bp within the eNOS intron-4 was determined by CE with specially designed tailed primers in Chinese Han patients with IS (n = 457) and matched elderly controls without IS (n = 457). Significant differences in BMI, WHR, hypertension, diabetes, smoking, TG, HDL, LDL, LDL, and FBG were observed between cases and controls. The distributions of eNOS VNTR polymorphism were not significantly associated with IS after adjustment for cardiovascular risk factors (OR = 1.18, 95% CI: 0.82–1.69). This finding was consistent with the further meta-analysis in Asians. The meta-analysis in Americans demonstrated that 4a/4b + 4a/4a genotype was significantly associated with IS risk with an OR of 1.54 (95% CI, 1.09–2.17) compared with the 4b/4b genotype. Our data suggests that BMI, WHR, hypertension, diabetes, smoking, TG, LDL, and FBG may increase the risk of IS. However, eNOS VNTR polymorphism may be not an independent major contributor for IS in Chinese Han population. The VNTR polymorphism might be associated with IS in Americans based on meta-analysis.     

Mitochondria: Role of citrulline and arginine supplementation in MELAS syndrome

Mitochondria are found in all nucleated human cells and generate most of the cellular energy. Mitochondrial disorders result from dysfunctional mitochondria that are unable to generate sufficient ATP to meet the energy needs of various organs. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a frequent maternally inherited mitochondrial disorder. There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS syndrome and results in impaired blood perfusion that contributes significantly to several complications including stroke-like episodes, myopathy, and lactic acidosis. Both arginine and citrulline act as NO precursors and their administration results in increased NO production and hence can potentially have therapeutic utility in MELAS syndrome. Citrulline raises NO production to a greater extent than arginine, therefore, citrulline may have a better therapeutic effect. Controlled studies assessing the effects of arginine or citrulline supplementation on different clinical aspects of MELAS syndrome are needed.     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.     

Leaf flavonoids of Amborella trichopoda

The leaf flavonoids of Amborella trichopoda were examined and two kaempterol glycosides were detected. Procyanidin was also present. These results are similar to the flavonoid pattern in other families of the Laurales and it is suggested that simple flavonol glycosides are a primitive feature in the order.     

Distribution of oxoacyl synthase homology sequences within Streptomyces DNA

Abstract A genomic DNA sequence of Streptomyces strain ISP 5485 was cloned, sequenced and compared with corresponding information from nucleic acid data banks. The DNA sequence was unique, but showed homology to DNA coding for the condensing enzyme, 2-oxoacyl synthase, of the deoxyerythronolide B synthase complex (DEBS) from Saccharopolyspora erythraea NRRL 2338. A subfragment of the sequenced DNA was used to construct a gene-specific probe that formed part of the putative 2-oxoacyl synthase gene. The PCR-amplified and labelled probe was used in hybridization experiments involving 33 streptomycete strains that produced different classes of antibiotics. The probe showed widespread homology with DNA considered to be part of analogous genes within genomes of different polyketide producers. The implications of the probe homology to bacterial chromosomal DNA are discussed.     

Glycogen metabolism in rat liver during transition form the fed to fasted states

Hepatic glycogen metabolism was studied in rats during the period of transition from the fed to fasted states. Glycogenic activity was measured in vivo based on the incorporation of [¹⁴C]glucose into liver glycogen. Its changes were almost parallel to the changes in glucogen synthase activity. Progressive accumulation of liver glycogen that occurred in the fed state was associated with a proportional increase in glycogenic activity. Within 4 h after the cessation of food intake, glycogenic activity showd a precipitous fall from the peak to its nadir without significant changes in glycogen content. Meanwhile, the glucose concentration in the portal vein decreased. Upon further development of fasting, glycogenic activity displayed a progressive regain, reciprocally as glycogen contents gradually decreased. The precipitous fall of glycogenic activity during the transition from the fed to fasted states was associated with a transient increase in plasma glucagon, and was partly overcome by the injection of anti-glucagon serum. It is concluded that the fall of portal venous concentration of glucose and secretion of glucagon act as a signal to initiate liver glycogen metabolism characteristics of the fasted or postabsorptive state.     

Field investigations of ammonia exchange between barley plants and the atmosphere. II. Nitrogen realiocation, free ammonium content and activities of ammonium-assimilating enzymes in different leaves

The activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) in different leaves of field-grown spring barley were measured during the reproductive growth phase in 2 consecutive years. Concurrently, the contents of soluble ammonium ions and free amides in the leaves were determined. The studies were carried out to investigate the relationship between variations in these parameters and emission of NH3 from the plant foliage. GS and GOGAT activities declined very rapidly with leafage. The decline in enzyme activities was followed by an increase in soluble ammonium ions and amides in the leaf tissues. During the same period, about 75% of leaf and stem nitrogen was reallocated to the developing ear. The amount of NH₃ volatilized from the foliage during the reproductive growth phase amounted to about 1% of the reallocated nitrogen. The experimental years were characterized by very favourable conditions for grain dry matter formation and for re-utilization of nitrogen mobilized from leaves and stems. Ammonia volatilization occurring under conditions with declining GS and GOGAT activities and increasing tissue concentrations of NH₄⁺ may be useful in protecting the plant from accumulation of toxic NH₃ and NH₄⁺ concentrations in the tissues.     

Asparagine-84, a regulatory allosteric site residue,helps maintain the quaternary structure of Campylobacter jejuni dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography.