Genetic Diversity of <Emphasis Type="Italic">Hevea</Emphasis> IRRDB’81 Collection Assessed by RAPD Markers

The majority of Hevea (Hevea brasiliensis Muell. Arg.) genetic resource in Vietnam derived from the IRRDB’81 germplasm collected in the Amazonian habitats of the genus. Random amplified polymorphic DNA (RAPD) analysis was used to examine the genetic diversity and structure of the IRRDB’81 germplasm. A total of 59 accessions from 13 different districts of the Brazilian states namely Acre, Rondonia, and Mato Grosso were brought into the study using six arbitrarily preselected primers. Sixty-five RAPD band patterns ranging in size from 0.2 to 3.0 kbp were scored for analysis. Differences in the level of DNA polymorphism among the districts and states were revealed. The percentage of the polymorphic DNA fragments calculated for 13 individual districts varied from 15.38 to 70.77%. The mean values of heterozygosity within the district varied from 0.064 to 0.264. Pairwise district Nei’s genetic distance values ranged from 0.046 for Catriquacu and Itanba of Mato Grosso to 0.304 for Tarauaca of Acre and Aracatuba of Mato Grosso. The estimated values of Shannon’s diversity index ranged from 0.093 for the Assis-Brasil district of Acre to 0.389 for the Jiparana district of Rondonia. The analysis of molecular variance (AMOVA) indicated that most of the genetic variations were found among accessions within the districts, while interdistrict variance component accounted for 14.1% only. The low interdistrict differentiation probably implied an extensive gene flow among them. Both the principal coordinate analysis and UPGMA cluster analysis based on genetic distance values revealed a varying degree of separation among the districts and that conformed to geographical origins of Hevea IRRDB’81 collection.     

A predominant β-CGTase G1 engineered to elucidate the relationship between protein structure and product specificity

Low reaction yields and the high cost of obtaining a single type of pure CD make γ-CD costly. Using rational design and with the aid of 3D modeling structures, recombinant CGTase from Bacillus sp. G1 was molecularly engineered with the aim of producing a higher percentage of γ-CD. A single mutation at subsite −3, denoted H43T, was found to increase γ-CD production from 10% to approximately 39% using tapioca starch. This novel increment was probably the result of reduced steric hindrance to the formation of γ-CD because of the shortened side chain together with the shortened loop at positions 86–89, at substrate-binding subsite −3. A mutation (Tyr188 → Trp) and a deletion at loop 139–144 showed little effect on product specificity; however, mutagenesis at these sites affected cyclization, coupling and hydrolysis activities as well as the kinetic properties of the mutant CGTase. Based on rational design, three further mutations of the mutant H43T (denoted H43T/Δ(139–144)/S134T/A137V/L138D/V139I, H43T/S85G and H43T/Y87F) were constructed and produced γ-CD with yields of 20%, 20% and 39%, respectively. The mutant H43T/Δ(139–144)/S134T/A137V/L138D/V139I had very low cyclization and coupling activities, however their hydrolysis activity was retained. Double mutation (H43T/S85G) caused the enzyme to exhibit higher starch hydrolysis activity, approximately 26 times higher than the native CGTase G1. Although the mutants H43T and H43T/Y87F could produce the same percentage (39%) of γ-CD, the latter was more efficient as the total amount of CD produced was higher based on the V_max and k_cat values.     

Interaction between the macrophyte <Emphasis Type="Italic">Stratiotes aloides</Emphasis> and filamentous algae: does it indicate allelopathy?

The aquatic macrophyte Stratiotes aloides Linnaeus, which has recently received attention in studies on allelopathy, has been shown to suppress phytoplankton growth. In the Netherlands, S. aloides often co-occurs with floating filamentous algae. However, filamentous algae are generally absent in close proximity to S. aloides, resulting in gaps in filamentous algae mats. We analyzed whether those gaps may be caused by allelopathic substances excreted by S. aloides or by nutrient depletion. We studied in a field survey the colonization of natural S. aloides by filamentous algae and determined in situ nutrient concentrations in natural S. aloides stands. To analyze the relative importance of allelopathy and nutrient competition in the interaction between S. aloides and filamentous algae, we carried out field experiments. Introduction of artificial (non-allelopathic) plants in natural S. aloides stands enabled us to compare the colonization by filamentous algae of both Stratiotes sp. and artificial plants. The filamentous algae were absent in close vicinity to S. aloides. Significantly lower concentrations of ortho-phosphate and potassium were observed close to S. aloides as compared with the filamentous algae. In the field experiments the artificial plants were rapidly colonized by filamentous algae, mainly Cladophera Kützing and Spirogyra Link, while all natural plants remained free of such algae. Additionally, most nutrient concentrations did not significantly differ in the proximity of artificial or natural stands of S. aloides. The concentrations of the major growth-limiting nutrients, phosphate and nitrate, were significantly higher and nonlimiting in natural Stratiotes stands. Our main conclusion is that, although allelopathic interactions between S. aloides and filamentous algae do occur under natural conditions, nutrient competition between the two can also be an important factor.     

Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5α,13α-diacetoxy-9α,10β-diol to generate taxadien-5α,10β,13α-tri-acetoxy-9α-ol and taxadien-5α,9α,13α-triacetoxy-10β-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5α- or the 13α-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.     

Interactive Effects of Three Ecosystem Engineers on Infiltration in a Semi-Arid Mediterranean Grassland

The redistribution of water in semi-arid environments is critical for the maintenance and survival of vegetation patches. We used a systems approach to examine the interactive effects of three engineers—Stipa tenacissima, biological soil crusts, and the European rabbit (Oryctolagus cuniculus)—on infiltration processes in a model gypseous semi-arid Mediterranean grassland. We measured the early (sorptivity) and later (steady-state infiltration) stages of infiltration at two supply potentials using disk permeameters, which allowed us to determine the relative effects of different engineers and soil micropores on water flow through large macropores. We detected few effects under tension when flow was restricted to matrix pores, but under ponding, sorptivity and steady-state infiltration adjacent to Stipa tussocks were 2–3 times higher than in intact or rabbit-disturbed biological soil crusts. Structural Equation Modeling (SEM) showed that both Stipa and biological soil crust cover exerted substantial and equal positive effects on infiltration under ponding, whereas indirectly, rabbit disturbance negatively affected infiltration by reducing crust cover. Under tension, when macropores were prevented from conducting water, Stipa had a direct negative effect and biological soil crust cover was relatively unimportant. More complex SEM models demonstrated that (1) Stipa primarily influenced biological soil crusts by reducing their richness, (2) rabbits exerted a small negative effect on crust richness, and (3) lichens were negatively, and mosses positively, correlated with a derived “infiltration” axis. Our results highlight the importance of biological soil crusts as key players in the maintenance of infiltration processes in Stipa grasslands, and demonstrate the modulating role played by rabbits through their surface disturbances.     

Multiligand specificity of pathogen-associated molecular pattern-binding site in peptidoglycan recognition protein

The peptidoglycan recognition protein PGRP-S is an innate immunity molecule that specifically interacts with microbial peptidoglycans and other pathogen-associated molecular patterns. We report here two structures of the unique tetrameric camel PGRP-S (CPGRP-S) complexed with (i) muramyl dipeptide (MDP) at 2.5 Å resolution and (ii) GlcNAc and β-maltose at 1.7Å resolution. The binding studies carried out using surface plasmon resonance indicated that CPGRP-S binds to MDP with a dissociation constant of 10⁻⁷ m, whereas the binding affinities for GlcNAc and β-maltose separately are in the range of 10⁻⁴ m to 10⁻⁵ m, whereas the dissociation constant for the mixture of GlcNAc and maltose was estimated to be 10⁻⁶ m. The data from bacterial suspension culture experiments showed a significant inhibition of the growth of Staphylococcus aureus cells when CPGRP-S was added to culture medium. The ELISA experiment showed that the amount of MDP-induced production of TNF-α and IL-6 decreased considerably after the introduction of CPGRP-S. The crystal structure determinations of (i) a binary complex with MDP and (ii) a ternary complex with GlcNAc and β-maltose revealed that MDP, GlcNAc, and β-maltose bound to CPGRP-S in the ligand binding cleft, which is situated at the interface of molecules C and D of the homotetramer formed by four protein molecules A, B, C, and D. In the binary complex, the muramyl moiety of MDP is observed at the C-D interface, whereas the peptide chain protrudes into the center of tetramer. In the ternary complex, GlcNAc and β-maltose occupy distinct non-overlapping positions belonging to different subsites.     

Management of Spontaneously Ruptured Hepatocellular Carcinomas in the Radiofrequency Ablation Era

Background and aim

Spontaneous rupture of hepatocellular carcinoma (HCC) carries a high mortality. The use of radiofrequency ablation (RFA) in recent years has enriched the armamentarium for hemostasis of spontaneously ruptured HCCs but its results have not been documented. This study investigated the prognosis and outcome of spontaneous rupture of HCC as well as the results of using RFA for hemostasis.

Patients and method

From January 1991 to December 2010, 5283 patients were diagnosed with HCC at our hospital, and 189 of them had spontaneous rupture of HCCs. They were grouped under two periods: period 1, 1991–2000, n = 70; period 2, 2001–2010, n = 119. RFA was available in period 2 only.

Results

Hepatitis B virus infection was predominant in both periods. Surgical hemostasis was mainly achieved by hepatic artery ligation in period 1 and by RFA in period 2. The 30-day hospital mortality after surgical treatment was 55.6% (n = 18) in period 1 and 19.2% (n = 26) in period 2 (p = 0.012). Multivariate analysis identified 4 independent factors for better overall survival, namely, hemostasis by transarterial chemoembolization (hazard ratio 0.516, 95% confidence interval 0.354–0.751), hemostasis by RFA (hazard ratio 0.431, 95% confidence interval 0.236–0.790), having surgery as a subsequent treatment (hazard ratio 0.305, 95% confidence interval 0.186–0.498), and a serum total bilirubin level <19 umol/L (hazard ratio 1.596, 95% confidence interval 1.137–2.241).

Conclusion

The use of RFA for hemostasis during laparotomy greatly reduced the hospital mortality rate when compared with conventional hepatic artery ligation.     

Folate Deficiency Triggers an Oxidative-Nitrosative Stress-Mediated Apoptotic Cell Death and Impedes Insulin Biosynthesis in RINm5F Pancreatic Islet β–Cells: Relevant to the Pathogenesis of Diabetes

It has been postulated that folic acid (folate) deficiency (FD) may be a risk factor for the pathogenesis of a variety of oxidative stress-triggered chronic degenerative diseases including diabetes, however, the direct evidence to lend support to this hypothesis is scanty. For this reason, we set out to study if FD can trigger the apoptotic events in an insulin-producing pancreatic RINm5F islet β cells. When these cells were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature proceeding through a mitochondria-dependent pathway. In addition to evoke oxidative stress, FD condition could also trigger nitrosative stress through a NF-κB-dependent iNOS-mediated overproduction of nitric oxide (NO). The latter compound could then trigger depletion of endoplasmic reticulum (ER) calcium (Ca²⁺) store leading to cytosolic Ca²⁺ overload and caused ER stress as evidence by the activation of CHOP expression. Furthermore, FD-induced apoptosis of RINm5F cells was found to be correlated with a time-dependent depletion of intracellular gluthathione (GSH) and a severe down-regulation of Bcl-2 expression. Along the same vein, we also demonstrated that FD could severely impede RINm5F cells to synthesize insulin and their abilities to secret insulin in response to glucose stimulation were appreciably hampered. Even more importantly, we found that folate replenishment could not restore the ability of RINm5F cells to resynthesize insulin. Taken together, our data provide strong evidence to support the hypothesis that FD is a legitimate risk factor for the pathogenesis of diabetes.