Alterations in tocopherol cyclase activity in transgenic and mutant plants of Arabidopsis affect tocopherol content, tocopherol composition, and oxidative stress

Tocopherol belongs to the Vitamin E class of lipid soluble antioxidants that are essential for human nutrition. In plants, tocopherol is synthesized in plastids where it protects membranes from oxidative degradation by reactive oxygen species. Tocopherol cyclase (VTE1) catalyzes the penultimate step of tocopherol synthesis, and an Arabidopsis (Arabidopsis thaliana) mutant deficient in VTE1 (vte1) is totally devoid of tocopherol. Overexpression of VTE1 resulted in an increase in total tocopherol of at least 7-fold in leaves, and a dramatic shift from alpha-tocopherol to gamma-tocopherol. Expression studies demonstrated that indeed VTE1 is a major limiting factor of tocopherol synthesis in leaves. Tocopherol deficiency in vte1 resulted in the increase in ascorbate and glutathione, whereas accumulation of tocopherol in VTE1 overexpressing plants led to a decrease in ascorbate and glutathione. Deficiency in one antioxidant in vte1, vtc1 (ascorbate deficient), or cad2 (glutathione deficient) led to increased oxidative stress and to the concomitant increase in alternative antioxidants. Double mutants of vte1 were generated with vtc1 and cad2. Whereas growth, chlorophyll content, and photosynthetic quantum yield were very similar to wild type in vte1, vtc1, cad2, or vte1vtc1, they were reduced in vte1cad2, indicating that the simultaneous loss of tocopherol and glutathione results in moderate oxidative stress that affects the stability and the efficiency of the photosynthetic apparatus.     

Malaria and urbanization in sub-Saharan Africa

There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria.     

Fasting-based estimates of insulin sensitivity in overweight and obesity: a critical appraisal

Objective: To identify simple methods to estimate the degree of insulin resistance. Research Methods and Procedures: The performance of a wide range of fasting‐based index estimates of insulin sensitivity was compared by receiver operating characteristic analysis (area under curves and their 95% confidence intervals) against the M value from euglycemic insulin clamp studies collected in the San Antonio (non‐Hispanic whites and Hispanic residents of San Antonio, TX) and European Group for the Study of Insulin Resistance (non‐diabetic white Europeans) databases (n = 638). Results: Insulin resistance differed substantially between lean (BMI < 25 kg/m²), overweight or obese (BMI ≥ 25 kg/m²), and type 2 diabetic individuals. Estimates of insulin resistance were, therefore, assessed in each group separately. In the overweight and obese subgroup (n = 302), the receiver operating characteristic performance of fasting‐based indices varied from 0.72 (0.62 to 0.82), in the case of the insulin/glucose ratio, to 0.80 (0.72 to 0.88) in the case of Belfiore free fatty acids. One superior method could not be identified; the confidence intervals overlapped, and no statistically significant differences emerged. All indices performed better when using the whole study population, with fasting plasma insulin, homeostatic model assessment, insulin/glucose ratio, quantitative insulin sensitivity check index, glucose/insulin ratio, Belfiore glycemia, revised quantitative insulin sensitivity check index, McAuley index, and Belfiore free fatty acids showing area under curves of 0.83, 0.90, 0.66, 0.90, 0.66, 0.90, 0.85, 0.83, and 0.86, respectively, because of the inclusion of very insulin sensitive (lean) and very insulin resistant cases (diabetic subjects). Discussion: In conclusion, a superior fasting‐based index estimate to distinguish between the presence and absence of insulin resistance in overweight and obesity could not be identified despite the use of the large datasets.     

Timing of malolactic fermentation inoculation in Shiraz grape must and wine: influence on chemical composition

Malolactic fermentation (MLF) is an integral step in red winemaking, which in addition to deacidifying wine can also influence the composition of volatile fermentation-derived compounds with concomitant affects on wine sensory properties. Long-established winemaking protocols for MLF induction generally involve inoculation of bacteria starter cultures post alcoholic fermentation, however, more recently there has been a trend to introduce bacteria earlier in the fermentation process. For the first time, this study shows the impact of bacterial inoculation on wine quality parameters that define red wine, including wine colour and phenolics, and volatile fermentation-derived compounds. This study investigates the effects of inoculating Shiraz grape must with malolactic bacteria at various stages of alcoholic fermentation [beginning of alcoholic fermentation (co-inoculation, with yeast), mid-alcoholic fermentation, at pressing and post alcoholic fermentation] on the kinetics of MLF and wine chemical composition. Co-inoculation greatly reduced the overall fermentation time by up to 6 weeks, the rate of alcoholic fermentation was not affected by the presence of bacteria and the fermentation-derived wine volatiles profile was distinct from wines produced where bacteria were inoculated late or post alcoholic fermentation. An overall slight decrease in wine colour density observed following MLF was not influenced by the MLF inoculation regime. However, there were differences in anthocyanin and pigmented polymer composition, with co-inoculation exhibiting the most distinct profile. Differences in yeast and bacteria metabolism at various stages in fermentation are proposed as the drivers for differences in volatile chemical composition. This study demonstrates, with an in-depth analysis, that co-inoculation of yeast and bacteria in wine fermentation results in shorter total vinification time and produces sound wines, thus providing the opportunity to stabilise wines more rapidly than traditional inoculation regimes permit and thereby reducing potential for microbial spoilage.     

Deep phylogeographic divergence and cytonuclear discordance in the grasshopper Oedaleus decorus

The grasshopper Oedaleus decorus is a thermophilic insect with a large, mostly south-Palaearctic distribution range, stretching from the Mediterranean regions in Europe to Central-Asia and China. In this study, we analyzed the extent of phylogenetic divergence and the recent evolutionary history of the species based on 274 specimens from 26 localities across the distribution range in Europe. Phylogenetic relationships were determined using sequences of two mitochondrial loci (ctr, ND2) with neighbour-joining and Bayesian methods. Additionally, genetic differentiation was analyzed based on mitochondrial DNA and 11 microsatellite markers using F-statistics, model-free multivariate and model-based Bayesian clustering approaches. Phylogenetic analyses detected consistently two highly divergent, allopatrically distributed lineages within O. decorus. The divergence among these Western and Eastern lineages meeting in the region of the Alps was similar to the divergence of each lineage to the sister species O. asiaticus. Genetic differentiation for ctr was extremely high between Western and Eastern grasshopper populations (F(ct)=0.95). Microsatellite markers detected much lower but nevertheless very significant genetic structure among population samples. The nuclear data also demonstrated a case of cytonuclear discordance because the affiliation with mitochondrial lineages was incongruent in Northern Italy. Taken together these results provide evidence of an ancient separation within Oedaleus and either historical introgression of mtDNA among lineages and/or ongoing sex-specific gene flow in this grasshopper. Our study stresses the importance of multilocus approaches for unravelling the history and status of taxa of uncertain evolutionary divergence.     

Dipeptides as Leaving Group in the Enzyme‐Catalyzed DNA Synthesis

Conjugates of 2′‐deoxyadenosine monophosphate with dipeptides have been synthesized and tested as substrates for several polymerases. Although the incorporation efficiency is not very high, it demonstrates that some of these dipeptides can be accommodated in the active site of polymerases and function as leaving groups in the enzymatic synthesis of DNA.     

Caspase-mediated cleavage of HuR in the cytoplasm contributes to pp32/PHAP-I regulation of apoptosis

The RNA-binding protein HuR affects cell fate by regulating the stability and/or the translation of messenger RNAs that encode cell stress response proteins. In this study, we delineate a novel regulatory mechanism by which HuR contributes to stress-induced cell death. Upon lethal stress, HuR translocates into the cytoplasm by a mechanism involving its association with the apoptosome activator pp32/PHAP-I. Depleting the expression of pp32/PHAP-I by RNA interference reduces both HuR cytoplasmic accumulation and the efficiency of caspase activation. In the cytoplasm, HuR undergoes caspase-mediated cleavage at aspartate 226. This cleavage activity is significantly reduced in the absence of pp32/PHAP-I. Substituting aspartate 226 with an alanine creates a noncleavable isoform of HuR that, when overexpressed, maintains its association with pp32/PHAP-I and delays the apoptotic response. Thus, we propose a model in which HuR association with pp32/PHAP-I and its caspase-mediated cleavage constitutes a regulatory step that contributes to an amplified apoptotic response.     

Purified mouse egg zona pellucida glycoproteins polymerize into homomeric fibrils under non-denaturing conditions

The mouse egg's zona pellucida (ZP) is composed of three glycoproteins, called ZP1, ZP2, and ZP3, that migrate as relatively broad, single bands on SDS-PAGE. The glycoproteins are organized within the ZP as a network of long interconnected fibrils that exhibit a structural periodicity. Here, ZP2 and ZP3 were purified by HPLC to homogeneity and analyzed by Blue Native- (BN-) PAGE and transmission electron microscopy (TEM), as well as by SDS-PAGE. As opposed to SDS-PAGE, BN-PAGE, and TEM permit analysis of ZP2 and ZP3 under non-denaturing conditions. ZP2 and ZP3 migrate on BN-PAGE, not as single bands, but as several discrete oligomers that give rise to larger structures which remain at the origin of the gel. Consistent with this, ZP2 and ZP3 are visualized by TEM as long interconnected fibrils that consist of contiguous beads. Therefore, under non-denaturing conditions both purified ZP2 and ZP3 polymerize into higher order structures. These findings are of interest since purified ZP3 inhibits binding of mouse sperm to eggs and induces sperm to undergo the acrosome reaction in vitro. Results presented here suggest that these biological effects of ZP3 are due to binding of homomeric fibrils of ZP3 to sperm.     

Pentoxifylline reduces fibrin deposition and prolongs survival in neonatal hyperoxic lung injury.

Bronchopulmonary dysplasia is a leading cause of mortality and morbidity in preterm infants despite improved treatment modalities. Pentoxifylline, a phosphodiesterase inhibitor, inhibits multiple processes that lead to neonatal hyperoxic lung injury, including inflammation, coagulation, and edema. Using a preterm rat model, we investigated the effects of pentoxifylline on hyperoxia-induced lung injury and survival. Preterm rat pups were exposed to 100% oxygen and injected subcutaneously with 0.9% saline or 75 mg/kg pentoxifylline twice a day. On day 10, lung tissue was harvested for histology, fibrin deposition, and mRNA expression, and bronchoalveolar lavage fluid was collected for total protein concentration. Pentoxifylline treatment increased mean survival by 3 days (P = 0.0018) and reduced fibrin deposition by 66% (P < 0.001) in lung homogenates compared with untreated hyperoxia-exposed controls. Monocyte chemoattractant protein-1 expression in lung homogenates was decreased, but the expressions of TNF-alpha, IL-6, matrix metalloproteinase-12, tissue factor, and plasminogen activator inhibitor-1 were similar in both groups. Total protein concentration in bronchoalveolar lavage fluid was decreased by 33% (P = 0.029) in the pentoxifylline group. Pentoxifylline treatment attenuates alveolar fibrin deposition and prolongs survival in preterm rat pups with neonatal hyperoxic lung injury, probably by reducing capillary-alveolar protein leakage.