Metabolic effects and the methylenetetrahydrofolate reductase (MTHFR) polymorphism associated with neural tube defects in southern Brazil

BACKGROUND: The importance of metabolic factors in neural tube defects (NTDs) has been the focus of many investigations. Several authors have suggested that abnormalities in homocysteine metabolism, such as hyperhomocysteinemia, folate deficiency, and low vitamin B12, may be responsible for these malformations and that both nutritional factors and genetic abnormalities are associated with them. METHODS: We conducted a case-control study to investigate the influence of biochemical and genetic factors in NTDs in infants in southern Brazil. Levels of folate, vitamin B12, total homocysteine (t-Hcy) and the 677C>T and 1298A>C polymorphisms of the MTHFR gene were analyzed in 41 NTD child-mother pairs and 44 normal child-mother control pairs. RESULTS: Subjects in the case group had a higher mean blood folate level than those in the control group. The level of vitamin B12 was lower in mothers in the NTD group than in control mothers (p = 0.004). The level of t-Hcy was not different in the two groups, but t-Hcy and vitamin B12 were correlated (p = 0.002). There was no difference in the genotype distribution for 677C>T and 1298A>C polymorphisms of MTHFR in the case and control pairs. The level of t-Hcy was correlated with 677TT. CONCLUSIONS: Despite the small sample in this study, we suggest that low vitamin B12 and, consequently, hyperhomocysteinemia are important risk factors for NTDs in our population.     

Automated apparatus for quantitation of social approach behaviors in mice

Mouse models of social dysfunction, designed to investigate the complex genetics of social behaviors, require an objective methodology for scoring social interactions relevant to human disease symptoms. Here we describe an automated, three chambered apparatus designed to monitor social interaction in the mouse. Time spent in each chamber and the number of entries are scored automatically by a system detecting photocell beam breaks. When tested with the automated equipment, juvenile male C57BL/6J mice spent more time in a chamber containing a stranger mouse than in an empty chamber (sociability), similar to results obtained by the observer scored method. In addition, automated scoring detected a preference to spend more time with an unfamiliar stranger than a more familiar conspecific (preference for social novelty), similar to results obtained by the observer scored method. Sniffing directed at the wire cage containing the stranger mouse correlated significantly with time spent in that chamber, indicating that duration in a chamber represents true social approach behavior. Number of entries between chambers did not correlate with duration of time spent in the chambers; entries instead proved a useful control measure of general activity. The most significant social approach behavior took place in the first five minutes of both the sociability and preference for social novelty tests. Application of these methods to C57BL/6J, DBA/2J and FVB/NJ adult males revealed that all three strains displayed tendencies for sociability and preference for social novelty. To evaluate the importance of the strain of the stranger mouse on sociability and preference for social novelty, C57BL/6J subject mice were tested either with A/J strangers or with C57BL/6J strangers. Sociability and preference for social novelty were similar with both stranger strains. The automated equipment provides an accurate and objective approach to measuring social tendencies in mice. Its use may allow higher-throughput scoring of mouse social behaviors in mouse models of social dysfunction.     

Analysis of RING finger genes required for embryogenesis in C. elegans

The RING finger motif exists in E3 ligases of the ubiquitination pathway. These ubiquitin ligases bind to target proteins, leading to their modification by covalent addition of ubiquitin peptides. Current databases contain hundreds of proteins with RING finger motifs. This study investigates the role of RING finger genes in embryogenesis of the nematode, Caenorhabditis elegans. We expand the previous list of RING finger-containing genes and show that there are 103 RING finger-containing genes in the C. elegans genome. DNA microarrays of these 103 genes were probed with various RNA samples to identify 16 RING finger genes whose expression is enriched in the germline. RNA interference (RNAi) analysis was then used to determine the developmental role of these genes. One RING finger gene, C32D5.10, showed a dramatic larval arrest upon RNAi. Three RING finger genes exhibited embryonic lethality after RNAi. These three genes include par-2, and two small RING finger proteins: F35G12.9 (an ortholog of APC11) and ZK287.5 (an ortholog of rbx1). Embryos from RNAi of the APC11 and rbx1 orthologs were arrested in the cell cycle, confirming the role of these particular RING finger proteins in regulation of the cell cycle. genesis 38:1-12, 2004.     

Plasma appearance and tissue accumulation of non-esterified, free astaxanthin in C57BL/6 mice after oral dosing of a disodium disuccinate diester of astaxanthin (Heptax)

Oral bioavailability of natural and synthetic carotenoids is generally poor in rodents, and this has limited the ability to test these antioxidant compounds in well-defined rodent models of human disease. Various strategies have been employed, with variable success, to increase the percentage of the total oral dose absorbed by the rodent GI tract. In the current study, a novel carotenoid derivative (the disodium disuccinate diester of astaxanthin; Heptax) was administered by oral gavage in a lipophilic emulsion to C57BL/6 mice. Plasma appearance and tissue accumulation of non-esterified, free astaxanthin was studied by HPLC over 72 h after single- and multiple-dose regimens. One-time dosing of Heptax in emulsion at 500 mg/kg resulted in significant appearance of free astaxanthin in plasma (Cmax=0.2 mg/l; 381 nM) and accumulation in solid organs (e.g. liver Cmax=0.9 mg/l; 1735 nM), levels not previously reported after single carotenoid doses in rodents. At each point in the concentration/time curve (AUC), free astaxanthin levels in liver were greater than the corresponding concentration in plasma, suggesting concentrative uptake by the liver. As the ED50 as an antioxidant for non-esterified, free astaxanthin in model systems is approximately 200 nM, the current results suggest that hepatoprotection against oxidative insults may be achieved after a single dose of Heptax in these animals. In humans, where the bioavailability of oral carotenoids ranges from 40 to 60% of the total dose when given in lipophilic vehicle, much smaller oral doses may be utilized for therapeutic benefit in a particular clinical application.     

Using lanthanide ions to align troponin complexes in solution: order of lanthanide occupancy in cardiac troponin C

The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy.     

Oxidative decomposition of vitamin C in drinking water

We have previously shown that vitamin C (ascorbic acid) can initiate hydroxyl radical formation in copper contaminated household drinking water. In the present study, we have examined the stability of vitamin C in copper and bicarbonate containing household drinking water. In drinking water samples, contaminated with copper from the pipes and buffered with bicarbonate, 35% of the added vitamin C was oxidized to dehydroascorbic acid within 15 min. After 3 h incubation at room temperature, 93% of the added (2 mM) ascorbic acid had been oxidized. The dehydroascorbic acid formed was further decomposed to oxalic acid and threonic acid by the hydrogen peroxide generated from the copper (I) autooxidation in the presence of oxygen. A very modest oxidation of vitamin C occurred in Milli-Q water and in household water samples not contaminated by copper ions. Moreover, addition of vitamin C to commercially sold domestic bottled water samples did not result in vitamin C oxidation. Our results demonstrate that ascorbic acid is rapidly oxidized to dehydroascorbic acid and further decomposed to oxalic- and threonic acid in copper contaminated household tap water that is buffered with bicarbonate. The impact of consuming ascorbic acid together with copper and bicarbonate containing drinking water on human health is discussed.     

Platelet sparing effect of COX II inhibition used with pegylated interferon alfa-2a for the treatment of chronic hepatitis C: a short term pilot study

The role of a cyclooxygenase (COX) II inhibitor in reducing microvascular inflammation and the platelet count associated with interferon (IFN) plus ribavirin therapy of chronic hepatitis C (HCV) was assessed. Three plasma mediators (biomarkers) associated with platelet activation, inflammation and fibrosis were measured. Eighteen IFN na?ve patients were studied. Nine were treated with pegylated IFN alfa-2a (PEG-IFN alpha-2a) plus ribavirin and rofecoxib; nine were treated with PEG-IFN alpha-2a plus ribavirin. A complete blood count, liver panel and HCV-RNA were assayed weekly. Human soluble P-selectin (hs-P-selectin), human interleukin-8 (IL-8), human interleukin-13 (IL-13) and human thrombopoietin (TPO) were assayed at 4 week intervals. The COX II inhibitor reduced the platelet reduction experienced with PEG-IFN alpha-2a treatment of HCV despite a reduction in the plasma TPO level. Hs-P-selectin was increased in both groups. In contrast, human IL-8 levels declined to undetectable levels in virologic responders. Similarly, human IL-13 levels declined with therapy (P < 0.001). These data suggest that: (1) a COX II inhibition is associated with an increase in the platelet count despite a reduction in the TPO level; (2) human IL-8 and human IL-13 but not hs-P-selectin levels decline in those who experience an early virologic response.     

Systemic host inflammatory and coagulation response in the Dengue virus primo-infection

BACKGROUND: Dengue virus infection has been rising in tropical countries. Clinical manifestations range from fever and general malaise to hemorrhagic manifestations and death. The role of endothelial damage and cytokines has not been well established for dengue infection. OBJECTIVE: Determine the profile of the pro-inflammatory cytokines and several markers of coagulopathy of dengue infection. METHODS: Patients admitted between September 2000 and April 2001, who met the WHO dengue diagnosis criteria, were enrolled. Blood samples were collected at 0, 6, 12, 24, 48, 72 h and 5 and 7 days after hospitalization. Profile of pro-inflammatory cytokines, markers of coagulopathy, protein C, protein S, d-dimer, prothrombin time, activated partial thromboplastin time, fibrinogen and activated protein C levels were determined. RESULTS: Thirty-three patients were enrolled. Median (range) age was 31 (13-70) years; 51.5% (17/33) were female. Ten of 33 (30%) presented with hemorrhagic manifestations. Patients were classified: Grade 1: 23/33 (70%), Grade II: 10/33 (30%). At study entry IL-6 was the most elevated, followed by IL-8 and TNF alpha. IL-10 was not elevated. No significant differences (P < 0.05) were demonstrated in the levels of any of the haemostatic or cytokine markers by disease severity (Grade I versus Grade II patients). CONCLUSION: The systemic host inflammatory and coagulation activation response occurs early in patients with dengue viral infection in the absence of severe hemorrhagic manifestations, and provides the basis for considering future clinical study in the use of recombinant human activated protein C to treat patients with severe sepsis from dengue infection.     

Effect of irradiance on the partitioning of assimilated carbon during the early phase of grain filling in rice

Low irradiance in the early phase of grain filling in rice often results in a low grain yield, but its effects on the partitioning of previously or recently assimilated carbon within the plant or panicle have not been seriously examined. The objective of this study was to demonstrate the effect of shading during the different stages in the early phase of grain filling on the partitioning of previously or recently assimilated carbon among constituent organs and into superior and inferior spikelets of the panicle in rice (Oryza sativa L. 'Sasanishiki') plants using 13C as a tracer. Plants were grown either under low (shading) or moderate (non-shading) irradiance (120 and 800 micromol quantum m(-2) s(-1)) for 3 or 4 d before or after the 13CO2 feeding at heading, full-heading or milky stages during the early phase of grain filling. Four days after the 13CO2 feeding, the proportion of labelled (previously assimilated) carbon partitioned into the panicle was 17% higher in plants grown under low irradiance compared with plants grown under moderate irradiance at the full-heading stage (7-11 d after heading), while the proportion partitioned into the culm was 13% lower. The light treatments for 3 d were conducted before the 13CO2 feeding and partitioning of the labelled (recently-assimilated) carbon into spikelets was examined 6 h after feeding. The amount of labelled carbon partitioned into the spikelets of the secondary branch (inferior grains) in the plants grown under low irradiance was only 31% when compared with plants grown under moderate irradiance at the full-heading stage, although the partitioning of labelled carbon into the apical spikelets of the primary branch (superior grains) was not affected by the light treatments. These results clearly indicate that preferential partitioning of assimilated carbon into the panicle occurs under low irradiance at around 7-11 d after heading and that the priority of superior spikelets for assimilated carbon intensifies. This phenomenon is thought to be an important strategy for such rice cultivars as used in this study to achieve a certain proportion of ripened grains even under light limited conditions.