Increased levels of cell-free hemoglobin,oxidation markers,and the antioxidative heme scavenger α1-microglobulin in preeclampsia

Preeclampsia is a major cause of morbidity and mortality during pregnancy. To date, the pathogenesis of the disease is not fully understood. Recent studies show that preeclampsia is associated with overexpression of the hemoglobin genes α2 and γ and accumulation of the protein in the vascular lumen of the placenta. Hypothesizing that cell-free hemoglobin leaks from the placenta into the maternal circulation and contributes to the endothelial damage and symptoms by inducing oxidative stress, we analyzed fetal and adult hemoglobin (HbF, HbA), haptoglobin, oxidation markers, and the heme scavenger and antioxidant α₁-microglobulin in plasma, urine, and placenta in preeclamptic women (n = 28) and women with normal pregnancy (n = 27). The mean plasma concentrations of HbF, HbA, protein carbonyl groups, membrane peroxidation capacity, and α₁-microglobulin were significantly increased in preeclamptic women. The levels of total plasma Hb correlated strongly with the systolic blood pressure. The plasma haptoglobin concentrations of women with preeclampsia were significantly depressed. Increased amounts of α₁-microglobulin mRNA and protein were found in placenta from preeclamptic women, and the levels of plasma and placenta α₁-microglobulin correlated with the plasma Hb concentrations. The heme-degrading form t-α₁-microglobulin was significantly increased in urine in preeclampsia. These results support the idea that hemoglobin-induced oxidative stress is a pathogenic factor in preeclampsia.     

Intralateral hypothalamic area injection of isoproterenol and propranolol affects food and water intake in broilers

The role played by adrenergic system in water intake, especially food intake, has long been known in mammals. In avian species, there have been many experiments exploring the effects of the adrenergic system in different sites in the central nervous system in meat- and egg-type poultry. This study was designed to examine the possible effects of intralateral hypothalamic area (ILHy) microinjections of a beta-adrenergic agonist, isoproterenol, and a beta-adrenoceptor blocker, propranolol, on food and water intake in 3-h food-deprived and 3-h water-deprived broiler cockerels. Our findings suggest that the β-adrenergic system directly affects food especially water intake in broilers. Although isoproterenol significantly (P ≤ 0.05) decreased food intake for the first 15 min, it reduced food intake during the experiment. Isoproterenol reduced water intake significantly (P ≤ 0.05), which was abolished by pretreatment with propranolol. It is proposed that β-adrenoceptors in LHy play a direct and indirect role in the regulation of food especially water intake in broiler cockerels.     

Dihydrofolate reductase gene variations in susceptibility to disease and treatment outcomes

Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate (THF). THF is needed for the action of folate-dependent enzymes and is thus essential for DNA synthesis and methylation. The importance of this reaction is demonstrated by the effectiveness of antifolate medications used to treat cancer by inhibiting DHFR, thereby depleting THF and slowing DNA synthesis and cell proliferation. Due to the pivotal role that DHFR plays in folate metabolism and cancer treatment, changes in the level of DHFR expression can affect susceptibility to a variety of diseases dependent on folate status such as spina bifida and cancer. Likewise, variability in DHFR expression can affect sensitivity to anti-cancer drugs such as the folate antagonist methotrexate. Alterations in DHFR expression can be due to polymorphisms in the DHFR gene. Several variations have recently been described in DHFR, including promoter polymorphisms, the 19-bp deletion allele and variations in 3'UTR. These polymorphisms seem to be functional, affecting mRNA levels through various interesting mechanisms, including regulation through RNA interference. Several groups have assessed the association of these polymorphisms with folate levels, risk of cancer and spina bifida as well as the outcome of diseases treated with MTX. The latter may lead to different treatment schedules, improving treatment efficacy and/or allowing for a reduction in drug side effects. This review will summarize present knowledge regarding the predictive potential of DHFR polymorphisms in disease and treatment.     

Amplification efficiency of thermostable DNA polymerases

The amplification efficiencies of several polymerase chain reaction (PCR) enzymes were compared using real-time quantitative PCR with SYBR Green I detection. Amplification data collected during the exponential phase of PCR are highly reproducible, and PCR enzyme performance comparisons based upon efficiency measurements are considerably more accurate than those based on endpoint analysis. DNA polymerase efficiencies were determined under identical conditions using five different amplicon templates that varied in length or percentage GC content. Pfu- and Taq-based formulations showed similar efficiencies when amplifying shorter targets (<900 bp) with 45 to 56% GC content. However, when amplicon length or GC content was increased, Pfu formulations with dUTPase exhibited significantly higher efficiencies than Taq, Pfu, and other archaeal DNA polymerases. We discuss the implications of these results.     

SLEB3 in systemic lupus erythematosus (SLE) is strongly related to SLE families ascertained through neuropsychiatric manifestations

Seizures and psychosis are neuropsychiatric (NP) manifestations of a large number of systemic lupus erythematosus (SLE) patients. Since NP manifestations were part of the SLE phenotype for some, but not all SLE affecteds, we hypothesized that those SLE patient families with NP manifestations might be more genetically homogeneous at loci important to NP-related SLE, and hence have increased power to detect linkage. We identified 23 families of European-American (EA) origin and 20 families of African-American (AA) origin, in which at least one SLE patient in each family was diagnosed with the presence of NP manifestations. A total of 318 microsatellite markers at an average marker density of 11 cM were genotyped. Uncertainty of the genetic model led us to perform the initial genome scan by a multipoint non-parametric allele sharing linkage method. Once the evidence of linkage was suggestive, we then performed parametric model-based linkage by maximizing the relevant parameters to define a parsimonious genetic model. We found the maximum multipoint parametric LOD score was 5.19 and the non-parametric linkage score (Zlr) was 3.12 ( P=9x10(-4)) for EA NP pedigrees at 4p16, previously identified as SLEB3. The segregation behavior of this linked locus suggests a dominant mode of inheritance with an almost 100% homogeneous genetic effect in these pedigrees. The results demonstrated a significant increase of LOD score to detect SLEB3 when the families were further ascertained through NP, compared with the analysis of all EA SLE families together.     

High-throughput SNP genotyping on universal bead arrays

We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.     

Carbohydrate-active enzymes involved in the secondary cell wall biogenesis in hybrid aspen

Wood formation is a fundamental biological process with significant economic interest. While lignin biosynthesis is currently relatively well understood, the pathways leading to the synthesis of the key structural carbohydrates in wood fibers remain obscure. We have used a functional genomics approach to identify enzymes involved in carbohydrate biosynthesis and remodeling during xylem development in the hybrid aspen Populus tremula x tremuloides. Microarrays containing cDNA clones from different tissue-specific libraries were hybridized with probes obtained from narrow tissue sections prepared by cryosectioning of the developing xylem. Bioinformatic analyses using the sensitive tools developed for carbohydrate-active enzymes allowed the identification of 25 xylem-specific glycosyltransferases belonging to the Carbohydrate-Active EnZYme families GT2, GT8, GT14, GT31, GT43, GT47, and GT61 and nine glycosidases (or transglycosidases) belonging to the Carbohydrate-Active EnZYme families GH9, GH10, GH16, GH17, GH19, GH28, GH35, and GH51. While no genes encoding either polysaccharide lyases or carbohydrate esterases were found among the secondary wall-specific genes, one putative O-acetyltransferase was identified. These wood-specific enzyme genes constitute a valuable resource for future development of engineered fibers with improved performance in different applications.     

Breast reconstruction with the DIEP flap or the muscle-sparing (MS-2) free TRAM flap: is there a difference?

The advantages of breast reconstruction using the deep inferior epigastric perforator (DIEP) flap and the muscle-sparing free transverse rectus abdominis musculocutaneous (TRAM) flap (MS-2) are well recognized. Both techniques optimize abdominal function by maintaining the vascularity, innervation, and continuity of the rectus abdominis muscle. The purpose of this study was to compare these two methods of breast reconstruction and determine whether there is a difference in outcome. The study considered 177 women who have had breast reconstruction using muscle-sparing flaps over a 4-year period. This includes 89 women who had an MS-2 free TRAM flap procedure, of which 65 were unilateral and 24 were bilateral, and 88 women who had a DIEP flap procedure, of which 66 were unilateral and 22 were bilateral. The total number of flaps was 223. Mean follow-up was 23 months (range, 3 to 49 months). For all MS-2 free TRAM flaps (n = 113), outcome included fat necrosis in eight (7.1 percent), venous congestion in three (2.7 percent), and total necrosis in two (1.8 percent). For the women who had an MS-2 free TRAM flap, an abdominal bulge occurred in three women (4.6 percent) after unilateral reconstruction and in five women (21 percent) after bilateral reconstruction. The ability to perform sit-ups was noted in 63 women (97 percent) after unilateral reconstruction and 20 women (83 percent) after bilateral reconstruction. For all DIEP flaps (n = 110), outcome included fat necrosis in seven (6.4 percent), venous congestion in five (4.5 percent), and total necrosis in three (2.7 percent) patients. For the women who had DIEP flap reconstruction, an abdominal bulge occurred in one woman (1.5 percent) after unilateral reconstruction and in one woman (4.5 percent) after bilateral reconstruction. The ability to perform sit-ups was noted in all women after unilateral reconstruction and in 21 women (95 percent) after bilateral reconstruction. These results demonstrate that there are no significant differences in fat necrosis, venous congestion, or flap necrosis after DIEP or MS-2 free TRAM flap reconstruction. The percentage of women who are able to perform sit-ups and the percentage of women who did not develop a postoperative abdominal bulge is increased after DIEP flap reconstruction; however, this difference is not statistically significant.