Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring

Down syndrome (DS) resulting from free trisomy 21 (FT21) has been largely associated with advanced maternal age. However, approximately 60% of FT21 cases are born to young couples. Thus, the etiological factors responsible for these FT21 children must differ from those proposed for maternal age-related FT21. These factors have not been defined. In this study, we analyzed the chromosomes of peripheral blood lymphocytes from three groups of couples aged ≤35 years, to identify chromosomal trisomies: Group I included 5 couples with normal offspring; Group II included 22 couples with one FT21 child; and Group III consisted of 3 couples with recurrent FT21. A total of 13,809 metaphases were analyzed with G-banding and 60,205 metaphases were analyzed with FISH using a 13/21 centromeric probe. Aneuploidy was significantly more frequent in Groups II and III. The frequencies of hyperdiploid cells were 0.19, 0.49 and 0.96% in Groups I–III, respectively. FISH analysis showed that trisomy 21 cell percentages were 0.08, 0.21 and 0.76 for Groups I–III, respectively, and were very similar to those obtained with G-banding. Trisomy 21 mosaicism was found in 2/22 couples with one FT21 offspring, and in 2/3 couples with recurrent FT21. Our data suggest that mosaicism is an important cause of FT21 offspring in young couples, and that aneuploidy is more frequent among couples with FT21 offspring. This may be related with age and other undetermined intrinsic and extrinsic factors.     

Modulation of histamine-induced Ca2+ release by protein kinase C. Effects on cytosolic and mitochondrial [Ca2+] peaks

In HeLa cells, histamine induces production of inositol 1,4,5-trisphosphate (InsP3) and release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ release is typically biphasic, with a fast and brief initial phase, followed by a much slower and prolonged one. In the presence of inhibitors of protein kinase C (PKC), including staurosporine and the specific inhibitors GF109203X and Ro-31-8220, the fast phase continued until the ER became fully empty. On the contrary, treatment with phorbol 12,13-dibutyrate inhibited Ca2+ release. Staurosporine had no effect on InsP3-induced Ca2+ release in permeabilized cells and did not modify either histamine-induced InsP3 production. These data suggest that histamine induces Ca2+ release and with a short lag activates PKC to down-regulate it. Consistently, Ca2+ oscillations induced by histamine were increased in amplitude and decreased in frequency in the presence of PKC inhibitors. We show also that mitochondrial [Ca2+] was much more sensitive to changes in ER-Ca2+ release induced by PKC modulation than cytosolic [Ca2+]. PKC inhibitors increased the histamine-induced mitochondrial [Ca2+] peak by 4-fold but increased the cytosolic [Ca2+] peak only by 20%. On the contrary, PKC activation inhibited the mitochondrial [Ca2+] peak by 90% and the cytosolic one by only 50%. Similarly, the combination of PKC inhibitors with the mitochondrial Ca2+ uniporter activator SB202190 led to dramatic increases in mitochondrial [Ca2+] peaks, with little effect on cytosolic ones. This suggests that activation of ER-Ca2+ release by PKC inhibitors could be involved in apoptosis induced by staurosporine. In addition, these mechanisms allow flexible and independent regulation of cytosolic and mitochondrial [Ca2+] during cell stimulation.     

Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

Thinning affects Pinus sylvestris needle decomposition rates and chemistry differently depending on site conditions

Changes in mass and chemical composition of Pinus sylvestris senescent needles were studied over a 5 year period in Mediterranean (MF) and Continental forests (CF) in the Pyrenees under varying levels of thinning (P0: reference, no thinning; P20: removal of 20% basal area, P30: removal of 30% basal area). Decomposition rates were higher in MF (k = 0.423 year^?1) than in CF (k = 0.245 year^?1). However, the maximum decomposition limit was higher in MF (87.9%) compared to CF (78.1%). The relative importance and timing of rainfall, and cellulose and lignin abundance on the decomposition process was similar among both sites. However, air temperature and degree-days only affected CF (the colder site) during the initial stages of decomposition, while litter moisture was significant only in MF (the drier site) in the latter stages of decomposition. Nutrient and carbon dynamics showed temporal patterns similar to those reported in higher latitudes (except for Ca), however, indicators of N mineralization such as C/N and lignin/N at the study sites were lower than values reported in the literature. Decreases in decomposition rates after thinning were higher in MF than in CF, indicating that this ecosystem could, in the short term, be more sensitive to human intervention. Thinning had similar temporary qualitative effects at both sites, slowing decomposition, increasing N and P immobilization and decreasing Ca immobilization. However, quantitative effects of thinning were site dependent in that the magnitude of nutrient immobilization was higher in CF. A conceptual model is presented to explain effects of thinning on litter N dynamics. These temporary changes are not trivial as nutrient immobilization and accumulated organic matter losses over a thinning cycle may affect tree growth particularly during short rotations and intensive fast-growing plantations. Under similar nutrient availability conditions, sites where nutrient release occurs faster may show higher post-thinning tree growth rates.     

Synchronous and early activation of planarian Hox genes and the re-specification of body axes during regeneration in Dugesia (G.) tigrina (Turbellaria; Tricladida)

Seven Hox cluster-related genes (Dthox-A to -G) have been isolated from the freshwater triclad Dugesia (G.) tigrina, their sequence compared to other Hox genes and their expression in intact and regenerating organisms analyzed by whole mount in situ hybridization. Sequence comparison analyses show high similarities of D. tigrina Hox genes to anterior and medial groups of coelomate Hox genes. Expression analyses show very early, synchronous, and overlapping expression of Dthox -A, -E, -G and -F in anterior, posterior and lateral regenerative tissues. At one hour of regeneration all Dthox genes studied showed a neat, clear expression at the wound boundary. Later, as the blastema grows, the expression area expands to more proximal regions covering the blastema and the distal postblastema regions. Blastemas formed by intercalary regeneration also show a synchronous expression of the same Hox genes though the onset of activation is much delayed. The finding that the same set of Hox genes is synchronously activated in anterior, posterior, intercalary and lateral regeneration is in sharp contrast to its well established role in specifying antero-posterior pattern during embryonic development. The implications of these results as regards ancestral versus co-opted roles of Hox genes in development and regeneration are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn²⁺-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.Abbreviations GS glutamine synthetase - MOPS 2-(N-morpholine) propane sulfonate - MSX l-methionine-d,l-sulfoximine     

Methyl cyanide induces α to β transition and aggregation at high concentrations in E-state of human serum albumin

We have studied the effect of 2,2,2-trifluoroethanol (TFE), an α-helix inducer, versus methyl cyanide (MeCN), a β-sheet inducer, on acid-denatured human serum albumin (HSA) using far-UV circular dichroism, intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate binding, and acrylamide quenching studies. Interestingly, at pH 2.0, where the recovery and resolution of the protein in reverse phase chromatography is high, its secondary structure remains unchanged even in the presence of very high concentration (76% v/v) of MeCN. Gain of 23 and 34% α-helicity was observed in the presence of 20 and 50% TFE, respectively. At pH 7.3, HSA aggregates in the presence of 40% MeCN, but it remains soluble up to 75% MeCN at pH 2.0. The results seem to be important for HSA isolation and purification.     

Combined Microbial-Fe(0) Treatment System to Remove Nitrate from Contaminated Groundwater

Experiments were conducted to delineate the applicability and limitations of biologically active Fe(0) barriers to remove nitrate under various geochemical and hydraulic conditions. Microcosm studies showed that, while no Fe(0) treatment was needed to remove nitrate from a high-carbon soil, adding Fe(0) to a low-carbon soil supplemented the electron donor pool and enhanced nitrate removal. Montmorillonite, an acidic aluminosilicate mineral, enhanced Fe(0) corrosion and nitrate removal (from about 1 to 3 mg/L NO₃-N per day), and reduced the transient accumulation of nitrite. Combining autotrophic denitrifiers (e.g., Paracoccus denitrificans) with Fe(0) significantly reduced the amount of nitrite eluted from aquifer columns, from up to 7 to less than 1 mg/L NO₂∼-N. Bacteria were observed to preferentially colonize the Fe(0) surface, which produces cathodic H₂ when corroded by water. The preferential colonization of Fe(0) suggests that hydrogenotrophic consortia are likely to develop around Fe(0) walls to exploit cathodic depolarization as a metabolic niche.