Size ofAT(n)Insertions in Promoter Region ModulatesGmhsp17.6-LmRNA Transcript Levels

During earlier experiments, an SSR molecular marker (176 Soy HSP) showing high correlation (70%) with resistance/susceptibility to javanese root-knot nematodeMeloidogyne javanicawas identified in soybean. After being sequenced, results indicated that the SSR 176 Soy HSP marker was inserted in the promoter region ofGmhsp17.6-Lgene. It was also detected in this region that resistant genotypes presented insertions betweenAT(31)andAT(33)in size and susceptible genotypes,AT(9).Gmhsp17.6-Lgene coding region presented a perfect match in amino acid sequence in all soybean genotypes. A ribonuclease protection assay showed thatGmhsp17.6-Lgene mRNA transcripts were present in all genotypes. A real-time relative quantification (qPCR) indicated in the resistant individuals higher mRNA transcripts levels, which presented in the sequencing moreAT(n)insertions. These results suggest that the number ofAT(n)insertions inside this promoter region could modulate up or down gene levels. Those findings can lead to the possibility of manipulating, between some limits, the mRNA transcripts levels using different sizes ofAT(n)insertions.

Prolonged administration of a dithiol antioxidant protects against ventricular remodeling due to ischemia-reperfusion in mice

The prolonged production of reactive oxygen species due to ischemia-reperfusion (I/R) is a potential cause of the pathological remodeling that frequently precedes heart failure. We tested the ability of a potent dithiol antioxidant, bucillamine, to protect against the long-term consequences of I/R injury in a murine model of myocardial infarction. After transiently occluding the left anterior descending coronary artery for 30 min, saline or bucillamine (10 μg/g body wt) was injected intravenously as a bolus within the first 5 min of reperfusion. The antioxidant treatment continued with daily subcutaneous injections for 4 wk. There were no differences in infarct sizes between bucillamine- and saline-treated animals. After 4 wk of reperfusion, cardiac hypertrophy was decreased by bucillamine treatment (ventricular weight-to-body weight ratios: I/R + saline, 4.5 ± 0.2 mg/g vs. I/R + bucillamine, 4.2 ± 0.1 mg/g; means ± SE; P < 0.05). Additionally, the hearts of bucillamine-treated mice had improved contractile function (echocardiographic measurement of fractional shortening) relative to saline controls: I/R + saline, 32 ± 3%, versus I/R + bucillamine, 41 ± 4% ( P < 0.05). Finally, I/R-induced injury in the saline-treated mice was accompanied by a fetal pattern of gene expression determined by ribonuclease protection assay that was consistent with pathological cardiac hypertrophy and remodeling [increased atrial natriuretic peptide, β-myosin heavy chain (MHC), skeletal α-actin; decreased sarco(endo)plasmic reticulum Ca2+ ATPase 2a, and α-MHC-to-β-MHC ratio]. These changes in gene expression were significantly attenuated by bucillamine. Therefore, treatment with a dithiol antioxidant for 4 wk after I/R preserved ventricular function and prevented the abnormal pattern of gene expression associated with pathological cardiac remodeling.

Chemokine detection in the cerebral tissue of patients with posttraumatic brain contusions

Object The clinical outcome of patients with severe head injuries is still critically dependent on their secondary injuries. Although hypoxia and hypotension appear to mediate a substantial proportion of secondary injuries, many studies associate secondary brain injury with neuroinflammatory responses. Chemokines have been detected in the cerebrospinal fluid but not in the brain tissue of patients with head trauma. This study was performed to determine if chemokines were expressed in pericontusional brain tissue in patients with moderate or severe head trauma who underwent surgical evacuation of their brain contusions. Methods Twelve patients with posttraumatic cerebral contusion requiring a surgical evacuation were studied. A 20- to 40-mg sample of white matter was removed from the surgical cavity in the pericontusional area. Two patients undergoing elective surgery for clip ligation of an unruptured aneurysm were used as controls. The median interval from trauma to biopsy procedure was 44 hours (range 3–360 hours). Total RNA was isolated from these samples and a ribonuclease protection assay was performed to measure the mRNA levels of several chemokines: CCL2, CCL3, CCL4, CCL5, CXCL8, CXCL10, and XCL1. Results The CCL2, a monocyte chemoattractant produced by activated astrocytes, was the most strongly expressed chemokine, followed by CXCL8, CCL3, and CCL4. The chemokines CXCL10 and CCL5 were expressed at very low levels, and XCL1 was not detected. Conclusions Chemokine activation occurs early after moderate or severe head trauma and is maintained for several days after trauma. This event may contribute to neuroinflammatory exacerbation of posttraumatic brain damage in the pericontusional brain tissue.

Elevated iron absorption in the neonatal rat reflects high expression of iron transport genes in the distal alimentary tract

Intestinal iron absorption is extremely high in neonatal mammals but falls rapidly to adult levels following weaning. The aim of this study was to investigate the molecular basis of this elevated neonatal absorption using the rat as an experimental model. RNA was extracted from various sections of the intestine of 10-, 15-, 20-, 25-, and 300-day-old rats and the expression of the genes encoding DMT1 ( Slc11a2), ferroportin ( Slc40a1), Cybrd1 ( Cybrd1), and hephaestin ( heph) determined by ribonuclease protection assay. The hepatic expression of Hamp was studied at the same ages. Iron absorption was examined by following 59Fe uptake in both whole animals and in isolated intestinal loops. Slc11a2, Slc40a1, and Cybrd1 mRNAs were highly expressed in all regions of the small intestine and colon studied in suckling rats. However, after weaning, when iron absorption declined significantly, strong expression was retained only in the duodenum. No change in hephaestin mRNA occurred in any part of the digestive tract. In the distal small intestine and colon, Slc40a1 expression most closely followed the change in absorption that occurred after weaning. Hamp expression was low during the neonatal period and increased to adult levels following weaning. Our results suggest that the distal small intestine and colon contribute significantly to the high intestinal iron absorption seen in neonatal animals and that this reflects increased expression of the iron transporters, particularly Slc40a1.

RNA expression as a prognostic tool in idiopathic nephrotic syndrome

A gyermekkori nephrosis 90%-a idiopathiás nephrosis szindróma. Az idetartozó három kórkép, a minimal change betegség, a mesangialis proliferatio és a focalis sclerosis hasonló klinikai képpel jelentkező, eltérő prognózisú és terápiás válaszú betegség. Dolgozatunk célja az idiopathiás nephrosis szindrómába tartozó kórképek kialakulásával, progressziójával összefüggő genetikai ismeretek, génexpressziós változások áttekintése és funkcionális csoportosítása. A génexpressziós változások meghatározásának eszközeként, dolgozatunk röviden összefoglalja a northern blot, a ribonuclease protection assay, az in situ RNS-hibridizáció, a kvantitatív RT-PCR és a microarray módszerek lényegét. Az eddig elvégzett vizsgálatok a DNS-szintézis és repair gének, növekedési faktorok, extracelluláris mátrix, extracelluláris ligandreceptorok, extracelluláris jelátvitel zavarai mellett kiemelik a metabolikus és transzporter gének, illetve az immunszabályozó gének molekuláris eltéréseit, amelyek összefüggésben vannak az idiopathiás nephrosis szindróma eddig megismert molekuláris hátterével. A chiptechnológia fejlődésével és elterjedésével ezek a markerek és a hagyományos vizsgálati módszerek párhuzamos alkalmazása rutindiagnosztikai szempontból is fontossá válhat.