Systemic delivery of adult stem cells improves cardiac function in spontaneously hypertensive rats

Heart regeneration after myocardial infarction (MI) can occur after cell therapy, but the mechanisms, cell types and delivery methods responsible for this improvement are still under investigation. In the present study, we evaluated the impact of systemic delivery of bone marrow cells (BMC) and cultivated mesenchymal stem cells (MSC) on cardiac morphology, function and mortality in spontaneously hypertensive rats (SHR) submitted to coronary occlusion. Female syngeneic adult SHR, submitted or not (control group; C) to MI, were treated with intravenous injection of MSC (MI + MSC) or BMC (MI + BM) from male rats and evaluated after 1, 15 and 30 days by echocardiography. Systolic blood pressure (SBP), functional capacity, histology, mortality rate and polymerase chain reaction for the Y chromosome were also analysed. Myocardial infarction induced a decrease in SBP and BMC, but not MSC, prevented this decrease. An improvement in functional capacity and ejection fraction (38 +/- 4, 39 +/- 3 and 58 +/- 2% for MI, MI + MSC and MI + BM, respectively; P < 0.05), as well as a reduction of the left ventricle infarcted area, were observed in rats from the MI + BM group compared with the other three groups. Treated animals had a significantly reduced lesion tissue score. The mortality rate in the C, MI + BM, MI + MSC and MI groups was 0, 0, 16.7 and 44.4%, respectively (P < 0.05 for the MI + MSC and MI groups compared with the C and MI + BM groups). The results of the present study suggest that systemic administration of BMC can improve left ventricular function, functional capacity and, consequently, reduce mortality in an animal model of MI associated with hypertension. We speculate that the cells transiently home to the myocardium, releasing paracrine factors that recruit host cells to repair the lesion.

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut

Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0-3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5-3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midguit cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3. (C) 2009 Elsevier Ltd. All rights reserved.

Stigma/style cell cycle inhibitor 1 (SCI1), a tissue-specific cell cycle regulator that controls upper pistil development

P>A cDNA encoding a small lysine-rich protein of unknown function was identified in a tobacco (Nicotiana tabacum) stigma/style suppression subtractive hybridization cDNA library. After its characterization, the corresponding gene was designated stigma/style cell cycle inhibitor 1 (SCI1). Fluorescence microscopy with an SCI1-GFP protein fusion demonstrated its nuclear localization, which was confined to the interchromatic region. Real-time RT-PCR and in situ hybridization experiments showed that SCI1 is stigma/style-specific and developmentally regulated. SCI1 RNAi knockdown and overexpression plants had stigmas/styles with remarkably enlarged and reduced areas, respectively, which was attributable to differences in cell numbers. These results indicate that SCI1 is a tissue-specific negative cell cycle regulator. The differences in cell division had an effect on the timing of the differentiation of the stigmatic papillar cells, suggesting that their differentiation is coupled to stigma cell divisions. This is consistent with a role for SCI1 in triggering differentiation through cell proliferation control. Our results revealed that SCI1 is a novel tissue-specific gene that controls cell proliferation/differentiation, probably as a component of a developmental signal transduction pathway.

Soybean and sunflower oil-induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable-oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 mu L) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB- and SF-induced insulin resistance was shown to involve GLUT4. In SB- and SF-treated animals, the GLUT4 protein expression was reduced similar to 20% and 10 min after an acute it? vivo stimulus with insulin, the plasma membrane GLUT4 content was similar to 60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid (similar to 150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections. Copyright (C) 2010 John Wiley & Sons, Ltd.

Renal ischemia in rats: Mitochondria function and laser autofluorescence

Ischemia-reperfusion injury is the major cause of organ dysfunction or even nonfunction following transplantation. It can attenuate the long-term survival of transplanted organs. To evaluate the severity of renal ischemia injury determined by histology, we applied laser(442 nm and 532 nm) induced fluorescence (LIF), mitochondria respiration, and membrane swelling to evaluate 28 Wistar rats that underwent left kidney warm ischemia for 20, 40, 60, or 80 minutes. LIF performed before ischemia (control) was repeated at 20, 40, 60, and 80 minutes thereafter. We harvested left kidney tissue samples immediately after LIF determination for histology and mitochondrial analyses: state 3 and 4 respiration, respiration control rate (RCR), and membrane swelling. The association of optic spectroscopy with histological damage showed: LIF, 442 nm (r(2) = 0.39, P < .001) and 532 nm, (r(2) = 0.18, P = .003); reflecting laser/fluorescence-induced, 442 nm (r(2) = 0.20, P = .002) and 532 nm (r(2) = 0.004, P = .67). The associations between mitochondria function and tissue damage were: state 3 respiration (r(2) = 0.43, P = .0004), state 4 respiration (r(2) = 0.03, P = 0.38), RCR (r(2) = 0.28, P = .007), and membrane swelling (r(2) = 0.02, P = .43). The intensity of fluorescence emitted by tissue excited by laser, especially at a wave length of 442 nm, was determined in real time. Mitochondrial state 3 respiration and respiratory control ratio also exhibited good correlations with the grade of ischemic tissue damage.