Exceptional complex chromosomal rearrangement and microdeletions at the 4q22.3q23 and 14q31.1q31.3 regions in a patient with azoospermia

In this report we describe the first patient ever found to have azoospermia in association with both exceptional complex chromosomal rearrangements and microdeletions at two translocation breakpoints. A 36-year-old male who had been suffering from male factor infertility was admitted to our clinic. The patient also displayed mild dysmorphia. An analysis of the patient's semen revealed azoospermia. GTG banding revealed the presence of an exceptional complex chromosomal rearrangement involving chromosomes 1, 4, 10 and 14. Using subtelomeric FISH analysis, the patient's karyotype was designated as 46,XY,t(1;10)(q43q44;q21q26.1)(CEB108/T7+,D1S3738-;10PTEL006+,D10S2290+, D1S3738+), ins(14;4) (q31.3;q23q33)(D14S1420+; D4S3359+, D4S2930+). Array-CGH analysis revealed two microdeletions at the 4q22.3q23 and 14q31.1q31.3 chromosomal regions. We suggest that microdeletions at the 4q22.3q23 and 14q31.1q31.3 chromosomal regions associated with both an exceptional complex chromosomal rearrangement and the Homo sapiens chromosome 4 open reading frame 37 (C4orf37) gene located at the 4q22.3q23 region might be associated with male factor infertility.     

Environmental acidification triggers oxidative stress and enhances globin expression in zebrafish gills

Animals in many aquatic ecosystems must cope with changing environmental parameters, such as temperature, oxygen availability or pH. We have investigated the molecular responses to acidification in the gills and body of zebrafish (Danio rerio) by means of quantitative real-time PCR. Expression levels of typical stress genes and genes for antioxidant defense were strongly enhanced in gills, and to lesser extents in the body, suggesting that acidification leads to oxidative stress. Surprisingly, the globins were found to be among the most prominent stress–responsive proteins in our study. Myoglobin showed the strongest response of all investigated genes in the gills, as confirmed by Western blotting. These findings agree with the role of globins in oxidative energy metabolism, but may also hint at a specific function in antioxidative defense.     

Effect of type-1 diabetes mellitus on the regulation of insulin and endothelin-1 receptors in rat hearts

This project assesses the treatment role with insulin and (or) angiotensin II receptor subtype-1 (AT1-R) blocker (ARB) on insulin receptor and endothelin-1 receptor subtype (ETA-R and ETB-R) regulation in rat hearts suffering from insulin-dependent diabetes mellitus (IDDM). Animals were divided into 6 groups: groups 1, 3, and 5 were controls consisting of normal, diabetic (streptozotocin-treated, once at 0 time), and diabetic supplemented daily with insulin, respectively, whereas groups 2, 4, and 6 were the controls treated daily with losartan. One month after enrollment, rats were sacrificed and samples of cardiac tissue were snapped frozen for immunostaining and Western blotting. Insulin receptor density was observed to be upregulated in the cardiomyocytes of diabetic animals, but downregulated with insulin supplementation alone. Cotreatment with insulin and an ARB resulted in drastic increase in insulin-receptor density in the diabetic rats. In addition, expression of ETA-R in cardiomyocytes was upregulated and was consistently maintained within the various treatment modalities. However, ETB-R expression was significantly reduced in the diabetic group treated with both insulin and an ARB. The changes in the expression of the insulin, the ETA-Rs, and the ETB-Rs at the various sites of the myocardium and the effect of both insulin treatment and blockade of the AT1-R explain the new benefits related to the halting of myocardial remodeling in IDDM rats.     

sFas levels increase in response to cisplatin-based chemotherapy in lung cancer patients

The Fas/Fas Ligand (FasL) system and survivin have counteracting roles in cell survival. Therefore, we explored the role of circulating soluble Fas (sFas) and the tissue levels of Fas and survivin with regard to response to chemotherapy in lung cancer patients. Serum samples from 52 lung cancer patients and 54 control subjects (19 benign lung disease and 35 healthy control subjects) were collected prior to and 24 and 48 h after chemotherapy. sFas was statistically significantly higher in the cancer group than that in the control groups (p < 0.001). Baseline (before chemotherapy) sFas values showed a statistically significant inverse correlation with overall survival (r = ?0.599, p < 0.001). There was a significant increase in serum sFas levels 24 h after treatment (p < 0.05). Contrarily, tissue levels of Fas and survivin were not changed following the chemotherapy (p > 0,05). In conclusion, increased sFas may be an indicator of poor outcome in lung cancer patients. However, cisplatin‐based chemotherapy may not be effective via neither the Fas/FasL system nor survivin pathway. Indeed, larger sample size is required for further evaluation. Copyright © 2010 John Wiley & Sons, Ltd.     

Arabidopsis lyrata Small RNAs: Transient MIRNA and Small Interfering RNA Loci within the Arabidopsis Genus

Twenty-one-nucleotide microRNAs (miRNAs) and 24-nucleotide Pol IV-dependent small interfering RNAs (p4-siRNAs) are the most abundant types of small RNAs in angiosperms. Some miRNAs are well conserved among different plant lineages, whereas others are less conserved, and it is not clear whether less-conserved miRNAs have the same functionality as the well conserved ones. p4-siRNAs are broadly produced in the Arabidopsis genome, sometimes from active hot spot loci, but it is unknown whether individual p4-siRNA hot spots are retained as hot spots between plant species. In this study, we compare small RNAs in two closely related species (Arabidopsis thaliana and Arabidopsis lyrata) and find that less-conserved miRNAs have high rates of divergence in MIRNA hairpin structures, mature miRNA sequences, and target-complementary sites in the other species. The fidelity of miRNA biogenesis from many less-conserved MIRNA hairpins frequently deteriorates in the sister species relative to the species of first discovery. We also observe that p4-siRNA occupied loci have a slight tendency to be retained as p4-siRNA loci between species, but the most active A. lyrata p4-siRNA hot spots are generally not syntenic to the most active p4-siRNA hot spots of A. thaliana. Altogether, our findings indicate that many MIRNAs and most p4-siRNA hot spots are rapidly changing and evolutionarily transient within the Arabidopsis genus.     

Thermodynamic and structural analysis of interactions between peptide ligands and SEB

Staphylococcal enterotoxin B (SEB) is an exotoxin produced by Staphylococcus aureus and commonly associated with food poisoning. In this study, SEB‐binding peptides were identified by screening a phage displayed peptide library. The binding of peptides to SEB was tested with isothermal titration calorimetry (ITC) and of the five selected peptides, three showed affinity to SEB, with one measured to have the highest affinity constant (10⁵ M^?1). ITC revealed that the interaction of peptide ligands with SEB was driven entropically and the binding was dominated by hydrophobic interactions. Circular dichroism (CD) measurements and molecular dynamics (MD) simulations, together, give a structural insight into the interaction of peptides with SEB. While SEB binding peptides showed random coil structure before binding, after complex formation they had more ordered structures. The peptide with highest affinity to SEB showed stable conformation during MD simulation. Taken together, our approach about thermodynamic and structural characterization of peptide ligands can be used to develop aptamers, with high affinity and selectivity, for biosensor applications. Copyright © 2009 John Wiley & Sons, Ltd.     

Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants

Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs.     

Purification,biochemical characterization and gene sequencing of a thermostable raw starch digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov.

This study reports the purification and biochemical characterization of a raw starch-digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov. (strain Pizzo^T). The molecular weight was estimated to be 58 kDa by SDS–PAGE. The enzyme was highly active over a wide range of pH from 4.0–10.0. The optimum temperature of the enzyme was 70°C. It showed extreme thermostability in the presence of Ca²⁺, retaining 50% of its initial activity after 90 h at 70°C. The enzyme efficiently hydrolyzed 20% (w/v) of raw starches, concentration normally used in starch industries. The α-amylase showed an high stability in presence of many organic solvents. In particular the residual activity was of 73% in presence of 15% (v/v) ethyl alcohol, which corresponds to ethanol yield in yeast fermentation process. By analyzing its complete amyA gene sequence (1,542 bp), the enzyme was proposed to be a new α-amylase.     

Matrix metalloproteinase 9 (MMP9) expression in preeclamptic decidua and MMP9 induction by tumor necrosis factor alpha and interleukin 1 beta in human first trimester decidual cells

Extravillous trophoblasts (EVTs) invade human decidua via sequential integrin-mediated binding and proteolysis of basement membrane proteins in the extracellular matrix (ECM). In preeclampsia, shallow EVT invasion impairs spiral artery and arteriole remodeling to reduce uteroplacental blood flow. Excess decidual cell-expressed matrix metalloproteinases (MMPs) 2 and 9, in response to preeclampsia-related interleukin 1 beta (IL1B) and tumor necrosis factor alpha (TNF), may inappropriately degrade these basement membrane proteins and impede EVT invasion. This study found significantly higher immunohistochemical MMP9 levels in decidual cells and adjacent interstitial trophoblasts in placental sections of preeclamptic versus gestational age-matched control women. In contrast, immunostaining for MMP2 and tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP1 and TIMP2) were similar in preeclamptic and control groups. First-trimester decidual cells were incubated with estradiol (E(2)) or E(2) + medroxyprogesterone acetate (MPA), with or without TNF or IL1B. As measured by ELISA, both cytokines elicited concentration-dependent increases in secreted MMP9 levels that were unaffected by MPA. In contrast, secreted levels of MMP2, TIMP1, and TIMP2 were unchanged in all treatment groups. Substrate gel zymography and Western blotting confirmed that each cytokine increased secreted levels of MMP9 but not MMP2. Similarly, quantitative RT-PCR found that TNF and IL1B enhanced MMP9, but not MMP2, mRNA levels. At the implantation site, inflammatory cytokine-enhanced MMP9 may promote preeclampsia by disrupting the decidual ECM to interfere with normal stepwise EVT invasion.