169 research outputs found
Superoxide dismutase analog (Tempol: 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine 1-oxyl) treatment restores erectile function in diabetes-induced impotence.
We hypothesized that the administration of the superoxide dismutase (SOD) mimetic Tempol (4-hydroxy-2, 2, 6, 6-tetramethylpiperidine 1-oxyl) may reverse diabetes-induced erectile dysfunction. To test this hypothesis, reactive oxygen species-related genes (SOD1, SOD2, GP x 1, CAT, NOS2, NOS3) were tested, erectile functional studies and immunohistochemical analysis were carried out in diabetic rats treated with or without Tempol. Thirty Sprague-Dawley (3-4 months old) rats were divided into three groups (n=10 each), 20 with diabetes (diabetic control and Tempol treatment) and 10 healthy controls. At 12 weeks after the induction of diabetes by streptozotocin and Tempol treatment, all groups underwent in vivo cavernous nerve stimulation. Rat crura were harvested and the expression of antioxidative defense enzymes were examined by semi-quantitative reverse transcriptase PCR (RT-PCR). To confirm the RT-PCR results, we carried out immunohistochemistry (IHC) for catalase (CAT) and iNOS (NOS2). Nitration of tyrosine groups in proteins was also examined by IHC. Mean intracavernous pressure in the diabetic group was significantly lower than in the healthy controls (P <0.001) and was reversed by Tempol treatment (P <0.0108). NOS2 protein expression was significantly increased in diabetic animals compared with healthy controls and Tempol restored NOS2 protein level. Nitrotyrosine was also higher in diabetic animals and although Tempol treatment decreased its formation, it remained higher than that found in healthy controls. This study suggests that Tempol treatment increased erectile function through modulating oxidative stress-related genes in diabetic rats. This is the first report about the relationship between diabetes-induced erectile dysfunction and oxidative stress, and antioxidative therapy using the superoxide dismutase mimetic, Tempol, to restore erectile function
Small RNAs Targeting Transcription Start Site Induce Heparanase Silencing through Interference with Transcription Initiation in Human Cancer Cells
Heparanase (HPA), an endo-h-D-glucuronidase that cleaves the heparan sulfate chain of heparan sulfate proteoglycans, is overexpressed in majority of human cancers. Recent evidence suggests that small interfering RNA (siRNA) induces transcriptional gene silencing (TGS) in human cells. In this study, transfection of siRNA against β9/+10 bp (siH3), but not β174/β155 bp (siH1) or β134/β115 bp (siH2) region relative to transcription start site (TSS) locating at 101 bp upstream of the translation start site, resulted in TGS of heparanase in human prostate cancer, bladder cancer, and gastric cancer cells in a sequence-specific manner. Methylation-specific PCR and bisulfite sequencing revealed no DNA methylation of CpG islands within heparanase promoter in siH3-transfected cells. The TGS of heparanase did not involve changes of epigenetic markers histone H3 lysine 9 dimethylation (H3K9me2), histone H3 lysine 27 trimethylation (H3K27me3) or active chromatin marker acetylated histone H3 (AcH3). The regulation of alternative splicing was not involved in siH3-mediated TGS. Instead, siH3 interfered with transcription initiation via decreasing the binding of both RNA polymerase II and transcription factor II B (TFIIB), but not the binding of transcription factors Sp1 or early growth response 1, on the heparanase promoter. Moreover, Argonaute 1 and Argonaute 2 facilitated the decreased binding of RNA polymerase II and TFIIB on heparanase promoter, and were necessary in siH3-induced TGS of heparanase. Stable transfection of the short hairpin RNA construct targeting heparanase TSS (β9/+10 bp) into cancer cells, resulted in decreased proliferation, invasion, metastasis and angiogenesis of cancer cells in vitro and in athymic mice models. These results suggest that small RNAs targeting TSS can induce TGS of heparanase via interference with transcription initiation, and significantly suppress the tumor growth, invasion, metastasis and angiogenesis of cancer cells
Hub-Centered Gene Network Reconstruction Using Automatic Relevance Determination
Network inference deals with the reconstruction of biological networks from experimental data. A variety of different reverse engineering techniques are available; they differ in the underlying assumptions and mathematical models used. One common problem for all approaches stems from the complexity of the task, due to the combinatorial explosion of different network topologies for increasing network size. To handle this problem, constraints are frequently used, for example on the node degree, number of edges, or constraints on regulation functions between network components. We propose to exploit topological considerations in the inference of gene regulatory networks. Such systems are often controlled by a small number of hub genes, while most other genes have only limited influence on the network's dynamic. We model gene regulation using a Bayesian network with discrete, Boolean nodes. A hierarchical prior is employed to identify hub genes. The first layer of the prior is used to regularize weights on edges emanating from one specific node. A second prior on hyperparameters controls the magnitude of the former regularization for different nodes. The net effect is that central nodes tend to form in reconstructed networks. Network reconstruction is then performed by maximization of or sampling from the posterior distribution. We evaluate our approach on simulated and real experimental data, indicating that we can reconstruct main regulatory interactions from the data. We furthermore compare our approach to other state-of-the art methods, showing superior performance in identifying hubs. Using a large publicly available dataset of over 800 cell cycle regulated genes, we are able to identify several main hub genes. Our method may thus provide a valuable tool to identify interesting candidate genes for further study. Furthermore, the approach presented may stimulate further developments in regularization methods for network reconstruction from data
Morphological and Functional Changes in the Retina after Chronic Oxygen-Induced Retinopathy
The mouse model of oxygen-induced retinopathy (OIR) has been widely used for studies of retinopathy of prematurity (ROP). This disorder, characterized by abnormal vascularization of the retina, tends to occur in low birth weight neonates after exposure to high supplemental oxygen. Currently, the incidence of ROP is increasing because of increased survival of these infants due to medical progress. However, little is known about changes in the chronic phase after ROP. Therefore, in this study, we examined morphological and functional changes in the retina using a chronic OIR model. Both the a- and b-waves in the OIR model recovered in a time-dependent manner at 4 weeks (w), 6 w, and 8 w, but the oscillatory potential (OP) amplitudes remained depressed following a return to normoxic conditions. Furthermore, decrease in the thicknesses of the inner plexiform layer (IPL) and inner nuclear layer (INL) at postnatal day (P) 17, 4 w, and 8 w and hyperpermeability of blood vessels were observed in conjunction with the decrease in the expression of claudin-5 and occludin at 8 w. The chronic OIR model revealed the following: (1) a decrease in OP amplitudes, (2) morphological abnormalities in the retinal cells (limited to the IPL and INL) and blood vessels, and (3) an increase in retinal vascular permeability via the impairment of the tight junction proteins. These findings suggest that the experimental animal model used in this study is suitable for elucidating the pathogenesis of ROP and may lead to the development of potential therapeutic agents for ROP treatment
Accommodating a Non-Conservative Internal Mutation by WaterMediated Hydrogen-Bonding Between Ξ²-Sheet Strands: A Comparison of Human and Rat Type B (Mitochondrial) Cytochrome b5
Mammalian type B (mitochondrial) cytochromes b5 exhibit greater amino acid sequence diversity than their type A (microsomal) counterparts, as exemplified by the type B proteins from human (hCYB5B) and rat (rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5B reported herein reveals a striking difference in packing involving the five-stranded Ξ²-sheet, attributable to fully buried residue 21 in strand Ξ²4. The greater bulk of Leu21 in hCYB5B in comparison to Thr21 in rCYB5B results in a substantial displacement of the first two residues in Ξ²5, and consequent loss of two of the three hydrogen bonds between Ξ²5 and Ξ²4. Hydrogen-bonding between the residues is instead mediated by two well-ordered, fully buried water molecules. In a 10 ns molecular dynamics simulation, one of the buried water molecules in the hCYB5B structure exchanged readily with solvent via intermediates having three water molecules sandwiched between Ξ²4 and Ξ²5. When the buried water molecules were removed prior to a second 10 ns simulation, Ξ²4 and Ξ²5 formed persistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observed conformation. Despite the apparently greater ease of water access to the interior of hCYB5B than of rCYB5B suggested by these observations, the two proteins exhibit virtually identical stability, dynamic and redox properties. The results provide new insight into the factors stabilizing the cytochrome b5 fold
Combined In Silico and In Vivo Analyses Reveal Role of Hes1 in Taste Cell Differentiation
The sense of taste is of critical importance to animal survival. Although studies of taste signal transduction mechanisms have provided detailed information regarding taste receptor calcium signaling molecules (TRCSMs, required for sweet/bitter/umami taste signal transduction), the ontogeny of taste cells is still largely unknown. We used a novel approach to investigate the molecular regulation of taste system development in mice by combining in silico and in vivo analyses. After discovering that TRCSMs colocalized within developing circumvallate papillae (CVP), we used computational analysis of the upstream regulatory regions of TRCSMs to investigate the possibility of a common regulatory network for TRCSM transcription. Based on this analysis, we identified Hes1 as a likely common regulatory factor, and examined its function in vivo. Expression profile analyses revealed that decreased expression of nuclear HES1 correlated with expression of type II taste cell markers. After stage E18, the CVP of Hes1β/β mutants displayed over 5-fold more TRCSM-immunoreactive cells than did the CVP of their wild-type littermates. Thus, according to our composite analyses, Hes1 is likely to play a role in orchestrating taste cell differentiation in developing taste buds
Isolation of Hox Cluster Genes from Insects Reveals an Accelerated Sequence Evolution Rate
Among gene families it is the Hox genes and among metazoan animals it is the insects (Hexapoda) that have attracted particular attention for studying the evolution of development. Surprisingly though, no Hox genes have been isolated from 26 out of 35 insect orders yet, and the existing sequences derive mainly from only two orders (61% from Hymenoptera and 22% from Diptera). We have designed insect specific primers and isolated 37 new partial homeobox sequences of Hox cluster genes (lab, pb, Hox3, ftz, Antp, Scr, abd-a, Abd-B, Dfd, and Ubx) from six insect orders, which are crucial to insect phylogenetics. These new gene sequences provide a first step towards comparative Hox gene studies in insects. Furthermore, comparative distance analyses of homeobox sequences reveal a correlation between gene divergence rate and species radiation success with insects showing the highest rate of homeobox sequence evolution
Coordinated spatial and temporal expression of Hox genes during embryogenesis in the acoel Convolutriloba longifissura
Background: Hox genes are critical for patterning the bilaterian anterior-posterior axis. The evolution of their clustered genomic arrangement and ancestral function has been debated since their discovery. As acoels appear to represent the sister group to the remaining Bilateria (Nephrozoa), investigating Hox gene expression will provide an insight into the ancestral features of the Hox genes in metazoan evolution. Results: We describe the expression of anterior, central and posterior class Hox genes and the ParaHox ortholog Cdx in the acoel Convolutriloba longifissura. Expression of all three Hox genes begins contemporaneously after gastrulation and then resolves into staggered domains along the anterior-posterior axis, suggesting that the spatial coordination of Hox gene expression was present in the bilaterian ancestor. After early surface ectodermal expression, the anterior and central class genes are expressed in small domains of putative neural precursor cells co-expressing ClSoxB1, suggesting an evolutionary early function of Hox genes in patterning parts of the nervous system. In contrast, the expression of the posterior Hox gene is found in all three germ layers in a much broader posterior region of the embryo. Conclusion: Our results suggest that the ancestral set of Hox genes was involved in the anteriorposterior patterning of the nervous system of the last common bilaterian ancestor and were later co-opted for patterning in diverse tissues in the bilaterian radiation. The lack of temporal colinearity of Hox expression in acoels may be due to a loss of genomic clustering in this clade or, alternatively, temporal colinearity may have arisen in conjunction with the expansion of the Hox cluster in the Nephrozoa
Epigenetic regulation of prostate cancer
Prostate cancer is a commonly diagnosed cancer in men and a leading cause of cancer deaths. Whilst the underlying mechanisms leading to prostate cancer are still to be determined, it is evident that both genetic and epigenetic changes contribute to the development and progression of this disease. Epigenetic changes involving DNA hypo- and hypermethylation, altered histone modifications and more recently changes in microRNA expression have been detected at a range of genes associated with prostate cancer. Furthermore, there is evidence that particular epigenetic changes are associated with different stages of the disease. Whilst early detection can lead to effective treatment, and androgen deprivation therapy has a high response rate, many tumours develop towards hormone-refractory prostate cancer, for which there is no successful treatment. Reliable markers for early detection and more effective treatment strategies are, therefore, needed. Consequently, there is a considerable interest in the potential of epigenetic changes as markers or targets for therapy in prostate cancer. Epigenetic modifiers that demethylate DNA and inhibit histone deacetylases have recently been explored to reactivate silenced gene expression in cancer. However, further understanding of the mechanisms and the effects of chromatin modulation in prostate cancer are required. In this review, we examine the current literature on epigenetic changes associated with prostate cancer and discuss the potential use of epigenetic modifiers for treatment of this disease
Evolution of CYP2J19, a gene involved in colour vision and red coloration in birds: positive selection in the face of conservation and pleiotropy
- β¦