DNA damage recognition in the normal epithelium of human prostate and seminal vesicles

Prostate cancer is one of the most prevalent cancer types in men. The development of prostate tumors is known to require androgen exposure, and several pathways governing cell growth are deregulated in prostate tumorigenesis. Recent genetic studies have revealed that complex gene fusions and copy - number alterations are frequent in prostate cancer, a unique feature among solid tumors. These chromosomal aberrations are though to arise as a consequence of faulty repair of DNA double strand breaks (DSB). Most repair mechanisms have been studied in detail in cancer cell lines, but how DNA damage is detected and repaired in normal differentiated human cells has not been widely addressed. The events leading to the gene fusions in prostate cancer are under rigorous studies, as they not only shed light on the basic pathobiologic mechanisms but may also produce molecular targets for prostate cancer treatment and prevention. Prostate and seminal vesicles are part of the male reproductive system. They share similar structure and function but differ dramatically in their cancer incidence. Approximately fifty primary seminal vesicle carcinomas have been reported worldwide. Surprisingly, only little is known on why seminal vesicles are resistant to neoplastic changes. As both tissues are androgen dependent, it is a mystery that androgen signaling would only lead to tumors in prostate tissue. In this work, we set up novel ex vivo human tissue culture models of prostate and seminal vesicles, and used them to study how DNA damage is recognized in normal epithelium. One of the major DNA - damage inducible pathways, mediated by the ATM kinase, was robustly activated in all main cell types of both tissues. Interestingly, we discovered that secretory epithelial cells had less histone variant H2A.X and after DNA damage lower levels of H2AX were phosphorylated on serine 139 (γH2AX) than in basal or stromal cells. γH2AX has been considered essential for efficient DSB repair, but as there were no significant differences in the γH2AX levels between the two tissues, it seems more likely that the role of γH2AX is less important in postmitotic cells. We also gained insight into the regulation of p53, an important transcription factor that protects genomic integrity via multiple mechanisms, in human tissues. DSBs did not lead to a pronounced activation of p53, but treatments causing transcriptional stress, on the other hand, were able to launch a notable p53 response in both tissue types. In general, ex vivo culturing of human tissues provided unique means to study differentiated cells in their relevant tissue context, and is suited for testing novel therapeutic drugs before clinical trials. In order to study how prostate and seminal vesicle epithelial cells are able to activate DNA damage induced cell cycle checkpoints, we used primary cultures of prostate and seminal vesicle epithelial cells. To our knowledge, we are the first to report isolation of human primary seminal vesicle cells. Surprisingly, human prostate epithelial cells did not activate cell cycle checkpoints after DSBs in part due to low levels of Wee1A, a kinase regulating CDK activity, while primary seminal vesicle epithelial cells possessed proficient cell cycle checkpoints and expressed high levels of Wee1A. Similarly, seminal vesicle cells showed a distinct activation of the p53 - pathway after DSBs that did not occur in prostate epithelial cells. This indicates that p53 protein function is under different control mechanisms in the two cell types, which together with proficient cell cycle checkpoints may be crucial in protecting seminal vesicles from endogenous and exogenous DNA damaging factors and, as a consequence, from carcinogenesis. These data indicate that two very similar organs of male reproductive system do not respond to DNA damage similarly. The differentiated, non - replicating cells of both tissues were able to recognize DSBs, but under proliferation human prostate epithelial cells had deficient activation of the DNA damage response. This suggests that prostate epithelium is most vulnerable to accumulating genomic aberrations under conditions where it needs to proliferate, for example after inflammatory cellular damage.

A Study of The Fatigue, Impact and fracture-Toughness Characteristics of Electroslag Refined IS-7670 Alloy

The nuclear, aerospace, naval and missile industries place emphasis on materials with high structural integrity and reliable performance so as to meet certain stringent requirements in service. Strength is not the only criterion for selection. Properties such as fatigue resistance. impact toughness and fracture toughness are equally important. Electroslag refining (ESR) has been used widely and successfully over the years for improving the fatigue resistance, creep resistance, impact strength and fracture toughness of steels and alloy steels. But application of ESR to aluminium alloys is only a recent endeavour. A high-strength aircraft aluminium alloy IS: 7670 was therefore chosen for studies on the fatigue strength and the impact and fracture toughness. The results indicate that the fatigue resistance is considerably improved after refining and that the impact strength and fracture toughness of the refined alloy are comparable with that of the unrefined alloy.

Metabolism and Translocation of Aminophospholipids in Mammalian Cells

In this study we investigated the metabolism, i.e. remodeling and translocation, of the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE). A new method for introduction of exogenous PS and PE molecular species to cultured cells was developed, and combined with mass spectrometry it enabled more detailed follow-up of the metabolism of single molecular species than previously. We found that I) exogenous PS and PE molecular species can be efficiently introduced to cultured cells without compromising cell integrity, II) PS and PE molecular species are remodeled by several phospholipases displaying selectivity based on phopholipid head group and acyl chain composition, III) PS decarboxylase (PSD) and Kennedy pathways provide a different PE molecular species composition to the cellular PE pool. In addition, PE species produced by these pathways are translocated from the site of synthesis to other cell compartments depending on their acyl chain composition. The data obtained in the present study helps to understand cellular phospholipid metabolism in more depth. The data show that effective labeling of cultured cells by exogenous phospholipids does not compromise cell viability and may be used to disturb cellular phospholipid composition to study lipid homeostasis. Remodeling and translocation of PS and PE molecular species is highly selective. The developed method and mass- spectrometric techniques may be used in future studies to understand disturbances in lipid homeostasis for example in diabetes mellitus, thus opening doors to optional scientific approaches to study mechanisms behind pathologies related to lipid disturbances.

Sport, body and power: Reassessing the myth of President Kekkonen

Former President of Finland Urho Kekkonen was not only a powerful politician but also a well-known sportsman and keep-fit enthusiast. The president’s sports hobbies were covered and celebrated in the media and thus became an integral part of his public persona. This paper looks at Kekkonen’s athletic and able-bodied image and its significance for his power from the perspective of gender. In his exercise activities, Kekkonen was able to display his bodily prowess and demonstrate his version of masculinity, which emphasized both physical and mental strength. The union of mind and muscle in turn buttressed his political ascendancy. Kekkonen’s athletic body served as a cornerstone of his dominance over his country and, simultaneously, as a shield protecting Finland from both internal and external threats. Furthermore, Kekkonen’s sports performances were essential elements in the myth that was created around the president during his term and which was carefully conserved after his fall from power. Drawing upon scholarship on men and masculinities, this paper reassesses the still-effective mythical image of Kekkonen as an invincible superman. The article reveals the performative nature of his athletic activities and shows that in part, his pre-eminence in them was nothing more than theatre enacted by him and his entourage. Thus, Kekkonen’s superior and super-masculine image was actually surprisingly vulnerable and dependent on the success of the performance. The president’s ageing, in particular, demonstrates the fragility of his displays of prowess, strength and masculinity, and shows how fragile the entanglement of body and power can be.

Thermomechanical generalization of pin joints in finite plates

The understanding of thermoelastic behaviour of joints is significant in order to ensure the integrity of large and complex structures exposed to a thermal environment, particularly in fields such as aerospace and nuclear engineering. Thermomechanical generalization of partial contact behaviour of a pin joint under combined in-plane mechanical loading and on-axis unidirectional heat flow has already been established by the authors for the analytically simpler domains of large plates. This paper successfully extends the on-going investigation to a single pin in a finite rectangular isotropic plate as a two-dimensional abstraction from a practical situation of a multipin fastener joint. The finite element method is used to analyse the joint problem under on-axis thermomechanical loading and unified load-contact relationships are established for a class of loading conditions.

Diamond-like carbon coating for optical windows

Hard, low stress diamond-like carbon films have been deposited by plasma assisted chemical vapour deposition technique, The various substrates include soft IR components like ZnS and ZnSe windows, Gaseous precursors such as propene, ethyl alcohol and acetone have been used to synthesize the films to study the nature of precursors in determining the film compatibility with the underlying component (substrate), The residual compressive stresses, the Young's modulus and the adhesion energy of the films have been estimated to be 10(10) dynes/cm(2), 10(10) N/m(2) and 1000 ergs/cm(2) respectively. To alleviate film failure, a study on the effects of additive gases such as hydrogen and the use of buffer layers such as ZrO2, has been undertaken, The diamond-like carbon films produced here are hard (5000 kg/mm(2)), specularly smooth in the wavelength region from 2.5 mu m to 20 mu m, with no microstructural features and have excellent adhesion on ZnS and ZnSe windows. The figure of merit of these films for aero-space applications has been evaluated by subjecting the film-buffer layer ZnS or ZnSe composite stack to wind, dust and rain erosion studies and by establishing the integrity of the specular IR transmittance of the stack upto 16 or 20 mu m as the case may be.

Analysis of a conformation-specific epitope of the alpha subunit of human chorionic gonadotropin: Study using monoclonal antibody probes

Monoclonal antibodies (MAbs) have been used extensively for identification of sequence-specific epitopes using either the ELISA or/and IRMA methods, However, attempts to use MAbs for identification of conformation-specific epitopes have been very few as they are considered very labile. We have investigated the stability of conformation-specific epitopes of human chorionic gonadotropin (hCG) using a quantitative solid-phase radioimmnunoassay (SPRIA) technique. Several epitopes are stable to mild modification (chemical and proteolytic) conditions, and epitopes show differential stability for these modifications. Based on these observations, a monoclonal antibody (MAb 16) for an a-subunit-specific epitope of hCG has been used to monitor changes at the epitopic site (identified as epitope 16) on modification of hCG, using SPRIA with immobilized MAb 16. Modifications of amino groups, hydroxyl group of tyrosine as well as carboxyl group of Asp/Glu all bring about sufficient changes in the epitope integrity. Peptide bond hydrolysis at lysine residues damages the epitope, but not at arginine residues, Hydrolysis at tyrosine does not affect the epitope, though modification of the side-chain of tyrosine inactivates the epitope. Destruction of the epitope occurs on reduction of the disulphide bonds. Partial retention of the epitope activity is seen on modification of carboxyl or the epsilon-amino groups of lysine. Based on these results four to six amino acids have been identified to be at the epitopic site, and the data suggest that two peptide segments are brought together by the disulphide bond Cys10-Cys60 to form the epitope.

A novel method for mapping assembled epitopes in batches: Identification of three epitopes at the receptor binding region of human chorionic gonadotropin

Identification of conformation-specific epitopes of hCG beta has been done using a simple batch method, Chemically or enzymatically-modified hCG beta has been prepared in a batch and the effect of modifications on the integrity of different epitope regions has been investigated in a quantitative manner using monoclonal antibodies (MAbs) immobilized on plastic tubes from culture supernatants. Based on the extent of damage done to different regions by different modifications, three conformation-specific epitopes of hCG beta have been identified. The method has been shown to have important advantages over the existing methods on many considerations, Using this approach, these epitopes have been shown to be at/near the receptor-binding region.

Interaction of sheep liver apo-serine hydroxymethyltransferase with pyridoxal-5'-phosphate: A physicochemical, kinetic, and thermodynamic study

Sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) is a homotetramer of M(r) 213,000 requiring pyridoxal-5'-phosphate (PLP) as cofactor, Removal of PLP from the holoenzyme converted the enzyme to the apo form which, in addition to being inactive, was devoid of the characteristic absorption spectrum. Upon the addition of PLP to the apoenzyme, complete activity was restored and the visible absorption spectrum with a maximum at 425 nm was regained. The interaction of PLP with the apoenzyme revealed two phases of reaction with pseudo-first-order rate constants of 20 +/- 5 s(-1) and 12.2 +/- 2.0 x 10(-3) s(-1), respectively. However, addition of PLP to the apoenzyme did not cause gross conformational changes as evidenced by circular dichroic and fluorescence spectroscopy. Although conformationally apoenzyme and holoenzyme were indistinguishable, they had distinct apparent melting temperatures of 51 +/- 2 and 58 +/- 2 degrees C, respectively, and the reconstituted holoenzyme was thermally as stable as the native holoenzyme. These results suggested that there was no apparent difference in the secondary structure of holoenzyme, apoenzyme, and reconstituted holoenzyme, However, sedimentation analysis of the apoenzyme revealed the presence of two peaks of S-20,S-w values of 8.7 +/- 0.5 and 5.7 +/- 0.3 S, respectively. A similar pattern was observed when the apoenzyme was chromatographed on a calibrated Sephadex G-150 column. The first peak corresponded to the tetrameric form (M(r) 200,000 +/- 15,000) while the second peak had a M(r) of 130,000 +/- 10,000. Reconstitution experiments revealed that only the tetrameric form of the apoenzyme could be converted into an active holoenzyme while the dimeric form could not be reconstituted into an active enzyme. These results demonstrate that PLP plays an important role in maintaining the structural integrity of the enzyme by preventing the dissociation of the enzyme into subunits, in addition to its function in catalysis. (C) 1996 Academic Press, Inc.

What makes a cell tick? The A, B and C of the matter

Nuclear lamina in an eukaryotic cell is primarily composed of the lamins A, B and C. The A type lamins are found only in differentiated cell types while the B type lamins are present both in differentiated and undifferentiated cells. Lamin B interacts with the inner nuclear membrane, In recent years there have been extensive studies on the relationship between the dynamic state of lamin B and the nuclear envelope integrity with respect to the fate of a particular cell, In this article, we have analysed the recent developments and have considered the sequence of events that might be contributing to the fate of a cell either to undergo normal cell division or uncontrolled cellular proliferation or apoptosis.

TolA mediates the differential detergent resistance pattern between the Salmonella enterica subsp enterica serovars Typhi and Typhimurium

The tol-pal genes are essential for maintaining the outer membrane integrity and detergent resistance in various Gram-negative bacteria, including Salmonella. The role of TolA has been well established for the bile resistance of Salmonella enterica subsp. enterica serovar Typhimurium. We compared the bile resistance pattern between the S. enterica serovars Typhi and Typhimurium and observed that Typhi is more resistant to bile-mediated damage. A closer look revealed a significant difference in the TolA sequence between the two serovars which contributes to the differential detergent resistance. The tolA knockout of both the serovars behaves completely differently in terms of membrane organization and morphology. The role of the Pal proteins and difference in LPS organization between the two serovars were verified and were found to have no direct connection with the altered bile resistance. In normal Luria broth (LB), S. Typhi Delta tolA is filamentous while S. Typhimurium Delta tolA grows as single cells, similar to the wildtype. In low osmolarity LB, however, S. Typhimurium Delta tolA started chaining and S. Typhi Delta tolA showed no growth. Further investigation revealed that the chaining phenomenon observed was the result of failure of the outer membrane to separate in the dividing cells. Taken together, the results substantiate the evolution of a shorter TolA in S. Typhi to counteract high bile concentrations, at the cost of lower osmotic tolerance.

Modelling of a patch repair to a thin cracked sheet

Integrity enhancement of damaged or design deficient structures through repairs is attracting considerable engineering attention. Bonded composite patch repairs to cracked metallic sheets offer various advantages over riveted doubler type, particularly for airframe applications. This paper first reviews the R&D activity in the area of structural repairs. It then approaches the problem of a composite patch repair to a cracked aluminium sheet with different finite element modelling strategies and compares their outcome. The efficient finite element modelling approach thus established is used to study the effect of patch material, patch size, patch symmetry and adhesive thickness on repair performance as the crack grows in the repair configuration. (C) 1999 Elsevier Science Ltd. All rights reserved.

Residual strength and fatigue life assessment of composite patch repaired specimens

Design, analysis and technology for the integrity enhancement of damaged or underdesigned structures continues to be an engineering challenge. Bonded composite patch repairs to metallic structures is receiving increased attention in the recent years. It offers various advantages over rivetted doubler, particularly for airframe repairs. This paper presents an experimental investigation of residual strength and fatigue crack-growth life of an edge-cracked aluminium specimen repaired using glass epoxy composite patch. The investigation begins with the evaluation of three different surface treatments from bond strength viewpoint. A simple thumb rule formula is employed to estimate the patch size. Cracked and repaired specimens are tested under static and fatigue loading. The patch appears to restore the original strength of the undamaged specimen and enhance the fatigue crack growth life by an order of magnitude. (C) 1999 Elsevier Science Ltd. All rights reserved.

Vasomodulatory effect of novel peroxovanadate compounds on rat aorta: Role of rho kinase and nitric oxide/cGMP pathway

The present study was undertaken to assess the role of reactive oxygen species (ROS) in rat aortic ring vasoreactivity and integrity by using various peroxovanadate (pV) compounds. All the pV compounds (1 nM-300 mu M) used in the present study exerted concentration-dependent contractions on endothelium intact rat aortic rings. All compounds with an exception of DPV-asparagine (DPV-asn) significantly altered vascular integrity as shown by diminished KCl responses. Phenylephrine (PE)-mediated contractions (3 nM-300 mu M) were unaltered in the presence of these compounds. Acetylcholine (Ach)-mediated relaxation in PE (1 mu M) pre-contracted rings was significantly reduced in presence of diperoxovanadate (DPV), poly (sodium styrene sulfonate-co-maleate)-pV (PSS-CoM-pV) and poly (sodium styrene 4-sulfonate)-pV (PSS-pV). However, no significant change in Ach-mediated responses was observed in the presence of poly (acrylate)-pV (PM-pV) and DPV-asn. DPV-asn was thus chosen to further elucidate mechanism involved in peroxide mediated modulation of vasoreactivity. DPV-asn (30 nM-300 mu M) exerted significantly more stable contractions, that was found to be catalase (100 U/ml) resistant in comparison with H(2)O(2) (30 nM-300 mu M) in endothelium intact aortic rings. These contractile responses were found to be dependent on extracellular Ca(2+) and were significantly inhibited in presence of ROS scavenger N-acetylcysteine (100 mu M). Intracellular calcium chelation by BAPTA-AM (10 mu M) had no significant effect on DPV-asn (30 nM-300 mu M) mediated contraction. Pretreatment of aortic rings by rho-kinase inhibitor Y-27632 (10 mu M) significantly inhibited DPV-asn-mediated vasoconstriction indicating role of voltage-dependent Ca(2+) influx and downstream activation of rho-kinase. The small initial relaxant effect obtained on addition of DPV-asn (30 nM-1 mu M) in PE (1 mu M) pre-contracted endothelium intact rings, was prevented in the presence of guanylate cyclase inhibitor, methylene blue (10 mu M) and/or nitric oxide synthase (NOS) inhibitor, L-NAME (100 mu M) suggesting involvement of nitric oxide and cGMP. DPV-asn, like H(2)O(2), exerted a response of vasoconstriction in normal arteries and vasodilation at low concentrations (30 nM-1 mu M) in PE-pre contracted rings with overlapping mechanisms. These findings suggest usefulness of DPV-asn having low toxicity, in exploring the peroxide-mediated effects on various vascular beds. The present study also convincingly demonstrates role of H(2)O(2) in the modulation of vasoreactivity by using stable peroxide DPV-asn and warrants future studies on peroxide mediated signaling from a newer perspective. (C) 2011 Published by Elsevier Ltd.

Influence of land use changes in river basins on diversity and distribution of amphibians

Land-use changes influence local biodiversity directly, and also cumulatively, contribute to regional and global changes in natural systems and quality of life. Consequent to these, direct impacts on the natural resources that support the health and integrity of living beings are evident in recent times. The Western Ghats being one of the global biodiversity hotspots, is reeling under a tremendous pressure from human induced changes in terms of developmental projects like hydel or thermal power plants, big dams, mining activities, unplanned agricultural practices,monoculture plantations, illegal timber logging, etc. This has led to the once contiguous forest habitats to be fragmented in patches, which in turn has led to the shrinkage of original habitat for the wildlife, change in the hydrological regime of the catchment, decreased inflow in streams,human-animal conflicts, etc. Under such circumstances, a proper management practice is called for requiring suitable biological indicators to show the impact of these changes, set priority regions and in developing models for conservation planning. Amphibians are regarded as one of the best biological indicators due to their sensitivity to even the slightest changes in the environment and hence they could be used as surrogates in conservation and management practices. They are the predominating vertebrates with a high degree of endemism (78%) in Western Ghats. The present study is an attempt to bring in the impacts of various land-uses on anuran distribution in three river basins. Sampling was carried out for amphibians during all seasons of 2003-2006 in basins of Sharavathi, Aghanashini and Bedthi. There are as many as 46 species in the region, one of which is new to science and nearly 59% of them are endemic to the Western Ghats. They belong to nine families, Dicroglossidae being represented by 14 species,followed by Rhacophoridae (9 species) and Ranidae (5 species). Species richness is high in Sharavathi river basin, with 36 species, followed by Bedthi 33 and Aghanashini 27. The impact of land-use changes, was investigated in the upper catchment of Sharavathi river basin. Species diversity indices, relative abundance values, percentage endemics gave clear indication of differences in each sub-catchment. Karl Pearson’s correlation coefficient (r) was calculated between species richness, endemics, environmental descriptors, land-use classes and fragmentation metrics. Principal component analysis was performed to depict the influence of these variables. Results show that sub-catchments with lesser percentage of forest, low canopy cover, higher amount of agricultural area, low rainfall have low species richness, less endemic species and abundant non-endemic species, whereas endemism, species richness and abundance of endemic species are more in the sub-catchments with high tree density, endemic trees, canopy cover, rainfall and lower amount of agriculture fields. This analysis aided in prioritising regions in the Sharavathi river basin for further conservation measures.