Oligomeric Hsp33 with enhanced chaperone activity: gel filtration, cross-linking, and small angle x-ray scattering (SAXS) analysis

Hsp33, an Escherichia coli cytosolic chaperone, is inactive under normal conditions but becomes active upon oxidative stress. It was previously shown to dimerize upon activation in a concentration- and temperature-dependent manner. This dimer was thought to bind to aggregation-prone target proteins, preventing their aggregation. In the present study, we report small angle x-ray scattering (SAXS), steady state and time-resolved fluorescence, gel filtration, and glutaraldehyde cross-linking analysis of full-length Hsp33. Our circular dichroism and fluorescence results show that there are significant structural changes in oxidized Hsp33 at different temperatures. SAXS, gel filtration, and glutaraldehyde cross-linking results indicate, in addition to the dimers, the presence of oligomeric species. Oxidation in the presence of physiological salt concentration leads to significant increases in the oligomer population. Our results further show that under conditions that mimic the crowded milieu of the cytosol, oxidized Hsp33 exists predominantly as an oligomeric species. Interestingly, chaperone activity studies show that the oligomeric species is much more efficient compared with the dimers in preventing aggregation of target proteins. Taken together, these results indicate that in the cell, Hsp33 undergoes conformational and quaternary structural changes leading to the formation of oligomeric species in response to oxidative stress. Oligomeric Hsp33 thus might be physiologically relevant under oxidative stress.     

Purification, and properties of a bovine uricase

Uricase from bovine kidney, purified to homogeneity level, had a molecular weight of 70 kDa. The apparent K(m) and V(max) values for uric acid hydrolysis were 0.125 mM and 102 IU mg(-1) protein respectively. The activation energy requirement for uric acid hydrolysis by uricase and inactivation of enzyme were 11.6 and 14.5 kJ/M respectively. Both enthalpy (Delta H*) and entropy of activation (Delta S*) for uricase activity were lower than those reported for some thermostable enzymes.     

Rac1 links integrin-mediated adhesion to the control of lactational differentiation in mammary epithelia

The expression of tissue-specific genes during mammary gland differentiation relies on the coincidence of two distinct signaling events: the continued engagement of beta1 integrins with the extracellular matrix (ECM) and a hormonal stimulus from prolactin (Prl). How the integrin and Prl receptor (PrlR) systems integrate to regulate milk protein gene synthesis is unknown. In this study, we identify Rac1 as a key link. Dominant-negative Rac1 prevents Prl-induced synthesis of the milk protein beta-casein in primary mammary epithelial cells cultured as three-dimensional acini on basement membrane. Conversely, activated Rac1 rescues the defective beta-casein synthesis that occurs under conditions not normally permissive for mammary differentiation, either in beta1 integrin-null cells or in wild-type cells cultured on collagen. Rac1 is required downstream of integrins for activation of the PrlR/Stat5 signaling cascade. Cdc42 is also necessary for milk protein synthesis but functions via a distinct mechanism to Rac1. This study identifies the integration of signals provided by ECM and hormones as a novel role for Rho family guanosine triphosphatases.     

Rv3303c of Mycobacterium tuberculosis protects tubercle bacilli against oxidative stress in vivo and contributes to virulence in mice

Ability of Mycobacterium tuberculosis to survive under oxidative stress in vivo is an important aspect of pathogenesis. Rv3303c gene from M. tuberculosis encodes an NAD(P)H quinone reductase. These enzymes have been shown to manage oxidative stress in other pathogenic bacteria. We have hypothesized that Rv3303c protein will remove reactive oxygen species released by the host and hence reduce oxidative stress to M. tuberculosis. rv3303c was PCR cloned and the purified recombinant enzyme reduced superoxide generator menadione. Antisense and sense RNA constructs of rv3303c were electroporated in M. tuberculosis H37Rv. The transformants were characterized by difference in expression of specific mRNA and protein. Antisense transformants were markedly reduced in virulence as compared to sense transformants as judged by several parameters such as weight and survival of infected mice, growth in vivo, colonization and histopathology of lungs. In the presence of menadione, the sense transformant was more resistant to killing in vitro than the antisense transformant. It may be concluded that the rv3303c gene contributes to virulence of M. tuberculosis in vivo and this might be mediated in part by increased resistance to reactive oxygen intermediates thereby enhancing intracellular growth and colonization.     

Phosphoinositide 3-kinase mediated signalling contributes to development of diabetes-induced abnormal vascular reactivity of rat carotid artery

Diabetes mellitus is associated with vascular complications, including an impairment of vascular function and alterations in the reactivity of blood vessels to vasoactive agents. Phosphatidylinositol 3-kinase (PI3K) is a signalling enzyme that plays key roles in vascular growth, proliferation and cellular apoptosis and is implicated in modulating vascular smooth muscle contractility. The aim of this study was to determine whether PI3K plays a role in development of diabetes-induced altered vascular reactivity to selected vasoconstrictors and vasodilators. The effect of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a selective PI3K inhibitor, on isolated segments of carotid arteries from streptozotocin (STZ)-diabetic rats was investigated. Ring segments of the isolated carotid arteries were mounted in organ baths to measure changes in isometric tension. Our results showed that STZ treatment produced an increase in the vasoconstrictor response to norepinephrine (NE), angiotensin II (Ang II) and endothelin-1 (ET-1) and an attenuated vasodilator response to carbachol and histamine in the isolated carotid arteries from STZ-diabetic animals. Diabetes-induced impaired vascular responsiveness to the vasoactive agonists was prevented by chronic inhibition of PI3K by LY294002 even though blood glucose levels remained high. This is the first study to show that selective inhibition of PI3K can attenuate the development of diabetes-induced abnormal vascular reactivity in the isolated carotid arteries of diabetic rats.     

Identification of novel SNPs in SYK gene of breast cancer patients: computational analysis of SNPs in the 5′UTR

Spleen tyrosine kinase (SYK) is a non receptor type tyrosine kinase and a known candidate tumor suppressor gene in breast carcinoma. Loss of Syk is associated with breast cancer invasion and increased cell mortality. The main goal of our study was to detect germ-line polymorphisms in SYK gene in breast cancer affected females of Pakistani origin, in order to understand the genetic basis of complex human breast cancer. Seven novel SYK gene SNPs were identified in breast cancer patients. Among these, three were identified in intronic region, one at the 5'splice donor site (5'SD) and three in 5'untranslated region (5'UTR) of SYK gene. Mutations at the 5'SD and at 5'UTR can be crucial and could be responsible for generation of mutated Syk protein. In silico analysis of the 5'UTR variations revealed that one of the mutations was responsible for generation of a more stable structure of 5'UTR. Such a change in pre-mRNA could potentially down regulate SYK expression. These findings add to the growing literature implicating dysfunctional SYK gene as a contributor to human breast cancer, and suggest that therapies targeting its molecular pathways could provide effective means of treating/preventing breast cancer.     

Angiotensin-(1–7) Via the Mas Receptor Alleviates the Diabetes-Induced Decrease in GFAP and GAP-43 Immunoreactivity with Concomitant Reduction in the COX-2 in Hippocampal Formation: An Immunohistochemical Study

We have previously shown that chronic treatment with angiotensin-(1?C7) [Ang-(1?C7)] can prevent diabetes-induced cardiovascular dysfunction. However, effect of Ang-(1?C7) treatment on diabetes-induced alterations in the CNS is unknown. The aim of this study was to test the hypothesis that treatment with Ang-(1?C7) can produce protection against diabetes-induced CNS changes. We examined the effect of Ang-(1?C7) on the number of cyclooxygenase-2 (COX-2) immunoreactive neurons and the glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes and assessed the changes in the neuronal growth-associated protein-43 (GAP-43) of the hippocampal formation in streptozotocin-induced diabetes in rats. Animals were sacrificed 30?days after induction of diabetes and/or treatment with Ang-(1?C7). Ang-(1?C7) treatment significantly prevented diabetes-induced decrease in the number of GFAP immunoreactive astrocytes and GAP-43 positive neurons in all hippocampal regions. Co-administration of A779, a selective Ang-(1?C7) receptor antagonist, inhibited Ang-(1?C7)-mediated protective effects indicating that Ang-(1?C7) produces its effects through activation of receptor Mas. Further, Ang-(1?C7) treatment through activation of Mas significantly prevented diabetes-induced increase in the number of the COX-2 immunolabeled neurons in all sub-regions of the hippocampus examined. These results show that Ang-(1?C7) has a protective role against diabetes-induced changes in the CNS.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.