Disulfide formation and stability of a cysteine-rich repeat protein from Helicobacter pylori

Helicobacter pylori cysteine-rich proteins (Hcps) are disulfide-containing repeat proteins. The repeating unit is a 36-residue, disulfide-bridged, helix-loop-helix motif. We use the protein HcpB, which has four repeats and four disulfide bridges arrayed in tandem, as a model to determine the thermodynamic stability of a disulfide-rich repeat protein and to study the formation and the contribution to stability of the disulfide bonds. When the disulfide bonds are intact, the chemical unfolding of HcpB at pH 5 is cooperative and can be described by a two-state reaction. Thermal unfolding is reversible between pH 2 and 5 and irreversible at higher pH 5. Differential scanning calorimetry shows noncooperative structural changes preceding the main thermal unfolding transition. Unfolding of the oxidized protein is not an all-or-none two-state process, and the disulfide bonds prevent complete unfolding of the polypeptide chain. The reduced protein is significantly less stable and does not unfold in a cooperative way. During oxidative refolding of the fully reduced protein, all the possible disulfide intermediates with a correct disulfide bond are formed. Formation of "wrong" (non-native) disulfide bonds could not be demonstrated, indicating that the reduced protein already has some partial repeating structure. There is a major folding intermediate with disulfides in the second, third, and fourth repeat and reduced cysteines in the first repeat. Disulfide formation in the first repeat limits the overall rate of oxidative refolding and contributes about half of the thermodynamic stability to native HcpB, estimated as 27 kJ mol(-1) at 25 degrees C and pH 7. The high contribution to stability of the first repeat may be explained by the repeat acting as a cap to protect the hydrophobic interior of the molecule.     

Studies on the removal of dyes from a synthetic textile effluent using barley husk in static-batch mode and in a continuous flow,packed-bed,reactor

Yarrowia lipolytica LGAM S(7)1 presented remarkable growth on industrial glycerol used as sole carbon substrate. Nitrogen-limited flask cultures were accompanied by restricted synthesis of reserve lipid, whilst amounts of citric acid were produced extracellularly. On the contrary, high amounts of reserve lipid (up to 3.5 g/l, 43% w/w of lipids in dry biomass) were produced in highly aerated continuous cultures. Lipid production was favoured at low specific dilution rates whilst fat-free material yield increased over the whole range of D (h(-1)). The maximum volumetric productivity obtained was 0.12 g lipid/1 h. Storage lipid composition did not present remarkable changes in the specific dilution rates tested. Oleate and linoleate were the dominant cellular fatty acids.     

Climate Adaptation and Policy-Induced Inflation of Coastal Property Value

Human population density in the coastal zone and potential impacts of climate change underscore a growing conflict between coastal development and an encroaching shoreline. Rising sea-levels and increased storminess threaten to accelerate coastal erosion, while growing demand for coastal real estate encourages more spending to hold back the sea in spite of the shrinking federal budget for beach nourishment. As climatic drivers and federal policies for beach nourishment change, the evolution of coastline mitigation and property values is uncertain. We develop an empirically grounded, stochastic dynamic model coupling coastal property markets and shoreline evolution, including beach nourishment, and show that a large share of coastal property value reflects capitalized erosion control. The model is parameterized for coastal properties and physical forcing in North Carolina, U.S.A. and we conduct sensitivity analyses using property values spanning a wide range of sandy coastlines along the U.S. East Coast. The model shows that a sudden removal of federal nourishment subsidies, as has been proposed, could trigger a dramatic downward adjustment in coastal real estate, analogous to the bursting of a bubble. We find that the policy-induced inflation of property value grows with increased erosion from sea level rise or increased storminess, but the effect of background erosion is larger due to human behavioral feedbacks. Our results suggest that if nourishment is not a long-run strategy to manage eroding coastlines, a gradual removal is more likely to smooth the transition to more climate-resilient coastal communities.     

Novel structurally designed vaccine for S. aureus α-hemolysin: protection against bacteremia and pneumonia

Staphylococcus aureus (S. aureus) is a human pathogen associated with skin and soft tissue infections (SSTI) and life threatening sepsis and pneumonia. Efforts to develop effective vaccines against S. aureus have been largely unsuccessful, in part due to the variety of virulence factors produced by this organism. S. aureus alpha-hemolysin (Hla) is a pore-forming toxin expressed by most S. aureus strains and reported to play a key role in the pathogenesis of SSTI and pneumonia. Here we report a novel recombinant subunit vaccine candidate for Hla, rationally designed based on the heptameric crystal structure. This vaccine candidate, denoted AT-62aa, was tested in pneumonia and bacteremia infection models using S. aureus strain Newman and the pandemic strain USA300 (LAC). Significant protection from lethal bacteremia/sepsis and pneumonia was observed upon vaccination with AT-62aa along with a Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE) that is currently in clinical trials. Passive transfer of rabbit immunoglobulin against AT-62aa (AT62-IgG) protected mice against intraperitoneal and intranasal challenge with USA300 and produced significant reduction in bacterial burden in blood, spleen, kidney, and lungs. Our Hla-based vaccine is the first to be reported to reduce bacterial dissemination and to provide protection in a sepsis model of S. aureus infection. AT62-IgG and sera from vaccinated mice effectively neutralized the toxin in vitro and AT62-IgG inhibited the formation of Hla heptamers, suggesting antibody-mediated neutralization as the primary mechanism of action. This remarkable efficacy makes this Hla-based vaccine a prime candidate for inclusion in future multivalent S. aureus vaccine. Furthermore, identification of protective epitopes within AT-62aa could lead to novel immunotherapy for S. aureus infection.     

EGFR inhibition by pentacyclic triterpenes exhibit cell cycle and growth arrest in breast cancer cells

Aims

Pentacyclic triterpenes are a group of molecules with promising anticancer potential, although their precise molecular target remains elusive. The current work aims to investigate the antiproliferative and associated mechanisms of triterpenes in breast cancer cells in vitro.

Main methods

Effect of triterpenes on cell cycle distribution, ROS and key regulatory proteins were analyzed in three breast cancer cells in vitro. Growth inhibition, new DNA synthesis, colony formation assays and Western blot analysis were performed to assess the EGFR inhibitory effect of triterpenes. Molecular docking was performed to study the interaction between EGFR and triterpenes.

Key findings

We have demonstrated the ability of dimethyl melaleucate (DMM), a pentacyclic triterpene to exhibit cell cycle arrest at G0/G1 phase by down-regulation of cyclin D1 through PI3K/AKT inhibition. Further, to identify the upstream target of DMM, potential EGFR inhibitory activity of DMM and three structurally related pentacyclic triterpenes, ursolic acid, 18α-glycyrrhetinic acid and carbenoxolone was investigated. Interestingly, pentacyclic triterpenes limit EGF mediated breast cancer proliferation through sustained inhibition of EGFR and its downstream effectors STAT3 and cyclin D1 in breast cancer lines. We also show pentacyclic triterpenes to bind at the ATP binding pocket of tyrosine kinase domain of EGFR leading to the hypothesis that pentacyclic triterpenes could be a novel class of EGFR inhibitors. In conclusion, pentacyclic triterpenes inhibit EGFR activation through binding with tyrosine kinase domain thereby suppressing breast cancer proliferation.

Significance

Pentacyclic triterpenes may serve as a potential platform for development of novel drugs against breast cancer.     

Phospholipid and cholesterol alterations accompany structural disarray in myelin membrane of rats with hepatic encephalopathy induced by thioacetamide

Fulminant hepatic failure is often associated with a wide range of neurological symptoms which are collectively referred to as hepatic encephalopathy. Fulminant hepatic failure with associated hepatic encephalopathy has a poor prognosis with the currently available sure treatment being only liver transplantation. This is largely owing to the lack of understanding of critical factors involved in the etiology of the condition. Lipid changes have been implicated in cerebral derangements characteristic of hepatic encephalopathy. About 79% of the brain lipid is concentrated in the myelin fraction where they play an important role in ion balance and conduction of nerve impulses. Hence, in the present study we aimed to investigate changes in myelin lipid composition and structure. Myelin was isolated by sucrose density gradient centrifugation from cerebral cortex of male Wistar rats (250-300 g body weight) treated with 300 mg/kg body weight thioacetamide administered twice at 24h interval to induce hepatic encephalopathy. Significant decrease was observed in the cholesterol and phospholipids content of myelin from treated rats. Sphingomyelin, phosphatidylserine and phosphatidylethanolamine content also decreased significantly following 18 h of thioacetamide administration. However, phosphatidylcholine levels remained unaltered. Transmission electron microscopic observation of myelin membrane from cerebral cortex sections showed considerable disorganization in myelin structure. Increase in malondialdehyde levels precede lipid changes leading to the speculation that oxidative damage may be the critical factor leading to decrease in the anionic phospholipids. Changes in myelin were evident only in later stages of hepatic encephalopathy indicating that myelin alteration may not play a role in early stages of hepatic encephalopathy. Nevertheless, myelin alteration may have a crucial role to play in various psycho-motor alterations during later stages of hepatic encephalopathy.