Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation

Recent studies have suggested that Rap1 and Rap2 small GTP-binding proteins are both expressed in human red blood cells (RBCs). In this work, we carefully examined the expression of Rap proteins in leukocytes- and platelets-depleted RBCs, whose purity was established on the basis of the selective expression of the beta2 subunit of the Na+/K+ -ATPase, as verified according to the recently proposed "beta-profiling test" [J.F. Hoffman, A. Wickrema, O. Potapova, M. Milanick, D.R. Yingst, Na pump isoforms in human erythroid progenitor cells and mature erythrocytes, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 14572-14577]. In pure RBCs preparations, Rap2, but not Rap1 was detected immunologically. RT-PCR analysis of mRNA extracted from highly purified reticulocytes confirmed the expression of Rap2b, but not Rap2a, Rap2c, Rap1a or Rap1b. In RBCs, Rap2 was membrane-associated and was rapidly activated upon treatment with Ca2+/Ca2+ -ionophore. In addition, Rap2 segregated and was selectively enriched into microvesicles released by Ca2+ -activated RBCs, suggesting a possible role for this GTPase in membrane shedding.     

Identification of the first archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix

The archaeon Aeropyrum pernix grows optimally at 90°C and derives energy primarily from aerobic degradation of complex proteinaceous substrates. The ability of these nutrients to sustain growth is generally associated with the presence of oligopeptide transport systems, such as the well-known protein-dependent ATP-binding cassette (ABC) transporters. This study is concerned with the isolation and characterisation of the first archaeal oligopeptide-binding protein (OppA_Ap) from the extracellular medium of A. pernix. The protein shows a pI of 3.9 and a molecular mass of about 90 kDa under native conditions. By using a proteomic approach, the OppA_Ap-encoding gene was identified (APE1583) and about 55% of the protein amino-acid sequence was validated. The extracellular purified protein was able to efficiently bind oligopeptide substrates such as Xenopsin. The amount of a liganded peptide to OppA_Ap was about 70% at 90°C using a 1/100 (w/w) OppA_Ap/substrate ratio. Sequence comparisons showed a weak but significant similarity of OppA_Ap with bacterial oligopeptide binding proteins. Furthermore, APE1583 neighbouring genes encode for the cognate components of an ABC transport system, suggesting that these ORFs are organised in an operon-like structure, with OppA_Ap as the extracellular component for the uptake of oligopeptides.     

Resistance and bioaccumulation of Cd2+, Cu2+, Co2+ and Mn2+ by thermophilic bacteria, Geobacillus thermantarcticus and Anoxybacillus amylolyticus

In this study, bioaccumulation and heavy metal resistance of Cd²⁺, Cu²⁺, Co²⁺ and Mn²⁺ ions by thermophilic Geobacillus thermantarcticus and Anoxybacillus amylolyticus was investigated. The bacteria, in an order with respect to metal resistance from the most resistant to the most sensitive, was found to be Mn²⁺ > Co²⁺ > Cu²⁺ > Cd²⁺ for both G. thermantarcticus and A. amylolyticus. It was determined that the highest metal bioaccumulation was performed by A. amylolyticus in Mn²⁺ (28,566 μg/g dry weight), and the lowest metal bioaccumulation was performed by A. amylolyticus in Co²⁺ (327.3 μg/g dry weight). The highest Cd²⁺ capacities of dried cell membrane was found to be 36.07 and 39.55 mg/g membrane for G. thermantarticus and A. amylolyticus, respectively, and the highest Cd²⁺ capacities of wet cell membrane was found to be 14.36 and 12.39 mg/g membrane for G. thermantarcticus and A. amylolyticus, respectively.     

Biosorption of Heavy Metals (Cd2+, Cu2+, Co2+, and Mn2+) by Thermophilic Bacteria,Geobacillus thermantarcticus and Anoxybacillus amylolyticus: Equilibrium and Kinetic Studies

ABSTRACT

Two strains of thermophilic bacteria, Geobacillus thermantarcticus and Anoxybacillus amylolyticus, were employed to investigate the biosorption of heavy metals including Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ ions. The effects of different biosorption parameters such as pH (2.0–10.0), initial metal concentrations (10.0–300.0 mg L^?1), amount of biomass (0.25–10 g L^?1), temperature (30–80°C), and contact time (15–120 min) were investigated. Concentrations of metal ions were determined by using an inductively coupled plasma optical emission spectrometry (ICP-OES). Optimum pHs for Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ biosorption by Geobacillus thermantarcticus were found to be 4.0, 4.0, 5.0, and 6.0, respectively. For Anoxybacillus amylolyticus, the optimum pHs for Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ biosorption were found to be 5.0, 4.0, 5.0, and 6.0, respectively. The Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ removals at 50 mg L^?1 in 60 min by 50 mg dried cells of Geobacillus thermantarcticus were 85.4%, 46.3%, 43.6%, and 65.1%, respectively, whereas 74.1%, 39.8%, 35.1%, and 36.6%, respectively, for Anoxybacillus amylolyticus. The optimum temperatures for heavy metal biosorption were near the optimum growth temperatures for both strains. Scatchard plot analysis was employed to obtain more compact information about the interaction between metal ions and biosorbents. The plot results were further studied to determine if they fit Langmuir and Freundlich models.     

Lipid composition modulates the interaction of peptides deriving from herpes simplex virus type I glycoproteins B and H with biomembranes

Lipid membranes play a key role in the viral life cycle. Enveloped viruses particularly require a sequence of fusion and fission events between the viral envelope and the target membranes for entry into the cell and egress from it. These processes are controlled by one or more viral glycoproteins that undergo conformational changes favoring the necessary micro- and mesoscopic lipid re-arrangements. Multiple regions from these glycoproteins are thought to interact with the membranes, according to a concerted mechanism, in order to generate the distortion necessary for fusion. In this work, we perform an EPR study on the role played by the membrane composition in tuning the interaction between lipid bilayers and two peptides, gH626-644 and gB632-650, that are highly fusogenic fragments of the gH and gB glycoproteins of herpes simplex virus. Our results show that both peptides interact with lipid bilayers, perturbing the local lipid packing. gH626-644 localizes close to the hydrophilic bilayer surface, while gB632-650 penetrates deeply into the membrane. Chain perturbation by the peptides increases in the presence of charged phospholipids. Finally, cholesterol does not alter the ability of gB632-650 to penetrate deeply in the membrane, whereas it limits penetration of the gH626-644 peptide to the more external layer. The different modes of interaction result in a higher fusogenic ability of gB632-650 towards cholesterol-enriched membranes, as demonstrated by lipid mixing assays. These results suggest that the mechanism of action of the gH and gB glycoproteins is modulated by the properties and composition of the phospholipid bilayer.     

Molasses as fermentation substrate for levan production by <Emphasis Type="Italic">Halomonas</Emphasis> sp.

Levan is a homopolymer of fructose with many outstanding properties like high solubility in oil and water, strong adhesiveness, good biocompatibility, and film-forming ability. However, its industrial use has long been hampered by costly production processes which rely on mesophilic bacteria and plants. Recently, Halomonas sp. AAD6 halophilic bacteria were found to be the only extremophilic species producing levan at high titers in semi-chemical medium containing sucrose, and in this study, pretreated sugar beet molasses and starch molasses were both found to be feasible substitutes for sucrose. Five different pretreatment methods and their combinations were applied to both molasses types. Biomass and levan concentrations reached by the Halomonas sp. AAD6 cells cultivated on 30 g/L of pretreated beet molasses were 6.09 g dry cells/L and 12.4 g/L, respectively. When compared with literature, Halomonas sp. was found to stand out with its exceptionally high levan production yields on available fructose. Molecular characterization and monosaccharide composition studies confirmed levan-type fructan structure of the biopolymers. Rheological properties under different conditions pointed to the typical characteristics of low viscosity and pseudoplastic behaviors of the levan polymers. Moreover, levan polymer produced from molasses showed high biocompatibility and affinity with both cancerous and non-cancerous cell lines.     

Role of proteomics to differentiate between benign and potentially malignant pancreatic cysts

Pancreatic cystic neoplasms represent 10-15% of primary cystic masses of the pancreas. While pancreatic cysts are detected with an increasing frequency due to the use of advanced imaging modalities in clinical practice, the diagnosis of pancreatic cystic neoplasms remains unsatisfactory because available diagnostic techniques proved not sensitive enough so far. This study was designed to characterize the proteomic pattern of pancreatic cyst fluids obtained from various cystic lesions. Cyst fluids were collected by direct puncture during open surgery to avoid any possible contamination from other tissues. CEA, CA-19-9, and amylase concentrations were measured using specific immunoassays. After immunodepletion and fractionation by SDS-PAGE, proteins were digested and analyzed by LC-MS/MS. Specific histological lesions were found to be associated with distinct protein patterns. Interestingly, some of these proteins have been proposed as biomarkers of pancreatic cancer. Immunoblots allowed for verifying the differential expression in specific cyst fluids of two selected proteins, olfactomedin-4 and mucin-18. Finally, immunohistochemistry was performed to correlate these data with the expression pattern of olfactomedin-4 and mucin-18 in pancreatic cyst tissues. Results from this study indicate that proteomic analysis of cyst fluid could provide reliable candidates for developing new biomarkers for the preoperative management of malignant and premalignant pancreatic cysts.