The Degree and Length of O-Glycosylation of Recombinant Proteins Produced in Pichia pastoris Depends on the Nature of the Protein and the Process Type

The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N-linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O-glycosylation. O-glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O-mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O-glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, P_GAP and P_AOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI-MS. In the samples expressed with P_GAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with P_AOX1. The results indicate that the O-glycosylation level is markedly higher when the protein is produced in a methanol-based expression system.     

Low dose computerized tomography examinations of coronary occlusive disease

Background: The aim of this study was to present an original technique of low-dose coronary computerized tomographyangiography (CCTA) for the evaluation and early diagnosis of coronary occlusive disease (COD) and to compare from this technique of CCTA with those resulting from the latest conventional angiography and multidetector computerized tomography units. Methodology: The study included 820 CCTA exams of patients with COD (average age 61 +/? 7 years), with a follow-up exam in 204 male (39%) and 62 female (20%) patients with hemodynamically insignificant coronary occlusion. Exams were performed using a 64-slice computerized tomography (CT) unit using electrocardiography (ECG)-triggering and individual settings (voltage of the x-ray tube and effective tube-current) based on each patient’s body mass index. Exponential dose for each exam was defined. Results:There was a statistically significant progression in the number of patients in whom occlusion of one of 3 coronary arteries occurred in hemodynamically significant occlusive disease (occlusion of more than 50% of lumen) — 60 of 204 males and 12 of 62 females (p<0.0001 and p<0.001). The mid-effective radiation dose during CCTA exams was 1.9+/-0.7mSv (range of 0.9 to 3.9 mSv). Conclusion: Prospective ECG-triggering allowed for low-dose CCTA exams while still enabling high diagnostic accuracy in evaluating patients with COD. The technique used in this study resulted in 2 times less the exponential dose than conventional angiography.     

Molecular dynamics study of the proposed proton transport pathways in [FeFe]-hydrogenase

Possible proton transport pathways in Clostridium pasteurianum (CpI) [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to evaluate the functional pathway and provide insight into the hydrogen bonding features defining an active proton transport pathway. Three pathways were evaluated, two of which consist of water wires and one of predominantly amino acid residues. Our simulations suggest that protons are not transported through water wires. Instead, the five-residue motif (Glu282, Ser319, Glu279, H₂O, Cys299) was found to be the likely pathway, consistent with previously made experimental observations. The pathway was found to have a persistent hydrogen bonded core (residues Cys299 to Ser319), with less persistent hydrogen bonds at the ends of the pathway for both H₂ release and H₂ uptake. Single site mutations of the four residues have been shown experimentally to deactivate the enzyme. The theoretical evaluation of these mutations demonstrates redistribution of the hydrogen bonds in the pathway, resulting in enzyme deactivation. Finally, coupling between the protein dynamics near the proton transport pathway and the redox partner binding regions was also found as a function of H₂ uptake and H₂ release states, which may be indicative of a correlation between proton and electron movement within the enzyme.     

Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin-Dendra2 fusion proteins

Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin-Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.     

Risk factors for cardiovascular disease in rural area of Croatia

Rural areas, where 47.6% of the Croatian population lives are not generally the focus of research; yet there are challenges which affect the rural population that often go unreported. These communities often exhibit disadvantages in many areas of health. The aim of this study is to examine the specific health needs and related determinants of rural populations influenced by transition that were affected by the consequences of war. The focus of the research is rural lifestyle, behaviour and cardiovascular risk factors in three villages of Sisacko-moslavacka County. Results show that participants generally understand that their own lifestyles influence their health, but they often neglect to change their behaviour to improve their health. This can be explained through complex socio-economic conditions and traditional values of their heritage. These results suggest a need for further research on health status, attitude, and behaviour of Croatia's rural population. Specific public health intervention and services for rural populations must be promoted.     

Mon2, a relative of large Arf exchange factors, recruits Dop1 to the Golgi apparatus

The protein Mon2 is distantly related to the guanine nucleotide exchange factors (GEFs) that activate Arf1 on Golgi membranes. However, unlike these "large" Arf GEFs, Mon2 lacks the Sec7 domain that catalyzes nucleotide exchange on Arf1. Here we report that yeast Mon2 shares extensive homology with the noncatalytic parts of both the BIG and Golgi brefeldin A resistance factor subfamilies of Arf GEFs and is located to the trans-Golgi. Moreover, we find that Mon2 forms a complex with Dop1, a large cytoplasmic protein conserved in evolution from humans to protozoa. Deletion of Mon2 results in mislocalization of Dop1 from the Golgi and defects in cycling between endosomes and the Golgi. However, unlike Mon2, Dop1 is essential for yeast viability. A conditional allele of Dop1 shows that loss of Dop1 activity not only affects endosome to Golgi transport but also causes a severe perturbation of the organization of the endoplasmic reticulum. Thus, it appears that Dop1 plays a widespread role in membrane organization, and Mon2 acts as a scaffold to recruit the Golgi-localized pool of Dop1.     

Regulation of cathepsins S and L by cystatin F during maturation of dendritic cells

In dendritic cells (DCs) cysteine cathepsins play a key role in antigen processing, invariant chain (Ii) cleavage and regulation of cell adhesion after maturation stimuli. Cystatin F, a cysteine protease inhibitor, is present in DCs in endosomal/lysosomal vesicles and thus has a potential to modulate cathepsin activity. In immature DCs cystatin F colocalizes with cathepsin S. After induction of DC maturation however, it is translocated into lysosomes and colocalizes with cathepsin L. The inhibitory potential of cystatin F depends on the properties of the monomer. We showed that the full-length monomeric cystatin F was a 12-fold stronger inhibitor of cathepsin S than the N-terminally processed cystatin F, whereas no significant difference in inhibition was observed for cathepsins L, H and X. Therefore, the role of cystatin F in regulating the main cathepsin S function in DCs, i.e. the processing of Ii, may depend on the form of the monomer present in endosomal/lysosomal vesicles. On the other hand, intact and truncated monomeric cystatin F are both potent inhibitors of cathepsin L and it is likely that cystatin F could regulate its activity in maturing, adherent DCs, controlling the processing of procathepsin X, which promotes cell adhesion via activation of Mac-1 (CD11b/CD18) integrin receptor.     

Anti-biofilm activities of essential oils rich in carvacrol and thymol against Salmonella Enteritidis

The aim of the present study was to determine the bioactive compounds in four essential oils (EO’s) from Origanum heracleoticum, Origanum vulgare, Thymus vulgaris and Thymus serpyllum and to assess their antimicrobial and anti-biofilm activity against Salmonella Enteritidis. Strains were previously characterized depending on the expression of the extracellular matrix components cellulose and curli fimbriae as rdar (red, dry and rough) and bdar morphotype (brown, dry and rough). This study revealed that the EO’s and EOC’s (carvacrol and thymol) investigated showed inhibition of biofilm formation at sub-minimum inhibitory concentration. Comparing the efficacy of EO’s and EOC’s in the inhibition of biofilm formation between the strains with different morphotype (rdar and bdar) did not show a statistically significant difference. Results related to the effectiveness of EO’s and EOC’s (the essential oil components, carvacrol and thymol) on eradication of preformed 48?h old biofilms indicated that biofilm reduction occurred in a dose-dependent manner over time.