Radiolabeling of the antibody IgG M75 for epitope of human carbonic anhydrase IX by Cu and Cu and its biological testing.

Human carbonic anhydrase IX is a membrane enzyme that is significantly expressed in some types of cancer cells, while copper radioisotopes offer wide range of diagnostic, therapeutic and theranostic properties. The work was focused on a new approach to the labelling of antibody IgG M75 for epitope human carbonic anhydrase IX with copper radioisotopes Cu and Cu and its in vivo testing in mice with inoculated colorectal cancer. Monoclonal antibody IgG M75 for epitope human carbonic anhydrase IX was successfully conjugated with copper-specific chelator "phosphinate" and labelled with Cu and Cu The obtained molecule has considerable potential as a radioimmuno pharmaceutical suitable for imaging of tumours expressing carbonic anhydrase IX by positron emission tomography (PET).

SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties.

Background: Serine proteases are important virulence factors for many pathogens. Recently, we discovered a group of trypsin-like serine proteases with domain organization unique to flatworm parasites and containing a thrombospondin type 1 repeat (TSR-1). These proteases are recognized as antigens during host infection and may prove useful as anthelminthic vaccines, however their molecular characteristics are under-studied.

Novel Structural Mechanism of Allosteric Regulation of Aspartic Peptidases via an Evolutionarily Conserved Exosite.

Pepsin-family aspartic peptidases are biosynthesized as inactive zymogens in which the propeptide blocks the active site until its proteolytic removal upon enzyme activation. Here, we describe a novel dual regulatory function for the propeptide using a set of crystal structures of the parasite cathepsin D IrCD1. In the IrCD1 zymogen, intramolecular autoinhibition by the intact propeptide is mediated by an evolutionarily conserved exosite on the enzyme core.

Activation route of the Schistosoma mansoni cathepsin B1 drug target: structural map with a glycosaminoglycan switch.

Cathepsin B1 (SmCB1) is a digestive protease of the parasitic blood fluke Schistosoma mansoni and a drug target for the treatment of schistosomiasis, a disease that afflicts over 200 million people. SmCB1 is synthesized as an inactive zymogen in which the N-terminal propeptide blocks the active site. We investigated the activation of the zymogen by which the propeptide is proteolytically removed and its regulation by sulfated polysaccharides (SPs).

Enzymatic activity and immunoreactivity of Aca s 4, an alpha-amylase allergen from the storage mite Acarus siro.

Background: Enzymatic allergens of storage mites that contaminate stored food products are poorly characterized. We describe biochemical and immunological properties of the native alpha-amylase allergen Aca s 4 from Acarus siro, a medically important storage mite.

Results: A.

Comparison of the influence of small GTPases Arl1 and Ypt6 on yeast cells' tolerance to various stress factors.

The GTPases Arl1 and Ypt6 are involved in the intracellular transport of vesicles and their fusion with the trans-Golgi network. This work is focused on comparing the roles of these GTPases in the tolerance of Saccharomyces cerevisiae cells to an increased concentration of alkali metal cations and other stress factors. We studied the phenotypes of arl1 or ypt6 deletions in combination with the deletions of genes encoding alkali-metal-cation transporters (ena1-4, nha1, nhx1, and kha1).

A genomewide screen for tolerance to cationic drugs reveals genes important for potassium homeostasis in Saccharomyces cerevisiae.

Potassium homeostasis is crucial for living cells. In the yeast Saccharomyces cerevisiae, the uptake of potassium is driven by the electrochemical gradient generated by the Pma1 H(+)-ATPase, and this process represents a major consumer of the gradient. We considered that any mutation resulting in an alteration of the electrochemical gradient could give rise to anomalous sensitivity to any cationic drug independently of its toxicity mechanism.

Mapping the pro-peptide of the Schistosoma mansoni cathepsin B1 drug target: modulation of inhibition by heparin and design of mimetic inhibitors.

Blood flukes of the genus Schistosoma cause the disease schistosomiasis that infects over 200 million people worldwide. Treatment relies on just one drug, and new therapies are needed should drug resistance emerge. Schistosoma mansoni cathepsin B1 (SmCB1) is a gut-associated protease that digests host blood proteins as source of nutrients.

Genetic interactions among the Arl1 GTPase and intracellular Na(+) /H(+) antiporters in pH homeostasis and cation detoxification.

The roles of intracellular GTPase Arl1 and organellar cation/H(+) antiporters (Kha1 and Nhx1) in Saccharomyces cerevisiae tolerance to various stress factors were investigated and interesting new phenotypes of strains devoid of these proteins were found. The role of Arl1 GTPase in their tolerance to various cations is not caused by an altered plasma-membrane potential. Besides the known sensitivity of arl1 mutants to high temperature, we discovered their sensitivity to low temperature.

New applications of pHluorin--measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis.

pHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pH(in)) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media.

Membrane hyperpolarization drives cation influx and fungicidal activity of amiodarone.

Cationic amphipathic drugs, such as amiodarone, interact preferentially with lipid membranes to exert their biological effect. In the yeast Saccharomyces cerevisiae, toxic levels of amiodarone trigger a rapid influx of Ca(2+) that can overwhelm cellular homeostasis and lead to cell death. To better understand the mechanistic basis of antifungal activity, we assessed the effect of the drug on membrane potential.

Comparative analyses of the 9 glycoprotein genes found in wild-type and vaccine strains of varicella-zoster virus.

The complete DNA sequences of wild-type and vaccine strains of varicella-zoster virus have been published and listed in GenBank. In this comparative genomic analysis, the sequences of the 9 glycoprotein open reading frames (ORFs) were compared. They included gE (ORF68), gI (ORF 67), gC (ORF14), gH (ORF37), gL (ORF60), gB (ORF31), gK (ORF5), gM (ORF50), and gN (ORF8 or ORF9A).

Applications of a microplate reader in yeast physiology research.

Microplate readers have been useful assistants of researchers for several decades. This work is focused on the applications of a simple absorbance microplate reader in yeast physiology research, and its advantages and limitations in comparison with alternative methods are discussed. The two main procedures involved are measuring growth curves and monitoring the pH changes of medium using two different pH indicators.

Cathepsin D propeptide: mechanism and regulation of its interaction with the catalytic core.

Propeptide blocks the active site in the inactive zymogen of cathepsin D and is cleaved off during zymogen activation. We have designed a set of peptidic fragments derived from the propeptide structure and evaluated their inhibitory potency against mature cathepsin D using a kinetic assay. Our mapping of the cathepsin D propeptide indicated two domains in the propeptide involved in the inhibitory interaction with the enzyme core: the active site "anchor" domain and the N-terminus of the propeptide.

Arabidopsis thaliana CHX17 gene complements the kha1 deletion phenotypes in Saccharomyces cerevisiae.

AtChx17p is a putative K(+)/H(+) exchanger from Arabidopsis thaliana, expressed in the roots and probably involved in K(+) acquisition and homeostasis. AtCHX17 cDNA complements the phenotypes of the kha1Delta mutation in S. cerevisiae cells: a growth defect at increased pH and hygromycin sensitivity.

Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance.

K+ is one of the cations (besides protons) whose transport across the plasma membrane is believed to contribute to the maintenance of membrane potential. To ensure K+ transport, Saccharomyces cerevisiae cells possess several types of active and passive transporters mediating the K+ influx and efflux, respectively. A diS-C3(3) assay was used to compare the contributions of various potassium transporters to the membrane potential changes of S.

Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants.

Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. In Saccharomyces cerevisiae, the high K+ content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters formerly believed to extrude K+ from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p and presumed as a putative plasma membrane K+/H+ antiporter.

Incorporation of three endocytosed varicella-zoster virus glycoproteins, gE, gH, and gB, into the virion envelope.

The cytoplasmic tails of all three major varicella-zoster virus (VZV) glycoproteins, gE, gH, and gB, harbor functional tyrosine-based endocytosis motifs that mediate internalization. The aim of the present study was to examine whether endocytosis from the plasma membrane is a cellular route by which VZV glycoproteins are delivered to the final envelopment compartment. In this study, we demonstrated that internalization of the glycoproteins occurred in the first 24 h postinfection but was reduced later in infection.

Regulation of varicella-zoster virus-induced cell-to-cell fusion by the endocytosis-competent glycoproteins gH and gE.

The gH glycoprotein of varicella-zoster virus (VZV) is a major fusogen. The realigned short cytoplasmic tail of gH (18 amino acids) harbors a functional endocytosis motif (YNKI) that mediates internalization in both VZV-infected and transfected cells (T. J.

Physiological characterization of osmotolerant yeast Pichia sorbitophila and comparison with a putative synonym Pichia farinosa.

The osmotolerant yeast Pichia sorbitophila was found to differ from other yeast species, not only from the conventional ones (Saccharomyces cerevisiae, Schizosaccharomyces pombe), but also from those widely known as osmotolerant (Debaryomyces hansenii, Zygosaccharomyces rouxii). P. sorbitophila was able to survive extremely high extracellular concentrations of salts (e.

A functional YNKI motif in the short cytoplasmic tail of varicella-zoster virus glycoprotein gH mediates clathrin-dependent and antibody-independent endocytosis.

The trafficking of varicella-zoster virus (VZV) gH was investigated under both infection and transfection conditions. In initial endocytosis assays performed in infected cells, the three glycoproteins gE, gI, and gB served as positive controls for internalization from the plasma membrane. Subsequently, we discovered that gH in VZV-infected cells was also internalized and followed a similar trafficking pattern.

Identification of the authentic varicella-zoster virus gB (gene 31) initiating methionine overlapping the 3' end of gene 30.

The varicella-zoster virus (VZV) gB sequence was re-examined in light of recent knowledge about unusually long gB signal peptides in other herpesviral gB homologs. Through mutational analysis, the discovery was made that the authentic initiating methionine for VZV gB is a codon beginning at genome nucleotide 56,819. The total length for the VZV gB primary translation product was 931 amino acids (aa) with a 71-aa signal sequence.

Immune response to E7 protein of human papillomavirus type 16 anchored on the cell surface.

To target the E7 protein of human papilloma virus 16 to the cell surface, a fusion gene was constructed. It encodes the signal peptide, part of the immunoglobulin (IgG)-like domain, the transmembrane anchor of vaccinia virus (VV) hemagglutinin (HA), and the complete E7-coding sequence. The fusion gene was expressed under the HA late promoter by a recombinant VV, designated VV-E7-HA.

Varicella-zoster Virus gB and gE coexpression, but not gB or gE alone, leads to abundant fusion and syncytium formation equivalent to those from gH and gL coexpression.

Varicella-zoster virus (VZV) is distinguished from herpes simplex virus type 1 (HSV-1) by the fact that cell-to-cell fusion and syncytium formation require only gH and gL within a transient-expression system. In the HSV system, four glycoproteins, namely, gH, gL, gB, and gD, are required to induce a similar fusogenic event. VZV lacks a gD homologous protein.