Migration of vesicular stomatitis virus glycoprotein to the nucleus of infected cells.

A new means of direct visualization of the early events of viral infection by selective fluorescence labeling of viral proteins coupled with digital imaging microscopy is reported. The early phases of viral infection have great importance for understanding viral replication and pathogenesis. Vesicular stomatitis virus, the best-studied rhabdovirus, is composed of an RNA genome of negative sense, five viral proteins, and membrane lipids derived from the host cell. The glycoprotein of vesicular stomatitis virus was labeled with fluorescein isothiocyanate, and the labeled virus was incubated with baby hamster kidney cells. After initiation of infection, the fluorescence of the labeled glycoprotein was first seen inside the cells in endocytic vesicles. The fluorescence progressively migrated to the nucleus of infected cells. After 1 h of infection, the virus glycoprotein was concentrated in the nucleus and could be recovered intact in a preparation of purified nuclei. These results suggest that uncoating of the viral RNA occurs close to the nuclear membrane, which would precede transcription of the leader RNA that enters the nucleus to shut off cellular RNA synthesis and DNA replication.

Myristate exchange on the Trypanosoma brucei variant surface glycoprotein.

The glycosyl-phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG) is unique in having exclusively myristate as its fatty acid component. We previously demonstrated that the myristate specificity is the result of two independent pathways. First, the newly synthesized free GPI, which is not myristoylated, undergoes fatty acid remodeling to replace both its fatty acids with myristate. Second, the myristoylated precursor, glycolipid A, undergoes a myristate exchange reaction, detected by the replacement of unlabeled myristate by [3H]myristate. Remodeling and exchange have different enzymatic properties and apparently occur in different subcellular compartments. We now demonstrate that the GPI anchor linked to VSG is the major substrate for myristate exchange. VSG can be efficiently labeled with [3H]myristate by exchange in the presence of cycloheximide, an inhibitor that prevents new VSG synthesis and thus anchor addition to protein. Not only is newly synthesized VSG subject to exchange, but mature VSG, possibly recycling from the cell surface, also undergoes myristate exchange.

Facile synthesis of rhodamine esters using acetyl chloride in alcohol solution

The rhodamines are a highly fluorescent class of compound used in many different fields of research, from the lasing medium in dye lasers to biological stains and markers for cellular drug resistance. In this study, esters (2-7) of rhodamine 110 (1) were conveniently prepared via the addition of acetyl chloride to a solution of the free acid (1) in the appropriate alcohol. This method conferred several advantages over previous preparations, namely that for low boiling alcohols, simple evaporation of the solution afforded the ester in quantitative yield with no need for purification. For higher boiling point alcohols, a method has been developed which allows the separation of longer chain esters from the alcohol solvent.

Ebola virus glycoprotein GP is not cytotoxic when expressed constitutively at a moderate level

Transient expression of Ebola virus (EBOV) glycoprotein GP causes downregulation of surface proteins, cell rounding and detachment, a phenomenon believed to play a central role in the pathogenicity of the virus. In this study, evidence that moderate expression of GP does not result in such morphological changes was provided. It was shown that GP continuously produced in 293T cells from the Kunjin virus replicon was correctly processed and transported to the plasma membrane without affecting the surface expression of beta 1 and alpha 5 integrins and major histocompatibility complex I molecules. The level of GIP expression in Kunjin replicon GP-expressing cells was similar to that observed in cells infected with EBOV early in infection and lower than that produced in cells transfected with plasmid DNA, phCMV-GP(1) expressing GP from a strong promoter. Importantly, transient transfection of Kunjin replicon GIP-expressing cells with GIP-coding plasmid DNA resulted in overexpression of GP, which lead to the downregulation of surface molecules and massive rounding and detachment of transfected cells. Here, it was also demonstrated that cell rounding and downregulation of the surface markers are the late events in EBOV infection, whereas synthesis and massive release of virus particles occur at early steps and do not cause significant cytotoxic effects. These findings indicate that the synthesis of EBOV GP in virus-infected cells is controlled well by several mechanisms that do not allow GP overexpression and hence the early appearance of its cytotoxic properties.

Mechanism of attenuation of skeletal muscle atrophy by zinc-α2-glycoprotein

The mechanism by which the adipokine zinc-a2-glycoprotein (ZAG) increases the mass of gastrocnemius, but not soleus muscle of diabetic mice, has been evaluated both in vivo and in vitro. There was an increased phosphorylation of both double-stranded RNA-dependent protein kinase and its substrate, eukaryotic initiation factor-2a, which was attenuated by about two-thirds in gastrocnemius but not soleus muscle of ob/ob mice treated with ZAG (50 µg, iv daily) for 5 d. ZAG also reduced the expression of the phospho forms of p38MAPK and phospholipase A2, as well as expression of the ubiquitin ligases (E3) muscle atrophy F-box/atrogin-1 and muscle RING finger protein, and the increased activity of both caspase-3 and casapse-8 to values found in nonobese controls. ZAG also increased the levels of phospho serine-threonine kinase and mammalian target of rapamycin in gastrocnemius muscle and reduced the phosphorylation of insulin receptor substrate-1 (Ser307) associated with insulin resistance. Similar changes were seen with ZAG when murine myotubes were incubated with high glucose concentrations (10 and 25 mm), showing that the effect of ZAG was direct. ZAG produced an increase in cAMP in murine myotubes, and the effects of ZAG on protein synthesis and degradation in vitro could be replicated by dibutyryl cAMP. ZAG increased cAMP levels of gastrocnemius but not soleus muscle. These results suggest that protein accretion in skeletal muscle in response to ZAG may be due to changes in intracellular cAMP and also that ZAG may have a therapeutic application in the treatment of muscle wasting conditions.

Antidiabetic properties of zinc-alpha2-glycoprotein in ob/ob mice

Zinc-alpha(2)-glycoprotein (ZAG) is an adipokine associated with fat loss in cancer cachexia. The purpose of this study was to evaluate the ability of recombinant human ZAG to attenuate type 2 diabetes in the ob/ob mouse model. ZAG (50 microg daily, iv) induced a progressive loss of body weight (3.5 g in 5 d), without an effect on food or water intake but with a 0.4 C rise in body temperature, suggesting an increased energy expenditure. Despite an increased plasma glycerol, indicative of increased lipolysis, levels of glucose, triglycerides, and nonesterified fatty acids were decreased by 17, 25, and 62%, respectively, due to an increased use of both glucose and lipids by muscle and brown adipose tissue. The weight of the latter increased 2-fold, and there was increased expression of uncoupling proteins-1 and -3. Plasma insulin levels were reduced by 36%, whereas pancreatic insulin was increased 4-fold, and there was a 53% decrease in the total area under the glucose curve in the glucose tolerance test and reduced insulin requirement. There was an increase in skeletal muscle mass due to an increase in protein synthesis and a decrease in protein degradation. These results suggest that ZAG may potentially be effective in the treatment of type 2 diabetes.

Selective in vitro glycosylation of recombinant proteins:semi-synthesis of novel homogeneous glycoforms of human erythropoietin

Background: A natural glycoprotein usually exists as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties, although details of structure–function relationships are poorly understood, because of the microheterogeneity of biological samples. Hence, there is clearly a need for synthetic methods that give access to natural and unnatural homogeneously glycosylated proteins. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine residues, placed by site-directed mutagenesis at desired glycosylation sites has been developed. This provides a general method for the synthesis of homogeneously glycosylated proteins that carry saccharide side chains at natural or unnatural glycosylation sites. Here, we have shown that the approach can be applied to the glycoprotein hormone erythropoietin, an important therapeutic glycoprotein with three sites of N-glycosylation that are essential for in vivo biological activity. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins. Results: Wild-type recombinant erythropoietin and three mutants in which glycosylation site asparagine residues had been changed to cysteines (His10-WThEPO, His10-Asn24Cys, His10-Asn38Cys, His10-Asn83CyshEPO) were overexpressed and purified in yields of 13 mg l−1 from Escherichia coli. Chemical glycosylation with glycosyl-β-N-iodoacetamides could be monitored by electrospray MS. Both in the wild-type and in the mutant proteins, the potential side reaction of the other four cysteine residues (all involved in disulfide bonds) were not observed. Yield of glycosylation was generally about 50% and purification of glycosylated protein from non-glycosylated protein was readily carried out using lectin affinity chromatography. Dynamic light scattering analysis of the purified glycoproteins suggested that the glycoforms produced were monomeric and folded identically to the wild-type protein. Conclusions: Erythropoietin expressed in E. coli bearing specific Asn→Cys mutations at natural glycosylation sites can be glycosylated using β-N-glycosyl iodoacetamides even in the presence of two disulfide bonds. The findings provide the basis for further elaboration of the glycan structures and development of this general methodology for the synthesis of semi-synthetic glycoproteins

Design, Synthesis And Biological Evaluation Of Hybrid Bioisoster Derivatives Of N-acylhydrazone And Furoxan Groups With Potential And Selective Anti-trypanosoma Cruzi Activity.

Hybrid bioisoster derivatives from N-acylhydrazones and furoxan groups were designed with the objective of obtaining at least a dual mechanism of action: cruzain inhibition and nitric oxide (NO) releasing activity. Fifteen designed compounds were synthesized varying the substitution in N-acylhydrazone and in furoxan group as well. They had its anti-Trypanosoma cruzi activity in amastigotes forms, NO releasing potential and inhibitory cruzain activity evaluated. The two most active compounds (6, 14) both in the parasite amastigotes and in the enzyme contain the nitro group in para position of the aromatic ring. The permeability screening in Caco-2 cell and cytotoxicity assay in human cells were performed for those most active compounds and both showed to be less cytotoxic than the reference drug, benznidazole. Compound 6 was the most promising, since besides activity it showed good permeability and selectivity index, higher than the reference drug. Thereby the compound 6 was considered as a possible candidate for additional studies.

Synthesis And Antiproliferative Activity Of 8-hydroxyquinoline Derivatives Containing A 1,2,3-triazole Moiety.

Twelve novel 8-hydroxyquinoline derivatives were synthesized with good yields by performing copper-catalyzed Huisgen 1,3-dipolar cycloaddition (click reaction) between an 8-O-alkylated-quinoline containing a terminal alkyne and various aromatic or protected sugar azides. These compounds were evaluated in vitro for their antiproliferative activity on various cancer cell types. Protected sugar derivative 16 was the most active compound in the series, exhibiting potent antiproliferative activity and high selectivity toward ovarian cancer cells (OVCAR-03, GI50 < 0.25 μg mL(-1)); this derivative was more active than the reference drug doxorubicin (OVCAR-03, GI50 = 0.43 μg mL(-1)). In structure-activity relationship (SAR) studies, the physico-chemical parameters of the compounds were evaluated and docking calculations were performed for the α-glucosidase active site to predict the possible mechanism of action of this series of compounds.

Lawsonia Inermis-mediated Synthesis Of Silver Nanoparticles: Activity Against Human Pathogenic Fungi And Bacteria With Special Reference To Formulation Of An Antimicrobial Nanogel.

Lawsonia inermis mediated synthesis of silver nanoparticles (Ag-NPs) and its efficacy against Candida albicans, Microsporum canis, Propioniabacterium acne and Trichophyton mentagrophytes is reported. A two-step mechanism has been proposed for bioreduction and formation of an intermediate complex leading to the synthesis of capped nanoparticles was developed. In addition, antimicrobial gel for M. canis and T. mentagrophytes was also formulated. Ag-NPs were synthesized by challenging the leaft extract of L. inermis with 1 mM AgNO₃. The Ag-NPs were characterized by Ultraviolet-Visible (UV-Vis) spectrophotometer and Fourier transform infrared spectroscopy (FTIR). Transmission electron microscopy (TEM), nanoparticle tracking and analysis sytem (NTA) and zeta potential was measured to detect the size of Ag-NPs. The antimicrobial activity of Ag-NPs was evaluated by disc diffusion method against the test organisms. Thus these Ag-NPs may prove as a better candidate drug due to their biogenic nature. Moreover, Ag-NPs may be an answer to the drug-resistant microorganisms.

Albumin Is Synthesized In Epididymis And Aggregates In A High Molecular Mass Glycoprotein Complex Involved In Sperm-egg Fertilization.

The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

Stereoselective Synthesis Of Aryl Cyclopentene Scaffolds By Heck-matsuda Desymmetrization Of 3-cyclopentenol.

A new enantioselective Heck-Matsuda desymmetrization reaction was accomplished by using 3-cyclopentenol to produce chiral five-membered 4-aryl cyclopentenol scaffolds in good yields and high ee's, together with some 3-aryl-cyclopentanones as minor products. Mechanistically, the hydroxyl group of 3-cyclopentenol acts as a directing group and is responsible for the cis- arrangement in the formation of the 4-aryl-cyclopentenols.

Design And Synthesis Of N-acylated Aza-goniothalamin Derivatives And Evaluation Of Their In Vitro And In Vivo Antitumor Activity.

Herein we describe the synthesis of a focused library of compounds based on the structure of goniothalamin (1) and the evaluation of the potential antitumor activity of the compounds. N-Acylation of aza-goniothalamin (2) restored the in vitro antiproliferative activity of this family of compounds. 1-(E)-But-2-enoyl-6-styryl-5,6-dihydropyridin-2(1H)-one (18) displayed enhanced antiproliferative activity. Both goniothalamin (1) and derivative 18 led to reactive oxygen species generation in PC-3 cells, which was probably a signal for caspase-dependent apoptosis. Treatment with derivative 18 promoted Annexin V/7-aminoactinomycin D double staining, which indicated apoptosis, and also led to G2 /M cell-cycle arrest. In vivo studies in Ehrlich ascitic and solid tumor models confirmed the antitumor activity of goniothalamin (1), without signs of toxicity. However, derivative 18 exhibited an unexpectedly lower in vivo antitumor activity, despite the treatments being administered at the same site of inoculation. Contrary to its in vitro profile, aza-goniothalamin (2) inhibited Ehrlich tumor growth, both on the ascitic and solid forms. Our findings highlight the importance of in vivo studies in the search for new candidates for cancer treatment.

Synthesis And Antitumor Activity Of Novel 1-substituted Phenyl 3-(2-oxo-1,3,4-oxadiazol-5-yl) β-carbolines And Their Mannich Bases.

A series of novel 1-(substituted phenyl)-3-(2-oxo-1,3,4-oxadiazol-5-yl) β-carbolines (4a-e) and the corresponding Mannich bases 5-9(a-c) were synthesized and evaluated for their in vitro antitumor activity against seven human cancer cell lines. Compounds of 4a-e series showed a broad spectrum of antitumor activity, with GI50 values lower than 15μM for five cell lines. The derivative 4b, having the N,N-dimethylaminophenyl group at C-1, displayed the highest activity with GI50 in the range of 0.67-3.20μM. A high selectivity and potent activity were observed for some Mannich bases, particularly towards resistant ovarian (NCI-ADR/RES) cell lines (5a, 5b, 6a, 6c and 9b), and ovarian (OVCAR-03) cell lines (5b, 6a, 6c, 9a, 9b and 9c). In addition, the interaction of compound 4b with DNA was investigated by using UV and fluorescence spectroscopic analysis. These studies indicated that 4b interact with ctDNA by intercalation binding.

Clozapine Promotes Glycolysis And Myelin Lipid Synthesis In Cultured Oligodendrocytes.

Clozapine displays stronger systemic metabolic side effects than haloperidol and it has been hypothesized that therapeutic antipsychotic and adverse metabolic effects of these drugs are related. Considering that cerebral disconnectivity through oligodendrocyte dysfunction has been implicated in schizophrenia, it is important to determine the effect of these drugs on oligodendrocyte energy metabolism and myelin lipid production. Effects of clozapine and haloperidol on glucose and myelin lipid metabolism were evaluated and compared in cultured OLN-93 oligodendrocytes. First, glycolytic activity was assessed by measurement of extra- and intracellular glucose and lactate levels. Next, the expression of glucose (GLUT) and monocarboxylate (MCT) transporters was determined after 6 and 24 h. And finally mitochondrial respiration, acetyl-CoA carboxylase, free fatty acids, and expression of the myelin lipid galactocerebroside were analyzed. Both drugs altered oligodendrocyte glucose metabolism, but in opposite directions. Clozapine improved the glucose uptake, production and release of lactate, without altering GLUT and MCT. In contrast, haloperidol led to higher extracellular levels of glucose and lower levels of lactate, suggesting reduced glycolysis. Antipsychotics did not alter significantly the number of functionally intact mitochondria, but clozapine enhanced the efficacy of oxidative phosphorylation and expression of galactocerebroside. Our findings support the superior impact of clozapine on white matter integrity in schizophrenia as previously observed, suggesting that this drug improves the energy supply and myelin lipid synthesis in oligodendrocytes. Characterizing the underlying signal transduction pathways may pave the way for novel oligodendrocyte-directed schizophrenia therapies.