Tunicamycin

In this report, we have investigated the contribution of primary sequence to the carbohydrate requirement for intracellular transport of two closely related glycoproteins, the G proteins of the San Juan and Orsay strains of vesicular stomatitis virus. We used site-directed mutagenesis of the coding sequence to eliminate the two consensus sites for glycosylation in the Orsay G protein. Whereas the nonglycosylated San Juan G protein required at least one of its two asparagine-linked oligosaccharides for transport to the plasma membrane at 37 degrees C, a fraction of the Orsay G protein was transported without carbohydrate. Of the 10 amino acid differences between these two proteins, residue 172 (tyrosine in San Juan, aspartic acid in Orsay) played the major role in determining the stringency for the carbohydrate requirement. The rates at which the glycosylated and nonglycosylated Orsay G proteins were transported to the cell surface were the same, although a smaller fraction of the no...

In T47D breast cancer cell line, progestin (R5020) induces de novo synthesis of an alkaline phosphatase enzyme. Based on inhibitor profiles and antigenic specificity, it is apparent that this enzyme belongs to the class of membrane-associated tissue-unspecific alkaline phosphatases. Enzyme induction was uniquely specific to progestins and not altered by other steroid hormones or synthetic analogues. The progestin induction of the tissue-unspecific alkaline phosphatase was time and dose dependent. The protein synthesis inhibitor cycloheximide blocks the enzyme synthesis and tunicamycin blocks the enzyme activity, showing that the induction was new synthesis of protein in its complete glycosylated form and not activation of a preexisting enzyme. To our knowledge this is the first report of progesterone-induced expression of a tissue-unspecific alkaline phosphatase gene of such magnitude (about 30- to 100-fold) in a progesterone-responsive tissue.

M. ZAMFIR, R. CALLEWAERT, P.C. CORNEA, L. SAVU, I. VATAFU and L. DE VUYST.1999.Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6·5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 °C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.

Objectives: To investigate the neuroprotective efficacy of  Valeriana
wallichii  DC rhizome extract against 1-methyl-4-phenlypyridinium
(MPP
+
) and  tunicamycin induced cell death.  Methods:  The 50%
methanolic extract of  V. wallichii  rhizome (VWE) was evaluated for
free radical scavenging properties. Cell viability analysis and IC50
determination for VWE in SH-SY5Y neuroblastoma cell line was
performed by MTT assay. The neuroprotective efficacy of VWE in
SH-SY5Y cells against the damages induced by the neurotoxin,
MPP
+
and the endoplasmic reticulum stress inducer, tunicamycin was
assessed by MTT assay and by morphological analysis with phase
contrast microscope. MTT assay was performed using 3 different
concentrations (0.1, 0.5 and 1 mg/mL) of VWE that were added at 0,
8 and 16 h post MPP
+
and tunicamycin treatment.  Results:  VWE
showed good free radical scavenging properties. From the cell
viability analysis the IC50
value of VWE was calculated as 2.207
mg/mL in SH-SY5Y cells. In MPP
+
treated cells 0.5mg and 1mg/mL
concentrations of VWE brought significant improvement in cell
viability at 0h, while at 8h and 16h post MPP
+
treatment the effect
was significant at 1mg/ml concentration. VWE exhibited significant
protective effect in tunicamycin treated cells only at 0h but not at 8h
and 16h post treatment. MPP
+
and tunicamycin induced cell
shrinkage and condensation were inhibited by treatment with
1mg/mL of VWE.  Conclusions:  The present work is the first study
that sheds light on the cytoprotective effect of  Valeriana wallichii
DC rhizome and provides lead for identification and isolation of
novel drugs for various neurodegenerative diseases

Bovine rotavirus grown in the presence or absence of tunicamycin was analyzed with respect to yield of infectious virus, the ratio of complete to incomplete particles, and polypeptide composition. Tunicamycin at a concentration of 1 microgram/ml reduced virus yields by 4 logs and completely prevented the incorporation of [3H]uridine into complete rotavirus particles, as determined by cesium chloride gradient analysis. Concomitant with a reduction in complete particles, three rotavirus polypeptides shifted in their relative position on polyacrylamide gels from 41,900-molecular-weight position (41.9K), 29.3K, and 16.1K to migrate at 35.5K, 22.7K, and 15.5K, respectively. Limited proteolysis indicated that the lower-molecular-weight polypeptides possessed the same constituent peptides as the larger polypeptides, suggesting that they represented the unglycosylated equivalents. These results suggest that interference with glycosylation prevents proper assembly of the outer coat proteins ...

Human bitter taste receptors of the TAS2R gene family play a crucial role as warning sensors against the ingestion of toxic food compounds. Moreover, the genetically highly polymorphic hTAS2Rs recognize an enormous number of structurally diverse toxic and non-toxic bitter substances, and hence, may substantially influence our individual eating habits. Heterologous expression in mammalian cells is a useful tool to investigate interactions between these receptors and their agonists. However, many bitter taste receptors are poorly expressed at the cell surface of heterologous cells requiring the addition of plasma membrane export promoting epitopes to the native receptor proteins. Currently, nothing is known about amino acid motifs or other receptor-intrinsic features of TAS2Rs affecting plasma membrane association. In the present study, we analyzed the Asn-linked glycosylation of hTAS2Rs at a consensus sequence in the second extracellular loop, which is conserved among all 25 hTAS2Rs....

When MA104 cells were infected with SA11 rotavirus, 12 protein classes, absent in mock-infected cells, could be distinguished by polyacrylamide gel electrophoresis. At least two of these proteins were glycosylated, and their synthesis could be blocked with tunicamycin. The oligosaccharides of both glycoproteins were cleaved by endo-beta-N-acetylglucosaminidase H, suggesting that they were residues of the "high-mannose" type. Of the 12 viral polypeptides observed in infected cells, 1 was probably the apoprotein of one of these glycoproteins; 5, including 1 glycoprotein, were structural components of the virions, whereas the other 6, including a second and possibly third glycoprotein, were nonstructural viral proteins. When the 11 double-stranded RNA genome segments of SA11 were translated, after denaturation, in an RNA-dependent cell-free translation system, at least 11 different polypeptides were synthesized. Ten of these polypeptides had electrophoretic migration patterns...

Purpose Aberrant protein glycosylation and disassembly of E-cadherin-mediated cell–cell adhesion are characteristics of epithelial cancer. However, the relationship between these two events in colorectal cancer remains to be defined. In this study, we analyzed whether N-glycan expression is crucial for the loss of E-cadherin-mediated cell–cell adhesion in human colorectal cancer cells. Methods Differentiated Caco-2 and undifferentiated HCT-116 colon cancer cells were used as models of stable and unstable adherens junctions (AJs), respectively. Complex-type N-glycans were detected using the lectins E-PHA (Phaseolus vulgaris E.) and L-PHA (Phaseolus vulgaris L.). To study E-cadherin-mediated AJ assembly, we examined the effects of swainsonine, an inhibitor of α-mannosidase II, and tunicamycin, a drug that inhibits the biosynthesis of N-glycans, via western blot, immunofluorescence, differential extraction in Triton X-100, and electron microscopy. Cell proliferation and apoptosis were examined by crystal violet staining and flow cytometry, respectively. Results We observed positive labeling for E-PHA and L-PHA lectins in both cell lines; however, HCT-116 cells had increased E-cadherin-linked complex-type N-glycans. Interestingly, tunicamycin, but not swainsonine, was able to induce functional E-cadherin-mediated cell–cell adhesion in undifferentiated HCT-116 cells, as shown by the increased association of E-cadherin with the actin cytoskeleton. Moreover, in HCT-116 cells, tunicamycin also induced the formation of tight cell–cell contacts, and it inhibited cell proliferation without triggering apoptosis. Conclusions Collectively, our results demonstrate for the first time that altered N-glycan expression plays an important role in the loss of AJ stability in undifferentiated colorectal cancer cells and that this loss may be associated with the progression of colorectal cancer.

Production of immuno and biologically active erythropoietin was documented to occur in the human hepatoblastoma cell line HepG-2. The expression of the erythropoietin gene was further verified by Northern blot analysis using a single stranded RNA probe. In vitro studies showed that erythropoietin production by these cells was not stimulated by hypoxia or cobalt chloride, but was related to the proliferative activity of the cells in culture. In addition it was found that the secretion of erythropoietin was almost completely abrogated by tunicamycin, an inhibitor of N-linked glycosylation. This effect of tunicamycin was also observed in a permanently transfected cell line that secretes erythropoietin in large quantities.

Adenylyl cyclases (ACs) are a family of critically important signaling molecules that are regulated by multiple pathways. Adenylyl cyclase 8 (AC8) is a Ca(2+) stimulated isoform that displays a selective regulation by capacitative Ca(2+) entry (CCE), the process whereby the entry of Ca(2+) into cells is triggered by the emptying of intracellular stores. This selectivity was believed to be achieved through the localization of AC8 in lipid raft microdomains, along with components of the CCE apparatus. In the present study, we show that an intact leucine zipper motif is required for the efficient N-linked glycosylation of AC8, and that this N-linked glycosylation is important to target AC8 into lipid rafts. Disruption of the leucine zipper by site-directed mutagenesis results in the elimination of N-glycosylated forms and their exclusion from lipid rafts. Mutants of AC8 that cannot be N-glycosylated are not demonstrably associated with rafts, although they can still be regulated by CCE; however, raft integrity is required for the regulation of these mutants. These findings suggest that raft localized proteins in addition to AC8 are needed to mediate its regulation by CCE.

Lipoprotein lipase (LPL) is the enzyme responsible for the hydrolysis of plasma triglycerides from apolipoprotein C-II-containing lipoproteins at the capillary endothelium and it is synthesized in parenchymal cells of several tissues. Intracellular LPL processing is a major aspect of LPL regulation. The present study aims to determine the intracellular accumulation site of the LPL that is not glycosylated at Asn43. Human LPL (hLPL) cDNA was mutated by site-directed mutagenesis. An Ala residue was substituted for Asn at position 43 of the protein generating N43A hLPL. Wild type hLPL and the mutant hLPL were expressed in COS1 cells. Using immunofluorescence and immunoelectron microscopy we found that wild type hLPL in addition to being secreted into the medium was present in the rough endoplasmic reticulum (ER), Golgi compartments, and vesicles. Neither LPL activity nor protein was found in medium of cells expressing the mutant hLPL and all detectable protein was present exclusively i...

Tetracyclines (doxycycline and minocycline) inhibit inducible NO synthase expression and augment cyclooxygenase (COX)-2 expression and PGE2 production. In contrast, chemically modified tetracyclines (CMTs), such as CMT-3 and -8 (but not CMT-1, -2, and -5), that lack antimicrobial activity, inhibit both NO and PGE2 production in LPS-stimulated murine macrophages, bovine chondrocytes, and human osteoarthritis-affected cartilage, which spontaneously produces NO and PGE2 in ex vivo conditions. Furthermore, CMT-3 augments COX-2 protein expression but inhibits net PGE2 accumulation. This coincides with the ability of CMT-3 and -8 to inhibit COX-2 enzyme activity in vitro. The action of CMTs is distinct from that observed with tetracyclines because 1) CMT-3-mediated inhibition of PGE2 production coincides with modification of COX-2 protein, which is distinct from the nonglycosylated COX-2 protein generated in the presence of tunicamycin, as observed by Western blot analysis and 2) CMT-3 an...