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Abstract The global COVID-19 pandemic caused by SARS-CoV2 infected millions of people and resulted in more than 4 million deaths worldwide. Apart from vaccines and drugs, RNA silencing is a novel approach for treating COVID-19. In the present study, siRNAs were designed for the conserved regions targeting three structural genes, M, N, and S, from forty whole-genome sequences of SARS-CoV2 using four different software, RNAxs, siDirect, i-Score Designer, and OligoWalk. Only siRNAs which were predicted in common by all the four servers were considered for further shortlisting. A multistep filtering approach has been adopted in the present study for the final selection of siRNAs by the usage of different online tools, viz., siRNA scales, MaxExpect, DuplexFold, and SMEpred. All these web-based tools consider several important parameters for designing functional siRNAs, e.g., target-site accessibility, duplex stability, position-specific nucleotide preference, inhibitory score, thermodynamic parameters, GC content, and efficacy in cleaving the target. In addition, a few parameters like GC content and dG value of the entire siRNA were also considered for shortlisting of the siRNAs. Antisense strands were subjected to check for any off-target similarities using BLAST. Molecular docking was carried out to study the interactions of guide strands with AGO2 protein. A total of six functional siRNAs (two for each gene) have been finally selected for targeting M, N, and S genes of SARS-CoV2. The siRNAs have not shown any off-target effects, interacted with the domain(s) of AGO2 protein, and were efficacious in cleaving the target mRNA. However, the siRNAs designed in the present study need to be tested in vitro and in vivo in the future.

The global COVID-19 pandemic caused by SARS-CoV2 infected millions of people and resulted in more than 4 million deaths worldwide. Apart from vaccines and drugs, RNA silencing is a novel approach for treating COVID-19. In the present study, siRNAs were designed for the conserved regions targeting three structural genes, M, N, and S, from forty whole-genome sequences of SARS-CoV2 using four different software, RNAxs, siDirect, i-Score Designer, and OligoWalk. Only siRNAs which were predicted in common by all the four servers were considered for further shortlisting. A multistep filtering approach has been adopted in the present study for the final selection of siRNAs by the usage of different online tools, viz., siRNA scales, MaxExpect, DuplexFold, and SMEpred. All these web-based tools consider several important parameters for designing functional siRNAs, e.g., target-site accessibility, duplex stability, position-specific nucleotide preference, inhibitory score, thermodynamic parameters, GC content, and efficacy in cleaving the target. In addition, a few parameters like GC content and dG value of the entire siRNA were also considered for shortlisting of the siRNAs. Antisense strands were subjected to check for any off-target similarities using BLAST. Molecular docking was carried out to study the interactions of guide strands with AGO2 protein. A total of six functional siRNAs (two for each gene) have been finally selected for targeting M, N, and S genes of SARS-CoV2. The siRNAs have not shown any off-target effects, interacted with the domain(s) of AGO2 protein, and were efficacious in cleaving the target mRNA. However, the siRNAs designed in the present study need to be tested in vitro and in vivo in the future. [ABSTRACT FROM AUTHOR]

High-throughput single-cell heterogeneity imaging and analysis is essential for understanding complex biological systems and for advancing personalized precision disease diagnosis and treatment. Here, we present a miniaturized lensless chemiluminescence chip for high-throughput single-cell functional imaging with subcellular resolution. With the sensitive chemiluminescence sensing and wide field of view of contact lensless imaging, we demonstrated the chemiluminescent imaging of over 1000 single cells, and their membrane glycoprotein and the high-throughput single-cell heterogeneity of membrane protein imaging were examined for precision analysis. Furthermore, the functional adhesion and heterogeneity of single live cells were imaged and explored. This miniaturized lensless on-chip CL-CMOS imaging platform enables high-throughput single-cell imaging and analysis with high sensitivity and subcellular resolution, providing new techniques for the cellular study of biological heterogeneity and has potential application in precision disease diagnosis and treatment at the point-of-care settings.

Novel coronavirus (SARS-CoV-2) leads to coronavirus disease 19 (COVID-19), declared as a pandemic that outbreaks within almost 225 countries worldwide. For the time being, numerous mutations have been reported that led to the generation of numerous variants spread more rapidly. This study aims to establish an efficient multi-epitope subunit vaccine that could elicit both T-cell and B-cell responses sufficient to recognize three confirmed surface proteins of the virus. The sequences of the viral surface proteins, e.g., an envelope protein (E), membrane glycoprotein (M), and S1 and S2 domain of spike surface glycoprotein (S), were analyzed by an immunoinformatic approach. Top immunogenic epitopes have been selected based on the assessment of the affinity with MHC class-I and MHC class-II, population coverage, along with conservancy among wild type and new variants of SARS-CoV-2 genomes. Molecular docking and molecular dynamic simulation suggest that the proposed top peptides have the potential to interact with the highest number of both the MHC class I and MHC class II. The epitopes were assembled by the appropriate linkers to form a multi-epitope vaccine. Epitopes used in the vaccine construct are conserved in all the variants evolved till now. This in silico-designed multi-epitope vaccine is highly immunogenic and induces levels of SARS-CoV2-neutralizing antibodies in mice, which is detected by inhibition of cytopathic effect in Vero cell monolayer. Further studies are required to improve its efficiency in the prevention of virus replication in lung tissue, in addition to safety validation as a step for human application to combat SARS-CoV-2 variants. Key points: • We discovered five T-cell epitopes from three surface proteins of SARS-CoV-2. • These are conserved in the wild-type virus and variants, e.g., beta, delta, and omicron. • The multi-epitope vaccine can induce IgG in mice that can neutralize the virus. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 is the virus responsible for causing COVID-19 disease in humans, creating the recent pandemic across the world, where lower production of Type I Interferon (IFN-I) is associated with the deadly form of the disease. Membrane protein or SARS-CoV-2 M proteins are known to be the major reason behind the lower production of human IFN-I by suppressing the expression of IFNβ and Interferon Stimulated Genes. In this study, 7,832 compounds from 32 medicinal plants of India possessing traditional knowledge linkage with pneumonia-like disease treatment, were screened against the Homology-Modelled structure of SARS-CoV-2 M protein with the objective of identifying some active phytochemicals as inhibitors. The entire study was carried out using different modules of Schrodinger Suite 2020-3. During the docking of the phytochemicals against the SARS-CoV-2 M protein, a compound, ZIN1722 from Zingiber officinale showed the best binding affinity with the receptor with a Glide Docking Score of −5.752 and Glide gscore of −5.789. In order to study the binding stability, the complex between the SARS-CoV-2 M protein and ZIN1722 was subjected to 50 ns Molecular Dynamics simulation using Desmond module of Schrodinger suite 2020-3, during which the receptor-ligand complex showed substantial stability after 32 ns of MD Simulation. The molecule ZIN1722 also showed promising results during ADME-Tox analysis performed using Swiss ADME and pkCSM. With all the findings of this extensive computational study, the compound ZIN1722 is proposed as a potential inhibitor to the SARS-CoV-2 M protein, which may subsequently prevent the immunosuppression mechanism in the human body during the SARS-CoV-2 virus infection. Further studies based on this work would pave the way towards the identification of an effective therapeutic regime for the treatment and management of SARS-CoV-2 infection in a precise and sustainable manner. [ABSTRACT FROM AUTHOR]

Genus Flavivirus contains several important human pathogens. Among these, the Zika virus is an emerging etiological agent that merits concern. One of its structural proteins, prM, plays an essential role in viral maturation and assembly, making it an attractive drug and vaccine development target. Herein, we have characterized ZikV-M as a potential viroporin candidate using three different bacteria-based assays. These assays were subsequently employed to screen a library of repurposed drugs from which ten compounds were identified as ZikV-M blockers. Mutational analyses of conserved amino acids in the transmembrane domain of other flaviviruses, including West Nile and Dengue virus, were performed to study their role in ion channel activity. In conclusion, our data show that ZikV-M is a potential ion channel that can be used as a drug target for high throughput screening and drug repurposing. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 is the virus responsible for causing COVID-19 disease in humans, creating the recent pandemic across the world, where lower production of Type I Interferon (IFN-I) is associated with the deadly form of the disease. Membrane protein or SARS-CoV-2 M proteins are known to be the major reason behind the lower production of human IFN-I by suppressing the expression of IFNβ and Interferon Stimulated Genes. In this study, 7,832 compounds from 32 medicinal plants of India possessing traditional knowledge linkage with pneumonia-like disease treatment, were screened against the Homology-Modelled structure of SARS-CoV-2 M protein with the objective of identifying some active phytochemicals as inhibitors. The entire study was carried out using different modules of Schrodinger Suite 2020-3. During the docking of the phytochemicals against the SARS-CoV-2 M protein, a compound, ZIN1722 from Zingiber officinale showed the best binding affinity with the receptor with a Glide Docking Score of −5.752 and Glide gscore of −5.789. In order to study the binding stability, the complex between the SARS-CoV-2 M protein and ZIN1722 was subjected to 50 ns Molecular Dynamics simulation using Desmond module of Schrodinger suite 2020-3, during which the receptor-ligand complex showed substantial stability after 32 ns of MD Simulation. The molecule ZIN1722 also showed promising results during ADME-Tox analysis performed using Swiss ADME and pkCSM. With all the findings of this extensive computational study, the compound ZIN1722 is proposed as a potential inhibitor to the SARS-CoV-2 M protein, which may subsequently prevent the immunosuppression mechanism in the human body during the SARS-CoV-2 virus infection. Further studies based on this work would pave the way towards the identification of an effective therapeutic regime for the treatment and management of SARS-CoV-2 infection in a precise and sustainable manner.

The pandemic of COVID-19 by SARS-CoV-2 has become a global disaster. However, we still don’t know how specific SARS-CoV-2-encoded proteins contribute to viral pathogenicity. We found that SARS-CoV-2-encoded membrane glycoprotein M could induce caspase-dependent apoptosis via interacting with PDK1 and inhibiting the activation of PDK1-PKB/Akt signaling. Our investigation further revealed that SARS-CoV-2-encoded nucleocapsid protein N could specifically enhance the M-induced apoptosis via interacting with both M and PDK1, therefore strengthening M-mediated attenuation of PDK1-PKB/Akt interaction. Furthermore, when the M-N interaction was disrupted via certain rationally designed peptides, the PDK1-PKB/Akt signaling was restored, and the boosting activity of N on the M-triggered apoptosis was abolished. Overall, our findings uncovered a novel mechanism by which SARS-CoV-2-encoded M triggers apoptosis with the assistance of N, which expands our understanding of the two key proteins of SARS-CoV-2 and sheds light on the pathogenicity of this life-threatening virus.

The pandemic of COVID-19 by SARS-CoV-2 has become a global disaster. However, we still don't know how specific SARS-CoV-2-encoded proteins contribute to viral pathogenicity. We found that SARS-CoV-2-encoded membrane glycoprotein M could induce caspase-dependent apoptosis via interacting with PDK1 and inhibiting the activation of PDK1-PKB/Akt signaling. Our investigation further revealed that SARS-CoV-2-encoded nucleocapsid protein N could specifically enhance the M-induced apoptosis via interacting with both M and PDK1, therefore strengthening M-mediated attenuation of PDK1-PKB/Akt interaction. Furthermore, when the M-N interaction was disrupted via certain rationally designed peptides, the PDK1-PKB/Akt signaling was restored, and the boosting activity of N on the M-triggered apoptosis was abolished. Overall, our findings uncovered a novel mechanism by which SARS-CoV-2-encoded M triggers apoptosis with the assistance of N, which expands our understanding of the two key proteins of SARS-CoV-2 and sheds light on the pathogenicity of this life-threatening virus. [ABSTRACT FROM AUTHOR]

Amidst infectious disease outbreaks, a practical tool that can quantitatively monitor individuals' antibodies to pathogens is vital for disease control. The currently used serological lateral flow immunoassays (LFIAs) can only detect the presence of antibodies for a single antigen. Here, we fabricated a multiplexed circular flow immunoassay (CFIA) test strip with YOLO v4-based object recognition that can quickly quantify and differentiate antibodies that bind membrane glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or hemagglutinin of influenza A (H1N1) virus in the sera of immunized mice in one assay using one sample. Spot intensities were found to be indicative of antibody titers to membrane glycoprotein of SARS-CoV-2 and were, thus, quantified relative to spots from immunoglobulin G (IgG) reaction in a CFIA to account for image heterogeneity. Quantitative intensities can be displayed in real time alongside an image of CFIA that was captured by a built-in camera. We demonstrate for the first time that CFIA is a specific, multi-target, and quantitative tool that holds potential for digital and simultaneous monitoring of antibodies recognizing various pathogens including SARS-CoV-2. • Unlike LFIAs, CFIAs allowed for concurrent measurements of multiple antibodies in a one reaction using only one sample. • Anti-SARS-CoV-2 membrane glycoprotein antibodies in immunized mice exhibited a purple spot normalized with anti-mouse IgG. • Intensities of purple spots corresponding to antibody titers on CFIAs were quantified by YOLO v4 in conjunction with ImageJ. • CFIAs can be used for on-site detection of antibodies to SARS-CoV-2 membrane glycoprotein and antibody differentiation. [ABSTRACT FROM AUTHOR]

Genus Flavivirus contains several important human pathogens. Among these, the Zika virus is an emerging etiological agent that merits concern. One of its structural proteins, prM, plays an essential role in viral maturation and assembly, making it an attractive drug and vaccine development target. Herein, we have characterized ZikV-M as a potential viroporin candidate using three different bacteria-based assays. These assays were subsequently employed to screen a library of repurposed drugs from which ten compounds were identified as ZikV-M blockers. Mutational analyses of conserved amino acids in the transmembrane domain of other flaviviruses, including West Nile and Dengue virus, were performed to study their role in ion channel activity. In conclusion, our data show that ZikV-M is a potential ion channel that can be used as a drug target for high throughput screening and drug repurposing.

Objective To explore the clinical significance of the changes of platelet membrane glycoprotein levels in peripheral blood of patients with gastric cancer and its influencing factors. Methods The levels of platelet membrane glycoproteins (CD62p and CD63) in peripheral blood of 87 patients with gastric cancer were measured by flow cytometry (FCM). 40 healthy people were served as control. Statistical analysis was performed by using the SPSS 10.0 software package. Results The levels of platelet membrane glycoproteins in peripheral blood in patients with gastric cancer [CD62p: (4.15 ± 1.42) % and CD63 (4.72 ± 1.54) %] were significantly higher than controls [CD62p (1.54 ± 0.44) % and CD63 (1.23 ± 0.34) %] P < 0.01. The levels of platelets membrane glycoproteins in peripheral blood in patients with gastric cancer were significantly associated with clinical stage [CD62p (2.80 ±0.63) versus (4.76 ± 1.25); CD63 (3.28 ± 0.70) versus (5.36 ±1.37], operation [(CD62p:3.49 ± 1.21) versus (4.86 ± 1.28), (3.98 ± 1.31) versus (5.51 ± 1.38)], metastasis [CD62p (4.94 ±1.07) versus (3.31 ± 1.25); CD63 (5.55 ± 1.19) versus (3.82 ± 1.38)], while were not influenced by gender and pathological types. Conclusion Detecting the levels of platelets membrane glycoprotein in peripheral blood may dynamically monitor the progression and prognosis of gastric cancer. [ABSTRACT FROM AUTHOR]

Abstract: Background: Vacancy of occupied N-glycosylation sites of glycoproteins is quite disruptive to a multicellular organism, as underlined by congenital disorders of glycosylation. Since a neuronal component is typically associated with this disease, we evaluated the impact of N-glycosylation processing of a neuronal voltage gated potassium channel, Kv3.1b, expressed in a neuronal-derived cell line, B35 neuroblastoma cells. Methods: Total internal reflection fluorescence and differential interference contrast microscopy measurements of live B35 cells expressing wild type and glycosylation mutant Kv3.1b proteins were used to evaluate the distribution of the various forms of the Kv3.1b protein in the cell body and outgrowths. Cell adhesion assays were also employed. Results: Microscopy images revealed that occupancy of both N-glycosylation sites of Kv3.1b had relatively similar amounts of Kv3.1b in the outgrowth and cell body while vacancy of one or both sites led to increased accumulation of Kv3.1b in the cell body. Further both the fully glycosylated and partially glycosylated N229Q Kv3.1b proteins formed higher density particles in outgrowths compared to cell body. Cellular assays demonstrated that the distinct spatial arrangements altered cell adhesion properties. Conclusions: Our findings provide direct evidence that occupancy of the N-glycosylation sites of Kv3.1b contributes significantly to its lateral heterogeneity in membranes of neuronal-derived cells, and in turn alters cellular properties. General significance: Our study demonstrates that N-glycans of Kv3.1b contain information regarding the association, clustering, and distribution of Kv3.1b in the cell membrane, and furthermore that decreased occupancy caused by congenital disorders of glycosylation may alter the biological activity of Kv3.1b. [Copyright &y& Elsevier]

Aim: CD147 is a membrane glycoprotein that is expressed in various cancer cells and is involved in tumor invasion and metastasis by inducing stromal fibroblastic cells to produce matrix metalloproteinases. This study was carried out to evaluate the correlation between CD147 expression and various clinicopathologic parameters, including histological grade and prognosis in a small sample set of human ovarian cancer patients. Material and Methods: Paraffin-embedded surgical tissue samples from 25 patients with ovarian serous and endometrioid adenocarcinoma were stained with anti-CD147 antibody (monoclonal antibody 12C3: MoAb 12C3) for immunohistochemical analysis. Results: CD147 protein was expressed in 84.0% (21 of 25 cases) of cancerous lesions, but not in normal lesions. CD147 expression by ovarian cancer cells was inversely correlated with overall survival. There was no correlation between CD147 expression and histological grade. Conclusions: These results suggest that measurement of CD147 expression may enhance the understanding of the pathophysiology of epithelial ovarian cancer. [ABSTRACT FROM AUTHOR]

Protein interactions play key roles throughout all subcellular compartments. In the present paper, we report the visualization of protein interactions throughout living mammalian cells using two oligomerizing MV (measles virus) transmembrane glycoproteins, the H (haemagglutinin) and the F (fusion) glycoproteins, which mediate MV entry into permissive cells. BiFC (bimolecular fluorescence complementation) has been used to examine the dimerization of these viral glycoproteins. The H glycoprotein is a type II membrane-receptor-binding homodimeric glycoprotein and the F glycoprotein is a type I disulfide-linked membrane glycoprotein which homotrimerizes. Together they cooperate to allow the enveloped virus to enter a cell by fusing the viral and cellular membranes. We generated a pair of chimaeric H glycoproteins linked to complementary fragments of EGFP (enhanced green fluorescent protein) - haptoEGFPs - which, on association, generate fluorescence. Homodimerization of H glycoproteins specifically drives this association, leading to the generation of a fluorescent signal in the ER (endoplasmic reticulum), the Golgi and at the plasma membrane. Similarly, the generation of a pair of corresponding F glycoprotein-haptoEGFP chimaeras also produced a comparable fluorescent signal. Co-expression of H and F glycoprotein chimaeras linked to complementary haptoEGFPs led to the formation of fluorescent fusion complexes at the cell surface which retained their biological activity as evidenced by cell-to-cell fusion. [ABSTRACT FROM AUTHOR]

In the present study, one of the targets present on the envelopes of coronaviruses, membrane glycoprotein (M) was chosen for the design of a multi-epitope vaccine by Immunoinformatics approach. The B-cell and T-cell epitopes used for the construction of vaccine were antigenic, nonallergic and nontoxic. An adjuvant, β-defensin and PADRE sequence were included at the N-terminal end of the vaccine. All the epitopes were joined by linkers for decreasing the junctional immunogenicity. Various physicochemical parameters of the vaccine were evaluated. Secondary and tertiary structures were predicted for the vaccine construct. The tertiary structure was further refined, and various parameters related to the refinement of the protein structure were validated by using different tools. Humoral immunity induced by B-cells relies upon the identification of antigenic determinants on the surface of the vaccine construct. In this regard, the vaccine construct was found to consist of several B-cell epitopes in its three-dimensional conformation. Molecular docking of the vaccine was carried out with TLR-3 receptor to study their binding and its strength. Further, protein-protein interactions in the docked complex were visualized using LigPlot+. Population coverage analysis had shown that the multi-epitope vaccine covers 94.06% of the global population. The vaccine construct was successfully cloned in silico into pET-28a (+). Immune simulation studies showed the induction of primary, secondary and tertiary immune responses marked by the increased levels of antibodies, INF-γ, IL-2, TGF-β, B- cells, CD4+ and CD8+ cells. Finally, the vaccine construct was able to elicit immune response as desired.Communicated by Ramaswamy H. Sarma.

In poorly differentiated hepatoma cells, a glycoprotein carrying lactosaminoglycans is identified, and the structure of its glycan moiety is proposed. After membrane solubilization, protein fractionation by gel filtration, and electroelution, this glycoprotein (GPIII) was identified by its affinity for Datura stramonium lectin and its content in large glycopeptides. As shown by PAGE, GPIII has an apparent molecular mass of 100 kDa and is highly giycosylated (36%). It appears as an integral membrane glycoprotein. It is absent from normal hepatocytes, in that no heavy glycopeptides could be detected that bound to Datura lectin or to specific antiserum. The glycan moiety of GPIII has been analyzed according to carbohydrate composition, glycosidase treatment, affinity chromatography on immobilized pokeweed, Datura and Griffonia lectins, and by NMR and methylation analyses. The glycan is a N-linked tetraantennary lactosaminoglycan of 6.6 kDa, containing Gal, GlcNAc, Man, and NeuNAc in a 16:14:3:4 molar ratio, with an average of throe repeating units/branch. Its β-Gal residues are in the penultimate position and are linked in β1-4 at least in four structural elements (three peripheral and one internal). It contains a very branched structure with Galα1-3Galβ1-4GlcNAc side chains linked in the C6 position to an inner Gal residue in a main branch, α-Gal and NeuNAc residues [mainly NeuNAcα(2-3) linkage] are expressed as the nonreducing terminal groups. A possible structural model is proposed for this heterogeneous lactosaminoglycan, although no definitive structure can be established. That this lactosaminoglycan-carrying glycoprotein GPIII is not expressed in hepatocytes suggests its expression to be linked to the undifferentiated and/or malignant state of this hepatoma. [ABSTRACT FROM AUTHOR]

We previously detected a fucosylagalactobiantenna with a bisecting GlcNAc residue (BA-2) and one lacking the GlcNAc residue linked to the Manal-3 residue of BA-2 (BA-1), which were enriched specifically in mouse brain [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334–338]. Pyridylamino sugar chains were prepared from mouse brains of various ages, and BA-1 and BA-2 were quantified after separation by HPLC. In cerebrum, BA-1 was scarcely expressed in newborn brain but gradually increased in amount during development, while expression of BA-2 reached a maximum 1 week after birth followed by a rapid decrease; in adult mice, the amount of BA-1 was almost the same as that of BA-2. In cerebellum, expression of BA-1 was lower than that of BA-2 at all stages. Glycoproteins with the BA-1 and BA-2 structures were enriched in the membrane fraction, and the glycoproteins solubilized were purified by lectin-affinity chromatography and gel filtration. The results indicated that BA-1 and BA-2 occurred in glycoproteins of more than 20 kDa in cerebellum, but most BA-1 and BA-2 were found in a 80–200 kDa fraction in cerebrum. These results show that the two brain-specific sugar chains are developmentally regulated and linked to the membrane-associated glycoproteins of subcellular organellas. [ABSTRACT FROM AUTHOR]

Rae-1 cDNA is one of the retinoic acid (RA)-inducible cDNA clones in mouse embryonal carcinoma F9 cells. Rae-1 mRNAs were detected in mouse early embryos, but not in various tissues of adult mice. RAE-1 protein apparently consists of 253 amino acids and is likely to be a glycoprotein consisting of a leader sequence, an extracellular domain, a serine, threonine, proline-rich domain, and a transmembrane domain. Interestingly, it has a weak, but significant homology with major histocompatibility complex (MHC) class I molecules and was immunocytochemically identified as a cell surface protein. By determining partial nucleotide sequences of 17 Rae-1 cDNAs isolated from the RA-induced F9 cells, at least three different kinds of Rae-1 cDNAs were identified and were named Rae-lα, Rae-1β, and Rae-1γ cDNAs, respectively. As the overall nucleotide sequence homology among these three cDNAs was about 98%, they constitute a novel gene family which is likely to be involved in early mammalian embryogenesis. [ABSTRACT FROM AUTHOR]

The aim of this study was to find out whether only resistant cells of the 'multidrug-resistant' phenotype show the described changes of plasma membrane glycoprotein (170 kDa) or whether resistant cells that do not express this phenotype reveal corresponding results. Doxorubicin-resistant (L1210) and daunorubicin-resistant L1210 ascites tumor cells (L1210) (multidrug-resistant tumor cells) were therefore compared with cytosine-arabinoside-resistant (L1210) and cyclophosphamide-resistant L1210 ascites tumor cells (L1210) (not multidrug-resistant tumor cells). The resistant cell lines were generated in vivo in tumor-bearing mice and the resistance to cytostatic agents was evaluated in vivo and in vitro. Using the accumulation assay with rhodamine-123, the multidrug resistance can be detected. In order to determine alterations in the plasma membranes we used the monoclonal antibodies 265/F4 and C219, which were prepared against the membrane glycoprotein P170 (170 kDa) in colchicin-resistant Chinese hamster ovary cells. The results demonstrate that L1210 and L1210 tumor cells show an intense immunostaining by the streptavidin/biotinylated-peroxidase-complex method and by the streptavidin/biotin/phycoerythrin immunofluorescence method. In contrast no specific immunostaining was observed in parental (sensitive) and L1210 or L1210 tumor lines. The results were confirmed by immunoblotting. To determine whether multidrug-resistant DNA sequences were expressed in the multidrug-resistant tumor cells, Northern blots with RNA od sensitive and resistant cells were performed using the clone pcDR1.5. Elevated RNA levels were detected only in resistant cells with the multidrug-resistant phenotype. Thus, the results of this study demonstrate that only resistant cells with the multidrug-resistant phenotype show an increased expression of the membrane 170-kDa glycoprotein. [ABSTRACT FROM AUTHOR]

We have established conditions for the study of membrane glycoprotein synthesis and turnover in cultured human malignant melanoma cell lines using the labeled precursor [H]glucosamine. Uptake of label increased parallel with cell growth, reaching a steady state in resting cultures. Fifteen to 30% of incorporated label can be released from the cells by trypsin treatment depending on the conditions of exposure to the enzyme, and about 50% of the incorporated label is spontaneously shed from the cells within 96 hr of incubation. Labeling in exhausted medium gave a 5- to 8-fold increase in uptake which was inhibited by addition of glucose (2 mg per ml) into the culture medium. The percentage of trypsin-releasable material was identical in fresh and exhausted medium; however, the percentage shed was less in cells initially labeled in exhausted medium. These data provide background information for further studies on the antigenic composition of the glycoproteins of cultured melanoma cells. [ABSTRACT FROM AUTHOR]

Peripheral resistance to insulin is a major component of non-insulin dependent diabetes mellitus. Defects in insulin receptor tyrosine kinase activity have been demonstrated in several tissues from insulin resistant subjects, but mutations in the insulin receptor gene occur in only a small fraction of cases. Therefore, other molecules that are capable of modulating the function of the insulin receptor are likely candidates in the search for the cellular mechanisms of insulin resistance. We have isolated an inhibitor of insulin receptor tyrosine kinase activity from cultured fibroblasts of an insulin resistant NIDDM patient and identified it as membrane glycoprotein PC-1. Subsequently we have demonstrated that expression of PC-1 is elevated in fibroblasts from other insulin resistant subjects, both with and without NIDDM. Studies in muscle, the primary site for insulin-mediated glucose disposal, have shown that the levels of PC-1 in this tissue are inversely correlated to insulin action both in vivo and in vitro. Transfection of PC-1 into cultured cells has confirmed that overexpression of PC-1 can produce impairments in insulin receptor tyrosine kinase activity and the subsequent cellular responses to insulin. Preliminary data suggests a direct interaction between PC-1 and the insulin receptor. However, the mechanisms whereby PC-1 inhibits insulin receptor signaling remain to be determined. [ABSTRACT FROM AUTHOR]

A plasma membrane protein common to nonmyelin-forming peripheral glia, including non-myelin-forming Schwann cells, satellite cells and enteric glia, is recognized and defined by monoclonal antibody A5E3. It is not detectable immunohistochemically on myelin-forming Schwann cells. The antigen is also present in large amounts on smooth muscle cells and perineurial cells, on some PNS neurons, and at lower levels on astrocytes of adult rat. In neonatal but not adult animals, the antigen is present on skeletal muscle fibres and myoblasts. In immunoblots and immune precipitation experiments on smooth muscle and Schwann cell extracts the antigen is a polypeptide with an apparent molecular weight of 130 kd. In being present in some non-neural tissues, albeit very highly restricted in cell type, this antigen resembles several other cell surface glycoproteins found in large amounts in the nervous system. [ABSTRACT FROM AUTHOR]