Bay K8644 like activity of an antibody against a 60 kDa tubular membrane protein

Partial purification of the dihydropyridine receptor from rat skeletal muscle demonstrated mainly a 60 kDa band in SDS-polyacrylamide gel. An antibody raised against that protein behaved as a calcium channel agonist viz. Bay K8644. The affinity purified antibody, when added to cultured heart cells, increased the beat rate 40-80% depending on the titer of the antiserum. The antibody also woke up the beats of the cells previously blocked with the channel antagonist, nifedipine. Immunoblot analysis indicated that the receptor of this antibody in heart cell membrane is also a 60 kDa protein.     

Interaction of the plant glycine-rich RNA-binding protein MA16 with a novel nucleolar DEAD box RNA helicase protein from Zea mays

The maize RNA-binding MA16 protein is a developmentally and environmentally regulated nucleolar protein that interacts with RNAs through complex association with several proteins. By using yeast two-hybrid screening, we identified a DEAD box RNA helicase protein from Zea mays that interacted with MA16, which we named Z. maysDEAD box RNA helicase 1 (ZmDRH1). The sequence of ZmDRH1 includes the eight RNA helicase motifs and two glycine-rich regions with arginine-glycine-rich (RGG) boxes at the amino (N)- and carboxy (C)-termini of the protein. Both MA16 and ZmDRH1 were located in the nucleus and nucleolus, and analysis of the sequence determinants for their cellular localization revealed that the region containing the RGG motifs in both proteins was necessary for nuclear/nucleolar localization The two domains of MA16, the RNA recognition motif (RRM) and the RGG, were tested for molecular interaction with ZmDRH1. MA16 specifically interacted with ZmDRH1 through the RRM domain. A number of plant proteins and vertebrate p68/p72 RNA helicases showed evolutionary proximity to ZmDRH1. In addition, like p68, ZmDRH1 was able to interact with fibrillarin. Our data suggest that MA16, fibrillarin, and ZmDRH1 may be part of a ribonucleoprotein complex involved in ribosomal RNA (rRNA) metabolism.     

Identification of a 48 kDa form of unconventional myosin 1c in blood serum of patients with autoimmune diseases

We searched for protein markers present in blood serum of multiple sclerosis (MS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients in comparison to healthy human individuals. We used precipitation/extraction methods and MALDI TOF/TOF mass spectrometry, and identified a protein with Mr ~46 kDa as a fragment of human unconventional myosin IC isoform b (Myo1C). Western blotting with specific anti-human Myo1C antibodies confirmed the identity. Screening of blood serum samples from different autoimmune patients for the presence of Myo1c revealed its high level in MS and RA patients, relatively low level in SLE patients, and undetected in healthy donors. These data are suggesting that the level of p46 Myo1C in blood serum is a potential marker for testing of autoimmune diseases.     

Latent membrane protein 1 of Epstein-Barr virus plays an important role in the serum starvation resistance of Epstein-Barr virus-immortalized B lymphocytes

We have previously shown that SNU-1103, which is a latency type III Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) that was developed from a Korean cancer patient, resists serum starvation-induced G(1) arrest. In this study, we examined the role of latent membrane protein-1 (LMP-1) in serum starvation resistance, since LMP-1 is known to be essential for EBV-mediated immortalization of human B lymphocytes. The LMP-1 gene from SNU-1103 was introduced into the EBV-negative BJAB cell line, and shown to be associated with resistance to G(1) arrest during serum starvation. Western blot analyses of the LMP-1-transfected cells revealed several protein alterations as compared to vector-transfected control cells. The expression of key cell-cycle regulatory proteins was affected in the G(1) phase: the expression of cyclin D3, CDK2, p27, and E2F-4 was up-regulated, and the expression of cyclin D2, CDK6, p21, and p103 was down-regulated during serum starvation. These results imply that of the several EBV viral genes expressed in EBV-negative B lymphoma cells, LMP-1 mediates resistance to serum starvation-induced G(1) arrest. However, we cannot rule out the possibility that other EBV genes are also involved in the cell-cycle progression of the EBV-transformed LCL during serum starvation, since the altered protein expression profile of the LMP-1 transfectants was distinct from that of the SNU-1103 cells that expressed all of the EBV viral proteins.     

Binding characteristics of bovine serum albumin encapsulated in sol-gel glasses: an alternative for protein interaction studies

Silica glasses doped with 500-700 microg of bovine serum albumin were prepared by the sol-gel method; two pH conditions (pH 5 and 7) were assayed for protein encapsulation. Both biomaterials showed a highly porous structure, with pore sizes in the range 5-28 nm. Columns packed with the ground biogels were on-line coupled to a C18 HPLC column for evaluation of the entrapped protein binding properties using propranolol. Binding capacities (at saturation) were approximately 3.7 and 7.1 microg of propranolol (drug-protein molar ratios 1.4 and 2.7) for the biogels prepared at pH 5 and 7, respectively. The significant difference indicates increased albumin denaturation upon encapsulation at pH 5. A frontal analysis study was then performed in cartridges packed with biogel prepared at pH 7 to evaluate the protein interaction with naproxen at low concentrations (相似文献   

The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture

Sericin is the silk protein that covers fibroin fibers and functions as a `glue' in the cocoons of silkworms, and its most abundant component, Ser1, contains repeats of Ser- and Thr-rich 38 amino acid residues. The viability of Sf9 insect cells was 20, 57 and 49% on the fifth day and 41, 91 and 70% on the ninth day after serum deprivation in the presence of no additives, 3000 g sericin hydrolysate and 350 g SerD (the peptide containing the two repetitive units) ml^–1, respectively. Thus, the sericin samples were useful in preventing cell death and promoting cellular growth after acute serum deprivation.     

Specificity studies of an antibody developed against a mucin-type glycoprotein

The specificity of a new anti-epiglycanin antibody (AE-3) which recognizes a mucin-type glycoprotein, the Human Carcinoma Antigen, found in the blood of patients with carcinomas, was studied. Information regarding the chemical nature of the antibody binding site was obtained by altering the structure of epiglycanin by chemical or enzymic means and testing the product in a competitive binding assay for inhibition of the binding of AE-3 to epiglycanin. The need for a high molecular weight antigen containing clustered T disaccharide, Gal1-3GalNAc, was demonstrated. The specificity was further explored by inhibition studies with glycopeptides having one to three mono- to disaccharides. The results were interpreted using computer graphics molecular modeling which predicted the specific recognition of hydroxyl groups on oligosaccharides on adjacent amino acids. Thus T antigen O-linked glycopeptide tumour markers can be designed to be distinguished by antibodies by the amount of clustering of their oligosaccharides.     

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.     

Culture of an epithelial cell line (MDCK) with a serum substitute

This paper reports the suitability of culturing a line of dog kidney epithelial cells, MDCK, in the presence of a serum substitute, Ultroser G. Serial subcultivation with this product was possible for at least 10 passages without any change in cell shape and size, saturation density, dome-forming ability, transepithelial resistance, and growth curve. Adhesion of newly plated cells to plastic was somewhat lower than in fetal calf serum but the trypsin-harvesting kinetics were essentially the same. However, the membrane ion transport systems was alterd: cell sodium influx was greatly diminished, suggesting a deep change in the amiloride-sensitive Na⁺ channels: sodium efflux was highly enhanced (both active and passive).     

Conversion of an apparent 100 kDa folate binding protein from human milk,choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C

Gel filtration studies in the presence of Triton X-100 showed that treatment with phosphatidylinositol-specific phospholipase C reduced the apparent molecular size of the 100 kDa folate binding protein from human milk, choroid plexus and semen to 25 kDa. Cleavage of a hydrophobic glycosly phosphatidylinositol domain (a membrane anchor) inserting the protein into Triton X-100 micelles could account for this phenomenon.     

Microinjection of an antibody against the cysteine-protease involved in male chromatin remodeling blocks the development of sea urchin embryos at the initial cell cycle

We reported recently that the inhibition of cysteine-proteases with E-64-d disturbs DNA replication and prevents mitosis of the early sea urchin embryo. Since E-64-d is a rather general inhibitor of thiol-proteases, to specifically target the cysteine-protease previously identified in our laboratory as the enzyme involved in male chromatin remodeling after fertilization, we injected antibodies against the N-terminal sequence of this protease that were able to inhibit the activity of this enzyme in vitro. We found that injection of these antibodies disrupts the initial zygotic cell cycle. As shown in this report in injected zygotes a severe inhibition of DNA replication was observed, the mitotic spindle was not correctly bipolarized the embryonic development was aborted at the initial cleavage division. Consequently, the injection of these antibodies mimics perfectly the effects previously described for E-64-d, indicating that the effects of this inhibitor rely mainly on the inhibition of the cysteine-protease involved in male chromatin remodeling after fertilization. These results further support the crucial role of this protease in early embryonic development.     

Proteins immunoreactive with antibody against a human leptin fragment are found in serum and tissues of the sea lamprey, Petromyzon marinus L

An affinity-purified, polyclonal antibody raised against a peptide corresponding to amino acids 137–156 at the carboxy terminus of human leptin (16 kD) was used to search for immunoreactive protein(s) in the lamprey, Petromyzon marinus. Immunoblots of serum from different phases of the life cycle showed the presence of a 65-kD immunoreactive protein in the larvae and all stages of metamorphosis but not in feeding juvenile and upstream migrant adults. Extracts of tissues known to store fat were also examined using the same antibody. Muscle and fat column from all phases tested (larvae, stage 2 and 4 metamorphosing animals, feeding juveniles and upstream migrants) showed 100- and 50-kD immunoreactive proteins. Extracts of nephric fold, the primary site of fat storage during metamorphosis, lacked the 100-kD protein but had the 50 kD; they also had a 16 kD immunoreactive protein not found in the other tissues. The immunoreactivity of the proteins of both serum and tissue extracts was blocked by pretreatment of the antibody with the leptin-derived antigen. The results indicate that P. marinus has proteins that share at least one epitope with mammalian leptin.     

Sequence of a plant cDNA from Vicia faba encoding a novel Ran-related GTP-binding protein

A clone obtained from a broad bean (Vicia faba) developing cotyledon cDNA library contained the complete coding sequence of a polypeptide with very high homology to the small GTP-binding proteins Ran from human cells and Spi1 from yeast. These proteins belong to the ras superfamily of proteins involved in different basic cellular processes. The Ran/Spi1 proteins interact with a protein bound to DNA (RCC1) and are thought to function in the regulation of the cell cycle. The amino acid sequence of the obtained plant Ran-homologue, designated Vfa-ran, is 74% and 76% identical to Ran and Spi1, respectively. The five functional, conserved domains of ras-related proteins are present in the Vfa-ran sequence. However, as in Ran/Spi1 the C-terminus of Vfa-ran is very acidic and lacks the Cys motif for isoprenylation.Northern blotting revealed a corresponding mRNA expression in broad bean roots, leaves, and cotyledons with the highest level in roots.     

Isolation and characterization of a monoclonal antibody containing an extra heavy-light chain Fab arm

Isolation and characterization of monoclonal antibody (mAb) variants to understand the impact of their structure on function is a typical activity during early-stage candidate selection that contributes to derisking clinical development. In particular, efforts are devoted to characterizing oligomeric variants, owing to their potential immunogenic nature. We report here a mAb variant consisting of a canonical mAb monomer associated in a non-covalent fashion with an antigen-binding fragment (Fab) arm amputated from its Fc domain. The truncated heavy chain is encoded in the cell line genome and is the likely product of a genomic recombination during cell line generation. The addition of the Fab arm results in severe loss of potency, indicating its interaction with the Fab domain of the monomer. The presence of such a variant can easily be mitigated by an adequate purification step.     

Expression of a 24 kDa GTP-binding protein (Gn24) is increased in lovastatin treated human erythroleukemia cells

A major 27 kDa particulate and a minor 24 kDa cytosolic GTP-binding protein was detected in HEL cells upon incubation with [-³²P]GTP of nitrocellulose blots containing polypeptides separated using SDS-PAGE. Addition of lovastatin (30 M) to HEL cells in culture inhibited protein synthesis by 35%. However, this treatment resulted in a 5-fold increase, as quantitated by [-³²P]GTP binding, in the amount of cytosolic 24 kDa GTP-binding protein. Addition of cycloheximide plus lovastatin to cells in culture abolished the observed increase in 24 kDa GTP-binding protein. Incubation of cells with lovastatin plus [R,S]-[5-³H]mevalonolactone resulted in the incorporation of radioactivity into several polypeptides in both the cytosolic and particulate fractions including a polypeptide of molecular mass of 24 kDa in the cytosol. The mobility of this 24 kDa isoprenylated protein on SDS-PAGE was identical to that of the GTP-binding protein increased in response to lovastatin. However, the 24 kDa protein remained in the cytosol after undergoing isoprenylation. The 24 kDa protein was distinct from the HEL cell, G25K/CDC42Hs GTP-binding protein and the GTP-binding protein that was a substrate for botulinum toxin C3 catalyzed ADP-ribosylation. Results demonstrate that lovastatin specifically increases the expression of a 24 kDa GTP-binding protein in HEL cells and that, isoprenylation of low molecular mass GTP-binding protein(s) may have function(s) in addition to its role in the targetting of these proteins to cell membrane.     

Uninfected red cells from malaria-infected blood: Alteration of fatty acid composition involving a serum protein: An in vivo and in vitro study

Summary Alteration of uninfected erythrocytes, fromPlasmodium (the malaria parasite)-infected blood remained an open question. In this study we compared the in vivo fatty acid compositions of control and uninfected monkey erythrocytes. A large (40%) increase in the linoleic acid level was observed, which was recovered mostly in neutral lipids. An in vitro system was developed to study medium-mediated alterations of cultured erythrocytes byPlasmodium falciparum. The increase in the linoleate level was reproduced in vitro and was also localized in the neutral lipid fraction, especially in triacylglycerols. Studies using proteolytic digestion and heat denaturation showed that a heat-labile serum protein is indispensable for the increase in the linoleate level of red cells treated with the supernatant, ofP. falciparum cultures. Both the function and the mechanism of this modification of uninfected erythrocytes still remain unknown. This work was supported by the UNDP/World Bank/WHO special program for Research and Training in Tropical Diseases (grant T16-181-M2-15B).     

Sequence of a canine cDNA clone encoding a Ran/TC4 GTP-binding protein

We report the isolation and characterization of a canine cDNA encoding a 216-amino acid GTP-binding protein of the Ras superfamily. The protein is almost identical to the human TC4 [Drivas et al., Mol. Cell. Biol. 10 (1990) 1793–1798] and Ran [Bischoffand Ponstingi, Proc. Natl. Acad. Sci. USA 88 (1991) 10830–10834; Nature 354 (1991) 80–82] proteins, the latter of which has been found to be involved in cell cycle control. Furthermore, the protein is highly similar to the fission yeast spi1 gene product [Matsumoto and Beach, Cell 66 (1991) 347–360]. The high degree of evolutionary conservation in this protein suggests that it plays a vital role in the eukaryotic cell.