Protein Of Apparent Molecular Weight Research Articles

Purification and partial characterization of a ribosome-inactivating protein from the latex of Euphorbia trigona Miller with cytotoxic activity toward human cancer cell lines

BackgroundThe objective of this study is to investigate the cytotoxic activity of three isolectins purified from the latex of Euphorbia trigona Miller. HypothesisAmong lectins are the ribosome-inactivating proteins (RIPs), which are potent inhibitors of protein synthesis in cells and in cell-free systems. ResultsThree isolectins, ETR1, ETR2 and ETR3, were purified by anion exchange chromatography. Both ETR1 and ETR3 yielded a single band on SDS–PAGE under reducing conditions, corresponding to a molecular weight of 32 g mol−1, while ETR2 yielded two bands corresponding to 31 and 33 g mol−1. When non-reducing conditions were used molecular weight decreased, indicating the presence of intrachain disulfide bonds. Size-exclusion chromatography revealed proteins of apparent molecular weight of 59–63 g mol−1, suggesting a dimeric nature, with subunits not being held together by disulfide linkage. ETR1, ETR2 and ETR3 hemagglutinated human, sheep and rat erythrocytes and this hemagglutination was specifically inhibited by galactose and its derivatives. The lectins studied were thermostable up to 60 °C and their observed activity was maintained across pH range 5–12. These lectins, from the latex of Euphorbia trigona, are potent inhibitors of eukaryotic protein synthesis in a cell-free system. Flow cytometry analysis revealed the antiproliferative activity of them toward A549, HeLa, H116, HL-60 cell lines. ConclusionEuphorbia trigona isolectins are RIPs with cytotoxic activity toward human cancer cell lines.

Studying the Stability of S-Layer Protein of Lactobacillus Acidophilus ATCC 4356 in Simulated Gastrointestinal Fluids Using SDS-PAGE and Circular Dichroism.

Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are now recognized as one of the most common outermost cell envelope components of prokaryotic organisms. The surface layer protein of Lactobacillus acidophilus ATCC4356 is composed of a single species of protein of apparent molecular weight of 43-46 KDa. Considering the Lactobacillus acidophilus ATCC4356 having the S-layer is stable in harsh gastrointestinal (GI) conditions, a protective role against destructive GI factors which has been proposed for these nanostructures. It opens interesting perspectives in the using and development of this S-layer as a protective coat for oral administration of unstable drug nanocarriers. To achieve this goal, it is necessary to study the in-vitro stability of the S-layers in the simulated gastrointestinal fluids (SGIF). This study was planned to evaluate the in-vitro stability of the extracted S-layer protein of Lactobacillus acidophilus ATCC4356 in SGIF using it as a protective coat in oral drug delivery. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy were used to study the stability of the S-layer protein incubated in SGIF. Both the SDS-PAGE and CD spectra results showed that Lactobacillus acidophilus ATCC4356 S-layer protein is stable in simulated gastric fluid (SGF) with pH = 2 up to 5 min. It is stable in SGF pH = 3.2 and above it, with and without pepsin. It is also stable in all the simulated intestinal fluids. This S-layer is also stable in all of the simulated intestinal fluids.

Isolation and partial characterization of a D-galactose-binding lectin from the latex of Synadenium carinatum

A lectin from the latex of Synadenium carinatum was purified by affinity chromatography on immobilized-D-galactose-agarose and shown to be a potent agglutinin of human erythrocytes. The haemagglutination of human red cells was inhibited by 3.0 mM N-acetyl-D-galactopyranoside, 6.3 mM methyl-beta-D-galactopyranoside, 50 mM methyl-alpha-D-galactopyranoside and 50 mM D-fucose but not by L-fucose, demonstrating an anomeric and a conformational specificity. According to SDS-PAGE analysis, the lectin appeared to be a glycoprotein composed of two polypeptide chains of ca. 28 and 30 kDa, but size exclusion chromatography (Sephadex G-100) and native PAGE revealed a protein of apparent molecular weight 120 - 130 kDa made up of 28 and 30 kDa subunits. The lectin was stable in the range pH 6 - 9, and 4 - 56ºC. The N-terminal sequence of the 30 kDa subunit contained the conserved consensus sequence GPN observed in other D-galactose-binding lectins found in latex of members of the Euphorbiaceae.

Expression and localization of myotonic dystrophy protein kinase in human skeletal muscle cells determined with a novel antibody: Possible role of the protein in cytoskeleton rearrangements during differentiation

Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.

Production of monoclonal antibodies for detection of Vibrio harvey

Monoclonal antibodies (MAbs) against Vibrio harveyi were produced from mice immunized with heat-killed and SDS-mercaptoethanol-treated highly virulent V. harveyi 639. Fifteen MAbs were selected and sorted into 6 groups according to their specificity to various proteins of apparent molecular weight ranging from 8 to 49 kDa. Some antibodies were used for detection of V. harveyi at concentrations as low as 10(4) CFU ml(-1) using immunodot blots. Most of the selected MAbs did not show cross-reactivity to other Vibrio species and other gram-negative bacteria tested. Only 1 MAb (VH39-4E) showed slight cross-reactivity to Aeromonas hydrophila. Another MAb (VH24-8H) bound lightly to V. harveyi 1526 but strongly to V. harveyi 639, allowing rapid differentiation. Two of the MAb groups were used to localize V. harveyi in tissues of infected black tiger shrimp Penaeus monodon by immunohistochemistry. This study demonstrates the versatility of a highly specific immunological tool for the detection of V. harveyi in aquaculture and opens the way for further development of convenient test kits.

Molecular cloning, expression of testicular transcript abundant in germ cells and immunobiological effects of the recombinant protein.

It has been well documented that antisperm antibodies can be causative factors for infertility. In this report we have identified a protein on human sperm referred as human sperm-associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm-specific protein--a candidate molecule for control of fertility. An immunoinfertile woman serum showing head-head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot. Anti-48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library. A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type-specific expression was done using in situ RNA hybridization studies. To obtain recombinant HSAP (r-HSAP), hsap cDNA was cloned in pET 22b(+) expression vector. r-HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified. Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies. In order to evaluate in vitro immunocontraceptive potential, the anti-r-HSAP antibodies were characterized by agglutination assay, zona-free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis. We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids. Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r-HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure. The anti-r-HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona-free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry. The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676. This study has put in evidence that novel sperm-specific r-HSAP has role in sperm function and may have application in the development of a contraceptive vaccine. The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen.

Identification, characterization, and cloning of a complementary DNA encoding a 60-kd house dust mite allergen (Der f 18) for human beings and dogs

Background: House dust mites of the Dermatophagoides genus are the most important cause of perennial allergic disease in both humans and companion animals. Although the major mite allergens for humans are proteins of relatively low molecular weight, this is not the case for dogs. Western blotting shows that canine anti-mite IgE responses are directed primarily toward proteins in the molecular weight range of 50 to 120 kd. Objective: The objectives of this study were to characterize a D farinae allergen with a molecular weight of approximately 60 kd and to isolate the cDNA coding for this allergen. Methods: A protein of apparent molecular weight of 60 kd was identified by Western blotting by using canine serum IgE from house dust mite-sensitized atopic dogs. The protein was purified from homogenized D farinae mite bodies by ammonium sulfate precipitation, followed by gel filtration and cation exchange HPLC. The presence of IgE directed to the 60-kd protein in sera from humans and dogs with dust mite allergy was measured by FcϵRIα-based ELISA. A cDNA encoding a full-length 60-kd protein was isolated from a D farinae cDNA library by a combination of both PCR amplification and hybridization screening. A panel of mAbs specific for the 60-kd protein was generated and used to localize the protein in whole body sections of D farinae mites. Results: ELISA showed that the purified protein bound IgE in 54% of the sera from patients with D farinae allergy. In addition, the 60-kd protein was able to bind IgE in 57% to 77% of D farinae -sensitized dogs. A cDNA was isolated that encoded a protein of 462 amino acids, consisting of a 25 amino acid signal sequence and a 437 amino acid mature protein. The calculated molecular weight of the mature protein is 50 kd, and the amino acid sequence contains a single N-glycosylation site. A protein database search showed homology with multiple chitinases. A mAb specific for the 60-kd chitinase recognized the allergen in the mite digestive system, but fecal pellets did not stain positively for this allergen. Conclusions: A 60-kd D farinae protein (Der f 18), with homology to chitinase, is a major allergen for humans and dogs sensitive to house dust mites. (J Allergy Clin Immunol 2003;112:79-86.)

Functional role of specific amino acid residues in human thiamine transporter SLC19A2: mutational analysis.

SLC19A2 is a membrane thiamine transporter expressed in a variety of human tissues, including the gastrointestinal tract. Little is currently known about the structure/function relationship of SLC19A2. We examined the effect of introducing mutations in SLC19A2 identical to those found in thiamine-responsive megaloblastic anemia syndrome (TRMA), on functional activity and membrane expression of the transporter. We also examined the effect of mutating the only conserved anionic residue (E138) in the transmembrane (TM) domains of the SLC19A2 and that of the putative glycosylation sites (N63, N314). Northern blot analysis showed SLC19A2 mRNA was expressed at the same level in HeLa cells transfected with wild-type or mutated SLC19A2. Introducing the clinically relevant mutations (D93H, S143F, G172D) or mutation at the conserved anionic residue (E138A) of SLC19A2 led to a significant (P < 0.01) inhibition of thiamine uptake. Mutations of the two potential N-linked glycosylation sites (N63Q, N314Q) of SLC19A2 did not affect functional activity; they did, however, lead to a noticeable reduction in apparent molecular weight of protein. Western blot analysis showed all proteins (except D93H) were expressed in the membrane (not the cytoplasmic) fraction of HeLa cells. These results provide direct confirmation that clinically relevant mutations in SLC19A2 observed in TRMA cause malfunctioning of the transporter and/or a defect in its translation/stability. Results also show conserved TM anionic residue of the SLC19A2 protein is critical for its function. Furthermore, native SLC19A2 is glycosylated, but this is not important for its function.

The upstream stem–loop domain of the 3′ untranslated region of apolipoprotein II mRNA binds the estrogen-regulated mRNA stabilizing factor

Apolipoprotein II (apoII), a component of the very low density lipoprotein (VLDL) particle, is a yolk protein expressed in the liver in response to estrogen. Its expression is modulated by estrogen-mediated stimulation of transcription as well as stabilization of its mRNA. This stabilization is due to the estrogen-regulated mRNA stabilizing factor (E-RmRNASF) [Cell. Mol. Biol. Res. 41 (1995) 583). E-RmRNASF protects apoII mRNA from targeted endonucleolytic degradation. The expression of E-RmRNASF itself is under estrogenic control. The hepatic expression of E-RmRNASF is also modulated by certain estrogenic and anti-estrogenic non-steroidal environmental xenobiotics [Biochem. Pharmacol. 53 (1997) 1425]. Studies involving RNA affinity-based depletion of mRNA stabilization activity indicated that E-RmRNASF binds to apoII mRNA. E-RmRNASF binds apoII mRNA in a region-specific manner. The region of binding has been narrowed down to the upstream domain of stem–loop secondary structure spanning nucleotides (nt) 402–558 in the 3′ untranslated region (3′UTR). A RNA affinity chromatography procedure using this portion of apoII mRNA was utilized for the purification of E-RmRNASF. A gel filtration (GF) chromatography step preceding the RNA affinity chromatography was required for additional enrichment of E-RmRNASF. A functional assay involving the in vitro stabilization of apoII mRNA from degradation was utilized to detect E-RmRNASF during chromatography. E-RmRNASF appears to be a protein of apparent molecular weight of 20–25 kDa visualized by SDS polyacrylamide gel electrophoresis.

Tachyzoite-specific isoform of Toxoplasma gondii lactate dehydrogenase is the target antigen of a murine CD4 + T-cell clone

In two-dimensionally separated Toxoplasma gondii lysate, mouse Th1 clone 3Tx15 detects two proteins of apparent molecular weight 40 000 and p I of 5.8 and 5.9. Microsequencing of peptide fragments from tryptic digestion of one of these proteins yielded partial sequences of T. gondii lactate dehydrogenase (LDH)1. As shown by Western blot, toxoplasmic LDH co-migrates in two-dimensional gel electrophoresis with both T-cell antigenic proteins. With synthetic peptides spanning the complete primary structure of T. gondii LDH1, the T-cell epitope was mapped to a nine amino acid partial sequence which exhibits a motif for binding to I-E k, the class II restriction element of antigen recognition by clone 3Tx15. From the two known isoforms of T. gondii LDH, clone 3Tx15 specifically recognises tachyzoite LDH1, but not bradyzoite LDH2, as shown with the corresponding epitope peptides and recombinant proteins. Antigen-presenting cells infected with live bradyzoites stimulate 3Tx15 T cells, while killed bradyzoites provide no antigenic stimulus. This finding implies that a transformation into the tachyzoite stage occurs in cells challenged with bradyzoites. Although LDH1 represents one major constituent of the tachyzoite proteome, the protein does not seem to be immunogenic in T. gondii infection of mice. This is evident from the lack of serum anti-LDH immunoreactivity and the failure of adoptively transferred 3Tx15 T cells to protect against lethal challenge. In conclusion, a T-cell-stimulatory Toxoplasma antigen is identified by means of a novel, high-resolution T-cell blot technique, the cloneˈs antigenic fine specificity allowing detection of parasite-stage conversion.

Characterization and cloning of a major high molecular weight house dust mite allergen (Der f 15) for dogs

Although house dust mites (HDM(s)) are important elicitors of canine allergy, the low molecular weight molecules defined as major allergens for humans do not appear to be major allergens for dogs. Western blotting of Dermatophagoides farinae ( D. farinae) extracts with sera from sensitized dogs showed that the majority of animals had IgE antibodies specific for two proteins of apparent molecular weights of 98 and 109 kDa (98/109 kDa). The N-terminal sequences of these two proteins were identical, suggesting they were very closely related, and sequencing of internal peptides showed the protein(s) to have homology with insect chitinases. A purified preparation of 98/109 kDa proteins elicited positive intradermal skin tests (IDST(s)) in a group of well-characterized atopic dogs sensitized to D. farinae, but not in normal dogs. A rabbit polyclonal antiserum raised against the purified proteins was used to immunoscreen a D. farinae cDNA library. The mature coding region of the isolated chitinase cDNA predicts a protein of 63.2 kDa; sequence analysis and glycan detection blotting suggest that the molecule is extensively O-glycosylated. Monoclonal antibodies made against the purified native protein were used to localize the chitinase in sections of whole D. farinae mites. The protein displayed an intracellular distribution in the proventriculus and intestine of the mite, suggesting that it has a digestive, rather than a moulting-related, function. The high prevalence of IgE antibodies to this antigen in canine atopic dermatitis makes it a major HDM allergen for dogs, and the protein has been formally designated Der f 15.

Identification of two human nuclear proteins that recognise the cytosine-rich strand of human telomeres in vitro.

Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)(n)repeat] with high affinity and bind double-stranded telomeric DNA with a 100xreduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66-64, 45 and 35 kDa, respectively. To identify these polypeptides we screened alambdagt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.

Cloning of Guinea Pig Tissue Factor cDNA: Comparison of Primary Structure among Six Mammalian Species

Tissue factor (TF) is a transmembrane glycoprotein that serves as an essential cofactor for plasma coagulation factor VII. TF procoagulant activity exhibits varying species specificity. In particular, guinea pig (GP) TF is unable to activate clotting in heterologous plasma systems, but the molecular basis for this phenomenon is not yet understood. The full-length GP TF cDNA was cloned and sequenced. The open reading frame encoded a predicted precursor protein of 289 amino acids (aa) which was expressed in a reticulocyte lysate system as a protein of apparent molecular weight of 34 kD. The identity of the predicted aa sequence of mature GP TF with rabbit, human, bovine, rat and mouse TF was 66.4, 64.4, 60.6, 53.2 and 52.2%, respectively. With a focus on sites of potential functional significance, we compared sequences within the known binding regions. The eleven residues at the interface region between the TF1 and TF2 modules, which bind to the EGF domain of VIIa, were perfectly conserved among the six species, with the exception of an isoleucine replacing a lysine in the guinea pig sequence. However, only four of the eleven binding residues in the TF1 module, known to interact with the catalytic domain of factor VII, and three of the five residues in the TF2 module, involved in binding the factor VII Gla domain, were conserved among species. We hypothesize that divergence at these regions contributes to the specificity and non-reciprocity of TF procoagulant activity between species.

Purification of P0 myelin glycoprotein by a Cu2+-immobilized metal affinity chromatography.

P0 is an abundant myelin glycoprotein of peripheral nerves of vertebrates. Various point mutations of this protein are responsible for hereditary neuropathies. In this paper we described purification of P0 glycoprotein using SDS and a metal chelate affinity chromatography. Purified myelin fraction from bovine spinal roots in 0.5% SDS, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.4 is filtered and applied directly to the Cu2+-immobilized affinity chromatography column, equilibrated with the same buffer. After eluting a void volume (or pass through) fraction, P0 protein was eluted by the same buffer but without salt. To remove contamination from the eluent, further purification is continued on a Concanavalin-A coupled agarose column. We purify within two days, 30 mg of P0 protein of apparent molecular weight 27 kDa. The method can be used to purify recombinant or mutated P0 protein found in severe pathologies.

Identification of actin as an estradiol 17-beta-stimulated protein in the human placenta.

Addition of estradiol 17-beta to first trimester human placental minces resulted in an increased synthesis of a protein of apparent molecular weight 45 kDa. The specific involvement of estrogen in the stimulation of this protein was established by demonstrating a reduction in the level of this protein by the addition of CGS 16949 A, an inhibitor of aromatase, a key enzyme in the biosynthesis of estradiol 17-beta and ICI 182,780, an estrogen receptor antagonist. The protein was purified to homogeneity and N-terminal sequencing of two of the internal peptides obtained by enzymatic digestion of the protein, as well as the absence of a free N-terminal indicated that it could be actin. This was confirmed by Western blotting using commercially available actin antiserum. The role of estradiol 17-beta in the stimulation of actin synthesis in human placenta was also established by monitoring the quantitative inhibition of DNase I by actin.

Two Novel Variants of the v-srcOncogene Isolated from Low and High Metastatic RSV-Transformed Hamster Cells

Four different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70–200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). All cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-srcprotein in cell lines was not correlated with their metastatic potentialin vivo.Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-srcisoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and v-srcLM: 62 kDa. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-srcsequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the oncoprotein. Both v-srcvariants and chimeric v-srcwith mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretina cells were associated with the mutations in the carboxy-terminal region of the v-srconcogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM.

Selective humoral immune response of Balb/C mice to Brucella abortus proteins expressed by vaccinia virus recombinants

Genes encoding Brucella abortus Cu Zn superoxide dismutase (SOD) and a 54 kDa Escherichia coli HtrA homologue were cloned into shuttle plasmids pUV-1 and pSC11, and transfected into vaccinia virus to develop recombinants vUBSOD and vSB54. Control vaccinia virus recombinants vUV-1 and vSC11, carrying only the β-gal reporter gene but no B. abortus DNA were also developed. Recombinants were analyzed in Western blotting using a polyclonal B. abortus immune serum. vUBSOD expressed a protein of apparent molecular weight of 28 kDa, composed of the 20 kDa B. abortus Cu Zn -SOD and a protein approximately 8 kDa encoded by a portion of the vaccinia virus TK gene. vSB54 expressed a 54 kDa protein corresponding to the 54 kDa HtrA homologue. Recombinants vUSV-1 and vSC11 did not express B. abortus proteins. Groups of mice were inoculated intraperitoneally with 10 7 TCID 50 of 1 of the 4 different recombinant vaccinia viruses and 5 weeks later their sera were analyzed for antibodies against vaccinia virus and B. abortus proteins. Each group of mice responded with antibodies to vaccinia virus. Sera of vSB54-inoculated mice recognized the 54 kDa HtrA homologue. vUBSOD did not induce a humoral immune response. These results represent the first report on the expression of B. abortus proteins by vaccinia virus recombinants and the first demonstrated immune response against a B. abortus protein expressed by such a recombinant.

Identification, Characterization, and Intracellular Distribution of Cofilin in Dictyostelium discoideum

We identified and purified an actin monomer-binding protein of apparent molecular weight of 15,000 from Dictyostelium discoideum. The 15-kDa protein depolymerized actin filaments in a pH-dependent manner. The protein also had an activity to decrease apparent viscosity of actin solutions in a dose-dependent manner. This activity was inhibited by phosphatidyl inositides. Molecular cloning of genes encoding this protein revealed that the protein is 42% identical in its primary sequence to yeast cofilin. We concluded that the 15-kDa protein is cofilin of this organism. D. discoideum cells contain two cofilin genes (DCOF1 and DCOF2) whose nucleotide sequences were entirely identical in their exsons while the promoter and intron regions were different. Promoter assay experiments revealed that DCOF1 is expressed both in vegetative and differentiating cells and that DCOF2 is not expressed under any conditions examined. Gene disruption experiments suggested that DCOF1 might be essential for the proliferation of D. discoideum cells whereas the disruption of DCOF2 was proven not to alter any phenotypes. Indirect immunofluorescence microscopic observations showed that cofilin is distributed diffusely throughout cytoplasm in vegetative cells. In flattened cells under starvation stress, cofilin localized at dramatically reorganizing actin-cytoskeletons in ruffling membranes of the leading edge, but not at rigid actin meshwork in focal adhesion plaques. These results suggest that cofilin may be involved in dynamic reorganization of membranous actin cytoskeletons.

Bombesin stimulates the in vitro growth of a human gastric cancer cell line.

Bombesin (BBS) and its mammalian equivalent, gastrin-releasing peptide (GRP), exhibit diverse biological functions, including that of a neurotransmitter, a regulator of gastrointestinal hormone release, and a trophic factor for various normal and neoplastic tissues. Bombesin stimulates the growth of normal cells of the stomach, pancreas, and bronchial epithelium as well as cells in breast cancer, gastrinoma, and small cell lung cancer. The purpose of this study was to determine whether BBS regulates the growth of a human gastric cancer cell line (SIIA) in vitro, and if so, to examine the mechanisms of signal-transduction that are involved. We found that BBS stimulated the growth of SIIA cells in vitro. The GRP receptor antagonists, BIM 26189 and BIM 26226, had no effect on growth of SIIA cells. Although these antagonists blocked the BBS-induced increase of [Ca2+]i, they failed to block the growth-stimulatory effect of BBS. BBS stimulated intracellular tyrosine phosphorylation of multiple proteins, with a predominant protein of apparent molecular weight of 125 kDa. Inhibition of intracellular tyrosine kinases by tyrphostin blocked the growth-stimulatory effect of BBS on SIIA cells. These results indicate that BBS exerts its trophic effect on SIIA cells through a receptor(s) linked to tyrosine kinase pathway, but not to the phospholipase C (PLC) pathway.

Affinity isolation of neuron‐reactive antibodies in MRL/lpr mice

Autoantibodies from the MRL/lpr mice react with numerous proteins on neuronal cell surfaces. The purpose of this study was to isolate and characterize a population of autoantibodies reactive preferentially or exclusively with nervous system tissue. Using a purified plasma membrane preparation from brain cortex of balb/c mice coupled to diaminopropylamine agarose gel, we affinity-isolated antineuronal antibodies from pooled MRL/lpr immunoglobulins. The isolated immunoglobulins reacted with brain cortex plasma membranes and neuroblastoma cells (but not liver, kidney, or fibroblasts) by Western blot and indirect immunofluorescence with confocal microscopy. By Western blot, the epitopes in the brain cortex were proteins of apparent molecular weights 101, 63, 53, 43, 39, and 33, kd; the epitopes in the neuroblastoma cells were 63, 57, and 53 kd. Lectin column isolation revealed that the 101 and 63 kd epitopes were glycosylated. Indirect immunofluorescence revealed that the antibodies bound to the cell soma more intensely than to the cell processes of viable cultured neuroblastoma cells. The cell surface localization of this binding was confirmed by confocal microscopy. Within the central nervous system the antibodies bound more intensely to primary cultures of isolated neurons from fetal cortex than to hippocampal or neostriatal cells. With these antibodies we can begin studies of their potential pathogenic effects.