The group C adenovirus tumor antigens: identification in infected and transformed cells and a peptide map analysis.

Serum from hamsters bearing group C adenovirus-induced tumors can be divided into two classes: first, a broad spectrum serum that contains antibodies to several early adenovirus proteins, immunoprecipitated from virus-infected cell extracts, with molecular weights of 72,000, 58,000, 44,000 and 17,000 daltons; and second, a narrow spectrum serum that contains antibodies to the 58,000 dalton protein from virus-infected cell extracts. Both types of sera have been used to immunoprecipitate specifically the 58,000 dalton protein from a type 2 adenovirus-transformed hamster cell line and a type 2 adenovirus-SV40 nondefective hybrid (Ad2⁺ND-1) transformed hamster cell line. In addition, the broad spectrum serum immunoprecipitates or co-precipitates a late adenovirus protein of 120,000 daltons from virus-infected, but not virus-transformed cells.Peptide maps of the 120,000 dalton antigen and the virus hexon structural protein (120,000 daltons) demonstrate that these proteins are closely related. The 72,000 dalton antigen has been shown to be the adenovirus single-strand-specific DNA binding protein. Peptide maps of this 72,000 dalton antigen demonstrate that it contains all the peptides found in the 44,000 dalton antigen. The 72,000 dalton antigen contains two additional peptide fragments not detected in the 44,000 dalton protein, indicating that this 44,000 dalton antigen is a proteolytic breakdown product of the 72,000 dalton protein. The 58,000 dalton adenovirus tumor antigen has a peptide map which is completely distinct from the 120,000, 72,000 and 44,000 dalton proteins. These data demonstrate that the 58,000 dalton antigen is chemically distinct from the 72,000–44,000 dalton early adenovirus proteins.     

Comparative studies on the infectivity of Theileria parva in ticks fed in vitro and those fed on cattle

Nymphs of the brown ear tick, Rhipicephalus appendiculatus, were fed on heparinised bovine blood infected with Theileria parva parasites in an in vitro feeding system consisting of rabbit skin membranes. The main feeding and development parameters such as the mean attachment rate, feeding duration and engorgement weights of membrane-fed ticks were not significantly different from nymphs fed on cattle. The moulting rate was also comparable although a slight significant difference was observed. Assessment of infection prevalence and abundance with T. parva in adults indicated that the membrane-fed ticks acquired infection to the same level as those fed on cattle. Stabilates prepared from both the membrane- and cattle-fed adult ticks were found to be infective and caused severe reactions in susceptible cattle. When the immunised cattle were challenged with a lethal homologous dose of T. parva (Marikebuni), they were found to be immune.     

Identification of lipoprotein-binding proteins in rat liver Golgi apparatus membranes

The low density lipoprotein (LDL) receptor has been shown to be a plasma membrane glycoprotein responsible for the cellular binding and endocytosis of plasma lipoproteins. Inasmuch as the Golgi apparatus has been shown to participate in glycoprotein processing and in the assembly of plasma lipoproteins by hepatic and intestinal epithelial cells, the present studies were designed to test the hypothesis that lipoprotein receptors are present within Golgi membranes. Utilizing ligand blotting with a variety of iodinated lipoproteins, several lipoprotein-binding proteins were identified in rat liver Golgi membranes at apparent molecular weights (Mr) 200,000, 160,000, 130,000, 120,000, 100,000, 80,000, and 70,000. The 130,000 protein was the most prominent and was identified as the mature LDL receptor by its binding characteristics and an Mr characteristic of the plasma membrane receptor. Enzymatic deglycosylation studies suggested that the 120,000 and 100,000 proteins were LDL receptor precursors lacking sialic acid. Antibody to the LDL receptor recognized all the bands on immunoblots except the 70,000 protein, with the 130,000 protein being the most prominent. Isolation of the Golgi fractions in the presence of protease inhibitors did not eliminate any of the proteins recognized by the antibody but did result in sharper bands on the blots. Additionally, we investigated the hypothesis that conditions that regulate plasma membrane LDL receptors also cause detectable changes in receptors in Golgi membranes. All the binding proteins were increased in Golgi membranes from rats treated with 17-alpha-ethynylestradiol. Colchicine caused an accumulation of 120,000 Mr protein, suggesting blockage of final sialylation in the trans Golgi. When protein synthesis was inhibited by cycloheximide, there was no reduction of mature LDL receptors in Golgi membranes, consistent with recycling of receptors through this organelle.     

Theileria annulata: cross-reactions between a cell culture schizont antigen and antigens of East African species in the indirect fluorescent antibody test

A schizont antigen for the indirect fluorescent antibody test was prepared from an in vitro culture suspension of lymphoid cells infected with Theileria annulata macroschizonts. Two cattle recovered from T. annulata infection showed marked rises in antibody titer to this schizont antigen, with peak titers of 1:40,960 and 1:2560. Using sera from these recovered cattle, T. annulata cell culture schizont antigen was shown to cross-react markedly with Theileria parva and Theileria lawrencei cell culture schizonts and with Theileria mutans piroplasms in the indirect fluorescent antibody test. In contrast, using high-titer antisera to T. parva, T. lawrencei, and T. mutans, serological cross-reactions with T. annulata schizonts were only detected with T. parva and T. lawrencei antisera, and in both instances these were minimal. The value of the indirect fluorescent antibody test in the differential diagnosis of Theileria species pathogenic for cattle is discussed.     

Identification and characterization of a heterogeneous population of growth hormone receptors in mouse hepatic membranes

Covalent cross-linking of radiolabeled mouse growth hormone (125I-mGH) with the homobifunctional cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) to microsomal membranes prepared from late pregnant mouse liver resulted in the labeling of three specific mGH binding proteins (receptors) with apparent Mr = 125,000, 62,000, and 56,000, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. These same three specifically labeled proteins were present, but with slightly lower apparent molecular weights, when samples were electrophoresed in the absence of reductant. Cross-linking of 125I-mGH to plasma membrane-enriched fractions of late pregnant mouse liver resulted only in the specific labeling of the two lower molecular weight receptors. Removal of all N-linked carbohydrate with peptide: N-glycosidase F resulted in decreasing the apparent molecular weights of the three receptor forms to 110,000, 50,000, and 46,000 for the 125,000, 62,000, and 56,000 molecular weight forms of the receptor, respectively. Smaller decreases in the molecular weights of all three receptor forms were also apparent after treatment with neuraminidase. However, the differences seen in the intact forms of the growth hormone receptor were also present in the deglycosylated receptors. The relationship between the three forms of the growth hormone receptor was further investigated by comparing the fragments produced by proteolytic digestion of the cross-linked receptors with Staphylococcus aureus protease and endoproteinase Lys-C. The fragments produced from all three receptor forms had very similar molecular weights, although there were slight molecular weight differences in the fragments produced by endoproteinase Lys-C digestion. The overall similarity of the fragments produced by the proteolytic digestions suggests that the three forms of the receptor are related.     

Growth of Theileria annulata and Theileria parva macroschizont-infected bovine cells in immunodeficient mice: effect of irradiation and tumour load on lymphocyte subsets.

Bovine cells infected with macroschizonts of the protozoan parasites Theileria annulata and Theileria parva formed solid tumours when injected into irradiated Balb/c and irradiated Balb/c nude mice. T. annulata tumours grew more vigorously than T. parva tumours, when initiated with similar doses of infected cells in mice exposed to the same doses of gamma-irradiation. In irradiated Balb/c mice, tumours of both species of parasites began to regress 2-3 weeks after injection of cells but grew without regression in irradiated Balb/c nude mice. Haemorrhage and necrosis of tumours, induced by macrophages and neutrophils, were seen in both mouse strains but were insufficient to cause regression in Balb/c nude mice. Theileria-infected bovine cells failed to establish in C57 beige mice, which lack functional natural killer (NK) cells. Flow cytometry, using monoclonal antibodies to murine leukocyte/lymphocyte antigens, showed that the radiation dose required to allow establishment of T. annulata tumours in Balb/c mice caused a severe depletion of splenic lymphocytes. B cells, helper T and cytotoxic T cells showed differing levels of susceptibility to irradiation. The presence of a tumour promoted the recovery of lymphocyte populations: this recovery was accompanied by destruction of the tumour.     

Molecular characteristics of receptors for atrial natriuretic factor

Specific, high-affinity receptors for atrial natriuretic factor (ANF) have been identified on membranes from a variety of tissues and cultured cells. By affinity labeling procedures, radioactivity from 125I-labeled ANF was specifically incorporated into three different polypeptides of ca. 120,000, 70,000, and 60,000 daltons, which may represent the binding subunits of ANF receptors. These polypeptides were present in varying amounts in different target tissues. In rat adrenal membranes, the 120,000- and 70,000-dalton peptides were specifically labeled whereas in A10 rat smooth muscle cells, only the 60,000-dalton peptide was labeled. Membranes from rat kidney and rabbit aorta contain all three peptides. Gel filtration chromatography of solubilized receptors suggested that intact ANF receptors are large molecular complexes with apparent molecular masses in the range of 250,000-350,000 daltons. The differential labeling pattern observed with the various tissues suggested that there might be at least two different receptors composed of unique ANF-binding polypeptides.     

A Remeasurement of the Molecular Weights of T-Even Bacteriophage Substructural Proteins

T-even bacteriophage substructural proteins were studied by using discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was found that tail fibers are composed of two major proteins of 155,000 and 120,000 daltons molecular weight and four minor proteins of 51,000, 38,000, 27,000, and 23,000 daltons. Tail tubes were composed of one predominant protein of 18,500 daltons and one minor protein of 35,000 daltons molecular weight. Tubular polyheads obtained from a T4D amber mutant and by treatment of T4B-infected cells with L-canavanine were also examined, and no significant differences were noted in the molecular weight of the P23 protein.     

Identification of a receptor for atrial natriuretic factor in rabbit aorta membranes by affinity cross-linking

Binding sites in rabbit aorta membranes for atrial natriuretic factor (ANF) have been specifically and covalently labeled by two methods. In the first, the photoreactive analog of ANF, 125I-azidobenzoyl-ANF, was synthesized and used to photoaffinity label ANF receptors. In the second, 125I-ANF was covalently attached to its binding site by treatment of the 125I-ANF-receptor complex with bifunctional cross-linking agents. Analysis of the labeled proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that by both methods the same three protein bands were labeled. These bands had apparent molecular masses of 60,000, 70,000, and 120,000 daltons. With the photoaffinity label, half-maximal inhibition of labeling of each of these bands was achieved when approximately 200 pM of unlabeled ANF was included in the binding assay. These results suggest that these three different polypeptides are specific components of ANF receptors in rabbit aorta membranes.     

Analysis of structural proteins of measles, canine distemper, and rinderpest viruses

Serological relationships among measles virus (MV), canine distemper virus (CDV), and rinderpest virus (RV), which constitute morbillivirus subgroup of paramyxoviridae, were investigated by immunoprecipitation and SDS-polyacrylamide gel electrophoresis for their major structural proteins, i.e., hemagglutinin (H), nucleocapsid (NC), fusion (F), and matrix (M) proteins. The molecular weights of the four structural proteins of MV and CDV were confirmed to correspond to those previously reported by several investigators. Structural proteins of RV were analyzed for the first time in the present study and found to have molecular weights of 74,000, 62,000, 44,000, and 40,000 for H, HC, F, and M proteins, respectively. By labeling with glucosamine, the presence of carbohydrate moiety was found in H protein for all the three viruses and in F protein of CDV. The serums from the convalescent animals infected with respective virus disclosed one-way cross pattern depending on the combinations of virus and antiserums, but failed to show the reciprocal cross reactivity. On the other hand, hyperimmune serums to respective virus showed the reciprocal cross-reactivity with the four structural proteins indicating that each of the major structural proteins possesses the antigen common to all three morbilliviruses.     

Theileria parva: CD4+ helper and cytotoxic T-cell clones react with a schizont-derived antigen associated with the surface of Theileria parva-infected lymphocytes.

Theileria parva is a protozoan parasite which infects and transforms bovine lymphocytes, resulting in a fatal lymphoproliferative disease. There is evidence that immunity to the intralymphocytic schizont stage is mediated by T cells. We have previously reported derivation of CD4+ T-cell clones which recognize parasite-derived antigens presented on the surface of infected cells in conjunction with MHC molecules and partial characterization of the antigens. The present study further evaluated one of these antigens, demonstrating that it could be derived from cells infected with different parasite stocks as well as from purified theilerial schizonts and that it was recognized by primed, but not unprimed, bovine lymphocytes including cytolytic CD4+ T cells. Using a cloned CD4+ cytolytic cell line, lysis of schizont-infected cells was shown to be MHC-restricted but not parasite-strain restricted. In addition we demonstrated that T cells which respond to the HSS antigen preparation were generated in cattle immunized with parasites from any of the three subspecies of T. parva. The antigenic material was fractionated by sequential subjection to anion-exchange chromatography, hydroxylapatite chromatography, and gel filtration using HPLC, which resulted in recovery of approximately 20% of the antigenic material with more than 10(6)-fold purification in selected fractions. To assess the molecular size of the proteins in the highly purified antigenic fractions, the T. parva-infected lymphocytes were metabolically labeled before fractionation with 3H-amino acids and the material was analyzed by SDS-polyacrylamide gel electrophoresis and liquid scintillation counting of gel slices. The major protein in these fractions had a molecular mass of 9-10 kDa.     

Secretory vesicle — Cytosol interactions in exocytosis: Isolation by Ca2+-dependent affinity chromatography of proteins that bind to the chromaffin granule membrane

Proteins from adrenal medullary cytosol that bind to chromaffin granule membranes in the presence of Ca²⁺ were isolated by affinity chromatography on granule membranes coupled to Sepharose 4B. Cytosol was applied to the affinity column in the presence of 2 mM free Ca²⁺. One group of proteins was eluted at 50 μM Ca²⁺ and had molecular weights of 60,000, 46,000, 36,000, 34,000, 32,000 and 26,000. At 0.1 μM Ca²⁺ additional proteins of molecular weights 70,000, 44,000 and 33,000 were eluted. Both groups of proteins aggregated isolated chromaffin granules in the presence of Ca²⁺. Since exocytosis involves cytosol-membrane interactions regulated by Ca²⁺, these proteins may have functional roles in this process. The term “chromobindins” is introduced to describe these proteins.     

Protein phosphorylation during 1-methyladenine-induced maturation of Asterias oocytes

Maturation was induced in Asterias oocytes with 1-methyladenine (1-MA) at a final concentration of 2 microM. At 5, 10, and 30 min of treatment, oocytes were homogenized and the cytosolic fraction was prepared. The cytosol was incubated with [gamma-32P]ATP and [gamma-32P]GTP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the radioactivity in the gels was determined by autoradiography. The cytosol prepared from 1-MA-treated oocytes incubated with [gamma-32P]ATP showed a marked increase in the radiolabeling of proteins with estimated molecular weights of 70,000 and 62,000 Da. With [gamma-32P]GTP a 56,000-Da protein showed increased radiolabeling. The present finding suggests that an early biochemical event of 1-MA-induced oocyte maturation in Asterias is the stimulation of phosphorylation of specific proteins.     

Baculovirus surface display of Theileria parva p67 antigen preserves the conformation of sporozoite-neutralizing epitopes

Theileria parva is an intracellular protozoan parasite that causes East Coast fever, a severe lymphoproliferative disease in cattle. Previous attempts to produce recombinant sporozoite surface antigen (p67) in bacterial or insect cells for vaccine purposes have not resulted in a correctly folded protein. Here, we report the expression of N- and C-terminal domains of p67 fused to the baculovirus envelope glycoprotein GP64 by cloning the appropriate p67 cDNA segments between the signal sequence and the major portion of GP64. To further advance the generation of such recombinants, existing surface display techniques were combined with bacmid technology. Chimeric proteins were present on the surface of budded viruses as judged by immunogold labelling and were exposed on the surface of insect cells, as concluded from immunofluorescence studies of infected, non-fixed insect cells. In non-denaturing dot blot experiments, a strong reaction was obtained between monoclonal TpM12 and baculovirus particles displaying the p67N-GP64 chimeric protein. This antibody, raised against native p67, also specifically recognized the surface of recombinant-infected cells. Apparently, a more native conformation was achieved than when p67 was expressed in E.coli or in conventional baculovirus expression systems. The baculovirus surface expression system, therefore, provides an improved way of expressing this T.parva sporozoite surface protein.     

Tick-Theileria interactions in response to immune activation of the vector

Watt, D. M., Walker, A. R., Lamza, K. A., and Ambrose, N. C. 2001. Tick-Theileria interactions in response to immune activation of the vector. Experimental Parasitology 97, 89-94. Immune mechanisms towards the haemoprotozoan parasite Theileria parva were investigated in their tick vector, Rhipicephalus appendiculatus. The exoskeletons of adult ticks were initially pierced with bacteria-coated, saline-coated, or sterile dry glass needles. Haemolymph was extracted from the ticks at 6, 24, 48, and 72 h postinjection and applied to bacterial plates to measure the growth inhibition effects. The inhibition zones were larger with all the injected groups compared to uninjected controls. The largest inhibition zones were seen 24 h after injection with bacteria-coated needles. An experiment was carried out to investigate whether antibacterial immune responses were relevant to the parasite/tick relationship and, if so, which parasite form was most vulnerable. R. appendiculatus nymphs were infected with T. parva by feeding on an infected calf and were then injected with needles on days 7, 13, 15, and 17 throughout their moult in an attempt to induce tick immune responses at the same time as different lifecycle forms of T. parva would be present. Salivary glands from the moulted adult ticks in the control and different treatment groups were dissected out and examined for the presence of T. parva sporoblasts. No difference in infection levels was seen in any of the treatment groups compared with the controls, suggesting that immune responses in R. appendiculatus, induced by bacterial injection, do not affect T. parva infections. The fecundity of injected ticks was compared with that of uninjected controls to ensure that the injection procedure itself was not detrimental to the ticks. Injected females had higher engorgement masses than controls but reduced levels of egg hatching.     

Stage-specific synthesis and fucosylation of plasma membrane proteins by mouse pachytene spermatocytes and round spermatids in culture

Little is known about the ability of mammalian spermatogenic cells to synthesize plasma membrane components in the presence or absence of Sertoli cells. In this study, purified populations (greater than 90%) of pachytene spermatocytes or round spermatids were isolated by unit gravity sedimentation and cultured for 20-24 h in the presence of [35S]methionine or [3H]fucose. Cell viabilities remained over 90% during the course of these experiments. Plasma membranes were purified from these cells and analyzed by two-dimensional gel electrophoresis. Qualitatively, the same plasma membrane proteins were synthesized by both cell types with the exception of the major Concanavalin A-binding glycoprotein, p151; the synthesis of p151 is greatly diminished or inhibited after meiosis. [3H]Fucose was incorporated into at least 6 common glycoproteins of both cells. Eight components fucosylated with molecular weights from 35,000 to 120,000 were specific to pachytene spermatocyte membranes. One fast-migrating fucosylated component may represent an uncharacterized lipid whose synthesis is terminated after meiosis. Round spermatids specifically fucosylated two components with molecular weights of 45,000 and 80,000. These results demonstrate the viability of germ cells of the male mouse in short-term culture and show that they are capable of synthesizing and fucosylating plasma membrane components in the absence of Sertoli cells.     

Characterization of the Blood-Brain Barrier: Protein Composition of the Capillary Endothelial Cell Membrane

Microvessels were isolated from canine cerebral cortex, and the composition of the endothelial cell membrane was investigated. Endothelial cell membranes were separated from the surrounding basement membrane, solubilized, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels. Staining with Coomassie Blue revealed a characteristic banding pattern of at least 12 major proteins with apparent molecular weights between 14,000 and 250,000. When proteins from red blood cell ghosts were run simultaneously, no similarities were observed, except for proteins at apparent molecular weights of 43,000 (band 3) and 35,000 (band 4). These two proteins migrated exactly to the positions of the erythrocyte proteins actin and glyceraldehyde 3-phosphate dehydrogenase, respectively. Membrane glycoproteins in gels were also examined by the use of fluorescent lectins. Of the fluorescein isothiocyanate-conjugated (FITC) lectins tested, only FITC-concanavalin A had an affinity for any membrane components. Diazotized [125I]iodosulfanilic acid, a membrane-impermeable reagent, was used to label the internal (lumen) cell surface and the external (antilumen) cell surface. Autoradiography and determination of radioactivity levels in gel slices showed that several proteins were specifically labeled, and that major differences in radioactivity of proteins existed in internal and external labeling experiments. It is concluded that the protein composition of the luminal membrane is different from that of the antiluminal membrane.     

Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms

Three forms (I, IIa and IIb) of phospholipase C, hydrolyzing specifically inositol phospholipids, were resolved from human platelet cytosol and partially purified by DEAE-cellulose, phenyl-Sepharose, Ultrogel ACA-44 and hydroxylapatite column chromatographies. All three forms exhibited pH optimum at 6.5 - 7.0 in the presence of deoxycholate and their molecular weights were 67,000 (form I), 120,000 (IIa) and 70,000 (IIb). These enzymes hydrolyzed both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in Ca2+-dependent manner; their maximal activities for phosphatidylinositol hydrolysis were obtained at 10(-4) to 10(-3) M Ca2+, whereas phosphatidylinositol 4,5-bisphosphate was preferentially hydrolyzed at lower concentration of Ca2+.     

Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A

The lamins are the major components of the nuclear matrix and are known as lamins A, B, and C with Mr 72,000, 68,000, and 62,000 when analysed by SDS PAGE. These three polypeptides are very similar, as determined by polypeptide mapping and immunological reactivity. Lamins A and C are so homologous that a precursor-product relationship has been proposed. Using an antiserum against nuclear matrix proteins that specifically immunoprecipitates the three lamins, we examined their synthesis in the rabbit reticulocytes lysate. Four bands of Mr 62,000, 68,000, 70,000, and 74,000 were specifically immunoprecipitated when polysomes or polyadenylated RNA were translated in vitro. By two-dimensional gel electrophoresis, the 68,000- and the 62,000-mol-wt proteins were identified as lamins B and C, respectively, and the 74,000-mol-wt polypeptide had properties of a precursor of lamin A. The mRNAs of lamin C and of the putative precursor of lamin A were completely separated by gel electrophoresis under denaturing conditions, and their respective sizes were determined. These results suggest that lamin A is not a precursor of lamin C.