In vitro IgE inhibition in B cells by anti-CD23 monoclonal antibodies is functionally dependent on the immunoglobulin Fc domain.

CD23, the low affinity receptor for IgE (FcvarepsilonRII), is involved in regulation of IgE synthesis by B-lymphocytes. Five monoclonal antibodies to human CD23 were generated from cynomolgus macaques immunized with purified soluble CD23 (sCD23). Four of the five primate antibodies blocked the binding of IgE complexes to CD23 positive cells and also inhibited the production of IgE in vitro by IL-4 induced human peripheral blood mononuclear cells (PBMC). The variable domains of several primate antibodies were utilized to construct chimeric macaque/human (PRIMATIZED((R))) monoclonal antibodies. PRIMATIZED((R)) p5E8G1, containing human gamma 1 constant region, inhibited IgE production in vitro as efficiently as the parent primate antibody, but the human gamma 4 constant version, PRIMATIZED((R)) p5E8G4, was not as effective in IgE inhibition. An F(ab')(2) of p5E8G1 did not inhibit IgE production but did interfere with IgE inhibition by the intact anti-CD23 antibody in a dose dependent fashion. The murine monoclonal antibody MHM6 recognizes human CD23 at a different epitope than primate antibody 5E8, and inhibits IgE production by IL-4 induced PBMC. As with the F(ab')(2) of p5E8G1, the F(ab')(2) of MHM6 also failed to inhibit IgE production. These data imply that the mechanism by which anti-CD23 antibodies inhibit IgE production requires cross-linking of CD23 to an IgG receptor. These data also imply that neither bivalent cross-linking of CD23 alone or inhibition of CD23 binding to its natural ligands is sufficient to inhibit IgE production.

Protection against feline leukemia virus infection by use of an inactivated virus vaccine.

The protective immunity induced by 3 experimental FeLV vaccines were evaluated: Prototype inactivated FeLV vaccine developed from a molecularly cloned FeLV isolate (FeLV-FAIDS-61E-A); a mixture of immunodominant synthetic peptides corresponding to regions of the FeLV-Gardner-Arnstein-B (FeLV-GA-B) envelope proteins; and an adjuvant-disrupted but non-activated virus prepared from a non-cloned FeLV field isolate comprised of subgroup A and B viruses (FeLV-05821-AB). Included as controls were parallel groups of cats inoculated with adjuvants alone or with an established commercial FeLV vaccine. After each inoculation and after virulent virus challenge exposure, sera from all cats were assayed for ELISA-reactive antibody against purified FeLV, FeLV neutralizing (VN) antibody, and FeLV antigenemia/viremia--viral p27 antigen in serum and within circulating leukocytes. Immunity was challenged by oral/nasal exposure of vaccinated and control cats with FeLV-FAIDS-61E-A or FeLV-05821-AB, an infective, noncloned, tissue-origin, FeLV field isolate containing subgroup-A and -B viruses. Vaccine-induced immunity was assessed by comparing the postchallenge-exposure incidence of persistent viremia and the pre- and postchallenge exposure titers of VN and ELISA antibody in cats of the control and vaccine groups. The percentage of cats, that resisted development of persistent viremia after FeLV challenge exposure and the preventable fraction (PF) for the vaccine groups (which adjusts for the severity of the challenge and the degree of innate resistance in the controls) were as follows: adjuvant controls, 26%; FeLV-FAIDS-61E-A inactivated virus vaccine, 95% (PF = 93.2%); FeLV-GA-B peptide vaccine, 5% (-28.4%); FeLV-05821-AB noninactivated vaccine, 67% (55.4%); and commercial FeLV vaccine, 35% (12.2%). The prechallenge exposure mean VN antibody titer for each group was: less than 1:8 in the adjuvant controls; 1:43 in the FeLV-FAIDS-61E-A-vaccinated cats; less than 1:8 in the peptide-vaccinated cats; 1:38 in the noninactivated virus-vaccinated cats group; and 1:12 in the cats vaccinated with the commercial vaccine. Thus, induction of VN antibody in the vaccinated cats, although modest, appeared to be correlated with induction of protective immunity as defined by resistance to FeLV challenge exposure. Results of these studies indicate that inoculation of cats with an experimental inactivated virus vaccine prepared from a molecularly cloned FeLV isolate was most effective in stimulating protective immunity against heterologous and homologous FeLV challenge exposure.

Biological activities of a synthetic peptide composed of two unlinked domains from a retroviral transmembrane protein sequence.

We report several biological activities of a synthetic peptide whose sequence contains the highly conserved region of feline leukemia virus transmembrane protein (TM) synthetically linked to another short TM-derived sequence particularly rich in polar positive residues. This 29-amino-acid peptide blocked [3H]thymidine uptake 30 to 50% by concanavalin A-stimulated CD4(+)--but not CD8(+)-enriched murine splenocytes. Maximal suppression was detected at 12.5 micrograms (3 microM) to 75 micrograms (19 microM) per ml of growth medium; stimulation of [3H]thymidine uptake was observed at higher peptide concentrations. The synthetic peptide inhibited but did not stimulate [3H]thymidine uptake by mitogen-activated thymocytes and antibody production by splenocytes as determined in a liquid hemolytic plaque assay. Similarities are reported between a consensus sequence of diverse retroviral TMs and a region of alpha interferons shown by others to be important for antiviral and cytostatic properties. The TM sequence-derived synthetic peptide blocked in a nontoxic and sequence-specific manner the release of murine leukemia virus from two chronically infected cell lines. We suggest that some of the biological effects of retroviral TM are mediated through a common pathway shared with alpha interferons.

Differential effects of tumor promoters on P210bcr-abl expression.

It is a widely held theory that the bcr-abl hybrid gene plays an active role in chronic myelogenous leukemia (CML). The bcr-abl gene product (P210bcr-abl) is a structurally altered and enzymatically activated form of the normal c-abl gene product. P210bcr-abl is expressed in two cell lines derived from CML patients in blast crisis: K562 and EM2. Activation of protein kinase C by the strong tumor promoter TPA induced dramatic changes in K562 cells. We have shown that exposure of K562 cells to low concentrations (10 nM) of TPA stopped cell division and sharply reduced the expression of P210bcr-abl. In contrast, similar treatment of EM2 cells resulted in a slightly increased proliferation rate and stimulation of P210bcr-abl expression. A second tumor promoter, mezerein, also dramatically reduced P210 levels in K562 cells and elevated them in EM2 cells. These observations establish that expression of the bcr-abl gene can be either increased or decreased, depending on the cell type, and that these effects correlate with the proliferative state of the cell. These results are consistent with the hypothesis that P210bcr-abl plays an important role in the maintenance of CML.

A novel 53 kDa protein complexed with P210bcr-abl in human chronic myelogenous leukemia cells.

Leukemic cells from patients with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) contain a 210 kDa protein (P210bcr-abl) with a protein tyrosine kinase activity that is a product of fused bcr and abl genes. We have prepared two monoclonal anti-peptide antibodies, one from each gene product, and have affinity purified each. Incubation of anti-abl (c-abl 51-64) immunoprecipitates of K562 cells with [gamma-32P]ATP in protein kinase assays resulted in the labeling of P210bcr-abl and a 53 kDa (ph-P53) protein. Increasing concentrations of antibody detected similar ratios of P210bcr-abl: ph-P53, suggesting the presence of a complex between the proteins. Several different anti-abl and anti-bcr antibodies detected the ph-P53/P210 complex. Sodium dodecyl sulfate (SDS) treatment without 2-mercaptoethanol eluted P210bcr-abl and ph-P53 from the monoclonal antibody in the form of complexes which migrated on 6% SDS-polyacrylamide gels and had apparent molecular weights of 275,000 and more than 500,000. Both complexes yielded ph-P53 and P210bcr-abl upon treatment with SDS-mercaptoethanol. Studies involving glycerol gradient centrifugation also detected complexes of P210bcr-abl and ph-P53. Our results indicate that ph-P53 is not a degraded product of P210bcr-abl, does not share antigenic determinants with P210bcr-abl since it is not recognized by anti-abl and bcr antibodies in immunoblots, is not the phosphorylated heavy chain of immunoglobulin G, and is different from p53 (the nonviral T protein) complexed to the large T antigen of simian virus 40. Previous studies (Maxwell et al., 1987) have shown that ph-P53 has a different peptide map than P210bcr-abl. Therefore, we conclude that ph-P53 is a distinct cellular protein complexed to P210bcr-abl in K562 cells.

Expression of c-abl in Philadelphia-positive acute myelogenous leukemia.

The identical cytogenetic marker, t(9;22)(q34;q11) (Philadelphia [Ph] translocation), is found in approximately 90%, 20%, and 2% of adult patients with chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL), and acute myelogenous leukemia (AML), respectively. In CML, the molecular events resulting from the Ph translocation include a break within the bcr locus on chromosome 22, transfer of the c-abl protooncogene from chromosome 9 to 22, and formation of an aberrant 210-kD bcr-abl fusion protein (p210bcr-abl). Recently, the absence of bcr rearrangement and expression of a distinct aberrant 190-kd abl protein (p190c-abl) has been described in Ph-positive ALL, with the suggestion that the two abl variants may be pathogenetically associated with myeloid v lymphoid leukemogenesis. Here we report that the genomic configuration and translation product of Ph-positive AML can be similar to that of Ph-positive ALL: the break at 22q11 may occur outside the 5.8 kb bcr region and result in expression of a 190-kD abl protein lacking these bcr sequences. Phosphokinase enzymatic activity, a fundamental property of p210bcr-abl, was also associated with AML-derived p190c-abl. Our current observations indicate that p190c-abl can be found in cells of lymphoid or myeloid lineage and is therefore unlikely to play a specific role in the development of lymphoid leukemias. Formation of p190c-abl instead of p210bcr-abl appears to be a characteristic of the acute rather than the chronic Ph-positive leukemic state.

Identification of molecular variants of p210bcr-abl in chronic myelogenous leukemia.

The aberrant abl protein product of a chronic myelogenous leukemia (CML) blast crisis cell line (K562) and of five Philadelphia chromosome-positive CML patients in blast crisis were analyzed by an immune complex kinase assay using two antipeptide sera generated against the hydrophilic domain of v-abl and a region within the third exon of the breakpoint cluster region (bcr) respectively. Both the anti-abl and anti-bcr sera detected a 210 kd band in extracts derived from K562 cells and from two CML patients with myeloid blast crisis. p210 was detected by the anti-abl but not the anti-bcr sera in three CML patients with myeloid (one patient) and lymphoid (two patients) blast crisis, indicating the absence of bcr exon 3 in this protein. Southern blot analysis on DNA derived from one of the patients in the latter group was consistent with the break on chromosome 22 occurring 5' to bcr exon 3. Our observations demonstrate that the Philadelphia translocation results in the generation of a chimeric bcr-abl protein with at least two molecular variants, both of which are enzymatically active as protein kinases.

Analysis of P210bcr-abl tyrosine protein kinase activity in various subtypes of Philadelphia chromosome-positive cells from chronic myelogenous leukemia patients.

An altered c-abl gene product (P210bcr-abl) possessing associated tyrosine protein kinase activity was recently been reported in several blast chronic myelogenous leukemia (CML) cell lines. We have examined different morphological types of leukocytes directly obtained from patients at the blast crisis stage of CML for expression of P210bcr-abl tyrosine protein kinase activity. Phosphorylation of P210bcr-abl in an immune complex kinase assay using an anti-v-abl peptide serum was observed in blast cells from four Philadelphia chromosome (Ph1)-positive CML patients in blast crisis. P210bcr-abl protein kinase activity was detected regardless of whether the blast cells were of myeloid, lymphoid, or undifferentiated morphology. P210bcr-abl protein kinase activity was not detected in immune complexes either from leukocytes of four Ph1-negative CML patients in blast crisis, of five acute myelogenous leukemia patients, or in the promyelocytic cell line HL-60. Mature myeloid cells are associated with an inhibitory factor for not only P210bcr-abl protein kinase activity, but also protein kinases in general. Therefore, analyses of Ph1-positive benign phase CML myeloid cells, the majority of which are well differentiated, could not be successfully performed. The inhibition of P210bcr-abl protein kinase activity is not a specific property of mature cells from CML patients since granulocytes from a normal volunteer also demonstrated a similar effect. However, extracts of Ph1-positive cultured B-lymphocytes from a patient in benign phase demonstrated active P210bcr-abl protein indicating that the P210bcr-abl protein is expressed in an enzymatically active form in the earlier phases of CML. In addition to the previously reported P210 and P190 abl-related proteins, a novel Mr 53,000 protein was found to undergo phosphorylation at serine and tyrosine in immune complex kinase assays of two blast crisis CML cell lines (K562 and EM2) and in samples from blast crisis patients in which P210bcr-abl was detected. Peptide mapping by the Cleveland technique suggested that Mr 53,000 protein is unrelated to P210bcr-abl. Immune complex kinase assays of K562 cells with an anti-src serum (GD-11) yielded active c-src kinase and a Mr 50,000 phosphorylated protein, both of which were resistant to alkaline hydrolysis. Peptide mapping suggested that Mr 53,000 protein is related to Mr 50,000 protein which is precipitated with P210bcr-abl as an Mr 300,000 protein complex.

A novel c-abl protein product in Philadelphia-positive acute lymphoblastic leukaemia.

Activation of cellular proto-oncogenes as a result of chromosomal abnormalities has been implicated in the development of some human malignancies. Perhaps one of the most striking examples of this association occurs in chronic myelogenous leukaemia, where the Philadelphia (Ph) translocation results in substitution of the 5' end of the c-abl proto-oncogene with bcr gene sequences. A unique hybrid bcr-abl message is produced. As the Ph translocation is also present in some patients with acute lymphoblastic leukaemia, we initiated studies to determine if similar genomic events occur in these two different forms of Ph-positive leukaemia. Here we report that the Ph translocation in acute lymphoblastic leukaemia can result in production of a novel aberrant c-abl protein that is distinct from the bcr-abl protein found in Ph-positive chronic myelogenous leukaemia. Our observations suggest that alternative mechanisms of activation of c-abl exist, and may be important in the development of human acute lymphoid rather than chronic myeloid malignancies.

Rearrangement in the breakpoint cluster region and the clinical course in Philadelphia-negative chronic myelogenous leukemia.

We have followed one patient with Philadelphia (Ph)-negative chronic myelogenous leukemia and identified an additional four patients from the literature who showed the rearrangement in the breakpoint cluster region (bcr) on chromosome 22 characteristic of Ph-positive chronic myelogenous leukemia. The clinical course of these five patients was similar to that of Ph-positive patients, with easily controlled leukocyte counts, a prolonged benign phase, and prolonged survival. Furthermore, we have shown, for the first time, that bcr rearrangement in Ph-negative chronic myelogenous leukemia can result in expression of the aberrant 210-kilodalton bcr-abl fusion protein, which has been strongly implicated in Ph-positive leukemogenesis. Research data pertaining to possible cytogenetic mechanisms leading to production of p210bcr-abl in the absence of the Ph chromosome are reviewed. Molecular analysis provides an important tool for classifying and predicting prognosis of some patients with Ph-negative chronic myelogenous leukemia.

Expression of epidermal growth factor receptor and associated glycoprotein on cultured human brain tumor cells.

The expression of epidermal growth factor (EGF-R) in normal glial and glioma cells grown in culture was examined by using several independent assays. Immunoprecipitation with the monoclonal antibody R1 of extracts from metabolically labeled glial and glioma cells revealed a protein of Mr approximately 170,000, with a migration in sodium dodecyl sulfate-polyacrylamide gels identical to the EGR-R of A431 epidermal carcinoma cells. Furthermore, in the majority of glioma extracts, a protein of Mr approximately 190,000 was specifically immunoprecipitated by this antibody. Similar results were obtained by immunoblotting with a second antibody directed against a synthetic peptide in the sequence of the v-erb-B oncogene. In cell lines expressing both proteins, each was specifically phosphorylated on tyrosine in immune complex kinase assays. The majority of glioma cells bound between 40,000 to 80,000 125I-labeled epidermal growth factor molecules per cell. These results suggest that the expression of EGF-R is common in cultured human glioma cells. In addition, a structurally related protein, is expressed in some of these cells.

Expression of p21ras in fresh primary and metastatic human colorectal tumors.

Activation of the cellular oncogene ras has been implicated in many types of human malignancies. In this study, the relative levels of p21 protein product of ras (p21ras) in primary and metastatic colon tumors were compared to those in adjacent normal tissues. Nine of the 17 primary tumors had substantially elevated levels of p21ras with respect to adjacent normal tissues. Eight of these tumors were from Dukes' B and C stages. Four of the five tumors classified as "D" stage (in which distant metastases are present) did not show elevated levels of p21ras. In metastases from primary colon tumors, nine of nine were considerably reduced in p21ras expression regardless of the site of metastasis. These data suggest that elevation of p21ras may be a common event in early stages of colon tumors, and tumor progression may lead to a more autonomous population of cells in which other growth factors supplant the role of this protein.

Further characterization of the P85gag-mos -associated protein kinase activity.

The gag-mos hybrid protein encoded by ts110 MoMuSV was shown to have an associated protein kinase activity which phosphorylated both P85gag-mos and P58gag when [gamma-32P]ATP and a manganese cofactor were added to an immune complex containing P85gag-mos. Immunoprecipitation and removal of P85gag-mos from the reaction mixture by either an anti-mos or anti-gag serum resulted in a subsequent elimination of in vitro P85gag-mos and P58gag phosphorylation. This kinase activity was shown to be either an intrinsic property of P85gag-mos or else a tightly bound cellular enzyme activity resistant to elution with 2.0 M NaCl, 0.5% deoxycholate, and 0.1% SDS. A correlation was made between the amount of kinase activity and the concentration of P85gag-mos. Viral gag antisera were also used to show immune complex phosphorylation of another gag-mos hybrid protein termed P100gag-mos, derived from a revertant of ts110. In vitro phosphorylation experiments derived from v-mos transformed MuSV 124 cells using viral gag antisera were completely negative which shows that the gag-mos kinase in 6m2 cells is not merely a gag-associated kinase that phosphorylates MuSV coded gag gene products. When shifting 6m2 cells from a permissive temperature to the nonpermissive temperature of 39 degrees for 2-4 hr, a noticeable change toward a more normal morphology occurs. NRK 54-5A4 cells infected with a revertant of ts110 with wild-type phenotype, showed little change in morphology between permissive and nonpermissive temperatures. In addition to the ts defect affecting P85gag-mos production previously reported, a second ts defect in ts110 is reported here which is functional in nature; it can be detected within 5 min after shift to 39 degrees by the heat lability of the P85-associated kinase activity. The P100gag-mos protein kinase from the wild-type revertant cells did not exhibit this heat sensitivity under similar conditions. The thermal inactivation of the P85 kinase was shown to precede events that occur as cells are shifted to the restricted temperature including morphological reversion to the normal phenotype, and the decrease in P85gag-mos concentration. Based on all of these observations, it is suggested that the P85-associated kinase activity is not merely an adherent cellular kinase, but actually a function of the gag-mos gene product.

Gag-mos Polyproteins encoded by variants of the Moloney strain of mouse sarcoma virus.

Two revertants of ts110 Moloney murine sarcoma virus (MuSV) with wild-type MuSV phenotype were examined for the presence of mos gene products, ts110 MuSV has a temperature-sensitive defect in a function required to maintain the transformed phenotype. The nonproducer 6m2 cell clone transformed by ts110 produces an 85,000-Da gag-mos protein (P85gag-mos) and a 58,000-Da gag protein (P58gag). A spontaneous revertant (clone 54-5A4) of the 6m2 cell clone produces a 100,000-Da protein (P100) recognized by antisera raised against murine leukemia virus p15, p12, and p30 but lacks determinants of p10, reverse transcriptase, and gp70. P100 was specifically recognized by antisera (anti-C3) prepared against a synthetic peptide representing the predicted C-terminal 12 amino acids of Moloney MuSV v-mos gene. Normal sera or anti-C3 blocked with excess synthetic peptide did not recognize P100. Thus, P100 is a product of the gag and mos genes. P100 was found to be phosphorylated. A second wild-type revertant (clone 204-3) was obtained by superinfection of ts110 nonproducer cells with Simian sarcoma associated virus (SSAV); it was also found to contain a phosphorylated P100gag-mos protein. The 204-3 cell clone also contained two gag polyproteins (Pr60gag and Pr55gag) of the size and antigenic properties of those found in SSAV-infected cells. These results provide two examples of P100 gag-mos proteins both derived from the P85gag-mos producing 6m2 cell clone. The P100 gag-mos polyproteins are made in amounts that are easily detected by radiolabeling experiments using [3H]leucine. The intracellular viral RNAs present in 6m2 cells and the two revertant clones were also examined. All three cell clones contained a 4.0 kb RNA hybridizing to v-mos sequences but only the 6m2 clone contained a 3.5 kb mos-containing RNA. Our findings indicate that the 3.5 kb RNA codes for P85gag-mos in cell-free translation experiments (Junghans et al., 1982, J. Mol. Biol. 161, 229). These findings as they relate to the mechanism that produces P100gag-mos instead of P85gag-mos are discussed.

P85: a gag-mos polyprotein encoded by ts110 Moloney murine sarcoma virus.

Antibody to a synthetic peptide (anti-C3 serum) with the predicted sequence of the C terminus of the Moloney murine sarcoma virus (strain 124) v-mos gene was used in immunoprecipitation experiments with cytoplasmic extracts of a clone of NRK cells infected with ts110 Moloney murine sarcoma virus, termed 6m2 cells. ts110 Moloney murine sarcoma virus codes for two viral proteins of 85,000 and 58,000 M(r), termed P85 and P58, respectively, in nonproducer 6m2 cells maintained at 33 degrees C. Anti-C3 serum specifically recognized [(3)H]leucine-labeled P85, but not P58, from infected cells maintained at 33 degrees C, whereas antiserum prepared against murine leukemia virus p12 recognized both proteins. Normal serum and anti-C3 serum pretreated with excess C3 peptide did not precipitate P85. Immunoprecipitation experiments after metabolic labeling of 6m2 cells with (32)P(i) showed that P85 is phosphorylated. Both anti-C3 and anti-p12 sera specifically detected (32)P-labeled P85. Cell-free translation of ts110 murine sarcoma virus/murine lukemia virus RNA produces P85, P58, and helper virus protein Pr63(gag). Anti-C3 serum specifically precipitated P85 but neither P58 nor Pr63(gag). We conclude from these studies that P85 is a product of both the gag and mos genes of ts110 murine sarcoma virus, and, therefore, it is referred to as P85(gag-mos). We have not detected any other v-mos gene product in ts110-infected cells.

P85gag-mos encoded by ts110 Moloney murine sarcoma virus has an associated protein kinase activity.

A protein identified as P85(gag-mos) was shown to be phosphorylated when immunoprecipitates from ts110 Moloney murine sarcoma virus transformed nonproducer cells (clone 6m2) were incubated with [gamma-(32)P]ATP. The in vitro-labeled 85,000-dalton phosphoprotein comigrated on NaDodSO(4)/polyacrylamide gels with authentic phosphorylated P85(gag-mos). Immunoprecipitates obtained with antisera prepared against Rauscher murine leukemia virus core protein p30 were active in the immune complex kinase assay but anti-murine leukemia virus p10 precipitates were not. Previous studies have shown that anti-p30 but not anti-p10 antisera recognize P85(gag-mos). The 6m2 clone has been shown to express P85(gag-mos) at 33 degrees C but not at 39 degrees C. Anti-p30 immune complexes from 6m2 cells maintained at 39 degrees C failed to phosphorylate the 85,000-dalton protein. Furthermore, the in vitro phosphorylated 85,000-dalton protein gave the same pattern of V8 protease-generated cleavage products as in vivo(32)P-labeled P85(gag-mos). We conclude from these results that P85(gag-mos) is phosphorylated in anti-p30 immune complex kinase reactions. Phosphoamino acid analyses indicated that the in vitro phosphorylated P85(gag-mos) contained phosphoserine and phosphothreonine. Our findings indicate that incubation of anti-p30 immunoprecipitates at 39 degrees C drastically reduced, in a specific way, the kinase activity associated with P85(gag-mos). This result and other data suggest that the kinase is virus-encoded. Because P85(gag-mos), but not Pr65(gag) is phosphorylated in anti-p30 immunoprecipitates from MuLV-MuSV ts110 producer cells, the kinase enzyme is associated with P85(gag-mos) and not gag gene products. A second major polypeptide of the size of P58(gag) was also phosphorylated in anti-p30 immunoprecipitates from cells maintained at 33 degrees C but not at 39 degrees C. Since 6m2 cells at 39 degrees C contain P58(gag), this is also consistent with the kinase activity being associated with P85(gag-mos).

Binding of retrovirus-associated protein kinase and proteins to Staphylococcus aureus.

Formalin-fixed Staphylococcus aureus strain Cowan, bearing protein A, routinely used for the absorption of antigen-antibody complexes, was found to bind protein kinase activity from disrupted Moloney murine leukaemia virus (Mo-MuLV). The Wood strain of S. aureus lacking protein A also bound the kinase with similar efficiency. About 50% of the bound kinase activity, as detected by phosphorylation of casein using [gamma-32P]ATP, could be eluted from the bacterial preparation with buffer containing 0 X 5 M-KC1. Similar results were obtained with Moloney murine sarcoma virus (Mo-MuSV) strain 349 and ts110 MuSV(MuLV). The bacterial preparation was also found to bind casein kinase activity from cellular extracts of uninfected, Rauscher murine leukaemia virus (R-MuLV)-infected and Mo-MuLV-infected cells. Analysis of [3H]leucine-labelled proteins from purified virus showed selective binding to S. aureus of only two major labelled virus proteins. One virus component bound to S. aureus had the relative mobility of p15; the other polypeptide co-migrated with virus p10. Upon exposure to increased salt concentration, most of the p10 but very little of the p15 proteins were released. The S. aureus-binding proteins from ts110 Mo-MuSV and MuSV-349 revealed similar binding and elution patterns of p10 and p15 molecules. The p10 and protein kinase activity eluted from Mo-MuLV-absorbed bacteria were separated by gel filtration into a high molecular weight species, containing p10 and kinase activity, and a low molecular weight p10 monomer lacking enzymic activity.

Comparative morphological and histochemical aspects of selected arteries in the chicken and rat.

Morphologic and histochemical characteristics of selected portions of normal arteries from two species known to differ in susceptibility to vascular disease were examined. Arteries were classified as predominantly elastic, muscular or complex. Species differences in the structural organization of the abdominal aortic segment were observed. Arterial mucopoly-saccharides were stained more intensely in the tunica intima and media of chicken vessels than within those of the rat, and tended to be most concentrated in proximity of the internal elastic membrane. Histochemical procedures for the demonstration of enzymatic activity revealed inter-and intraspecies variations in vascular metabolism. Pronounced differences in reaction intensity for hydroxybutyrate dehydrogenase and malic enzyme, affecting chicken and rat coronary arteries, were noted. In contrast, theses vessels displayed only minimal activity for acid phosphatase. Marked endothelial deposition of alkaline phosphatase reaction products in the arteries of the chicken was demonstrated, while this enzyme's activity in the vessels of the rat was restricted to the tunica adventitia. The implications of these structural and histochemical factors with regard to vascular susceptibility to disease were discussed.