Increases in tyrosine phosphorylation are detectable before phospholipase C activation after T cell receptor stimulation.

Antiphosphotyrosine immunoblots were used to characterize tyrosine phosphorylated proteins after stimulation of the human TCR. Increased tyrosine phosphorylation was evident on at least 12 substrates within 2 min after ligation of the TCR with mAb. Analysis of the time course for increased tyrosine phosphorylation revealed distinct patterns. Increased phosphorylation of 135-kDa and 100-kDa substrates was evident within 5 s, whereas increased phosphorylation of the TCR-zeta-chain required several minutes after treatment with anti-CD3 mAb. This rapid cellular tyrosine phosphorylation occurred independent of the cell cycle, as it occurred after stimulation of resting T cells, T cell blasts, and the Jurkat T cell leukemia line. When the TCR complex was cross-linked together with the CD4 receptor by heteroconjugate anti-CD3/CD4 mAb, an increased magnitude of tyrosine phosphorylation occurred, although no new substrates could be detected. The increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates was specific in that anti-HLA class I, anti-CD6, anti-CD7, and anti-CD28 antibodies did not cause increased tyrosine phosphorylation. Anti-CD4 stimulation of resting T cells did not cause increased tyrosine phosphorylation of pp100 and pp135, suggesting that the CD4-associated kinase, lck, does not account for the tyrosine phosphorylation observed after TCR stimulation. Similarly, pharmacologic treatment of cells with phorbol ester and calcium ionophore did not cause increased tyrosine phosphorylation of these substrates, indicating that activation of protein kinase C or phospholipase C does not account for these early increases in tyrosine phosphorylation. The time of onset of pp100 phosphorylation, and the magnitude of phosphorylation correlated with the magnitude of calcium mobilization when cells were stimulated with different forms of TCR stimulation. When cells were labeled with [3H]myoinositol and analyzed after stimulation by anti-CD3 mAb, increased tyrosine phosphorylation of the 135-kDa and 100-kDa substrates preceded the activation of phospholipase C, as measured by the appearance of inositol 1,4,5-trisphosphate. This occurred in both T cell blasts and in the Jurkat T cell line. Thus, these findings show that increased tyrosine phosphorylation is the earliest yet detected signal observed after ligation of the TCR complex, and furthermore suggest that tyrosine phosphorylation might link the TCR to the phosphatidylinositolbisphosphate hydrolysis signaling pathway.     

CD28 is an inducible T cell surface antigen that transduces a proliferative signal in CD3+ mature thymocytes

The rearrangement of TCR genes during thymic ontogeny creates a repertoire of T cell specificities that is refined to ensure the deletion of autoreactive clones and the MHC restriction of T cell responses. Signals delivered via the accessory molecules CD2, CD4, and CD8 have a crucial role in this phase of T cell differentiation. Recently, CD28 has been identified as a signal transducing molecule on the surface of most mature T cells. Perturbation of the CD28 molecule stimulates a novel pathway of T cell activation regulating the production of a variety of lymphokines including IL-2. We have studied the expression and function of CD28 during thymic ontogeny, and in resting and activated PBL. A variable percentage of resting thymocytes were CD28+ (3 to 25%, n = 8), but it was found in high density only on mature CD3+(bright) CD4/CD8 cells. Both unseparated thymocytes and isolated CD3-CD28-/dull cells proliferated when stimulated with PMA plus IL-2 or PMA plus ionomycin. PMA treatment also rapidly up-regulated CD28 expression in the CD3- subset as these cells became CD3-CD28+(bright). Despite the ability of PMA to induce high density CD28 expression in CD3- cells, CD3- thymocytes did not proliferate in response to PMA plus anti-CD28 mAb, in contrast to unseparated cells. CD3+ thymocytes stimulated with immobilized anti-CD3 mAb also failed to proliferate in culture. However, the addition of either IL-2 or anti-CD28 mAb supported proliferation, suggesting that only CD3+ cells could respond to CD28 signaling. The comitogenic effect of anti-CD3 and anti-CD28 mAb was IL-2 dependent as it was abrogated by an anti-IL-2R mAb. Interestingly, the expression of CD28 on the cell surface of CD3+ cells was also inducible, as flow cytometric analysis demonstrated a 10-fold increase in cell surface CD28 by 24 to 48 h after anti-CD3 stimulation of both CD3+ thymocytes and peripheral blood T cells. This increase was accounted for by a commensurate increase in CD28 mRNA levels. Together, these results suggest that CD28 is an inducible T cell antigen in both CD3- and CD3+ cells. In addition, stimulation of the CD28 pathway can provide a second signal to support the growth of CD3+ thymocytes stimulated through the TCR/CD3 complex, and may therefore represent a mechanism for positive selection during thymic ontogeny.     

Development of Amblyospora campbelli (Microsporida: Amblyosporidae) in the mosquito Culiseta incidens (Thomson)

The complete life cycle of Amblyospora campbelli (Kellen and Wills, 1962) (Microsporida: Amblyosporidae) requires a two-host system involving the mosquito host, Culiseta incidens (Thomson), and an obligatory intermediate copepod host. The parasite has dimorphic spore development producing meiospores (haploid condition) and binucleated spores (diploid condition), either as an exclusive infection or simultaneously (within females only). This is the 1st known report of concurrent spore development within an adult mosquito host, and, therefore, shows the Amblyospora campbelli system to be uniquely different from other Amblyospora spp. cycles previously described. The significance of dimorphic spore development is discussed. In females, diplokaryotic meronts may invade oenocytes, causing a benign-type of infection. A blood-meal is required to initiate sporulation of the binucleate spore. The binucleate spore contains the sporoplasm involved in transovarial transmission. A 2nd sporulation sequence, primarily in adipose tissue, may involve both males and females. In this sequence, repeated merogonic division greatly increased the density of diplokaryotic meronts and generally involved most of the body of the host. Production of meiospores, unlike that for the binucleate spore, appeared to be spontaneous (i.e. no obligatory blood meal). Survivorship of male and female larval mosquitoes was nearly equal. Adult females spread the parasite in three ways: transovarial, transovum, and by meiospore deposition.     

Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms

Full length cDNAs for a variety of human N-CAM isoforms have been transfected into mouse L-cells and/or 3T3 cells. Three independent clones of each cell line that were shown to express human N-CAM were tested for their ability to support the morphological differentiation of sensory neurons. The cell surface expression of N-CAM isoforms, linked to the membrane directly by an integral transmembrane spanning domain or indirectly via covalent attachment to a glycosyl-phosphatidylinositol moiety, were consistently found to be associated with a significant increase in the morphological differentiation of both human and rat dorsal root ganglion neurons. Modification of the extracellular structure of both classes of N-CAM, consequent to the expression of a glycosylated 37-amino acid sequence normally found expressed exclusively in muscle N-CAM isoforms did not obviously affect the ability of transfected cells to support increased neuronal differentiation. 3T3 cells that were transfected with a full length cDNA encoding a secreted N-CAM isoform, and that have previously been shown to secrete N-CAM into the growth media rather than link it to the membrane did not significantly differ from control cells in their ability to support neuronal differentiation. These data provide direct evidence for both transmembrane and lipid-linked N-CAM isoforms being components of the regulatory machinery that determines neuronal morphology and process outgrowth.     

Production and characterization of mammary-derived growth factor 1 in mammary epithelial cell lines.

A mammary-derived growth factor, MDGF1, which stimulates collagen synthesis and proliferation in mammary epithelial cells was previously detected and purified from human milk and primary human breast tumors. MDGF1 binds to putative cell-surface receptors of 120-140 kDa and stimulates proliferation of normal and malignant human mammary epithelial cells. Partial protein sequence (N-terminal 18 amino acid sequence) shows that MDGF1 has no homology to any other known growth-promoting peptides. Polyclonal antiserum raised against this synthetic peptide recognizes native milk-derived MDGF1. We hypothesize that MDGF1 might be an autocrine or paracrine factor produced by and acting on normal and malignant human breast epithelial cells possessing MDGF1 receptors. As a first step in testing this possibility, we examined whether human breast epithelial cells in culture produce the growth factor. A protein with the size of MDGF1 was immunologically detected in the concentrated conditioned medium prepared from human breast cancer cell line MDA-MB 231, the mammary-derived but nontumorigenic HBL-100 line, and the normal reduction mammoplasty-derived, nonimmortalized 184 cell strain. A competitive radioreceptor assay (RRA) was used to estimate the level of MDGF1 in the conditioned medium. MDGF1 was present in the nanogram range per 1 million cells. A 62-kDa protein was detected in the above cell lysates by Western immunoblotting or by immunoprecipitation of metabolically labeled cell-conditioned media. The polyclonal antisera directed against the 18 amino acid peptide sequence from milk-derived MDGF1 could adsorb MDGF1 biological activity from conditioned medium. In vitro translation of cell mRNA yielded a protein of 55 kDa which was immunoprecipitated by anti-MDGF1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhipicephalus appendiculatus feeding on rabbits and cattle: Salivary-gland responses to varying host resistance

AdultRhipicephalus appendiculatus ticks were fed as three sequential infestations on both rabbits and cattle. The feedings at first infestation on naive hosts were optimum for the ticks, whereas at third infestation the hosts were resistant, as expressed by reduced tick feeding performance. Ticks from naive and resistant hosts were examined for histological differences of salivary glands. In ticks fed on resistant rabbits there was a large increase in the synthesis of glycoprotein secretory granules in thec ₁ cells compared with ticks fed on naive rabbits. In ticks fed on naive and resistant cattle, the activity of thec ₁ cells was less than in ticks fed on naive and resistant rabbits. It was concluded that the salivary glands are able to respond selectively to conditions at the feeding site, and that this may be advantageous to the tick.     

Mutations Modulating Raf Signaling in Drosophila Eye Development

The R7 fate is specified during Drosophila eye development by an inductive signal transduced intracellularly via the Raf kinase. We have performed a genetic screen for dominant mutations that alter the efficiency with which cells respond to a constitutively activated Raf kinase. Such mutations may affect genes involved in signal transduction downstream of Raf. We have isolated 44 mutations that define eight genes. One of these encodes a mitogen-activated protein kinase homologue; another is a putative target gene of this signaling pathway. We present the results of this screen in detail, as well as a preliminary genetic analysis of the six loci still to be characterized molecularly.