In vitro studies of rickettsia-host cell interactions: ultrastructural changes induced by Rickettsia rickettsii infection of chicken embryo fibroblasts.

We have compared the tyrosine kinase activity of pp60c-src isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp60c-src and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60c-src at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471-1477, 1985) suggest that vanadate may enhance a cellular regulatory mechanism that inhibits the activity of pp60c-src in normal cells. A likely candidate for this mechanism is phosphorylation at a tyrosine residue distinct from tyrosine 416, probably tyrosine 527 in the carboxyl-terminal sequence of amino acids unique to pp60c-src. The regulatory role, if any, of serine phosphorylation in pp60c-src remains unclear. The 36-kilodalton phosphoprotein, a substrate of pp60v-src, showed a significant phosphorylation at tyrosine after treatment of normal chicken embryo fibroblasts with vanadate. Assuming that pp60c-src is inhibited intracellularly by vanadate, either another tyrosine kinase is stimulated by vanadate (e.g., a growth factor receptor) or the 36-kilodalton phosphoprotein in normal cells is no longer rapidly dephosphorylated by a tyrosine phosphatase in the presence of vanadate.

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Identification and Characterization of Cryptosporidium parvum Clec, a Novel C-Type Lectin Domain-Containing Mucin-Like Glycoprotein

Infection and Immunity ◽

10.1128/iai.00436-13 ◽

2013 ◽

Vol 81 (9) ◽

pp. 3356-3365 ◽

Cited By ~ 21

Author(s):

Seema Bhalchandra ◽

Jacob Ludington ◽

Isabelle Coppens ◽

Honorine D. Ward

Keyword(s):

Host Cell ◽

Transmembrane Domain ◽

Cell Interactions ◽

Apical Region ◽

Content Type ◽

Lectin Domain ◽

Loop Region ◽

Wide Range ◽

Host Cell Interactions

ABSTRACTCryptosporidiumspecies are waterborne apicomplexan parasites that cause diarrheal disease worldwide. Although the mechanisms underlyingCryptosporidium-host cell interactions are not well understood, mucin-like glycoproteins of the parasite are known to mediate attachment and invasionin vitro. We identifiedC. parvumClec (CpClec), a novel mucin-like glycoprotein that contains a C-type lectin domain (CTLD) and has orthologs inC. hominisandC. muris. CTLD-containing proteins are ligand-binding proteins that function in adhesion and signaling and are present in a wide range of organisms, from humans to viruses. However, this is the first report of a CTLD-containing protein in protozoa and inApicomplexa. CpClec is predicted to be a type 1 membrane protein, with a CTLD, an O-glycosylated mucin-like domain, a transmembrane domain, and a cytoplasmic tail containing a YXXϕ sorting motif. The predicted structure ofCpClec displays several characteristics of canonical CTLD-containing proteins, including a long loop region hydrophobic core associated with calcium-dependent glycan binding as well as predicted calcium- and glycan-binding sites.CpClec expression duringC. parvuminfectionin vitrois maximal at 48 h postinfection, suggesting that it is developmentally regulated. The 120-kDa mass of nativeCpClec is greater than predicted, most likely due to O-glycosylation.CpClec is localized to the surface of the apical region and to dense granules of sporozoites and merozoites. Taken together, these findings, along with the known functions ofC. parvummucin-like glycoproteins and of CTLD-containing proteins, strongly implicate a significant role forCpClec inCryptosporidium-host cell interactions.

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The v-Src SH3 domain binds phosphatidylinositol 3'-kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5225 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5225-5232 ◽

Cited By ~ 114

Author(s):

X Liu ◽

L E Marengere ◽

C A Koch ◽

T Pawson

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Chicken Embryo ◽

Sh3 Domain ◽

Sh2 Domain ◽

Src Tyrosine Kinase ◽

Amino Terminal ◽

Embryo Fibroblasts ◽

Chicken Embryo Fibroblasts

Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.

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Ubiquitin is a heat shock protein in chicken embryo fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.949-956.1985 ◽

1985 ◽

Vol 5 (5) ◽

pp. 949-956

Author(s):

U Bond ◽

M J Schlesinger

Keyword(s):

Heat Shock ◽

Chicken Embryo ◽

Recombinant Dna ◽

Protein Product ◽

Amino Acid Sequences ◽

In Vitro Translation ◽

Bovine Ubiquitin ◽

Embryo Fibroblasts ◽

Chicken Embryo Fibroblasts

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.

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Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector

Molecular and Cellular Biology ◽

10.1128/mcb.6.5.1545-1551.1986 ◽

1986 ◽

Vol 6 (5) ◽

pp. 1545-1551

Author(s):

S Kornbluth ◽

F R Cross ◽

M Harbison ◽

H Hanafusa

Keyword(s):

Chicken Embryo ◽

Mammalian Cells ◽

Cell Transformation ◽

T Antigen ◽

Rous Sarcoma ◽

Embryo Fibroblasts ◽

Polyomavirus Middle T Antigen ◽

Middle T Antigen ◽

Chicken Embryo Fibroblasts

The middle T antigen of polyomavirus transformed primary chicken embryo fibroblasts when expressed from a replication-competent avian retrovirus. This in vitro-constructed retrovirus, SRMT1, is a variant of Rous sarcoma virus that encodes the middle T antigen in place of v-src. Inoculation of SRMT1 into 1-week-old chickens rapidly induced hemangiomas and hemangiosarcomas. As shown with mammalian cells infected with polyomavirus, polyomavirus middle T antigen appears to be associated with p60c-src in chicken cells infected with SRMT1. When lysates of SRMT1-infected cells immunoprecipitated with either a monoclonal antibody against p60src or anti-T serum were assayed in an in vitro kinase reaction, the middle T antigen was heavily phosphorylated. To see whether an excess of p60c-src could alter the extent of phosphorylation of the middle T protein or the process of cell transformation by middle T, cells were doubly infected with SRMT1 and NY501, a virus which overexpresses p60c-src. Doubly infected chicken embryo fibroblasts transformed with the same kinetics and were morphologically indistinguishable from chicken embryo fibroblasts infected with SRMT1 alone. Phosphorylation of the middle T antigen was elevated two- to fivefold relative to cells infected only with SRMT1.

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Opposite replication polarities of transcribed and nontranscribed histone H5 genes.

Molecular and Cellular Biology ◽

10.1128/mcb.8.4.1657 ◽

1988 ◽

Vol 8 (4) ◽

pp. 1657-1663 ◽

Cited By ~ 21

Author(s):

J P Trempe ◽

Y I Lindstrom ◽

M Leffak

Keyword(s):

Chicken Embryo ◽

Dot Blot ◽

Replication Assay ◽

Relative Enrichment ◽

Nuclease Digestion ◽

Embryo Fibroblasts ◽

Histone H5 ◽

Endogenous Nuclease ◽

Chicken Embryo Fibroblasts

We used an in vitro nuclear runoff replication assay to analyze the direction of replication of the active and inactive histone H5 genes in avian cells. In embryonic erythrocytes the transcribed histone H5 gene displayed sensitivity to endogenous nuclease cleavage. In contrast, this gene was insensitive to endogenous nuclease digestion under the same conditions in nuclei of the lymphoblastoid cell line MSB-1, and histone H5 gene transcripts were not detectable by dot-blot analysis of MSB-1 cell RNA. When nuclei were isolated from embryonic erythrocytes and incubated with bromodeoxyuridine triphosphate, runoff replication from endogenous nuclease cleavage sites led to a relative enrichment for fragments near the 3' end of the histone H5 gene in the density-labeled DNA. In nuclei of MSB-1 cells or chicken embryo fibroblasts, however, runoff replication from restriction enzyme-cut sites (or induced endogenous nuclease-cut sites in MSB-1 nuclei) led to a relative enrichment for fragments near the 5' end of the H5 gene in dense DNA. Based on the enhanced incorporation of bromodeoxyuridine into origin-distal regions of DNA during the in vitro runoff replication assay, we conclude that the active histone H5 gene in embryonic erythrocytes is preferentially replicated in the transcriptional direction from an origin in the 5'-flanking DNA, whereas its inactive counterparts in MSB-1 cells and chicken embryo fibroblasts are preferentially replicated in the opposite direction.

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