Association between fibronectin receptor and the substratum: Spare receptors for cell adhesion

We have investigated the association of the recently described 140-kDa cell membrane receptor for fibronectin with the cytoskeleton or with substratum-bound fibronectin. Using a monospecific polyclonal antibody to the 140-kDa receptor, we have demonstrated that most of the receptor molecules are soluble in nonionic detergent either in suspension culture CHO cells or in CHO cells attached to and spread on a fibronectin-coated substratum. This may suggest that putative linkages of the receptor either to fibronectin or to detergent-insoluble cytoskeletal components are labile to nonionic detergent and thus are rather weak. Alternatively, it may mean that only a small fraction of the cell's receptors are needed to mediate adhesion. In order to explore this latter concept, we have coated substrata with various concentrations of PB1, a monoclonal antibody with a high affinity for fibronectin receptor. We demonstrate that coating the substratum with increasing concentrations of PB1 results in increasing amounts of 140-kDa receptor becoming bound to the substratum in detergent-insoluble form. However, the amount of receptor bound does not necessarily correlate with the degree of cell adhesion and spreading. Thus, coating the substratum with 5 μg/ml of PB1 results in essentially complete attachment and spreading of CHO cells, but only a small fraction of the 140-kDa receptor becomes substratum bound. Coating with 50 μg/ml of PB1 produces no further increase in cell adhesion and spreading, but results in the detergent-stable association of a large fraction of the total receptor pool with the substratum. These observations suggest the possibility of there being “spare” receptors for cell adhesion processes.     

The 140-kDa fibronectin receptor complex is required for mesodermal cell adhesion during gastrulation in the amphibian Pleurodeles waltlii

We have studied the localization and function of a 140-kDa glycoprotein complex implicated in cell adhesion to fibronectin- and laminin-rich extracellular matrices in Pleurodeles waltlii gastrulae. In particular, we have shown that antibodies directed against highly purified avian fibronectin (FN) receptor complex cross-react with two major polypeptides of apparent molecular weights of 140,000 and 100,000 and a third minor component of 90,000. Using sections of embryos or whole mounts, we have also discovered that the putative FN receptor is widely distributed on the early embryonic cell surface. We have also found that the basal surface of the roof of the blastocoel, a region particularly enriched in an extracellular matrix consisting of fibronectin- and laminin-rich fibrils, is rich in receptor complex. We have prepared monovalent Fab' fragments of this antibody and have found that they cause detachment of cells previously attached to substrata coated with fibronectin, and they also arrest gastrulation when injected into the blastocoel of early gastrulae. Thus, it appears that the fibronectin receptor complex plays a significant functional role in cell attachment of gastrula-stage cells in vitro and in cell migration in vivo during gastrulation.     

Adhesion of lymphoid cells to the carboxyl-terminal heparin-binding domains of fibronectin

Previously, we have shown that some lymphoid cell lines adhere to fibronectin (FN)-coated substratum, whereas others do not. In this study, the adhesion of five adherent lymphoid cell lines to different FN domains was examined. These cell lines ranged in their adherence to substratum coated with FN, the cell-binding domain (CBD) fragment, or the heparin-binding domain (HBD) fragments. None of the cell lines adhered to substratum coated with the gelatin-binding domain fragment. Three of the lymphoid cell lines adhered preferentially to HBD over CBD, whereas two other lymphoid cell lines and BHK fibroblasts adhered preferentially to CBD. These results suggest that two distinct adhesive interactions occur between cells and FN and that the pattern of interaction varies among cell types. Using MOPC 315 (which adheres preferentially to HBD) as a cell model to study the cell-HBD interaction, the HBD-promoted adhesion was found to be independent of the RGD sequence and could be inhibited by anti-FN antibodies. Moreover, the MOPC 315-HBD interaction had the following characteristics: (1) adhesion was temperature dependent, (2) presence of divalent cations was necessary, (3) integrity of cellular microfilaments but not microtubules was required, (4) inhibition of protein synthesis abolished adhesion, (5) pretreatment of cells with trypsin inhibited adhesion, and (6) the adhesion was mediated by the carboxyl-terminal HBD.     

Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor

Affinity chromatography was used to identify a putative cell surface receptor for fibronectin. A large cell-attachment-promoting fibronectin fragment was used as the affinity matrix, and specific elution was effected by using synthetic peptides containing the sequence Arg-Gly-Asp, which is derived from the cell recognition sequence in the fibronectin cell attachment site. A 140 kd protein was bound by the affinity matrix from octylglucoside extracts of MG-63 human osteosarcoma cells and specifically eluted with the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro. The 140 kd protein was labeled by cell surface specific radioiodination and became incorporated into liposomes at a high efficiency. Liposomes containing this protein showed specific affinity toward fibronectin-coated surfaces, and this binding could be selectively inhibited by the synthetic cell-attachment peptide but not by inactive peptides. Affinity chromatography on wheat germ agglutinin-Sepharose showed that the 140 kd protein is a glycoprotein and, in combination with the fibronectin fragment chromatography, gave highly enriched preparations of the 140 kd protein. These properties suggest that the 140 kd glycoprotein is a membrane-embedded cell surface protein directly involved in the initial step of cell adhesion to fibronectin substrates.     

Biochemical characterization and biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus-transformed human B and T lymphoid cell lines

The Hodgkin-associated Ki-1 antigen was analyzed in different cell lines. In Hodgkin analogous L428 cells, biosynthetically labeled with radioactive amino acids, the Ki-1 antibody precipitated three glycoproteins with 90, 105, and 120 kDa, respectively. Surface-labeling revealed that the two larger components were membrane-associated forms of the Ki-1 antigen, although the 90-kDa molecule was shown in pulse-chase experiments to be the precursor of the 105- and 120-kDa forms. All three forms of the Ki-1 antigen possess a tunicamycin-sensitive 6-kDa N-linked carbohydrate moiety. O-Linked oligosaccharides could not be detected. Thus, the differences in m.w. are probably not due to glycosylation. The ionophore monensin prevented the appearance of the membrane-associated molecules, which demonstrated that they are assembled between the transcompartment of the Golgi complex and their insertion into the cell membrane. The 90-kDa precursor molecule cannot be generated by disulfide reduction from the two larger forms. After internal labeling with P-32, only the 105- and 120-kDa bands became visible, indicating that the Ki-1 molecule is phosphorylated after its processing into the two larger membrane-associated forms. Analysis of the Ki-1 antigens from other cell lines demonstrated that after external labeling of two other Hodgkin-derived cell lines, six Epstein-Barr virus lymphoblastoid cell lines and one human T leukemia virus I-positive T cell line, both the 105- and the 120-kDa membrane molecules could be detected, regardless of the presence or type of virus integrated.     

Adhesion to the substratum improves the motility ofAmoeba proteus in the absence of a cell nucleus

Summary Anucleated fragments ofAmoeba proteus obtained by dissection and kept on an untreated glass surface fail to adhere to this substratum, lose motor polarity, and stop moving, at least for several hours. If they are transferred after the operation to a highly adhesive surface (polylysine-coated glass), they adhere to the substratum, although locomotion is not spontaneously restored. However, after exposure to a light-shade difference along their body they start moving towards the shaded area and continue locomotion as long as the photic stimulus is acting. Disorganisation of the F-actin cytoskeleton of anucleated fragments was observed on the untreated glass but reorganization on the polylysine-coated surface. The anucleated fragments can show transient recovery of slight spontaneous motor activity and react promptly to external stimuli after up to several days on untreated glass. These intermittent activity periods are enabled by reconstruction of F-actin cytoskeleton in the anucleated fragments during their temporary adhesion to the glass. It is concluded that the injurious effect of cell nucleus removal on the locomotor capacity of amoebae can be compensated by the simultaneous enhancement of cell adhesion and application of a stimulus restoring the motor polarity of the cell. The compensation is achieved by cytoskeletal reorganization.     

Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum.

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.     

Adhesion of B cell lines to endothelial cells from human lymphoid tissue modulates tyrosine phosphorylation and endothelial cell activation

Through the production of cytokines and growth factors the endothelium of secondary lymphoid organs plays a crucial role in controlling lymphocyte migration to the lymphoid microenvironment, an essential step in the initiation of the immune response. Here we demonstrate that direct contact of B cell lines with tonsil-derived human endothelial cells resulted in changes in the phosphorylation state of endothelial cells, causing their functional activation. We found a rapid (<15-s) and transient dephosphorylation, followed by a rapid rephosphorylation of tyrosine residues of the focal adhesion kinase, paxillin, and ERK2. Maximal rephosphorylation occurred after 15-30 min of B cell contact. Preincubation of lymphoid B cells with an adhesion-blocking Ab directed against alpha(4)beta(1) integrin abrogated adhesion-mediated changes of endothelial cell tyrosine phosphorylation, suggesting that cell contact was essential. Similar patterns of tyrosine phosphorylation, but with slightly different kinetics were induced after cross-linking of beta(1) integrin or CD40 on endothelial cells. Functional activation of endothelial cells by B cell adhesion was confirmed by the production of IL-6, IL-8, monocyte chemoattractant protein-1, M-CSF, and macrophage inflammatory protein-1beta mRNA. However, direct cross-linking of beta(1) integrin and CD40 failed to accomplish the same functional activation. These data indicate that direct contact of lymphoid B cells with the endothelium from lymphoid tissue induce endothelial cell signaling, resulting in chemokine and cytokine production. This phenomenon may provide a mechanism for the remodeling of the endothelium from lymphoid tissues, thus contributing to the free migration of lymphocytes and other cells into the lymphoid organs.     

Expression and function of a putative cell surface receptor for fibronectin in hamster and human cell lines

We have previously reported the use of monoclonal antibodies to identify a 140-kD cell surface glycoprotein in mammalian cells that is specifically involved in fibronectin-mediated cell adhesion. We now report the purification of this molecule using immunoaffinity chromatography and the subsequent generation of polyclonal antibodies that selectively immunoprecipitate 140-kD putative fibronectin receptor glycoprotein (gp140) extracted from rodent or human cells; these antibodies also specifically block fibronectin-mediated cell adhesion but not adhesion mediated by other factors in serum. Expression of gp140-like molecules was detected on the surfaces of several adherent human cell lines (HDF, WISH, and EFC) but not on erythrocytes; however, gp140 was also detected on a nonadherent human lymphoid line (DAUDI). Analysis of gp140 on nonreducing SDS gels revealed two closely migrating bands. Protease digestion and peptide mapping suggests that the two bands are closely related polypeptides.     

Biochemical and physiological characterization of the efrotomycin fermentation

Summary An efrotomycin fermentation was characterized through physical, chemical and biochemical studies. Growth of the actinomycete,Nocardia lactamdurans occurred during the first 50 h of the fermentation cycle at the expense of glucose, protein, and triglycerides. The initiation of efrotomycin biosynthesis was observed when glucose dropped to a low concentration. Upon glucose depletion, cell growth ceased and a switch in the respiratory quotient occurred. Efrotomycin biosynthesis was supported by the utilization of soybean oil and starch. Analysis of triglyceride metabolism showed that no diglycerides or monoglycerides accumulated during the fermentation. The activity of extracellular enzymes (lipase, protease, and amylase) increase during the cell growth phase and decreased significantly after 150 h. The concentrations of DNA, tetrahydro-vitamin K₂ (a membrane component), and free amino acids in the supernatant increased dramatically late in the fermentation cycle (225 h), indicating massive cell lysis. During this same time period, a reduction in cellular respiratory activity and efrotomycin biosynthesis were observed.     

The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin.

Binding of the N-terminal 70-kDa (70K) fragment of fibronectin to fibroblasts blocks assembly of intact fibronectin and is an accurate indicator of the ability of various agents to enhance or inhibit fibronectin assembly. Such binding is widely thought to be to already assembled fibronectin. We evaluated this hypothesis with fibronectin-null mouse fibroblasts plated on laminin-1 in the absence of intact fibronectin. As a proteolytic fragment or recombinant protein, 70K bound fibronectin-null cells specifically in linear arrays that extended outwards from the periphery of spread cells. At early time points, these arrays were similar to those formed by intact fibronectin. 70K arrays formed within 5 min following ligand addition at concentrations as low as 5 nM, indicating rapid and high affinity binding. Bound 70K was extractable with Triton X-100 or deoxycholate but became insoluble when cross-linked with a membrane-impermeable agent into large SDS-stable complexes. Intact fibronectin, in contrast, became progressively non-extractable in the absence of cross-linking. The detergent-resistant arrays of cross-linked 70K localized to tips of cellular extensions and partially overlapped with alpha6 and beta1 integrin subunits at the base of the extensions. alpha5 did not localize with 70K arrays, but became progressively co-localized with assemblies of intact fibronectin over time. These results support a model in which the 70-kDa region of fibronectin binds to linearly arrayed cell surface molecules of adherent cells to initiate assembly, display of the arrays is controlled by the integrin that mediates adhesion, and fibronectin-binding integrins promote fibronectin-fibronectin interactions during progression of assembly.     

Diploid and haploid states of the glucocorticoid receptor gene of mouse lymphoid cell lines

A glucocorticoid-sensitive mouse thymoma line, W7, is compared to the mouse lymphoma line S49 which has been extensively used in studies of steroid action. Glucocorticoid-resistant variants are known to arise spontaneously at high rate from S49 (3.5 × 10^?6 per cell per generation) and at a frequency orders of magnitude lower in the case of W7 (<3 × 10^?9). The receptors of both cell lines have the same affinity for dexamethasone (K_d = 1.3 ± 0.3 × 10^?8 M), but W7 cells contain twice the amount of glucocorticoid receptors present in S49 and are measurably more sensitive than S49 cells to dexamethasone. By selection for resistance to low concentrations of dexamethasone, derivatives of W7 have been isolated which are similar to S49 in that they have a higher resistance than the parental W7 line and approximately half the receptor content. Moreover, like S49, the partially resistant variants of W7 give rise to fully resistant derivatives at a high rate (2 × 10^?6 per cell per generation). These results suggest that a structural gene (r) coding for the receptor is present in two functional copies in W7 (r^?,+), but in only one functional copy (r^+/?) in partially resistant derivatives of W7 and in S49. The gene dosage effect observed in these pseudodiploid lines indicates that the receptor gene, r, is autosomal, and that the inactivation of the r gene is a recessive genetic event. Consequences of the homozygous and heterozygous states of the receptor locus are discussed.     

Properties and fate of plasma fibronectin bound to the tissue culture substratum

Plasma fibronectin (pFn) is a serum protein which, when adsorbed to a glass or plastic substratum, mediates the adhesion of fibroblasts in culture. We have studied some of the details of its adsorption and subsequent fate. By using ¹²⁵l-labeled pFn, we show that a substratum incubated with pFn adsorbs approximately 0.4 μg/cm² pFn (a monomolecular layer), and one incubated with medium containing serum adsorbs approximately 7 ng/cm² pFn (a 12-fold enrichment relative to a random selection of the soluble proteins). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) suggests the bound serum proteins (eluted with SDS) are primarily BSA and β-globulins. The bound pFn adheres so tightly, though, that most resists elution, as assayed (1) with pFn radioiodinated before binding, (2) with pFn radioiodinated after binding, or (3) by the cell spreading activity of the bound pFn retained after SDS treatment. Under culture conditions, there is a continuous “turnover” of substratum-bound pFn: soluble pFn can bind to a serum-coated substratum, while bound pFn is gradually removed by incubation with serum proteins. The presence of fibroblasts increases the rate of this removal several-fold. By SDS-PAGE the material removed (as well as that eluted from the substratum with SDS after cell detachment) is intact pFn or large (possibly proteolytically generated) fragments. Thus, pFn binds preferentially to the tissue culture substratum, but can be removed subsequently by the combined action of cells and other serum proteins.     

Biochemical characterization of murine lymphoid alloantigen Ly-m20.2, a cell surface marker controlled by a gene linked to the Mls locus

The lymphoid alloantigen Ly-m20.2 is expressed on the majority of B cells and a wide variety of hemopoietic cells including stem cells. However, it is not detectable on T lymphocytes. Genetic studies indicate that expression of Ly-m20.2 is controlled by a gene(s) closely linked to the M1s locus. Our present biochemical analysis shows that Ly-m20.2 is a monomeric glycoprotein of 55,000 to 60,000 daltons, with no detectable intramolecular disulfide bonds. The Ly-m20.2 molecules of tissues and clonal cell lines exhibit size and charge heterogeneity that can be eliminated by the complete removal of N-linked sugars with the enzyme endo-F or by inhibiting glycosylation with tunicamycin. The resulting unglycosylated Ly-m20.2 molecule migrates as a single band of 40,000 daltons in SDS-gels and behaves as a single charge species in IEF. The Ly-m20.2 antigen was compared biochemically with two other alloantigens: LyM-1, an alloantigen whose expression is also controlled by gene(s) tightly linked to the M1s locus, and Ly-17.1, an alloantigen serologically allelic to the Ly-m20.2 antigen. Immunoprecipitates obtained with the respective LyM-1 and Ly-17.1 antisera yielded similar 55,000 to 60,000 dalton molecules from cells of the appropriate mouse strains. In the case of LyM-1, sequential immunoprecipitation provided evidence that Ly-m20.2 and LyM-1 are identical.     

Isolation and characterization of Chinese hamster ovary cell variants defective in adhesion to fibronectin-coated collagen

Variant clones of Chinese hamster ovary (CHO) cells were selected for reduced adhesion to serum-coated tissue culture plates. These clones also displayed reduced adhesion to substrata composed of collagen layers coated with bovine serum or with fibronectin (cold-insoluble globulin). Wild-type (WT) and adhesion variant (ADv) cells grew at comparable rates in suspension culture, but the adhesion variants could not be grown in monolayer culture because of their inability to attach to the substratum. The adhesion deficit in these cells was not corrected by raising the concentration of divalent cations or of serum to levels 10-fold greater than those normally utilized in cell culture. However, both WT and ADv clones could adhere, spread, and attain a normal CHO morphology on substrata coated with concanavalin A or poly-L- lysine. In addition, the adhesion variants could attach to substrata coated with "footpad" material (substratum-attached material) derived from monolayers of human diploid fibroblasts or WT CHO cells. These observations suggest that the variant clones may have a cell surface defect that prevents them from utilizing exogeneous fibronectin as an adhesion-promoting ligand; however the variants seem to have normal cytoskeletal and metabolic capacities that allow them to attach and spread on substrata coated with alternative ligands. These variants should be extremely useful in studying the molecular basis of cell adhesion.     

Biochemical and physiological tests as aids to identification of Verticillium section Nigrescentia

An account is given of several biochemical and physiological techniques which were evaluated as tools to assist in identification of different strains of five species in Verticillium section Nigrescentia, including the important pathogens V. albo-atrum and V. dahliae. Although many of the tests gave results that varied between individual strains of the same species certain enzymatic activity tests provide a means of characterising the individual species studied.     

Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.     

Adhesion of neutrophils to fibronectin: role of the cd66 antigens

Adhesion of neutrophils to substrate is initiated by receptor-ligand interactions that induce outside-in signaling. Inside-out signals and lateral interactions between surface molecules further fine tune the response. This study investigates the role of CD66 in adhesion of neutrophils to fibronectin, using domain-mapped monoclonal antibodies to CD66. Neutrophils express CD66a, CD66b, and CD66c on their surface. The neutrophil surface molecules that bind to fibronectin are the alpha(4)beta(1) and alpha(5)beta(1) integrins. Our results show that the monoclonal antibody Kat4c, which recognizes the AB domain of CD66a, b, and c and the polyclonal anti-CD66 (anti-carcinoembryonic antigen), augments neutrophil adhesion to fibronectin, while monoclonal antibodies to the individual CD66 antigens, the Fab fragment of Kat4c, and a mixture of the individual antibodies to CD66 antigens were unable to affect the adhesion. Thus heterodimerization of CD66a, b, and c is required for promoting neutrophil adhesion to fibronectin. The increased adhesion in presence of Kat4c was inhibited by antibodies to the beta(1) and beta(2) integrins. Antibody ligation of CD66 antigens causes their clustering and concomitant coclustering of the alpha(M) subunit of the beta(2) integrin, thereby activating the integrin. The sugar alpha-methyl mannoside inhibited anti-CD66-mediated clustering, indicating that a carbohydrate-lectin interaction may exist between CD66 and alpha(M) integrin. It also reduced the increased adhesion of neutrophils to fibronectin, suggesting that beta(2) integrin activation precedes beta(1) integrin activation. Further, the anti-CD66-mediated adhesion to fibronectin is accompanied by increased localization of Src family kinases (lyn and hck) to the cytoskeleton and an increase in their kinase activity. These results suggest that crosslinking of CD66a, CD66b, and CD66c promotes activation of the beta(2) integrin and in turn an alteration in the affinity of the beta(1) integrin, which enhances the adhesion of neutrophils to fibronectin.     

Fish cell lines: Establishment and characterization of nine cell lines from salmonids

Summary Nine permanent cell lines have been established from five species of salmonids native to America's Pacific Northwest. With the exception of a hepatoma from an adult trout, the lines were derived from normal tissues of embryonic or juvenile fish. Cells were routinely grown in Eagle's minimum essential medium with 10% fetal bovine serum. Optimum growth temperatures for these lines ranged from 21 to 24°C. All survived storage for at least 1 yr at −65°C and at least 5 yr in liquid nitrogen. Six of the lines were demonstrably free of any microbial contamination but mycoplasmas were found in three. Eight of the lines were heteroploid. The morphology of only one was fibroblastic. All the lines effectively replicated one or more of the common salmonid viruses. Isozyme patterns were consistent with those of the species of origin. These cell lines have significant application in fish virology. This work is a result of research sponsored in part by the Oregon State University Sea Grant College Program supported by NOAA Office of Sea Grant, U.S. Department of Commerce, under Grant NA79AA-D-0016 and by the Oregon Department of Fish and Wildlife under PL-89304 Anadromous Fish Act and is Oregon Agricultural Experiment Station Technical Paper 6857.     

Biochemical characterization and physiological properties of Escherichia coli UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase

The UDP-N-acetylmuramate:L-alanyl-gamma-D-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide L-Ala-D-Glu and L-Ala very poorly. Replacement of meso-diaminopimelic acid by L-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the LD-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.