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PubMed2001

Differential localization of non-muscle myosin II isoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts.

We investigated the localization of non-muscle myosin II isoforms and mono- (at serine 19) and diphosphorylated (at serine 19 and threonine 18) regulatory light chains (RLCs) in motile and non-motile MRC-5 fibroblasts. In migrating cells, myosin IIA localized to the lamella and throughout the posterior region. Myosin IIB colocalized with myosin IIA to the posterior region except at the very end. Diphosphorylated RLCs were detected in the restricted region where myosin IIA was enriched. In non-motile cells, myosin IIA was enriched in peripheral stress fibers with diphosphorylated RLCs, but myosin IIB was not. Our results suggest that myosin IIA may be highly activated by diphosphorylation of RLCs and primarily involved in cell migration.

Cell Movement↗

PubMed1989

In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C.

Treatment of human platelets with 162 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in phosphorylation of a number of peptides, including myosin heavy chain and the 20-kDa myosin light chain. The site phosphorylated on the myosin heavy chain was localized by two-dimensional peptide mapping to a serine residue(s) in a single major tryptic phosphopeptide. This phosphopeptide co-migrated with a tryptic peptide that was produced following in vitro phosphorylation of platelet myosin heavy chain using protein kinase C. The sites phosphorylated in the 20-kDa myosin light chain in intact cells were analyzed by two-dimensional mapping of tryptic peptides and found to correspond to Ser1 and Ser2 in the turkey gizzard myosin light chain. In vitro phosphorylation of purified human platelet myosin by protein kinase C showed that in addition to Ser1 and Ser2, a third site corresponding to Thr9 in turkey gizzard myosin light chain is also phosphorylated. The phosphorylatable myosin light chains from human platelets were found to consist of two major isoforms present in approximately equal amounts, but differing in their molecular weights and isoelectric points. A third, minor isoform was also visualized by two-dimensional gel electrophoresis. Following treatment with TPA, both the mono- and diphosphorylated forms of each isoform could be visualized, and the sites of phosphorylation were identified. The phosphate content rose from negligible amounts found prior to treatment with TPA to 1.2 mol of phosphate/mol of myosin light chain and 0.7 mol of phosphate/mol of myosin heavy chain following treatment. These results suggest that TPA mediates phosphorylation of both myosin light and heavy chains in intact platelets by activation of protein kinase C.

Blood Platelets↗

PubMed1984

Cysteines in the transmembrane region of major histocompatibility complex antigens are fatty acylated via thioester bonds.

Exogenous radioactive palmitic acid is incorporated post-translationally into the HLA-B and -DR heavy chains, but not HLA-A heavy chains or -DR light chains of the human B lymphoblastoid cells JY and T51 . Protease digestions localize the label to the transmembrane region of the B7 heavy chain. Both B7 and DR heavy chains have a cysteine in the transmembrane hydrophobic region, while the A2 heavy chain and DR light chains have none. Palmitic acid is covalently linked to these transmembrane cysteines via a thioester bond since: 1) the label is not removed by organic extraction or boiling in sodium dodecyl sulfate and dithiothreitol, but is released at room temperature by methanolic KOH as methyl palmitate, and by hydroxylamine as palmitohydroxamate . 2) The pH sensitivity and kinetics of release by hydroxylamine and Tris are similar to those of palmitoyl-CoA (thioester linkage) and unlike those of methyl palmitate and palmitoyllysophosphatidylcholine ( hydroxyester linkages). 3) Neutral hydroxylamine treatment (but not neutral Tris treatment) generates sites that can be reduced and alkylated in the transmembrane region of B7 heavy chain and to a lesser extent in DR heavy chain. 4) Organic extraction of pronase digests of labeled B7 yields peptides containing palmitate and cysteine (but not serine or threonine) which co-migrate by thin layer chromatography. A population of beta 2-microglobulin molecules not associated with heavy chains is palmitylated , but not via a thioester linkage.

Cysteine↗

PubMed2000

Ultrastructural and molecular analysis of Bowman's layer corneal dystrophies: an epithelial origin?

PURPOSE: Two mutations (R555Q and R124L) in the BIGH3 gene have been described in anterior or Bowman's layer dystrophies (CDB). The clinical, molecular, and ultrastructural findings of five families with CDB was reviewed to determine whether there is a consistent genotype:phenotype correlation. METHODS: Keratoplasty tissue from each patient was examined by light and electron microscopy (LM and EM). DNA was obtained, and exons 4 and 12 of BIGH3 were analyzed by polymerase chain reaction and single-stranded conformation polymorphism/heteroduplex analysis. Abnormally migrating products were analyzed by direct sequencing. RESULTS: In two families with type I CDB (CDBI), the R124L mutation was defined. There were light and ultrastructural features of superficial granular dystrophy and atypical banding of the "rod-shaped bodies" ultrastructurally. Patients from three families with "honeycomb" dystrophy were found to carry the R555Q mutation and had characteristic features of Bowman's dystrophy type II (CDBII). CONCLUSIONS: There is a strong genotype:phenotype correlation among CBDI (R124L) and CDBII (R555Q). LM and EM findings suggest that epithelial abnormalities may underlie the pathology of both conditions. The findings clarify the confusion over classification of the Bowman's layer dystrophies.

Adult↗

PubMed1999

Why is erythropoietin present in human milk? Studies of erythropoietin receptors on enterocytes of human and rat neonates.

Erythropoietin receptors (Epo-R) are expressed on cells in the small bowel of human fetuses, but their function has not been defined. We hypothesized that intestinal Epo-R are present postnatally, and that recombinant erythropoietin (rEpo) would increase enterocyte migration and decrease cytokine-induced apoptosis. We used reverse transcriptase-polymerase chain reaction and immunohistochemistry to evaluate the presence of Epo-R mRNA and protein in rat intestinal epithelial cells (IEC-6), and in postnatal human and rat bowel. The effect of rEpo on rates of cell migration and proliferation were established in IEC-6 cells by using cell counting and incorporation of bromodeoxyuridine. To determine whether rEpo affects response to injury, cells were pretreated with rEpo, then were damaged with 25 or 50 ng/mL tumor necrosis factor-alpha plus 2.5 microg/mL cycloheximide. Cell death was determined by colorimetric bioassay. We found that Epo-R mRNA and protein were expressed by IEC-6 cells and by enterocytes of postnatal rat and human small bowel. Cells that had been exposed to 0.05 or 5.00 U/mL rEpo migrated faster than did the controls (p < 0.05), but no difference was noted in cell proliferation. Treatment of IEC-6 cells with rEpo before or at the time of injury resulted in a lower percentage of cell death, and this effect was neutralized by anti-Epo antibody. We conclude that Epo-R is expressed in enterocytes postnatally in rats and humans. Recombinant Epo increases the rate of migration of IEC-6 cells and decreases cytokine-induced apoptosis. These studies suggest that Epo within human milk has actions on neonate's intestinal function.

Animals↗

PubMed1990

Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells.

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.

Binding, Competitive↗

PubMed2006

[Investigation of discrepant proteins between two breast cancer cell lines with different metastatic abilities].

BACKGROUND & OBJECTIVE: Metastasis associated proteins play important roles in metastasis. This study was to investigate proteins which may be involved in breast cancer cell metastasis and further explore the potential mechanisms. METHODS: LM-MCF-7 and MCF-7, two breast cancer cell lines with different metastatic potentials, derived from the same parent cell line, were used in our study. Proteomics and Western blot were applied to identify differentially expressed proteins. Wound healing assay was performed to observe the effect of survivin gene on breast cancer cell migration by transfecting pcDNA3-Sur plasmid into MCF-7 cells. RESULTS: Eight differently expressed proteins, which were correlated with cell structures, cellular metabolism, apoptosis, protein enfold or interaction, were identified. Protein expression of nm23 and p27 was relatively higher in MCF-7 cells; while the expression of survivin, Bcl-2 and myosin light chain kinase was relatively higher in LM-MCF-7 cells. Increased migration ability was observed in MCF-7 cells which were transfected with pcDNA3-Sur. CONCLUSION: Metastasis associated proteins exist in breast cancer cell lines with different metastatic abilities. Survivin is closely related to the metastasis in breast cancer cells.

Blotting, Western↗

PubMed1991

Activation of the fibrinogen receptor on human platelets exposed to alpha chymotrypsin. Relationship with a major proteolytic cleavage at the carboxyterminus of the membrane glycoprotein IIb heavy chain.

The serine proteinase alpha chymotrypsin from bovine pancreas (CT) is known to expose fibrinogen binding sites on the surface of human platelets in the absence of cell activation and granular secretion. This is accompanied by the appearance of membrane-bound chymotryptic fragments of both glycoprotein (GP) IIb and GPIIIa, the two subunits of the platelet fibrinogen receptor, the GPIIb-IIIa complex. However, no clear relationship between discrete proteolytic event(s) within GPIIb-IIIa and fibrinogen-binding-site expression has yet been established. We have now evaluated the proteolysis of GPIIb-IIIa by CT by Western blot analyses using a panel of polyclonal and monoclonal antibodies against GPIIb or GPIIIa. The different proteolytic events were then correlated with the kinetics of the expression of active fibrinogen binding sites on platelets, as measured through the binding of 125I-labelled purified fibrinogen and to the capacity of CT-treated platelets to aggregate. Treatment of platelets with CT at 22 degrees C resulted in the expression of fibrinogen binding sites prior to cleavage of GPIIIa (Mr approximately 90,000) into a previously described, major membrane-bound fragment with Mr 60,000. In contrast, fibrinogen receptor expression closely paralleled a proteolytic cleavage at the carboxy terminus of the GPIIb heavy chain (Mr approximately 120,000), which was converted into a faster migrating species with Mr approximately 115,000). This proteolysis resulted in the release of a soluble peptide with an expected molecular mass of less than 3.7 kDa. Quantitation of this peptide using a competitive immunoenzymatic assay, confirmed that its release from the platelet surface correlated with the expression of fibrinogen binding sites and aggregability. When platelets were exposed to CT at 37 degrees C, a prompt increase in fibrinogen binding sites and platelet aggregability was observed, whereas the GPIIb heavy chain was rapidly converted into the carboxy-terminal-cleaved form. However, incubation at 37 degrees C for longer than 10 min resulted in extensive and simultaneous degradation of both the GPIIb heavy and light chains and of GPIIIa, with the latter being converted into the 60-kDa fragment. These later events were associated with a sharp decline of platelet aggregability and a reduction in the number of fibrinogen binding sites. These data allow us to propose that an early and limited proteolytic processing of the GPIIb component of the platelet fibrinogen receptor is associated with a shift of this receptor complex into a state which expresses specific binding sites for fibrinogen. Further cleavage of GPIIIa to generate the 60-kDa fragment results in loss of receptor activity.

Amino Acid Sequence↗

PubMed1998

Requirement of the IL-2 receptor beta chain for the development of Vgamma3 dendritic epidermal T cells.

Vgamma3 TCR cells develop in the fetal thymus and migrate to the skin as dendritic epidermal T cells (DETC). Fetal Vgamma3 thymocytes differentiate from immature heat stable antigen (HSA)high cells to mature HSAlow cells and the latter subset predominantly expresses IL-2 receptor beta chain (IL-2Rbeta). In this study, the role of IL-2Rbeta in the development of Vgamma3 cells was determined in IL-2Rbeta-deficient mice. There was a moderate reduction of mature HSAlow Vgamma3 thymocytes in IL-2Rbeta-deficient mice. Small numbers of Vgamma3 DETC were detected in the fetal skin of IL-2Rbeta-deficient mice, but they were absent in newborn and adult mice. These results suggest that IL-2Rbeta may transduce the crucial signal for survival and/or expansion of Vgama3 cells in the fetal thymus and in the fetal skin. In normal mice, IL-15 but not IL-2 mRNA was expressed in the fetal epidermis and exogenous addition of low concentration of IL-15 to fetal skin organ culture induced proliferation of Vgamma3 DETC. The dependence of fetal Vgamma3 DETC on the expression of IL-2Rbeta and the presence of IL-15 mRNA in the fetal epidermis imply an essential role of IL-15 signaling through IL-2Rbeta in the selective localization of this gammadelta T cell subpopulation in the skin.

Animals↗

PubMed1987

2.5 A structure of aspartate carbamoyltransferase complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate.

In an X-ray diffraction study using the method of multiple isomorphous replacement, the structure of aspartate carbamoyltransferase (EC 2.1.3.2) complexed with the bisubstrate analog N-(phosphonacetyl)-L-aspartate (PALA) has been solved to 2.5 A. Ten rounds of model building and 123 cycles of restrained reciprocal space refinement have resulted in a model containing 94.4% of the theoretical atoms of the protein-inhibitor complex with an R-factor of 0.231. The fit of the model to the density is excellent, except for occasional side-chains and two sections of the regulatory chains that may be disordered. The electron density for the PALA molecule is readily identifiable for both catalytic (c) chains of the asymmetric unit and bonding interactions with several important residues including Ser52, Arg54, Thr55, Ser80, Lys84, Arg105, His134, Arg165, Arg229 and Gln231 are apparent. The carboxylate groups of the PALA molecule are in a nearly cis conformation. Gross quaternary changes between the T and R forms are noted and in agreement with earlier work from this laboratory. Namely, in the new structure the catalytic trimers move apart by 12 A along the 3-fold axis of the enzyme and relocate by 10 degrees relative to each other, adopting a more eclipsed position. The regulatory (r) chains in the new structure reorient about their 2-fold axis by 15 degrees. Large tertiary changes that include domain migration and rearrangement are also present between these two forms. In the R form both domains of the catalytic chain relocate closer to each other in order to bind to the inhibitor. The polar domain seems to bind primarily to the carbamoyl phosphate moiety of PALA, and the equatorial domain binds primarily to the L-aspartate moiety. Other changes in tertiary structure bring the 80s loop (from an adjacent catalytic chain) and the 240s loop into a position to interact with the PALA molecule. Changes have been searched for in all interface regions of the enzyme. While the C1-C4 and C1-R4 regions have been completely altered, most of the other interchain interfaces are similar in the T and R forms. The intrachain interfaces, between domains of the same catalytic chains, have undergone some reorganization as these domains move closer to each other when the inhibitor is bound. This new structure allows a reinterpretation of genetic and chemical modification studies done to date.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed2005

Mast cell and neutrophil peptidases attack an inactivation segment in hepatocyte growth factor to generate NK4-like antagonists.

Hepatocyte growth factor (HGF) is a plasminogen-like protein with an alpha chain linked to a trypsin-like beta chain without peptidase activity. The interaction of HGF with c-met, a receptor tyrosine kinase expressed by many cells, is important in cell growth, migration, and formation of endothelial and epithelial tubes. Stimulation of c-met requires two-chain, disulfide-linked HGF. Portions of an alpha chain containing an N-terminal segment and four kringle domains (NK4) antagonize HGF activity. Until now, no physiological pathway for generating NK4 was known. Here we show that chymases, which are chymotryptic peptidases secreted by mast cells, hydrolyze HGF, thereby abolishing scatter factor activity while generating an NK4-like antagonist of HGF scatter factor activity. Thus, chymase interferes with HGF directly by destroying active protein and indirectly by generating an antagonist. The site of hydrolysis, Leu480, lies in the alpha chain on the N-terminal side of the cysteine linking the alpha and beta chains. This site appears to be specific for HGF because chymase does not hydrolyze other plasminogen-like proteins, such as macrophage-stimulating protein and plasminogen itself. Mast cell/neutrophil cathepsin G and neutrophil elastase generate similar fragments of HGF by cleaving near the chymase site. Mast cell and neutrophil peptidases are secreted during tissue injury, infection, ischemia, and allergic inflammation, where they may oppose HGF effects on epithelial repair. Thus, HGF possesses an "inactivation segment" that serves as an Achilles' heel attacked by inflammatory proteases. This work reveals a potential physiological pathway for inactivation of HGF and generation of NK4-like antagonists.

Amino Acid Sequence↗

PubMed1992

Prediction of migration behavior of oligonucleotides in capillary gel electrophoresis.

The influence of the primary structure (base composition) on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary polyacrylamide gel electrophoresis was investigated using homo- and heterooligomers under denaturing and non-denaturing conditions. Homooligodeoxyribonucleotides of equal chain lengths but of different base composition showed significant differences in mobility. In addition, the migration properties of heterooligomers were found to be highly dependent on their base composition. A simple equation is presented for predicting relative migration times using denaturing and non-denaturing polyacrylamide capillary gel electrophoresis. Orange-G was used as an internal standard and as the basis of the relative migration time calculations. Examples are presented using homo- and heterooligomers in the 10-20-mer range to show the correlation of the primary structure and their predicted and observed migration rates.

Base Sequence↗

PubMed1995

A minimal motor domain from chicken skeletal muscle myosin.

The myosin head (S1) consists of a wide, globular region that contains the actin- and nucleotide-binding sites and an alpha-helical, extended region that is stabilized by the presence of two classes of light chains. The essential light chain abuts the globular domain, whereas the regulatory light chain lies near the head-rod junction of myosin. Removal of the essential light chain by a mild denaturant exposes the underlying heavy chain to proteolysis by chymotrypsin. The cleaved fragment, or "motor domain" (MD), migrates as a single band on SDS-polyacrylamide gel electrophoresis, with a slightly greater mobility than S1 prepared by papain or chymotrypsin. Three-dimensional image analysis of actin filaments decorated with MD reveals a structure similar to S1, but shorter by an amount consistent with the absence of a light chain-binding domain. The actin-activated MgATPase activity of MD is similar to that of S1 in Vmax and Km. But the ability of MD to move actin filaments in a motility assay is considerably reduced relative to S1. We conclude that the globular, active site region of the myosin head is a stable, independently folded domain with intrinsic motor activity, but the coupling efficiency between ATP hydrolysis and movement declines markedly as the light chain binding region is truncated.

Actins↗

PubMed1994

p53 mutations and overexpression in locally advanced breast cancers.

Alterations in the p53 gene were analysed in 39 patients with locally advanced breast cancers (LABCs) (stage III-IV) with inflammatory signs in most cases (UICC stage T4d = 32 patients) by molecular and immunohistochemical (IHC) approaches. All patients were included in the same therapy protocol. Using polymerase chain reaction (PCR) and a single-strand conformational polymorphism migration technique (SSCP), the presence of mutations in exons 2-11, covering the entire coding sequence of the p53 gene, was evaluated. Using the mouse specific anti-human p53 monoclonal antibody (PAb 1801), we also looked for overexpression of the p53 protein in tissue sections. In 16 cases shifted bands were reproducibly identified by PCR-SSCP, and all but one (localised to exon 10) were in exons 5-8, the usual mutational hotspots. Fifteen of these 16 samples were sequenced and 14 of the suspected mutations (36%) were confirmed. Most of them (12) were single nucleotide substitutions, and transitions were more frequent (eight cases) than transversions (four cases). Fourteen of the tumour samples were positively stained with the monoclonal antibody PAb 1801, 11 with nuclear staining only, two with mixed cytoplasmic and nuclear staining and one with cytoplasmic staining only. Staining patterns were very heterogeneous in terms of the percentage of positive cells (10-75%) and their distribution in the tissue section (isolated foci or dispersed cells). In 11 of the 14 mutated cases a positive immunostaining was observed. The presence of a p53 mutation was significantly associated with larger tumour diameter (chi 2 = 7.490, P = 0.0062) and the presence of clinical metastases (stage IV) (chi 2 = 10.113, P = 0.0015). A non-statistically significant trend of association was observed between p53 mutation, negative oestrogen receptors and lower response rate to therapy. Our results in this group of patients and the heterogeneity of the staining of tumour cells in tissue sections suggest that p53 mutations could be a late event in this non-familial form of breast cancer.

Base Sequence↗

PubMed1997

Macrophage stimulating protein (MSP) binds to its receptor via the MSP beta chain.

Macrophage stimulating protein (MSP) is a 78-kDa disulfide-linked heterodimer belonging to the plasminogen-related kringle protein family. MSP activates the RON receptor protein-tyrosine kinase, which results in cell migration, shape change, or proliferation. A structure-activity study of MSP was performed using pro-MSP, MSP, MSP alpha and beta chains, and a complex including the first two kringles and IgG Fc (MSP-NK2). Radioiodinated MSP and MSP beta chain both bound specifically to RON. The Kd of 1.4 nM for MSP beta chain is higher than the reported Kd range of 0.6-0.8 nM for MSP. Pro-MSP, MSP alpha chain, and MSP-NK2 did not bind. Only MSP stimulated RON autophosphorylation. Although the beta chain bound to RON and partially inhibited MSP-induced RON phosphorylation in kidney 293 cells, it did not induce RON phosphorylation. Pro-MSP, MSP alpha chain, or MSP-NK2 failed to activate RON, consistent with their inability to bind to the RON receptor. Functional studies showed that only MSP induced cell migration, and shape change in resident macrophages, and growth of murine keratinocytes. Our data indicate that the primary receptor binding domain is located in a region of the MSP beta chain, in contrast to structurally similar hepatocyte growth factor, in which the receptor binding site is in the alpha chain. However, full activation of RON requires binding of the complete MSP disulfide-linked alphabeta chain heterodimer.

3T3 Cells↗

PubMed1991

Charge forms of serum and whey transferrin in rat differ in the sialic acid content of their glycan chains: immunological implications.

Transferrin has been purified from both rat whey and rat serum. Both proteins migrated as the same size on electrophoresis in the presence of sodium dodecyl sulphate but when electrophoresed under native conditions showed the presence of multiple charge forms. The patterns observed were not altered by iron binding or removal and all forms were shown to bind iron. Treatment with neuraminidase tended to condense all bands into a single species common to both transferrins whereas treatment with a peptide N-glycosidase resulted in a complex pattern of bands consistent with removal of one of the two glycan chains of the transferrins. A sensitive enzyme-linked immunosorbent assay (ELISA) was set up to examine the transferrins. Iron binding and removal and treatment with peptide N-glycosidase all modified the ELISA signal obtained. Neuraminidase, however, did not affect the ELISA signal.

Animals↗

PubMed2003

Mycelial incompatibility operative in pairings between single basidiospore isolates of Helicobasidium mompa.

Helicobasidium mompa is a clampless basidiomycete and binucleate both in secondary and primary hyphae. Single basidiospore isolates were paired in sibling and non-sibling combinations. We did not observe any alterations in colony morphology, such as tuft formation, but mycelial incompatibility, as indicated by the presence of a dark demarcation line between colonies, occurred in a unifactorial manner. Techniques using DNA molecular markers, such as internal transcribed spacer-restriction fragment-length polymorphism (ITS-RFLP), inter-simple-sequence-repeat polymerase chain reaction (PCR), and universally primed PCR, failed to identify nuclear migration to opposite single basidiospore isolates in all but one of the 92 pairings. These results imply that single basidiospore isolates of H. mompa may be incompetent to mate under laboratory conditions and that a single mycelial incompatibility factor operates in single basidiospore isolates.

Basidiomycota↗

PubMed2000

Bone marrow-derived cells as carriers of recombinant immunomodulatory cytokine genes to lymphoid organs.

The purpose of this study was to determine the feasibility of cytokine gene delivery to lymphatic tissue using transduced bone marrow-derived cells. MBAE and pBABE retroviral vectors carrying the genes for murine interleukin-4 and the selection marker neomycin phosphotransferase (neo) were used to transduce bone marrow-derived dendritic cells (DC) and hematopoietic stem cells (HSC). A transduction efficiency of 11-33% for HSC and 2-10% for DC was achieved. Transduced HSC and DC released 55-170 pg of recombinant interleukin-4 per 1 x 10(6) cells/mL in vitro. To study the migration of the cells in vivo, we introduced the transduced cells into syngenic mice. DC were injected subcutaneously into the front limbs of unconditioned mice and HSC were intravenously administered to irradiated mice. The distribution of the transduced cells was studied by quantitative polymerase chain reaction for the neo gene as a marker. After 3 days, DC migrated to the axillary lymph nodes in the drainage area of the injection site and were detectable up to 5 days. After intravenous administration of transduced HSC, the neo gene could be found in up to 100 copies/5 x 10(3) cells in mesenterial lymph node, spleen, bone marrow, thymus, and liver. The distribution of the transduced cells was heterogenous: in different mice, different organs showed high copies of the neo gene after 10 and 13 days. After 39 days, two of three mice were negative for neo in all organs analyzed. In conclusion, bone marrow-derived cells can be genetically engineered ex vivo to deliver recombinant cytokine genes to lymphoid organs in vivo. In particular, DC might be candidate cells for use in immunomodulatory gene therapy for autoimmune diseases and cancer.

Animals↗