Isolation and characterization of restriction endonuclease in Plesiomonas shigelloides and Aeromonas species.

Five restriction endonucleases (ENases) and one ENase were found in a screen of 196 strains of Plesiomonas shigelloides and 147 strains of Aeromonas species. Plesiomonas and Aeromonas species are classified as Vibrionaceae, identified as food-poisoning bacteria, are closely genetically related to each other, and their ENases producing abilities have not bee reported. ENases were detected at relatively low frequencies in these species as compared to those in other species, such as Salmonella species and Vibrio parahaemolyticus.

A 31-kDa recombinant fibronectin cell-binding domain fragment: its binding to receptor, cell adhesive activity, and fusion proteins.

The binding of fibronectin to fibronectin receptor was studied using a recombinant 31-kDa cell-binding domain fragment of fibronectin (C279), which consisted of three type III repeats (III8-III9-III10). Fibronectin receptor in several cell lysates was bound to a column of C279-immobilized Sepharose HP and obtained in a highly purified form by elution with a synthetic peptide, GRGDSP. alpha 5 beta 1-Integrin was detected in the GRGDSP-eluted fraction by immunoblotting.

A novel Streptomyces restriction endonuclease, Sse1825I, cleaving at 5'-GG/GWCCC-3'.

We isolated and characterized from a Streptomyces species a new class-II restriction endonuclease, which recognizes the palindromic heptanucleotide sequence [sequence: see text] (where W = A or T) and cleaves double-stranded DNA after the second G in this sequence. This Sse1825I enzyme cleaves phage lambda DNA at one site, adenovirus type 2 DNA at eight sites, but does not cleave pBR322, SV40, ColE1, pUC18 and pUC19, and replicative forms of M13mp18 and M13mp19, and phiX174 DNAs.

Role of the heparin-binding domain of chimeric peptides derived from fibronectin in cell spreading and motility.

Cellular responses to fibronectin (FN) are likely to have a complex molecular basis involving the interactions between multiple functional domains of FN and specific cell surface molecules. We have utilized several types of recombinant FN fragments and their chimeric fragments to examine the regulatory mechanisms of the spreading and chemotactic migration of HT1080 human fibrosarcoma cells on FN. The CH-271 fusion fragment, in which the cell-binding domain (C-274) of FN is adjacent to the heparin-binding domain (H-271), promoted cell spreading more efficiently than C-274, H-271, or their mixture (C-274 + H-271) over a 60-min incubation.

Molecular cloning and characterization of two complementary DNAs encoding putative peroxidases from rice (Oryza sativa L.) shoots.

PCR with oligonucleotide primers that corresponded to two highly homologous regions, in terms of amino acid sequence, of plant peroxidases was used to amplify a specific DNA fragment from a mixture of rice (Oryza sativa L.) cDNAs. We then screened a cDNA library prepared from mRNAs of rice shoots utilizing the product of PCR as probe.

Angiogenic activity of a fusion protein of the cell-binding domain of fibronectin and basic fibroblast growth factor.

We constructed a fusion protein of the cell-binding domain of human fibronectin and human basic fibroblast growth factor, and prepared a polypeptide with both cell-adhesive activity and growth factor activity. A human gene fragment coding for basic fibroblast growth factor was amplified by the polymerase chain reaction, and introduced into the expression vector pTF7520, which encodes the cell-binding domain of human fibronectin. The resulting plasmid encoded a fusion protein in which basic fibroblast growth factor was added covalently to the C-terminal end of the fibronectin fragment.

Gene structure and expression of the MboI restriction--modification system.

The genes from Moraxella bovis encoding the MboI restriction--modification system were cloned and expressed in Escherichia coli. Three open reading frames were found in the sequence containing the genes. These genes, which we named mboA, mboB, and mboC, had the same orientation in the genome.

Engineering of artificial cell adhesion proteins by grafting the Arg-Gly-Asp cell adhesive signal to a calpastatin segment.

A new artificial cell adhesive protein was engineered by grafting the Arg-Gly-Asp (RGD) sequence, the minimal recognition signal of fibronectin for interaction with integrins, to a calpastatin segment by in vitro mutagenesis. The mutagenized protein showed cell adhesive activity in addition to calpain inhibitory activity. The RGD signal grafted to the calpastatin segment was recognized by the vitronectin receptor but not by the fibronectin receptor.

Purification of restriction endonuclease EcoO128I produced by an enteropathogenic Escherichia coli O128Ly3.

Restriction endonuclease EcoO128I, an isoschizomer of BstEII, was purified from a rough mutant of Escherichia coli O128Ly3. EcoO128I should be more convenient for recombinant DNA applications than BstEII, because of its improved cleavage activity at 37 degrees C.

Cloning and expression of the HpaI restriction-modification genes.

The genes from Haemophilus parainfluenzae encoding the HpaI restriction-modification system were cloned and expressed in Escherichia coli. From the DNA sequence, we predicted the HpaI endonuclease (R.HpaI) to have 254 amino acid residues (Mr 29,630) and the HpaI methyltransferase (M.

Construction and characterization of a fusion protein with epidermal growth factor and the cell-binding domain of fibronectin.

An efficient expression system was constructed for C-EGF, a fusion protein made of a fragment of the cell-binding domain of human fibronectin (FN) bound with epidermal growth factor (EGF). C-EGF was produced in Escherichia coli HB101 cells carrying the recombinant plasmid pCE102 as inclusion bodies, which were solubilized and refolded after purification. C-EGF had both cell-adhesive and EGF activities, so it might be more effective than EGF in therapeutic applications.

Inhibitory effect of antimetastatic fusion polypeptide of human fibronectin on tumor cell adhesion to extracellular matrices.

We investigated the inhibitory mechanism of liver metastasis by using recombinant fragments with cell- and/or heparin-binding domains (C-274, H-271 or the fusion fragment CH-271). Intravenous co-injection of L5178Y-ML25 cells with CH-271 was more effective for the inhibition of liver metastasis than C-274, H-271 or C-274 + H-271. Reduction of the arrest and retention of the radiolabeled tumor cells in the liver of mice was found when CH-271 was co-injected with tumor cells.

Recombinant fusion polypeptide with cell- and heparin-binding domains of fibronectin inhibits liver metastasis of L5178Y-ML25 lymphoma cells.

We have investigated the effect of recombinant polypeptides with cell-binding domain (C-274) or with heparin-binding domain (H-271) and their fusion polypeptide (CH-271) on liver metastasis of murine lymphoid tumor. The polypeptides containing heparin-binding domain, H-271 and CH-271, were able to inhibit liver metastasis when co-injected i.v.

Inhibitory effect of fibronectin and its recombinant polypeptides on the adhesion of metastatic melanoma cells to laminin.

We have utilized recombinant fibronectin fragments with cell-binding domain (C-274), heparin-binding domain (H-271) or CS1 peptide in type III connecting segment (IIICS) and their fusion polypeptides such as CH-296 (containing C-274, H-271 and CS1), CH-271 (containing C-274 and H-271) and C-CS1 (containing C-274 and CS1) to investigate the mechanism of the fibronectin-mediated inhibitory effect on tumor cell adhesion to laminin as well as fibronectin. These fragments retained cell adhesion-promoting and/or heparin-binding properties when they were immobilized on a surface. Pretreatment of tumor cells with CH-296 or CH-271 suppressed cell adhesion to both laminin and fibronectin.

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13.

Role of type III homology repeats in cell adhesive function within the cell-binding domain of fibronectin.

Recombinant fibronectin (FN) fragments and their mutant proteins were produced to elucidate the role of type III homology repeats in cell adhesive activity within the cell-binding domain of FN. Cell adhesive activity of the 11.5-kDa fragment, the cell attachment site of the cell-binding domain, was less than 0.

Inhibition of lung metastasis by synthetic and recombinant fragments of human fibronectin with functional domains.

We have investigated the antimetastatic effect of synthetic or recombinant peptides containing the functional domains of fibronectin on experimental and spontaneous lung metastases of murine tumor cells. CS1 peptide which is present within type III homology connecting segment (IIICS) as well as C-274 (cell-binding domain) were able to inhibit experimental lung metastasis when co-injected intravenously (iv) with B16-BL6 melanoma cells, while H-271 (heparin-binding domain) could not. In the spontaneous metastasis model, multiple iv administrations of CS1 or C-274 after surgical excision of primary tumors caused a significant reduction of metastatic colonies in the lung.

Modulation of haptotactic migration of metastatic melanoma cells by the interaction between heparin and heparin-binding domain of fibronectin.

We utilized recombinant fibronectin polypeptides with cell-binding domain and heparin-binding domains (referred to as C-274 and H-271, respectively) and their fusion polypeptide (CH-271) to examine the role of sulfated polysaccharide heparin and/or the functional domains of fibronectin in modulating tumor cell behavior. Both C-274 and CH-271 polypeptides with cell-binding domains promoted the adhesion and migration of B16-BL6 melanoma cells, whereas H-271 did not. Heparin bound to the immobilized polypeptides with heparin-binding domain (H-271, CH-271, and a mixture of C-274 and H-271 or fibronectin) but did not affect the tumor cell adhesion to the substrates.

Characterization of a functional domain of human calpastatin.

Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli.

Isolation and characterization of two monoclonal antibodies that recognize remote epitopes on the cell-binding domain of human fibronectin.

Two monoclonal anti-fibronectin antibodies that inhibit fibronectin-mediated cell adhesion have been established and characterized. One antibody, FN12-8, inhibited attachment of rat kidney fibroblasts on the fibronectin-coated substrate in a concentration-dependent manner, attaining a maximal inhibition of greater than 85% at 850 micrograms/ml. Another antibody, FN30-8, caused about 70% inhibition at a concentration as low as 0.

Primary structure and functional expression of h-caldesmon complementary DNA.

Recently, the two Mr forms of caldesmon (Mr's in the range of 120-150kDa and 70-80kDa as judged by SDS-PAGE) have been identified. h-Caldesman (high Mr 120-150kDa caldesmon) is predominantly expressed in smooth muscles, and l-caldesmon (low Mr 70-80kDa caldesmon) in non-muscle cells. In this paper, we report the nucleotide sequence of chick embryo gizzard h-caldesmon cDNA and its translation into amino acid sequence.

The primary structure of porcine liver acylamino acid-releasing enzyme deduced from cDNA sequences.

Two overlapping cloned cDNAs encoding the entire amino acid sequences of the subunits of acylamino acid-releasing enzyme (AARE) [EC 3.4.19.

A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide.

A tetrapeptide Arg-Gly-Asp-Ser (RGDS) has been shown to be a versatile cell recognition signal of extracellular matrix components for the interaction with cells. We introduced the RGDS tetrapeptide into a truncated form of protein A, a staphylococcal immunoglobulin-binding protein, by inserting an oligonucleotide cassette encoding the tetrapeptide into the coding region of the protein A expression vector pRIT2T. The mutagenized protein was capable of not only binding to immunoglobulin G but also mediating cell attachment and spreading onto an inert substrate.