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At least 91 records · Page 5Linked to original sources
PubMed2000

[Construction of an expression plasmid vector for accomplishing in vivo delivery of recombinant biologically active proteins. 2. Synthesis of HBcAG in a vaccine strain of Salmonella choleraesuis].

The capacity of a previously described plasmid vector pAZ to deliver bioactive proteins to targets in vivo has been studied. This vector molecule has a strong constitutive promoter, is extremely stable in cells of vaccinal S. choleraesuis strain, and encodes the synthesis of marker protein beta-galactosidase which helps monitor the vector's fate in the host. The gene encoding hepatitis B virus core antigen (HBcAg) has been inserted into pAZ under its constitutive promoter. The resultant recombinant plasmid p19-24 has been used to transform Enterobacteriaceae (E. coli and S. choleraesuis) cells. Transformed cells produce immunologically active HBcAg. p19-24 was stable in S. choleraesuis cells during their culturing and during this strain persistence in mice. Triple oral immunization of rabbits in a dose of 1 x 10(9) S. choleraesuis cells TC177 induced the production of virus-specific antibodies. Successful transformation of cells of another vaccinal strain S. abortus ovis by this plasmid extends the potentialities of the vector. The results demonstrate good prospects of using pAZ vector for the construction of live oral vaccines.

Amino Acid Sequence↗
PubMed1997

Separation used for purification of recombinant proteins.

The purification of molecules from recombinant cells may be strongly influenced by the molecular biology of gene isolation and expression. At the beginning of the process there may be a demand for information on the minute amounts of proteins and thus for ever increasingly sensitive techniques. Purification of recombinant proteins can differ from conventional purifications in several ways, depending on the solubility of the protein, occurrence in inclusion bodies, creation of fusion proteins with tags that enable simpler purification. Sometimes a (re)naturation step is required to get a bioactive protein. On the other hand, the techniques used in separation are essentially the same as for purification from the natural source and environment.

Chromatography, Liquid↗
PubMed2001

Site-directed mutagenesis of heparin-binding EGF-like growth factor (HB-EGF): analysis of O-glycosylation sites and properties.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a 22 kDa, O-glycosylated protein. HeLa cells infected with a recombinant vaccinia virus expressing human HB-EGF produced a secreted, bioactive protein, with Mr 22,000 that was decreased to 14,000 by treatment with O-glycanase. Site-directed mutagenesis of HB-EGF cDNA using oligonucleotide- and PCR-directed techniques was performed to change the potential glycosylation sites, Thr75 and Thr85, to alanine residues to prevent O-glycosylation. Purification and characterization of the mutant proteins demonstrated that: (i) both O-glycosylation sites of HB-EGF are utilized, (ii) HB-EGF secretion does not require O-glycosylation, (iii) removal of O-glycans does not affect proteolytic cleavage of the HB-EGF precursor, nor does it influence HB-EGF intracellular trafficking or subcellular localization, and (iv) HB-EGF produced by HeLa cells is heavily sialylated. Comparisons between glycosylation mutants and wild-type HB-EGF revealed no significant apparent differences in receptor binding activity.

Alanine↗
PubMed1998

[Molecular design of polymer-conjugated cytokines and its application for drug delivery systems].

In recent years, clinical application of recombinant cytokines has been expected as a novel drug for various diseases. However, cytokines have generally poor stability in vivo, so they required of very high doses to achieve sufficient clinical effect. In addition, because cytokines have pleiotropic functions, they would cause unfavourable side-effects. Therefore an drug delivery system (DDS) is necessary for clinical use, which stabilizes the cytokines and potentiates only the expected function from other unfavorable ones in vivo. Then we performed the chemical modification of cytokines with water-soluble polymers to overcome such problems as poor stability and pleiotropic activities. This approach includes some advantages that decrease renal excretion rate of proteins and prevent the degradation by proteases. This would result in prolonging the half-lives of bioactive proteins and potentiating their clinical effects. Interestingly, we found that the polymer-conjugated cytokines, that we named hybrid-cytokines, for instance, polyethylene glycol-modified interleukin-6 (PEG-IL-6), were able to increase selectively in their function of promoting platelet production, but not in other unfavourable functions. This effects were suggested for a result of the change in the systemic distribution pattern by pegylation of proteins. In this review, we proposed that the DDS using hybrid-cytokines would be able to increase the stability and regulate the spectrum of the functions of the cytokines by controlling the pharmacokinetics and pharmacodynamics in vivo. This will provide a fundamental information enabling us to design the hybrid-proteins applicable to therapeutic use.

Cytokines↗
PubMed1997

Inclusion bodies and purification of proteins in biologically active forms.

Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved.

Animals↗
PubMed2006

Properties and exploitation of oleosins.

Oleosins stabilize oil bodies in seeds and other tissues and contain a unique hydrophobic domain which appears to be inserted into the oil matrix as an alpha-helical hairpin. The oleosin proteins may be exploited to stabilize emulsions while the ease of oil body preparation has led to the expression of bioactive proteins as oleosin fusions in molecular farming.

Arabidopsis Proteins↗
PubMed2005

Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells.

The protein delivery across cellular membranes or compartments is limited by low biomembrane permeability because of the hydrophobic characteristics of cell membranes. Usually the delivery processes utilize passive protein channels or active transporters to overcome the membrane impediment. In this report, we demonstrate that arginine-rich intracellular delivery (AID) peptide is capable of efficiently delivering fused fluorescent proteins unpreferentially into different plant tissues of both tomato (a dicot plant) and onion (a monocot plant) in a fully bioactive form. Thus, cellular internalization via AID peptide can be a powerful tool characterized by its simplicity, non-invasion and high efficiency to express those bioactive proteins in planta or in plant cells in vivo. This novel method may alternatively provide broader applications of AID chimera in plants without the time-consuming transgenic approaches.

Allium↗
PubMed1988

Purification of a paracrine factor, P-Mod-S, produced by testicular peritubular cells that modulates Sertoli cell function.

A testicular paracrine factor, P-Mod-S, was purified from conditioned medium obtained from serum-free cultures of peritubular cells. Stimulation of testicular transferrin production by cultured Sertoli cells was utilized as a bio-assay for P-Mod-S. A bioactive protein with an apparent molecular weight of 50,000 under physiological conditions was isolated by high pressure size exclusion chromatography. P-Mod-S was found to have an affinity for heparin and bound to a heparin affinity column. Two forms of P-Mod-S were purified with reverse-phase chromatography. The less hydrophobic form was referred to as P-Mod-S (A) and is a 56,000 molecular weight protein. The more hydrophobic form was referred to as P-Mod-S (B) and is a 59,000 molecular weight protein. Purification of P-Mod-S (A) and P-Mod-S (B) from peritubular cell-radiolabeled secreted proteins revealed that both proteins contain radioactivity. This result demonstrates active synthesis and secretion of P-Mod-S by peritubular cells. Although the amino acid composition of the two proteins indicates distinct differences in the content of several amino acids, the relationship of P-Mod-S (A) and P-Mod-S (B) is unknown at present. A greater than 1000-fold increase in the specific activity of P-Mod-S was achieved with the purification procedure utilized. P-Mod-S can account for essentially all the bioactivity present in crude peritubular cell-secreted protein preparations. The effects of the two forms of P-Mod-S on both transferrin and androgen-binding protein production by Sertoli cells was examined. Purified forms of P-Mod-S were found to have a greater effect on Sertoli cell function than any individual regulatory agent previously known to influence the cell, including follicle-stimulating hormone. The significance of peritubular cell-Sertoli cell interactions mediated via P-Mod-S to spermatogenesis and testicular function is discussed, as well as insight provided into general mesenchymal-epithelial cell interactions.

Amino Acids↗
PubMed1996

Recombinant human milk proteins - an opportunity and a challenge.

Several human milk proteins have physiologic functions in infants. These proteins are involved in defense against infectious agents and in the optimization of nutrient uptake from milk. Therefore, interest in producing recombinant human milk proteins to use in infant formula has been growing. Microorganisms and transgenic animals can now be used for the production of bioactive proteins. However, the benefits of each protein must be evaluated in cells, animal models, and infants before claims can be made that adding them to formula improves the health or nutrition of infants. Once benefits are shown, proper manufacturing conditions must be developed for introducing the protein or proteins into formula. Processing conditions must be evaluated to ensure that biologic activity is maintained. Dry blending, aseptic processing, sterile filtration, and other techniques will likely be necessary for introducing proteins that require specific tertiary structure for activity. The importance of posttranslational modifications must also be considered: some proteins may require proper glycosylation or phosphorylation for physiologic activity.

Animals↗
PubMed2004

[Development of novel DDS technologies for pharmacoproteomic-based drug discovery and development].

With the success of the Human Genome Project, the focus of life science research has shifted to the functional and structural analyses of proteins, such as proteomics and structural genomics. These novel approaches to the analysis of proteins, including newly identified ones, are expected to help in the identification and development of protein therapies for various diseases. Thus pharmacoproteomic-based drug discovery currently has a very high profile. Nevertheless, the use of bioactive proteins in the clinical setting is not straightforward because in vivo these proteins have low stability and pleiotropic action. To promote pharmacoproteomic-based drug discovery and development, we have attempted to establish a system for creating functional mutant proteins (muteins) with the desired properties and to develop a site-specific bioconjugation system for further improving their therapeutic potency. These innovative protein-drug systems are discussed in this review.

Drug Delivery Systems↗
PubMed2005

Phage display and PEGylation of therapeutic proteins.

With the success of the human genome project, the focus of life science research has shifted to the functional and structural analyses of proteins, such as disease proteomics and structural genomics. These novel approaches to the analyses of proteins, including newly identified ones, are expected to help in the identification and development of protein therapies for various diseases. Thus, disease proteomic-based drug discovery has a very high profile. Nevertheless, the use of bioactive proteins in the clinical setting is not straightforward because, in vivo, these proteins have a low stability and a pleiotropic action. To promote disease proteomic-based drug discovery and development, we have attempted to establish a system for creating functional mutant proteins (muteins) with the desired properties, and also to develop a site-specific polymer-conjugation system for further improving their therapeutic potency. These innovative protein-drug systems are discussed in this review.

Animals↗
PubMed2004

Milleporin-1, a new phospholipase A2 active protein from the fire coral Millepora platyphylla nematocysts.

Stings of fire corals, potent hydroids common in the Red Sea, are known to cause severe pain and they develop burns and itching that lasts few hours after contact. Nematocyst venom of Millepora platyphylla (Mp-TX) was isolated according to a recent method developed in our laboratory to conduct a previous investigation on the nematocyst toxicity of Millepora dichotoma and M. platyphylla. In this study, Mp-TX was fractionated by using both gel filtration and ion exchange chromatography. Simultaneous biological and biochemical assays were performed to monitor the hemolytic (using washed human red blood cells, RBCs) and phospholipase A2 (using radiolabeled sn-2 C14-arachidonyl phosphatidylcholine as a substrate) active venom fractions. The magnitude of both hemolysis and phospholipase A2 activity was found in a fraction rich of proteins of molecular masses approximately 30,000-34,000 Daltons. The former fraction was purified by ion exchange chromatography, and a major bioactive protein factor (approx. 32,500 Daltons , here named milleporin-1) was recovered. Milleporin-1 enzymatic activity showed a significant contribution to the overall hemolysis of human RBCs. This activity, however, could not be completely inhibited using phospholipid substrates. Melliporin-1 fraction retained about 30% hemolysis, until totally rendered inactive when boiled for 3 min. The overall mechanism of action of milleporin-1 to impact the cellular membrane was discussed; however, it is pending more biochemical and pharmacological future studies.

Animals↗
PubMed2000

Microglia and macrophages are the major source of tumor necrosis factor in permanent middle cerebral artery occlusion in mice.

The proinflammatory cytokine tumor necrosis factor (TNF) is known to be expressed in brain ischemia; however, its cellular and temporal appearance is not fully settled. In this study, nonradioactive in situ hybridization for murine TNF mRNA was performed on brain sections from adult C57x129 mice at 6 hours, 12 hours, 24 hours, 2 days, 5 days, or 10 days (six to eight mice per group) after induction of permanent focal cerebral ischemia. Cortical infarct volumes were estimated, and TNF mRNA-expressing cells were counted within the infarct and infarct border using Cast-Grid analysis. At 12 hours, a peak of 19.2 +/- 5.1 TNF mRNA-expressing cells/mm2 was counted, contrasting two to three times lower values at 6 and 24 hours (6.4 +/- 4.6 and 9.2 +/- 3.4 cells/mm2, respectively) and <2 cells/mm2 at 48 hours and later stages. The TNF mRNA-expressing cells were distributed along the entire rostrocaudal axis of the cortical infarcts and occasionally within the caudate putamen. At all time points, TNF mRNA colocalized with Mac-1-positive microglia/macrophages but not with Ly-6G (Gr-1)-positive polymorphonuclear leukocytes. Similarly, combined in situ hybridization for TNF mRNA and immunohistochemistry for glial fibrillary acidic protein at 12 and 24 hours revealed no TNF mRNA-expressing astrocytes at these time points. Translation of TNF mRNA into bioactive protein was demonstrated in the neocortex of C57B1/6 mice subjected to permanent middle cerebral artery occlusion. In summary, this study points to a time-restricted microglial/macrophage production of TNF in focal cerebral ischemia in mice.

Animals↗
PubMed2006

Monocyte chemotactic protein-1 is a potential player in the negative cross-talk between adipose tissue and skeletal muscle.

Adipose tissue is a major secretory and endocrine active organ producing a variety of bioactive proteins that may regulate energy metabolism and insulin sensitivity. In several studies, we have already shown that adipocyte-secretory products induce skeletal muscle insulin resistance. However, the precise nature of these factors has remained elusive. Human adipocytes were found to secrete various cytokines including IL-6, IL-8, macrophage inflammatory protein-1alpha/beta, and monocyte chemotactic protein-1 (MCP-1). Among these candidates, MCP-1 alone impaired insulin signaling in skeletal muscle cells at doses similar to its physiological plasma concentrations (200 pg/ml), whereas IL-6, IL-8, and macrophage inflammatory protein-1beta were effective at very high concentrations only. In addition, MCP-1 significantly reduced insulin-stimulated glucose uptake in the myocytes. Expression analysis of chemokine receptors in skeletal muscle cells revealed the presence of chemokine CXC motif receptor 1/2 and chemokine CC motif receptor 1/2/4/5/10. The action of MCP-1 on insulin signaling in skeletal muscle cells occurs via ERK1/2 activation but does not involve activation of the nuclear factor kappaB pathway. In conclusion, our data show that adipocytes secrete various adipokines that may be involved in the negative cross-talk between adipose tissue and skeletal muscle. Human skeletal muscle cells are highly sensitive toward MCP-1, which impairs insulin signaling and glucose uptake at concentrations even below that found in the circulation. However, other cytokines that are released by adipocytes impair insulin action only at supraphysiological concentrations. Therefore, MCP-1 may represent a molecular link in the negative cross-talk between adipose tissue and skeletal muscle assigning a completely novel important role to MCP-1 besides inflammation.

Adipocytes↗
PubMed2004

[DDS and Me].

With the success of the human genome project, the focus of life science research has shifted to the functional and structural analyses of proteins, such as proteomics and structural genomics. These analyses of proteins including newly identified proteins are expected to contribute to the identification of therapeutically applicable proteins for various diseases. Thus, pharmaco-proteomic-based drug discovery and development for protein therapies, including gene therapy, cell therapy, and vaccine therapy, is attracting current attention. However, there is clinical difficulty in using almost all bioactive proteins, because of their very low stability and pleiotropic actions in vivo. To promote pharmaco-proteomic-based drug discovery and development, we have attempted to develop drug delivery systems (DDSs), such as the protein-drug innovation system and the optimal cell therapeutic system. In this review, we introduce our original DDSs.

Animals↗
PubMed2002

[Isolation and identification of proteins with anti-tumor and fibrinolysogen kinase activities from Eisenia foetida].

Proteins from Eisenia foetida possess many biological activities. A group of proteins precipitated by ethanol were isolated and purified by Sephadex G-75 and HiPrep 16/60 DEAE columns, then identified by one- or two- dimensional electrophoresis and mass spectrometry. 2D gel experiments displayed that the pI of proteins from Eisenia foetida were mainly from 3.0 to 4.0. Anti-tumor and kinase activities were determined by in vitro experiments. The enthanol fraction D2(8) showed both of the activities. These ethanol-precipitated proteins were identified further by native polyacrylamide electrophoresis, the protein spots were cut off from gels and digested by trypsin, the peptide mass fingerprints (PMFs) were determined by mass spectrometry. PMF, molecular weight, amino acid composition and N-terminus of 6 proteins were characterized, and band 9 was identified as D2(8). The results suggested that there exist proteins in Eisenia foetida possessed both anti-tumor and fibrinolysogen kinase activities. These methods can be used for identification of the natural bioactive proteins.

Amino Acids↗
PubMed2006

Clinical applications of bioactive factors in sports medicine: current concepts and future trends.

The ability to biologically manipulate musculoskeletal healing and augment bone and soft tissue repair and regeneration holds great promise. Advances in the basic science study and clinical application of bioactive proteins and growth factors continues to evolve. Improvement in the surgical resurfacing of articular cartilage defects and tendon and ligament repair through the addition of bioactive polypeptides is currently underway. The purpose of this article is to review the present array of biologically active materials that may be clinically applicable in sports medicine and arthroscopy. Mechanisms for biologic augmentation of tissue repair and regeneration will be discussed. Current limitations and future considerations will be reviewed particularly as they relate to practical clinical approaches.

Arthroscopy↗