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PubMed2004

Copolymers of 2-methacryloyloxyethyl phosphorylcholine (MPC) as biomaterials.

Copolymers of 2-methacryloyloxyethyl phosphorylcholine (MPC) showed good hemocompatibility as hypothesized. The hypothesis was surfaces having phosphorylcholine groups by polymerization of MPC could accumulate phospholipids from blood stream and show good blood compatibility. We designed and prepared a methacylate having a phosphorylcholine group. While it was possible to introduce them by polymer reactions, polymer reaction is not always good method to prepare the desired pure surface. This must be very important point to consider biomaterials, as we have to apply them in our body without any adverse effects. The hypothesis was confirmed by changing copolymer composition. The adsorption amount of phospholipids on the surfaces increased with increasing the MPC units in the copolymers. On the other hand, increasing MPC units in MPC copolymers decreased adsorption amount of peptides. There is limitation in blood compatibility tests in vitro due to unstable characteristics of blood itself. We evaluated them with series of blood compatibility tests, in vitro, ex vivo and in vivo, on coated PMMA beads, modified hollow fibers for hemodialysis and 2 mm small diameter blood vessels, respectively. These data suggested MPC is a promising methacrylate to develop good blood contacting devises, which may not require systemic anticoagulation. Conventional blood compatible biomaterials were not suitable to make permeable membranes. But MPC is soluble in water and we could prepare permeable membranes to various solutes by the copolymerization. Introduction of MPC copolymers on cellulose and polysulfone hollow fiber membranes gave them nonthrombogenicity but it did not give adverse effect on their permeability. These data suggested that it is possible to apply them to hemodialyzers, oxygenators and percutaneous glucose sensors to keep diabetic patients easier. MPC surfaces are good hydrogel to minimize damage on tissues by lubricating between organs and the coated devices. They do not induce denaturation of peptides, which is beneficial to keep activities of enzymes longer. And poly-MPC dissolved is applicable to stabilize several bioactive peptides in aqueous phase. So MPC polymers are useful to minimize fouling by inhibiting the adsorption of bioactive proteins. MPC has high potential to develop many varieties of new biomaterials useful in so-called biotechnology. MPC and their copolymers are commercially available from NOF (Tokyo, Japan) and Biocompatibles (UK, as PC technology).

Biocompatible Materials↗

PubMed2006

Using recombinant platelet-derived growth factor to facilitate wound healing.

Recombinant human platelet-derived growth factor (rhPDGF-BB) is a bioactive protein that has been combined with bone graft materials in an effort to regenerate the periodontium to its prediseased state more predictably. The combination of a biocompatible scaffold with this highly purified, recombinant human growth factor capitalizes on PDGF's stimulatory effects on cellular migration and proliferation, with subsequent matrix formation, to drive the healing process toward regeneration. When using a growth factor such as rhPDGF-BB with broad wound healing activity, an added clinical benefit is the more rapid soft tissue wound healing pattern observed at the surgical site. This also may be seen as a patient-centered benefit because it allows dentists to provide for a patient's prosthetic care on a timely basis, leading to a faster return to normal function. Also, the patient may resume plaque control efforts earlier, which further facilitates the wound-healing process. The purpose of these 2 case reports is to document the use of rhPDGF-BB as a wound-healing agent in 2 clinical situations and to provide a rationale for its use in this capacity.

Aged↗

PubMed1991

A simple nonisotopic in vitro bioassay for LT and TNF employing sodium fluoride-treated L-929 target cells that detects picogram quantities of LT and TNF and is as sensitive as TNF assays done with ELISA methodology.

We have developed a highly sensitive in vitro bioassay for lymphotoxin (LT) and tumor necrosis factor (TNF). This system employs a monolayer of 20,000 L-929 target cells in each well of 96-well microplates that are incubated in dilutions of LT and TNF at 37 degrees C for 20-24 h, after which the viability of the L-929 target cells is assessed by spectrophotometric techniques. We find that incorporation of 2 mM NaF increases the sensitivity of L-929 cells to lysis by 10(3)-10(4) x for LT and 10(4)-10(5) x for TNF. This method allows the regular detection of less than 1-5 pg of bioactive protein. ELISA assays did not detect synthetic TNF and TNF fragments that were not biologically active in this assay. The L-929 bioassay requires no isotopes, is very reproducible, is as sensitive as ELISA techniques, and detects only biologically active LT and TNF.

Animals↗

PubMed1990

Colloidal bismuth subcitrate inhibits peptic degradation of gastric mucus and epidermal growth factor in vitro.

The effect of an antiulcer drug, colloidal bismuth subcitrate (De-Nol), on the proteolytic activity of pepsin toward gastric mucus and salivary epidermal growth factor was investigated. Samples of pig gastric mucus and mouse epidermal growth factor were incubated with pepsin in the absence and in the presence of De-Nol, and the released alpha-amino acid residues were quantified. Results of analysis revealed that, in the absence of De-Nol, the apparent Km value of pepsin toward gastric mucus was 1.4 g/L and that towards epidermal growth factor 120 microM. Introduction of De-Nol to the incubation mixtures led, in both cases, to reduction of the rates of proteolysis. With gastric mucus, the rate of proteolysis inhibition was proportional to the De-Nol concentration up to 1 x 10(-2) g/L, at which point a 54% reduction in mucus proteolysis occurred, whereas, with epidermal growth factor, this concentration of De-Nol caused nearly 52% inhibition in the rate of proteolysis. The apparent Ki value for peptic degradation of gastric mucus in the presence of De-Nol was 2.1 x 10(-4) g/L and that for peptic degradation of epidermal growth factor 1.8 x 10(-2) g/L. The results suggest that among the beneficial effects of colloidal bismuth subcitrate on ulcer healing is its ability to interfere with peptic digestion of the protective gastric mucus coat and of such important bioactive protein as epidermal growth factor.

Animals↗

PubMed1989

In vivo dynamics of murine tumor necrosis factor-alpha gene expression. Kinetics of dexamethasone-induced suppression.

Tumor necrosis factor-alpha (TNF) has been implicated as an important, proximal mediator of many of the pathophysiologic effects observed during septic shock. In vitro studies have demonstrated that the glucocorticoid dexamethasone (Dex) will suppress the production of TNF; yet, clinical studies have shown that glucocorticoids are not protective in septic shock. In this paper we described the in vivo effects of lipopolysaccharide (LPS) on the kinetics of local and systemic TNF production, the time dependent expression of TNF mRNA, and the suppression of both TNF mRNA and bioactive protein using a defined treatment protocol of Dex. Peritoneal macrophages were elicited by CBA/J mice in the injection of complete Freunds adjuvant and the mice challenged with an intraperitoneal injection of LPS 2 weeks later. Kinetic studies showed that the peak of TNF production occurred 1 hour post LPS injection and reached a maximum of 775 units/ml within the ascites and 26 units/ml within the plasma. Northern blot analysis of mRNA extracted from peritoneal cells showed a peak of mRNA 30 minutes post LPS challenge. Dose-response studies disclosed that 10 micrograms of LPS/mouse produced maximal TNF within the ascites fluid, and half-maximal stimulation occurred at 70 ng LPS/mouse. Mice treated with Dex in vivo before LPS challenge showed a dramatic reduction in TNF production within both the ascites and plasma, and Northern blot analysis showed a corresponding reduction in the TNF specific mRNA. Further studies revealed that mice treated with 4 mg/kg of Dex intraperitoneally 4 hours before, or at the time of LPS challenge, had dramatic reductions in TNF levels within both the ascites and plasma. However, delaying the treatment only 20 minutes after LPS injection failed to significantly reduce TNF in either compartment. These data may provide a rationale why glucocorticoids are not clinically efficacious in the treatment of septic shock, since there is rapid upregulation of LPS-induced TNF gene expression. By the time patients develop clinical signs and symptoms of septic shock there are already preformed, circulating levels of TNF.

Animals↗

PubMed1986

Prostaglandin-induced storage and secretion of esteroproteases in the mouse submaxillary gland.

The submaxillary glands of adult C3H mice which received intraperitoneal injections of prostaglandins F2 alpha and E2 (PGF2 alpha and PGE2) were examined biochemically and ultrastructurally. Results indicated that the specific activity of esteroprotease in an homogenate of submaxillary glands was significantly increased when mice were treated with PGF2 alpha (96 or 480 micrograms/kg), and decreased when they were treated with PGE2 (96 or 480 micrograms/kg). Ultrastructural findings were correlated with these biochemical data. Thus, it appeared that PGF2 alpha stimulated the secretion and synthesis of bioactive proteins, and that PGE2 stimulated only the secretion.

Animals↗

PubMed1994

Adenovirus-mediated cytokine gene transfer at tissue sites. Overexpression of IL-6 induces lymphocytic hyperplasia in the lung.

The biologic function of cytokines may be best studied in the context of a defined tissue site. To establish a model for studying the function of IL-6 at local tissue sites, we targeted the IL-6 transgene into the bronchial epithelium in the lung of Sprague-Dawley rats by intratracheal administration of a recombinant human type 5 adenovirus with rat IL-6 cDNA incorporated into the E3 region of the viral genome. This approach led to a highly compartmentalized overexpression of the IL-6 transgene and production of bioactive protein within the lung for about 7 days post-infection. Associated with this overexpression of IL-6 was the development of profound local lymphocytic hyperplasia around day 7, characterized by the dramatic expansion of bronchial associated lymphoid aggregates and massive lymphocytic infiltration in the pulmonary parenchyma. Concurrently, there were strikingly increased numbers of lymphocytes in bronchoalveolar lavage fluids. The majority of these lymphocytes were found to be CD3+CD8+ cytotoxic T and CD3+CD4+ helper T cells with the remaining being primarily a small number of CD45R+ B cells. In addition, there was moderate bronchial and alveolar epithelial hyperplasia associated with lymphocytic hyperplasia. However, all of these changes subsided concomitant with the decrease in IL-6 expression and the lung seemed normal at 12 to 14 days post-infection. Thus, our study provides a tissue-specific transient transgene model for investigating cytokine functions in vivo and demonstrates that IL-6 has a profound stimulatory effect on the local lymphoid tissue in the lung.

Adenoviruses, Human↗

PubMed1994

Soft-tissue augmentation and replacement in the head and neck. General considerations.

It is clear that many of the factors that affect wound healing when natural biomaterials are used also affect the long-term success of semisynthetic and synthetic alloplasts. The development of a new generation of biomaterials that combine synthetic components with bioactive proteins are just now beginning to move from the laboratory into clinical testing. Within the next decade, biologically active growth proteins, in combination with resorbable synthetic alloplasts, may allow missing tissues to be regenerated rather than just "filled in" by permanent implants. There will also be a steady drift away from materials that show any risk of being carcinogenic or immunoreactive over time. As promising as these new alloplasts appear to be, we should learn from past experience. Implants that initially seem biocompatible can fail in unpredictable ways when exposed to the hostile environment of the body. As surgeons, we well ultimately be held responsible for the biomaterials we implant. Our selection of biomaterials should be based on sound scientific and surgical knowledge, and not solely upon the recommendations of manufacturers. The authors hope that the information in this monograph will help to integrate reconstructive options so that autologous tissues are used whenever possible and synthetic materials are appropriately selected and applied when necessary.

Biocompatible Materials↗

PubMed1998

Antitumor activity of tumor necrosis factor alpha conjugated with divinyl ether and maleic anhydride copolymer on solid tumors in mice.

The purpose of this study is to further explore the usefulness of conjugation with functional polymeric modifiers for clinical application of bioactive proteins and to increase the therapeutic efficacy of tumor necrosis factor alpha (TNF-alpha) by conjugation in vivo. We synthesized TNF-alpha conjugated with the copolymer of divinyl ether and maleic anhydride (DIVEMA), which has intrinsic antitumor activity as a synthetic biological response modifier. The synthesis of DIVEMA-TNF-alpha could be controlled by the addition of 2,3-dimethylmaleic anhydride (DMMAn), which binds to or separates from amino groups when the pH is changed. The specific activity of DIVEMA-TNF-alpha (+) synthesized with DMMAn was hardly decreased in vitro. However, DIVEMA-TNF-alpha (-), which is conjugated without blocking by DMMAn, had a markedly diminished specific activity. DIVEMA-TNF-alpha (+) caused a dramatic hemorrhagic necrotic effect on the tumor when compared to native TNF-alpha 24 h after i.v. injection into mice bearing Sarcoma-180 solid tumors. In addition, DIVEMA-TNF-alpha (+) at a dose of only 100 Japan reference units per mouse revealed a dramatic antitumor effect that is approximately 100 times greater than native TNF-alpha and that could induce complete regression in all five mice bearing Meth-A solid tumors without any apparent side effects. Because neither DIVEMA alone nor a mixture of TNF-alpha and DIVEMA caused antitumor activity with i.v. administration, the increase in antitumor potency of TNF-alpha may be caused by the covalent conjugation with DIVEMA. DIVEMA-TNF-alpha at low dose revealed dramatic antitumor potency. Because TNF-alpha injected in vivo is extremely low-dose, concentration of intrinsic TNF-alpha in vivo is not influenced. Therefore, the cytokine network in vivo is not destroyed. These results suggest that DIVEMA is a useful polymeric modifier for conjugation of TNF-alpha to increase its antitumor activity.

Animals↗

PubMed2004

Carboxyl terminus truncated human papillomavirus type 58 L1 protein maintains its bioactivity and ability to form virus-like particles.

To prepare carboxyl terminus truncated human papillomavirus type 58 L1 (HPV58L1) protein and evaluate its ability to form virus-like particles, the baculovirus and Sf-9 insect cells was used to express HPV58L1 protein, and pFastBac-Htb containing HPV58L1 gene sequence of carboxyl terminus truncation was generated. Then Sf-9 cells were infected with recombinant baculovirus. After being cultured, the post-infected cells expressing--HPV58L1 protein were harvested and analyzed by SDS-PAGE and Western blot. The ProBond purification system was used for protein purification. The bio-activity of purified protein was identified by mouse erythrocyte hemagglutination assay, and the VLP formation was examined with transmission electron microscope. Our results showed that the recombinant baculovirus was generated and the Sf-9 cells was infected with the recombinant baculovirus, and after collecting, total cellular proteins were extracted. Truncated HPV58L1 protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The purified L1 proteins under native condition could cause mouse erythrocytes to agglutinate and form VLP. It is concluded that HPV58L1 protein with carboxyl terminus truncation could be efficiently expressed. In baculovirus Sf-9 cells expression system, the purified protein could self-assemble into virions in vitro, and induce agglutination of mouse erythrocytes, indicating that carboxyl terminus truncation does not interfere with the bioactivity of HPV58L1 protein.

Baculoviridae↗

PubMed2004

Multifunctional peptides encrypted in milk proteins.

Many bioactivities of milk are latent in that they are inactive within the protein sequence, requiring enzymatic proteolysis for release of bioactive peptides from milk proteins precursors. Bioactivities of peptides encrypted in major milk proteins are latent until released and activated, e.g. during gastrointestinal digestion or food processing. Bioactive peptides can be produced in vivo following intake of milk proteins, and the proteolytic system of bacterial species used in the production of fermented milk products and cheese can contribute to the liberation of bioactive peptides or precursors thereof. Activated peptides are potential modulators of various regulatory processes in the living system: immunomodulatory peptides stimulate the activities of cells of the immune system and several cytomodulatory peptides inhibit cancer cell growth, antimicrobial peptides kill sensitive microorganisms, angiotensin-I-converting enzyme (ACE)-inhibitory peptides exert an hypotensive effect, opioid peptides are opioid receptor ligands which can modulate absorption processes in the intestinal tract, mineral binding peptides may function as carriers for different minerals, especially calcium. Many milk-derived peptides reveal multifunctional properties, i.e. specific peptide sequences having two or more different biological activities have been reported. Milk protein-derived bioactive peptides are claimed to be health enhancing components that can be used to reduce the risk of disease or to enhance a certain physiological function.

Animals↗

PubMed2026

Protein isolation markedly enhances in vitro digestibility, nutritional quality, and bioactivity of fungal mycelial proteins.

Fungal mycelial proteins are promising sustainable protein sources, yet their nutritional utilization is often limited by structural constraints. This study systematically evaluated the effects of protein isolation on the proteomic composition, gastrointestinal digestion behavior, amino acid utilization, and bioactivity of Pleurotus citrinopileatus mycelial proteins. Quantitative proteomics identified 3591 proteins, of which 3374 were shared between mycelial flour (PCMF) and protein isolate (PCMPI), indicating that PCMPI primarily represents the soluble proteome fraction. In vitro digestion revealed that PCMPI exhibited significantly higher digestibility (93.98%) than PCMF (42.98%) (p < 0.05), reaching levels comparable to whey protein isolate. Enhanced enzymatic accessibility in PCMPI promoted rapid peptide generation during the gastric phase and efficient amino acid release during the intestinal phase, resulting in higher peptide (634.76 mg/g) and free amino acid levels (341.69 mg/g) at the digestion endpoint. Consequently, PCMPI achieved a balanced amino acid profile with a PDCAAS of 1.0. Moreover, its digestion products exhibited stronger antioxidant activity (IC₅₀ = 8.36 mg/mL) and ACE inhibitory activity (IC₅₀ = 15.65 mg/mL) compared with PCMF. Mechanistically, protein isolation disrupted the cell wall matrix, shifting digestion from a structure-limited to an accessibility-driven regime. Collectively, these findings demonstrate that protein isolation markedly enhances the digestibility, nutritional quality, and functional potential of mycelial proteins, supporting their application as high-value sustainable protein ingredients.

Digestion↗

PubMed2006

Expression of bioactive bone morphogenetic proteins in the subacromial bursa of patients with chronic degeneration of the rotator cuff.

Degeneration of the rotator cuff is often associated with inflammation of the subacromial bursa and focal mineralization of the supraspinatus tendon. Portions of the supraspinatus tendon distant from the insertion site could transform into fibrous cartilage, causing rotator-cuff tears owing to mechanical instability. Indirect evidence is presented to link this pathology to ectopic production and secretion of bioactive bone morphogenetic proteins (BMPs) from sites within the subacromial bursa. Surgically removed specimens of subacromial bursa tissue from patients with chronic tears of the rotator cuff were analyzed by immunohistochemistry and reverse transcription-PCR. Bioactive BMP was detected in bursa extracts by a bioassay based on induction of alkaline phosphatase in the osteogenic/myogenic cell line C2C12. Topical and differential expression of BMP-2/4 and BMP-7 mRNA and protein was found in bursa tissue. The bioassay of C2C12 cells revealed amounts of active BMP high enough to induce osteogenic cell types, and blocking BMP with specific antibodies or soluble BMP receptors Alk-3 and Alk-6 abolished the inductive properties of the extract. Sufficient information was gathered to explain how ectopic expression of BMP might induce tissue transformation into ectopic bone/cartilage and, therefore, promote structural degeneration of the rotator cuff. Early surgical removal of the subacromial bursa might present an option to interrupt disease progression.

Adult↗

PubMed1994

Effect of lipopolysaccharide (LPS) chain length on interactions of bactericidal/permeability-increasing protein and its bioactive 23-kilodalton NH2-terminal fragment with isolated LPS and intact Proteus mirabilis and Escherichia coli.

The target-specific cytotoxicity for gram-negative bacteria and the endotoxin-neutralizing activity of the 55-kDa bactericidal/Permeability-increasing protein (BPI) and its bioactive 23-kDa NH2-terminal fragment depend on the strong attraction of BPI for the lipid A region of lipopolysaccharides (LPS). We have shown before that smooth gram-negative bacteria with long-chain LPS are more resistant to BPI (especially holo-BPI) than are rough strains. It has been suggested that the high BPI resistance of some gram-negative bacteria, such as Proteus mirabilis, might also reflect the structural diversity of lipid A. To explore this possibility, we compared the antibacterial activity and binding of natural and recombinant holo-BPI and a recombinant NH2-terminal fragment (rBPI-23) to an isogenic rough (Re-LPS chemotype) and a smooth (S-LPS chemotype) strain of P. mirabilis and to LPS isolated from the two strains. Holo-BPI and rBPI-23 were both potently active against the Re strain of P. mirabilis (90% lethal dose, 20 nM). In contrast, the smooth strain was > or = 100 times more resistant to holo-BPI but only 10 times more resistant to rBPI-23. rBPI-23 was also more potent against several Escherichia coli strains from clinical bacteremia isolates. Differences in the antibacterial potency of BPI toward the Re and S strains of P. mirabilis correlated with differences in the binding of holo-BPI and rBPI-23 to these bacteria. In contrast, the binding of biosynthetically (in vitro transcribed and translated) 35S-labeled holo-BPI and NH2-terminal fragment to isolated Re- and S-LPS from P. mirabilis in solution was similar. Moreover, in the Limulus amebocyte lysate assay, holo-BPI and rBPI-23 potently neutralized both forms of LPS with equal effectiveness. Together, these results strongly suggest that BPI recognizes Proteus lipid A and that the relative resistance of (smooth) P. mirabilis to holo-BPI is due to the inhibitory effect of long polysaccharide chains of tightly packed LPS in the envelope.

Antimicrobial Cationic Peptides↗

PubMed2000

Hepatitis C virus glycoprotein E2 binding to CD81: the role of E1E2 cleavage and protein glycosylation in bioactivity.

The hepatitis C virus glycoproteins E1 and 2 have been expressed using recombinant baculoviruses following fusion to the carrier protein glutathione S-transferase (GST). Proteins were expressed singly and as an E1E2 polyprotein with and without an N-terminal affinity tag. Expression of the E1E2 polyprotein, even when preceded by GST, led to processing in insect cells and detection of an E1E2 complex that could be specifically purified by glutathione affinity chromatography. Baculovirus expressed E2 and a purified GST-E1E2 protein bound to the second extracellular loop of CD81 (EC2), a reported ligand for the molecule, but not to a truncated derivative of CD81 consisting of only the central domain of the loop. Purified GST-E2, however, failed to bind to CD81 suggesting a requirement for a free E2 amino terminus for biological activity. The binding to CD81 by baculovirus expressed E2 protein was comparable to that observed for E2 derived from mammalian cells when detected by a monoclonal antibody sensitive to protein conformation. Furthermore, E2 protein expressed in insect cells in the presence of N-butyldeoxynojirimycin, an inhibitor of terminal glucose residue processing, formed complexes with E1 and bound to CD81-EC2 similarly to untreated protein. Together these data suggest that although hyperglucosylation of E2 does not have a major effect on bioactivity, polyprotein processing to reveal the free amino terminus is required.

1-Deoxynojirimycin↗

PubMed1991

Ethanol-induced enhancement of cocaine bioactivation and irreversible protein binding: evidence against a role of cytochrome P-450IIE1.

Chronic ethanol consumption potentiates cocaine-induced liver injury in rodents. Since cocaine has to be bioactivated by a cytochrome P-450-dependent N-oxidative pathway to exert its hepatotoxic effects, we studied the role of the ethanol-inducible P-450IIE1 for cocaine metabolism. Male Sprague-Dawley rats were pretreated with either a liquid diet containing ethanol (30% of calories) for 4 weeks or injected with pyrazole (200 mg/kg/day, ip, for 3 days). Both agents induced microsomal p-nitrophenol hydroxylation which is a probe for the catalytic activity of P-450IIE1. However, only ethanol, but not pyrazole, increased both microsomal cocaine N-demethylase activity (by 47%) and the extent of irreversible binding of [3H]-cocaine to microsomal proteins (by 100%), which was taken as a quantitative endpoint for the formation of a reactive metabolite. Cocaine N-demethylation and irreversible protein binding of cocaine were not inhibited by P-450IIE1 isozyme-selective substrates, nor was the rate of cocaine metabolism and binding decreased by functionally active polyclonal anti-rat P-450IIE1 antibodies. Furthermore, pyrazole pretreatment sensitized cultured hepatocytes to the glutathione-dependent cytotoxic effects of nontoxic concentrations of cocaine. These results indicate that (a) cocaine is not a major substrate for the ethanol-inducible P-450IIE1, (b) the enhancing effects of ethanol on cocaine bioactivation may be due to induction of other P-450 isoforms, and (c) induction of P-450IIE1 may potentiate cocaine-induced hepatocellular toxicity in vitro independently of cocaine metabolism, e.g., by P-450IIE1-dependent oxidative stress.

Animals↗

PubMed2002

Computer automated prediction of potential therapeutic and toxicity protein targets of bioactive compounds from Chinese medicinal plants.

Understanding the molecular mechanism and pharmacology of bioactive compounds from Chinese medicinal plants (CMP) is important in facilitating scientific evaluation of novel therapeutic approaches in traditional Chinese medicine. It is also of significance in new drug development based on the mechanism of Chinese medicine. A key step towards this task is the determination of the therapeutic and toxicity protein targets of CMP compounds. In this work, newly developed computer software INVDOCK is used for automated identification of potential therapeutic and toxicity targets of several bioactive compounds isolated from Chinese medicinal plants. This software searches a protein database to find proteins to which a CMP compound can bind or weakly bind. INVDOCK results on three CMP compounds (allicin, catechin and camptotecin) show that 60% of computer-identified potential therapeutic protein targets and 27% of computer-identified potential toxicity targets have been implicated or confirmed by experiments. This software may potentially be used as a relatively fast-speed and low-cost tool for facilitating the study of molecular mechanism and pharmacology of bioactive compounds from Chinese medicinal plants and natural products from other sources.

Animals↗

PubMed2003

Delivery of bioactive, gel-isolated proteins into live cells.

The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein.

Animals↗