A Glucose binding lectin from Leucaena leucocephala seeds and its mitogenic activity against human lymphocytes.

Lectins are a specialized group of proteins with immense biological properties and applications. This study describes the purification and characterization of a lectin from Leucaena leucocephala seeds, a plant belonging to the Fabaceae family. Leucaena leucocephala lectin (LLL) was purified by a two-step purification method involving DEAE-cellulose anion exchange chromatography and Sephadex G-75 size exclusion chromatography. The isolated lectin displayed a high haemagglutination titre upon treatment with rabbit erythrocytes. SDS-PAGE and Reverse-Phase High performance liquid chromatography (RP-HPLC) analysis experimentally revealed the presence of three bands corresponding to 37, 27 and 20 kDa indicating the presence of isolectins. Periodic Acid Schiff's (PAS) staining of LLL confirmed the presence of glycoprotein. Various biochemical parameters were analysed to study its effect on the haemagglutination activity. Sugar inhibition studies experimentally revealed that Glucose was the most potent inhibitor. Fluorescence spectrometric analysis of LLL and Glucose indicated a strong interaction with an association constant of 0.159 × 103 M-1. Circular Dichroism spectroscopy indicated a higher alpha helical content (25.27%). LLL was observed to possess mitogenic activity against Peripheral blood mononuclear cells (PBMC). The present investigation reports the isolation of a novel lectin from this plant which could contribute towards the diagnostic studies of certain diseases and for its therapeutic potential.

Stable Fibroblast Growth Factor 2 Dimers with High Pro-Survival and Mitogenic Potential.

Fibroblast growth factor 2 (FGF2) is a heparin-binding growth factor with broad mitogenic and cell survival activities. Its effector functions are induced upon the formation of 2:2 FGF2:FGFR1 tetrameric complex. To facilitate receptor activation, and therefore, to improve the FGF2 biological properties, we preorganized dimeric ligand by a covalent linkage of two FGF2 molecules. Mutations of the FGF2 WT protein were designed to obtain variants with a single surface-exposed reactive cysteine for the chemical conjugation via maleimide-thiol reaction with bis-functionalized linear PEG linkers. We developed eight FGF2 dimers of defined topology, differing in mutual orientation of individual FGF2 molecules. The engineered proteins remained functional in terms of FGFR downstream signaling activation and were characterized by the increased stability, mitogenic potential and anti-apoptotic activity, as well as induced greater migration responses in normal fibroblasts, as compared to FGF2 monomer. Importantly, biological activity of the dimers was much less dependent on the external heparin administration. Moreover, some dimeric FGF2 variants internalized more efficiently into FGFR overexpressing cancer cells. In summary, in the current work, we showed that preorganization of dimeric FGF2 ligand increased the stability of the growth factor, and therefore, enhanced its biological activity.

Automated Dicentric Aberration Scoring for Triage Dose Assessment: 60Co Gamma Ray Dose-response at Different Dose Rates.

The objective of this study was to establish radiation dose-response calibration curves using automated dicentric scoring to support rapid and accurate cytogenetic triage dose-assessment. Blood was drawn from healthy human volunteers and exposed to Co gamma rays at several dose rates (i.e., 1.0, 0.6, and 0.1 Gy min). After radiation, the blood was placed for 2 h in a 37 °C incubator for repair. Blood was then cultured in complete media to which a mitogen (i.e., phytoghemagglutinin, concentration 4%) was added for 48 h. Colcemid was added to the culture at a final concentration of 0.2 µg mL after 24 h for the purpose of arresting first-division metaphase mitotics. Cells were harvested at the end of 48 h. Samples were processed using an automated metaphase harvester and automated microscope metaphase finder equipped with a suite of software including a specialized automated dicentric scoring application. The data obtained were used to create dose-response tables of dicentric yields. The null hypothesis that the data is Poisson-distributed could not be rejected at the significance level of α = 0.05 using results from a Shiny R Studio application (goodness-of-fit Poisson). Calibration curves based on linear-quadratic fits for Co gamma rays at the three different dose rates were generated using these data. The calibration curves were used to detect blind test cases. In conclusion, using the automated harvester and automated microscope metaphase finder with associated automated dicentric scoring software demonstrates high-throughput with suitable accuracy for triage radiation dose assessment.

Purification and characterisation of a xylose-specific mitogenic lectin from Fusarium sambucinum.

A xylose-specific intracellular lectin, showing hemagglutination only with rabbit erythrocytes was purified from mycelium of Fusarium sambucinum which was designated as FSL. An array of anion exchange chromatography on Q-Sepharose and gel-exclusion chromatography on Sephadex G-100 resulted in 84.21% yield and 53.99-fold purification of lectin with specific activity of 169.53 titre/mg. Molecular weight of FSL determined by SDS-PAGE was 70.7 kDa, which was further confirmed by gel-exclusion chromatography. Native-PAGE analysis of FSL showed its monomeric nature. FSL was observed to be a glycoprotein containing 2.9% carbohydrate. Hapten inhibition profile of FSL displayed its strong affinity towards D-xylose (MIC 1.562 mM), L-fucose (MIC 6.25 mM), D-mannose (MIC 3.125 mM), fetuin (MIC 15.62 µg/mL), asialofetuin (MIC 125 µg/mL) and BSM (MIC 3.125 µg/mL). Affinity of FSL towards xylose is rare. FSL was found stable over a pH range 6.0-7.5 and upto 40 °C temperature. Hemagglutination activity of FSL remained unaffected by divalent ions. Lectin concentration of 5 µg/mL was found sufficient to stimulate proliferation of murine spleen cells and its concentration 75 µg/mL exhibited highest mitogenic potential. FSL exhibited maximum mitogenic stimulatory index of 14.35. The purification, characterisation and mitogenicity of F. sambucinum lectin has been reported first time.

Purification of a potent mitogenic homodimeric Penicillium griseoroseum lectin and its characterisation.

Penicillium griseoroseum lectin was 80-fold purified by successive DEAE Sepharose anion exchange and Sephadex G-100 gel permeation chromatography. P. griseoroseum lectin exhibited haemagglutination activity towards protease-treated rabbit erythrocytes. It showed specificity towards various carbohydrates such as d-mannose, N-acetyl-d-glucosamine, mucins, and so forth. P. griseoroseum lectin was found as a glycoprotein with glycan content of 4.33%. Purified P. griseoroseum lectin is homodimeric having a molecular mass of 57 kDa with subunit molecular mass of 28.6 kDa. Haemagglutination activity of purified P. griseoroseum lectin was completely stable from 25°C to 35°C at a pH range of 6-7.5. Lectin activity was not influenced by divalent metal ions and denaturants. P. griseoroseum lectin manifested mitogenicity towards mice splenocytes and activity reached a peak at 75 µg/ml of lectin concentration. P. griseoroseum lectin in microgram concentrations stimulated proliferation of mice splenocytes. Thus, P. griseoroseum lectin exhibits potential mitogenicity, which can be exploited for further biomedical applications.

Purification and characterization of a mitogenic lectin from Penicillium duclauxii.

Lectins are proteins/glycoproteins of non-immune origin which interact specifically and non-covalently with carbohydrate moieties on the cell surface. In this study, a lectin was purified from Penicillium duclauxii by ion-exchange chromatography on DEAE-Sepharose and gel filtration chromatography on a Sephadex G-100 column. An overall recovery of 94.11% and 60-fold purification was achieved. The purified lectin had a molecular weight of 54.9 kDa and was found to be heterogeneous as revealed by double band of sub-units with molecular mass of 21.13 kDa and 33.26 kDa, under reducing conditions. It is a glycoprotein with carbohydrate content of 3.95%. Lectin induced haemagglutination of erythrocytes was inhibited strongly by glycoproteins such as bovine submaxillary mucin, porcine stomach mucin and fetuin. The maximum haemagglutinating activity of P. duclauxii lectin was maintained after incubation at a temperature and pH range of 20-35 °C and 6.0-8.0, respectively. The haemagglutinating activity of P. duclauxii lectin was unaffected by EDTA and various metal ions. The purified P. duclauxii lectin exhibited maximum mitogenic activity towards mouse splenocytes at a concentration of 75 µg/mL. This manuscript reports a novel lectin from P. duclauxii with potent mitogenic activity towards mouse splenocytes.

Uncoupling the Mitogenic and Metabolic Functions of FGF1 by Tuning FGF1-FGF Receptor Dimer Stability.

The recent discovery of metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis has expanded the functions of this classically known mitogen. To dissect the molecular basis for this functional pleiotropy, we engineered an FGF1 partial agonist carrying triple mutations (FGF1ΔHBS) that diminished its ability to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1ΔHBS exhibited a severely reduced proliferative potential, while preserving the full metabolic activity of wild-type FGF1 in vitro and in vivo. Hence, suboptimal FGFR activation by a weak FGF1-FGFR dimer is sufficient to evoke a metabolic response, whereas full FGFR activation by stable and sustained dimerization is required to elicit a mitogenic response. In addition to providing a physical basis for the diverse activities of FGF1, our findings will impact ongoing drug discoveries targeting FGF1 and related FGFs for the treatment of a variety of human diseases.

Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus.

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure.

Enhanced activation of periodate by iodine-doped granular activated carbon for organic contaminant degradation.

In this study, iodine-doped granular activated carbon (I-GAC) was prepared and subsequently applied to activate periodate (IO4-) to degrade organic contaminants at ambient temperature. The physicochemical properties of GAC and I-GAC were examined using scanning electron microscopy, N2 adsorption/desorption, Raman spectroscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. No significant difference was observed between the two except for the existence of triiodide (I3-) and pentaiodide (I5-) on I-GAC. The catalytic activity of I-GAC towards IO4- was evaluated by the degradation of acid orange 7 (AO7), and superior catalytic performance was achieved compared with GAC. The effects of some influential parameters (preparation conditions, initial solution pH, and coexisting anions) on the catalytic ability were also investigated. Based on radical scavenging experiments, it appeared that IO3 was the predominant reactive species in the I-GAC/IO4- system. The mechanism underlying the enhanced catalytic performance of I-GAC could be explained by the introduction of negatively charged I3- and I5- into I-GAC, which induced positive charge density on the surface of I-GAC. This accelerated the interaction between I-GAC and IO4-, and subsequently mediated the increasing generation of iodyl radicals (IO3). Furthermore, a possible degradation pathway of AO7 was proposed according to the intermediate products identified by gas chromatography-mass spectrometry.

Sustained mitogenic effect on K562 human chronic myelogenous leukemia cells by dietary lectin, jacalin.

Dietary lectins have been shown to affect the proliferation of human cancer cell lines. The anti-proliferative effects of lectins from varied sources have been extensively studied and in some cases, the underlying mechanism has been explored. Except for peanut agglutinin (PNA), the mitogenic effects of no other lectins have been studied in detail. In the present study, we have shown that jacalin, lectin purified from jackfruit (Artocarpus integrifolia) seeds act as a mitogen for K562, the Bcr-Abl expressing erythroleukemia cell line (K562) and the effect was found to be dose dependent. K562 cells remained in the proliferative state for a longer period even after the withdrawal of jacalin stimulation, thus jacalin was found to induce sustained mitogenic effect on K562 cells. Further, conditioned media from K562 cells treated with jacalin were observed to have the similar mitogenic effect even in the presence of galactose. Importantly, galactose which is a known ligand for jacalin will interact with functionally active jacalin present in the conditioned media and neutralise its effect. In addition, jacalin treatment also resulted in increased mRNA expression levels of pro-inflammatory cytokines including IL-1ß, IL-6 and IFN-γ. Our results indicate that jacalin induces secretion of soluble molecules, which maybe responsible for this observed increased proliferation of K562 cells.

Engineering a therapeutic lectin by uncoupling mitogenicity from antiviral activity.

A key effector route of the Sugar Code involves lectins that exert crucial regulatory controls by targeting distinct cellular glycans. We demonstrate that a single amino-acid substitution in a banana lectin, replacing histidine 84 with a threonine, significantly reduces its mitogenicity, while preserving its broad-spectrum antiviral potency. X-ray crystallography, NMR spectroscopy, and glycocluster assays reveal that loss of mitogenicity is strongly correlated with loss of pi-pi stacking between aromatic amino acids H84 and Y83, which removes a wall separating two carbohydrate binding sites, thus diminishing multivalent interactions. On the other hand, monovalent interactions and antiviral activity are preserved by retaining other wild-type conformational features and possibly through unique contacts involving the T84 side chain. Through such fine-tuning, target selection and downstream effects of a lectin can be modulated so as to knock down one activity, while preserving another, thus providing tools for therapeutics and for understanding the Sugar Code.

Purification and Characterization of a Lectin from Green Split Peas (Pisum sativum).

Lectins have captured the attention of a large number of researchers on account of their various exploitable activities, including antitumor, immunomodulatory, antifungal, as well as HIV reverse transcriptase inhibitory activities. A mannose/glucose-specific lectin was isolated from green split peas (a variety of Pisum sativum) and characterized. The purification step involved anion-exchange chromatography on a DEAE-cellulose column, cation-exchange chromatography on an SP-Sepharose column, and gel filtration by fast protein liquid chromatography (FPLC) on Superdex 200. The purified lectin had a native molecular mass of around 50 kDa as determined by size exclusion chromatography. It appeared as a heterotetramer, composed of two distinct polypeptide bands with a molecular mass of 6 and 19 kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The N-terminal sequence of green split pea lectin shows some degree of homology compared to lectins from other legume species. Its hemagglutinating activity was inhibited by glucose, mannose, and sucrose, and attenuated at pH values higher than 12 or lower than 3. Hemagglutinating activity was preserved at temperatures lower than 80 °C. The lectin did not show antifungal activity toward fungi including Fusarium oxysporum, Botrytis cinerea, and Mycosphaerella arachidicola. Green split pea lectin showed a mitogenic effect toward murine splenocytes and could inhibit the activity of HIV-1 reverse transcriptase.

Purification, characterization, and mitogenic potential of a mucin-specific mycelial lectin from Aspergillus sparsus.

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which is responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Due to their carbohydrate specificity, lectins have been used for purification and characterization of glycoproteins like antibodies, cytokines, tumor-associated glycoproteins, hormones, inhibitors, growth factors, various enzymes, membrane proteins (receptors), or even toxins and viruses. In the present study, a mycelial lectin from Aspergillus sparsus was purified, characterized, and evaluated for its mitogenic potential. Lectin could be effectively purified 17.8-fold in a single-step using affinity chromatography on mucin-sepharose column. Lectin migrated as a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular mass of 100.2 kDa. Lectin showed pH optima of 6.5-8.0, and optimum temperature was determined to be 20-30 °C. Lectin was stable within a pH range of 5.5-10.0 and showed fairly good thermostability. Lectin activity was unaffected in the presence of EDTA, while activity reduced upon interaction with denaturants. MTT assay revealed strong mitogenic potential of A. sparsus lectin at a concentration up to 100 µg/ml.

The effect of IL-4 gene polymorphisms on cytokine production in patients with chronic periodontitis and in healthy controls.

Chronic periodontitis (CP) is an inflammatory disease of the teeth-supporting tissues in which genetic predisposition, dental plaque bacteria, and immune mechanisms all play important roles. The aim of this study was to evaluate the occurrence of IL-4 gene polymorphisms in chronic periodontitis and to investigate the association between polymorphisms and cytokines production after bacterial stimulation. Sixty-two subjects (47 CP patients and 15 healthy controls) with detected two polymorphisms in the IL-4 gene (-590C/T and intron 3 VNTR) were examined. Production of cytokines (IL-1α, IL-1ß, IL-4, IL-5, IL-6, IL-10, IL-17, TNFα, INFγ, and VEGF) was studied after in vitro stimulation of isolated peripheral blood by mitogens (Pokeweed mitogen, Concanavalin A), dental plaque bacteria (Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Prevotella intermedia), and Heat Shock Protein (HSP) 70 by the Luminex multiplex cytokine analysis system. The results were correlated with IL-4 genotypes in patients with CP and healthy controls. The mononuclear cells isolated from peripheral blood of CP patients with selected IL-4 polymorphisms significantly altered the production of IFNγ, IL-10, IL-1ß, IL-1α, TNFα, and IL-6 after stimulation by HSP 70 or selected bacteria (from P < 0.001 to P < 0.05). IL-4 gene polymorphisms may influence the function of mononuclear cells to produce not only interleukin-4 but also other cytokines, especially in patients with CP.

Mitogenic effects of phosphatidylcholine nanoparticles on MCF-7 breast cancer cells.

Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design.

Structural characterization and antitumor and mitogenic activity of a lectin from the gill of bighead carp (Aristichthys nobilis).

In this study, we investigated the gross structure, secondary structure, and antitumor and mitogenic activity of GANL, a lectin from the gill of bighead carp (Aristichthys nobilis). We used infrared spectroscopy, ß-elimination, and circular dichroism spectroscopy to determine the structure of GANL. We measured antiproliferation activity against six human tumor cell lines and mitogenic activity against murine splenocytes using the MTT assay. Based on infrared spectroscopy and ß-elimination, we conclude that GANL is a glycoprotein. The protein and carbohydrate moieties are joined by O-glycosidic linkage. A circular dichroism spectroscopic analysis revealed that the secondary structure of GANL consists of α-helices (34.8 %), ß-sheets (12.1 %), ß-turns (24.5 %), and unordered structures (33.0 %). GANL exerted potent antitumor activity against the HeLa cell line (IC(50) = 11.86 µg/mL) and a mitogenic effect on murine splenocytes in the MTT assay. GANL, a lectin that is isolated from the gills of bighead carp, is a glycoprotein with potent antitumor and mitogenic activity.

Solid-phase N-terminus PEGylation of recombinant human fibroblast growth factor 2 on heparin-sepharose column.

Recombinant fibroblast growth factor-2 (FGF-2) has been extensively studied and used in several clinical applications including wound healing, bone regeneration, and neuroprotection. Poly(ethylene glycol) (PEG) modification of recombinant human FGF-2 (rhFGF-2) in solution phase has been studied to increase the in vivo biostabilities and therapeutic potency. However, the solution-phase strategy is not site-controlled and the products are often not homogeneous due to the generation of multi-PEGylated proteins. In order to increase mono-PEGylated rhFGF-2 level, a novel solid-phase strategy for rhFGF-2 PEGylation is developed. RhFGF-2 proteins were loaded onto a heparin-sepharose column and the PEGylaton reaction was carried out at the N-terminus by PEG20 kDa butyraldehyde through reductive alkylation. The PEGylated rhFGF-2 was purified to near homogeneity by SP sepharose anion-exchange chromatography and the purity was more than 95% with a yield of mono-PEGylated rhFGF-2 of 58.3%, as confirmed by N-terminal sequencing and MALDI-TOF mass spectrometry. In vitro biophysical and biochemical measurements demonstrated that PEGylated rhFGF-2 has an unchanged secondary structure, receptor binding activity, cell proliferation, and MAP kinase stimulating activity, and an improved bio- and thermal stability. Animal assay showed that PEGylated rhFGF-2 has an increased half-life and reduced immunogenicity. Compared to conventional solution-phase PEGylation, the solid-phase PEGylation is advantageous in reaction time, production of mono-PEGylated protein, and improvement of biochemical and biological activity.

Mitogenic activity of sulfated chitosan and cellulose derivatives is related to protection of FGF-2 from proteolytic cleavage.

Novel chitosan (CHS) and cellulose sulfates (CSs) are studied regarding their mitogenic activity and their protective effect against proteolytic digestion of FGF-2. An intermediate degree of sulfation (DS(S) ) and lower concentration of CHS have superior effect on 3T3 cell growth while the mitogenic activity of CS increases with DS(S) and concentration. Experiments with trypsin as model proteinase show that protection of FGF-2 from proteolytic digestion depends on DS(S) and the concentration of derivatives in the same manner as cell growth. Studies on stability of FGF-2 added to cultures of 3T3 cells show that the FGF-2 concentration remains higher in the presence of derivatives. Results indicate that the mitogenic activity of CHS and CS is due to protection of FGF-2 from proteolytic cleavage.

GDNF restores human blood-nerve barrier function via RET tyrosine kinase-mediated cytoskeletal reorganization.

Endoneurial microvessels and the perineurium are responsible for maintaining homeostasis in peripheral nerves. Endoneurial endothelial cells form the blood-nerve barrier (BNB). The molecular pathways responsible for endoneurial microvascular barrier formation in humans are not fully understood. We tested the effect of different mitogens on the transendothelial electrical resistance (TEER) of confluent primary human endoneurial endothelial cell (pHEndEC) cultures following serum withdrawal (mimicking diffuse endothelial injury) in vitro. We show that glial-derived neurotrophic factor (GDNF, 1 ng/mL) sufficiently induced a maximal 114.2% recovery in TEER over basal conditions 48 h after serum withdrawal. Solute permeability to high molecular weight dextran was reduced by 52.4% following GDNF treatment. GDNF-mediated increase in TEER was dependent on RET tyrosine-kinase signaling pathways and mildly enhanced by cyclic adenosine monophosphate in combination with maximal concentrations of multiple redundant mitogens. There was no significant increase in adherens or tight junction proteins ß-catenin, VE-Cadherin, zona occludens-1 and occludin following GDNF treatment. GDNF induced a small increase in total claudin-5 protein expression without significant increase in messenger RNA or modulation in tyrosine phosphorylation following serum withdrawal. Indirect immunocytochemistry revealed membrane relocation of longitudinal F-actin cytoskeletal filaments in pHEndECs following GDNF treatment, resulting in more continuous intercellular contacts that formed adherens and tight junctions. Together, these results demonstrate a sufficient role for GDNF in human BNB recovery following serum withdrawal in vitro, facilitated primarily by endothelial cell cytoskeletal reorganization. These observations provide insights into the regulation of human BNB function during recovery from peripheral nerve injury.

Molecular cloning of a mitogenic proteinase from Carica candamarcensis: its potential use in wound healing.

Cysteine proteinases from the Caricaceae belong to the C1 family of the CA clan and display papain-like structured, the archetype enzyme for this group of proteins. Carica candamarcensis, also named Vasconcellea cundinamarcensis, a member of Caricaceae family common to many areas in South America, contains cysteine proteinases with proteolytic activity five to eight-fold higher than those from latex of Carica papaya. The cysteine protease CMS2MS2 from C. candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In this study, the cDNA cloning, expression and evaluation of biological activity of a CMS2MS2-like protein from C. candamarcensis is reported. The 650 bp fragment was cloned in bacteria and the DNA sequence confirmed a cysteine-proteinase similar to CMS2MS2. The recombinant protein is 30 kDa, induces a mitogenic response, and enhances ERK1/2 phosphorylation, like the non-recombinant enzyme, but lacks either amidase or caseinolytic activity. The mitogenic activity of this protein and its lack of proteolytic activity underscore a potential for use in wound healing treatment.