Cytotoxic and anticancer properties of the Malaysian mangrove pit viper (Trimeresurus purpureomaculatus) venom and its disintegrin (purpureomaculin)

The Asiatic pit vipers from the Trimeresurus complex are medically important venomous snakes. These pit vipers are often associated with snakebite that leads to fatal coagulopathy and tissue necrosis. The cytotoxic venoms of Trimeresurus spp.; however, hold great potential for the development of peptide-based anticancer drugs. Methods: This study investigated the cytotoxic effect of the venom from Trimeresurus purpureomaculatus, the mangrove pit viper (also known as shore pit viper) which is native in Malaysia, across a panel of human cancer cell lines from breast, lung, colon and prostate as well as the corresponding normal cell lines of each tissue. Results: The venom exhibited dose-dependent cytotoxic activities on all cell lines tested, with median inhibition concentrations (IC50) ranging from 0.42 to 6.98 µg/mL. The venom has a high selectivity index (SI = 14.54) on breast cancer cell line (MCF7), indicating that it is significantly more cytotoxic toward the cancer than to normal cell lines. Furthermore, the venom was fractionated using C18 reversed-phase high-performance liquid chromatography and the anticancer effect of each protein fraction was examined. Fraction 1 that contains a hydrophilic low molecular weight (approximately 7.5 kDa) protein was found to be the most cytotoxic and selective toward the breast cancer cell line (MCF7). The protein was identified using liquid chromatography-tandem mass spectrometry as a venom disintegrin, termed purpureomaculin in this study. Conclusion: Taken together, the findings revealed the potent and selective cytotoxicity of a disintegrin protein isolated from the Malaysian T. purpureomaculatus venom and suggested its anticancer potential in drug discovery.(AU)

Cell migration inhibition activity of a non-RGD disintegrin from Crotalus durissus collilineatus venom

In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line. Methods: Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-TricineSDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively. Results: Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity (15% and 17%, at 3 and 6 µg/mL, respectively) after 24 h of incubation and in the wound-healing assay, the disintegrin (3 µg/mL) was able to significantly inhibit cell migration (24%, p < 0.05), compared to negative control. Conclusion: Thus, our results demonstrate that non-RGD disintegrin from C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.(AU)

Effects of blocking αvβ3 integrin by a recombinant RGD disintegrin on remodeling of wound healing after induction of incisional hernia in rats

PURPOSE: To investigate the changes induced by DisBa-01 on repair of wound healing after induced incisional hernia (IH) in rats. METHODS: Thirty two male albino rats were submitted to IH and divided into four experimental groups: G1, placebo control; G2, DisBa-01-treated; G3, anti-αvβ3 antibodies-treated and G4, anti-α2 antibodies-treated. Histological, biochemical and extracellular matrix remodeling analysis of abdominal wall were evaluated. RESULTS: After 14 days, 100% of the G2 did not present hernia, and the hernia ring was closed by a thin membrane. In contrast, all groups maintained incisional hernia. DisBa-01 also increased the number macrophages and fibroblasts and induced the formation of new vessels. Additionally, MMP-2 was strongly activated only in G2 (p<0.05). Anti- αvβ3-integrin antibodies produced similar results than DisBa-01 but not anti-α2 integrin blocking antibodies. CONCLUSION: DisBa-01 has an important role in the control of wound healing and the blocking of this integrin may be an interesting therapeutically strategy in incisional hernia. .

Effects of Blocking αvβ3 integrin by a recombinant RGD disintegrin on remodeling of wound healing after induction of incisional hernia in rats

PURPOSE: Incisional hernia (IH) is characterized by defective wound healing process. Disba-01, a αvb3 integrin blocker has shown to control the rate of wound repair and therefore it could be a target for new wound healing therapies.The objective of the study was to determine the changes induced by Disba-01 on repair of wound healing after induced IH in rats. METHODS: Thirty two male albino rats were submitted to IH and divided into 4 experimental groups: G1, placebo control; G2, DisBa-01-treated; G3, anti-αvβ3 antibodies-treated and G4, anti-α2 antibodies-treated. Histological. biochemical and extracellular matrix remodeling analysis of abdominal wall were evaluated. RESULTS: After 14 days, 100% of the G2 did not present hernia, and the hernia ring was closed by a thin membrane. In contrast, all groups maintained incisional hernia. DisBa-01 also increased the number macrophages and fibroblasts and induced the formation of new vessels. Additionally, MMP-2 was strongly activated only in G2 (P<0.05). Anti- αvβ3-integrin antibodies produced similar results than Disba-01 but not anti-α2 integrin blocking antibodies. CONCLUSION: These results strongly indicate that Disba-01 has an important role in the control of wound healing and the blocking of this integrin may be an interesting therapeutical strategy in IH. .

Genetic polymorphisms and asthma: findings from a case - control study in the Madeira island population

BACKGROUND: Asthma is a complex disease influenced by multiple genetic and environmental factors. While Madeira has the highest prevalence of asthma in Portugal (14.6%), the effect of both genetic and environmental factors in this population has never been assessed. We categorized 98 asthma patients according to the Global Initiative for Asthma (GINA) guidelines, established their sensitization profile, and measured their forced expiratory volume in 1second (FEV1) and forced vital capacity (FVC) indexes. Selected single nucleotide polymorphisms (SNPs) were analysed as potential markers for asthma susceptibility and severity in the interleukin 4 (IL4), interleukin 13 (IL13), beta-2-adrenergic receptor (ADRB2), a disintegrin and metalloprotease 33 (ADAM33), gasdermin-like (GSDML) and the signal transducer and activator of transcription 6 (STAT6) genes comparatively to a population reference set. RESULTS: Although mites are the major source of allergic sensitization, no significant difference was found amongst asthma severity categories. IL4-590*CT/TT and IL4-RP2*253183/183183 were found to predict the risk (2-fold) and severity (3 to 4-fold) of asthma and were associated with a lower FEV1 index. ADRB2-c.16*AG is a risk factor (3.5-fold), while genotype GSDML-236*TT was protective (4-fold) for moderate-severe asthma. ADAM33-V4*C was associated to asthma and mild asthma by the transmission disequilibrium test (TDT). Finally, ADAM33-V4*CC and STAT6-21*TT were associated with higher sensitization (mean wheal size ≥10mm) to house dust (1.4-fold) and storage mite (7.8-fold). CONCLUSION: In Madeira, IL4-590C/T, IL4-RP2 253/183, GSDML-236C/T and ADAM33-V4C/G SNPs are important risk factors for asthma susceptibility and severity, with implications for asthma healthcare management.

disintegrin and metalloproteinase 33 [ADAM33] gene polymorphism association with asthma in Egyptian children

A disintegrin and metalloproteinase-encoding gene [ADAM33], was recently identified as an asthma susceptibility gene. ADAM33 protein is expressed in smooth muscle cells of bronchi and pulmonary fibroblasts, playing a major role in airway remodeling. Earlier studies, have mostly confirmed a link between ADAM33 and asthma as well as bronchial hyperresponsiveness. This work studied a group of Egyptian asthmatic children for 3 ADAM33 single nucleotide polymorphisms [SNPs], previously identified as putative risk alleles: T1 G > A[rs2280091], T2 A > G[rs2280090], V4 G > C[rs2787094] using Polymerase Chain Reaction - restriction fragment length polymorphism [PCRRFLP] with emphasis on their relation to clinical [severity, smoking, family history, and atopic manifestations] and laboratory data [Ig Immunoglobulin E [Ig E] level and absolute eosinophilia] and pulmonary functions. Sixty [3-12 years old] asthmatic children and 32 matched controls were recruited. The genotype distribution for the SNPs showed no significant difference between the patients and the controls. A higher frequency of the [AA] genotype of T1 polymorphism was found in controls [75%] than in patients [41%], while the [AG] variant was higher in cases [46.6%] than in controls [21.9%] but with no statistically significant difference. Also the [GG] genotype was higher in cases [11.6%] than in controls [3.1%] but with no statistical significance. The allelic frequencies of T1 showed a higher [A] allele in controls [85.93%] than cases [65%] and higher [G] allele in cases [35%] than controls [14.06%], showing a high significant difference. No correlation was found between [T1, T2, and V4] and the demographic, clinical and laboratory parameters, except SNP T1 showing a positive correlation with Ig E level, and SNP V4 showing a positive correlation with passive smoking as a precipitating factor and borderline significance with absolute eosinophilia. In conclusion, no significant association was detected between these SNPs and asthma susceptibility in this study

Uso potencial de componentes del veneno de serpiente en el tratamiento del cáncer / Potential use of snake venom components in cancer treatment

El desarrollo del cáncer es posible en la medida que las células tumorales proliferen, se dispersen e invadan otros tejidos del cuerpo. Las integrinas son una familia de receptores heterodiméricos de superficie celular que cumplen un papel crucial en el desarrollo de la angiogénesis, crecimiento y metástasis de un tumor señalándolas como un atractivo blanco terapéutico. Los venenos de serpientes contienen péptidos de bajo peso molecular conocidos como desintegrinas, las que se unen con una alta afinidad a las integrinas e inhiben su accionar en un proceso cancerígeno. En el siguiente articulo revisamos los resultados de investigaciones, tanto in vitro como in vivo, que han mostrado resultados promisorios, por lo cual el uso de las desintegrinas podrían constituir una alternativa promisoria para el tratamiento de diversas neoplasias.

Cancer can develop to the extent tumor cells grow, divide and grow into other body tissues. Integrins are a family of cell-surface heterodimeric receptors that play an important role in the development of tumor angiogenesis, growth and metastasis, thus being recognized as an attractive therapeutic target. Snake venom contains low-molecular weight peptides known as “disintegrins” that bind to integrins with high affinity, and prevent their action in cancer. In the next article, we go over the results of investigations, both in vitro and in vivo, which have shown promising results, thus revealing that the use of disintegrins could be a promising alternative for the treatment of different neoplasias.

Second trimester screening for trisomy 21 using ADAM12-S as a maternal serum marker / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the value of a disintegrin and metalloproteinase 12 secreting form (ADAM12-S) as a maternal serum marker in second trimester screening for trisomy 21 (Down syndrome, DS), and to develop an appropriate prenatal DS screening protocol.</p><p><b>METHODS</b>Serum samples were collected from 53 pregnant women carrying a trisomy 21 fetus and 621 pregnant women with matched gestational age and weight carrying a healthy fetus. ADAM12-S concentrations were determined with a time-resolved fluorescence immunoassay (TRFIA). Curve fitting by weighted regression and other statistical methods were conducted, and the model was optimized for prenatal trisomy 21 screening program in second trimester. ADAM12-S alone or in combination with other two- or three-combination test was selected as a serum marker for prenatal second-trimester screening of trisomy 21 by calculation of detection rate (DR) and false positive rate (FPR).</p><p><b>RESULTS</b>By comparison, the median multiple of the median (MoM) value of ADAM12-S in DS pregnancy group was higher than that of the control group (P< 0.01). When FPR = 5%, the DR of ADAM12-S was 28.3%, and the positive and negative likelihood ratios were 5.66 and 0.75, respectively. The DR of three-combination test of ADAM12-S, alpha-fetoprotein (AFP) and free beta subunit of human chorionic gonadotropin (β-HCG) has increased to 52.80% from 39.62% of the conventional two-combination test (AFP and free β-HCG). For women with a risk between 1/300 and 1/1000 by two-combination test for DS, the DR has increased from 39.62% to 47.12%, but FPR only increased by 0.8% after adding ADAM12-S as a maternal serum marker.</p><p><b>CONCLUSION</b>Considering the increased DR of pregnancies with a risk between 1/300 and 1/1000 in second trimester, ADAM12-S may provide a feasible maternal serum maker when combined with AFP and free β-HCG. The cost-effectiveness ratio is reasonable.</p>

Púrpura trombótica trombocitopenica en adultos / Trombocitopenica trombótica purple in adults

Se revisa la presentación de 6 pacientes adultos con púrpura trombótica trombocitopénica (PTT), que fueron atendidos en el Servicio de Clínica Médica del Hospital Escuela de Corrientes durante un período de 11 años. La púrpura trombótica trombocitopénica es una anemia hemolítica microangiopática caracterizada por una pentada clínica típica: trombocitopenia, anemia hemolítica microangiopática, fallo renal, fiebre y manifestaciones neurológicas. Todos los pacientes presentaron trombocitopenia y anemia hemolítica microangiopática. El compromiso neurológico fue la segunda característica más frecuente. Todos los pacientes fueron tratados con plasmaféresis. Tres pacientes se recuperaron completamente y 3 fallecieron. El pilar del tratamiento es la transfusión de plasma y la plasmaféresis. Se hace hincapié en su implementación inmediata dada la alta mortalidad de esta patología.

Evaluating kinetic and physiological features of rCHO-K1 cells cultured on microcarriers for production of a recombinant metalloprotease/disintegrin

We present kinetic and physiological data regarding the culturing of rCHO-K1 cells on various microcarriers, to evaluate the potential of this culture strategy for mass production of these cells and expression of a recombinant disintegrin. Cultures were performed in 500 mL spinner flasks in DMEM culture medium with 10 percent v/v fetal calf serum, gently shaken at 37 C, pH 7.4, in a 10 percent v/v CO2 atmosphere. The following values were obtained, respectively, for the adhesion time-constant Ka (h) and specific growth rate micron max (d-1) on each microcarrier: Cytodex 1 (0.91, 0.45), Cultispher S (0.28, 0.34), Immobasil FS (0.85, 0.52) and Pronectin F (5.12, 0.67). Metabolic characteristics showed some variation among the cultures with the four microcarriers, the most significant being the higher production of ammonia with microcarriers coated with adhesive molecules (Cultispher S and Pronectin F) relative to the uncoated carriers (Cytodex 1 and Immobasil FS). Experiments where the DMEM medium was gradually replaced by the serum-free medium (CHO-SFM-II) revealed important advantages over media containing serum, not only for assay purposes, but also for purification of the disintegrin. Altogether these results demonstrate that cultures on microcarriers, especially on Pronectin F, show good potential for larger scale cultures of rCHO-K1 cell.

effect of snake venom metalloproteinase/disintegrin like activity on liver of albino white mice

This is a study on the effect of snake venom metalloproteinase/ Disintegrin like action [SVMP] purified from Cerastes cerastes venom on expression of TNF-alpha, and Heme-oxygenase-1 in white mice liver tissue injected intraperitoneally with the purified fraction. Hepato-protective effect of the fraction against some hepatotoxins as CCU was investigated histo-pathologically. This hepatoprotection coincides with expression of TNF-alpha and HO-1 genes in liver tissue. This study is supported by grant no. 28 from Professor Dr. M. F. EL-Asmar

potential of the crude snake venom in treatment of human breast cancer with and without combination of Anticancer drugs

Clinical usefulness of the snake venom has been worked out since the last many decades to treat blood pressure, cancer of breast, ovary and etc. we wanted to evaluate the effect of natural compounds of cobra snake venom in vitro on nucleic acid in normal and breast cancer tissues in combination with and without anticancer drugs. Surgically removed tissue from cancerous and non cancerous of same human breast were homogenized and extraction of nucleic acids were prepared. All homogenate samples were incubated with and without snake venom and antineoplastic drugs [Cyclophosophamide and Mitomycin-C] for 30 min at 37 °C and measured at 490 m micro for DNA and at 660 m micro for RNA by spectrophotometer [Sspectron-21[R]]. The effect of the snake venom was compared singly as well as in combination with the two chemotherapeutic agents viz mitomycin-C [an antibiotic] and Cyclophosophamide [an alkylating agent] on the nucleic acids [RNA and DNA]. In human breast cancer tissue shows significant reduction in nucleic acids contains when treated with venom [25 micro g/ml] in combination and compared with singly use anticancer drugs. Cyclophosophamide and Mitomycin produced some effect in human breast tissue at 10 micro g/ml on the DNA only for short time and eventually destroyed. However snake venom does have potential and is more effective with or without anticancer drugs on both nucleic acids [RNA and DNA] suggests wide range of receptors to act. Both drugs could not produce inhibition or potentiating effect on normal and cancerous tissues in vitro by all different doses. Anticancer drugs [Cyclophosophamide and Mitomycin-C], specific medicines for solid tumour [Ca. Breast] used in this study showed no significant reduction at the same dose of snake venom [25 micro g/ml] in vitro in comparison to snake venom. However drugs at 10 micro g/ml has shown some effect on the DNA but only for short time and possibly, eventually are destroyed. However significant and promising anticancer effect of snake venom seems to have a better future as an alternative. None toxic dose of snake venom affects on both DNA and RNA

Alternagin-C, a disintegrin-like protein from the venom of Bothrops alternatus, modulates alpha2ß1 integrin-mediated cell adhesion, migration and proliferation

The alpha2ß1 integrin is a major collagen receptor that plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Alternagin-C (ALT-C), a disintegrin-like protein purified from the venom of the Brazilian snake Bothrops alternatus, competitively interacts with the alpha2ß1 integrin, thereby inhibiting collagen binding. When immobilized in plate wells, ALT-C supports the adhesion of fibroblasts as well as of human vein endothelial cells (HUVEC) and does not detach cells previously bound to collagen I. ALT-C is a strong inducer of HUVEC proliferation in vitro. Gene expression analysis was done using an Affimetrix HU-95A probe array with probe sets of 10,000 human genes. In human fibroblasts growing on collagen-coated plates, ALT-C up-regulates the expression of several growth factors including vascular endothelial growth factor, as well as some cell cycle control genes. Up-regulation of the vascular endothelial growth factor gene and other growth factors could explain the positive effect on HUVEC proliferation. ALT-C also strongly activates protein kinase B phosphorylation, a signaling event involved in endothelial cell survival and angiogenesis. In human neutrophils, ALT-C has a potent chemotactic effect modulated by the intracellular signaling cascade characteristic of integrin-activated pathways. Thus, ALT-C acts as a survival factor, promoting adhesion, migration and endothelial cell proliferation after binding to alpha2ß1 integrin on the cell surface. The biological activities of ALT-C may be helpful as a therapeutic strategy in tissue regeneration as well as in the design of new therapeutic agents targeting alpha2ß1 integrin.

Disintegrins: integrin selective ligands which activate integrin-coupled signaling and modulate leukocyte functions

Extracellular matrix proteins and cell adhesion receptors (integrins) play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp) motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.

Desintegrinas en veneno de serpientes: un nuevo enfoque en el tratamiento de las enfermedades cardiovasculares cáncer-diábetes / Desintegrings in snakes venoms: a new focus in the treatment cardiovascular, cancer, diabetes disease

Las integrinas mediante la adhesión de células a matriz extracelulares, o a células-células a través de ligandos. La adherencia célula a célula puede ser una agregación homotipica de células semejantes o una agregación heterotípica de diversos tipos de celulares. Se plantea, que la función de las integrinas implica mucho más que la adherencia celular. Las integrinas están implicadas en la transducción, la cual desempeña un papel importante en muchos procesos fisiológicos tales como el desarrollo embrionario, diferenciación, migración de la célula, actividades curativas de la inflamación, la coagulación y la tumorigénesis. Las serpientes de la familia Viperidae tienen una clase de proteínas de bajo peso molecular llamadas desintegrinas, en sus venenos. Estas moléculas han mostrado ser inhibidores potentes de la unión del fibrinógeno a las plaquetas activadas. Se ha demostrado que las desintegrinas se pueden unir a los receptores de las integrinas en las plaquetas y otros tejidos, por ello, podrían tener aplicación potencial en medicina. Las desintegrinas tienen una homología notable con la secuencia y selectividad en sus instrucciones con los receptores de las integrinas celulares

Cloning of human angiogenesis inhibitor METH1 and its expression in mammalian cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To get the full length of human METH1 cDNA and express it steadily in mammalian cell stably.</p><p><b>METHODS</b>METH1 was amplified by RT-PCR, and cloned into pCDNA3.0 after confirmed by sequence analysis. HepG2 cells were transfected by Lipofectamine reagent and then selected in medium with G418. The expression level of METH1 was detected by RT-PCR and Western blot.</p><p><b>RESULTS</b>METH1 with expected length was effectively amplified, and completely matched the published sequence of encoding mature peptide [GI:5725505] as shown by sequence analysis. Eukaryotic vector expressing METH1 was obtained by gene cloning, cells expressing METH1 was got by selection with G418 at 3 weeks after transfection. RT-PCR and Western blot showed high level expression of METH1.</p><p><b>CONCLUSION</b>Full length of human METH1 gene is cloned successfully and expressed in HepG2 steadily, The results set up a basis for the study of effects of METH1 on hypertrophic scar angiogenesis.</p>

Isolation of bothrasperin, a disintegrin with potent platelet aggregation inhibitory activity, from the venom of the snake Bothrops asper

The venom of Bothrops asper induces severe coagulation disturbances in accidentally envenomed humans. However, only few studies have been conducted to identify components that interact with the hemostatic system in this venom. In the present work, we fractionated B. asper venom in order to investigate the possible presence of inhibitors of platelet aggregation. Using a combination of gel filtration, anion-exchange chromatography, and reverse-phase high performance liquid chromatography, we isolated an acidic protein which shows a single chain composition, with a molecular mass of approximately 8 kDa, estimated by SDS-polyacrylamide gel electrophoresis. Its N-terminal sequence has high similarity to disintegrins isolated from different snake venoms, which are known to bind to cellular integrins such as the GPIIb/IIIa fibrinogen receptor on platelets. The purified protein exerted potent aggregation inhibitory activity on ADP-stimulated human platelets in vitro, with an estimated IC50 of 50 nM. This biological activity, together with the biochemical characteristics observed, demonstrate that the protein isolated from B. asper venom is a disintegrin, hereby named bothrasperin. This is the first disintegrin isolated from Central American viperid snake species.

Caracterização de disintegrinas de venenos viperídeos como ferramentas seletivas na detecção ou inibição da função de integrinas / Characterization of disintegrins of Viperidae venoms as selective tools in the detection or inhibition of the function of integrins

As integrinas são proteínas de membrana envolvidas em diversos processos biológicos como embriogênese, inflamação e agregação plaquetária. A alteração de seu padrão de expressão está relacionada com processos patológicos como trombose e câncer, fazendo das integrinas ótimos indicadores da progressão destas doenças. Assim a integrina `alfa 2'ß1 pode ser considerada como indicadora de angiogênese, sendo expressa nas células endoteliais durante o crescimento de novos vasos. Já a ausência da `alfaIIb'ß3 em plaquetas está relacionada com a doença de Glanzmann, caracterizada por uma agregação plaquetária deficiente, e sua presença em outros tipos celulares está relacionada com processos de metástase e invasão de tecidos...

Molecular cloning and sequence analysis of ussurin, a new metalloproteinases/disintegrin from Gloydius ussuriensis / 生物工程学报

The metalloproteinases/disintegrins in the snake venom act as platelet aggregation inhibitor by an antagonism against integrin on platelet through its RGD sequence and may play other important role in cell-cell fusion, cell matrix interaction and other cellular function. Ussurin is a new metalloproteinase/disintegrin that was cloned from Gloydius ussuriensis. Poly (A+) RNA was purified from the total RNA preparation from venom gland of a single G. ussuriensis using the poly (A+) tract-mRNA isolation system. A cDNA library was constructed with the SMART PCR cDNA library construction kit. The cDNA library was screened and the positive clones were selected. The full-length cDNA of Ussurin was obtained. The cDNA encoding the Ussurin precursor has a 51bp 5'-UTR, the open reading frame of Ussurin and a 490 bp 3'-UTR, the open reading frame of Ussurin cDNA nucleotide sequence is 1434 bp and codes for 478 amino acids with a predicted molecular mass of 53.2 kD and an isoelectric point of 5.37. There is no potential N-glycosylation site in the deduced sequence region. Its deduced amino acid sequence consists of four region, a signal sequence of 18 amino acid residues, a zymogen pro-peptide of 171 amino acid residues with a cysteine switch motif (PK-MCGVT) in it, a central metalloproteinase domain of 201 amino acid residues containing a conserved zinc-chelating sequence (HEXXHXXGXXH) and a methionine-turn CIM involving zinc banding also, a space sequence between metalloproteinase domain and disintegrin domain of 15 amino acid residues with a conserved T392, T397, S400, which is specific residues of the P-II snake venom metalloproteinases, a disintegrin domain of 73 amino acid residues with a characteristic RGD region and six-disulfide bonds. Ussurin belongs to P-II class. The cDNA sequence and deduced amino acid sequence of Ussurin precursor were compared with homologous sequence in the GenBank database, the result reveals high degree of homology in sequence and organization pattern of domain with metalloproteinase/disintegrin gene family of other snake species. Compared with the alignment of amino acid sequence of metalloproteinase/disintegrin member, hypervariable regions of this member were revealed, besides they share higher homologous in the zymogen domain. It suggests that the hypervariable regions are the counterparts directly suitable for interacting with different domain of receptors, different receptors or substrates.

Integrins and disintegrins: the candidate molecular players in sperm-egg interaction.

Fertilization includes sperm-egg recognition, adhesion, binding, fusion and egg activation. Integrin (ITG) receptors which are adhesion molecules are expressed on mouse, hamster and human gametes and their potential ligands also have been identified. Role of ITGs during fertilization is supported by inhibition of sperm-egg adhesion and/or fusion by means of anti-ITG mAbs, Arg-Gly-Asp (RGD) or disintegrin-like peptides. This review includes the current understanding of the molecular mechanism that regulates sperm-egg interaction and implications of this knowledge for assessing the fertility potential of men and immunocontraceptive method.