Alternagin-C binding to 21 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of 21 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an 21 integrin. Herein, we used ALT-C as a 21 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The 21, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to 21 integrin...

Alternagin-C binding to 21 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of 21 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an 21 integrin. Herein, we used ALT-C as a 21 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The 21, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to 21 integrin...(AU)

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

Background: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-Tricine-SDS-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...(AU)

Contribution of flagella and motility to gut colonisation and pathogenicity of Salmonella Enteritidis in the chicken

ABSTRACT Salmonella Enteritidis causes fowl paratyphoid in poultry and is frequently associated to outbreaks of food-borne diseases in humans. The role of flagella and flagella-mediated motility into host-pathogen interplay is not fully understood and requires further investigation. In this study, one-day-old chickens were challenged orally with a wild-type strain Salmonella Enteritidis, a non-motile but fully flagellated (SE motB) or non-flagellated (SE fliC) strain to evaluate their ability to colonise the intestine and spread systemically and also of eliciting gross and histopathological changes. SE motB and SE fliC were recovered in significantly lower numbers from caecal contents in comparison with Salmonella Enteritidis at early stages of infection (3 and 5 dpi). The SE motB strain, which synthesises paralysed flagella, showed poorer intestinal colonisation ability than the non-flagellated SE fliC. Histopathological analyses demonstrated that the flagellated strains induced more intense lymphoid reactivity in liver, ileum and caeca. Thus, in the present study the flagellar structure and motility seemed to play a role in the early stages of the intestinal colonisation by Salmonella Enteritidis in the chicken.(AU)

Roles of quorum sensing molecules from Rhizobium etli RT1 in bacterial motility and biofilm formation

ABSTRACT Strain RT1 was isolated from root nodules of Lens culinaris (a lentil) and characterized as Rhizobium etli (a Gram-negative soil-borne bacterium) by 16S rDNA sequencing and phylogenetic analysis. The signaling molecules produced by R. etli (RT1) were detected and identified by high-performance liquid chromatography coupled with mass spectrometry. The most abundant and biologically active N-acyl homoserine lactone molecules (3-oxo-C8-HSL and 3-OH-C14-HSL) were detected in the ethyl acetate extract of RT1. The biological role of 3-oxo-C8-HSL was evaluated in RT1. Bacterial motility and biofilm formation were affected or modified on increasing concentrations of 3-oxo-C8-HSL. Results confirmed the existence of cell communication in RT1 mediated by 3-oxo-C8-HSL, and positive correlations were found among quorum sensing, motility and biofilm formation in RT1.(AU)

Primordial germ cell migration and the Wnt signaling pathway

A critical step in the development of gametes is the migration of their primordial germ cell (PGC) precursors to the forming gonads. Although the location and mode of PGC specification differs between organisms, the formation of a committed germline before organogenesis creates a need for migration through the growing embryo in order to reach the site of gonadogenesis. Failure of PGC migration can, in many cases, compromise fertility or conversely lead to the formation of teratomas in sites outside of the gonad. Here we review the mechanism of migration employed by PGCs and compare the timing and routes across several model organisms. We summarize recent work on the role of the Wnt signaling pathway in cell migration and the lineage specific function in PGCs, mainly through the ligand Wnt5a and its receptor Ror2.

Primordial germ cell migration and the Wnt signaling pathway

A critical step in the development of gametes is the migration of their primordial germ cell (PGC) precursors to the forming gonads. Although the location and mode of PGC specification differs between organisms, the formation of a committed germline before organogenesis creates a need for migration through the growing embryo in order to reach the site of gonadogenesis. Failure of PGC migration can, in many cases, compromise fertility or conversely lead to the formation of teratomas in sites outside of the gonad. Here we review the mechanism of migration employed by PGCs and compare the timing and routes across several model organisms. We summarize recent work on the role of the Wnt signaling pathway in cell migration and the lineage specific function in PGCs, mainly through the ligand Wnt5a and its receptor Ror2.(AU)

Sperm motility, fertilization, and larval development of silver catfish (Rhamdia quelen) in copper-contaminated water / Motilidade espermática, fertilização e desenvolvimento inicial de jundiá (Rhamdia quelen) em água contaminada com cobre

The objective of this study was to evaluate the effect of copper-contaminated water on sperm motility, fertilization, and embryonic and larval development of silver catfish (Rhamdia quelen). A randomized experimental design with five treatments and four replicates was used. Two experiments were carried out: (1) controlled fertilization was performed under different levels of copper contamination and egg hatching was performed in clean water; and (2) copper-contaminated water was used for both fertilization and hatching assays. The time of sperm motility and sperm motility rates linearly decreased with increasing copper concentration in the water. Fertilization and hatching rates were also affected when the concentrations of copper in the water were above 0.0979 mg Cu+2 L-1 and 0.0331 mg Cu+2 L-1, respectively. Gamete exposure to levels between 15 mg Cu+2 L-1 and 60 mg Cu+2 L-1 for short periods of time negatively affected sperm motility, oocyte fertilization, and egg hatching rates. In addition, when gametes and embryos were exposed at levels above 0.03 mg Cu+2 L-1 during long periods of time, egg hatching rates were reduced, and at levels between 0.05 mg Cu+2 L-1 and 0.20 mg Cu+2 L-1 the number of abnormal larvae increased.(AU)

O objetivo deste estudo foi avaliar o efeito da água contaminada de cobre na motilidade espermática, fertilização e desenvolvimento embrionário e larval de jundiá (Rhamdia quelen). O delineamento experimental foi inteiramente casualizado, com cinco tratamentos e quatro repetições. Dois ensaios foram conduzidos: (1) a fertilização controlada foi realizada com diferentes níveis de água contaminada com cobre e a incubação do ovo realizada em água limpa; e (2) a contaminação da água foi utilizada tanto na fertilização e quanto na incubação dos ovos. O tempo de motilidade de espermatozóides e as taxas de motilidade do esperma foram reduzidas com o aumento da concentração de cobre na água. As taxas de fertilização e eclosão também foram afetados quando as concentrações de cobre na água estavam acima de 0,0979 mg Cu+2 L-1 e 0,0331 mg Cu+2 L-1, respectivamente. A exposição dos gametas por um curto período de tempo em níveis entre 15 ou 60 mg de Cu+2 L-1 promoveu danos sobre a motilidade dos espermatozóides, a fertilização de oócitos e a eclosão dos ovos. Além disso, quando os gametas e embriões foram expostos a níveis acima de 0,03 mg de Cu+2 L-1 por um longo período de tempo, a eclosão dos ovos foi reduzida, e em níveis de 0,05 a 0.20mg Cu+2 L-1 o anormalidade larval aumentou.(AU)

Sperm motility, fertilization, and larval development of silver catfish (Rhamdia quelen) in copper-contaminated water / Motilidade espermática, fertilização e desenvolvimento inicial de jundiá (Rhamdia quelen) em água contaminada com cobre

The objective of this study was to evaluate the effect of copper-contaminated water on sperm motility, fertilization, and embryonic and larval development of silver catfish (Rhamdia quelen). A randomized experimental design with five treatments and four replicates was used. Two experiments were carried out: (1) controlled fertilization was performed under different levels of copper contamination and egg hatching was performed in clean water; and (2) copper-contaminated water was used for both fertilization and hatching assays. The time of sperm motility and sperm motility rates linearly decreased with increasing copper concentration in the water. Fertilization and hatching rates were also affected when the concentrations of copper in the water were above 0.0979 mg Cu+2 L-1 and 0.0331 mg Cu+2 L-1, respectively. Gamete exposure to levels between 15 mg Cu+2 L-1 and 60 mg Cu+2 L-1 for short periods of time negatively affected sperm motility, oocyte fertilization, and egg hatching rates. In addition, when gametes and embryos were exposed at levels above 0.03 mg Cu+2 L-1 during long periods of time, egg hatching rates were reduced, and at levels between 0.05 mg Cu+2 L-1 and 0.20 mg Cu+2 L-1 the number of abnormal larvae increased.

O objetivo deste estudo foi avaliar o efeito da água contaminada de cobre na motilidade espermática, fertilização e desenvolvimento embrionário e larval de jundiá (Rhamdia quelen). O delineamento experimental foi inteiramente casualizado, com cinco tratamentos e quatro repetições. Dois ensaios foram conduzidos: (1) a fertilização controlada foi realizada com diferentes níveis de água contaminada com cobre e a incubação do ovo realizada em água limpa; e (2) a contaminação da água foi utilizada tanto na fertilização e quanto na incubação dos ovos. O tempo de motilidade de espermatozóides e as taxas de motilidade do esperma foram reduzidas com o aumento da concentração de cobre na água. As taxas de fertilização e eclosão também foram afetados quando as concentrações de cobre na água estavam acima de 0,0979 mg Cu+2 L-1 e 0,0331 mg Cu+2 L-1, respectivamente. A exposição dos gametas por um curto período de tempo em níveis entre 15 ou 60 mg de Cu+2 L-1 promoveu danos sobre a motilidade dos espermatozóides, a fertilização de oócitos e a eclosão dos ovos. Além disso, quando os gametas e embriões foram expostos a níveis acima de 0,03 mg de Cu+2 L-1 por um longo período de tempo, a eclosão dos ovos foi reduzida, e em níveis de 0,05 a 0.20mg Cu+2 L-1 o anormalidade larval aumentou.

Evaluation of the anti-inflammatory and analgesic effects of green tea (Camellia sinensis) in mice

PURPOSE: To evaluate the anti-inflammatory and analgesic effects of green tea (Camellia sinensis) in mice.METHODS:The anti-inflammatory effect of alcoholic extracts of green tea (AE) was evaluated in a cell migration assay with four groups of six Swiss mice receiving 0.07g/Kg or 0.14g/Kg EA (treatment groups), saline (negative control) or 10mg/Kg indomethacin (positive control) by gavage. One hour later 300 µg carrageen an was administered intraperitoneally or subcutaneously. The analgesic effect was evaluated using four groups of six animals receiving 0.07g/Kg or 0.14g/Kg EA, saline or 10mg/Kg indomethacin subcutaneously, followed 30 minutes later by 1% acetic acid.RESULTS: When administered subcutaneously at either dose (0.07g/Kg and 0.14g/Kg), AE inhibited carrageenan-induced cell migration (p 0.05). However, when administered by gavage, only the latter (0.14 g/Kg) was efficient (p 0.05). AE at both doses (0.07g/Kg and 0.14g/Kg) inhibited abdominal contortions (p 0.05), but the effect was not dose-dependent.CONCLUSION:Green tea was shown to have analgesic and anti-inflammatory properties and may constitute a natural treatment option in chronic inflammatory disorders.(AU)

Avaliação da migração das células progenitoras após terapia da tendinite equina / Migration evaluation of progenitor cells after treatment of equine tendinitis

A terapia celular vem sendo utilizada com resultados promissores no tratamento da tendinite equina, entretanto ainda existem dúvidas quanto à persistência e ao comportamento dessas células quando implantadas no local da lesão, e quanto à sua migração para outros focos inflamatórios. O objetivo deste estudo foi avaliar a marcação das células-tronco mesenquimais (CTMs) com nanocristal antes e após o implante em lesões tendíneas experimentais do tendão flexor digital superficial (TFDS) de equinos, bem como observar a possibilidade de migração das CTMs marcadas para outro foco de lesão, o membro contralateral do mesmo animal. Para isso, foi realizada a indução de lesão experimental no TFDS em ambos os membros torácicos de cinco equinos e, após sete dias, foram implantadas as CTMs autólogas marcadas com o nanocristal Qtracker 655 em um dos membros dos animais. Após sete dias do implante, foi realizada a biópsia tendínea para posterior avaliação histopatológica, utilizando-se microscopia com fluorescência. Também foi realizado o teste de viabilidade celular antes e após a incubação com o nanocristal. As CTMs marcadas e injetadas no tecido tendíneo mantiveram sua fluorescência sete dias após seu implante, e não ocorreu migração para o membro contralateral. O uso do nanocristal para a marcação das CTMs derivadas da medula óssea equina mostrou-se efetivo pelo fato de essa nanopartícula não ter alterado a viabilidade celular e por ela ter permanecido ativa durante o período implantado.(AU)

Cell therapy has been used with promising results in the treatment of equine tendinitis. However, there are still doubts about the persistence and behavior of these cells implanted in the injured tissue and their migration to other inflamed sites. The aim of this study was to evaluate the labeling of mesenchymal stem cells (MSCs) with nanocrystals before and after implantation in experimental tendinitis of the superficial digital flexor tendon (SDFT) of horses, observing the migration possibility of MSCs marked to another lesion, performed on the contralateral limb of the same animal. An experimental lesion was induced in SDFT in both forelimbs of five horses, and after seven days autologous MSCs labeled with Qtracker(r) 655 were implanted in one member of the animals. Tendon biopsy was performed for subsequent histopathological evaluation using fluorescence microscopy seven days after the implant. Cell viability test was also performed before and after incubation with the cell labeling kit. MSCs labeled and injected into the tendon tissue maintained their fluorescence seven days after their implantation and there was no migration to the contralateral limb. The use of nanocrystals for labeling MSCs was effective because it does not alter cell viability and remains active during the experimental period.(AU)

Cell migration induced by Leishmania (Leishmania) amazonensis, Leishmania (Leishmania) major and Leishmania (Viannia) braziliensis into the peritoneal cavity of BALB/c mice

In American cutaneous leishmaniasis, the initial infection phase is characterized by recruitment of neutrophils and monocytes. The migration of these cells in response to the presence of Leishmania in the peritoneum of affected animals remains unclear. The objective of this study was to investigate cell migration to the peritoneum of BALB/c mice after infection with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) major. Initially, Leishmania spp. was intraperitoneally inoculated in five groups of six animals each and the cell migration was analyzed 0, 3, 6, 12, 24 and 48 hours after infection. Different cell counts were performed with a staining kit and showed a higher percentage of polymorphonuclear than mononuclear cells in all three species studied. The total cell count revealed peak migration in L. (L.) amazonensis and L. (L.) major at six hours, and in L. (V.) braziliensis at 12 hours. These results suggest that factors released from different cell types probably act by attracting polymorphonuclear cells, with the peak migration most likely depending on the species of Leishmania inoculated into the host.(AU)

Análises histológica e morfométrica do uso de membrana biossintética de celulose em trocleoplastia experimental de cães / Histological and morphometric analysis for the use of a biosynthetic cellulose membrane in experimental trochleopasty

O objetivo deste trabalho foi avaliar a aplicação de membrana biossintética de celulose, de fabricação nacional, após a realização da trocleoplastia experimental, com intuito de verificar se o uso desta poderia favorecer a migração de células com potencial condrogênico. Foram utilizados 12 cães adultos, de ambos os sexos, sadios e sem alterações no aparelho locomotor. Os animais foram submetidos ao procedimento de trocleoplastia em ambos os membros pélvicos, após tranquilização e anestesia epidural. Na trocleoplastia do membro esquerdo foi aplicada membrana biossintética à base de celulose (grupo tratado, GT), fixada à cartilagem por meio de pontos simples separados com Poliglactina 910 6-0; no membro direito, foi realizada apenas a trocleoplastia, constituindo o grupo controle (GC). Os animais foram subdivididos em quatro subgrupos de acordo com o período final de avaliação aos 15, 30, 60 e 90 dias do pós-operatório. Após artrotomia exploratória nos momentos pré-estabelecidos, foi realizada biópsia da região da trocleoplastia para avaliação histológica e morfométrica do tecido de reparação. Aos 30 e 60 dias do pós-operatório, notou-se a presença de maior número de células semelhantes a condrócitos nas lesões tratadas com celulose em relação ao membro contra-lateral, apesar do aspecto imaturo. Aos 90 dias, o tecido de reparação era do tipo fibrocartilaginoso maduro, não havendo diferenças entre os dois grupos. No GC houve aumento progressivo do número de células até o período final de avaliação. Por outro lado no grupo tratado verificou-se que, em relação ao período inicial (15 dias), houve aumento do número de células até os 60 dias, com subseqüente retorno aos valores iniciais aos 90 dias. Dos 15 aos 60 dias o número de células foi maior no GT em relação ao GC. Inicialmente, o tecido de reparação neoformado foi mais espesso no grupo tratado. Dessa forma, conclui-se que a membrana de celulose acelerou o processo de reparação tecidual...(AU)

The aim of this study was to evaluate the use of a locally made biosynthetic cellulose membrane after experimental trochleoplasty, in order to verify whether its use could support migration of chondrogenic cells. Twelve male and female adult healthy dogs and without claudication were used. All dogs were submitted to trochleoplasty in both pelvic limbs after sedation and epidural anesthesia. In the left hind limb, the biosynthetic cellulose membrane was fixed with simple suture using Polyglactin 910 6-0 after performing trochleoplasty (treated group); whereas in the right limb (control group) only trochleoplasty was performed. The dogs were subdivided into 4 subgroups for postoperative evaluation at 15, 30, 60 and 90 days post-surgery. Biopsy was performed after exploratory arthrotomy for histopathologic and morfometric evaluation. At 30 and 60 days post-surgery, more condrocyte-like cells of immature aspect were observed in lesions treated with the cellulose membrane. At 90 days post-surgery the reparative tissue was characterized as mature fibrocartilage-like tissue without difference between the groups. In the control group there was a progressive increase of the number of cells until the end of the evaluation period. Otherwise, when compared to the initial period (15 days), there was an increase in the number of cells until 60 days, followed by a return the initial values at 90 days in the treated group. In comparison to controls, the number of cells was greater in the treated group from 15 to 60 days. Initially, the neoformed repair tissue was thicker in the treated group. From the results of this study, it was concluded that the cellulose membrane shortened the initial tissue repair process in the trochleoplasty area, showing good integration of the neoformed tissue with the adjacent cartilage.(AU)