Using crystallographic water properties for the analysis and prediction of lectin-carbohydrate complex structures.

Understanding protein-ligand interactions is a fundamental question in basic biochemistry, and the role played by the solvent along this process is not yet fully understood. This fact is particularly relevant in lectins, proteins that mediate a large variety of biological processes through the recognition of specific carbohydrates. In the present work, we have thoroughly analyzed a nonredundant and well-curated set of lectin structures looking for a potential relationship between the structural water properties in the apo-structures and the corresponding protein-ligand complex structures. Our results show that solvent structure adjacent to the binding sites mimics the ligand oxygen structural framework in the resulting protein-ligand complex, allowing us to develop a predictive method using a Naive Bayes classifier. We also show how these properties can be used to improve docking predictions of lectin-carbohydrate complex structures in terms of both accuracy and precision, thus developing a solid strategy for the rational design of glycomimetic drugs. Overall our results not only contribute to the understanding of protein-ligand complexes, but also underscore the role of the water solvent in the ligand recognition process. Finally, we discuss our findings in the context of lectin specificity and ligand recognition properties.

Protein content of antivenoms and relationship with their immunochemical reactivity and neutralization assays.

CONTEXT: Therapy for snakebites relies on the application of antivenoms, which may be produced with different immunogenic mixtures of venom and possess different pharmaceutical characteristics. For these reasons, immunological cross-reactivity and heterologous neutralization were analyzed relative to the protein content of three antivenoms used in the Americas. METHODS: The antivenoms studied were composed of equine F(ab')2 fragments from animals immunized with Crotalinae venoms. The antivenoms were tested against venoms of seven pit viper species from Argentina, seven from Mexico, one from Costa Rica, and one from Colombia. RESULTS: Immunoblotting showed high cross-reactivity of all major protein bands with all the antivenoms tested. ELISA results also showed high cross-reactivity among the different venoms and antivenoms, and a high heterologous neutralization was observed. The results can be interpreted in different ways depending on whether the reactivity is considered in terms of the volume of antivenom used or by the amount of protein contained in this volume of antivenom. The antivenoms with high immunochemical reactivity and neutralizing capacity were those with higher protein content per vial; but when doses were adjusted by protein content, antivenoms of apparently lower neutralizing capacity and immunochemical reactivity showed at least similar potency and reactivity although volumetrically at higher doses. CONCLUSION: Protein content relative to neutralization potency of different products must be taken into account when antivenoms are compared, in addition to the volume required for therapeutic effect. These results show the importance of obtaining high-affinity and high-avidity antibodies to achieve good neutralization using low protein concentration and low-volume antivenoms.

Ros production by endogenously generated Protoporphyrin IX in murine leukemia cells.

Endogenous production of Protoporphyrin IX (PpIX) is successfully exploited for photodynamic therapy (PDT) on malignant cells, following 5-aminolevulinic acid (ALA) administration and light irradiation. This treatment kills cancer cells by damaging organelles and impairing metabolic pathways via cellular reactive oxygen species (ROS) generation. We studied the efficiency of PpIX synthetized from ALA on ROS generation, in the Vincristine resistant (LBR-V160), Doxorubicin resistant (LBR-D160) and sensitive (LBR-) murine leukemia cell lines. Cells were incubated 4 hr with 1 mM ALA and then irradiated during different times with fluorescent light. One hour later, production of ROS was analyzed by flow cytometry using different fluorescent probes: Hydroethidine (HE) for superoxide anion, 2',7' Dichlorodihydrofluorescein diacetate (DCFH-DA) for hydrogen peroxide; mitochondrial damage was examined with 3,3' Dihexyloxacarbocyanine iodide (DiOC6). We found that superoxide anion production in the three cell lines increased with irradiation time whereas no peroxide hydrogen was detected. Mitochondrial damage also increased in an irradiation time dependent manner, being higher in the Vincristine resistant line. Previous studies have demonstrated that apoptotic cell death increased with irradiation time, which is consistent with these results, indicating that ROS are critical in ALA-PDT efficiency to kill malignant cells.

Murine abortion is associated with enhanced hyaluronan expression and abnormal localization at the fetomaternal interface.

The remodelling of the endometrial architecture is fundamental to create a suitable environment for the establishment of pregnancy. During this process, substantial alterations in the composition of maternal extracellular matrix play an important role by providing a prosperous medium for implantation as well as modulating trophoblast invasion leading to the formation of a functional placental unit. Hyaluronan is a conspicuous component of the extracellular matrix, particularly in remodelling tissues undergoing regeneration and repair. During gestation, changes in HA deposition and distribution indicate that this molecule may participate in preparation of the endometrial stroma for reception and implantation of the embryo. However, little is known about the role of hyaluronan at the fetomaternal interface, specially regarding its influence in pregnancy outcome. In the present study we show increased decidual hyaluronan levels in spontaneous abortion compared with normal pregnancy mice on gestation day 7.5. Both in normal and pathologic pregnancies, high molecular size hyaluronan was found at the fetomaternal unit. However, hyaluronan metabolism (which results from the activity of hyaluronan synthases and hyaluronidases) seems to be altered in spontaneous abortion as shown by a decrease in Hyal-3 expression as well as by differences in hyaluronan molecular size spectrum. This alteration in hyaluronan metabolism in spontaneous abortion could explain its increased concentration observed in decidua and the abnormal distribution of hyaluronan around the embryo implantation crypt. Thus, increased decidual hyaluronan levels resulting from abnormal deposition and turn over may contribute to the pathogenesis of pregnancy failure.

CD44 and hyaluronic acid regulate in vivo iNOS expression and metalloproteinase activity in murine air-pouch inflammation.

OBJECTIVE: To evaluate the effects of anti-CD44 IM7.8.1 antibody, HMW-HA and LMW-HA on leukocyte migration and adhesion, and the induction of proinflammatory mediators, in mouse air-pouch inflammation induced by zymosan. METHODS: Leukocytes were obtained from zymosan-air pouches after the intra-pouch injection of anti-CD44 IM7.8.1, isotype control, HMW-HA, LMW-HA or PBS. TNF-alpha, IL-1beta and iNOS mRNA were estimated in leukocytes by semi-quantitative RT-PCR. Matrix metalloproteinases (MMPs) from exudates were evaluated by zymography and Western Blot. Adhesion and migration of leukocytes were evaluated in HA-coated plates and Boyden chambers respectively. RESULTS: IM7.8.1 decreased iNOS mRNA levels and the activity of both MMP-9 and MMP-2 eight h after injection into zymosan air pouch while IM7.8.1, HMW-HA and LMW-HA had no effect on IL1-beta or TNF-alpha mRNA levels. Leukocytes from air pouch adhered to and migrated in vitro against both HMW-HA and LMW-HA. LMW-HA increased the number of leukocytes in the air pouch and iNOS mRNA levels as compared to PBS injection. In contrast, HMW-HA decreased leukocyte count and reduced iNOS mRNA levels. Paradoxically, the activity of both MMP-9 and MMP-2 was increased by HMW-HA and decreased by LMW-HA. CONCLUSIONS: Both CD44 and HA can modulate leukocyte migration and induction of proinflammatory mediators in mouse zymosan air pouch inflammation. IM7.8.1 had consistent anti-inflammatory effects, reducing iNOS, MMP-9 and MMP-2. HMW-HA and LMW-HA were able to modulate both the induction of proinflammatory mediators and leukocyte count in the air pouch.

Immunobiological features of the galactoside lectin L-Lc isolated from the Argentine mistletoe Ligaria cuneifolia.

Ligaria cuneifolia has been used in Argentine folk medicine and is currently employed as substitute for the European mistletoe (Viscum album) as hypotensor agent. Extracts from V. album are widely used in cancer therapy and the antineoplasic effect is attributed to their cytostatic/cytotoxic and immunomodulatory actions. When studying immunomodulatory effects of L. cuneifolia extracts (Lc extracts), they inhibited proliferation of murine mitogen-activated lymphocytes, leukaemic lymphocytes (LB) and breast tumour cells (MMT). The aim of this work was to isolate and identify lectins from Lc extracts and investigate their immunobiological actions. A galactoside lectin (L-Lc) of 57 kDa was isolated. A polyclonal antiserum obtained against Lc extract recognised both L-Lc and MLI (V. album lectin), suggesting the possibility of shared epitopes. Treatment of LB tumour cells with L-Lc (0.01 and 0.1 microg/ml) produced up to 40.0+/-6.9% inhibition of cell growth, which seems partly mediated by apoptosis (apoptosis of L-Lc treated cells 58.4+/-10.3% versus non-treated cells 38.1+/-8.8%; P<0.05), analysed by acridine orange and ethidium bromide staining. Inhibitory effect on ConA stimulated splenocyte growth was non-significant, while a mitogenic effect was observed on normal murine splenocytes and MMT cells. L-Lc in non-cytotoxic concentrations (250 ng/ml) modified mRNA expression of IL-10 but neither that of TGF-beta nor of IL-2 produced by LB cells. In addition, 43.9+/-0.5% reduction in NO production by LPS-stimulated murine macrophages was found. Finally, survival rates of LB tumour-bearing mice treated or not with Lc extract or L-Lc failed to show significant differences.

Immunomodulating properties of Argentine plants with ethnomedicinal use.

Five Argentine medicinal plants selected according to folk traditional or ethnomedical use, references and primary pharmacological screening; were chosen to elucidate their immunomodulating properties. Dichloromethane, methanolic and aqueous extracts of the aerial parts of Achyrocline flaccida (A. flaccida), Eupatorium arnottianum (E. arnottianum) and Eupatorioum buniifolium (E. buniifolium), leaves of Lithraea molleoides (L. molleoides) and leaves and stems of Phyllanthus sellowianus (P. sellowianus) were analyzed to disclose their effects on murine normal and tumor cell growth as well as on complement hemolytic activity. Modulation of cell growth was evaluated by tritiated thymidine incorporation while inhibition of complement activity was measured on both classical and alternative complement pathways (CP and AP respectively). The results obtained show that most of the extracts exerted inhibitory effect on tumor as well as on mitogen activated normal spleen cell growth. On tumor cells, IC50 ranged between 1-75 microg/ml for most of the extracts with the exception of dichloromethane of L. molleoides and P. sellowianus which required concentrations higher than 100 microg/ml to produce the effect. On mitogenic activated splenocytes, IC50 ranged between < 1 to 85 microg/ml with the exception of methanolic extract of E. buniifolium or P. sellowianus which were not effective on ConA or LPS stimulated splenocytes respectively. Only E. buniifolium was active on murine normal splenocytes proliferation (IC50 0.5-1.5 microg/ml). Finally, one (7%) of 15 extracts showed inhibition of complement activity on CP and 6 extracts (40%) presented moderate activity on CP. The dichloromethane extract of E. arnottianum was the most active (IC50 5 microg/ml), although remarkable effect was also obtained with dichloromethane and methanolic extracts of P. sellowianus (IC50 11.2 and 17.3 microg/ml respectively). Besides, 2 extracts (13%), dichloromethane extract of E. arnottianum and aqueous extract of P. sellowianus, showed moderate inhibition on AP.

Correlation between decreased apoptosis and multidrug resistance (MDR) in murine leukemic T cell lines.

Cancer cells may frequently develop cross-resistance to structurally dissimilar chemotherapeutic agents. However, the molecular mechanisms for sensitivity and resistance of tumor cells towards chemotherapy are still partially understood. Antineoplasic drugs have been shown to induce apoptosis in chemosensitive leukemias and solid tumors. In this work, cross-resistance among vincristine (VCR), doxorubicin (DOX) and other antineoplasic agents commonly used in the treatment of leukemia such as etoposide (VP-16), methotrexate (MTX), cyclophosphamide (CTX), dexamethasone (DEX), cytarabine (Ara-C) and L-asparaginase on vincristine resistant (LBR-V160), doxorubicin resistant (LBR-D160) and sensitive (LBR-) murine leukemic T cell lines, was determined. The effect of antineoplasic agents was assayed by tritiated thymidine incorporation. Our results showed that VCR exhibited cross-resistance with DOX, VP-16, DEX and MTX, while DOX demonstrated cross-resistance with VCR, VP-16 and MTX. Ara-C failed to present cross-resistance with any cell line. Apoptosis induced by the above drugs on the same cell lines was analyzed by acridine orange and ethidium bromide staining, DNA hypoploidy (flow cytometry) and oligonucleosomal fragmentation of nuclear DNA showing that therapeutic concentrations of these chemotherapeutic agents induced apoptosis in the LBR- cell line. Our results demonstrated that, except for DEX, none of the drugs presenting cross-resistance were able to induce cell death on LBR-V 160 or LBR-D 160 cell lines.

Chromosome studies of murine T-cell lymphoid leukemia and derived cell lines.

Several cell lines were previously established from a spontaneous murine T-cell leukemia (LB). The aim of this study was to analyze the G- and C-banded karyotypes of the parental LB tumor cells and the derived cell lines. A sensitive cell line (LBL) from which two sublines originated, as well as Vincristine (LBR-V160) and Doxorubicin (LBR-D160) resistant cell lines, were used. Our results showed that LB cells had a pseudo-diploid karyotype with 40 acrocentric chromosomes in which trisomy of chromosome 14 was the most relevant alteration. The sensitive cell line showed this alteration in all metaphases studied; no changes in karyotypes were observed in either subline, despite their dissimilar morphology and growth patterns. In contrast, both resistant lines displayed a more heterogeneous karyotype with no common markers, except for the finding that chromosome 5 was involved in a trisomy in LBR-V160 and in a translocation with chromosome 12 in LBR-D160. Taking into account that the mdr genes are located in chromosome 5, these results suggest a possible association between such alterations and the acquisition of drug resistance.

Zymosan modulates CD44 isoform expression in a murine model of inflammation resembling rheumatoid arthritis synovitis.

OBJECTIVE: To study local inflammation induced by zymosan in the murine air pouch, considered a model of synovial-like tissue inflammation, we investigated the time-course synthesis of CD44 and tumor necrosis factor-alpha (TNF-alpha) mRNA and established a relationship with leukocyte migration into the air pouch and CD44 expression on the leukocyte membrane. METHODS: Leukocytes from the air pouch exudate were collected and counted at 1, 4, 12, 24, 48, and 72 h after zymosan or saline injection. CD44 and TNF-alpha mRNA were studied by RT-PCR. CD44 variable exon analysis was assessed by Southern blot and CD44 membrane expression by flow cytometry. RESULTS: Leukocyte accumulation after zymosan injection was significantly higher than in saline injected controls. CD44 standard and variable isoforms including at least variable exons v6 and v9 were highly expressed in leukocytes from the zymosan air pouch exudate. In contrast, only the CD44 mRNA standard isoform was present in leukocytes from saline air pouch. Maximal TNF-alpha mRNA level was observed at 48 h after zymosan injection, whereas CD44 mRNA was constantly expressed throughout the whole term of the experiment, although variations in leukocyte count and relative formula were observed. CONCLUSION: Expression of CD44 variable isoform in leukocytes was specifically induced by zymosan, since none was detected in saline controls. TNF-alpha mRNA expression and leukocyte count at every time point served as markers for local inflammation. The presence of variable isoforms, including at least exons v6 and v9, consistently expressed throughout the assay suggests that they could play a role in this arthritis-like inflammation induced under zymosan stimulus.

Expression of CD44 splice variants in spontaneous murine tumors.

CD44 is a widely distributed set of cell surface glycoproteins expressed in several types of cells and tissues, implicated in cell-cell and cell-substrate interactions. This molecule plays a major role in cell differentiation, development and activation and has also been described as a potential marker of malignancy and metastasis. In the present study we investigated by RT-PCR followed by exon specific amplification the expression of CD44 splice variants in four different murine tumors as well as in the invaded organs in order to correlate the expression of CD44 variants with potential tumor invasiveness and their implications for growth. Our data showed deregulation in the expression of CD44 isoforms but no discernible correlation in isoform expression pattern. However, in all tumors studied isoforms presented by the primary tumor were detected in the invaded organs before metastasis could be demonstrated by histopathological analysis.

Induction of apoptosis in murine lymphoma cells by cyclosporin A.

The aim of this study was to investigate if CsA could induce apoptosis in the murine T-lymphoma cell line LBC, whose growth is inhibited by this immunosuppressive drug. CsA induced programmed cell death in LBC cells with typical features of apoptosis demonstrated by exposure of phosphatidyl serine residues on the cell membrane, the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Apoptosis was evident within 12 h after CsA incubation, with a maximal effect at 48 h, in a time and dose-dependent fashion. In addition, the role of apoptosis inhibitors (Bcl-2 and Bcl-x) and the apoptosis inducer (Bax) in CsA induced-apoptosis was evaluated. The expression of Bcl-2 and Bax proteins were high in LBC cells and following CsA treatment the expression of these proteins as well as Bcl-XL decreased. In this work we demonstrated that cell growth inhibition following CsA treatment in LBC was paralleled by the induction of apoptosis thus providing an interesting animal model to identify the mechanism participating in the regulation of apoptotic genes by CsA in T-cell neoplasms and to assess preclinical in vivo trials of T-cell lymphoma-related disorders.

Modulator activity of PSC 833 and cyclosporin-A in vincristine and doxorubicin-selected multidrug resistant murine leukemic cells.

Multidrug resistance (MDR) lines from a murine T-cell leukemia were selected in increasing vincristine (VCR) or doxorubicin (DOX) concentrations. Daunorubicin (DNR) efflux was evidenced after 25 additional passages with constant 160 ng ml(-1) of either VCR or DOX, an effect that was inhibited by verapamil, cyclosporin-A (CsA) and PSC 833. The expression of Pgp was not evidenced in the resistant cell lines using anti-human Pgp antibodies. Cell proliferation assay showed that cell lines resistant to VCR (LBR-V160) or DOX (LBR-D160) required higher doses of either drug to produce GI50 compared with control cell line obtained after culture in the absence of VCR or DOX. When resistant cell lines were maintained during 60 days in the absence of either VCR or DOX, MDR phenotype reversal was obtained in LBR-D160 while LBR-V160 remained resistant to the drug, as shown by cell proliferation assays and by drug efflux pump functionality. When VCR or DOX were used together with either CsA or PSC 833, the latter was more effective to produce reversal of resistance than the former, whereas CsA presented greater cytotoxic effect than PSC 833 for sensitive and resistant cells. Cross-resistance was found between VCR, DOX and other antineoplasic agents on murine leukemic cell line. VCR was more effective to induce MDR since the resistant cell lines were more stable to the MDR phenotype.

Splice variant expression of CD44 in patients with breast and ovarian cancer.

CD44 is an adhesion molecule involved in many biological functions and has been described to play a role in tumor progression as well as in promotion of metastasis. It has also been suggested that expression of certain CD44 isoforms could be useful for breast and ovarian cancer screening, detection or staging. However, many other reports document no correlation between CD44 isoform expression and tumor malignancy. In light of such contradictory findings, we evaluated by exon-specific RT-PCR whether the expression of CD44 isoforms in breast and ovarian tumors correlated with any of the diagnostic criteria used to assess these diseases. We found a deregulation in the CD44 expression pattern in malignant tumors of both type of cancer compared with the one in benign tumors or normal tissue. However, we could not find a clear correlation between this deregulation or a given CD44 isoform and any diagnostic criteria evaluated, such as age, clinical data, tumor size, hormone receptor status, histological grade or aggressiveness.

Some toxic and enzymatic activities of Bothrops ammodytoides (yarará ñata) venom.

Bothrops ammodytoides, the smallest representative of this genus, is found only in Argentina. Venom was extracted from thirty adult specimens (35-70 cm in length, 90-300 g in weight) captured in the Province of Buenos Aires and kept in captivity. Venom yield was 3-30 mg. SDS-PAGE showed strong bands at 14.0; 23-25; 45; 54 and 63 kDa and weak bands at 17.0; 30.0; 40.0 and 85.0 kDa. Toxic activities were: LD50 (intravenous, mice) 0.5+/-0.2 microg/g; minimal procoagulant dose on human plasma (MPD-P) 35+/-2 mg/l; and minimal defibrinogenating dose (MDD, mice) 6-12 microg. Hemorrhagic and/or necrotic activities appear to play a major role in lethality; minimal hemorrhagic dose (MHD, mice) is 10+/-2 microg/g and minimal necrotizing dose (MND, mice) is 38+/-5 microg. The LD50, MPD-P and MND are among the lowest in venoms from Bothrops species found in Argentina. B. ammodytoides venom exhibited high proteolytic and phospholipase A2 activities. Most of the B. ammodytoides venom components cross-react with Bivalent Bothropic antivenom (Instituto Nacional de Producción de Biológicos ANLIS Dr. G. Malbrin, against B. alternatus and B. neuwiedii venoms). One ml of antivenom neutralizes 1.2 mg of B. ammodytoides venom.

[Differential binding of hyaluronic acid in 2 CD44+ sublines. Relationship with tumor infiltration]. / Unión diferencial de ácido hialurónico en dos sublíneas celulares CD44+. Posible implicancia en la infiltración tumoral.

We have established and characterized a cell line (LBL) from a spontaneous murine T lymphoma LB. Histopathological analysis has demonstrated LB primary tumor infiltration in spleen, lymph nodes, liver, thymus, bone marrow and lung. However LBL cells infiltrated all these organs except lung. Two sublines with different growth behavior were derived from LBL cell line. One of them grew in suspension as clusters (LBLc) while the other one grew as adherent monolayers (LBLa). Growth rate, response to mitogenic stimuli and apoptosis induction were different among the parental cell line and the derived sublines. CD44 was expressed constitutively in LBL and LBLa cells. In contrast LBLc cells only expressed similar levels of this molecule when stimulated with PMA. LBLa cells showed hyaluronic acid (HA) binding properties, while LBL and LBLc cells were not able to bind HA even when activated with PMA. We postulate that differences in HA binding could be related with different infiltration behaviors.

[Isoforms modulation of CD44 adhesion molecule in a murine model of ischemia and intestinal reperfusion]. / Modulación de isoformas de la molécula de adhesión CD44 en un modelo murino de isquemia y reperfusión intestinal.

Gut ischemia-reperfusion (G-IR) induces a systemic inflammatory response, in which leukocyte contribution to this injury in distant organs is important. ICAM-1 as well as CD11/CD18 have been involved in leukocyte infiltration in liver and lungs. CD44 adhesion molecule plays an essential role in other inflammatory processes such as rheumatoid arthritis and allergic contact dermatitis, however its implication in G-IR has not been described. In order to establish a possible role of CD44 in the development of systemic inflammation by G-IR, we have studied CD44 mRNA expression by RT-PCR in a murine model of gut ischemia reperfusion. Animals subjected to G-IR showed an increased number of CD44 variable isoforms expressed in liver and spleen compared to non-treated animals or animals subjected to laparotomy. This finding indicates that G-IR specifically induces the expression of different CD44 variable isoforms. Liver CD44 upregulation in animals subjected to G-IR suggests a contribution of this molecule to lymphocyte activation and migration to this injured organ. Moreover, increased isoform expression in spleen may be induced by the proinflammatory environment resulting from a systemic depuration activity.