Cold Agglutinin Disease in a Patient of Pulmonary Tuberculosis.

We report a case of secondary cold agglutinin disease (CAD) due to pulmonary tuberculosis in a 68-year male patient who presented with acrocyanosis involving both upper limbs and greater portion of lower limbs. Direct Coombs' test was positive with mild anemia and the cold agglutinin titer was high. Sputum showed numerous acid fast bacilli per high power field. The patient was given standard anti-tuberculous treatment (ATT) and his symptoms gradually improved. After nine months of ATT, his haemoglobin improved, acrocyanosis resolved completely and cold agglutinin titer decreased in level.

Anti-leptospirosis agglutinins in Brazilian capybaras (hydrochoerus hydrochaeris)

The interest in commercial use of wild animals is increasing, especially regarding raising of capybaras. Although this wild species is potentially lucrative for the production of meat, oil and leather, it is suggested as a probable reservoir of leptospires. Methods Due to the economic importance of this species and the lack of studies concerning leptospirosis, the presence of anti-leptospirosis agglutinins was assayed in 55 serum samples of capybaras (Hydrochoerus hydrochaeris) from commercial and experimental breeding flocks located in São Paulo state, Paraná state, and Rio Grande do Sul state, Brazil. Samples were obtained through cephalic or femoral venipunction (5 to 10 mL). Microscopic agglutination test was used according to the Brazilian Health Ministry considering as cut-off titer of 100. Results Out of the 55 samples analyzed, 23 (41.82 %) tested positive. The most prevalent serovar was Icterohaemorrhagiae (56.52 %) in 13 samples, followed by Copenhageni in nine samples (39.13 %), Pomona in four samples (17.39 %), Djasiman and Castellonis in three samples each (13.04 %), Grippotyphosa, Hardjo, Canicola, and Cynopteri in two samples each (8.7 %), and Andamana and Bratislava in one sample each (4.34 %). Conclusions These results suggest the evidence of exposure toLeptospira spp. and the need of new studies to evaluate a higher number of capybaras from different regions to better understand the importance of leptospirosis infection in these animals and verify the zoonotic role of this species as a possible source of infection to humans and other animals.

Anti-leptospirosis agglutinins in Brazilian capybaras (hydrochoerus hydrochaeris)

Abstract Background The interest in commercial use of wild animals is increasing, especially regarding raising of capybaras. Although this wild species is potentially lucrative for the production of meat, oil and leather, it is suggested as a probable reservoir of leptospires. Methods Due to the economic importance of this species and the lack of studies concerning leptospirosis, the presence of anti-leptospirosis agglutinins was assayed in 55 serum samples of capybaras (Hydrochoerus hydrochaeris) from commercial and experimental breeding flocks located in São Paulo state, Paraná state, and Rio Grande do Sul state, Brazil. Samples were obtained through cephalic or femoral venipunction (5 to 10 mL). Microscopic agglutination test was used according to the Brazilian Health Ministry considering as cut-off titer of 100. Results Out of the 55 samples analyzed, 23 (41.82 %) tested positive. The most prevalent serovar was Icterohaemorrhagiae (56.52 %) in 13 samples, followed by Copenhageni in nine samples (39.13 %), Pomona in four samples (17.39 %), Djasiman and Castellonis in three samples each (13.04 %), Grippotyphosa, Hardjo, Canicola, and Cynopteri in two samples each (8.7 %), and Andamana and Bratislava in one sample each (4.34 %). Conclusions These results suggest the evidence of exposure toLeptospira spp. and the need of new studies to evaluate a higher number of capybaras from different regions to better understand the importance of leptospirosis infection in these animals and verify the zoonotic role of this species as a possible source of infection to humans and other animals.

Síndrome de aglutininas frías y púrpura trombocitopénica autoinmune: Un caso inusual de síndrome de Evans / Cold agglutinins syndrome and autoimmune thrombocytopenic purpura. An unusual case of Evans syndrome

El síndrome de Evans es un trastorno poco frecuente en el que se observan trombocitopenia y anemia, ambas de etiología autoinmune; las que pueden ocurrir de manera simultánea o sucesiva. Se presenta un caso poco usual de anemia hemolítica autoinmune por anticuerpos fríos asociada a púrpura trombocitopénica autoinmune. Paciente femenina de 22 años de edad con diagnóstico de púrpura trombocitopénica autoinmune, después de 7 años de evolución y un año en remisión, presentó una anemia hemolítica autoinmune por anticuerpos fríos, refractaria al tratamiento con esteroides y alcaloides de la Vinca, que requirió transfusiones de concentrado de eritrocitos y logró la remisión con la administración de anticuerpo monoclonal anti CD 20. Los restantes estudios de autoinmunidad fueron negativos. Actualmente se mantiene asintomática y sin tratamiento inmunosupresor(AU)

Evans syndrome is a rare disorder in which thrombocytopenia and anemia are observed, both of autoimmune aetiology, which may occur simultaneously or successively. A rare case of cold autoimmune hemolytic anemia associated to autoimmune thrombocytopenic purpura is presented. A 22-year-old female patient with diagnosis of autoimmune thrombocytopenic purpura, after 7 years of evolution and one year in remission, has a cold autoimmune hemolytic anemia, refractory to steroid treatment and vinca alkaloids, which requires transfusions of packed erythrocytes and achieves remission with anti CD 20 monoclonal antibody. The remaining studies of autoimmunity are negative. Currently the patient is asymptomatic and without immunosuppressive therapy(AU)

Structural basis for carbohydrate binding properties of a plant chitinase-like agglutinin with conserved catalytic machinery.

A new chitinase-like agglutinin, RobpsCRA, related to family GH18 chitinases, has previously been identified in black locust (Robinia pseudoacacia) bark. The crystal structure of RobpsCRA at 1.85Å resolution reveals unusual molecular determinants responsible for the lack of its ancestral chitinase activity. Unlike other chitinase-like proteins, which lack chitinase catalytic residues, RobpsCRA has conserved its catalytic machinery. However, concerted rearrangements of loop regions coupled to non-conservative substitutions of aromatic residues central to the chitin-binding groove explain the lack of hydrolytic activity against chitin and the switch toward recognition of high-mannose type N-glycans. Identification of close homologs in flowering plants with conservation of sequence motifs associated to the structural adaptations seen in RobpsCRA defines an emerging class of agglutinins, as emphasized by a phylogenetic analysis, that are likely to share a similar carbohydrate binding specificity for high-mannose type N-glycans. This study illustrates the recent evolution and molecular adaptation of a versatile TIM-barrel scaffold within the ancestral GH18 family.

Identification of the hydroxyapatite-binding domain of salivary agglutinin.

The salivary agglutinin glycoprotein (SAG) is present in saliva but is also part of the salivary pellicle, playing a seemingly paradoxical role with regard to bacterial homeostasis. On the one hand, SAG aggregates bacteria in solution, thereby preventing bacterial colonization. On the other hand, when bound to the tooth surface, SAG facilitates bacterial colonization and microbial growth. The protein part of SAG is predominantly composed of conserved scavenger receptor cysteine-rich (SRCR) domains. Previously it was found that bacterial binding and aggregation is mediated via a single peptide loop, designated SRCRP2 (P2), within the SRCR domains of SAG. The current data suggest that the SRCR domains also harbour a hydroxyapatite (HA)-binding moiety, SRCRP3 (P3). The observation that P2 and P3 individually play unique roles in the function of SAGs contributes to our understanding of the dual role of SAGs in bacterial binding. Inspired by the bacterial-modulating capacity of SAGs, we created a P3-polyethylene glycol (PEG) conjugate. It was found that a P3 coating resulted in an increased antifouling activity of 20% compared with the uncoated surface in vitro. An additional PEG moiety resulted in an antifouling activity of up to 40% and 30% for Streptococcus mutans and Staphylococcus epidermidis, respectively.

Modifications influencing Widal test reactivity in a novel microplate assay.

Reliability of the Widal tube agglutination test has been the subject of many controversies over the years. This study was performed to assess the effect of certain modifications on the performance of Widal test in a novel microplate assay. Sera from 37 patients (21 males; 16 females) (mean age 28 +/- 7 years) were tested in the Immunology Unit at King Khalid University Hospital, Riyadh. Among them were 26 patients with suspected typhoid fever and 11 had bacteriologically confirmed diagnosis of Salmonella infection. The modifications included either the use of 0.5% bovine serum albumin (BSA), absorption of sera with sheep red blood cells (SRBC) or heat inactivation of sera. Compared with Widal tube agglutination test, microplate assay with SRBC absorption of the sera from patients with suspected typhoid fever was not only associated with enhancement of detection titers for both H (p < or = 0.001) and O (p < or = 0.005) Salmonella agglutinins but also the percentage of reactivity. The presence of BSA augmented detection titers for Salmonella H agglutinins (p < or = 0.02) only. Heat inactivation of sera however was found to be associated with reduction in the detectable titers for both H (p < or = 0.03) and O (p < or = 0.01) agglutinins. Increased titers of Salmonella agglutinins were also evident in 11 patients with confirmed diagnosis of Salmonella infection. The novel microplate agglutination assay using the SRBC absorption was associated with enhancement in Widal test reactivity and appears to be a useful alternative for the diagnosis of Salmonella infection.

Rat liver carbohydrate alterations in streptozotocin-induced diabetic rats

Diabetes mellitus (DM) currently belongs to the most widespread human pathologies, affecting about 4% of the world adult population. Despite the pivotal role of the liver in the development of metabolic disorders, the influence of DM on hepatic glycoconjugates remains obscure. The aim of the present investigation was to use a set of lectins with different carbohydrate affinities to investigate impairment in rat liver glycoconjugates influenced by streptozotocin-induced diabetes mellitus. The lectin panel included 7 conventional lectins - Con A, SNA, RCA, WGA, PNA, SBA, and HPA, supplemented with the original fucose-specific lectin preparation from Laburnum anagyroides bark (LABA). Tissue samples were fixed in 4% neutral formalin, embedded in paraffin, and subjected to lectin-peroxidase-diaminobenzidine staining. In control rats a strong reactivity against Con A, LABA, SBA and SNA with cytoplasmic granularities of hepatocytes was detected, while RCA, WGA and HPA showed a strong reactivity with vascular endothelium, and WGA and HPA with bile capillaries. Experimental diabetes was associated with a redistribution of Con A and LABA receptor sites from centrolobular hepatocytes to hepatocytes with peripheral localization. Among the most remarkable observations was DMinduced exposure of lectin reactivity with hepatocyte and endothelial cell nuclei. The endothelial lining of sinusoidal hemocapillaries, of central veins, and portal tract vessels also displayed a significant and differential rearrangement of carbohydrate determinants when influenced by DM. Diabetes-induced activation of Kupffer cells was accompanied by the expression of SNA, PNA and SBA receptor sites within the cytoplasm of these cells, which was lectin-negative in control specimens. The results reported provide a new insight into the pathogenesis of DM-induced impairment of hepatic carbohydrates, and demonstrate the applicability of the original fucose-specific lectin preparation to experimental histopathology (AU)

No disponible

Yersinia pestis autoagglutination factor is a component of the type six secretion system.

Autoagglutination (AA) is a protective phenotypic trait facilitating survival of bacteria in hostile environments and in the host during infection. Autoagglutination factors (AFs) that possess self-associating ability are currently characterized in many Gram-negative bacteria, but Yersinia pestis AFs are still a matter of debate. Previously, we have shown that AF of Hms(-) strain Y. pestis EV76 is a complex of the 17,485-kDa protein and a low-molecular-weight component with siderophore activity. Here, we identified the protein moiety of AF and examined its role in AA of Hms(+) and Hms(-)Y. pestis strains. Using MALDI-TOF MS of trypsin-hydrolyzed AF, we unambiguously identified the protein as YPO0502, which belongs to a family of Hcp-proteins forming pilus-like structures of the type six secretion system (T6SS). To address the role of YPO0502 in AA, we cloned ypo0502 in E. coli, overexpressed it in Y. pestis and constructed its knock-out mutant in Y. pestis. However, all these approaches failed: YPO0502 was not secreted in E. coli, formed inclusion bodies when overexpressed in Y. pestis, and could probably be compensated by other Hcp-like proteins in Y. pestis. In contrast, downregulation of ypo0502 expression by its antisense RNA supported the contribution of YPO0502 in AA of Hms(+) and Hms(-)Y. pestis strains. The results of the present study indicate that the Hcp-like component of T6SS encoded by ypo502 is involved in Y. pestis AA and suggest that at least one (ypo0499-0516) of the 6 T6SS clusters of Y. pestis is involved in bacterial interaction.

Trasplante renal de vivo ABO incompatible / No disponible

No disponible

Trasplante renal ABO incompatible: de un sueño a una realidad. Experiencia del Hospital Clínic de Barcelona / ABO incompatible living donor kidney transplantation: a dream come true. Experience of Hospital Clínic of Barcelona

Introducción: En los últimos años se ha mantenido estable el número de pacientes en lista de espera para un trasplante renal. El trasplante renal de donante vivo representa actualmente una vía para aumentar el pool de donantes, pero hay un grupo de pacientes que presentan incompatibilidad de grupo sanguíneo ABO, lo que contraindicaba hasta ahora que pudiera llevarse a cabo el trasplante. Nuestro objetivo consiste en describir nuestra experiencia con el programa de trasplante renal de donante vivo con incompatibilidad de grupo ABO. Material y métodos: Se trata de un estudio de retrospectivo-descriptivo de los primeros 11 pacientes sometidos a trasplante renal de donante vivo ABO incompatible en el Hospital Clínic de Barcelona desde octubre de 2006 a enero de 2009. Se utilizó un protocolo de acondicionamiento basado en inmunoadsorción específica (con número sesiones necesarias hasta conseguir títulos de isoaglutininas aceptables pretrasplante), inmunoglobulina policlonal inespecífica y anticuerpo monoclonal anti-CD20, seguido del tratamiento inmunosupresor adaptado a cada receptor. Se determinaron títulos de isoaglutininas antes del tratamiento de acondicionamiento, pretrasplante y postrasplante durante las primeras 2 semanas. La valoración inmunológica, médica y quirúrgica fue la habitual en el programa de trasplante renal de donante vivo. Resultados: La edad media de los donantes y receptores fue de 47,8 ± 12,4 y 44,4 ± 14,1 años, respectivamente. Un 90,1% de los donantes fue mujer y un 72,7% de los receptores, hombres. El tiempo de seguimiento medio fue de 10,2 ± 10,2 meses. Hermanos y esposos fueron las relaciones más frecuentes (n = 4, 36,4%, respectivamente), al igual que la causa de nefropatía fueron la glomerulopatía, poliquistosis y el síndrome de Alport (n = 2, 18,2% para cada enfermedad renal primaria). Todos los pacientes adquirieron un título de isoaglutininas correctos pretrasplante (<8) y requirieron 5,54 ± 2,6 sesiones de inmunoadsorción pretrasplante y 2,82 sesiones postrasplante. Un paciente no requirió ninguna sesión de inmunoadsorción (única con incompatibilidad anti-B) y otro requirió recambios plasmáticos, en vez de inmunoadsorciones, por tratarse de un potencial receptor hipersensibilizado con crossmatch por citometría de flujo positivo. Los títulos de isoaglutininas postrasplante se mantuvieron a títulos bajos. Dos pacientes presentaron un episodio de rechazo agudo celular (Banff IA e IB), con buena respuesta al tratamiento. La supervivencia de paciente y del injerto fue de un 90,9% en el primer año y se mantuvo estable a lo largo del seguimiento. Únicamente se registró una pérdida del injerto por fallecimiento en relación con una complicación hemorrágica en las primeras 72 horas sin relación con la incompatibilidad de grupo ABO. La función de injerto renal al año es excelente, con valores de creatinina sérica de 1,3 ± 0,8 mg/dl, con aclaramiento de creatinina ajustado a superficie corporal 62,6 ml/min/1,73 m2 y proteinuria de 244,9 mg/orina de 24 horas. Conclusiones: El trasplante renal de donante vivo con incompatibilidad de grupo sanguíneo representa una alternativa eficaz y segura en determinados pacientes en lista de espera de trasplante renal, obteniendo resultados excelentes de supervivencia de paciente e injerto y con una buena función de injerto renal (AU)

Introduction: During the last years the number of patients on waiting list for kidney transplantation has been stable. Living donor kidney transplantation is nowadays a chance to increase the pool of donors. However, there are a group of patients with ABO incompatibility, making impossible the transplant until now. The aim of the present study is to describe the experience of Hospital Clinic Barcelona on ABO incompatible living transplantation. Material and methods: A retrospective-descriptive study was made based on 11 living donor kidney recipients with ABO incompatibility in Hospital Clinic of Barcelona from October’06 to January’09. Selective blood group, antibody removal with specific immunoadsortion, immunoglobulin and anti-CD20 antibody were made until the immunoglobulin (IgG) and isoaglutinine (IgM) antibody titters were 1/8 or lower. Immunosuppressive protocol was adjusted to particular recipient characteristics. Isoaglutinine titters were set before, during and post desensitization treatment and two weeks after transplant. Immunological, medical and surgical evaluation was the standard in living donor kidney transplant program. Results: Medium age of donors and recipients were 47.8 ±12.4 and 44.4 ± 14.1 years, respectively. 90% of donors were females and 73% of recipients males. Follow-up time was 10.2 ±10.2 months. Siblings and spouses were the most frequent relation (n = 4, 36.4%, respectively). Chronic glomerulonephritis, adult polycystic kidney disease and Alport syndrome, the most frequent cause of end-stage renal disease. All the patients acquire appropriate isoaglutinine titters pre transplant (<1/8), requiring 5.54 ± 2.6 immunoadsorption sessions pretransplant and 2.82 postransplant. One patient didn´t need any immunoadsorption session (incompatibility blood group B) and another patient plasma exchange instead of immunoadsorption for being hipersensitized with positive flow cytometry crossmath. Postransplant isoaglutinine titters remained low. Two patients had cellular acute rejection episode (type IA and IB of Banff classification) with good response to corticosteroid treatment. Patient and graft survival were 91% at first year and remain stable during the follow-up. A graft lost by death of patient in relation to haemorrhagic shock developed within the first 72 hours after transplantation. Renal graft function at first year was excellent with serum creatinina of 1.3 ± 0.8 mg/dl, creatinine clearance of 62.6 ml/min/1.73 m2 and proteinuria of 244.9 mg/U 24 h. Conclusion: ABO incompatible living donor kidney transplantation represent an effective and safe alternative in certain patients on waiting list for renal transplant, obtaining excellent results in patient and graft survival, with good renal graft function (AU)

The role of the elution in antibody investigations.

BACKGROUND: The direct antiglobulin test (DAT) commonly detects immunoglobulin G (IgG) molecules or complement fragments on the red blood cell (RBC) surface. If IgG antibodies are present then elution procedures can be performed to identify the specificity of these antibodies. Our reference laboratory performs elutions on the RBCs of those patients who have received cellular blood products in the past 30 days and have either a newly identified positive DAT with anti-IgG or the agglutination strength is increased over a previous DAT and if ordered by a clinician regardless of transfusion history. This study questioned how frequently elutions contributed novel serologic information under our reference laboratory's current policy or whether elutions should be performed in more selective serologic conditions. STUDY DESIGN AND METHODS: Recipients whose RBCs underwent eluate testing were identified from the blood bank's database and information about the antecedent DAT and antibody detection test and eluate was recorded. RESULTS: In total 648 eluates were evaluated and 82 of 648 (12.7%) revealed a novel antibody not present in the serum (an informative eluate). In 2 of 82 informative eluates non-anti-A/B alloantibodies that were not present in the serum were detected: one example each of anti-D and anti-E. Both were associated with a microscopically positive antecedent DAT. The rate of an informative eluate was higher when the antibody detection test was negative. CONCLUSION: The strength of the DAT does not indicate the likelihood of an informative eluate. Performing an eluate when the antibody detection test is positive has limited value.

Serum antileptospiral agglutinins in freshwater turtles from Southern Brazil / Aglutininas séricas anti-leptospira em tartarugas de água doce no sul do Brasil

In this study, we observed the presence of antileptospiral agglutinins in freshwater turtles of two urban lakes of Pelotas, Southern Brazil. Forty animals (29 Trachemys dorbigny and 11 Phrynops hilarii) were captured and studied. Attempts to isolate leptospires from blood and urine samples were unsuccessful. Serum samples (titer > 100) reactive to pathogenic strains were observed in 11 animals. These data encourage surveys of pet turtles to evaluate the risk of transmission of pathogenic leptospires to humans.

Neste estudo, observamos a presença de aglutininas anti-Leptospira em tartarugas de água doce de dois lagos urbanos de Pelotas, Sul do Brasil. Quarenta animais (29 Trachemys dorbigny e 11 Phrynops hilarii) foram capturados e estudados. Esforços para isolar leptospiras do sangue e urina não foram bem sucedidos. Amostras de soro positivas (títulos > 100), reativas para cepas patogênicas, foram observadas em 11 animais. Estes dados encorajam inquéritos para avaliação de tartarugas como potenciais transmissoras de leptospiras patogênicas para humanos.

Capillary zone electrophoresis method development for the analysis of Hippeastrum hybrid agglutinin samples.

A capillary zone electrophoresis (CZE) method was developed aiming the analysis of Hippeastrum hybrid agglutinin (HHA) samples. HHA is presently being tested as a vaginal microbicide to prevent HIV transmission. It acts by direct binding to mannose residues that are abundantly present on the HIV gp120 envelope and so interrupts the virus entry process. The final CZE method employs 50mM sodium tetraborate (pH 9.9) as background electrolyte. In this condition, a cluster of about 30 isoform peaks is obtained, with very repeatable patterns. RSDs in the order of 0.2% for the migration time and detection sensitivity in the order of 70microgml(-1) were achieved.

Freqüência de aglutininas anti-Leptospira interrogans em eqüídeos, em Minas Gerais, 2003 a 2004 / Frequency of anti-Leptospira interrogans agglutinins in equidae in Minas Gerais, Brazil, from 2003 to 2004

The goal of this research was to find the frequency and spatial distribution of infection caused by Leptospira interrogans in equidae in Minas Gerais State from September 2003 to March 2004. Samples of blood serum (6,475) were analyzed by microscopic agglutination test. From the total, 381 samples were positive (5.9 percent), with title equal or superior to 1:200 for one or more serovars of leptospira. The most frequent serovars were Hardjo (Norma), Pomona, Bratislava, and Batavie. The higher frequency of equidae reagents were recorded at the North and Northeast region of Minas Gerais, followed by Triângulo Mineiro and Alto Paranaíba, then Central, West, Metropolitan area of Belo Horizonte, and South/Southwest.

Evaluation of different serological techniques in laboratory diagnosis of brucellosis.

Diagnosis of brucellosis is vital for early institution of proper therapy as untreated cases may progress to chronic stage. Though the demonstration of the causative agent in blood is considered as the most conclusive test in the diagnosis of brucellosis, isolation of brucella organism by blood culture is relatively low. Hence a number of sensitive and rapid serological tests have been introduced for the diagnosis of brucellosis. In the present study, an attempt was made to compare the efficacies of existing serological tests such as agglutination reaction with newer rapid tests which help in the detection of either specific antigen or antibody. The study included specimens from 80 patients clinically suspected to be suffering from brucellosis and 20 apparently healthy controls. All serum samples were subjected for evidence of brucellosis by five serological tests viz, standard tube agglutination test, 2-mercaptoethanol test, modified antiglobulin test, counter immuno-electrophoresis and passive haemagglutination test for antibody detection and two serological tests viz, counter immunoelectrophoresis and latex agglutination test for antigen detection. Eighty blood samples were processed for microbiological evidence of brucellosis and yielded only 8 isolates of Brucella melitensis of biotype 1. By standard tube agglutination test, 25 sera showed titre of brucella agglutinins equal to more than the diagnostic titre (i.e., more than or equal to 160 IU). Counter immuno-electrophoresis test and latex agglutination showed presence of antigen in 3 and 4 blood culture negative cases respectively.

Prevalence of Salmonella antibodies among goats slaughtered for chevon in Bareilly (Northern India).

We screened serum samples of 1024 goats slaughtered for chevon in Bareilly in Northern India for Salmonella antibodies with indirect ELISA, MAT-H (microagglutination test using flagellar antigens e, n, x and 1, 5) and MAT-O (microagglutination test using somatic antigens 4, 12 and 3, 10, 15). Salmonella antibodies were detected in 48, 8 and 40%, goats using Salmonella-cytotoxi-I ELISA, MAT 'H' and MAT 'O', respectively. After adjusting for test accuracy, the seroprevalence were highest for Salmonella-cytotoxi-I ELISA (46%) followed by agglutinins against 'O' 3, 10, 15 (15%) and negligible for other agglutinins. With all 5 tests, prevalence of Salmonella antibodies was significantly higher in females than in males. No significant difference was evident in prevalence of Salmonella antibodies to different antigens in different age groups of male goats except for e, n, x agglutinins that were significantly more prevalent in young adult (<6-18 months) males than in adult (>18 months of age) or young (< or =6 months of age) goats. On the other hand, in females, prevalence of Salmonella-cytotoxin-I antibodies and e, n, x agglutinins differed significantly among three age groups, being the most prevalent in adult goats. As expected, the results of different tests had little or no correlation because the different tests targeted antibodies to different antigens.

Characterization of vascular-specific RSs1 and rolC promoters for their utilization in engineering plants to develop resistance against hemipteran insect pests.

Rice sucrose synthase1, RSs1 (isolated from rice) and rolC (isolated from Agrobacterium rhizogenes) promoters were evaluated by binding analyses of their respective cis-elements with host nuclear transcription factors. The expression profile of an insecticidal protein driven by these promoters in transgenic plants was monitored. Motif-search analysis with available phloem-specific promoter sequences revealed the presence of two BoxII elements in RSs1. An octopine synthase element, a stem-specific, a root-specific and a light-responsive element were found in the rolC promoter, whereas the ASL box, GATA and 13 bp motifs were detected in both promoters. Binding analysis of these cis-elements (both in native and mutant forms) with the trans-factors present in the nuclear extracts from rice, tobacco and chickpea, followed by electrophoretic mobility shift assay, documented a highly specific cis-trans interaction. Both promoters were utilized to express Allium sativum leaf agglutinin (ASAL) gene in the three aforementioned plant systems. By immunohistochemistry and immunohistofluorescence, specific patterns of ASAL accumulation were detected in vascular tissues of single copy transgenic plants. Transgenic plants expressing ASAL in a phloem-specific manner demonstrated about 60-65% more insecticidal activity than control plants. The two promoters, which evolved independently from two distinctly unrelated origins, were found to maintain their functionality in a conserved manner. They were able to express the insecticidal protein coding ASAL as transgene both in monocot and dicot hosts. Thus, the two promoters are valuable as prospective phloem-specific promoters for use in plant biotechnological programmes.

Actividades biológicas del extracto acuoso de la esponja Aplysina lacunosa (Porifera: Aplysinidae) / Biological activities of the aqueous extract of the sponge Aplysina lacunosa (Porifera: Aplysinidae)

Biological activity of an aqueons extract of the sponge Aplysina lacunosa (Porifera: Aplysinidae). The aqueous extract and protein precipitate of Aplysina lacunosa (Pallas, 1776) were studied to assess their hemagglutinating, hemolysing, antibacterial, and antifungal activities. Specimens of the marine sponge were collected in El Morro de Tigüitigüe, Santa Fe, Sucre state, Venezuela. The active protein was separated by molecular exclusion chromatography and its molar mass estimated by SDS-PAGE electrophoresis. The sponge A. lacunosa has a protein with a molar mass of about 43 000 Daltons which is capable of agglutinating human erythrocytes of the blood groups A, B, and O in a strong and unspecific mode. The assayed samples did not evidence any hemolysing activity. As for the antibacterial assay, only the aqueous extract was able to inhibit the growth of Enterococcus faecalis, Bacillus cereus, Escherichia coli, and Salmonella enteritidis, with inhibition halos of 24, 20, 24, and 22 mm, respectively. None of the samples exhibited antifungal activity. The chemical analysis of the aqueous extract revealed the presence of several secondary metabolites. It is presumed that its hemagglutinating activity is mediated by agglutinative proteins. The antibacterial activity could be attributed to the presence of saponins, alkaloids, tannins, and polyphenols, which are highly antimicrobial compounds. Poriferans are a rich source of bioactive compounds that can be used in the development of new drugs potentially useful in medicine. Rev. Biol. Trop. 54 (Suppl. 3): 189-200. Epub 2007 Jan. 15.

Evaluamos el extracto acuoso y precipitado de proteínas de Aplysina lacunosa, en relación con su actividad hemaglutinante, hemolizante, antibacteriana y antimicótica. Los ejemplares de la esponja marina fueron recolectados en el Morro de Tigüitigüe, Santa Fe, Estado Sucre, Venezuela. La proteína activa fue separada por cromatografía de exclusión molecular; y su masa molar fue estimada por electroforesis SDS-PAGE. La esponja A. lacunosa posee una proteína con masa molar aproximada de 4.000 Daltons capaz de aglutinar fuertemente y de manera inespecífica los eritrocitos humanos de los grupos sanguíneos A, B y O. No se observó actividad hemolizante por parte de las muestras ensayadas. Únicamente el extracto acuoso fue capaz de inhibir el crecimiento de Enterococcus faecalis, Bacillus cereus, Escherichia coli y Salmonella enteritidis con halos de inhibición de 24, 20, 24, 22 mm, respectivamente; ninguna de las muestras exhibió actividad antifúngica. El análisis químico del extracto acuoso reveló la presencia de diversos metabolitos secundarios. Se presume que la actividad hemaglutinante se deba a la presencia de proteínas aglutinantes. La actividad antibacteriana podría atribuirse a la presencia de saponinas, alcaloides, taninos y polifenoles, compuestos altamente antimicrobianos. Los poríferos constituyen una fuente rica de compuestos bioactivos que pueden ser utilizados para el desarrollo de nuevos fármacos.

Silver and gold glyconanoparticles for colorimetric bioassays.

The color changes associated with the aggregation of metal nanoparticles has led to the development of colorimetric-based assays for a variety of target species. We have examined both silver- and gold-based nanoparticles in order to establish whether either metal exhibits optimal characteristics for bioassay development. These silver and gold nanoparticles have been stabilized with a self-assembled monolayer of a mannose derivative (2-mercaptoethyl alpha-d-mannopyranoside) with the aim of inducing aggregation by exploiting the well-known interaction between mannose and the lectin Concanavalin A (Con A). Both metal glyconanoparticles were determined to be ca. 16 nm in diameter (using TEM measurements). Aggregation was observed on addition of Con A to both silver and gold nanoparticles resulting in a shift in the surface plasmon absorption band and a consequent color change of the solution, which was monitored using UV-visible spectrophotometry. Mannose-stabilized silver nanoparticles at a concentration of 3 nM provide an assay for Con A with the largest linear range (between 0.08 and 0.26 microM). Additionally, the kinetic rate of aggregation of the silver-nanoparticle-based bioassay was significantly greater than that of the gold-nanoparticle system. However, in terms of sensitivity, the mannose-stabilized gold-nanoparticle-based assay was optimum with a limit of detection of 0.04 microM Con A, as compared with a value of 0.1 microM obtained for the mannose-stabilized silver nanoparticles. Additionally, a lactose derivative (11-mercapto-3,6,9-trioxaundecyl beta-D-lactoside) was used to stabilize gold nanoparticles to induce aggregation upon addition of the galactose specific lectin Ricinus communis agglutinin (RCA(120)). To examine the specificity of the bioassay, lactose-stabilized gold nanoparticles were mixed with a solution of mannose-stabilized silver nanoparticles to give an aggregation assay capable of detecting two different lectins. When either Con A or RCA(120) was added to the mixed glyconanoparticles, selective recognition of the respective natural ligand was shown by aggregation of a single metal nanoparticle. Centrifugation and removal of the aggregated species enabled further bioassay measurements using the second glyconanoparticle system.