Deteção de Mycobacterium tuberculosis em amostras clínicas por reação em cadeia da polimerase utilizando primers baseados na região intergênica plcB-plcC / Using polymerase chain reaction with primers based on the plcB-plcC intergenic region to detect Mycobacterium tuberculosis in clinical samples

OBJECTIVE: To develop a system for the molecular diagnosis of tuberculosis by polymerase chain reaction (PCR), constructing primers based on the difference in gene organization of the intergenic region of phospholipase C (plcB-plcC region), which differentiates Mycobacterium tuberculosis from other mycobacteria. METHODS: A PCR product of the expected size (432 bp) was obtained from M. tuberculosis and M. africanum only. A total of 33 mycobacterial isolates and 273 clinical samples from patients suspected of having tuberculosis were examined. These were used in the comparative study of the PCR technique versus culture. RESULTS: For PCR versus culture, the data showed 93.8 percent accuracy (p < 0.0001), 93.1 percent sensitivity (CI: 88.7-96.0), and 96.4 percent specificity (CI: 96.1-99.4). The Kappa value (0.82) shows that there was a near-perfect concordance between the two tests. CONCLUSION: The use of the plcB-plcC region in PCR amplification was found to be an important and reliable tool for the specific diagnosis of tuberculosis in the samples analyzed.

OBJETIVO: Desenvolver um sistema para o diagnóstico molecular da tuberculose por reação em cadeia da polimerase, do inglês polymerase chain reaction (PCR), pela construção de primers baseados na diferença da organização de uma região intergênica da fosfolipase (phospholipase) C (região plcB-plcC), que diferencia Mycobacterium tuberculosis das outras micobactérias. MÉTODOS: Um produto de PCR com o tamanho esperado (432 pb) foi obtido somente de M. tuberculosis e M. africanum. Um total de 33 isolados micobacterianos e 273 amostras clínicas de pacientes com suspeita de tuberculose foram examinados. Estes foram submetidos ao estudo comparativo da técnica de PCR contra o cultivo. RESULTADOS: Os dados mostraram 93,8 por cento de exatidão para PCR contra o cultivo (p < 0,0001), 93,1 por cento de sensibilidade (IC: 88,7-96,0) e especificidade de 96,4 por cento (IC: 96,1-99,4). O valor de Kappa foi de 0,82, demonstrando um alinhamento perfeito para a verificação do grau de concordância entre os testes. CONCLUSÃO: O uso da região plcB-plcC para a amplificação por PCR é mostrado como uma ferramenta importante e de confiança para o diagnóstico específico de tuberculose nas amostras clínicas analisadas.

Adenosine A1 receptor-mediated inhibition of in vitro prolactin secretion from the rat anterior pituitary

In previous studies, we demonstrated biphasic purinergic effects on prolactin (PRL) secretion stimulated by an adenosine A2 agonist. In the present study, we investigated the role of the activation of adenosine A1 receptors by (R)-N6-(2-phenylisopropyl)adenosine (R-PIA) at the pituitary level in in vitro PRL secretion. Hemipituitaries (one per cuvette in five replicates) from adult male rats were incubated. Administration of R-PIA (0.001, 0.01, 0.1, 1, and 10 æM) induced a reduction of PRL secretion into the medium in a U-shaped dose-response curve. The maximal reduction was obtained with 0.1 æM R-PIA (mean ± SEM, 36.01 ± 5.53 ng/mg tissue weight (t.w.)) treatment compared to control (264.56 ± 15.46 ng/mg t.w.). R-PIA inhibition (0.01 æM = 141.97 ± 15.79 vs control = 244.77 ± 13.79 ng/mg t.w.) of PRL release was blocked by 1 æM cyclopentyltheophylline, a specific A1 receptor antagonist (1 æM = 212.360 ± 26.560 ng/mg t.w.), whereas cyclopentyltheophylline alone (0.01, 0.1, 1 æM) had no effect. R-PIA (0.001, 0.01, 0.1, 1 æM) produced inhibition of PRL secretion stimulated by both phospholipase C (0.5 IU/mL; 977.44 ± 76.17 ng/mg t.w.) and dibutyryl cAMP (1 mM; 415.93 ± 37.66 ng/mg t.w.) with nadir established at the dose of 0.1 æM (225.55 ± 71.42 and 201.9 ± 19.08 ng/mg t.w., respectively). Similarly, R-PIA (0.01 æM) decreased (242.00 ± 24.00 ng/mg t.w.) the PRL secretion stimulated by cholera toxin (0.5 mg/mL; 1050.00 ± 70.00 ng/mg t.w.). In contrast, R-PIA had no effect (468.00 ± 34.00 ng/mg t.w.) on PRL secretion stimulation by pertussis toxin (0.5 mg/mL; 430.00 ± 26.00 ng/mg t.w.). These results suggest that inhibition of PRL secretion after A1 receptor activation by R-PIA is mediated by a Gi protein-dependent mechanism.

The effect of hydrostatic pressure on membrane-bound proteins

Many cellular proteins are bound to the surfaces of membranes and participate in various cell signaling responses. Interactions between this group of proteins are in part controlled by the membrane surface to which the proteins are bound. This review focuses on the effects of pressure on membrane-associated proteins. Initially, the effect of pressure on membrane surfaces and how pressure may perturb the membrane binding of proteins is discussed. Next, the effect of pressure on the activity and lateral association of proteins is considered. We then discuss how pressure can be used to gain insight into these types of proteins.

Purificacão da ALPHA - Toxina (fosfolipase C) obtida a partir de Clostridium perfringens tipo A utilizando sistemas de duas fases aquosas de modo descontínuo e contínuo / Purification ALPHA - Toxin (phospholipase C) obtained by Clostridium perfringens type A using aqueous two-phase systems of discontinue and continue mode

O objetivo deste trabalho foi a purificacao da toxina alfa produzida por Clostridium perfringens tipo A em sistemas de duas fases aquosas (SDFA) PEG/fosfato nos modos contínuo e descontínuo. Dois planejamentos fracionários seqüenciais foram aplicados para estudar a partição da toxina alfa em SDFA, em função de 4 variáveis: massa molar e concentração do PEG, concentração do fosfato e pH. Os melhores resultados para o fator de purificação, rendimento em atividade e coeficiente de partição foram obtidos com PEG 8000 g/mol (15 porcento, w/w), fosfato 20 porcento (w/w) e pH 8.0...

Regulation of capacitative and non-capacitative Ca2+ entry in A7r5 vascular smooth muscle cells

A capacitative Ca2+ entry (CCE) pathway, activated by depletion of intracellular Ca2+ stores, is thought to mediate much of the Ca2+ entry evoked by receptors that stimulate phospholipase C (PLC). However, in A7r5 vascular smooth muscle cells, vasopressin, which stimulates PLC, empties intracellular Ca2+ stores but simultaneously inhibits their ability to activate CCE. The diacylglycerol produced with the IP3 that empties the stores is metabolized to arachidonic and this leads to activation of nitric oxide (NO) synthase, production of NO and cyclic GMP, and consequent activation of protein kinase G. The latter inhibits CCE. In parallel, NO directly activates a non-capacitative Ca2+ entry (NCCE) pathway, which is entirely responsible for the Ca2+ entry that occurs in the presence of vasopressin. This reciprocal regulation of two Ca2+ entry pathways ensures that there is sequential activation of first NCCE in the presence of vasopressin, and then a transient activation of CCE when vasopressin is removed. We suggest that the two routes for Ca2+ entry may selectively direct Ca2+ to processes that mediate activation and then recovery of the cell.

Análise de polimorfismos genéticos afetando os quatro genes de fosfolipase C (plc) em isolados clínicos do complexo Mycobacterium tuberculosis / Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

0 genoma de Mycobacterium tuberculosis contém quarto genes relacionados, que apresentam significante similaridade com os genes de fosfolipase C de Pseudomonas aeruginosa. Três destes genes, plcA, plcB, e plcC são organizados em "tandem" (locus plcABC). 0 quarto gene, plcD, está localizado em uma região diferente. Este estudo investiga variações nos genes plcABC e pleD em isolados clínicos de M. tuberculosis (MTB), M. africanum (MAF) e M. canettii (MC). Polimorfismos genéticos foram examinados por PCR, Southern blot, seqüenciamento e RT-PCR. Sete isolados MTB contém inserções do elemento IS6110 nos genes plcA, plcC, ou plcD. Em 19 de 25 isolados de MTB examinados, foram identificadas deleções genômicas, resultando na perda parcial ou completa dos genes plcABC e/ou pleD. Deleção completa do gene plcD foi observada em um isolado de MAR Em cada caso, as deleções foram associadas à presença de uma cópia do elemento IS6110 e, em todos os casos a 156110 apresenta a mesma orientação. 0 mecanismo de deleção resultou da recombinação homóloga entre duas cópias de IS6110 e foi reconhecido em um grupo de isolados de MTB geneticamente relacionados. Cinco isolados de MTB apresentaram extenso polimorfismo nas regiões plcABC e plcD, com perda de expressão afetando os quarto genes plc. Fosfolipase C é um fator de virulência bem conhecido em bactérias. 0 papel preciso da fosfolipase C na patogenicidade de M. tuberculosis não está estabelecido, mas considerando o potencial que os genes plc podem ter na virulência do bacilo da tuberculose, o estudo de isolados provenientes de pacientes com tuberculose ativa contendo variações genéticas afetando estes genes podem fornecer indícios da significância da fosfolipase C na patogênese da tuberculose

Mecanismos de acción de xenobióticos en ovocitos de anfibios / Xenobiotic mechanisms of action in amphibian oocytes

Comprender los procesos que determinan la toxicidad de diversas sustancias a nivel celular constituye un desafío para el desarrollo tecnológico actual. Los ovocitos de diversas especies son utilizados con distintos propósitos en investigación. El tamaño de los ovocitos de anfibios y la facilidad con que puede obtenerse un gran número de ellos como células aisladas, hacen de los mismos el sistema de elección para múltiples propósitos, tales como: estudios de las etapas tempranas de la reproducción, técnicas de fertilización in vitro, expresión y caracterización de receptores de membranas plasmática, mecanismos de acción de agonistas, vías metabólicas, etc. El objetivo del presente trabajo con ovocitos de sapo Bufo arenarum es estudiar los mecanismos de acción de sustancias xenobióticas a nivel bioquímico y molecular, en relación con la capacidad de éstas gametas de ser fertilizadas y avanzar a través de los estadios tempranos del desarrollo embrionario. Se ha trabajado con diversos biomarcadores que permitieron reconocer los mecanismos de toxicidad de compuestos organoclorados y más recientemente de algunos metales pesados. Se demostró que la activación de una fosfolipasa C específica por el Dieldrin provoca depleción de mediadores lipídicos y como consecuencia inhibición de la fertilización (desarrollo hasta 4-8 células). Actualmente se estudian los efectos del Zn sobre la fertilización y su probable relación con alteraciones en la actividad de enzimas de la vía de las pentosas, directamente ligada a los mecanismos de detoxificación en presencia de stress oxidativo. El estudio de los mecanismos de citotoxicidad de diversas sustancias resulta de gran utilidad cuando se lleva a cabo en gametas, porque permite también evaluar los efectos de dichas sustancias sobre la reproducción de la especie. Los ovocitos de anfibios son además células de gran interés, por su utilidad en el estudio de la citotoxicidad de una amplia variedad de contaminantes hídricos

Clonagem, expressäo e estudo de alguns cDNAs codificando proteínas estruturalmente relacionadas às alfa neurotoxinas da glândula de veneno da cobra coral Micrurus corallinus (Serpentes, Elapidae) / Cloning, expression and study of some cDNAs conding proteins structurally related to alpha-neurotoxins of venom gland coral snake Micrurus corallinus (Serpentes, Elapidae)

De uma bibliteca de cDNA da glândula de veneno da cobra coral brasileira Micrurus corallinus foi isolada uma seqüência denominada NXH8. Esta seqüência de cDNA apresenta similaridade estrutural com a família de toxinas de serpentes em "três dígitos" ricas em pontes dissulfeto. A subclasse melhor conhecida nesta família säo as alfa-neurotoxinas. Uma outra seqüência distinta, denominada NXH1 e suas isoformas NXH3 e NXH7, foram isoladas anteriormente, pertencem à mesma família de toxinas e estäo presentes na mesma biblioteca. Alguns resultados da caracterizaçäo da NXH1, säo utilizados neste estudo, em comparaçäo com NXH8. Algumas características estruturais tornam a seqüência NXH8 diferente da classe usual...

La aspirina vs. extracción dental / Aspirin vs. tooth extraction

Se describen los efectos de la aspirina vinculada a la extracción dental simple, para lo cual se expone una revisión bibliográfica con fines didácticos. Se exponen los efectos fisiopatológicos, con el fin de conocer y así poder llegar a determinar un posible enfoque terapéutico

Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis

Signal transduction induced in Trypanosoma cruzi metacyclic trypomastigotes during the invasion of mammalian cells

Penetration of Trypanosoma cruzi into mammalian cells depends on the activation of the parasite's protein tyrosine kinase and on the increase in cytosolic Ca2+ concentration. We used metacyclic trypomastigotes, the T. cruzi developmental forms that initiate infection in mammalian hosts, to investigate the association of these two events and to identify the various components of the parasite signal transduction pathway involved in host cell invasion. We have found that i) both the protein tyrosine kinase activation, as measured by phosphorylation of a 175-kDa protein (p175), and Ca2+ mobilization were induced in the metacyclic forms by the HeLa cell extract but not by the extract of T. cruzi-resistant K562 cells; ii) treatment of parasites with the tyrosine kinase inhibitor genistein blocked both p175 phosphorylation and the increase in cytosolic Ca2+ concentration; iii) the recombinant protein J18, which contains the full-length sequence of gp82, a metacyclic stage surface glycoprotein involved in target cell invasion, interfered with tyrosine kinase and Ca2+ responses, whereas the monoclonal antibody 3F6 directed at gp82 induced parasite p175 phosphorylation and Ca2+ mobilization; iv) treatment of metacyclic forms with phospholipase C inhibitor U73122 blocked Ca2+ signaling and impaired the ability of the parasites to enter HeLa cells, and v) drugs such as heparin, a competitive IP3-receptor blocker, caffeine, which affects Ca2+ release from IP3-sensitive stores, in addition to thapsigargin, which depletes intracellular Ca2+ compartments and lithium ion, reduced the parasite infectivity. Taken together, these data suggest that protein tyrosine kinase, phospholipase C and IP3 are involved in the signaling cascade that is initiated on the parasite cell surface by gp82 and leads to Ca2+ mobilization required for target cell invasion.

Hidróxido de cálcio em endondontia: suposiçöes x comprovaçäo científica / The calcium hydroxide in endodontics: supposition x cientifc proof

Várias propriedades biológicas têm sido atribuídas ao hidróxido de cálcio, tais como: açäo antiinflamatória, açäo antimicrobiana, solvente de matéria orgânica, inibiçäo da reabsorçäo e induçäo de reparo mineralizado. Neste trabalho, estas propriedades säo discutidas baseado em achados científicos

Efeito protetor da criocardioplegia cristalóide na isquemia global e reperfusäo durante circulaçäo extracorpórea: um mecanismo dependente do endotélio? / Protective effect of crystalloid cryocardioplegia in the global ischemia and reperfusion during extracorporeal circulation: an endothelium-dependent mechanism?

Estudos prévios demonstraram que o comprometimento da produçäo de EDRF/NO mediada por receptores, após isquemia global e reperfusäo, possa ser devido a uma disfunçäo de G-proteínas que liga os receptores da célula endotelial à via da síntese de EDRF/NO. O presente trabalho experimental sugere que a criocardioplegia cristalóide, associada a hipotermia tópica, previne ou pode reverter, em parte, a disfunçäo endotelial nas mesmas condiçöes. Mais estudos seräo necessários para conclusöes mais definitivas, pois as análises estatísticas mais acuradas sugeriram aumento da amostragem. Este detalhe talvez seja devido às grandes dificuldades de uniformizaçäo relacionada a este tipo de experimentos. Além disso, demonstrou-se pela primeira vez que a hipotermia, por si só, pode estimular a liberaçäo de EDRF/NO pelo endotélio vascular. Isto sugere que o endotélio possa ser um importante sensor de mudanças da temperatura sangüínea e tem importantes implicaçöes para o entendimento da fisiologia da CEC e dos mecanismos locais de auto-regulaçäo.

Cardioplegia cristalóide, barotrauma e funçäo endotelial: consideraçöes experimentais / Crystalloid cardioplegia, barotrauma and endothelium function: experimental considerations

O presente ensaio experimental estudou o efeito da infusao de soluçao cardioplégica cristalóide a altas pressoes sobre a funçao endotelial de artérias epicárdicas de caes. Nao se encontraram alteraçoes a nível de receptores (curvas dose-respostas à ACH e ADP; da transduçao do sinal iniciado nos receptores/sitema de G-proteínas (fluoreto de sódio) e nos processos intracelulares da produçao de EDRF/NO (fosfolipase C e ionóforo do cálcio A23187). A funçao da musculatura lisa vascular nao foi afetada quando se analisaram as respostas relaxantes (nitroprussiato de sódio e isoproterenol) e contráteis (KCI e prostaglandina 2alfa). Estes achados permitem as seguintes consideraçoes especulativas: a) O barotrauma produzido pela infusao da cardioplegia cristalóide a altas pressoes ocorreria apenas em circulaçoes coronarianas previamente doentes? b) Uma vez que as infusoes duraram de 2 a 3 minutos, seria o barotrauma coronariano um fenômeno dependente do tempo de infusao? c) Para que ocorra o barotrauma seriam necessários níveis mais elevados de Potássio? d) Questionara existência do fenômeno do barotrauma coronariano produzido pela infusao de soluçoes cadioplégicas pelo menos nas condiçoes experimentais utilizadas. e) A metodologia empregada estuda apenas as reatividades vasculares de artérias coronarias epicárdicas. estas artérias seriam menos sensíveis aos efeitos da pressao de infusao da cardioplegia do que a microcirculaçao coronariana? f) Seria a circulaçao coronária do cao menos sensível a altas pressoes do que do homem? Estas observaçoes experimentais sugerem que a infusao de cardioplegia cristalóide, moderadamente hipocalêmica, a altas pressoes em um tempo de 2 a 3 minutos, nao interfere com a produçao de EDRF/NO pelo endotélio de coronárias epicárdicas do cao.

Effects of alpha-MSH and cholinergic agents on cGMP and IP3 levels in rat brain slices

On one hand, it has been demonstrated that the exposure of rat brain slices containing caudate putamen and accumbens nuclei to alpha-MSH brings about an increase in cAMP. This increase is affected when dopamine is present in the incubation medium. On the other hand, an interaction of melanotropinergic-like peptides with acetylcholinergic drugs has been showed to be similar to the one observed with dopamine. In this study we have intended to measure cGMP Or IP3 in response to alpha-MSH, and also to study the interaction with cholinergic drugs by measuring the second messengers recently mentioned. cGMP and IP3 have been measured in tissues and medium in their response to the effect of alpha-MSH alone or in the presence of the peptide plus pilocarpine (selective muscarinic agonist) or atropine (selective muscarinic antagonist). None of them modified the cGMP levels when compared with lhe control group. The exposure of rat brain slices containing CP and Acc nuclei to alpha-MSH resulted in an increase in IP3 levels. Pilocarpine by itself brought about an increase of IP3 only when the highest doses was used. Atropine did not modify the IP3 content. However, when slices were exposured to both alpha-MSH and pilocarpine, IP3 content was similar to control values. The blockage of the muscarinic receptor with atropine blocked the IP3 increase induced by alpha-MSH as well. Therefore, we assume that alpha-MSH does not induce changes in cGMP but it does change the IP3 levels, probably acting at the muscarinic receptor level.

Characterization of a phospholipase C-resistant inositol containing glycolipid from Trypanosoma cruzi

Since glycosylphosphatidylinositol is the most common form of attachment of proteins to membranes in T. cruzi, and that this parasite depends on surface-mediated interactions for survival within the vector and mammalian host, it is probable that a drug which interfers with the metabolism of glycosylphosphatidylinositol (GPI) could be successfully employed in chemotherapy. Over the last few years several groups have been characterizing this mode of attachment in T. cruzi and more recently we have been concentrating our efforts on the identification of candidate precursors for protein anchors in metacyclic trypomastigotes. Previously detected GPI heterogeneity regarding solubilization of a major stage-specific antigen (1G7-Ag) by phospholipase C led us to investigate whether biosynthetic precursors with similar properties could also be identified. Two glycolipid species whose migration properties resemble glycolipids A and C of T. brucei were amenable to biosynthetic radiolabelling with palmitic acid, inositol, ethanolamine, glucosamine and mannose. Following purification, these species were submitted to classical GPI diagnostic treatments. In both cases digestion with GPI-specific phospholipase D (GPIPLD) produced phosphatidic acid and treatment with either mild base or phospholipase A2 (PLA2) produced free fatty acid, indicating an acylation at least at position 2 of the glycerol. The glycolipid A-like species proved to be susceptible to solubilization by PIPLC of B. thuriengiensis and by GPIPLC of T. brucei and the glycolipid C-like material proved to be fully resistant to both lipases. Although the glycolipid A-like species indeed presents these and other properties compatible with a precursor for the chemically characterized 1G7-Ag anchor, the PLC-resistant species which is completely insensitive to nitrous acid deamination might be an exception to the general finding of a non-acetylated glucosamine in the GPI moieties so far described

Free and protein-linked glycoinositolphospholipids in Trypanosoma cruzi

Two glycoinositol phospholipids (GIPL A and GIPL B) have been purified from epimastigotes of Trypanosoma cruzi at the logarithmic phase of growth (2 days). The GIPLs differ mainly in the lipid moiety and are similar to the lipopeptidophosphoglycan (LPPG) previously isolated from epimastigotes at the stationary phase (4-5 days). [3H]-palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in GIPL A and into a sphinganine ceramide with palmitic acid and lignoceric acid as the fatty acids in GIPL B. The lipids could be released by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) or glycosylphosphatidylinositol phospholipase D (GPI-PLD) from rat serum. The oligosaccharides share the common core structure of the glycosylphosphatidilinositol (GPI) membrane anchors. Microheterogeneity was demonstrated, as well as substitution by galactose, which is mainly in the furanose configuration as was previously described for the LPPG. However, methylation analysis indicated that 20 percent of the galactose is present as terminal pyranose units. In infective trypomastigotes, [3H]-palmitic acid was incorporated into the anchor of the Tc-85 glycoprotein. The lipid cleaved by phospholipase C digestion was identified as 1-O-hexadecylglycerol and the main oligosaccharide has the structure of the conserved core of all GPI anchors. [3H]-palmitic acid-labelled Tc-85 released into the culture medium as membrane vesicles showed 80 percent resistance to the action of PI-PLC. However, after mild alkaline hydrolysis, part of the radioactivity was released by the enzyme

Inositolphosphoglycan second messengers

The mechanisms by which cellular receptors can elicit different biological responses in a maturation state-dependent manner is one of the central problems in cell differentiation which remains to be resolved. The signals generated are likely to be due to additional (as yet unknown) transmembrane signalling pathways. In addition, the recent observation that a single growth factor receptor can activate a whole family of different putative second messengers and that the combinatorial interactions and stoichiometric ratios between the different messengers determine the resulting biological activities has opened up a whole new area of cell biology. It has been proposed that membrane GPI-anchors may function in signal transduction. We have recently confirmed the presence of a family of inositolphosphoglycan second messengers. Partial structural data suggest that these second messengers are not derived from known GPI membrane anchors and may thus constitute a novel class of non-protein-conjugated GPI

The role of GPI-PLC in Trypanosoma brucei

The glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) from trypanosoma brucei exhibits exquisite specificity for the GPI-anchor of the variant specific glycoprotein (VSG). However the evidence that it is involved in VSG metabolism in the living trypanosome is circunstantial; it shows the same life cycle stage regulated expression as the VSG, no feasible alternative substrate has been identified, and it metabolises the VSG efficiently in vitro and in vivo on hypotomic lysis. Against these considerations are the observations that the GPI-PLC is found on the cytoplasmic face of vesicles so it could not gain access to the VSG through normal vesicle fusion and that the accelerated loss of VSG from bloodstream forms on differentiation to procyclic forms occurs through the action of a protease. To try to determine the role of the GPI-PLC, a homozygous mull mutant T. brucei has been constructed. The null mutant was created by replacement of the entire gene at both alleles with selectable antibiotic resistance markers in procyclic form trypanosomes. The GPI-PLC gene is not usually expressed in procyclic forms and so, as would be expected, the null procyclics display no obvious phenotype. The null procyclics have been used to infect tsetse flies and it remains to be seen whether it is possible for them to differentiate to bloodstream forms and, if so, what the antigenic variation phenotype of the null bloodstream forms would be