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Chemical genetics and a global comparative analysis of phosphorylation and phospholipids in vivo shows that PKG is the upstream regulator that induces calcium signals that enables Plasmodium to progress through its complex life cycle., Many critical events in the Plasmodium life cycle rely on the controlled release of Ca2+ from intracellular stores to activate stage-specific Ca2+-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca2+ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca2+ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca2+ effectors, PKG emerges as a unifying factor to control multiple cellular Ca2+ signals essential for malaria parasite development and transmission., Author Summary Malaria, caused by Plasmodium spp. parasites, is a profound human health problem. Plasmodium parasites progress through a complex life cycle as they move between infected humans and blood-feeding mosquitoes. We know that tight regulation of calcium ion levels within the cytosol of the parasite is critical to control multiple signalling events in their life cycle. However, how these calcium levels are controlled remains a mystery. Here, we show that a single protein kinase, the cGMP-dependent protein kinase G (PKG), controls the calcium signals that are critical at three different points of the life cycle: (1) for the exit of the merozoite form of the parasite from human erythrocytes (red blood cells), (2) for the cellular activation that happens when Plasmodium sexual transmission stages are ingested by a blood-feeding mosquito, and (3) for the productive gliding of the ookinete, which is the parasite stage that invades the mosquito midgut. We provide initial evidence that the universal role of PKG relies on the production of lipid precursors which then give rise to inositol (1,4,5)-trisphosphate (IP3), a messenger molecule that serves as a signal for the release of calcium from stores within the parasite. This signalling pathway provides a potential target to block both malaria development in the human host and transmission to the mosquito vector.

Spermidine is a natural polyamine involved in many important cellular functions, whose supplementation in food or water increases life span and stress resistance in several model organisms. In this work, we expand spermidine's range of age-related beneficial effects by demonstrating that it is also able to improve locomotor performance in aged flies. Spermidine's mechanism of action on aging has been primarily related to general protein hypoacetylation that subsequently induces autophagy. Here, we suggest that the molecular targets of spermidine also include lipid metabolism: Spermidine-fed flies contain more triglycerides and show altered fatty acid and phospholipid profiles. We further determine that most of these metabolic changes are regulated through autophagy. Collectively, our data suggests an additional and novel lipid-mediated mechanism of action for spermidine-induced autophagy. Publisher PDF

The substrate specificities of Trypanosoma brucei and human (HeLa) GlcNAc-PI de-N-acetylases were determined using 24 substrate analogues. The results show the following. (i) The de-N-acetylases show little specificity for the lipid moiety of GlcNAc-PI. (ii) The 3'-OH group of the GlcNAc residue is essential for substrate recognition whereas the 6'-OH group is dispensable and the 4'-OH, while not required for recognition, cannot be epimerized or substituted. (iii) The parasite enzyme can act on analogues containing betaGlcNAc or aromatic N-acyl groups, whereas the human enzyme cannot. (iv) Three GlcNR-PI analogues are de-N-acetylase inhibitors, one of which is a suicide inhibitor. (v) The suicide inhibitor most likely forms a carbamate or thiocarbamate ester to an active site hydroxy-amino acid or Cys or residue such that inhibition is reversed by certain nucleophiles. These and previous results were used to design two potent (IC50 = 8 nM) parasite-specific suicide substrate inhibitors. These are potential lead compounds for the development of anti-protozoan parasite drugs.

When white Australians celebrated two hundred years of colonization in 1988 the work of art that was widely heralded as central to the event was a large mosaic set in the forecourt of the new Houses of Parliament in the capital city, Canberra. Designed by Michael Jagamara Nelson, it was based on traditional designs of his Central Desert people, the Walpiri, particularly on those shapes that signified a meeting place for profoundly important ceremony. Installed with the maximum of European-derived ceremony, opened by the Queen of England and Australia herself, it seemed to symbolize the racial reconciliation so strongly desired by many Australians. A quintessential work of public art, the mosaic seemed also to be a confirming embodiment of a broad cultural process, unfolding since 1970, through which works of art made by Aborigines have led the way in securing political gains for Aboriginal people. Much has changed in relations between the races in Australia since 1988-great advances, cataclysmic setbacks. Public art has continued to be a significant player. In September 1993, Nelson returned to Canberra and, in the glare of media attention, symbolically removed the central image-a number of snakelike lines converging on a set of concentric circles signifying peoples meeting-from his mosaic (fig. 1). He said

Clinical outcomes were assessed in 68 consecutive patients with multiple myeloma of high or intermediate tumor mass that had responded to VAD or dexamethasone-based therapy and were consolidated with early intensive therapy and autologous stem cell transplantation. Results were compared with those of 50 comparable patients who refused or were unable to receive intensive treatment for socioeconomic reasons. Following high-dose therapy, the rate of CR increased from 6 to 37%, with median survival prolonged by 10 months. Survival of 21 patients with disease converted from PR to CR (median 8.3 years) was significantly longer than that of similarly-treated patients who remained in PR (median 5.0 years). CR of myeloma represents the major surrogate marker of long survival and the primary goal of myeloablative treatment for patients in PR. Twelve of 18 patients with rapid reduction of myeloma protein (T(1/2) < 0.5 months), and myeloma protein reduction to

BACKGROUND Inflammatory breast carcinoma (IBC) is a rare but aggressive form of breast carcinoma. Anthracycline-based regimens represent the standard of treatment for IBC. Reports of significant clinical activity of paclitaxel in metastatic breast carcinoma led the authors to investigate the role of this drug in the management of IBC. METHODS Forty-four patients with IBC were enrolled between February 1994 and January 1998. The treatment plan consisted of induction chemotherapy (IC), mastectomy, adjuvant chemotherapy, and radiotherapy. Forty-two patients received IC with four cycles of fluorouracil, doxorubicin, and cyclophosphamide. If the clinical response was less than partial, patients were “crossed over” to paclitaxel before mastectomy. All patients received adjuvant paclitaxel. Patients unresectable after paclitaxel were offered high-dose chemotherapy with autologous peripheral blood progenitor cell support. RESULTS Thirty-four patients (81%) achieved an objective clinical remission; 3 patients (7%) achieved a clinical complete remission, 31 (74%) a partial remission. Six patients (14%) achieved pathologic complete remission. Sixteen patients were treated with paclitaxel, 7 of them (44%) were able to undergo mastectomy. Median time to progression (TTP) was 22 months. Median overall survival (OS) was 46 months. Concordance between clinical and pathologic response was documented in only 8 patients (24%). No differences in TTP and OS compared with a historical group of 178 IBC patients treated with anthracycline-based regimens. CONCLUSIONS Paclitaxel improves tumor resectability in anthracycline-refractory IBC. The impact of paclitaxel on the prognosis of IBC needs to be better evaluated in future trials using more dose-intensive schedules of administration. New imaging modalities may contribute to improve assessment of response to IC. Cancer 2001;92:1775–82. © 2001 American Cancer Society.

Summary Background African trypanosomiasis is a fatal disease caused by protozoan parasites of the species Trypanosoma brucei . The disease has reached epidemic dimensions in various countries of central Africa. Treatment of the second stage is long and complicated, and is hampered by severe adverse reactions to the first-line drug, melarsoprol. Despite these problems, melarsoprol is likely to remain the drug of choice for the next decade. We therefore did a randomised trial comparing the standard treatment schedule with a new, concise regimen. Methods The safety and efficacy of the new schedule were assessed in patients presenting to a hospital in Kwanza Norte, Angola with sleeping sickness. The control group followed the 26-day standard Angolan schedule of three series of four daily injections of melarsoprol at doses increasing from 1·2 to 3·6 mg/kg within each series, with a 7-day interval between series. The new treatment schedule comprised 10 daily injections of 2·2 mg/kg. Primary outcomes assessed were elimination of parasites, deaths attributed to treatment, and rate of encephalopathy. Analysis was by intention to treat. Findings Of 767 patients with second-stage disease, 500 were enrolled: 250 were assigned the standard schedule, and 250 the new schedule. 40 patients on the standard schedule and 47 on the new schedule had adverse events which resulted in treatment disruption or withdrawal. 50 patients on the standard regimen deviated or withdrew from treatment, compared with two on the new regimen. Parasitological cure 24 h after treatment was 100% in both groups; there were six deaths (all due to encephalopathy) 30 days after treatment in each group. The number of patients with encephalopathic syndromes was also the same in each group (14). Skin reactions were more common with the new treatment, but all could be resolved by additional medication or withdrawal of treatment. Interpretation Considering the economic and practical advantages of the new 10-day schedule over the standard 26-day treatment schedule, and the similarity of treatment outcome, the new schedule is a useful alternative to the present standard, especially in epidemic situations and in locations with limited resources.

Background: We have observed that molecular response, as defined by a PCR-negative status during the first year of therapy, along with β2-microglobulin (β2M), was the most important variable associated with failure-free survival (FFS) in follicular lymphoma (FL). Herein, we present an update of the previously published MDACC series. Patients and methods: A total of 116 patients (male: female ratio 64; 52; median age: 52 years) with indolent FL and BCL-2 rearrangement (at MBR or mcr breakpoints) assessable in peripheral blood (pb) by PCR prior to treatment, and with two or more PCR determinations during the first year, were selected for the present study. Results: Of the 116 patients, 4 who presented with progression and I who died of unrelated causes during the first year were excluded from the landmark analysis. One hundred patients (86%) achieved clinical CR and 80 (69%) achieved a negative PCR status within first year. Median FFS was 6.4 years. Five-year FFS was 73% and 28% for molecular responders and nonresponders, respectively (P = 0.001). In spite of this strikingly higher FFS favoring molecular responders, no clearcut plateau was evident in this group. Molecular response assessed in pb (P = 0.001) and serum p2M (P < 0.001) were the most important factors to predict FFS in the multivariate analysis. In the subset of patients with normal β2M and molecular CR, there was a trend for a plateau in the FFS curve. No significant difference between the groups has been observed so far in terms of survival. Conclusions: Molecular response assessed in pb using a PCR technique is, along with β2M, the most important factor to predict FFS in FL.

Synthetic analogues of D-GlcNalpha1-6D-myo-inositol-1-HPO(4)-3(sn-1, 2-diacylglycerol) (GlcN-PI), with the 2-position of the inositol residue substituted with an O-octyl ether [D-GlcNalpha1-6D-(2-O-octyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-octyl) PI] or O-hexadecyl ether [D-GlcNalpha1-6D-(2-O-hexadecyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-hexadecyl)PI], were tested as substrates or inhibitors of glycosylphosphatidylinositol (GPI) biosynthetic pathways using cell-free systems of the protozoan parasite Trypanosoma brucei (the causative agent of human African sleeping sickness) and human HeLa cells. Neither these compounds nor their N-acetyl derivatives are substrates or inhibitors of GPI biosynthetic enzymes in the HeLa cell-free system but are potent inhibitors of GPI biosynthesis in the T.brucei cell-free system. GlcN-(2-O-hexadecyl)PI was shown to inhibit the first alpha-mannosyltransferase of the trypanosomal GPI pathway. The N-acetylated derivative GlcNAc-(2-O-octyl)PI is a substrate for the trypanosomal GlcNAc-PI de-N-acetylase and this compound, like GlcN-(2-O-octyl)PI, is processed predominantly to Man(2)GlcN-(2-O-octyl)PI by the T.brucei cell-free system. Both GlcN-(2-O-octyl)PI and GlcNAc(2-O-octyl)PI also inhibit inositol acylation of Man(1-3)GlcN-PI and, consequently, the addition of the ethanolamine phosphate bridge in the T.brucei cell-free system. The data establish these substrate analogues as the first generation of in vitro parasite GPI pathway-specific inhibitors.

Glycosylphosphatidylinositols (GPIs) are synthesized in the endoplasmic reticulum (ER) via the sequential addition of monosaccharides, fatty acid, and phosphoethanolamine(s) to phosphatidylinositol (PI). While attempting to establish a mammalian cell-free system for GPI biosynthesis, we found that the assembly of mannosylated GPI species was impaired when purified ER preparations were substituted for unfractionated cell lysates as the enzyme source. To explore this problem we analyzed the distribution of the various GPI biosynthetic reactions in subcellular fractions prepared from homogenates of mammalian cells. The results indicate the following: (i) the initial reaction of GPI assembly, i.e. the transfer of GlcNAc to PI to form GlcNAc-PI, is uniformly distributed in the ER; (ii) the second step of the pathway, i.e. de-N-acetylation of GlcNAc-PI to yield GlcN-PI, is largely confined to a subcompartment of the ER that appears to be associated with mitochondria; (iii) the mitochondria-associated ER subcompartment is enriched in enzymatic activities involved in the conversion of GlcN-PI to H5 (a singly mannosylated GPI structure containing one phosphoethanolamine side chain; and (iv) the mitochondria-associated ER subcompartment, unlike bulk ER, is capable of the de novo synthesis of H5 from UDP-GlcNAc and PI. The confinement of these GPI biosynthetic reactions to a domain of the ER provides another example of the compositional and functional heterogeneity of the ER. The implications of these findings for GPI assembly are discussed.

The t(14;18) translocation, which involves the bcl-2oncogene, occurs in follicular lymphomas (FL) at two common sites: the major breakpoint region (MBR) and the minor cluster region (mcr). The biological and clinical significance of these breakpoints is unknown. The bcl-2 breakpoint site was determined in 247 previously untreated patients (49% men; median age 52 years) with indolent FL (155 grade I, 83 grade II, and 8 grade III) to correlate it with pretreatment characteristics, response, and outcome. The bcl-2 breakpoint site was determined by a polymerase chain reaction method of peripheral blood (all cases), bone marrows (149 cases), and fresh lymph node biopsy specimens (68 cases). The breakpoint site occurred at MBR in 175 cases (71%) and atmcr in 27 (11%). In 45 cases (18%), no breakpoint was detected (germline). No significant relationship was found between the rearrangements and the expression of BLC-2 and BAXproteins. Patients’ germline for MBR and mcr tended to present more frequently with stage IV disease and higher β2-microglobulin (β2M) levels, whereas mcr-rearranged patients presented more frequently with early stage and normal β2M. The complete response rate of germline patients was significantly lower than that of MBR and mcr patients. An estimated 3-year failure-free survival (FFS) for mcr, MBR, and germline cases was 95%, 76%, and 57%, respectively (P

Most patients with follicular lymphoma (FL) achieve a complete response (CR) after treatment, but eventually most of them, particularly those with stage IV, relapse due to minimal residual disease (MRD). The t(14;18) gives rise to a rearrangement of the bcl-2 oncogene that constitutes an excellent target for detection of MRD by polymerase chain reaction (PCR). One hundred ninety-four previously untreated patients with indolent FL and detectable bcl-2 rearrangement were studied. The PCR assay was used to detect bcl-2–rearranged cells in blood and marrow before and after treatment. Molecular response rate was 37%, 53%, 56%, and 66% at 3 to 5, 6 to 8, 9 to 14, and 15 to 18 months from the start of therapy, respectively. Although molecular response was higher among clinical CRs, one third of partial responders at 3 to 5 months also achieved a molecular response. Patients who achieved a molecular response during the first year of treatment had a significantly longer failure-free survival (FFS) than those who did not (4-year FFS: 76% v 38%, respectively; P < .001). Similar results were also observed in the subset of patients in clinical CR 1 year after treatment. By multivariate analysis, β2-microglobulin (β2-M; P < .01), and molecular response (P < .001) were the most important variables associated with outcome. When we combined β2-M and molecular response, three prognostic groups emerged: (1) low β2-M and molecular responders, (2) low β2-M and nonresponders or high β2-M and responders, and (3) high β2-M and nonresponders. The 4-year FFS of these 3 groups were 86%, 65%, and 23%, respectively. Finally, patients who achieved molecular response and sustained it had better FFS than those who either reverted back to PCR-positive or who never achieved molecular response. Serial PCR analysis to determine the molecular response in FL correlates well with outcome especially when combined with pretreatment β2-M.

In order to determine the activity of paclitaxel in patients with relapsed or refractory non-Hodgkin's lymphoma (NHL), we conducted a phase II clinical trial in which eligible patients received paclitaxel 200 mg/m2 intravenously over 3 h. Treatment was repeated every 3 weeks. Patients achieving complete or partial responses after two courses of paclitaxel continued to receive therapy for a maximum of eight courses, otherwise they were removed from the study. Of 96 evaluable patients, 45 (47%) had primary refractory disease, and 51 (53%) had relapsed lymphoma. The median number of prior treatment regimens was two (range one to 10 regimens). 45 patients had lowgrade, 44 had intermediate-grade, and seven had mantle cell lymphoma. 24/96 patients responded (10 complete and 14 partial remissions) for an overall response rate of 25% (95% CI 17-35%). Patients with relapsed lymphoma had a higher response rate than those with primary refractory disease (19/51 = 37% v 5/45 = 11%; P0.01), and patients with relapsed intermediate-grade lymphoma had a higher response than those with relapsed low-grade lymphoma (9/18 = 50% v 10/31 = 32%; P = 0.22). The treatment was very well tolerated with the most common side-effects being alopecia (100%), peripheral neuropathy (35% ofor = grade II), and arthralgia/myalgia (25% ofor = grade II). After the first course of paclitaxel, grade III/IV thrombocytopenia and neutropenia were observed in 21% and 23% of the patients respectively. 23 episodes of neutropenic fever developed after 250 courses of paclitaxel therapy (8%). We conclude that paclitaxel, at this dose and schedule, is an active new drug for the treatment of non-Hodgkin's lymphoma. The activity of paclitaxel combination programmes are currently under investigation.

The synthesis of two cell-permeable analogues related to a GPI intermediate is described for studies with living trypanosomes and human (HeLa) cells. One of the analogues is metabolised by the former GPI pathway and is toxic to the parasite Trypanosoma brucei but not to human cells.

It is difficult to imagine a theme more relevant to contemporary concerns than that of this conference.1 The imagining of worlds within the World is a topic that has concerned me for some time, to the extent of nominating it in recent publications as one that is at the core of contemporary art, as it is of current being-in-the-world, and thus definitive of our contemporaneity. For example, I conclude a recent article, 'The State of Art History: Contemporary Art', with the following remarks:Placemaking, world picturing and connectivity are the most common concerns of artists these days because they are the substance of contemporary being. Increasingly, they override residual distinctions based on style, mode, medium and ideology. They are present in all art that is truly contemporary. Distinguishing, precisely, this presence in each artwork is the most important challenge to an art criticism that would be adequate to the demands of contemporaneity. Tracing the currency of each artwork within the larger forces that are shaping this present is the task of contemporary art history.2These are, I know, bold claims, but I am encouraged in pursuing them because I know them to be accurate pointers to the main (but of course not the only) concerns of contemporary artists. They are also a straightforward-if rather summary-description of the enterprise of many art critics, historians and theorists of visual cultures working throughout the world today.The second issue of the journal World Art is devoted to contemporary art. I have contributed an essay that attempts to answer the question: Can we say that contemporary art is-perhaps for the first time in history-truly an art of the world? I argue along the following lines:As biennales have for decades attested, art now comes from the whole world, from a growing accumulation of art-producing localities that no longer depend on the approval of a metropolitan centre and are, to an unprecedented degree, connected to each other in a multiplicity of ways, not least regionally and globally. Geopolitical change has shifted the world picture from presumptions about the inevitability of modernisation and the universality of EuroAmerican values to recognition of the coexistence of difference, of disjunctive diversity, as characteristic of our contemporary condition. Contemporary life draws increasing numbers of artists to imagine the world-here understood as comprising a number of contemporaneous 'natures': the natural world, built environments ('second nature'), virtual space ('third nature'), and lived interiority ('human nature')-as a highly differentiated yet inevitably connected whole. In this sense, from what we might call a planetary perspective, contemporary art may be becoming an art for the world-for the world as it is now, and as it might be.3My essay is accompanied by an insightful commentary by Marsha Meskimmon that adds much depth to my raw intuitions about the affective dimensions of the kinds of coeval ethics called for in contemporary conditions: the world 'as it might be'. Ian McLean has also contributed an important essay that ties, in his unique way, the world to Australian perceptions of it.Two ideas from within the passage I have just quoted attract attention as being key topics of relevance to this conference. For over 50 years now, a world historical shift from EuroAmerican hegemony to a declining dominance within an increasingly differentiated world geopolitical and economic order has been underway-recently, at an accelerating pace. It has found expression in art, I suggest, as a shift from various kinds of practices oriented for, against or at oblique angles to modernisation (not all of which are modernisi) to practices that arise out of contemporary conditions. I will return in my conclusion to some implications for art historical writing of this world-wide shifting from modern to contemporary art.Worlds-Within-The-WorldI want to focus the main part of my remarks on the kinds of worlds that are envisaged in the passage I just quoted. …

In this study we interrogate ~630 compounds of the Maybridge Rule of 3 Fragment Library for compounds that interact with, and inhibit TbCK. The Maybridge Rule of 3 Fragment Library is a small collection of quantifiable diverse, pharmacophoric rich, chemical entities that comply with the following criteria; MW ≤ 300, cLogP ≤ 3, H-Bond Acceptors ≤ 3, H-Bond Donors ≤ 3, Rotatable bonds (Flexibility Index) ≤ 3, Polar Surface Area ≤ 60 A2 and aqueous solubility ≥ 1 mM using LogS and high purity (≥ 95%). Comparisons between two different screening methods, a coupled enzyme activity assay and differential scanning fluorimetry, has allowed identification of compounds that interact and inhibit the T. brucei choline kinase, several of which possess selective trypanocidal activity. Screening of a comparatively small fragment library by two different screening methods has allowed identification of several compounds that interact with and inhibit TbCK, a genetically validated drug target against African sleeping sickness. Some of the inhibitory fragments were also selectively trypanocidal, considering these are relatively simple molecules with no optimization, finding low μΜ inhibitors is very encouraging. Moreover some of the morphological phenotypes of these trypanocidal compounds include cell-cycle arrests similar to those observed for the TbCK conditional knockout grown under permissive conditions.

This work was supported by a Wellcome Trust Programme grant to KM and by a Wellcome Trust Strategic award to the Centre for Immunity, Infection and Evolution at the University of Edinburgh. SM was supported by a studentship from the Medical Research Council, UK. The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically 'slender forms' to transmissible 'stumpy forms' through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms), irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms. Publisher PDF

This study examines small intestinal absorption-permeability, intestinal inflammation and ileal structure and function in HIV-positive male homosexuals.Thirty HIV-seropositive male homosexuals at various stages of disease underwent intestinal absorption permeability and 111indium leukocyte studies (for quantification of intestinal inflammation). Twenty-six men with AIDS had a dual radioisotopic ileal function test (whole body retention of tauro 23-[75Se]-selena 25-homocholic acid and 58cobalt-labelled cyanocobalamine), and 17 underwent ileocolonoscopy with terminal ileal biopsy.Well, HIV-infected, subjects had normal intestinal absorption-permeability, but both functions were impaired upon the development of AIDS. The median faecal excretion of 111indium in well patients (0.66%) did not differ significantly (P0.5) from controls (0.46%), but subjects with AIDS who were well or who had diarrhoea had significant (P0.005) intestinal inflammation (1.33% and 2.18%, respectively). The median 7-day retention of tauro 23-[75Se]-selena 25-homocholic acid in well patients with AIDS (38.9%) did not differ significantly (P0.2) from controls (39.3%), whereas the absorption of 58cobalt-labelled cyanocobalamine was significantly (P0.05) lower than controls (32.1% and 59.4%). Patients with AIDS-diarrhoea had significant (P0.001) malabsorption of both the bile acid (7.7%) and vitamin B12 (8.9%) which was more severe than in Crohn's ileitis (14.2% and 30.3%, respectively). Morphometric analyses of ileal biopsies were unremarkable in AIDS.These studies demonstrate a low-grade enteropathy in patients with AIDS, severe ileal malabsorption in patients with AIDS diarrhoea and relatively minor ileal morphologic changes. Malabsorption of bile acids may play a pathogenic role in patients with AIDS and diarrhoea.

PURPOSE Hematopoietic growth factors have been shown to ameliorate the side effects of chemotherapy. Here we assess the ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to increase the dose-intensity and reduce the side effects of escalated methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) chemotherapy. PATIENTS AND METHODS A prospective randomized trial to compare escalated MVAC versus escalated MVAC with rhGM-CSF was conducted. All patients were treated at The University of Texas M.D. Anderson Cancer Center (UTMDACC) and had a metastatic or unresectable urothelial tumor. Forty-eight patients were randomized (25 to MVAC with rhGM-CSF and 23 to escalated MVAC alone). The clinical characteristics of the study populations were similar (ie, degree of tumor dissemination and performance status). RESULTS The dose-intensity in the two arms of the study did not differ significantly. No difference in the frequency of bacteriologically documented infections occurred between the two study arms. CONCLUSION The use of the hematopoietic growth factor rhGM-CSF did not result in an increased dose-intensity of escalated MVAC. The inability to increase the dose-intensity of MVAC further was a result of nonhematologic side effects of the chemotherapy. Escalation of treatment delivered at its median-tolerated dose is unlikely to result in additional therapeutic benefit for patients with common solid tumors. Future development of therapy may require the development of new agents or concepts, rather than modification of existing therapies.

γ-Glutamyl transpeptidase, an enzyme of central significance in glutathione metabolism, is inactivated by iodoacetamide, which esterifies an active site carboxyl group identified here as that of Asp-422. Treatment of the inactivated enzyme with hydroxylamine leads to de-esterification and to restoration of enzymatic activity. N-Acetylimidazole, which also inactivates the enzyme, acetylates several amino acid residues. Acetylation exposes Cys-453, which is buried in the native enzyme, to reaction with iodoacetamide. Incubation of the acetylated enzyme with glutamine produces a stabilized γ-glutamyl-enzyme form which is (a) located exclusively on the light subunit, (b) more labile to base than to acid, (c) destabilized by denaturation of the enzyme with guanidinium ions, and (d) reactive with hydroxylamine to form γ-glutamylhydroxamate. Stabilization of the γ-glutamyl-enzyme appears to be associated with acetylation of lysine residues (including Lys-99). These and other findings suggest that the α-amino group of the γ-glutamyl substrate is linked electrostatically to Asp-422 so as to facilitate reaction of the γ-carbonyl of the substrate with an enzyme hydroxyl group to form a γ-glutamyl-enzyme.

Acivicin is a potent inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), an enzyme of importance in glutathione metabolism. Acivicin inhibition and binding are prevented by gamma-glutamyl substrates and analogs (e.g., serine plus borate), consistent with the previous postulate that acivicin and substrates bind to the same enzyme site. Inactivation of rat kidney transpeptidase by acivicin leads to its binding as an ester to Thr-523. The pig enzyme, which has Ala-523 in place of Thr-523, is inhibited by acivicin with esterification at Ser-405. The human enzyme has Thr-524 (corresponding to Thr-523 in rat); its inactivation leads to esterification of Ser-406 (corresponding to Ser-405 in rat and pig). Hydroxylamine treatment of the acivicin-inactivated enzymes restores activity and releases the acivicin-derived threo-beta-hydroxyglutamate moiety. The findings indicate that there are significant structural differences between the active site region of the rat enzyme and the active site regions of the human and pig. Human mutant enzymes in which Thr-524 and Ser-406 were replaced by Ala, separately and together, are enzymatically active, indicating that these amino acid residues are not required for catalysis. However, esterification of these residues (and of another near the active site) effectively blocks the active site or hinders its function. Acivicin can bind at enzyme sites that are close to that at which gamma-glutamylation occurs; it may bind at the latter site and then be transesterified to another enzyme site.

The Hubble Space Telescope made systematic observations of the split comet P/Shoemaker-Levy 9 (SL9) (P designates a periodic comet) starting in July 1993 and continuing through mid-July 1994 when the fragments plunged into Jupiter's atmosphere. Deconvolutions of Wide Field Planetary Camera images indicate that the diameters of some fragments may have been as large as approximately 2 to 4 kilometers, assuming a geometric albedo of 4 percent, but significantly smaller values (that is,1 kilometer) cannot be ruled out. Most of the fragments (or nuclei) were embedded in circularly symmetric inner comae from July 1993 until late June 1994, implying that there was continuous, but weak, cometary activity. At least a few nuclei fragmented into separate, condensed objects well after the breakup of the SL9 parent body, which argues against the hypothesis that the SL9 fragments were swarms of debris with no dominant, central bodies. Spectroscopic observations taken on 14 July 1994 showed an outburst in magnesium ion emission that was followed closely by a threefold increase in continuum emission, which may have been caused by the electrostatic charging and subsequent explosion of dust as the comet passed from interplanetary space into the jovian magnetosphere. No OH emission was detected, but the derived upper limit on the H2O production rate of approximately 10(27) molecules per second does not necessarily imply that the object was water-poor.

Summary Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of prodrugs widely used to treat bacterial and trypanosome infections, but despite relative specificity, 5-nitrofurans often cause serious toxic side effects in people. Here, we use yeast and zebrafish, as well as human in vitro systems, to assess the biological activity of 5-nitrofurans, and we identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome prodrug, is dependent on zebrafish Aldh2 and is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5-nitrofuran treatment., Graphical Abstract Highlights ► Zebrafish provide a viable assay for the biological toxicity of 5-nitrofurans ► ALDH2 inhibitors prevent 5-nitrofuran toxicity in zebrafish and yeast ► Genetic dependence on ALDH2 for 5-nitrofuran toxicity in zebrafish and yeast systems ► 5-Nitrofurans bind to and are substrates of human ALDH2, 5-nitrofurans are antibiotics activated by pathogen specific enzymes, however, less is known about what happens in the host. Zhou et al. identify aldehyde dehydrogenase 2 as a 5-nitrofuran activating enzyme that has implications for managing some of the toxicity associated with 5-nitrofuran treatment.

The fluid mosaic model has recently been amended to account for the existence of membrane domains enriched in certain phospholipids. In rod-shaped bacteria, the anionic phospholipid cardiolipin is enriched at the cell poles but its role in the morphogenesis of the filamentous bacterium Streptomyces coelicolor is unknown. It was impossible to delete clsA (cardiolipin synthase; SCO1389) unless complemented by a second copy of clsA elsewhere in the chromosome. When placed under the control of an inducible promoter, clsA expression, phospholipid profile and morphogenesis became inducer dependent. TLC analysis of phospholipid showed altered profiles upon depletion of clsA expression. Analysis of cardiolipin by mass spectrometry showed two distinct cardiolipin envelopes that reflected differences in acyl chain length; the level of the larger cardiolipin envelope was reduced in concert with clsA expression. ClsA-EGFP did not localize to specific locations, but cardiolipin itself showed enrichment at hyphal tips, branch points and anucleate regions. Quantitative analysis of hyphal dimensions showed that the mycelial architecture and the erection of aerial hyphae were affected by the expression of clsA. Overexpression of clsA resulted in weakened hyphal tips, misshaped aerial hyphae and anucleate spores and demonstrates that cardiolipin synthesis is a requirement for morphogenesis in Streptomyces.

Acivicin analogues with an increased affinity for CTP synthetase (CTPS) were designed as potential new trypanocidal agents. The inhibitory activity against CTPS can be improved by increasing molecular complexity, by inserting groups able to establish additional interactions with the binding pocket of the enzyme. This strategy has been pursued with the synthesis of α-amino-substituted analogues of Acivicin and N1-substituted pyrazoline derivatives. In general, there is direct correlation between the enzymatic activity and the invitro anti-trypanosomal efficacy of the derivatives studied here. However, this cannot be taken as a general rule, as other important factors may play a role, notably the ability of uptake/diffusion of the molecules into the trypanosomes. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sphingolipids are ubiquitous in eukaryotic cells where they have been attributed a plethora of functions from the formation of structural domains to polarized cellular trafficking and signal transduction. Recent research has identified and characterised many of the key enzymes involved in sphingolipid metabolism and this has lead to a heightened interest in the possibility of targeting these processes for therapies against cancers, Alzheimer’s disease and numerous important human pathogens. In this review we outline the major pathways in eukaryotic sphingolipid metabolism and discuss these in relation to disease and therapy for both chronic and infectious conditions. Publisher PDF

Inactivation of gamma-glutamyl transpeptidase by acivicin (L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid) is rapid, thought to be irreversible, and associated with binding of close to 1 mol of inhibitor/mol of enzyme. Previous studies with [3-14C]acivicin indicated binding (prevented by substrate) to a specific hydroxyl group (threonine 523) of the rat kidney enzyme. In the present work, we found that such inactivation can be reversed by treating the inhibited enzyme with hydroxylamine. Reactivation (more than 85% complete) is associated with release from the inactivated enzyme of compounds that exhibit the properties of threo-beta-hydroxy-L-gamma-glutamyl hydroxamate and 3-hydroxypyrrolidone-2-carboxylate. We found that the enzyme acts very slowly on acivicin, at a rate that is about 10(-9) that of its normal catalytic rate with glutathione, to form threo-beta-hydroxy-L-glutamate and hydroxylamine. The findings indicate that inhibition by acivicin involves its transformation on the enzyme to an inhibitory species which is attached, apparently by ester linkage, to a specific hydroxyl group of the enzyme. The very slow rate of release of this intermediate appears to account for the observed inhibition.

One hundred twenty-seven adults with advanced hematologic malignancies received thiotepa 450-750 mg/m2, busulfan 10 or 12 mg/kg, and cyclophosphamide 120 or 150 mg/kg as a preparative regimen for autologous (86 patients) or allogeneic (41 patients) marrow transplantation. Early regimen-related toxicity (RRT) was scored according to the Seattle toxicity grading system. Grade 1-4 RRT occurred in 94% of the patients. Grade 3-4 RRT was noted in 19 patients (9% of the autologous and 27% of the allogeneic marrow recipients) and included 6% hepatic, 5% pulmonary, 3% renal, 2% mucosal, 2% bladder, 2% cardiac, and 1% CNS toxicity at the grade 3 or 4 level. No patient experienced life-threatening or fatal gastrointestinal or cutaneous toxicity. A stepwise logistic regression analysis suggested that the higher busulfan dose, Zubrod performance status of 2 or 3, and ten or more previous cycles of chemotherapy were factors predictive of grade 3-4 RRT. The regimen-related mortality for all patients was 8% (95% Cl 4-14%). The incidence and spectrum of RRT for this novel drug combination are similar to those reported for the standard preparative regimens. Heavily pretreated patients with poor performance status receiving the higher busulfan dose have a higher incidence of severe or fatal RRT.

PURPOSE To use the polymerase chain reaction (PCR) technique for molecular assessment of the results of myeloablative treatment of follicular lymphoma with autologous bone marrow transplantation. PATIENTS AND METHODS Seventy-six patients with follicular or transformed follicular lymphoma were treated with cyclophosphamide 60 mg/kg x 2 and total-body irradiation 12 Gy, supported by autologous bone marrow transplantation. The bone marrow mononuclear cell fraction was treated in vitro with CD20 monoclonal antibody and baby rabbit complement. The PCR technique was used to identify 50 patients with amplifiable t(14; 18) translocations in biopsy material from lymph nodes or bone marrow infiltrated by lymphoma. RESULTS Following treatment of the harvested bone marrow in vitro, 29 samples were tested by PCR to assess the efficacy of purging. In 25 cases, the same t(14; 18) sequences were amplified as from the patients' original biopsies, while in four cases, the marrow became PCR-negative. Three of the four patients treated with PCR-negative marrow subsequently developed recurrent lymphoma, compared with 11 of 25 in the PCR-positive group. Bone marrow and peripheral-blood mononuclear cell samples from 27 patients were studied during the follow-up period. All but one had the presence of the lymphoma-related t(14; 18) clone detectable by PCR and confirmed by direct sequencing from at least one sample between 3 months and 7 years after reinfusion of the bone marrow. With a median follow-up duration of 3 years, 13 patients developed recurrent disease, 13 remained in remission with the t(14; 18) still detectable, and one died of acute myeloid leukemia. CONCLUSION This form of therapy does not eliminate the lymphoma-related t(14; 18)-bearing clone of cells, although the significance of its continued presence is uncertain. Improved methods for both treatment of the bone marrow in vitro and treatment of the lymphoma in vivo are required.

The Hubble Space Telescope observed the fragmented comet P/Shoemaker-Levy 9 (1993e) (P indicates that it is a periodic comet) on 1 July 1993. Approximately 20 individual nuclei and their comae were observed in images taken with the Planetary Camera. After subtraction of the comae light, the 11 brightest nuclei have magnitudes between approximately 23.7 and 24.8. Assuming that the geometric albedo is 0.04, these magnitudes imply that the nuclear diameters are in the range approximately 2.5 to 4.3 kilometers. If the density of each nucleus is 1 gram per cubic centimeter, the total energy deposited by the impact of these 11 nuclei into Jupiter's atmosphere next July will be approximately 4 x 10(30) ergs ( approximately 10(8) megatons of TNT). This latter number should be regarded as an upper limit because the nuclear magnitudes probably contain a small residual coma contribution. The Faint Object Spectrograph was used to search for fluorescence from OH, which is usually an excellent indicator of cometary activity. No OH emission was detected, and this can be translated into an upper limit on the water production rate of approximately 2 x 10(27) molecules per second.

Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of acyl carrier protein (ACP), a key player in this fatty acid synthase pathway. We found that ACP depletion not only caused small changes in cellular phospholipids but also, surprisingly, caused changes in the kinetoplast. This structure, which contains the mitochondrial genome in the form of a giant network of several thousand interlocked DNA rings (kinetoplast DNA [kDNA]), became larger in some cells and smaller or absent in others. We observed the same pattern in isolated networks viewed by either fluorescence or electron microscopy. We found that the changes in kDNA size were not due to the disruption of replication but, instead, to a defect in segregation. kDNA segregation is mediated by the tripartite attachment complex (TAC), and we hypothesize that one of the TAC components, a differentiated region of the mitochondrial double membrane, has an altered phospholipid composition when ACP is depleted. We further speculate that this compositional change affects TAC function, and thus kDNA segregation.

International audience; OBJECTIVES: to investigate the anti-kinetoplastid activity of choline-derived analogues with previously reported antimalarial efficacy. METHODS: from an existing choline analogue library, seven antimalarial compounds, representative of the first-, second- and third-generation analogues previously developed, were assessed for activity against Trypanosoma and Leishmania spp. Using a variety of techniques, the effects of choline analogue exposure on the parasites were documented and a preliminary investigation of their mode of action was performed. RESULTS: the activities of choline-derived compounds against Trypanosoma brucei and Leishmania mexicana were determined. The compounds displayed promising anti-kinetoplastid activity, particularly against T. brucei, to which 4/7 displayed submicromolar EC(50) values for the wild-type strain. Low micromolar concentrations of most compounds cleared trypanosome cultures within 24-48 h. The compounds inhibit a choline transporter in Leishmania, but their entry may not depend only on this carrier; T. b. brucei lacks a choline carrier and the mode of uptake remains unclear. The compounds had no effect on the overall lipid composition of the cells, cell cycle progression or cyclic adenosine monophosphate production or short-term effects on intracellular calcium levels. However, several of the compounds, displayed pronounced effects on the mitochondrial membrane potential; this action was not associated with production of reactive oxygen species but rather with a slow rise of intracellular calcium levels and DNA fragmentation. CONCLUSIONS: the choline analogues displayed strong activity against kinetoplastid parasites, particularly against T. b. brucei. In contrast to their antimalarial activity, they did not act on trypanosomes by disrupting choline salvage or phospholipid metabolism, instead disrupting mitochondrial function, leading to chromosomal fragmentation.

Inositol-3-phosphate synthase (INO1) has previously been genetically validated as a drug target againstTrypanosoma brucei, the causative agent of African sleeping sickness. Chemical intervention of this essential enzyme could lead to new therapeutic agents. Unfortunately, no potent inhibitors of INO1 from any organism have been reported, so a screen for potential novel inhibitors ofT. bruceiINO1was undertaken. Detection of inhibition ofT. bruceiINO1 is problematic due to the nature of the reaction. Direct detection requires differentiation between glucose-6-phosphate and inositol-3-phosphate. Coupled enzyme assays could give false positives as potentially they could inhibit the coupling enzyme. Thus, an alternative approach of differential scanning fluorimetry to identify compounds that interact withT. bruceiINO1 was employed to screen ~670 compounds from the MayBridge Rule of 3 Fragment Library. This approach identified 38 compounds, which significantly altered the Tmof TbINO1. Four compounds showed trypanocidal activity with ED50s in the tens of micromolar range, with 2 having a selectivity index in excess of 250. The trypanocidal and general cytotoxicity activities of all of the compounds in the library are also reported, with the best having ED50S of ~20 μM againstT. brucei.

Two hundred sixty-four patients with chronic lymphocytic leukemia were treated with fludarabine 30 mg/m2 intravenously for 30 minutes each day for 5 days and with prednisone 30 mg/m2 orally each day for 5 days. Courses were repeated monthly. Of the 264 patients. 125 patients (47%) had Rai stage III-IV disease; 169 patients (64%) were previously treated with a median of 3 prior regimens; and 138 of them (82%) were refractory to therapy with alkylating agents. The overall response (OR) and complete response (CR) rates in the 169 previously-treated patients were 52% and 37%; these were 74% and 63%, respectively, in Rai stage O- II patients and declined to 64% and 46%, respectively, in Rai III-IV disease. Among the previously untreated patients, the OR and CR rates were 79% and 63%, these being 85% and 70%, respectively, in Rai O-II patients, and declining to 64% and 46%, respectively, in Rai III-IV disease. The incidence of minor infections or fever of unknown origin was similar in all patient groups and occurred in 22% of courses. The incidence of sepsis and/or pneumonia was significantly correlated with the extent of prior therapy and with Rai stage, and ranged from 3% of courses in the previously untreated Rai O-II patients, to 13% of courses in the previously treated Rai III-IV patients. Listeria sepsis or Pneumocystis carinii pneumonia was noted in 14 patients. With therapy, CD4 levels were uniformly depressed from a median 1,015/microL pretreatment to a median 159/microL after 3 months of fludarabine therapy. Median time to progression in previously treated patients was 22 months. In previously untreated patients, median time to progression was 30 months for patients who achieved a partial remission and has not been reached in patients who achieved a CR with a median follow-up of 2 years. The median survival was 18 months for previously treated patients and has not been reached for previously untreated patients. Response rates in previously treated and untreated patients, as well as infection rates, were identical to those seen in 110 patients treated with the same dose schedule of fludarabine alone. Logistic regression analysis selected 4 factors to be significantly associated with worse response: Rai III-IV stage disease, prior therapy, older age, and low albumin levels. The regression equation was used to derive a probability of response based on the 4 characteristics. When the model was applied to the same population, patients could be divided into 4 prognostic groups with different outcomes.

Purpose: In recent years, increasing numbers of patients with asymptomatic multiple myeloma have been diagnosed by chance and followed without any therapy. Those at risk for early or late disease progression should be distinguished in order to prevent complications. This study defined prognostic factors that would predict the need for early treatment. Patients and Methods: We followed 95 patients with asymptomatic multiple myeloma without chemotherapy between 1974 and 1991. Magnetic resonance imaging (MRI) of the spine was conducted in 23% of patients with normal radiographs. An increase in serum myeloma protein to more than 50 g/L or new lytic bone lesions justified the institution of chemotherapy. Response to treatment and survival were assessed, and prognostic factors were defined for early or late disease progression by standard techniques. Results: The median time to progression in all patients was 26 months. The 25 patients with either a lytic bone lesion, or both serum peak greater than 30 g/L and Bence Jones proteinuria, had the shortest median time to progression of 10 months; the 27 patients without any harmful factor remained stable for a median of 61 months. MRI confirmed bone or marrow disease in half of the patients with normal radiographs and may assist in the prognostic staging. Despite the markedly different times of disease stability, the response rates and survival after chemotherapy were similar for all groups of patients. Conclusion: Among asymptomatic patients with multiple myeloma, the extent of disease at diagnosis and the subsequent rate of disease evolution were major factors in the total survival time. These patients are a markedly heterogeneous group who may benefit from different approaches to treatment according to defined risk factors.

Deoxy and other analogues of d -glucosaminylphosphatidylinositol 1 have been synthesised and tested as substrates or inhibitors of a de- N -acetylase and mannosyltransferase (MT-1) involved in the biosynthesis of the glycosylphosphatidylinositol (GPI) membrane anchor of the parasite Trypanosoma brucei.

Supported by grants from EPSRC (EP/F011458/1) and The Wellcome Trust (086658). A stereocontrolled total synthesis of (+)-chamuvarinin, isolated from the root extract of Uvaria Chamae, utilizes a convergent modular strategy to construct the adjacently linked C15−C28 ether array, followed by a late-stage Julia−Kocienski olefination to append the butenolide motif. This constitutes the first total synthesis of (+)-chamuvarinin, defining the relative and absolute configuration of this unique annonaceous acetogenin. Publisher PDF

Phospholipase A(1) (PLA(1)) is an enzyme that hydrolyzes phospholipids and produces 2-acyl-lysophospholipids and fatty acids. This lipolytic activity is conserved in a wide range of organisms but is carried out by a diverse set of PLA(1) enzymes. Where their function is known, PLA(1)s have been shown to act as digestive enzymes, possess central roles in membrane maintenance and remodeling, or regulate important cellular mechanisms by the production of various lysophospholipid mediators, such as lysophosphatidylserine and lysophosphatidic acid, which in turn have multiple biological functions.

The first convenient synthesis of enantiomerically pure (αS,5S)-α-amino-3-bromo-4,5-dihydroisoxazol-5-yl acetic acid (3-bromoacivicin) is described. We demonstrate that 3-bromoacivicin is a CTP synthetase inhibitor three times as potent as its 3-chloro analogue, the natural antibiotic acivicin. Because CTP synthetase was suggested to be a potential drug target in African trypanosomes, the in vitro/in vivo antitrypanosomal activity of 3-bromoacivicin was assessed in comparison with acivicin. Beyond expectation, we observed a 12-fold enhancement in the in vitro antitrypanosomal activity, while toxicity against mammalian cells remained unaffected. Despite its good in vitro activity and selectivity, 3-bromoacivicin proved to be trypanostatic and failed to completely eradicate the infection when tested in vivo at its maximum tolerable dose.

SUMMARYThe biological membranes ofTrypanosoma bruceicontain a complex array of phospholipids that are synthesizedde novofrom precursors obtained either directly from the host, or as catabolised endocytosed lipids. This paper describes the use of nanoflow electrospray tandem mass spectrometry and high resolution mass spectrometry in both positive and negative ion modes, allowing the identification of ~500 individual molecular phospholipids species from total lipid extracts of cultured bloodstream and procyclic formT. brucei. Various molecular species of all of the major subclasses of glycerophospholipids were identified including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol as well as phosphatidic acid, phosphatidylglycerol and cardolipin, and the sphingolipids sphingomyelin, inositol phosphoceramide and ethanolamine phosphoceramide. The lipidomic data obtained in this study will aid future biochemical phenotyping of either genetically or chemically manipulated commonly used bloodstream and procyclic strains ofTrypanosoma brucei. Hopefully this will allow a greater understanding of the bizarre world of lipids in this important human pathogen.

Sugar-nucleotides such as GDP-mannose, GDP-fucose and UDP-glucose are important biomolecules with a central role in carbohydrate and glycoconjugate biosynthesis, metabolism and cell signalling. Analogues and mimics of naturally occurring sugar-nucleotides are sought after as chemical tools and inhibitor candidates for sugar-nucleotide-dependent enzymes including glycosyltransferases. Many sugar-nucleotides bind to their target glycosyltransferases via coordination of the diphosphate group to a divalent metal cofactor in the active site. The identification of uncharged, chemically stable surrogates for the diphosphate group, with the ability to coordinate to a divalent metal, is therefore an important design criteria for the development of sugar-nucleotide mimics. Here, we describe the rational design and synthesis of a novel class of sugar-nucleotide mimics based on a squaryldiamide scaffold, an uncharged phosphate isostere. We demonstrate by comprehensive NMR titration experiments that the new sugar-nucleotide mimics coordinate efficiently to Mg(2+), and provide results from biological studies with a therapeutically relevant mannosyltransferase from Trypanosoma brucei. Our findings suggest that squaryldiamides are a promising template for the development of sugar-nucleotide mimics, and illustrate the considerable potential of the squarylamide group as a fragment for inhibitor design.

The glycerophospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) account for greater than 50% of the total phospholipid species in eukaryotic membranes and thus play major roles in the structure and function of those membranes. In most eukaryotic cells, PC and PE are synthesized by an aminoalcoholphosphotransferase reaction, which uses sn-1,2-diradylglycerol and either CDP-choline or CDP-ethanolamine, respectively. This is the last step in a biosynthetic pathway known as the Kennedy pathway, so named after Eugene Kennedy who elucidated it over 50 years ago. This review will cover various aspects of the Kennedy pathway including: each of the biosynthetic steps, the functions and roles of the phospholipid products PC and PE, and how the Kennedy pathway has the potential of being a chemotherapeutic target against cancer and various infectious diseases.

Elevated serum beta-2 microglobulin (beta 2M) levels are associated with poor prognosis in several lymphoproliferative disorders including multiple myeloma and lymphoma. Their prognostic relevance in acute lymphocytic leukemia (ALL) is unknown. We analyzed the associations of serum beta 2M levels at diagnosis with pretreatment characteristics and with prognosis in adult ALL.One hundred fifty-nine adults with newly diagnosed ALL were investigated. Serum beta 2M levels were determined at diagnosis, on fresh peripheral blood samples, using a radioimmunoassay, the Pharmacia beta 2 Micro RIA (Pharmacia Diagnostics, Uppsala, Sweden). Statistical correlations were assessed by standard methods, and further independent prognostic value of serum beta 2M was determined by multivariate analysis.Patients with beta 2M levels of 4.0 mg/L or above had a lower complete response rate (61% versus 80%; p = 0.02), a significantly worse survival (p0.01), and a significantly higher association with development of central nervous system (CNS) leukemia (p0.01). High beta 2M levels were more common among patients with older age, with elevated creatinine, bilirubin, and alkaline phosphatase levels, with low albumin levels, and with B-cell disease. Multivariate analysis for survival indicated the beta 2M level to be an independent prognostic variable (after adjusting for pretreatment creatinine level and age). The evaluation of beta 2M levels within low- and high-risk groups for CNS disease suggested an association of elevated beta 2M levels with a worse incidence of CNS disease in the high-risk patients.Monitoring serum beta 2M levels may provide significant prognostic information in adults with ALL and should be included in their pretreatment evaluation. Its importance in childhood ALL requires investigation.

Pteridine reductase (PTR1) is a target for drug development against Trypanosoma and Leishmania species, parasites that cause serious tropical diseases and for which therapies are inadequate. We adopted a structure-based approach to the design of novel PTR1 inhibitors based on three molecular scaffolds. A series of compounds, most newly synthesized, were identified as inhibitors with PTR1-species specific properties explained by structural differences between the T. brucei and L. major enzymes. The most potent inhibitors target T. brucei PTR1, and two compounds displayed antiparasite activity against the bloodstream form of the parasite. PTR1 contributes to antifolate drug resistance by providing a molecular bypass of dihydrofolate reductase (DHFR) inhibition. Therefore, combining PTR1 and DHFR inhibitors might improve therapeutic efficacy. We tested two new compounds with known DHFR inhibitors. A synergistic effect was observed for one particular combination highlighting the potential of such an approach for treatment of African sleeping sickness.

Background\ud \ud The African trypanosome Trypanosoma brucei is covered with a dense layer of Variant Surface Glycoprotein (VSG), which protects it from lysis by host complement via the alternative pathway in the mammalian bloodstream. Blocking VSG synthesis by the induction of VSG RNAi triggers an unusually precise precytokinesis cell-cycle arrest.\ud \ud \ud Methodology/Principal Findings\ud \ud Here, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi) from the VSG221 expression site. Metabolic labeling of the arrested cells showed that blocking VSG synthesis triggered a global translation arrest, with total protein synthesis reduced to less than 1–4% normal levels within 24 hours of induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. Polysome analysis showed a drastic decrease in polysomes in the arrested cells. No major changes were found in levels of transcription, total RNA transcript levels or global amino acid concentrations in the arrested cells.\ud \ud \ud Conclusions\ud \ud The cell-cycle arrest phenotype triggered by the induction of VSG221 RNAi is not caused by siRNA toxicity, as this arrest can be alleviated if a second different VSG is inserted downstream of the active VSG221 expression site promoter. Analysis of polysomes in the stalled cells showed that the translation arrest is mediated at the level of translation initiation rather than elongation. The cell-cycle arrest induced in the presence of a VSG synthesis block is reversible, suggesting that VSG synthesis and/or trafficking to the cell surface could be monitored during the cell-cycle as part of a specific cell-cycle checkpoint.