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A highly polymorphic DNA probe (3′HVR) with genetic linkage to the locus of autosomal dominant polycystic kidney disease was used for screening. Families with subjects at risk were from the Po river delta region (Northern Italy), where the disease accounts for 24% of the demands for dialysis. 3′HVR alleles were investigated in white blood cell DNA from 142 members of 18 families. The genomic marker was found informative in 88% of cases. Two recombinations between the marker and the disease locus were observed in 79 meioses. In 42 of the subjects at risk the results of DNA analysis and renal ultrasonography were compared. In 36 subjects the tests confirmed each other (18 were positive). In the other six subjects (all under 20 years of age and four under 10) only DNA analysis could diagnose the inheritance of cystic disease in the absence of demonstrable cysts. The findings indicate that in the population of the Po river delta the presymptomatic detection of adult polycystic kidney disease by 3′HVR linkage analysis is feasible in 88% of cases with approximately 95% reliability. [ABSTRACT FROM PUBLISHER]

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway. It has been extensively studied by biochemical and structural techniques. 13 X-ray crystal structures and five electron cryo-microscopy structures in the PDB are focused on in this topical review. Two F 420 -dependent glucose-6-phosphate dehydrogenase (FGD) structures are also reported. The significant differences between human and parasite G6PDs can be exploited to find selective drugs against infections such as malaria and leishmaniasis. Furthermore, G6PD is a prognostic marker in several cancer types and is also considered to be a tumour target. On the other hand, FGD is considered to be a target against Mycobacterium tuberculosis and possesses a high biotechnological potential in biocatalysis and bioremediation., (open access.)

Plain language summary Pyrazolo[3,4- d ]pyrimidines are chemical compounds possessing remarkable versatility and significance in both biological and chemical contexts. These compounds are composed of specific arrangements of atoms, forming a unique ring structure, which is able to form bonds in a similar way as purines do. In the realm of chemistry, pyrazolo[3,4- d ]pyrimidines showcase impressive flexibility due to their ability to easily react with various molecules, opening avenues for the creation of novel compounds with diverse properties for potential applications in medicinal chemistry. In a biological context, pyrazolo[3,4- d ]pyrimidines play a crucial role due to their interaction with proteins such as enzymes. In fact, these compounds can impact various biological processes, including cancer cell proliferation, oxidative stress and inflammation. This has led to investigations into their potential as therapeutic agents: by designing pyrazolo[3,4- d ]pyrimidines with specific biological targets in mind, new drugs can be developed for the effective treatment of a range of medical conditions. Finally, novel administration tools (e.g., nanomaterials and functionalized liposomes) are being studied as effective ways to overcome the main unwanted characteristics of pyrazolo[3,4- d ]pyrimidines (scarce solubility and off-target side effects), thereby increasing their efficacy and specificity toward cell targets. In conclusion, pyrazolo[3,4- d ]pyrimidines are fascinating molecules with a dual role in chemistry and biology. Their adaptability in chemical reactions makes them valuable building blocks for designing new compounds with diverse applications. Additionally, their interaction with biological molecules holds promise for the development of innovative medicines. Ongoing research into the properties and behaviors of these compounds could lead to significant advancements in both scientific fields.

Introduction: Leishmaniasis is a major health problem and its diagnosis still represents a challenge. Since consistent evidence on the comparison of serological methods is lacking, our work aims to compare five serological tests for the diagnosis of visceral and asymptomatic leishmaniasis in southern France, a region where leishmaniasis is endemic., Methodology: Serum samples from 75 patients living in Nice, France were retrospectively analyzed. They included patients affected by visceral leishmaniasis (VL; n = 25), asymptomatic carriers (AC; n = 25) and negative controls (n = 25). Each sample was tested using two immunochromatographic tests (ICT; IT LEISH® and TruQuick IgG/IgM®), an indirect fluorescent antibody test (IFAT) and two Western Blotting (WB; LDBio BIORAD® and an in-house method)., Results: Diagnosis of VL with IFAT and TruQuick® showed the highest diagnostic performance parameters. IFAT had 100% sensitivity and specificity, while TruQuick had 96% sensitivity and 100% specificity. Finally, the two tests showed high accuracy (100% for IFAT and 98% for TruQuick) for the AC group. WB LDBio® was the only method able to detect Leishmania latent infection, with a sensitivity of 92%, and a specificity of 100%, with a Negative Predictive Value (NPV) of 93%. This performance is reflected in the high accuracy of the test., Conclusions: The data obtained with TruQuick® supports its application in the rapid diagnosis of leishmaniasis in endemic areas, a feature not shown by IFAT despite its high diagnostic performance. Regarding the diagnosis of asymptomatic leishmaniasis, the best results were obtained with WB LDBio®, confirming previous studies., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2023 Martina Maritati, Alessandro Trentini, Gregory Michel, Stefania Hanau, Matteo Guarino, Roberto De Giorgio, Christelle Pomares, Pierre Marty, Carlo Contini.)

While human and animal leishmaniasis affect several millions of people worldwide, L. infantum is the species responsible for visceral leishmaniasis in Europe, Middle East, and America. Antileishmanial drugs present issues associated with drug toxicity and increasing parasite resistance. Therefore, the study of this parasite with a focus on new potential drug targets is extremely useful. Accordingly, we purified and characterized a transglutaminase (TGase) from L. infantum promastigotes. While Tgases are known to be involved in cell death and autophagy, it appears that these functions are very important for parasites' virulence. For the first time, we showed a Ca 2+ - and GTP-dependent TGase in Leishmania corresponding to a 54 kDa protein, which was purified by two chromatographic steps: DEAE-Sepharose and Heparin-Sepharose. Using polyclonal antibodies against a 50-amino-acid conserved region of the catalytic core of human TGase 2, we revealed two other bands of 66 and 75 kDa. The 54 kDa band appears to be different from the previously reported TGase, which was shown to be Ca 2+ - independent. Future research should address the identification of the purified enzyme sequence and, subsequently, its cloning to more comprehensively investigate its pathophysiological function and possible differences from mammal enzymes.

Background: Development and worldwide availability of safe and effective vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to fight severe symptoms of coronavirus disease 2019 (COVID-19) and block the pandemic have been a great achievement and stimulated researchers on understanding the efficacy and duration of different vaccine types. Methods: We investigated the levels of anti-SARS-CoV-2 antibodies (IgG) and neutralizing antibodies (NAbs) in 195 healthy adult subjects belonging to the staff of the University-Hospital of Ferrara (Italy) starting from 15 days up to 190 days (about 6 months) after the second dose of the BNT162b2 (Pfizer-BioNTech) mRNA-based vaccine (n = 128) or ChAdOx1 (AstraZeneca) adenovirus-based vaccine (n = 67) using a combined approach of serological and genomics investigations. Results: A strong correlation between IgG and NAb levels was detected during the 190 days of follow-up ( r 2 = 0.807; p < 0.0001) and was confirmed during the first 90 days (T1) after vaccination ( r 2 = 0.789; p = 0.0001) and 91-190 days (T2) after vaccination ( r 2 = 0.764; p = 0.0001) for both vaccine types ( r 2 = 0.842; p = 0.0001 and r 2 = 0.780; p = 0.0001 for mRNA- and adenovirus-based vaccine, respectively). In addition to age ( p < 0.01), sex ( p = 0.03), and type of vaccine ( p < 0.0001), which partially accounted for the remarkable individual differences observed in the antibody levels and dynamics, interesting genetic determinants appeared as significant modifiers of both IgG and NAb responses among the selected genes investigated ( TP53 , rs1042522; APOE , rs7412/rs429358; ABO , rs657152; ACE2 , rs2285666; HLA-A rs2571381/rs2499; CRP , rs2808635/rs876538; LZTFL1 , rs35044562; OAS3 , rs10735079; SLC6A20 , rs11385942; CFH , rs1061170; and ACE1 , ins/del, rs4646994). In detail, regression analysis and mean antibody level comparison yielded appreciable differences after genotype stratification (P 1 and P 2 , respectively, for IgG and NAb distribution) in the whole cohort and/or in the mRNA-based vaccine in the following genes: TP53 , rs1042522 (P 1 = 0.03; P 2 = 0.04); ABO , rs657152 (P 1 = 0.01; P 2 = 0.03); APOE , rs7412/rs429358 (P 1 = 0.0018; P 2 = 0.0002); ACE2 , rs2285666 (P 1 = 0.014; P 2 = 0.009); HLA-A , rs2571381/rs2499 (P 1 = 0.02; P 2 = 0.03); and CRP , rs2808635/rs876538 (P 1 = 0.01 and P 2 = 0.09). Conclusion: High- or low-responsive subjects can be identified among healthy adult vaccinated subjects after targeted genetic screening. This suggests that favorable genetic backgrounds may support the progression of an effective vaccine-induced immune response, though no definite conclusions can be drawn on the real effectiveness ascribed to a specific vaccine or to the different extent of a genotype-driven humoral response. The interplay between data from the polygenic predictive markers and serological screening stratified by demogeographic information can help to recognize the individual humoral response, accounting for ethnic and geographical differences, in both COVID-19 and anti-SARS-CoV-2 vaccinations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gemmati, Longo, Gallo, Silva, Secchiero, Zauli, Hanau, Passaro, Pellegatti, Pizzicotti, Serino, Singh and Tisato.)

6-Phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) catalyses the oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate in the context of the oxidative part of the pentose phosphate pathway. Depending on the species, it can be a homodimer or a homotetramer. Oligomerization plays a functional role not only because the active site is at the interface between subunits but also due to the interlocking tail-modulating activity, similar to that of isocitrate dehydrogenase and malic enzyme, which catalyse a similar type of reaction. Since the pioneering crystal structure of sheep liver 6PGDH, which allowed motifs common to the β-hydroxyacid dehydrogenase superfamily to be recognized, several other 6PGDH crystal structures have been solved, including those of ternary complexes. These showed that more than one conformation exists, as had been suggested for many years from enzyme studies in solution. It is inferred that an asymmetrical conformation with a rearrangement of one of the two subunits underlies the homotropic cooperativity. There has been particular interest in the presence or absence of sulfate during crystallization. This might be related to the fact that this ion, which is a competitive inhibitor that binds in the active site, can induce the same 6PGDH configuration as in the complexes with physiological ligands. Mutagenesis, inhibitors, kinetic and binding studies, post-translational modifications and research on the enzyme in cancer cells have been complementary to the crystallographic studies. Computational modelling and new structural studies will probably help to refine the understanding of the functioning of this enzyme, which represents a promising therapeutic target in immunity, cancer and infective diseases. 6PGDH also has applied-science potential as a biosensor or a biobattery. To this end, the enzyme has been efficiently immobilized on specific polymers and nanoparticles. This review spans the 6PGDH literature and all of the 6PGDH crystal structure data files held by the Protein Data Bank., (open access.)

The current study was designed to identify new compounds as potential antiproliferative drug candidates. Synthesis of heteroaromatic bicyclic and monocyclic derivatives as purine bioisosters was employed. Their antiproliferative activity was studied against U937 cancer cells. The most effective compounds were evaluated for their selectivity against cancer cells, the possible mechanism of cell death, and their interference with DNA replication. Among the synthesized compounds, only three ( 4b , 4j and 4l ) demonstrated a value of IC 50 less than 20 μM. However, two of them ( 4b and 4l ) were specific against cancer cells, with 4l presenting high selectivity. The presence of substituted pyrazolo[3,4- d ]pyrimidine core is as essential for this activity as the presence of substituents at the thiol function in 6-position.

Background: Oxidative stress (OxS) has been linked to several pregnancy-related complications. Previous studies demonstrated that lactoferrin (LF) has the ability to modulate inflammation, OxS and the immune function. Therefore, we aimed to observe whether vaginal LF administration was able to decrease OxS in the amniotic fluid (AF) of pregnant women undergoing mid-trimester genetic amniocentesis. Methods: In this open-label clinical study, 60 pregnant women were divided into three groups: CONTROLS ( n = 20), not treated with LF; LACTO 4HRS ( n = 20), treated with LF 4 h prior to amniocentesis; LACTO 12HRS ( n = 20), treated with LF 12 h prior to amniocentesis. Thiobarbituric acid reactive substances (TBARS), total antioxidant status (TAS) and oxidative stress index (OSI) were measured in AF samples. In addition, the in vitro antioxidant activity of LF on a cell line was tested. Results: LF decreased the concentration of TBARS in the AF, with LACTO 4HRS demonstrating the lowest value compared with CONTROLS ( P < 0.0001). LACTO 4HRS had higher TAS and lower OSI than CONTROLS ( P < 0.0001 for both). In vitro , LF was effective against the oxidative challenge regardless of the time of pretreatment. Conclusion: In conclusion, LF decreased both in vivo and in vitro OxS. LF administration may represent an intriguing clinical solution as an adjuvant to treat complications of pregnancy related to inflammation and OxS. Trial Registration: Clinicaltrials.gov, NCT02695563. Registered 01 March 2016-Retrospectively registered, https://clinicaltrials.gov/show/NCT02695563., (Copyright © 2020 Trentini, Maritati, Rosta, Cervellati, Manfrinato, Hanau, Greco, Bonaccorsi, Bellini and Contini.)

The aim of this study was to evaluate the influence of sex on serum paraoxonase-1 (PON1) activities and on its relationship with cardiovascular disease risk factors such as overall and central obesity. Arylesterase and lactonase activities of PON1 were assessed in 374 women and 92 men. Both arylesterase and lactonase activities were significantly higher in women compared to men ( p <0.001), irrespectively of confounders such as high density lipoprotein-cholesterol, age, smoking and body mass index or waist circumference. Sex also strongly influenced the interplay between PON1 and both fat measures, with only the arylesterase showing a significant and independent inverse correlation with the former parameter ( r  = -0.248, p <0.001) and the risk of overall obesity (odds ratio: 0.559, 95% confidence interval: 0.340-0.919) in women, but not in men; conversely, neither of the two activities remained associated with waist circumference in men or women after full adjustment. Noteworthy, the association between arylesterase and BMI in the female subsample was significant among women younger than forty-five years ( r  = -0.453, p <0.001, R 2  = 0.207). In conclusion, our study suggests that sex might chiefly influence PON1 activity and its contribution to cardiovascular disease risk. Further studies are needed to confirm and clarify our preliminary findings., Competing Interests: No potential conflicts of interest were disclosed.

Introduction: Statin therapy is often associated with muscle complaints and increased serum creatine kinase (CK). However, although essential in determining muscle damage, this marker is not specific for skeletal muscle. Recent studies on animal models have shown that slow and fast isoforms of skeletal troponin I (ssTnI and fsTnI, respectively) can be useful markers of skeletal muscle injury. The aim of this study was to evaluate the utility of ssTnI and fsTnI as markers to monitor the statin-induced skeletal muscle damage., Materials and Methods: A total of 51 patients (14 using and 37 not using statins) admitted to the intensive care unit of the University of Ferrara Academic Hospital were included in this observational study. Serum activities of CK, aldolase, alanine aminotransferase and myoglobin were determined by spectrophotometric assays or routine laboratory analysis. Isoforms ssTnI and fsTnI were determined by commercially available ELISAs. The creatine kinase MB isoform (CK-MB) and cardiac troponin I (cTnI) were evaluated as biomarkers of cardiac muscle damage by automatic analysers., Results: Among the non-specific markers, only CK was significantly higher in statin users (P = 0.027). Isoform fsTnI, but not ssTnI, was specifically increased in those patients using statins (P = 0.009) evidencing the major susceptibility of fast-twitch fibres towards statins. Sub-clinical increase in fsTnI, but not CK, was more frequent in statin users (P = 0.007). Cardiac markers were not significantly altered by statins confirming the selectivity of the effect on skeletal muscle., Conclusions: Serum fsTnI could be a good marker for monitoring statin-associated muscular damage outperforming traditional markers., Competing Interests: Conflict of interest: None declared.

Purpose: Climate changes and immunosuppression are influencing the spread of leishmaniasis and re-emergence in Northern Italy, respectively. We evaluated the prevalence of subclinical leishmaniasis in patients from a Northern Italian region with chronic inflammatory rheumatism (CIRD) receiving biological drugs (BD) and correlated it to the area of residence., Methods: DNA from PBMC of patients affected by CIRD treated with either BD for at least 5 years (Group A) or other immunosuppressive drugs (Group B) was investigated by a qPCR for Leishmania infantum kDNA and compared to healthy subjects (Group C). Variables such as sex and age, rural areas, dog ownership, type of BD administered and association between BD and steroids, were evaluated by statistical analysis., Results: A higher proportion of L. infantum DNA positivity was found in Group A than in Group C (p < 0.05), while no parasite DNA was detected in Group B. In Group A, 18/50 patients (36%) had higher rates of parasite DNA (from 1 to 136 to 1.000.000 copies/ml) than Group C (from 1 to 10 copies/ml). 14/18 (77.7%) of positive patients from Group A lived in rural areas, but no statistical differences occurred in relation to dog ownership or BD type (p < 0.0003)., Conclusions: We can speculate that exposure to rural areas appears to be a factor closely linked with the risk of developing Leishmania subclinical infection. A screening with molecular methods in patients with CIRD treated with BD living in these areas and monitoring Leishmania DNA during such therapies, would be mandatory to prevent delay in diagnosis should VL symptoms appear.

6-Aminonicotinamide (6AN) is an antimetabolite used to inhibit the NADPH-producing pentose phosphate pathway (PPP) in many cellular systems, making them more susceptible to oxidative stress. It is converted by a NAD(P) + glycohydrolase to 6-aminoNAD and 6-aminoNADP, causing the accumulation of PPP intermediates, due to their inability to participate in redox reactions. Some parasites like Plasmodium falciparum and Coccidia are highly sensitive but not all cell types showed a strong responsiveness to 6AN, probably due to the different targeted pathway. For instance, in bacteria the main target is the Preiss-Handler salvage pathway for NAD + biosynthesis. We were interested in testing 6AN on the kinetoplastid protozoan Leishmania as another model to clarify the mechanisms of action of 6AN, by using metabolomics. Leishmania promastigotes, the life-cycle stage residing in the sandfly, demonstrated a three order of magnitude higher EC50 (mM) compared to P. falciparum and mammalian cells (μM), although pre-treatment with 100 μM 6AN prior to sub-lethal oxidative challenge induced a supra-additive cell kill in L. infantum. By metabolomics, we did not detect 6ANAD/P suggesting that NAD + glycohydrolases in Leishmania may not be highly efficient in catalysing transglycosidation as happens in other microorganisms. Contrariwise to the reported effect on 6AN-treated cancer cells, we did not detect 6-phosphogluconate (6 PG) accumulation, indicating that 6ANADP cannot bind with high affinity to the PPP enzyme 6 PG dehydrogenase. By contrast, 6AN caused a profound phosphoribosylpyrophosphate (PRPP) decrease and nucleobases accumulation confirming that PPP is somehow affected. More importantly, we found a decrease in nicotinate production, evidencing the interference with the Preiss-Handler salvage pathway for NAD + biosynthesis, most probably by inhibiting the reaction catalysed by nicotinamidase. Therefore, our combined data from Leishmania strains, though confirming the interference with PPP, also showed that 6AN impairs the Preiss-Handler pathway, underlining the importance to develop compounds targeting this last route., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Matrix Metalloproteases (MMPs) and cytokines have been involved in the pathogenesis of multiple sclerosis (MS). However, no studies have still explored the possible associations between the two families of molecules. The present study aimed to evaluate the contribution of active MMP-9, active MMP-2, interleukin- (IL-) 17, IL-18, IL-23, and monocyte chemotactic proteins-3 to the pathogenesis of MS and the possible interconnections between MMPs and cytokines. The proteins were determined in the serum and cerebrospinal fluid (CSF) of 89 MS patients and 92 other neurological disorders (OND) controls. Serum active MMP-9 was increased in MS patients and OND controls compared to healthy subjects (p < 0.001 and p < 0.01, resp.), whereas active MMP-2 and ILs did not change. CSF MMP-9, but not MMP-2 or ILs, was selectively elevated in MS compared to OND (p < 0.01). Regarding the MMPs and cytokines intercorrelations, we found a significant association between CSF active MMP-2 and IL-18 (r = 0.3, p < 0.05), while MMP-9 did not show any associations with the cytokines examined. Collectively, our results suggest that active MMP-9, but not ILs, might be a surrogate marker for MS. In addition, interleukins and MMPs might synergistically cooperate in MS, indicating them as potential partners in the disease process.

Objective: At present, there is growing demand for alternative, or additional, treatments to hormone replacement therapy for menopause-related hot flashes (HF). Antioxidant supplements have been recently proposed as possible candidates for this purpose, regardless of the absence of clear evidence in support of a link between these vasomotor symptoms and oxidative stress (OxS). The aim of our study was to evaluate the association between HF and OxS serum markers in a large sample of middle-aged women., Materials and Methods: We conducted a cross-sectional study on 245 perimenopausal and early postmenopausal women (age 45-60 years). The variables examined were presence of self-reported HF and levels of 8-iso-prostaglandin F2α, 8-OH-deoxy-2'-guanosine, advanced oxidation protein products, total antioxidant power, uric acid, thiols, and paroxonase-1., Results: Seventy-six women (31%) reported to suffer from HF (either medium or high intensity). None of the peripheral markers of OxS examined was found to be significantly associated with the presence of HF., Conclusion: Taken together, our data suggest that systemic OxS might not be implicated with the onset of the climacteric vasomotor symptoms that most commonly affect women experiencing perimenopause and early postmenopause., (Copyright © 2015. Published by Elsevier B.V.)

The reductive carboxylation of ribulose-5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (6PGDH) from Candida utilis was investigated using kinetic isotope effects. The intrinsic isotope effect for proton abstraction from Ru5P was found at 4.9 from deuterium isotope effects on V and V/K and from tritium isotope effects on V/K. The presence of 6-phosphogluconate (6PG) in the assay mixture changes the magnitude of the observed isotope effects. In the absence of 6PG (D)(V/K) and (D)(V) are 1.68 and 2.46, respectively, whereas the presence of 6PG increases (D)(V/K) to 2.84 and decreases (D)(V) to 1.38. A similar increase of (T)(V/K) is observed as 6PG builds up in the reaction mixture. These data indicate that in the absence of 6PG, a slow step, which precedes the chemical process, is rate-limiting for the reaction, whereas in the presence of 6PG, the rate-limiting step follows the isotope-sensitive step. Kinetic analysis of reductive carboxylation shows that 6PG at low concentrations decreases the K(m) of Ru5P, whereas at higher concentrations, the usual competitive pattern is observed. These data indicate that full activity of 6PGDH is achieved when one subunit carries out the catalysis and the other subunit carries an unreacted 6PG. Thus, 6PG is like an allosteric activator of 6PGDH.

6-Phosphogluconate dehydrogenase is a potential target for new drugs against African trypanosomiasis. Phosphorylated aldonic acids are strong inhibitors of 6-phosphogluconate dehydrogenase, and 4-phospho-d-erythronate (4PE) and 4-phospho-d-erythronohydroxamate are two of the strongest inhibitors of the Trypanosoma brucei enzyme. Binding of the substrate 6-phospho-d-gluconate (6PG), the inhibitors 5-phospho-d-ribonate (5PR) and 4PE, and the coenzymes NADP, NADPH and NADP analogue 3-amino-pyridine adenine dinucleotide phosphate to 6-phospho-d-gluconate dehydrogenase from T. brucei was studied using isothermal titration calorimetry. Binding of the substrate (K(d) = 5 microm) and its analogues (K(d) =1.3 microm and K(d) = 2.8 microm for 5PR and 4PE, respectively) is entropy driven, whereas binding of the coenzymes is enthalpy driven. Oxidized coenzyme and its analogue, but not reduced coenzyme, display a half-site reactivity in the ternary complex with the substrate or inhibitors. Binding of 6PG and 5PR poorly affects the dissociation constant of the coenzymes, whereas binding of 4PE decreases the dissociation constant of the coenzymes by two orders of magnitude. In a similar manner, the K(d) value of 4PE decreases by two orders of magnitude in the presence of the coenzymes. The results suggest that 5PR acts as a substrate analogue, whereas 4PE mimics the transition state of dehydrogenation. The stronger affinity of 4PE is interpreted on the basis of the mechanism of the enzyme, suggesting that the inhibitor forces the catalytic lysine 185 into the protonated state.

New drugs are urgently required for Human African Trypanosomiasis (sleeping sickness), a disease which has re-emerged as a major health threat in Sub-Saharan Africa. The third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase, has been shown to be a good target for drugs. The enzyme is essential to the trypanosomes that causes sleeping sickness and structural differences when compared to its mammalian counterpart allow for selective inhibition. Three series of inhibitors have been designed, these include phosphorylated carbohydrate substrate and transition state analogues, non-carbohydrate substrate analogues and also triphenylmethane-based compounds. All have shown selective inhibition of the trypanosomal 6-phosphogluconate dehydrogenase and representatives of each have trypanocidal activity.

Rat striatal synaptosomes and slices were used to investigate the responsiveness of different populations of nerve terminals to 3-nitropropionic acid (3-NP), a suicide inhibitor of the mitochondrial enzyme succinate dehydrogenase, and to elucidate the ionic mechanisms involved. 3-NP (0.3-3 mm) stimulated spontaneous gamma-aminobutyric acid (GABA), glutamate and [3H]-dopamine efflux but left unchanged acetylcholine efflux from synaptosomes. This effect was associated with a >70% inhibition of succinate dehydrogenase, as measured in the whole synaptosomal population. The facilitation was not dependent on extracellular Ca2+ but relied on voltage-dependent Na+ channel opening, because it was prevented by tetrodotoxin and riluzole. 3-NP also elevated spontaneous glutamate efflux from slices but in a tetrodotoxin-insensitive way. To investigate whether energy depletion could change the responsiveness of nerve endings to a depolarizing stimulus, synaptosomes were pretreated with 3-NP and challenged with pulses of KCl evoking 'quasi-physiological' neurotransmitter release. 3-NP potentiated the K+-evoked GABA, glutamate and [3H]-dopamine release but inhibited the K+-evoked acetylcholine release. The 3-NP induced potentiation of GABA release was Ca2+-dependent and prevented by tetrodotoxin and riluzole whereas the 3-NP-induced inhibition of acetylcholine release was tetrodotoxin- and riluzole-insensitive but reversed by glipizide, an ATP-dependent K+ channel inhibitor. We conclude that the responsiveness of striatal nerve endings to 3-NP relies on activation of different ionic conductances, and suggest that the selective survival of striatal cholinergic interneurons following chronic 3-NP treatment (as in models of Huntington's disease) may rely on the opening of ATP-dependent K+ channels, which counteracts the fall in membrane potential as a result of mitochondrial impairment.