Thermodynamic characterization of substrate and inhibitor binding to Trypanosoma brucei 6-phosphogluconate dehydrogenase

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

A Calcium- and GTP-Dependent Transglutaminase in Leishmania infantum

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 Ca2+- 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 Ca2+- 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

Oxidative stress and menopause-related hot flashes may be independent events.

Abstract 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

Host genetics impact on SARS-CoV-2 vaccine-induced immunoglobulin levels and dynamics: The role of TP53, ABO, APOE, ACE2, HLA-A, and CRP genes

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 (P1 and P2, respectively, for IgG and NAb distribution) in the whole cohort and/or in the mRNA-based vaccine in the following genes: TP53, rs1042522 (P1 = 0.03; P2 = 0.04); ABO, rs657152 (P1 = 0.01; P2 = 0.03); APOE, rs7412/rs429358 (P1 = 0.0018; P2 = 0.0002); ACE2, rs2285666 (P1 = 0.014; P2 = 0.009); HLA-A, rs2571381/rs2499 (P1 = 0.02; P2 = 0.03); and CRP, rs2808635/rs876538 (P1 = 0.01 and P2 = 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

Glyoxylate for affinity labelling of 6-phosphogluconate dehydrogenase

Inactivation of 6PGDH by glyoxilate requires the presence of a carboxyl group, an aldehydic group and a specific reducing agent, cyanoborohydride. It appears that the inactivation is due to an affinity labelling, but a lysine nearby is required. Other enzymes binding anionic ligands are inactivated but two enzymes, which use glyoxilate as a substrate, are not inactivated by glyoxilate and cyanoborohydride. Glyoxilate is thus useful to identify specific lysines at the carboxyl binding sites in proteins

Cyclospora and Cystoisospora

Cyclospora cayetanensis and Cystoisospora belli (formerly Isospora belli) are unicellular coccidian parasites causing gastrointestinal disease, mainly in the tropical and subtropical climates. Ingestion of sporulated oocysts in fecal contaminated food, water or other transmission vehicle causes infection in susceptible subjects. Thus, they are related to poor sanitation areas, affecting young children, the elderly, and immunocompromised people, albeit in most of the cases infections are self-limiting. Also, they are among parasites causing “traveler's diarrhea” hitting persons never exposed to the parasite before, which have gone to endemic countries. Cases of cyclosporiasis in North America and Europe were also associated to importation of contaminated fresh food. Hence, globalization can increase the number of infections overworld, along with climate change. Both cyclosporiasis and cystoisosporiasis are more severe in immunosuppressed persons, notably in HIV-infected patients, being considered as opportunistic. C. belli and C. cayetanensis, together with Cryptosporidium, may also be found in the biliary system. For both C. belli and C. cayetanensis first line treatment is based on trimethoprim-sulfamethoxazole. In immunocompromised persons chronic cystoisosporiasis may also involve lymph nodes, liver, pancreas and spleen, and requires indefinite prophylaxis. Along with epidemiological and clinical features, treatment, control and prevention, parasites biology and diagnostic methods are illustrated

6-Phosphogluconate dehydrogenase and its crystal structures

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 -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

INTRACEREBRAL TOXOPLASMA AND CRYPTOCOCCAL COINFECTION IN IMMUNOCOMPROMISED PATIENT WITH SISTEMIC LUPUS ERYTHEMATOSUS.

Objectives: Despite a significant increase in the survival rate of patients with systemic lupus erythematosus (SLE), opportunistic infections represent a significant cause of morbidity and mortality. Risk factors include immunosuppressive theraphies as well as some manifestations of active SLE itself. Clinicians need to be aware about the possibility of polymicrobial infections which may cause diagnostic and therapeutic delay. Methods: We report a case of intracerebral coinfection with Cryptococcus neoformans and Toxoplasma gondii in a 29-years-old man with SLE and congenital IgA deficit, under treatment with steroids. The patient, suffering from one week of fever, vomit and diarrhea, was admitted to our hospital for a syncopal episod following which did not regain consciousness. A neurological consultancy took over stiffness in all four limbs and lockjaw, coma with decortication reaction to painful stimuli, mydriatic pupils. Brain MRI with gadolinium contrast showed the presence of interhemispheric multiple focal lesions enhancement. Results: Cerebrospinal fluid (CSF) examinations revealed 24 white blood cells/μl, glucose 1 mg/dl and protein 266 mg/dl. An India-ink preparation of CSF disclosed mucinous capsule of Cryptococcus as a translucent halo surrounding budding yeast. A latex agglutination test was positive for cryptococcal antigen at a diluition of 1:4096 and CSF culture grew C. neoformans. HIV serology was negative but lymphocyte and CD4 lymphocyte count were 308/μl and 190/μl, respectively. CD4/CD8 ratio was 2.00. He was treated with liposomal amphotericin B (2 mg/kg/die) and dexamethasone (32 mg/die). Because of severe immunosuprression, other opportunistic pathogens were investigated. A qualitative in house PCR resulted positive for T. gondii DNA (B1 gene) in CSF, peripheral blood mononuclear cells and serum. Anamnestic T. gondii serology was negative. The therapeutic regimen was promptly strengthened with cotrimoxazole (20mg/kg/die+100mg/Kg/die), but a new CT scan demonstrated a diffuse cerebral swelling. Because of severe general impairment, the patient died two days later. Conclusions: Patients with severe SLE under immunosuppressive treatment with steroids may undergo acute infections including those from virus, common bacteria and Mycobacteria, fungi (i.e C. neoformans) and parasites (i.e T. gondii) which can occur alone or rarely in combination. In this regard, only one case of intracerebral coinfection with Burkholderia pseudomalley and C. neoformans has been recently described. To our knowledge, the presented case is the first describing an intracerebral coinfection with both C. neoformans and T. gondii in a patient with SLE. Monitoring CD4 lymphocyte count is highly recommended in patients with SLE under immunosuppressive treatment; if lower than 200/μl, they should assume cotrimoxazole prophylaxis, as HIV+ patients. In case of detection of brain lesions, rapid molecular diagnosis for more than one opportunistic infection should be taken into account as failure to timely recognition of coinfections may lead to insufficient treatment and affect outcome