Reversible self-phosphorylation of asparaginase complex in Leptosphaeria michotii: characterization of associated protein kinase and protein phosphatase activities

Regulation of the asparaginase activity rhythm in L. michotii has previously been shown to be dependent on a reversible phosphorylation process. Asparaginase was isolated as a purified protein complex having self-phosphorylating capacities, which were analyzed. In vivo phosphorylation of asparaginase complex was performed synchronously with the rhythm of asparaginase activity. In vitro self-phosphorylation of asparaginase complex resulted from the activity of an ATP-Mg2+-dependent protein kinase, which phosphorylated protein at threonine residues and was not dependent on cyclic AMP, Ca2+ or calmodulin. Dephosphorylation of this complex was due to a Mg2+-Zn2+-dependent protein phosphatase, molybdate inhibited, the specificity of which, for low-molecular-weight nonprotein phosphoesters, was broad.     

L-Asparaginase activity in Leptosphaeria michotii. Isolation and properties of two forms of the enzyme

The properties of L-asparaginase (EC 3.5.1.1) in Leptosphaeria michotii (West) Sacc., which has previously been shown to have an activity rhythm, were analyzed. Two forms of L-asparaginase were isolated from acetic acid and ammonium sulfate fractionations followed by DEAE-Sephacel chromatography. The activity of L-asparaginase changed rhythmically with the same period as that of crude extracts, but the rhythms of the two enzyme forms were out of phase. The two asparaginase forms differed in their isoelectric points and the substrate concentrations for attaining half-maximal velocity; non-Michaelis-Menten kinetics for hydrolysis of L-asparagine were observed. Analyses of asparaginase form II by polyacrylamide gel electrophoresis showed that four proteins, irrespective of the phase of the activity rhythm at which the enzyme was extracted, could be detected: asparaginase oligomer (M_r 130 000 to 140 000), its dimer, an aggregate (M_r 500 000 to 600 000) having a low asparaginase activity, and a protein (M_r 60 000) without asparaginase activity; the same proteins were found in asparaginase form I. These results indicate that L. michotii asparaginase could be implicated in a protein complex.     

FANCI Binds Branched DNA and Is Monoubiquitinated by UBE2T-FANCL

FANCI is integral to the Fanconi anemia (FA) pathway of DNA damage repair. Upon the occurrence of DNA damage, FANCI becomes monoubiquitinated on Lys-523 and relocalizes to chromatin, where it functions with monoubiquitinated FANCD2 to facilitate DNA repair. We show that FANCI and its C-terminal fragment possess a DNA binding activity that prefers branched structures. We also demonstrate that FANCI can be ubiquitinated on Lys-523 by the UBE2T-FANCL pair in vitro. These findings should facilitate future efforts directed at elucidating molecular aspects of the FA pathway.Fanconi anemia (FA)⁴ is characterized by developmental defects, bone marrow failure, and a strong predisposition to cancer. FA cells exhibit exquisite sensitivity to DNA cross-linking agents and marked genomic instability, indicative of a failure to repair damaged DNA (1–3). Thirteen FA proteins have been identified, of which eight, FANC-A, -B, -C, -E, -F, -G, -L, and -M, form part of a nuclear core complex that is required to monoubiquitinate two other FA proteins, FANCD2 and FANCI. When monoubiquitinated, FANCD2 and FANCI become chromatin-associated in foci that contain various factors, including the RAD51 recombinase BRCA2 (also known as FANCD1) and PALB2 (also called FANCN), which mediate DNA repair via RAD51-catalyzed homologous recombination (4).Monoubiquitination of FANCD2 appears to be a key event for proper repair of exogenous DNA damage but also occurs during an unperturbed S phase, likely in response to stalled replication forks (4–7). FANCD2 monoubiquitination depends on the E3 ligase activity of FANCL (8) and on the E2 ubiquitin-conjugating enzyme, UBE2T (9). In vitro, FANCL and UBE2T can monoubiquitinate chicken FANCD2 (10).FANCI was identified recently as a target protein for the ATM/ATR kinase. FANCI is also monoubiquitinated, in a manner that is dependent on the FA core complex (11). In cells, a fraction of FANCD2 and FANCI associates in a complex. Moreover, the amount and monoubiquitination of these two FA proteins are co-dependent in human cells, i.e. the quantity and monoubiquitination of FANCD2 are diminished in FANCI-deficient cells and vice versa (11–14). These observations suggest that FANCI and FANCD2 form a complex integral to cellular DNA repair capacity. Mutating the ubiquitinated target lysine of FANCI (Lys-523) renders cells sensitive to DNA damage and impairs the assembly of DNA damage-induced nuclear foci of FANCD2 and FANCI (11, 14). Herein, we document studies that reveal several biochemical attributes of FANCI, including DNA binding, and its monoubiquitination, that are relevant for understanding the biological role of this key FA protein.     

Alanine aminotransferase in Leptosphaeria michotii. Isolation, properties and cyclic variations of its activity in relation to polypeptide level

Alanine aminotransferase (EC 2.6.1.2) was obtained from the fungus Leptosphaeria michotii (West) Sacc, and enriched 714-fold by a 5-step purification procedure as a dimer of M_r 110000, associated with a polypeptide of M_r 25000. Its isoelectric point was 5.25. The enzyme was active from pH 3.5 to 9.5 with a maximum at pH 7.5. Its specific activity was 6000 nkat (mg protein)⁻¹; the K_m was 6.85 m M for L-alanine and 0.2 m M for 2-oxoglutarate. The enzyme did not show any detectable activity in the presence of L-aspartate, cysteine sulfinate, α-aminobutyrate or cyclic amino acids as substrates. It did not express alanine:glyoxylate aminotransferase activity. Alanine aminotransferase in L. michotii has previously been shown to have an activity rhythm in constant temperature and darkness. The enzyme level was quantified along the activity rhythm by enzyme-linked immunosorbent assay (ELISA), using a monospecific polyclonal antibody against the purified enzyme. The cyclic variations of alanine aminotransferase activity were correlated with cyclic variations in the enzyme level.     

Changes in protein kinase and protein phosphatase properties during the cycle of asparaginase activity in Leptosphaeria michotti

Regulation of the cyclic activity of asparaginase (obtained as a purified protein complex) by a reversible auto-phosphorylation process has been previously reported in the fungus Leptosphaeria michotii (West) Sacc. In the present study, the protein complex was purified in the presence of either a mixture of 3 protein phosphatase inhibitors (fluoride, vanadate and molybdate) or EGTA, during the cycle of asparaginase activity, and the protein kinase and protein phosphatase activities characterized. (I) At the phase of increasing asparaginase activity, a Ca²⁺/calmodulin-dependent kinase activity was identified by (a) its inhibition by calmidazolium, reversed by calmodulin, and its inhibition by EGTA, but not by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole or polylysine (b) an increasing level of calmodulin bound to the complex, as estimated by enzyme-linked immunosorbent assay (ELISA). (2) At the phase of decreasing asparaginase activity, the Ca²⁺-calmodulin-dependent kinase activity disappeared and a little calmodulin remained associated with the complex: phosphorylation of the complex was increased several-fold by 1 nM okadaic acid and 25 nM inhibitor-2, and was not affected by EGTA, indicating a protein phosphatase-2A-like activity. (3) When asparaginase activity was low, a little calmodulin was bound to the complex. The kinase could phosphorylate casein and phosvitin. was inhibited by poly(Glu/Tyr 4:1)n. dichloro-(ribofuranosyl)-benzimidazole and heparin, stimulated by polylysine and not affected by calmidazolium or EGTA, just as a casein kinase 2. A Ca²⁺-dependent but calmodulin-independent protein phosphatase activity, not affected by okadaic acid and inhibitor-2. was then identified. We postulate the presence in the complex, of (a) only one protein kinase and one protein phosphatase, whose properties could change during the cycle of asparaginase activity: (b) two Ca²⁺/-binding proteins: first calmodulin, which could bind to Ca²⁺ and the casein kinase-2 form to give a Ca²⁺/calmodulin-dependent kinase, which could become Ca²⁺/calmodulin-independent following an auto-phosphorylation process: second a protein homologous to calmodulin, able to bind to the protein phosphatase-2A catalytic subunit to give a protein phosphatase-2B catalytic subunit.     

Modelling habitat distributions for multiple species using phylogenetics

In this paper, we describe an empirical approach to model community structure using phylogenetic signals. That approach combines information about the species (i.e. traits and phylogeny) with information about the habitat (i.e. environmental conditions and spatial distribution of sampling sites) and their interactions to predict the species responses (e.g. the local densities). As an application, we use the approach to model fish densities in rivers. In the model, the different species and size classes were described using a functional trait, body length, and phylogenetic eigenvectors maps whereas the sites were described using water velocity, depth, substrate composition, macrophyte cover, degree‐days, total phosphorus, and spatial eigenvector maps. The model (estimated using a regularised Poisson‐family generalised linear modelling approach) fitted the data well (likelihood‐based R²_adj = 0.512) and showed fair predictive power (likelihood‐based cross‐validation R² = 0.283) to predict the density of fish pertaining to 48 species totalling 143 combinations of species and size classes in 15 unregulated Canadian rivers. Using the model as a baseline to estimate the effect of flow regulation on community composition, we found that, with few exceptions, the densities of most fish species were lower in regulated than in unregulated rivers. Phylogenetics have been proposed to study community structure, but this is, to our knowledge, the first time phylogenetic information is used explicitly for numerical habitat modelling. We expect that models of that type will be in increasing demand now that development projects are routinely assessed through impact studies.     

Prevalence and genotypes of GB virus C/hepatitis G virus among blood donors in Central Brazil

A survey was conducted in a blood donor population of Central Brazil aiming to investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) infection and also to analyze the virus genotypes distribution. A total of 241 voluntary blood donors were interviewed at the State Blood Bank in Goiania, State of Goiás, Brazil. Blood samples were collected and serum samples tested for GBV-C/HGV RNA by polymerase chain reaction. Genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. Seventeen samples were GBV-C/HGV RNA-positive, resulting in a prevalence of 7.1% (95% CI: 4.2-11.1). A significant trend of GBV-C/HGV RNA positivity in relation to age was observed, with the highest prevalence in donors between 29-39 years old. Ten infected individuals were characterized by reporting parenteral (30%), sexual (18%), both (6%) and intrafamiliar (6%) transmission. However, 7 (40%) GBV-C/HGV RNA-positive donors did not mention any potential transmission route. RFLP analysis revealed the presence of genotypes 1 and 2 of GBV-C/HGV; more precisely, 10 (58.9%) samples were found belonging to the 2b subtype, 4 (23.5%) to the 2a subtype, and 3 (17.6%) to genotype 1. The present data indicate an intermediate endemicity of GBV-C/HGV infection among this blood donor population, and a predominant circulation of genotype 2 (subtype 2b) in Central Brazil.     

Effect of PGE1 on gluconeogenesis and glycerol esterification in perfused liver of fasted rats

Using perfused livers of rats fasted for 48 hours, glucose production and incorporation of 2-¹⁴C pyruvate (trace dose) into perfusate glucose were studied. Both were found to be inhibited by PGE₁ (infused at a concentration of 0.5 μg/min) by about 60 %. The incorporation of 1-¹⁴C glycerol into perfusate glucose and into glycerol-glyceride part of the liver glycerides were also studied, using the same test conditions. The former incorporation was significantly inhibited (56%) and the latter strongly stimulated (360 %) by PGE₁. PGE₁ had no effect on glucose production in a perfusate overloaded with sodium pyruvate, nor on pyruvate carboxylase and phospho-enolpyruvate carboxykinase activity. This was in contrast with the results obtained in perfusions with a trace dose of 2-¹⁴C pyruvate. The results showed that PGE₁, at the physiological concentration used, stimulated the incorporation of 1-¹⁴C glycerol into glycerol-glyceride part of liver glycerides and, when there was no overload of pyruvate present in the perfusion medium, inhibited gluconeogenesis at some point, possibly, but perhaps not exclusively, between the glycerol and glucose steps.     

Antimonial resistance in Leishmania donovani is associated with increased in vivo parasite burden

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis (VL). Antimonials (SSG) have long been the first-line treatment against VL, but have now been replaced by miltefosine (MIL) in the Indian subcontinent due to the emergence of SSG-resistance. Our previous study hypothesised that SSG-resistant L. donovani might have increased in vivo survival skills which could affect the efficacy of other treatments such as MIL. The present study attempts to validate these hypotheses. Fourteen strains derived from Nepalese clinical isolates with documented SSG-susceptibility were infected in BALB/c mice to study their survival capacity in drug free conditions (non-treated mice) and in MIL-treated mice. SSG-resistant parasites caused a significant higher in vivo parasite load compared to SSG-sensitive parasites. However, this did not seem to affect the strains' response to MIL-treatment since parasites from both phenotypes responded equally well to in vivo MIL exposure. We conclude that there is a positive association between SSG-resistance and in vivo survival skills in our sample of L. donovani strains which could suggest a higher virulence of SSG-R strains compared to SSG-S strains. These greater in vivo survival skills of SSG-R parasites do not seem to directly affect their susceptibility to MIL. However, it cannot be excluded that repeated MIL exposure will elicit different adaptations in these SSG-R parasites with superior survival skills compared to the SSG-S parasites. Our results therefore highlight the need to closely monitor drug efficacy in the field, especially in the context of the Kala-azar elimination programme ongoing in the Indian subcontinent.