Purification, stability and kinetic properties of highly purified adenosine deaminase from Bacillus cereus NCIB 8122

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Bacillus cereus NCIB 8122 has been purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration through Sephadex G-100, DEAE-Sephadex A-50 chromatography and ion-exchange HPLC on DEAE-Polyol. The enzyme activity is stabilized (at temperatures from 0 degrees C to 40 degrees C) by 50 mM NH4+ or K+, while it is irreversibly lost in the absence of these or a few other monovalent cations. Glycerol (24% by volume) helps the cation in stabilizing the enzyme activity above 40 degrees C, but also exerts per se a noticeable protecting effect at room temperature. B. cereus adenosine deaminase displays the following properties: Mr on Sephadex G-200, 68,000; Mr in SDS-polyacrylamide gel electrophoresis, 53,700; optimal pH-stability (in the presence of 50 mM KCl) over the range 8-11 at 4 degrees C, and maximal catalytic activity at 30 degrees C between pH 7 and 10; Km for adenosine around 50 microM over the same pH range and Km for 2'-deoxyadenosine around 400 microM.     

Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli.

The Trp phosphorescence spectrum, intensity and decay kinetics of apo-aspartate aminotransferase, pyridoxamine-5P-aspartate-aminotransferase and pyridoxal-5P-aspartate aminotransferase were measured over a temperature range 160-273 K. The fine structure of the phosphorescence spectra in low-temperature glasses, with 0-0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine-5P-enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal-5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0-0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp-sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer. All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal-5P enzyme shows a decrease in lifetime-normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal-5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.     

Electroencephalographic characteristics of sleep in subjects with paleo and neocerebellar lesions

The research was performed in order to study: 1) paleo and neocerebellar contributions in the sleep organization and 2) the electrical sleep activities at different time intervals during the functional compensation which follows the cerebellar lesion. Polygraphic sleep records (EEG, EMG, EOG) were performed on four subjects with surgical lesions more than 6 months old in cerebellar cortex (two subjects in paleo and two in neocerebellum). Another subject was studied before a surgical paleocerebellar lesion and at different time intervals after that (8th, 30th, 60th, and 90th day). Paleo and neocerebellar lesions showed different sleep abnormalities. The former induced both quantitative and qualitative alterations in the cyclic sleep organization, the latter did not show significant alterations in this organization but rather in transition between sleeping and waking and in sleep maintenance. The acute paleocerebellar lesion showed at the 8th and 30th day a strong reduction of the synchronized sleep (SS) and an increase of the desynchronized one (DS). In the successive records, 30th and 90th day, the SS/DS ratio increased to the values observed in the chronic paleocerebellar lesioned subjects.     

Discovery of the first small molecule inhibitor of human DDX3 specifically designed to target the RNA binding site: towards the next generation HIV-1 inhibitors

Efficacy of currently approved anti-HIV drugs is hampered by mutations of the viral enzymes, leading invariably to drug resistance and chemotherapy failure. Recent data suggest that cellular co-factors also represent useful targets for anti-HIV therapy. Here we describe the identification of the first small molecules specifically designed to inhibit the HIV-1 replication by targeting the RNA binding site of the human DEAD-Box RNA helicase DDX3. Optimization of a easily synthetically accessible hit (1) identified by application of a high-throughput docking approach afforded the promising compounds 6 and 8 which proved to inhibit both the helicase and ATPase activity of DDX3 and to reduce the viral load of peripheral blood mononuclear cells (PBMC) infected with HIV-1.     

Two phenylalanine ammonia lyase isoforms are involved in the elicitor-induced response of rice to the fungal pathogen Magnaporthe oryzae

Suspension cultured cells of a blast-resistant rice genotype (Oryza sativa L. cv. Gigante Vercelli) were treated with cell wall hydrolysates prepared from the fungal pathogen Magnaporthe oryzae. As a consequence, a complex pattern of phenylalanine ammonia lyase time course specific activity levels was evident. Ion-exchange chromatographic fractionation of crude extracts suggested that the early (6 h) and the late (48-72 h after elicitation) increase of activity relied upon the sequential induction of two different isoenzymes. The relative expression levels of 11 genes putatively coding for a phenylalanine ammonia lyase were measured by semi-quantitative capillary gel electrophoresis of RT-PCR products. Two genes were indeed found to be induced by treatments with the hydrolysate, and data were validated by real-time PCR. Conversely, only the early-responsive enzyme form was observed following elicitation in a blast-sensitive rice genotype (cv. Vialone nano). Therefore, the late-responsive isoform may represent a candidate gene to select for decreased sensitivity to blast.     

β-Peptoids: synthesis of a novel dimer having a fully extended conformation

Chiral imines 1a,b, already synthesized in our laboratory, were converted in good yield by reduction into the corresponding N-benzyl-γ-lactams 2a,b. Desilylation followed by oxidation of the hydroxymethyl functionality gave the N-benzyl-β-amino acids 5a,b in good yield and high purity. Starting from compound 6a, the corresponding β-peptoid dimer 8 was prepared, together with its derivatives 9 and 10, these latter displaying conformational restriction about the peptide bond, as evidenced by NMR data.     

Reliability of an incremental exercise test to evaluate acute blood lactate,heart rate and body temperature responses in Labrador retrievers

Thirteen healthy Labrador retrievers underwent a 5-stage incremental treadmill exercise test to assess its reliability. Blood lactate (BL), heart rate (HR), and body temperature (BT) were measured at rest, after each stage of exercise, and after a 20-min recovery. Reproducibility was assessed by repeating the test after 7 days. Two-way MANOVAs revealed significant differences between consecutive stages, and between values at rest and after recovery. There was also a significant reduction in physiological strain between the first and second trial (learning effect). Test reliability expressed as typical error (BL = 0.22 mmol/l, HR = 9.81 bpm, BT = 0.22°C), coefficient of variation (BL = 19.3%, HR = 7.9% and BT = 0.6%) and test–retest correlation (BL = 0.89, HR = 0.96, BT = 0.95) was good. Results support test reproducibility although the learning effect needs to be controlled when investigating the exercise-related problems commonly observed in this breed.     

Development of substituted 6-[4-fluoro-3-(piperazin-1-ylcarbonyl)benzyl]-4,5-dimethylpyridazin-3(2H)-ones as potent poly(ADP–ribose) polymerase-1 (PARP-1) inhibitors active in BRCA deficient cells

We describe an extensive SAR study in the 6-[4-fluoro-3-(substituted)benzyl]-4,5-dimethylpyridazin-3(2H)-one series which led to the identification of potent PARP-1 inhibitors, capable of inhibiting the proliferation of BRCA-1 deficient cancer cells in the low nanomolar range, and displaying >100-fold selectivity over the BRCA wild type counterparts. The series of compounds was devoid of hERG channel activity, and CYP inhibition and induction liabilities. Several analogs were stable in rat and human liver microsomes and displayed moderate rat clearance, with urinary excretion of parent as the major route of elimination.     

Simultaneous quantification of 8-iso-prostaglandin-F2α and 11-dehydro thromboxane B2 in human urine by liquid chromatography-tandem mass spectrometry

Both F₂-isoprostanes (8-iso-PGF_2α), a well-known marker of oxidative stress, and thromboxanes A₂ (TXA₂) are involved in atherosclerosis through LDL oxidation and platelet activation. Different aspects of the pathology can be described by 8-iso-PGF_2α and TXA₂ so it is important to determine both their concentrations to monitor the disease progression and/or therapy effects. We developed a simple and sensitive method based on liquid chromatography-tandem mass spectrometry, using electrospray ionization in negative-ion mode, for the simultaneous measurement of the concentration of 8-iso-PGF_2α and 11-dehydro thromboxane B₂ (11-DH-TXB₂), a TXA₂ metabolite. This method was applied to analyze urine samples collected overnight from 15 atherosclerotic patients, with documented carotid artery sclerosis (CAS), and from 20 controls. The detection limit was 0.097 pg/μL for 8-iso-PGF_2α and 0.375 pg/μL for 11-DH-TXB₂, with a linear range of 0.78-25 pg/μL; the inter- and intraday imprecision was <5% for both metabolites. These analytes were higher in CAS (P < 0.005 vs controls) and were positively correlated in patients but not in controls, even after adjustment for age and gender (r = 0.60; P = 0.032). This highly sensitive, precise, and rapid method allows for the simultaneous determination of 8-iso-PGF_2α and 11-DH-TXB₂ in human urine samples in order to evaluate oxidative stress and platelet aggregation.