Isolation and partial characterization of sheep plasma α1-mucoprotein

1. 1. Sheep plasma α1-mucoprotein was isolated in an electrophoretically homogeneous state by a combination of ammonium sulphate saturation, isoelectric precipitation and preparative agar electrophoresis in a yield of approx. 150 mg/l of plasma. 2. 2. The mucoprotein was water-soluble, non-coagulable on heating at 100°, not precipitable by 1.8 M perchloric acid, 10% trichloroacetic acid but precipitable by saturated ammonium sulphate solution, 0.6 M sulfosalicylic acid and 5% phosphotungstic acid in 2 N HCl. It had E1 cm1 % value of 9.57 at 278 mμ in water, refractive-index index increment 1.9·10-4 (g/l) in water, isoelectric point at pH 4.45 (sodium acetate-acetic acid buffer) and was homogeneous in pH range 4.0-11.5 but at pH values 2.6 and 3.5 showed some dissociation. 3. 3. The mucoprotein had the following chemical composition: Nitrogen, 12.4%; polypeptide, 77.4%; total hexose (only mannose and galactose), 7.1%; fucose, 1.0%; glucosamine, 4.9% and sialic acid, 4.8%. It had no N-terminal amino acid.

Isolation and characterization of a naturally occurring inhibitor from mung bean (Vigna radiata) seedlings for serine hydroxymethyltransferase

A naturally occurring inhibitor of serine hydroxymethyltransferase (EC2.1.2.1) in mung bean seedlings extracts was purified by ammonium sulphate precipitation, phenyl-Sepharose chromatography followed by heating to release the inhibitor bound to the protein. The inhibitor had an absorption maximum at 200 nm, was not precipitated by trichloroacetic acid, was dialysable and resistant to inactivation by heating at 98-degrees-C for 4 hr, protease and ribonuclease digestion; but was acid labile. The chromatographically pure preparation inhibited both mung bean and sheep liver SHMT. Qualitative and quantitative analyses indicated that it contained a carbohydrate moiety, an O-amino and vicinal diol groups. Paper electrophoresis at pH 4.3 suggested that the inhibitor was positively charged.

Effect of calcium depletion on the secretion of newly synthesised human chorionic gonadotropin by first trimester human placenta

Depletion of calcium in the extracellular medium used to incubate first trimester human placental minces resulted in a significant decrease in the quantity of immuno-reactive hCG in the medium and a corresponding increase in the tissue. In contrast, when secretion of newly synthesised hCG was monitored in the absence of calcium by using a radioactive amino acid precursor, a significant increase in the secretion of newly synthesised hCG in the medium was noticed. This was true of secretion of other proteins also as evidenced by the increase in the trichloroacetic acid precipitable radioactivity in the medium in the absence of calcium. These results suggest that newly synthesised hCG is preferentially released over stored hormone in the absence of calcium.

Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): Insights from kinetic and crystallographic studies

Short-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the gamma-phosphate of ATP to propionate during L-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate > acetate approximate to butyrate), nucleotides (ATP approximate to GTP > CTP approximate to TTP; dATP > dGTP > dCTP) and metal ions (Mg2+ approximate to Mn2+ > Co2+). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, alpha-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO4 (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235-290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the gamma-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer. (C) 2013 Elsevier B.V. All rights reserved.

Spin dynamics of pulsed nuclear magnetic double resonance in solids

In the first part of this thesis, experiments utilizing an NMR phase interferometric concept are presented. The spinor character of two-level systems is explicitly demonstrated by using this concept. Following this is the presentation of an experiment which uses this same idea to measure relaxation times of off-diagonal density matrix elements corresponding to magnetic-dipole-forbidden transitions in a ^(13)C-^1H, AX spin system. The theoretical background for these experiments and the spin dynamics of the interferometry are discussed also.

The second part of this thesis deals with NMR dipolar modulated chemical shift spectroscopy, with which internuclear bond lengths and bond angles with respect to the chemical shift principal axis frame are determined from polycrystalline samples. Experiments using benzene and calcium formate verify the validity of the technique in heteronuclear (^(13)C-^1H) systems. Similar experiments on powdered trichloroacetic acid confirm the validity in homonuclear (^1H- ^1H) systems. The theory and spin dynamics are explored in detail, and the effects of a number of multiple pulse sequences are discussed.

The last part deals with an experiment measuring the ^(13)C chemical shift tensor in K_2Pt(CN)_4Br_(0.3) • 3H_2O, a one-dimensional conductor. The ^(13)C spectra are strongly affected by ^(14)N quadrupolar interactions via the ^(13)C - ^(14)N dipolar interaction. Single crystal rotation spectra are shown.

An appendix discussing the design, construction, and performance of a single-coil double resonance NMR sample probe is included.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of alpha-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg(-1), 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle-regulated by both diet and CB1 receptor activity-through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB1-/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.

The nuclear matrix of Euglena gracilis (Euglenophyta): A stage of nuclear matrix evolution?

Euglena gracilis cell was extracted sequentially with CSK-Triton buffer, RSB-Magik solution and DNase-As solution. DGD embedment-free electron microscopy showed that in the extracted nucleus there was a residual non-chromatin fibrous network. That it could not be removed by hot trichloroacetic acid further supported the idea that it was a non-histone, non-chromatin fibrous protein network, and should be the internal network of the nuclear matrix. After the sequential extraction, the nuclear membrane was removed, leaving behind a layer of lamina; the chromatin was digested and eluted from the dense chromosomes and residual chromosomal structures that should be chromosomal scaffold were revealed. Western blot analysis with antiserum against rat lamins showed that nuclear lamina of the cell possessed two positive polypeptides, a major one and a minor one, which had molecular masses similar to lamin B and lamin A, respectively. Comparing these data with those of the most primitive eukaryote Archezoa and of higher eukaryotes, it was suggested that the lower unicellular eukaryote E. gracillis already had the nuclear matrix structure, and its nuclear matrix (especially the lamina) might represent a stage of evolutionary history of the nuclear matrix. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Study of competitive binding of enantiomers to protein by affinity capillary electrochromatography

Affinity capillary electrochromatography (CEC) with zonal elution method was used to probe the competitive interactions of enantiomers with protein. In this approach, a known concentration of a competing agent is continuously applied to a CEC column with bovine serum albumin (BSA) physically adsorbed on SAX packing while injections of a small amount of analyte are made. The binding sites of solutes on the BSA molecule were determined by the changes in the retention factors of the solutes resulted from the addition of competitive agent. By using D- or L-tryptophan as competitive agents and D-, L-tryptophan and benzoin enantiomers as injected analytes showed that BSA molecule has a primary site to strongly bind L-tryptophan, but D-tryptophan dose not bind at this site; D- and L-tryptophan share a weak binding site on the BSA molecule. Benzoin enantiomers do not share any binding sites with either D- or L-tryptophan. Non-chiral compounds of trichloroacetic acid and n-hexanoic acid were applied as the competitive agents to study the binding of warfarin enantiomers to BSA, it was observed that trichloroacetic acid and n-hexanoic acid had a same binding site for warfarin enantiomers binding to BSA molecule. (C) 2002 Elsevier Science B.V. All rights reserved.

The role of metabolism in the pathogenesis of adherent invasive Escherichia coli (AIEC)

Crohn’s disease (CD) is a chronic, relapsing inflammatory condition affecting the gastrointestinal tract of humans, of which there is currently no cure. The precise etiology of CD is unknown, although it has become widely accepted that it is a multifactorial disease which occurs as a result of an abnormal immune response to commensal enteric bacteria in a genetically susceptible host. Recent studies have shown that a new group of Escherichia coli, called Adherent Invasive Escherichia coli (AIEC) are present in the guts of CD patients at a higher frequency than in healthy subjects, suggesting that they may play a role in the initiation and/or maintenance of the inflammation associated with CD. Two phenotypes define an AIEC and differentiate them from other groups of E. coli. Firstly, AIEC can adhere to and invade epithelial cells; and secondly, they can replicate in macrophages. In this study, we use a strain of AIEC which has been isolated from the colonic mucosa of a CD patient, called HM605, to examine the relationship between AIEC and the macrophage. We show, using a systematic mutational approach, that while the Tricarboxylic acid (TCA) cycle, the glyoxylate pathway, the Entner-Doudoroff (ED) pathway, the Pentose Phosphate (PP) pathway and gluconeogenesis are dispensable for the intramacrophagic growth of HM605, glycolysis is an absolute requirement for the ability of this organism to replicate intracellularly. We also show that HM605 activates the inflammasome, a major driver of inflammation in mammals. Specifically, we show that macrophages infected with HM605 produce significantly higher levels of the pro-inflammatory cytokine IL-1β than macrophages infected with a non-AIEC strain, and we show by immunoblotting that this is due to cleavage of caspase-1. We also show that macrophages infected with HM605 exhibit significantly higher levels of pyroptosis, a form of inflammatory cell death, than macrophages infected with a non-AIEC strain. Therefore, AIEC strains are more pro-inflammatory than non-AIEC strains and this may have important consequences in terms of CD pathology. Moreover, we show that while inflammasome activation by HM605 is independent of intracellular bacterial replication, it is dependent on an active bacterial metabolism. Through the establishment of a genetic screen aimed at identifying mutants which activate the inflammasome to lower levels than the wild-type, we confirm our observation that bacterial metabolism is essential for successful inflammasome activation by HM605 and we also uncover new systems/structures that may be important for inflammasome activation, such as the BasS/BasR two-component system, a type VI secretion system and a K1 capsule. Finally, in this study, we also identify a putative small RNA in AIEC strain LF82, which may be involved in modulating the motility of this strain. Overall this works shows that, in the absence of specialised virulence factors, the ability of AIEC to metabolise within the host cell may be a key determinant of its pathogenesis.

Los diferentes tipos de láser y sus aplicaciones en podología

Los diferentes tipos de láseres, sobre todo el láser de diodo, irrumpen en la terapéutica podológica para proporcionar una alternativa más de tratamiento en muchas patologías que son el día a día de las consultas. El buen manejo y el conocimiento de sus características son requisitos imprescindibles para no tener efectos secundarios indeseados y poder llevar a cabo tratamientos poco dolorosos, minimizando el tiempo total, y muchas veces proporcionando una solución a diversas patologías.

A minor catalase/peroxidase from Burkholderia cenocepacia is required for normal aconitase activity

The opportunistic bacterium Burkholderia cenocepacia C5424 contains two catalase/peroxidase genes, katA and katB. To investigate the functions of these genes, katA and katB mutants were generated by targeted integration of suicide plasmids into the katA and katB genes. The catalase/peroxidase activity of the katA mutant was not affected as compared with that of the parental strain, while no catalase/peroxidase activity was detected in the katB mutant. However, the katA mutant displayed reduced resistance to hydrogen peroxide under iron limitation, while the katB mutant showed hypersensitivity to hydrogen peroxide, and reduced growth under all conditions tested. The katA mutant displayed reduced growth only in the presence of carbon sources that are metabolized through the tricarboxylic acid (TCA) cycle, as the growth defect was abrogated in cultures supplemented with glucose or glycerol. This phenotype was also correlated with a marked reduction in aconitase activity. In contrast, aconitase activity was not reduced in the katB mutant and parental strains. The authors conclude that the KatA protein is a specialized catalase/peroxidase that has a novel function by contributing to maintain the normal activity of the TCA cycle, while KatB is a classical catalase/peroxidase that plays a global role in cellular protection against oxidative stress.

Study of predictive biomarkers of manganese toxicity : control and prevention of neurotoxicity associated with parenteral nutrition

Tese de doutoramento, Farmácia (Toxicologia), Universidade de Lisboa, Faculdade de Farmácia, 2014

Full-scale biological phosphorus removal: quantification of storage polymers, microbial performance and metabolic modelling

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímica

(A) BODIPY as a versatile fluorophore (B) Synthesis of PNAs via Staudinger reaction

(A) In recent years, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores have attracted considerable interest due to their unique photochemical properties. However detailed studies on the stability of BODIPY and analogues under acidic and basic conditions have been lacking. Thus the stability of a series of BODIPY analogues in acidic (di- and trichloroacetic acid) and basic (aqueous ammonium hydroxide) conditions was investigated using 11B NMR spectroscopy. Among the analogues tested, 4,4-diphenyl BODIPY was the most stable under the conditions used in the experiments. It was found that reaction of 4,4-dimethoxy BODIPY with dichloroacetic acid gave mixed anhydride 4,4-bis(dichloroacetoxy) BODIPY in good yields. Treatment of the latter mixed anhydride with alcohols such as methanol and ethanol in the presence of a base afforded corresponding borate esters, whereas treatment with 1,2-diols such as ethylene glycol and catechol in the presence of a base gave corresponding cyclic borate esters. Furthermore treatment of 4,4-difluoro-8-methyl-BODIPY with secondary amines in dihalomethane resulted in carbon–carbon bond formation at the meso-methyl position of BODIPY via Mannich-type reactions. The resulting modified BODIPY fluorophores possess high fluorescent quantum yields. Five BODIPY analogues bearing potential ion-binding moieties were synthesized via this Mannich-type reaction. Among these, the BODIPY bearing an aza-18-crown-5 tether was found to be selective towards copper (II) ion, resulting in a large blue shift in absorption and sharp fluorescent quenching, whereas aza-15-crown-4 analogue was selected towards fluoride ion, leading to effective florescent quenching and blue shift. (B) Peptide nucleic acids (PNA), as mimics of natural nucleic acids, have been widely applied in molecular biology and biotechnology. Currently, the preparation of PNA oligomers is commonly achieved by a coupling reaction between carboxyl and amino groups in the presence of an activator. In this thesis attempts were made towards the synthesis of PNA through the Staudinger ligation reactions between C-terminal diphenylphosphinomethanethiol thioesters and N-terminal α-azido PNA building blocks.

Effect of chymosin and salt reduction on the quality of ultrafiltrated white-salted cheese

This study demonstrated that both chymosin and salt-in-moisture (SM) were important factors for proteolysis in the manufacture of ultrafiltrated white-salted cheese, with significant effects on water-soluble nitrogen and nitrogen soluble in trichloroacetic acid. In contrast, the levels of free amino acids were not significantly affected by chymosin and salt treatments. The cheeses made using high levels of chymosin with low SM had lower levels of residual α(s1)- and β-casein at the end of ripening. On texture profile analysis, the hardness and fracturability of the cheeses significantly increased with SM and decreased during ripening. Increases in chymosin significantly contributed to the overall weakening of the structure throughout ripening. Bitter flavour was detected after 12 weeks in the cheese made with the higher chymosin level and lower SM, which could be the result of accumulation of γ-casein fractions. The sensory data indicated that the hedonic responses for low chymosin with low SM cheeses were good and acceptable in flavour, which may be due to the moderate levels of proteolysis products.