Orosomucoid (alpha-1 acid glycoprotein) phenotyping by use of immobilized pH gradients with 8 M urea and immunoblotting. A new variant encountered in a population study.

Orosomucoid (ORM) phenotyping has been performed on 329 unrelated Swiss subjects, using immobilized pH gradients with 8 M urea and 2% v/v 2-mercaptoethanol followed by immunoblotting. After desialylation the band patterns of ORM confirmed that the polymorphism of the structural locus ORM1 is controlled by three codominant autosomal alleles (ORM1*F1, ORM1*S and ORM1*F2). One rare and one new allele were detected. The rare variant, tentatively assigned to the second structural locus ORM2, is observed in a cathodal position and named ORM2 B1. The new variant, tentatively assigned to the first structural locus ORM1, is observed in a region located between ORM1 S and ORM1 F2, and named ORM1 F3. Moreover, the pI values of the ORM variants have been measured accurately with Immobiline Dry Plates (LKB): they were found to be within the pH range 4.93-5.14.

Oxidized oils and dietary zinc and alpha-tocopheryl acetate supplementation: effects on rabbit plasma, liver and meat fatty acid composition and meat Zn, Cu, Fe and Se content

The effects of the addition of heated oils to feeds (3%, w/w) and the dietary supplementation with a-tocopheryl acetate (TA; 100 mg/kg) and Zn (200 mg/kg) on rabbit tissue fatty acid (FA) composition and on the Zn, Cu, Fe and Se content in meat were assessed. Heating unrefined sunflower oil (SO) at 558C for 245 h increased its content in primary oxidation products and reduced its a-tocopherol content. However, this did not significantly affect tissue FA composition. Heating SO at 1408C for 31 h increased its content in secondary oxidation products and in some FA isomers asc9,t11-CLA and di-trans CLA. This led to increases in di-trans CLA in liver and in t9,c12-18:2 in meat. The c9,t11-CLA was the most incorporated CLA isomer in tissues. The dietary supplementation with a-TA did not affect the FA composition of plasma, liver or meat. The cooking of vacuum-packed rabbit meat at 788C for 5 min reduced significantly but slightly its polyunsaturated FA content. The dietary supplementation with Zn did not modify the content of Zn, Fe or Se in meat, but it reduced its Cu content. On the other hand, it increased the content of some FAs in meat when SO heated at 1408C for 31 h was added to feeds.

Circadian variation of salivary immunoglobin A, alpha-amylase activity and mood in response to repeated double-poling sprints in hypoxia.

PURPOSE: To assess the circadian variations in salivary immunoglobin A (sIgA) and alpha-amylase activity (sAA), biomarkers of mucosal immune function, together with mood during 2 weeks of repeated sprint training in hypoxia (RSH) and normoxia (RSN). METHODS: Over a 2-week period, 17 competitive cross-country skiers performed six training sessions, each consisting of four sets of five 10-s bouts of all-out double-poling under either normobaric hypoxia (FiO2: 13.8 %, 3000 m) or normoxia. The levels of sIgA and sAA activity and mood were determined five times during each of the first (T1) and sixth (T6) days of training, as well as during days preceding (baseline) and after the training intervention (follow-up). RESULTS: With RSH, sIgA was higher on T6 than T1 (P = 0.049), and sAA was increased on days T1, T6, and during the follow-up (P < 0.01). With RSN, sIgA remained unchanged and sAA was elevated on day T1 only (P = 0.04). Similarly, the RSH group demonstrated reduced mood on days T1, T6, and during the follow-up, while mood was lowered only on T1 with RSN (P < 0.01). CONCLUSIONS: The circadian variation of sIgA and sAA activity, biomarkers of mucosal immune function, as well as mood were similar on the first day of training when repeated double-poling sprints were performed with or without hypoxia. Only with RSH did the levels of sIgA and sAA activity rise with time, becoming maximal after six training sessions, when mood was still lowered. Therefore, six sessions of RSH reduced mood, but did not impair mucosal immune function.

Anti-TNF alpha medications and neuropathy.

We studied the clinical, electrophysiological, and pathological features, outcome, and frequency of anti-tumor necrosis factor alpha (a-TNF) medications-induced neuropathies (ATIN) in patients with inflammatory disorders. Of 2,017 patients treated with a-TNF medication, 12 patients met our inclusion criteria for a prevalence of 0.60% and an incidence of 0.4 cases per 1,000 person-years. The median time from a-TNF medication treatment to ATIN was 16.8 months (range 2-60 months). Six patients had focal or multifocal peripheral neuropathies. The other six had generalized neuropathies. For all, a-TNF medication was stopped. Seven patients received immunoglobulin infusions. ATIN outcome was favorable in all but one patient. ATINs are rare and heterogeneous neuropathies. In 10 patients, the neuropathy was "inflammatory", suggesting that it could be due to systemic pro-inflammatory effects of a-TNF agents.

Reações de inserção intramolecular de diazo compostos polifuncionais catalisadas por ródio(II): síntese de oxetan-3-ona-2-carboxilato e outros heterociclos funcionalizados

gamma-Hydroxy-alpha-diazo-beta-ketoesters are key intermediates in the chemistry of penicilin-based antibiotics and natural products. The method developed here for the synthesis of ethyl 2-diazo-4-hydroxy-3-oxo-butanoate 17 (in two steps from the diazo mercurial 2) compares very favorably with those reported in the literature for similar compounds. The Rh2(OAc)4-mediated intramolecular OH-insertion reaction of the diazo hydroxy ester 17 was investigated, furnishing the oxetan-3-one-2-carboxilate 18 in good yield. When the diazo ester lacks a free hydroxyl group as in the case of the phenoxy diazo ester 11 an intramolecular CH-insertion takes place, affording the 2H-chromene 20 in almost quantitative yield. The behavior of other functionalized diazo esters towards Rh2(OAc)4 was also investigated.

Metodologias de síntese de 2-arilcicloexanonas

Several methodologies concerning the preparation of 2-aryl and 2-heteroarylcyclohexanones are presented. The use of these intermediates in the synthesis of chemically and biologically interesting organic compounds is also discussed.

Síntese de novos herbicidas derivados do 1,2alfa,4alfa,5-tetrametil-8-oxabiciclo[3.2.1]oct-6-en-3-ona

In this paper we report the synthesis of biologically active compounds through a [3+4] cycloaddition reaction to produce the main frame structure, followed by several conventional transformations. The 1,2alpha,4alpha,5-tetramethyl-8-oxabicyclo[3.2.1]oct-6-en-3-one (11) obtained from a [3+4] cycloaddition reaction was converted into 1,2alpha,4alpha,5-tetramethyl-6,7-exo-isopropylidenedioxi-8 -oxabicyclo[3.2.1]octan-3-one (13) in 46% yield. This was further converted into the alcohols 1,2alpha,4alpha,5-tetramethyl-6,7-exo-isopropylidenedioxi-8-oxabicyclo[3.2.1]octan-3 alpha-ol (14), 1,2alpha,4alpha,5-tetramethyl-6,7-exo-isopropylidenedioxi-8 -oxabicyclo[3.2.1]octan-3beta-ol (15), 1,2alpha,4alpha,5-tetramethyl-3-butyl-6,7-exo-isopropylidenedioxi-8-oxabicyclo[3.2.1]octan-3 alpha-ol (17), 1,2alpha,4alpha,5-tetramethyl-3-hexyl-6,7-exo-isopropylidenedioxi-8-oxabicyclo[3.2.1]octan-3 alpha-ol (18) and 1,2alpha,4alpha,5-tetramethyl-3-decyl-6,7-exo-isopropylidenedioxi-8-oxabicyclo[3.2.1]octan-3 alpha-ol (19). Dehydration of 17, 18 and 19 with thionyl chloride in pyridine resulted in the alkenes 20, 21 and 22 in ca. 82% - 89% yields from starting alcohols. The herbicidal activity of the compounds synthesized was evaluated at a concentration of 100 µg g-1. The most active compound was 21 causing 42,7% inhibition against Cucumis sativus L.

Síntese de novas fitotoxinas derivadas do 8-oxabiciclo[3.2.1]oct-6-en-3-ona

The [4+3] cycloaddition was utilized in order to prepare 8-oxabicyclo[3.2.1]oct-6-en-3-one (1) derivatives. The correspondent acetonide 6 was converted into several alcohols (11-16). Addition of aryllithium reagents to 6 resulted in 3-(2-fluorophenyl)-6,7-exo-isopropylidenedioxy -8-oxabicyclo[3.2.1]octan-3alpha-ol (11, 72%) and 3-(2,4-dimethoxyphenyl)-6,7-exo-isopropylidenedioxy-8-oxabicyclo[3.2.1]octan -3alpha-ol (16, 20%). The 3-butyl-6,7-exo-isopropylidenedioxy-8-oxabicyclo[3.2.1]octan-3 alpha-ol (15, 56%) was obtained through a Grignard reaction. Reduction of 6 resulted in 6,7-exo-isopropylidenedioxy-8-oxabicyclo[3.2.1]octan-3 beta-ol (7, 62%) and 6,7-exo-isopropylidenedioxy-8-oxabicyclo[3.2.1]octan-3 alpha-ol (8, 20%). The alcohols were treated with thionyl chloride in pyridine, and the corresponding alkenes were obtained with 31-80% yield. The effect of these compounds on the development of radicle and aerial parts of Sorghum bicolor was evaluated.

Uma nova síntese de alfa-bromo-acetofenonas e sua aplicação na obtenção de 2-benzoil-benzofuranas

alpha-Bromoacetophenones are important in organic synthesis. They have been widely used as precursors of several natural products. Several methods of bromination aiming at their synthesis have been described, however they furnish a mixture of starting material 10, mono 8 and dibromide 11 products. We developed a novel, simple and efficient synthesis of these compounds with applications in the synthesis of benzoylbenzofurans 9, compounds with important pharmacological properties, such as the ability of dilating the coronary artery and analgesic action. Such compounds have also been used as key intermediates to obtain quinone systems.

Electrical and magnetic properties of new compounds of the conductor anion [Ni(dmit)2]- and nitronyl nitroxide magnetic radicals

Three compounds have been synthesized with formulae [3-MeRad][Ni(dmit)2] (1), [4-MeRad][Ni(dmit)2] (2) and [4-PrRad][Ni(dmit)2] (3) where [Ni(dmit)2]- is an anionic pi-radical (dmit = 1,3-dithiol-2-thione-4,5-dithiolate) and [3-MeRad]+ is 3-N-methylpyridinium alpha-nitronyl nitroxide, [4-MeRad]+ is 4-N-methylpyridinium alpha-nitronyl nitroxide and [4-PrRad]+ is 4-N-propylpyridinium alpha-nitronyl nitroxide. The temperature-dependent magnetic susceptibility of 1 revealed that an antiferromagnetic interaction operates between the 3-MeRad+ radical cations with exchange coupling constants of J1 = - 1.72 cm-1 and antiferromagnetism assigned to the spin ladder chains of the Ni(dmit)2 radical anions. Compound 1 exhibits semiconducting behavior and 3 presents capacitor behavior in the temperature range studied (4 - 300 K).

Monoterpenos di-hidroxilados e hidróxi-nitrogenados 1,2 e 1,3 como ligantes quirais em reação de reformatsky assimétrica

This study describes the use of three (-)-alpha-pinene derivatives, one diol-1,2 [(-)-(1R, 2R, 3S, 5R)-2,6,6-trimethylbicyclo[3.1.1]heptane-2,3-diol 4] and two piridine-hydroxy derivatives [(+)-(1R,2S,3R,5S)-2,6,6-trimethyl-3-(2-pyridinylmethyl)bicyclo[3.1.1]heptan-3-ol 7 and (-)-(1R,2S,3R,5S)-2,6,6-trimethyl-3-[2-(2-pyridinyl) ethyl]bicyclo[3.1.1]heptan-3-ol 8]; one diol-1,3 [(-)-(1S,2R,5S)-2-(1-hydroxy-1-methylethyl)-5-methylcyclohexanol 5] derived from (+)-isopulegol 2 and one diol-1,3 [(+)-(1R,2R,5R)-2-(1-hydroxy-1-methylethyl)-5-methylcyclohexanol 6] derived from (+)-neo-isopulegol 3, as ligands in the asymmetric Reformatsky reaction. The best enantiomeric excess of beta-hydroxy ester obtained in the Reformatsky asymmetric reaction was 18% using ligand 6, and the chemical yield of the reactions was 65% on average.

Prognostic Markers and Prolonged Interferon-alpha Therapy in Renal Cell Carcinoma

Molekyylimarkkerit ja pitkäaikainen alfainterferonihoito munuaissyövässä Munuaissyöpäpotilaiden viiden vuoden elossaololuku on noin 50 %. Aikaisempien tutkimuksien mukaan viiden vuoden elossaololuku metastasoituneessa munuaissyövässä on 3-16 %, kun käytettiin alfainterferonia sisältävää hoitoa. Tyypillisesti alfainterferonia on käytetty vähemmäin kuin 6 kuukautta. Avoimia kysymyksiä ovat alfainterferonin optimaalinen hoitoannos ja hoidon kesto yksin tai yhdessä uusien täsmähoitojen kanssa. Tärkeimmät tavoitteet olivat tutkia 1) jaksotetun pitkäaikaisen alfainterferonihoidon tehoa ja siedettävyyttä metastasoituneessa munuaissyövässä ja 2) p53-, Ki-67- ja COX-2-proteiinituotannon ennusteellista merkitystä munuaissyövässä. Tutkimuksessa 117 metastasoituneelle munuaissyöpää sairastaneelle potilaalle etsittiin yksilöllinen hänen sietämänsä maksimaalinen hoitoannos rekombinanttia alfa2a-interferonia (Roferon-ATM). Hoitoa pyrittiin jatkamaan 24 kuukauden ajan. Kolmen hoitoviikon jälkeen pidettiin yhden viikon tauko. Hoito lopetettiin, jos ilmaantui vakavia haittavaikutuksia tai tauti eteni. Toisessa tutkimuksessa proteiinituotanto analysoitiin immunohistokemiallisesti munuaissyöpäpotilaiden kasvainnäytteistä, joita oli säilytetty parafiinissa. Kasvainnäytteet oli otettu talteen munuaisen poistoleikkauksen yhteydessä. Nämä potilaat jaettiin kolmeen eri ryhmään: metastasointi primaarivaiheessa (n=29), metastasointi myöhemmin (n=37) ja ei metastasointia (n=51). Keskimääräinen alfainterferonihoidon kesto oli 11 kuukautta (kk) [0,5 – 32 kk]. Objektiivinen hoitovaste todettiin 17 %:lla, tautitilanne pysyi ennallaan 42 %:lla ja myöhäinen vaste (yli 12 kk:tta hoidon aloittamisesta) todettiin 3 %:lla. Aika vasteen saavuttamisesta taudin etenemiseen oli keskimäärin 8 kk ja elinaika 19,1 kk. Viiden vuoden elossaololuku oli 16 %. Jos metastasoituneella munuaissyöpäpotilaalla oli keuhkometastasointi, hän selvisi todennäköisemmin viisi vuotta kuin muut potilaat. Henkeä uhkaavia sivuvaikutuksia ei todettu. Yli 12 kk:n ajan kestävä alfainterferonihoito on hyödyllistä niille potilaille, jotka ovat saaneet objektiivisen hoitovasteen tai tautitilanne on pysynyt ennallaan. Positiivinen p53- ja Ki-67-ekspressio yhdessä viittaavat suureen metastasoinnin todennäköisyyteen. Positiivinen COX-2-ekspressio viittaa viivästyneeseen metastaasien ilmaantumiseen. Metastasoituneilla potilailla positiiviset p53- ja Ki-67-ekspressiot viittaavat huonoon ennusteeseen, mutta positiivinen COX-2 ekspressio viittaa suotuisaan ennusteeseen. Positiivinen COX-2- ja negatiivinen Ki-67-ekspressio yhdessä viittaavat parantuneeseen ennusteeseen metastasoituneessa munuaissyövässä.

Alpha2-adrenoceptors: Structure and ligand binding properties at the molecular level

Alpha₂-Adrenoceptors: structure and ligand binding properties at the molecular level The mouse is the most frequently used animal model in biomedical research, but the use of zebrafish as a model organism to mimic human diseases is on the increase. Therefore it is considered important to understand their pharmacological differences from humans also at the molecular level. The zebrafish Alpha_2-adrenoceptors were expressed in mammalian cells and the binding affinities of 20 diverse ligands were determined and compared to the corresponding human receptors. The pharmacological properties of the human and zebrafish Alpha_2--adrenoceptors were found to be quite well conserved. Receptor models based on the crystal structures of bovine rhodopsin and the human Beta₂-adrenoceptor revealed that most structural differences between the paralogous and orthologous Alpha_2--adrenoceptors were located within the second extracellular loop (XL2). Reciprocal mutations were generated in the mouse and human Alpha_2--adrenoceptors. Ligand binding experiments revealed that substitutions in XL2 reversed the binding profiles of the human and mouse Alpha_2--adrenoceptors for yohimbine, rauwolscine and RS-79948-197, evidence for a role for XL2 in the determination of species-specific ligand binding. Previous mutagenesis studies had not been able to explain the subtype preference of several large Alpha_2--adrenoceptor antagonists. We prepared chimaeric Alpha_2--adrenoceptors where the first transmembrane (TM1) domain was exchanged between the three human Alpha_2--adrenoceptor subtypes. The binding affinities of spiperone, spiroxatrine and chlorpromazine were observed to be significantly improved by TM1 substitutions of the Alpha_2a--adrenoceptor. Docking simulations indicated that indirect effects, such as allosteric modulation, are more likely to be involved in this phenomenon rather than specific side-chain interactions between ligands and receptors.

Characterisation of Human Neutral and Lysosomal alpha-Mannosidases

Neutral alpha-mannosidase and lysosomal MAN2B1 alpha-mannosidase belong to glycoside hydrolase family 38, which contains essential enzymes required for the modification and catabolism of asparagine-linked glycans on proteins. MAN2B1 catalyses lysosomal glycan degradation, while neutral α-mannosidase is most likely involved in the catabolism of cytosolic free oligosaccharides. These mannose containing saccharides are generated during glycosylation or released from misfolded glycoproteins, which are detected by quality control in the endoplasmic reticulum. To characterise the biological function of human neutral α-mannosidase, I cloned the alpha-mannosidase cDNA and recombinantly expressed the enzyme. The purified enzyme trimmed the putative natural substrate Man9GlcNAc to Man5GlcNAc, whereas the reducing end GlcNAc2 limited trimming to Man8GlcNAc2. Neutral α-mannosidase showed highest enzyme activity at neutral pH and was activated by the cations Fe₂+, Co2+ and Mn₂+, Cu2+ in turn had a strong inhibitory effect on alpha-mannosidase activity. Analysis of its intracellular localisation revealed that neutral alpha-mannosidase is cytosolic and colocalises with proteasomes. Further work showed that the overexpression of neutral alpha-mannosidase affected the cytosolic free oligosaccharide content and led to enhanced endoplasmic reticulum associated degradation and underglycosylation of secreted proteins. The second part of the study focused on MAN2B1 and the inherited lysosomal storage disorder α-mannosidosis. In this disorder, deficient MAN2B1 activity is associated with mutations in the MAN2B1 gene. The thesis reports the molecular consequences of 35 alpha-mannosidosis associated mutations, including 29 novel missense mutations. According to experimental analyses, the mutations fall into four groups: Mutations, which prevent transport to lysosomes are accompanied with a lack of proteolytic processing of the enzyme (groups 1 and 3). Although the rest of the mutations (groups 2 and 4) allow transport to lysosomes, the mutated proteins are less efficiently processed to their mature form than is wild type MAN2B1. Analysis of the effect of the mutations on the model structure of human lysosomal alpha-mannosidase provides insights on their structural consequences. Mutations, which affect amino acids important for folding (prolines, glycines, cysteines) or domain interface interactions (arginines), arrest the enzyme in the endoplasmic reticulum. Surface mutations and changes, which do not drastically alter residue volume, are tolerated better. Descriptions of the mutations and clinical data are compiled in an α-mannosidosis database, which will be available for the scientific community. This thesis provides a detailed insight into two ubiquitous human alpha-mannosidases. It demonstrates that neutral alpha-mannosidase is involved in the degradation of cytosolic oligosaccharides and suggests that the regulation of this α-mannosidase is important for maintaining the cellular homeostasis of N-glycosylation and glycan degradation. The study on alpha-mannosidosis associated mutations identifies multiple mechanisms for how these mutations are detrimental for MAN2B1 activity. The α-mannosidosis database will benefit both clinicians and scientific research on lysosomal alpha‑mannosidosis.

Salivary and serum cortisol levels, salivary alpha-amylase and unstimulated whole saliva flow rate in pregnant and non-pregnant

PURPOSE: To compare salivary and serum cortisol levels, salivary alpha-amylase (sAA), and unstimulated whole saliva (UWS) flow rate in pregnant and non-pregnant women. METHOD: A longitudinal study was conducted at a health promotion center of a university hospital. Nine pregnant and 12 non-pregnant women participated in the study. Serum and UWS were collected and analyzed every trimester and twice a month during the menstrual cycle. The salivary and serum cortisol levels were determined by chemiluminescence assay and the sAA was processed in an automated biochemistry analyzer. RESULTS: Significant differences between the pregnant and non-pregnant groups were found in median [interquartile range] levels of serum cortisol (23.8 µL/dL [19.4-29.4] versus 12.3 [9.6-16.8], p<0.001) and sAA (56.7 U/L [30.9-82.2] versus 31.8 [18.1-53.2], p<0.001). Differences in salivary and serum cortisol (µL/dL) and sAA levels in the follicular versus luteal phase were observed (p<0.001). Median UWS flow rates were similar in pregnant (0.26 [0.15-0.30] mL/min) and non-pregnant subjects (0.23 [0.20-0.32] mL/min). Significant correlations were found between salivary and serum cortisol (p=0.02) and between salivary cortisol and sAA (p=0.01). CONCLUSIONS: Serum cortisol and sAA levels are increased during pregnancy. During the luteal phase of the ovarian cycle, salivary cortisol levels increase, whereas serum cortisol and sAA levels decline.