Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Effects of t-butyl-4-hydroxyanisole and other phenolic antioxidants on tumoral cells and Trypanosoma parasites

The antioxidant food additives 2(3)-tert-butyl-4-hydroxyanisole (BHA), 2,6-di(tert-butyl)-p-cresol (BHT) and the methyl and propyl esters of gallic acid inhibited Trypanosoma cruzi culture growth and oxygen consumption. The I50 values for growth and oxygen uptake with BHA were 0.284 and 0.400 and for BHT 0.083 and 0.235 mM, respectively. Moreover, BHA inhibited the respiration of several tumor cells, as well as of the procyclic and bloodstream trypomastigote forms of T. brucei brucei, with I50 in the range 0.29-0.52 mM. Inhibition of the parasites' oxygen uptake by BHA was not of the pure Michaelis-Menten type, but may be of a mixed form. It is postulated that these compounds are inhibitors because they resemble ubiquinone.     

In vitro and in vivo studies of Trypanosoma cruzi DNA polymerase

One major DNA polymerase has been purified and characterized from Trypanosoma cruzi. The enzyme has a sedimentation coefficient of 6.8 S corresponding to an approximate molecular weight of 180,000 assuming a globular shape. The enzyme recognizes activated DNA very efficiently, as well as synthetic polydeoxynucleotides, whereas poly rA-dT12 is very poorly utilized. Trypanosoma cruzi DNA polymerase is not inhibited at all by aphidicolin, while araCTP inhibits the enzyme very slightly. The purified enzyme is strongly inhibited by N-ethyl maleimide, dideoxyTTP, ethidium bromide and berenil. All our attempts to find a DNA polymerase sensitive to aphidicolin in vitro have failed, nor have we been able to find a low molecular weight DNA polymerase in this organism. However, when DNA synthesis was studied in whole trypanosomes, aphidicolin was shown to inhibit DNA synthesis more efficiently than ethidium bromide and berenil.     

Role of glutathione in the susceptibility of Trypanosoma cruzi to drugs

1. Glutathione (G-SH) concentration, gamma-glutamyltranspeptidase and glutathione S-transferase activities were studied in several strains of T. cruzi epimastigotes. GSH varied from 1.04 mM for the LQ strain to 0.61 mM for the Tulahuen strain. 2. Cultures of the LQ strain presented more resistance to drugs than those of the Tulahuen. It was necessary a concentration of nifurtimox 4 times higher and one of benznidazole 10 times higher in order to inhibit approximately to 50% the growth of LQ strain cultures when compared with the Tulahuen strain. 3. Buthionine sulfoximine decreased the concentration of glutathione to about 50% in the LQ and Tulahuen strains and potentiated the toxicity of nifurtimox and benznidazole in T. cruzi epimastigote cultures. These results suggest that glutathione is an important factor in the resistance of T. cruzi to nifurtimox and benznidazole.     

Teratogenic effect of the cholesterol synthesis inhibitor AY 9944 on rat embryos in vitro.

AY 9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] is an amphiphilic cationic molecule. This chemical is an established inhibitor of cholesterol synthesis and is teratogenic in rats. The mechanisms of this teratogenicity remain to be clarified. This study used cultured rat whole embryos to ascertain whether AY 9944 had a direct effect on embryos, or whether its action was indirect, via the maternal cholesterol metabolism. Four experimental conditions were investigated: (A) controls; (B) 10 day untreated embryos were cultured in serum of treated rats; (C) 10 day untreated embryos were cultured in serum containing added AY 9944 (0-1,000 micrograms/ml); and (D) 10 day embryos from females treated on day 4 of gestation were cultured in normal serum. In group B there was no growth retardation; some slight nonspecific abnormalities were not significant. In group C, direct addition of AY 9944 to culture medium retarded growth and differentiation in a dose-dependent manner. No malformation was observed, but histological examinations showed numerous areas of cell necrosis, especially in the CNS. In group D, not only was growth retardation observed, but also characteristic malformations of AY 9944 teratogenesis, including pituitary agenesis. These results show that AY 9944 teratogenicity is initiated prior to day 10.     

In vitro interaction between ceftazidime and vancomycin/teicoplanin in the presence of azithromycin againstPseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen, intrinsically resistant to many antibiotics and prone to acquire resistance against many drugs. It is assumed that agents that disorganise the structure of the outer membrane might allow the passage of other drugs into cell. To verify this hypothesis, ceftazidime (CAZ) has been tested in association with glycopeptides (GLYs) and azithromycin (AZI). Time-kill experiments were performed on a representative strain. CAZ in combination with GLYs showed 99, 90 and 10% of CFU/ml reduction in 33.9,52.5 and 13.6% of the cases, respectively; the addition of AZI increased the incidence of 99% CFU/ml reduction to 42% of the cases. Indifference was the most common finding, and additive/synergism in the other cases. Present findings demonstrated that CAZ favourably reacted with GLYs in the presence of AZI.     

Evaluation of total reactive antioxidant potential (TRAP) of tissue homogenates and their cytosols

This study evaluates the possibility of obtaining total reactive antioxidant potential (TRAP) indexes in homogenates and their cytosolic fractions by a procedure based on the quenching of luminol luminescence induced by the thermolysis of 2,2'-azo-bis(2-amidinopropane). Measurements were performed in rat brain, liver, kidney, and heart homogenates. TRAP indexes can be easily determined both in homogenates and their cytosolic fractions. The results obtained indicate that heart homogenates are the least and liver homogenates the most protected of the systems considered. Glutathione is the measured antioxidant that contributes the most to TRAP values, while uric acid makes a significant contribution only in liver. A calculation of theoretical TRAP values from the measured concentrations of the main antioxidants (glutathione, uric acid, ascorbic acid, and alpha-tocopherol) for the different homogenates shows that, in most tissues (liver, brain, and kidney), nearly 50% of the experimentally determined TRAP values are not accounted for. This difference is mainly due to the contribution of proteins to the measured TRAP.     

Effects of buthionine sulfoximine nifurtimox and benznidazole upon trypanothione and metallothionein proteins in Trypanosoma cruzi

Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.     

Increased expression of costimulatory markers CD134 and CD80 on interleukin-17 producing T cells in patients with systemic lupus erythematosus

Introduction  

There is growing evidence that interleukin 17 (IL-17) producing T cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). Previous studies showed that increased percentages of T-cell subsets expressing the costimulatory molecules CD80 and CD134 are associated with disease activity and renal involvement in SLE. The aim of this study was to investigate the distribution and phenotypical characteristics of IL-17 producing T-cells in SLE, in particular in patients with lupus nephritis, with emphasis on the expression of CD80 and CD134.