Inhibition of adenylate cyclase activity by 5-aminolevulinic acid in rat and human brain

The effect of the haem precursor 5-aminolevulinic acid (ALA) on the production of cyclic adenosine-monophosphate (cAMP) by rat cerebellar membranes was investigated. It was found that ALA dose-dependently decreased cAMP levels (maximal inhibition of 38%, at 1 mM), due to an inhibition of basal adenylate cyclase activity. ALA also inhibited fluoride- and Gpp(NH)p-stimulated, but not the forskolin-stimulated adenylate cyclase activity. 5-Aminovaleric acid (an inhibitor of GABA(B) receptors) did not prevent the inhibition, indicating that it was not mediated by the activation of the G(i)-protein coupled GABA(B) receptor. In addition, the nucleotide binding site of G-protein appeared not to be affected by ALA since it did not inhibit [3H]Gpp(NH)p binding to our membrane preparation. Antioxidants (glutathione, ascorbate and trolox) completely prevented the inhibition indicating that ALA effect was mediated by an oxidative damage of adenylate cyclase. ALA also inhibited the activity of adenylate cyclase in membranes isolated from rat cortex and striatum and from human cortex. These results may be of value in understanding the neurochemical mechanisms underlying the neurotoxic effects of ALA.     

Discoidin domain receptor 1 is activated independently of beta(1) integrin

Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor (DDR) tyrosine kinases, DDR1 and DDR2. It is presently unclear whether collagen-induced DDR receptor activation, which occurs with very slow kinetics, involves additional proteins with kinase activity or membrane-anchored proteins serving as coreceptors. In particular, the role of the collagen-binding integrins alpha(1)beta(1) or alpha(2)beta(1) in the DDR activation process is undefined. Here, we provide three lines of evidence suggesting that DDR1 signaling is distinct from integrin activation. First we demonstrate that the enzymatic activity of DDR1 is essential for receptor tyrosine phosphorylation. Collagen-induced DDR receptor autophosphorylation can be blocked either by a dominant negative mutant or by a preparation of recombinant extracellular domain. Second, we show DDR1 signals independent of the epidermal growth factor (EGF) receptor. In cells that endogenously express both DDR1 and the EGF receptor, stimulation with EGF does not induce DDR activation. Third, we detected full DDR1 activation after collagen stimulation in cells that have been treated with blocking antibodies for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers.     

Effect of overall feedback inhibition in unbranched biosynthetic pathways

We have determined the effects of control by overall feedback inhibition on the systemic behavior of unbranched metabolic pathways with an arbitrary pattern of other feedback inhibitions by using a recently developed numerical generalization of Mathematically Controlled Comparisons, a method for comparing the function of alternative molecular designs. This method allows the rigorous determination of the changes in systemic properties that can be exclusively attributed to overall feedback inhibition. Analytical results show that the unbranched pathway can achieve the same steady-state flux, concentrations, and logarithmic gains with respect to changes in substrate, with or without overall feedback inhibition. The analytical approach also shows that control by overall feedback inhibition amplifies the regulation of flux by the demand for end product while attenuating the sensitivity of the concentrations to the same demand. This approach does not provide a clear answer regarding the effect of overall feedback inhibition on the robustness, stability, and transient time of the pathway. However, the generalized numerical method we have used does clarify the answers to these questions. On average, an unbranched pathway with control by overall feedback inhibition is less sensitive to perturbations in the values of the parameters that define the system. The difference in robustness can range from a few percent to fifty percent or more, depending on the length of the pathway and on the metabolite one considers. On average, overall feedback inhibition decreases the stability margins by a minimal amount (typically less than 5%). Finally, and again on average, stable systems with overall feedback inhibition respond faster to fluctuations in the metabolite concentrations. Taken together, these results show that control by overall feedback inhibition confers several functional advantages upon unbranched pathways. These advantages provide a rationale for the prevalence of this control mechanism in unbranched metabolic pathways in vivo.     

Coinfection by Strongyloides stercoralis in blood donors infected with human T-cell leukemia/lymphoma virus type 1 in São Paulo City, Brazil

The frequency of coinfection with Strongyloides stercoralis and human T-cell leukemia/lymphoma virus type 1 (HTML-1) was determined in 91 blood donors examined at the blood bank of a large hospital in S?o Paulo city, Brazil. As control group 61 individuals, not infected by HTLV-1, were submitted to the same techniques for the diagnosis of S. stercoralis infection. In HTLV-1 infected patients the frequency of S. stercoralis infection was 12.1%; on the other hand, the control group showed a frequency significantly lower of S. stercoralis infection (1.6%), suggesting that HTLV-1 patients should be considered as a high risk group for strongyloidiasis in S?o Paulo city.     

Procedures to characterize and study P2Z/P2X7 purinoceptor: flow cytometry as a promising practical, reliable tool

The expression of P2Z/P2X7 purinoceptor in different cell types is well established. This receptor is a member of the ionotropic P2X receptor family, which is composed by seven cloned receptor subtypes (P2X1 - P2X7). Interestingly, the P2Z/P2X7 has a unique feature of being linked to a non-selective pore which allows the passage of molecules up to 900 Da depending on the cell type. Early studies of P2Z/P2X7 purinoceptor were exclusively based on classical pharmacological studies but the recent tools of molecular biology have enriched the analysis of the receptor expression. The majority of assays and techniques chosen so far to study the expression of P2Z/P2X7 receptor explore directly or indirectly the effects of the opening of P2Z/P2X7 linked pore. In this review we describe the main techniques used to study the expression and functionality of P2Z/P2X7 receptor. Additionally, the increasing need and importance of a multifunctional analysis of P2Z/P2X7 expression based on flow cytometry technology is discussed, as well as the adoption of a more complete analysis of P2Z/P2X7 expression involving different techniques.     

Amino acid sequences of proteins from Leptospira serovar pomona

This report describes a partial amino acid sequences from three putative outer envelope proteins from Leptospira serovar pomona. In order to obtain internal fragments for protein sequencing, enzymatic and chemical digestion was performed. The enzyme clostripain was used to digest the proteins 32 and 45 kDa. In situ digestion of 40 kDa molecular weight protein was accomplished using cyanogen bromide. The 32 kDa protein generated two fragments, one of 21 kDa and another of 10 kDa that yielded five residues. A fragment of 24 kDa that yielded nineteen residues of amino acids was obtained from 45 kDa protein. A fragment with a molecular weight of 20 kDa, yielding a twenty amino acids sequence from the 40 kDa protein.     

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.      

Synthesis and biodistribution studies of carbohydrate derivatives radiolabeled with technetium-99m

Three carbohydrate derivatives, MAG₃-Gl, MAG₃-Ga, MAG₃-NG, were synthesized and radiolabeled in high yields. These substances were injected in health Swiss mice and their biodistribution were evaluated. Among them, ^99mTc-MAG₃-Ga displayed higher accumulation in hepatic tissue, due to the presence of specific receptors in the liver for this carbohydrate. Thus, the use of ^99mTc-MAG₃-Ga to assess hepatic function can be considered.