Trypanosoma cruzi: surface antigenic determinants

A fraction (FAd) capable of inhibiting specific agglutination reactions of anti-epimastigote sera (anti-LE) was obtained by extracting the sediment of lyophilized epimastigote lysates (LE) with 0.05 M phosphate buffered saline, at 37 degrees C for 1 h. These conditions favored the action of parasite proteinase whose presence was detected by tandem-crossed immunoelectrophoresis experiments. As expected from the proteinase properties, the addition of 2-mercaptoethanol or sodium iodoacetate to the extracting solution resulted, respectively, in either increased or decreased amounts of protein in the resulting FAd. FAd components could be precipitated by the addition of Concanavalin A, methylated albumins or 0.1 N HCl. This fraction presented a single component when subjected to electrophoresis in 1% agarose gel with an electrophoretic mobility 1.2 times higher than that of human albumin. FAd component(s) were unable to penetrate 15% polycrylamide gel matrix unless 1% SDS was used. Under this condition four glycopeptide components, with Rm of 0.5, 0.55, 0.6 and 0.86, were detected. The antigenic determinants present in FAd resisted heating at 100 degrees C for 30 min and the prolonged action of pronase. However, these determinants were completely destroyed by the action of 25 mM sodium periodate, thus suggesting polysaccharide characteristics. Immunization of rabbits with FAd induced the production of antibodies that were unable to precipitate with either FAd or with parasite proteinase. These antibodies exhibited positive agglutination reactions with epimastigote forms and positive immunofluorescence and immunoperoxidase reactions with trypomastigote and amastigote forms of the different strains tested. FAd was able to inhibit these reactions as well as those obtained with anti-LE and anti-FA immune sera, whereas purified proteinase was unable to inhibit any of these reactions.     

Effect of season on hormonal profile and some biochemical parameters at different stages of estrous cycles in Baladi goats

Understanding the seasonal patterns of estrus cycle in caprine is crucial to develop the efficient breeding plans in the subtropics. Thus, the aim of the current research was to evaluate the effects of breeding season on hormonal profile and blood biochemical indices at different stages of estrus cycle in normal breeding or out-of-season breeding in goats. Forty-four Baladi goats were monitored for a period of eight months (July–February). Baladi goats exhibit a normal seasonal breeding (NS) at midsummer and continue through the autumn season (68%), with a considerable percentage of females having estrus signs during out-of-season (OS) period (32%). At the mid and late luteal phases of estrus cycles, the NS breeding group had significantly higher serum progesterone level than that reported in the OS group (p = 0.013 and 0.039, respectively). At the estrus and late luteal phases of estrus cycles, the NS breeding group had significantly higher serum β-estradiol level than that reported in the OS group (p = 0.022 and 0.001, respectively). Compared to the OS group, the NS breeding group had significantly higher serum cholesterol at the mid and late luteal phases of estrus cycle (p = 0.001 and 0.016, respectively), and higher serum glucose level in the early luteal phase of the cycle (p = 0.009). In conclusion, the NS breeding goats had superior progesterone (mid-luteal and late luteal phases) and estradiol (estrus and late luteal phases) profiles than that reported in the OS group. This may indicate specific approaches to maintain the breeding efficiency of goats during the out-of-season period.     

4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate

Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called “Radical-SAM.” These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX₃CX₂C motif, and S-adenosyl-l-methionine (SAM) to generate a 5′-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.     

Fibrillation of human insulin A and B chains

Human insulin, which consists of disulfide cross-linked A and B polypeptide chains, readily forms amyloid fibrils under slightly destabilizing conditions. We examined whether the isolated A and B chain peptides of human insulin would form fibrils at neutral and acidic pH. Although insulin exhibits a pH-dependent lag phase in fibrillation, the A chain formed fibrils without a lag at both pHs. In contrast, the B chain exhibited complex concentration-dependent fibrillation behavior at acidic pH. At higher concentrations, e.g., >0.2 mg/mL, the B chains preferentially and rapidly formed stable protofilaments rather than mature fibrils upon incubation at 37 degrees C. Surprisingly, these protofilaments did not convert into mature fibrils. At lower B chain concentrations, however, mature fibrils were formed. The explanation for the concentration dependence of B chain fibrillation is as follows. The B chains exist as soluble oligomers at acidic pH, have a beta-sheet rich conformation as determined by CD, and bind ANS strongly, and these oligomers rapidly form dead-end protofilaments. However, under conditions in which the B chain monomer is present, such as low B chain concentration (<0.2 mg/mL) or in the presence of low concentrations of GuHCl, which dissociates the soluble oligomers, mature fibrils were formed. Thus, both A and B chain peptides can form amyloid fibrils, and both are likely to be involved in the interactions leading to the fibrillation of intact insulin.     

The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.     

Structural and spectroscopic studies of tripodal [MgL]2+ chelates containing only nitrogen donor atoms: alkaline earth metal complexes as potential drug delivery agents

Several tripodal diimine ligands, tris(2-(2-thiazolyl)methyliminoethyl)amine, 2-Tatren, tris(2-(4-(5-methyl)imidazolyl)methyliminoethyl)amine, 5-Me-4-Imtren, tris(2-(4-imidazolyl)methyliminoethyl)amine, 4-Imtren, tris(2-(2-imidazolyl)methyliminoethyl)amine, 2-Imtren, and their Mg(2+) complexes were prepared and characterized. X-ray diffraction studies show that the Mg(2+) ions are six-coordinate, with three acyclic imine N atoms and three imidazolyl or thiazolyl N atoms coordinated with the general formula [Mg(L)](ClO(4))(2) (L=4-Imtren (1), 2-Imtren (2), 2-Tatren (3), and 5-Me-4-Imtren (4)). These complexes are chiral with both Delta and Lambda isomers present in the unit cell. (1)H NMR titrations reveal that complexes also form in solution and that the chirality is maintained. Variable temperature (1)H NMR reveals that the Delta and Lambda isomers interconvert in the intermediate to slow time scale. The interconversion rate slows with increasing pK(a) of the ligand heterocycle, suggesting that interconversion proceeds through a partially dissociated state. These complexes undergo trans-metallation by Zn(2+), indicating that their ligands can be released in a kinetically facile manner to form more stable metal ion complexes.     

Guanidinium chloride- and urea-induced unfolding of the dimeric enzyme glucose oxidase

We have carried out a systematic study on the guanidinium chloride- and urea-induced unfolding of glucose oxidase from Aspergillus niger, an acidic dimeric enzyme, using various optical spectroscopic techniques, enzymatic activity measurements, glutaraldehyde cross-linking, and differential scanning calorimetry. The urea-induced unfolding of GOD was a two-state process with dissociation and unfolding of the native dimeric enzyme molecule occurring in a single step. On the contrary, the GdmCl-induced unfolding of GOD was a multiphasic process with stabilization of a conformation more compact than the native enzyme at low GdmCl concentrations and dissociation along with unfolding of enzyme at higher concentrations of GdmCl. The GdmCl-stabilized compact dimeric intermediate of GOD showed an enhanced stability against thermal and urea denaturation as compared to the native GOD dimer. Comparative studies on GOD using GdmCl and NaCl demonstrated that binding of the Gdm(+) cation to the enzyme results in stabilization of the compact dimeric intermediate of the enzyme at low GdmCl concentrations. An interesting observation was that a slight difference in the concentration of urea and GdmCl associated with the unfolding of GOD was observed, which is in violation of the 2-fold rule for urea and GdmCl denaturation of proteins. This is the first report where violation of the 2-fold rule has been observed for a multimeric protein.     

Modeling carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS): a trinuclear nickel complex employing deprotonated amides and bridging thiolates

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) utilizes a unique Ni-M bimetallic site in the biosynthesis of acetyl-CoA, where a square-planar Ni ion is coordinated to two thiolates and two deprotonated amides in a Cys-Gly-Cys motif. The identity of M is currently a matter of debate, although both Cu and Ni have been proposed. In an effort to model ACSs unusual active site and to provide insight into the mechanism of acetyl-CoA formation and the role of each of the metals ions, we have prepared and structurally characterized a number of Ni(II)–peptide mimic complexes. The mononuclear complexes Ni(II) N,N-bis(2-mercaptoethyl)oxamide (1), Ni(II) N,N-ethylenebis(2-mercaptoacetamide) (2), and Ni(II) N,N-ethylenebis(2-mercaptopropionamide) (3) model the Ni(Cys-Gly-Cys) site and can be used as synthons for additional multinuclear complexes. Reaction of 2 with MeI resulted in the alkylation of the sulfur atoms and the formation of Ni(II) N,N-ethylenebis(2-methylmercaptoacetamide) (4), demonstrating the nucleophilicity of the terminal alkyl thiolates. Addition of Ni(OAc)₂·4H₂O to 3 resulted in the formation of a trinuclear species 5, while 2 crystallizes as an unusual paddlewheel complex (6) in the presence of nickel acetate. The difference in reactivity between the similar complexes 2 and 3 highlights the importance of ligand design when synthesizing models of ACS. Significantly, 5 maintains the key features observed in the active site of ACS, namely a square-planar Ni coordinated to two deprotonated amides and two thiolates, where the thiolates bridge to a second metal, suggesting that 5 is a reasonable structural model for this unique enzyme.Ø. Hatlevik and M.C. Blanksma contributed equally to this work