Synthesis and hydrolysis by cathepsin B of fluorogenic substrates with the general structure benzoyl-X-ARG-MCA containing non-natural basic amino acids at position X

We synthesized one series of fluorogenic substrates for cathepsin B derived from the peptide Bz-F-R-MCA (Bz=benzoyl, MCA=7-methyl-coumarin amide) substituting Phe at the P(2) position by non-natural basic amino acids that combine a positively charged group with aromatic or aliphatic radicals at the same side chain, namely, 4-aminomethyl-phenylalanine, 4-guanidine-phenylalanine, 4-aminomethyl-N-isopropyl-phenylalanine, 3-pyridyl-alanine, 4-piperidinyl-alanine, 4-aminomethyl-cyclohexyl-alanine, 4-aminocyclohexyl-alanine, and N(im)-dimethyl-histidine. Bz-F-R-MCA was the best substrate for cathepsin B but also hydrolyzed Bz-R-R-MCA with lower efficiency, since the protease accepts Arg at S(2) due to the presence of Glu(245) at the bottom of this subsite. The presence of the basic non-natural amino acids at the P(2) position of the substrate partially restored the catalytic efficiency of cathepsin B. All the kinetic parameters for hydrolysis of the peptides described in this paper are in accordance with the structures of the S(2) pocket previously described. In addition, the substrate with 4-aminocyclohexyl-alanine presented the highest affinity to cathepsin B although the peptide was obtained from a mixture of cis/trans isomers of the amino acid and we were not able to separate them. For comparison all the obtained substrates were assayed with cathepsin L and papain.     

Proteasome inhibitors block a late step in lysosomal transport of selected membrane but not soluble proteins

The ubiquitin-proteasome pathway acts as a regulator of the endocytosis of selected membrane proteins. Recent evidence suggests that it may also function in the intracellular trafficking of membrane proteins. In this study, several models were used to address the role of the ubiquitin-proteasome pathway in sorting of internalized proteins to the lysosome. We found that lysosomal degradation of ligands, which remain bound to their receptors within the endocytic pathway, is blocked in the presence of specific proteasome inhibitors. In contrast, a ligand that dissociates from its receptor upon endosome acidification is degraded under the same conditions. Quantitative electron microscopy showed that neither the uptake nor the overall distribution of the endocytic marker bovine serum albumin-gold is substantially altered in the presence of a proteasome inhibitor. The data suggest that the ubiquitin-proteasome pathway is involved in an endosomal sorting step of selected membrane proteins to lysosomes, thereby providing a mechanism for regulated degradation.     

Detection of cocaine and cocaethylene in sweat by solid-phase microextraction and gas chromatography/mass spectrometry

In the present work, a semi-quantitative method was developed to detect simultaneously cocaine (COC) and cocaethylene (CE) (transesterification product of the coingestion of COC with ethanol) in sweat. Sweat samples were collected by means of a non-occlusive sweat patch device supplied by PharmChek. The method was based on the dissolution of COC and CE incorporated into the patch, with 0.2 M sodium acetate buffer (pH 5.0) and the extraction of the analytes by solid-phase microextraction (SPME). Gas chromatography/mass spectrometry (GC-MS) was used to detect the analytes in selected ion monitoring mode (SIM). The method showed to be very simple, rapid and sensitive. The limits of detection were 5 ng/ml for COC and CE (12.5 ng/patch). Good inter and intra-assay precision was also observed (coefficient of variation <8%) with the use of deuterated internal standards.     

Breeding system and floral morphometry of distylous Psychotria L. species in the Atlantic rain forest, SE Brazil

General patterns of floral morphology and incompatibility mechanisms have been described for many distylous plants. The absence of these patterns in typically distylous groups, as observed especially in tropical environments, is interpreted as atypical distyly, or as a new reproductive strategy derived from it. Data are presented here on the morphological and compatibility relations between floral morphs of four Psychotria dimorphic species in the Atlantic rain forest in SE Brazil: Psychotria jasminoides, P. birotula, P. mapourioides, and P. pubigera. When significant differences were found, floral parts were larger in thrum flowers. Results of controlled crosses showed that most incompatible pollen tubes were arrested in the stigma, and only in a low proportion in the upper parts of the style. We conclude that, at the study site, the majority and most important morphological and mating features of typical distyly seem to be conserved in P. jasminoides and P. mapourioides, which presented reciprocal herkogamy, self and intramorph incompatibility, and a balanced morph ratio in the population. Typical distyly in P. birotula is supported by floral morphology, pollen tube data and morph ratio and, in P. pubigera, only by floral morphology and pollen tube data.     

A monomeric mutant of restriction endonuclease EcoRI nicks DNA without sequence specificity

We have mutated the monomer-monomer interface of the restriction endonuclease EcoRI in order to destabilize the homodimer and to stabilize heterodimers. Mutations of Leu158 to charged amino acid residues result in strong destabilization of the dimer. The largest effect was detected for the L158D mutant which is monomeric even at higher concentrations. It unspecifically degrades DNA by cleaving both single strands independently every 15 nucleotides on the average. Although cleavage is reproducible, it is not determined by nucleotide sequence but by general properties like conformation or deformability as has been found for other unspecific nucleases. Mutations of Ile230, which is in direct contact with Leu158 of the other subunit, cause structural changes with the loss of about ten percent alpha-helix content, but interfere only marginally with homodimerization and double strand cleavage. Again the mutation to aspartate shows the strongest effects. Mixtures of single mutants, one containing aspartate at one of the two positions and the other lysine at the corresponding position, form heterodimers. These are mainly stabilized compared to the homodimers by re-establishment of the wild-type hydrophobic interaction at the not mutated residues while an interaction of aspartate and lysine seems energetically unfavorable in this structural context.     

uvsZ1 mutation shows epistatic relations with uvsD153 and uvsJ1 mutations without any involvement with checkpoint control in Aspergillus nidulans

The participation of the recently described uvsZ1 mutation in checkpoint control and the identification of epistatic relations between uvsZ1 mutation and uvsD153 and uvsJ1 mutations are provided. The effect of mutation uvsZ1 in mitotic exchanges into paba-bi (chromosome I) and cho-nic (chromosome VII) genetic intervals has also been evaluated. The mutation uvsZ1 was epistatic with regard to uvsD153 and uvsJ1 mutations, with no involvement with checkpoint control. In contrast to mutations in UvsB and UvsF groups, the uvsZ1 mutation failed to cause any changes in the frequencies of mitotic crossing-over. The distinct phenotypic traits given by mutation uvsZ1 suggest the presence of complex interactions among the different DNA repair pathways. Interaction may be an additional cell strategy of DNA damage response.     

Enantioselectivity in the steady-state pharmacokinetics and transplacental distribution of pindolol at delivery in pregnancy-induced hypertension

Nine patients taking oral doses of 10 mg/12 h rac-pindolol as part of their treatment for hypertension in pregnancy were recruited for the study. Maternal and fetal gestational age ranged from 20-38 years and 28-41 weeks, respectively. Blood was collected from the umbilical cord vein and from the mother from zero to 12 h after drug administration. Urine was collected for 12 h after rac-pindolol administration at the following intervals: 0-3, 3-6, 6-9, and 9-12 h. Plasma and urine concentrations of the pindolol enantiomers were determined by HPLC using a Chiralpak AD chiral column and fluorescence detection. The data were fitted to a one-compartment model and differences between (+)-R and (-)-S enantiomers were compared by the paired t-test (P < 0.05). Mean results are reported. The disposition of pindolol in maternal plasma was stereoselective, with higher AUC(SS)0-12 (84.34 vs. 95.69 ng.h/ml) and Cl(R) values (9.16 vs. 10.85 L/h) and lower Vd/f (251.38 vs. 225.17 L) and Cl/f (62.48 vs. 55.74 L/h) for the (+)-R pindolol. The transplacental distribution of pindolol was not stereoselective. Cord, plasma, and presumably fetal, concentrations of the pindolol enantiomers were 56% of the maternal plasma concentrations up to 6 h after the last dose.     

Abnormal neurovascular control during exercise is linked to heart failure severity

The purpose of this study was to determine if abnormalities of sympathetic neural and vascular control are present in mild and/or severe heart failure (HF) and to determine the underlying afferent mechanisms. Patients with severe HF, mild HF, and age-matched controls were studied. Muscle sympathetic nerve activity (MSNA) and forearm vascular resistance (FVR) in the nonexercising arm were measured during mild and moderate static handgrip. MSNA during moderate handgrip was higher at baseline and throughout exercise in severe HF vs. mild HF (peak MSNA 67 +/- 3 vs. 54 +/- 3 bursts/min, P < 0.0001) and higher in mild HF vs. controls (33 +/- 3 bursts/min, P < 0.0001), but the change in MSNA was not different between the groups. The change in FVR was not significantly different between the three groups during static exercise. During isolation of muscle metaboreceptors, MSNA and blood pressure remained elevated in normal controls and mild HF but not in severe HF. During mild handgrip, the increase in MSNA was exaggerated in severe HF vs. controls and mild HF, in whom MSNA did not increase. In summary, the increase in MSNA during static exercise in severe HF appears to be attributable to exaggerated central command or muscle mechanoreceptor control, not muscle metaboreceptor control.     

The signal transduction of the growth hormone receptor is regulated by the ubiquitin/proteasome system and continues after endocytosis

The growth hormone receptor (GHR) intracellular domain contains all of the information required for signal transduction as well as for endocytosis. Previously, we showed that the proteasome mediates the clathrin-mediated endocytosis of the GHR. Here, we present evidence that the proteasomal inhibitor MG132 prolongs the GH-induced activity of both GHR and JAK2, presumably through stabilization of GHR and JAK2 tyrosine phosphorylation. If proteasomal inhibitor was combined with ligand in an endocytosis-deficient GHR mutant, the same phenomenon occurred indicating that proteasomal action on tyrosine dephosphorylation is independent of endocytosis. Experiments with a GHR-truncated tail mutant (GHR-(1-369)) led to a prolonged JAK2 phosphorylation caused by the loss of a phosphatase-binding site. This raised the question of what happens to the signal transduction of the GHR after its internalization. Co-immunoprecipitation of GH.GHR complexes before and after endocytosis showed that JAK2 as well as other activated proteins are bound to the GHR not only at the cell surface but also intracellularly, suggesting that the GHR signal transduction continues in endosomes. Additionally, these results provide evidence that GHR is present in endosomes both in its full-length and truncated form, indicating that the receptor is down-regulated by the proteasome.     

Comparative studies of two transthyretin variants with protective effects on familial amyloidotic polyneuropathy: TTR R104H and TTR T119M

Recently, a new nonpathogenic transthyretin (TTR) variant-TTR R104H (TTR H104)-has been described in heterozygotic and compound heterozygotic individuals from a Japanese family with familial amyloidotic polyneuropathy (FAP). The compound heterozygotic individual, a carrier of TTR V30M (TTR M30) and TTR R104H (TTR M30/H104) presented a very mild form of FAP with slow progression of the disease. TTR and retinol binding protein (RBP) levels were found to be increased in serum from TTR H104 carriers. These characteristics are very similar to those found in compound heterozygotic carriers of TTR V30M-T119M (TTR M30/M119). To structurally compare these variants, we performed stability and thyroxine (T(4)) binding studies. TTR M30/H104 showed an increased resistance to dissociation into monomers similar to TTR M30/M119. This suggests that the His104 substitution has the same stabilizing effect on tetrameric TTR as the Met119 substitution. Concerning T(4) binding, TTR H104 presents a T(4) binding affinity lower than that of TTR M119, but still higher than normal TTR. However, TTR from the compound heterozygotic carrier of TTR M30/H104 presented a T(4) binding affinity lower than normal. The results indicate that the His 104 substitution induces structural alterations that increase the stability of the tetramer in compound heterozygotes for TTR M30 despite a lower affinity for T(4) binding. Thus, stability of TTR and binding affinity for T(4) may not be related. More detailed characterization of these variants is needed to clarify the structural alterations responsible for their increased stability.