Urinary elastase 1 in chronic pancreatic disease

Serum and urine elastase 1, its renal output and clearance and urinary gamma-glutamyltransferase and ribonuclease excretions were measured in 16 patients with pancreatic cancer, 23 with chronic pancreatitis and in 22 healthy controls in order to evaluate elastase 1 plasma-urine transfer in chronic pancreatic disease and to investigate any factors that might influence the clearance of this enzyme. In an additional group of 17 patients with different pancreatic diseases the serum molecular size distribution of elastase 1 after chromatography was ascertained. An increased urinary elastase 1 output was found in 4/16 patients with pancreatic cancer and in 6/23 with chronic pancreatitis. No correlation was found between circulating elastase 1 and its urinary output; a negative correlation was detected between the serum levels of this enzyme and its clearance. The excretion of ribonuclease and gamma-glutamyltransferase was correlated with elastase 1 output and clearance. While the majority of elastase 1 in serum was accounted for by high molecular forms, probably the expression of complexes with serum inhibitors, free circulating enzyme was present in all patients with high serum elastase 1. Our findings suggest that elastase 1 urinary excretion increases in some patients with chronic pancreatic disease regardless of the neoplastic or inflammatory nature of the illness. Although the availability of different amounts of ultrafiltrable enzyme may play a role in influencing elastase 1 plasma-urine transfer, renal tubular damage appears to be the most important factor influencing the increase in the urinary output of elastase 1.     

Characterization of two glycoproteins of human pancreatic juice: P35, a truncated protease E and P19, precursor of protein X

Four glycoproteins were separated by SDS-polyacrylamide gel electrophoresis of proteins of human pancreatic juice devoid of free proteolytic activity. The two low molecular weight glycoproteins were isolated and characterized. Protein P19, the precursor family of protein X, was analyzed by its carbohydrate content which seemed to play an important role in protein solubility at pH 8.0. Protein P35 was found to be a Con A-binding protein rich in mannose. Its N-terminal amino acid sequence covering 33 residues revealed a strong homology with human protease E without the dipeptide Val-Val. Is P35 a protein homologous to the subunit III of bovine procarboxypeptidase A?     

Exoantigens of Trypanosoma cruzi. I. Conditions for Their Detection and Immunogenic Properties in Experimental Infections1

ABSTRACT Exoantigens of Trypanosoma cruzi were produced in experimentally infected BALB/c mice. The exoantigens were detected by the counterimmunoelectrophoresis method (CIE), with antisera raised in rabbits by immunization with total homogenates of culture forms of ***T. cruzi in plasma from ***field animals obtained by centrifugation and filtration. Control experiments indicated that exoantigens are not somatic components of T. cruzi leaked during the preparative procedure. Exoantigens were detected in male and female mice, 11-90 days old, between 6 and 60 days of infection, and in all mice with patent parasitemia. After 13 days of infection, mice developed antibodies to exoantigens; by CIE up to three populations of antibodies were revealed in different groups of animals. In mice between 13 and 60 days of infection, the coexistence of exoantigens and homologous antibodies was also observed. The exoantigens are not strain specific since a cross reactivity between antigens from three strains of T. cruzi (Tulahuén, Higueras, and Alejandro) was seen. Finally, the presence of antibodies to exoantigens in humans with chronic Chagas’ disease was demonstrated.     

Deep soil heterogeneity and fine root distribution in forests and pastures of eastern Amazonia

Little is known about deep soil heterogeneity, or its relationship with fine root distribution. Beneath a mature, closed-canopy forest of eastern Amazonia, and the pastures and secondary forests that are derived from this forest, soil soft spots and hollow chambers occur to at least 9 meters depth. We measured the vertical distribution of these soil patches, and compared chemical characteristics, mycorrhizal infection, and root density of soil soft spots with the surrounding matrix of more homogeneous soil. Soil soft spots and chambers varied little with depth, but occupied the greatest soil volume (0.8 to 1.2%) from 4 to 6 m depth in the mature forest. Soft spots had lower pH, P availability and arbuscular mycorrhizal infection, and higher K availability than surrounding soil. Root length density was 2 to 15 times higher in soft spots than in surrounding soil. In the pastures, roots were found only in soil soft spots at depths of >3 m. Pastures and secondary forest had more soil chambers in the upper meter of soil than mature forest, but were otherwise indistinguishable in their patterns of deep soil heterogeneity. Soil soft spots may be vestiges of cutter ant nest chambers, while hollow chambers are cutter ant chambers and root channels. Chambers may act as conduits for root penetration and water penetration to deep soil.Abbreviations AM arbuscular mycorrhizae - RLD root length density (root length per unit of soil volume)     

Identification and characterization of the proBA operon of Streptococcus bovis.

A genomic DNA library of the rumen bacterium Streptococcus bovis was constructed in Escherichia coli, and recombinant plasmids able to complement proA and proB mutations of the host were found. Southern hybridization and restriction analysis showed that a 3.5-kb fragment of S. bovis DNA contained two genes, organized in an operon and coding for enzymes functionally similar to the glutamyl phosphate reductase-glutamyl kinase enzyme complex that in E. coli catalyzes the first step of proline biosynthesis. Complementation of the E. coli mutations was observed with the fragment inserted in both orientations, which suggested that the S. bovis proBA operon was transcribed from its own promoter. Genetic and biochemical data suggested that the proline biosynthetic pathway of S. bovis is similar to the one previously characterized for E. coli.     

Successful synthesis of a glial-specific blood–brain barrier shuttle peptide following a fragment condensation approach on a solid-phase resin

Successful manual synthesis of the TD2.2 peptide acting as a blood–brain barrier shuttle was achieved. TD2.2 was successfully synthesised by sequential condensation of four protected peptide fragments on solid-phase settings, after several unsuccessful attempts using the stepwise approach. These fragments were chosen to minimise the number of demanding amino acids (in terms of coupling, Fmoc removal) in each fragment that are expected to hamper the overall synthetic process. Thus, the hydrophobic amino acids as well as Arg(Pbf) were strategically spread over multiple fragments rather than having them congested in one fragment. This study shows how a peptide that shows big challenges in the synthesis using the common stepwise elongation methodology can be synthesised with an acceptable purity. It also emphasises that choosing the right fragment with certain amino acid constituents is key for a successful synthesis. It is worth highlighting that lower amounts of reagents were required to synthesise the final peptide with an identical purity to that obtained by the automatic synthesiser.     

Selective incorporation of 5-hydroxytryptophan blocks long range electron transfer in oxalate decarboxylase

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.     

Phytochemical Characterization of Cannabis sativa L. Roots from Northeastern Brazil

This article describes the phytochemical study of Cannabis sativa roots from northeastern Brazil. The dried plant material was pulverized and subjected to exhaustive maceration with ethanol at room temperature, obtaining the crude ethanolic extract (Cs-EEBR). The volatile compounds were analyzed by gas chromatography coupled with mass spectrometry (GC/MS), which allowed to identify 22 compounds by comparing the linear retention index (LRI), the similarity index (SI) and the fragmentation pattern of the constituents with the literature. By this technique the major compounds identified were: friedelan-3-one and β-sitosterol. In addition, two fractions were obtained from Cs-EEBR by classical column chromatography and preparative thin layer chromatography. These fractions were analyzed by NMR and IR and together with the mass spectrometry data allowed to identify the compounds: epifriedelanol, friedelan-3-one, β-sitosterol and stigmasterol. The study contributed to the phytochemical knowledge of Cannabis sativa, specifically the roots, as there are few reports on the chemical constituents of this part of the plant.