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.     

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.     

Change in social status and risk of low birth weight in Denmark: population based cohort study.

OBJECTIVE: To estimate the risk of having a low birthweight infant associated with changes in social, environmental, and genetic factors. DESIGN: Population based, historical cohort study using the Danish medical birth registry and Statistic Denmark''s fertility database. SUBJECTS: All women who had a low birthweight infant (< 2500 g) (index birth) and a subsequent liveborn infant (outcome birth) in Denmark between 1980 and 1992 (exposed cohort, n = 11,069) and a random sample of the population who gave birth to an infant weighing > or = 2500 g and to a subsequent liveborn infant (unexposed cohort, n = 10,211). MAIN OUTCOME MEASURES: Risk of having a low birthweight infant in the outcome birth as a function of changes in male partner, area of residence, type of job, and social status between the two births. RESULTS: Women in the exposed cohort showed a high risk (18.5%) of having a subsequent low birthweight infant while women in the unexposed cohort had a risk of 2.8%. After adjustment for initial social status, a decline in social status increased the absolute risk of having a low birthweight infant by about 5% in both cohorts, though this was significant only in the unexposed cohort. Change of male partner did not modify the risk of low birth weight in either cohort. CONCLUSION: Having had a low birthweight infant and a decline in social status are strong risk factors for having a low birthweight infant subsequently.     

Distribution of transposable elements in neotropical species of Drosophila

The phylogenetic distribution of transposable families, P, gypsy, hobo, I, and mariner has been analyzed in 33 species of 11 groups of neotropical Drosophila and a Drosophilidae species Zygotrica vittimaculosa, using squash blot and dot blot. Genomic DNA of almost all neotropical species tested hybridized with gypsy probe and some species showed a particularly strong hybridization signal, as D. gaucha, D. virilis, and species of flavopilosa group. The hobo element was restricted to melanogaster group and some strains of D. willistoni. Only D. simulans DNA showed hybridization to mariner probe in all species tested and D. simulans and D. melanogaster showed hybridization with I element probe. P element homologous sequence was present in D. melanogaster and all species and strains of the willistoni and saltans groups tested. The presence of at least one P-homologous sequence was detected in Drosophila mediopunctata. This one was the only P-bearing species of all six tested from the tripunctata group. Four different pairs of primers homologous to segments of the canonical sequence of D. melanogaster's P were used to amplify specific sequences from D. mediopunctata DNA, showing the occurrence of seemingly well-conserved P-homologous sequences. This revised version was published online in July 2006 with corrections to the Cover Date.