Mucosal layers and related nerve fibres in non-chagasic and chagasic human colon—a quantitative immunohistochemical study

Chagasic megacolon is accompanied by extensive myenteric and, simultaneously, moderate submucosal neuron loss. Here, we examined changes of the innervation pattern of the lamina propria (LP) and muscularis mucosae (MM). Two alternating sets of cryosections were taken from seven non-chagasic colonic and seven chagasic megacolonic specimens (the latter included both the dilated megacolonic and the non-dilated transitional oral and anal zones) and were immunohistochemically triple-stained for smooth-muscle actin (SMA), synaptophysin (SYN) and glial acid protein S100 and, alternatively, for SMA, vasoactive intestinal peptide (VIP) and somatostatin (SOM). Subsequent image analysis and statistical evaluation of nervous tissue profile areas revealed that, in LP, the most extreme differences (i.e. increase in thickness or decrease in nerve, glia and muscle tissue profile area, respectively) compared with control values occurred in the dilated megacolonic zone itself. In contrast, the most extreme differences in the MM were in the anal-to-megacolonic zone (except the profile area of muscle tissue, which was lowest in the megacolonic zone). This parallels our previous results in the external muscle coat. A partial and selective survival of VIP-immunoreactive in contrast to SOM-immunoreactive nerve fibres was observed in both mucosal layers investigated. Thus, VIPergic nerve elements might be crucial for the maintenance of the mucosal barrier. The differential changes of neural tissue parameters in LP and MM might reflect a multifactorial rather than a pure neurogenic development of megacolon in chronic Chagas’ disease.     

An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp

The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonella typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-1 and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.     

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop

Abstract A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: I. RELATIONSHIP BETWEEN TUBER GROWTH RATE AND ENZYME ACTIVITIES OF THE STARCH METABOLISM

Potato plants (Solanum tuberosum L.) were grown in water culturein a controlled environment. Cooling (+8°C) of individualtubers decreased their growth rates and increased the growthrates of non-cooled tubers of the same plant. The carbohydrateconcentration in non-cooled and cooled tubers did not differsignificantly, but ¹⁴C-import from labelled photosynthate waslower in cooled than in non-cooled tubers. The markedly lowerconversion rate of ethanol-soluble ¹⁴C to starch in cooled,in comparison to non-cooled tubers, was not associated withsignificant differences in the in vitro activities of starchsynthase, ADPG-pyrophosphorylase and starch phosphorylase understandard assay conditions (+30°C). However, the Q₁₀-valuesof the enzymes differed in vitro in the temperature range between30°C and 8°C, leading to a marked decrease in the activityratio of ADPG-pyrophosphorylase/starch phosphorylase in cooledtubers. In tubers differing in growth rates without manipulation, 14d after tuber initiation significant positive correlations werefound between ¹⁴C-concentration of tuber tissue and the in vitroactivities of starch synthase and ADPG-pyrophosphorylase anda significant negative correlation between ¹⁴C-concentrationand starch phosphorylase. In contrast, in tubers which wereanalysed 5 d after initiation, there were only small differencesbetween tubers in growth rate, ¹⁴C import and the activity ratioADPG-pyrophosphorylase/starch phosphorylase. From various directand indirect evidence it is concluded that the growth rate ofindividual tubers, and thus the sink strength, is at least inpart controlled by the activity of starch synthesizing enzymes. Key words: Potato tuber, cooling, starch synthesizing enzymes     

Transfer of [14C] cholesterol and its metabolites between adult male and female worms of Schistosoma mansoni

Adult male and female worms of Schistosoma mansoni are able to incorporate [14C]cholesterol and convert it into several metabolites. Schistosomula on the other hand cannot convert the incorporated cholesterol. Male worms are able to transfer the [14C]cholesterol and labelled metabolites to female worms but labelled female worms can transfer cholesterol but are unable to transfer the conversion products of cholesterol to male worms. Uptake by female worms of labelled products shed by male worms seems to occur more efficiently in the presence of serum than in its absence.     

Linkage disequilibrium analysis in Machado-Joseph disease patients of different ethnic origins

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration originally described in families of Portuguese-Azorean ancestry. The hypothesis that its present world distribution could result from the spread of an original founder mutation has been raised. To test this possibility we have conducted a linkage disequilibrium study of markers segregating with the MJD1 locus in a total of 64 unrelated families of different geographical origins. Significant association was detected between the MJD1 locus and marker alleles at loci D14S280, D14S1050 and D14S81. All affected individuals, except one Chinese family, had allele 3 (237 bp) at D14S280. This finding is consistent with a founder effect in our MJD population. However, distinct haplotypes were observed in patients originating from the two Azorean islands showing the highest disease prevalence; therefore, the possible existence of more than one founder mutation can not be excluded with the markers currently available. Received: 27 February 1996 / Revised: 4 June 1996     

Activation of Dictyostelium myosin light chain kinase A by phosphorylation of Thr166.

Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in non-muscle cells. Unlike most myosin light chain kinases (MLCKs), MLCK-A from Dictyostelium is not activated by Ca2+/calmodulin. Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism. Here, we show that MLCK-A is autophosphorylated on Thr289, which is C-terminal to the catalytic domain. Phosphorylation of MLCK-A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK-A. However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue. Based on comparisons with other protein kinases, we tested whether phosphorylation of Thr166 drives activation of MLCK-A. Our data indicate that phosphorylation of Thr289 occurs in vivo, but is not associated with conA-induced activation, whereas phosphorylation of Thr166 by some as yet unidentified kinase is associated with activation. Replacement of Thrl66 with glutamate results in a 12-fold increase in activity as compared with the wild-type enzyme, supporting the idea that phosphorylation of Thr166 increases MLCK-A activity.     

Studies on Stenostomum grande Child, 1902 (Platyhelminthes,Catenulida): fine structure of the digestive tract and the endocytotic activity of the gastrodermis

Abstract The digestive tract and its endocytotic activity in the catenulid Stenostomum grande were studied by electron microscopy. The pharynx was typical of the simplex type. At the mouth, between the integumental epithelium and the pharyngeal epithelium proper, was a transition zone. Among the epithelial cells of this transition were monociliated sensory cells and the necks of bucco-pharyngeal secretory cells of two types. The pharyngeal epithelium proper was densely ciliated, with long ciliary rootlets and mitochondria. It was surrounded by two layers of muscles. The gastrodermis consisted of phagocytes and typical secretory Minotian cells. It was underlain by a delicate basal lamina and muscle fibers. Distinctive of the phagocytes was the presence of differentiated cilia, cup-shaped mitochondria, and vacuoles with dense inclusions. Morphological differences between pharyngeal and gastrodermal cilia suggest functional differences. Experiments using latex beads as tracers and the identification of acid phosphatase in cytoplasmic vacuoles pointed to a high level of endocytotic and digestive activity in the phagocytes. Our data demonstrate that the basic structure of the digestive tract in S. grande conforms well to that of other free-living platyhelminths, but it does have ultrastructural peculiarities.     

Neurocircuitry of the basal ganglia studied by monitoring neurotransmitter release

The neurocircuitries of the basal ganglia are studied with in vivo microdialysis, with special consideration to dopamine transmission and its interaction with other neurotransmitter systems. The aim is to develop experimental models to study the pathophysiology and therapy of neurodegenerative disorders of the basal ganglia, as well as to develop models to study the short- and long-term consequences of perinatal asphyctic lesions. A main goal of these studies is to find and to characterize new treatments for these disorders.     

Microquantification of proteins with low chromophore content

The advantages of the intrinsic fluorescence of the tryptophan and the absorbance of the methionine residues of the 18 kDa-hsp - a recombinant protein from Mycobacterium leprae - was exploited here to develop a sensitive and low costs method for protein assaying. They presented linearity between 3 and 1000 g of protein. The correlations between intrinsic fluorescence or absorbance at 230 nm and protein contents were both superiors to 0.99. These methods can be extended to others proteins with low aromatic residue contents.