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.     

Nature and properties of humic acid prepared from different sources and its effect on nutrient availability

Summary Effect of sources, moisture levels and extractants on extraction, analytical properties, IR-spectra, macromolecular characteristics, interaction with cations and physiological properties of the extracted material (humic acid) were studied. Among the three variables, extractant influenced the degree of humification, IR-spectra, nature and properties of humic acids to a greater extent though the other two had significant contribution. Sodium pyrophosphate found to be an ideal extractant for humic acid. Reduced viscosity of humic acids increased with dilution particularly below 0.1 g/dl and decreased with increasing H⁺ concentration between pH 6.5 and 8.5 irrespective of source, moisture level and extractant. The amount of proton released and drop in initial pH of humic acids on addition of different metal ions indicated formation of mono-, di-hydroxy metal-HA complexes which broke down at pH 8.0 and above. HA-Fe²⁺ and HA-Cu²⁺ complexes were found to be most stable. Humic acids from different sources varied significantly in their effect on available nutrient status of soils though, the latter is main determining factor particularly for calcium and magnesium.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

P-factor,a developmental hormone of Penicillium cyclopium?

From the mycelium of Penicillium cyclopium a biologically active fraction (P-factor) was isolated, which increases conidiation and the formation of the benzodiazepine alkaloids cyclopenin and cyclopenol. Its activity was determined by measuring the increase of alkaloid formation in strain SM 72. On a preparative scale P-factor preparations were obtained from fermenter-grown hyphae of mutant dev 63 by extraction with water at 120°. P-factor is strongly hydrophilic but it is not a protein. It was active if added during conidiospore germination and early growth phase, causing an acceleration of protein biosynthesis. The action on alkaloid biosynthesis and sporulation is indirect and resembles that of a developmental hormone.     

THE INORGANIC CARBON REQUIREMENTS OF CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) FOR CELL DEVELOPMENT IN SYNCHRONOUS CULTURES1

The time in the cell cycle when CO₂ provision was required for cell development and division was determined in synchronous cultures of Chlamydomonas segnis Ettl bubbled with air (0.03% CO₂) or air enriched with 5% CO₂ under continuous light at 25°C and pH 7. Provision of CO₂ (% in air v/v) during the G₁-phase was found to be essential for the completion of the cell cycle. There was no demand for CO₂ supply throughout the S-phase and mitosis. Using cultures adapted to CO₂ concentrations ranging from 0.03 to 5% in air, the apparent CO₂ concentration (K_m) required for the cells to develop during the G_-1-phase and to attain one half the maximal rates of photo-synthetic O₂ evolution was calculated as 0.05%. This value increased to 0.1 and 0.5% during the S-phase. For total protein and carbohydrate accumulation, which would reflect inorganic carbon (CO₂+ HCO₃^?) assimilation, the K_m (% CO₂) were ca. 0.1 and 0.14 throughout the cell cycle, respectively. The CO₂ concentration at which the cells exhibited the shortest generation time (6.7 h) was 0.1%. These results showed that during development, cells photosynthesizing (evolving O₂) at maximal rates but accumulating protein and carbohydrate at one half the maximal rates or less would complete their vegetative life cycle in the shortest time.     

Use of activated clotting time for monitoring anticoagulation during cardiopulmonary bypass in infants and children with congenital heart disease

The use of a fixed dosage schedule was compared with the use of activated clotting time (ACT) for determining heparin and protamine dosages during and after cardiopulmonary bypass disease. Use of the ACT resulted in a statistically significant increase in heparin dosage and a statistically significant reduction of postoperative blood loss. With ACT use, chest tubes were retained for a shorter period of time, and the incidence of serious postoperative hemorrhage was reduced from 44% to 18%. These results confirm the superiority of the ACT method for monitoring intraoperative anticoagulation in pediatric patients with congenital heart disease.     

Exosomes surf on filopodia to enter cells at endocytic hot spots,traffic within endosomes,and are targeted to the ER

Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery.