Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to β-lactamase inhibitors

Abstract A novel procedure was used to purify a cytosolic chitinase from Candida albicans to electrophoretic homogeneity. The results represent the first demonstration of the purification of a fungal intracellular chitinase using the criterion of a single band detected following silver-staining of a polyacrylamide gel run under denaturing conditions. Purified chitinase had pH and temperature optima of 5.0 and 50°C, respectively. Inhibition of enzyme activity by allosamidin was pH-dependent occuring maximally at pH 8.0. Phospholipids had similar marked and highly specific effects on the activities of both the purified soluble enzyme and a solubilized microsomal chitinase from C. albicans . Evidence is provided for the existence of a complex chitinolytic system in this organism.     

Telolysomes in cultured iris epithelial cells and in the TVI cell-line.

Iris epithelial cells of adult newts, which are fully differntiated melanocytes and non-dividing, become dedifferentiated and converted into lens cells when put in culture. A recent study shows that this dedifferentiation is based on an autophagic process which is associated with proliferation and mainly affects melanosomes. The present report shows that in primary culture of iris epithelial cells after the majority of melanosomes have disappeared, myelinoid bodies, which are interpreted to be telolysosomes of autophagic nature, appear in high frequencies. This suggest that in these cells autophagy persists after the loss of melanosomes. A possible connection of this type of autophagy with the differentation of lens fiber which occurs in this culture is discussed. In the TVI cell line which is believed to be derived from the same cell type, but devoid of melanosomes, similar myelinoid bodies are a characteristic cell component, suggesting that the tendency for autophagy is inherited in theis cell line.     

Intestinal perfusion indicates high reliance on paracellular nutrient absorption in an insectivorous bat Tadarida brasiliensis

Flying vertebrates have been hypothesized to have a high capacity for paracellular absorption of nutrients. This could be due to high permeability of the intestines to nutrient-sized molecules (i.e., in the size range of amino acids and glucose, MW 75–180 Da). We performed intestinal luminal perfusions of an insectivorous bat, Tadarida brasiliensis. Using radio-labeled molecules, we measured the uptake of two nutrients absorbed by paracellular and transporter-mediated mechanisms (l-proline, MW 115 Da, and d-glucose, MW 180 Da) and two carbohydrates that have no mediated transport (l-arabinose, MW 150 Da, and lactulose, MW 342 Da). Absorption of lactulose (0.61 ± 0.06 nmol min^? 1 cm^? 1) was significantly lower than that of the smaller arabinose (1.09 ± 0.04 nmol min^? 1 cm^? 1). Glucose absorption was significantly lower than that of proline at both nutrient concentrations (10 mM and 75 mM). Using the absorption of arabinose to estimate the portion of proline absorption that is paracellular, we calculated that 25.1 ± 3.0% to 66.2 ± 7.8% of proline absorption is not transporter-mediated (varying proline from 1 mM to 75 mM). These results confirm our predictions that 1) paracellular absorption is molecule size selective, 2) absorption of proline would be greater than glucose absorption in an insectivore, and 3) paracellular absorption represents a large fraction of total nutrient absorption in bats.     

Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei

In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei—the causative agent of Human African Trypanosomiasis—by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)₂), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)₂ redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC–MS/MS analysis was established to quantify intra-cellular CYN, T(SH)₂, GSH, as well as GS-CYN and T(S-CYN)₂ adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)₂ pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)₂ redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)₂ redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.     

Anthropogenic indicators in pollen diagrams in eastern France: a critical review

The focus of palaeoenvironmental sciences on past human activities and their impact on the environment necessitates a precise understanding of the history and functioning of past and present anthropogenic ecosystems. A process is outlined which uses palaeoenvironmental and historical documentation as well as present-day observations of vegetational changes from two different plant communities, which are characterised by a very specific anthropogenic flora: arable weeds and ruderals. This study is coupled with modern pollen deposition data to deduce a set of pollen types characteristic of the range of human activities practised in eastern France, a region rich in pollen data. First, phytogeographical analysis of the evolution of these plant communities since the Neolithic enables the comparison to be validated. By distinguishing between native plants and aliens introduced long ago (archaeophytes), or more recently (neophytes), and by refining their ecological characteristics, the method also enables identification of species that are strong indicators of human activities. Next, local pollen deposition in these vegetation types is examined with a number of statistical analyses (PCIA, Davis indices), confirming the relationships between a given vegetation community, its theoretical pollen rain and its actual pollen rain, thus distinguishing local and regional pollen indicators. Lastly, comparison of the results obtained by these two approaches leads to a critical synthesis of the traditional anthropogenic pollen indicators (Behre’s “indicator species”) in the study area and to the establishment of more specific local pollen indicators.     

The assessment of total energy expenditure of female farmers under field conditions.

The paper reviews methods, and their difficulties, in the measurement of the daily energy expenditure of rural women under field conditions in developing countries. Since all methods need to be validated against a reference method which is usually based on indirect calorimetry, examples of the use of this technique are given. The energy costs of most agricultural and daily tasks of rural women in developing countries have been measured. Large intra- and inter-individual variations in the cost of a single activity occur, so repeated measurements are needed to obtain a valid mean energy cost for a specific activity for a homogeneous group of individuals. Much work remains to be done on the assessment of the duration and the intensity of the physical activity of the rural adolescent and adult female population. Studies indicate that the workload of most rural women in developing countries is excessive and frequently associated with acute poverty.