Intestinal protozoa and epithelial cell kinetics, structure and function

Intestinal protozoa are not only common enteric pathogens in the tropics but also the high incidence of infection among immunocompromised patients in northern countries has evoked an increased interest in these parasites. Although enteric protozoa are a major cause of diarrhea and malabsorption in humans and other animals, the pathophysiology of gut disturbances caused by them remains poorly understood. Clinical signs related to enteric protozoan disease commonly involve malabsorption, diarrhea, weight loss or retarded weight gain and anorexua. Since these infections are most prevalent and most severe in the young, this may translate into considerable illness among children and significant loss to the agricultural economy where domestic animals are prone to infection. In this review we describe the effects of intestinal protozoan diseases on the structure, kinetics and function of absorptive intestinal cells and other epithelial cells, and correlate morphological injury with physiological alterations in the parasitized gut. Some of the interactions between immune responses and pathophysiology will be discussed, but in-depth discussion of intestinal immunity has recently been undertaken by other authors.     

Effect of high pressure on the photochemical reaction center from Rhodobacter sphaeroides R26.1

High-pressure studies on the photochemical reaction center from the photosynthetic bacterium Rhodobacter sphaeroides, strain R26.1, shows that, up to 0.6 GPa, this carotenoid-less membrane protein does not loose its three-dimensional structure at room temperature. However, as evidenced by Fourier-transform preresonance Raman and electronic absorption spectra, between the atmospheric pressure and 0.2 GPa, the structure of the bacterial reaction center experiences a number of local reorganizations in the binding site of the primary electron donor. Above that value, the apparent compressibility of this membrane protein is inhomogeneous, being most noticeable in proximity to the bacteriopheophytin molecules. In this elevated pressure range, no more structural reorganization of the primary electron donor binding site can be observed. However, its electronic structure becomes dramatically perturbed, and the oscillator strength of its Q(y) electronic transition drops by nearly one order of magnitude. This effect is likely due to very small, pressure-induced changes in its dimeric structure.     

Effect of Culture Conditions on Ergosterol as an Indicator of Biomass in the Aquatic Hyphomycetes

Ergosterol is a membrane component specific to fungi that can be used to estimate fungal biomass using appropriate factors of conversion. Our objectives were to determine the limits of use of ergosterol content as a measure of biomass for aquatic hyphomycetes, and to evaluate a previously established ergosterol-to-biomass conversion factor. We varied inoculum quality, growth medium, and degree of shaking of four aquatic hyphomycete species. In cultures inoculated with homogenized mycelium, we found a significant effect of shaking condition and culture age on ergosterol content. In liquid cultures with defined medium, ergosterol content reached 10 to 11 μg/mg of mycelium (dry mass) and varied by factors of 2.2 during exponential growth and 1.3 during stationary phase. The increase in ergosterol content during exponential phase could be attributed, at least in part, to rapid depletion of glucose. Oxygen availability to internal hyphae within the mycelial mass is also responsible for the differences found between culture conditions. Ergosterol concentration ranged from 0.8 to 1.6 μg/mg in static cultures inoculated with agar plugs. Ergosterol content varied by a factor of 4 in two media of different richnesses. For different combinations of these parameters, strong (r² = 0.83 to 0.98) and highly significant (P 0.001) linear relationships between ergosterol and mycelial dry mass (up to 110 mg) were observed. Overall, the ergosterol content varied by a factor of 14 (0.8 to 11 mg/g). These results suggest that care must be taken when the ergosterol content is used to compare data generated in different field environments.     

Probing the binding sites of exchanged chlorophyll a in LH2 by Raman and site-selection fluorescence spectroscopies

In this work we have selectively released the 800 nm absorbing bacteriochlorophyll a molecules of the LH2 protein from the photosynthetic bacterium Rhodopseudomonas acidophila, strain 10050, and replaced them with chlorophyll a (Chla). A combination of low-temperature electronic absorption, resonance Raman and site-selection fluorescence spectroscopies revealed that the Chla pigments are indeed bound in the B800 binding site; this is the first work that formally proves that such non-native chlorins can be inserted correctly into LH2.     

Nef-induced CD4 downregulation: a diacidic sequence in human immunodeficiency virus type 1 Nef does not function as a protein sorting motif through direct binding to beta-COP

The Nef protein from the human immunodeficiency virus (HIV) induces CD4 cell surface downregulation by interfering with the endocytic machinery. It has been recently proposed that binding of HIV type 1 Nef to the beta subunit of COPI coatomers participated in the Nef-induced CD4 downregulation through recognition of a novel diacidic motif found in the C-terminal disordered loop of Nef (V. Piguet, F. Gu, M. Foti, N. Demaurex, J. Gruenberg, J. L. Carpentier, and D. Trono, Cell 97:63-73, 1999). We have mutated the glutamate residues which formed this motif in order to document this observation. Surprisingly, mutation of the diacidic sequence of Nef did not significantly affect its ability (i) to interact with beta-COP, (ii) to downregulate CD4 cell surface expression, and (iii) to address an integral resident membrane protein containing Nef as the cytoplasmic domain to the endocytic pathway. Our results indicate that these acidic residues are not involved in the connection of Nef with the endocytic machinery through binding to beta-COP. Additional studies are thus required to characterize the residues of Nef involved in the binding to beta-COP and to evaluate the contribution of this interaction to the Nef-induced perturbations of membrane trafficking.     

Conus venoms: a source of toxins which interact with membrane- potential-dependent sodium channels

Marine snails of the genus Conus, as they are carnivorous predators, have a venom apparatus used to capture their prey. The toxins contained in the venoms of Conidae, called conotoxins, are of a particular high degree of diversity and represent powerful tools in the neuroscience field. Indeed, these toxins specifically bind with a high affinity to receptors and ionic channels. Therefore, they provide original pharmacological tools which receive increasing investigation both to identify and study some functions of the nervous systems and to characterize new types and closely related subtypes of receptors or ionic channels. The voltage-gated sodium channel, because of its fundamental role in cell membrane excitability, is the specific target of a large number of animal and vegetal toxins. Actually, at least seven toxin receptor sites have been identified on this channel-protein. These toxins, and in particular conotoxins, are used to precise the role of different types and/or closely related subtypes of sodium channels in the peripheral and central nervous systems. The focus of the present review is to summarize our current knowledge of the consequences of physiological interactions between different conotoxin families and sodium channels.     

Effects of mutagenesis on the detailed structure of spheroidenone in the Rhodobacter sphaeroides reaction centre examined by resonance Raman spectroscopy

The effect of mutagenesis on the detailed conformation of the carotenoid cofactor of the bacterial reaction centre has been examined using resonance Raman spectroscopy. Four single site mutations were made, removing polar residues that line the binding pocket for spheroidenone in the reaction centre from Rhodobacter sphaeroides. All of the mutations caused changes in the relative intensity of bands in the ₂ frequency region of the carotenoid resonance Raman spectrum, suggesting a change in the geometry of the central 15,15-cis bond of the spheroidenone. In addition, increased splitting of the ₁ vibrational modes in two of the mutant RCs indicated a reduction of the effective conjugation length of the spheroidenone, possibly due to an increased distortion from a planar geometry along the C=C backbone of the spheroidenone. These changes in the detailed conformation of the reaction centre carotenoid do not affect the optical properties o f the cofactor, and are beyond the limits of detection of X-ray crystallography as currently applied to the bacterial reaction centre.     

Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.