Cell wall analysis

The cell wall is a rigid structure essential for survival of the fungal cell. Because of its absence in mammalian cells, the cell wall is an attractive target for antifungal agents. Thus, for different reasons, it is important to know how the cell wall is synthesized and how different molecules regulate that synthesis. The Schizosaccharomyces pombe cell wall is mainly formed by glucose polysaccharides and some galactomannoproteins. Here, we describe a fast and reliable method to analyze changes in S. pombe cell wall composition by using specific enzymatic degradation and chemical treatment of purified cell walls. This approach provides a powerful means to analyze changes in (1,3)beta-glucan and (1,3)alpha-glucan, two main polysaccharides present in fungal cell walls. Analysis of cell wall polymers will be useful to search for new antifungal drugs that may inhibit cell wall biosynthesis and/or alter cell wall structure.     

Revisiting the mouse mitochondrial DNA sequence

The existence of reliable mtDNA reference sequences for each species is of great relevance in a variety of fields, from phylogenetic and population genetics studies to pathogenetic determination of mtDNA variants in humans or in animal models of mtDNA-linked diseases. We present compelling evidence for the existence of sequencing errors on the current mouse mtDNA reference sequence. This includes the deletion of a full codon in two genes, the substitution of one amino acid on five occasions and also the involvement of tRNA and rRNA genes. The conclusions are supported by: (i) the re-sequencing of the original cell line used by Bibb and Clayton, the LA9 cell line, (ii) the sequencing of a second L-derivative clone (L929), and (iii) the comparison with 12 other mtDNA sequences from live mice, 10 of them maternally related with the mouse from which the L cells were generated. Two of the latest sequences are reported for the first time in this study (Balb/cJ and C57BL/6J). In addition, we found that both the LA9 and L929 mtDNAs also contain private clone polymorphic variants that, at least in the case of L929, promote functional impairment of the oxidative phosphorylation system. Conse quently, the mtDNA of the strain used for the mouse genome project (C57BL/6J) is proposed as the new standard for the mouse mtDNA sequence.     

Impact of aging on myocardial metabolic response to dobutamine

In humans, under resting conditions there is an age-related decrease in myocardial fatty acid utilization (MFAU) and oxidation (MFAO) and a relative increase in myocardial glucose utilization (MGU). The impact of age on an individual's myocardial metabolic response to catecholamines is not well defined. Sixteen younger (mean age, 26 +/- 5 yr) and 14 older (mean age, 69 +/- 4 yr) volunteers underwent positron emission tomography to measure myocardial blood flow, myocardial oxygen consumption (M.VO2), MFAU, MFAO, and MGU both under resting conditions and during dobutamine infusion. In response to dobutamine administration, the rate-pressure product, myocardial blood flow, and M.VO2 measurements increased by similar amounts in both groups. No age-related differences were noted in the responses of plasma insulin, glucose, fatty acid, or lactate levels to dobutamine. With dobutamine infusion, MFAU and MFAO increased by a similar extent in both younger and older volunteers (age/dobutamine interactions, P = 0.62 and 0.75, respectively). In contrast, MGU increased with dobutamine administration in the younger (from 149 +/- 71 to 209 +/- 78 nmol.g(-1).min(-1); P = 0.04) but not in the older (from 235 +/- 147 to 176 +/- 84 nmol.g(-1).min(-1); P = 0.23; age/dobutamine interaction, P = 0.03) group. With dobutamine infusion, hearts in both younger and older volunteers responded by increasing their MFAU and MFAO values. Whereas younger hearts also responded with an increase in MGU, older hearts did not. Although the clinical significance of these findings awaits further study, these results may partially explain the impaired contractile reserve and the increased incidence of cardiovascular disease in older individuals.     

How FMN binds to anabaena apoflavodoxin: a hydrophobic encounter at an open binding site

Molecular recognition begins when two molecules approach and establish interactions of certain strength. The mechanisms of molecular recognition reactions between biological molecules are not well known, and few systems have been analyzed in detail. We investigate here the reaction between an apoprotein and its physiological cofactor (apoflavodoxin and flavin mononucleotide) that binds reversibly to form a non-covalent complex (flavodoxin) involved in electron transfer reactions. We have analyzed the fast binding reactions between the FMN cofactor (and shorter analogs) and wild type (and nine mutant apoflavodoxins where residues interacting with FMN in the final complex have been replaced). The x-ray structures of two such mutants are reported that show the mutations are well tolerated by the protein. From the calculated microscopic binding rate constants we have performed a Phi analysis of the transition state of complex formation that indicates that the binding starts by interaction of the isoalloxazine-fused rings in FMN with residues of its hydrophobic binding site. In contrast, the phosphate in FMN, known to contribute most to the affinity of the final holoflavodoxin complex, is not bound in the transition state complex. Both the effects of ionic strength and of phosphate concentration on the wild type complex rate constant agree with this scenario. As suggested previously by nmr data, it seems that the isoalloxazine-binding site may be substantially open in solution. Interestingly, although FMN is a charged molecule, electrostatic interactions seem not to play a role in directing the binding, unlike what has been reported for other biological complexes. The binding can thus be best described as a hydrophobic encounter at an open binding site.     

Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection

The most common cause of infant mortality is diarrhea; the most common cause of bacterial diarrhea is Campylobacter jejuni, which is also the primary cause of motor neuron paralysis. The first step in campylobacter pathogenesis is adherence to intestinal mucosa. We found that such binding was inhibited in vitro by human milk and, with high avidity, by alpha1,2-fucosylated carbohydrate moieties containing the H(O) blood group epitope (Fuc alpha 1,2Gal beta 1,4GlcNAc em leader ). In studies on the mechanism of adherence, campylobacter, which normally does not bind to Chinese hamster ovary cells, bound avidly when the cells were transfected with a human alpha1,2-fucosyltransferase gene that caused overexpression of H-2 antigen; binding was specifically inhibited by H-2 ligands (lectins Ulex europaeus and Lotus tetragonolobus and H-2 monoclonal antibody), H-2 mimetics, and human milk oligosaccharides. Human milk oligosaccharides inhibited campylobacter colonization of mice in vivo and human intestinal mucosa ex vivo. Campylobacter colonization of nursing mouse pups was inhibited if their dams had been transfected with a human alpha1,2-fucosyltransferase gene that caused expression of H(O) antigen in milk. We conclude that campylobacter binding to intestinal H-2 antigen is essential for infection. Milk fucosyloligosaccharides and specific fucosyl alpha1,2-linked molecules inhibit this binding and may represent a novel class of antimicrobial agents.     

Asynchronous development of stigmatic receptivity in the pear (Pyrus communis; Rosaceae) flower

While stigma anatomy is well documented for a good number of species, little information is available on the acquisition and cessation of stigmatic receptivity. The aim of this work is to characterize the development of stigma receptivity, from anthesis to stigma degeneration, in the pentacarpellar pear (Pyrus communis) flower. Stigma development and stigmatic receptivity were monitored over two consecutive years, as the capacity of the stigmas to offer support for pollen germination and pollen tube growth. In an experiment where hand pollinations were delayed for specified times after anthesis, three different stigmatic developmental stages could be observed: (1) immature stigmas, which allow pollen adhesion but not hydration; (2) receptive stigmas, which allow proper pollen hydration and germination; and (3) degenerated stigmas, in which pollen hydrates and germinates properly, but pollen tube growth is impaired soon after germination. This developmental characterization showed that stigmas in different developmental stages coexist within a flower and that the acquisition and cessation of stigmatic receptivity by each carpel occur in a sequential manner. In this way, while the duration of stigmatic receptivity for each carpel is rather short, the flower has an expanded receptive period. This asynchronous period of receptivity for the different stigmas of a single flower is discussed as a strategy that could serve to maximize pollination resources under unreliable pollination conditions.     

Brain microvessel endothelin type A receptors are coupled to ceramide production

Treatment of brain microvessels with endothelin-1 evoked an early decrease in the sphingomyelin levels concomitantly with an increase in those of ceramides. These responses were time- and concentration-dependent. Evidence also shows that endothelin type A receptors are involved. This is the first report on the involvement of an agonist in the regulation of the ceramide signal transduction system on blood-brain barrier and shows a new pathway likely involved in the regulation of the cerebral microvascular functioning.     

Unusual characteristics of ciliate actins

Actin is a cytoskeletal protein that is ubiquitous in eukaryotes, hence the corresponding genes and proteins have been isolated from numerous organisms as different as animals, plants, fungi and protozoa. Several atomic models are available for the monomeric as well as the filamentous form, and more than 70 proteins that bind actin and control filament dynamics have been isolated from diverse eukaryotes. Moreover, the function and dynamics of the actin cytoskeleton in several eukaryotic systems have been depicted in depth. Unlike other protozoa, such as amoeba, actin is not an abundant protein in ciliates, whose cytoskeleton is mainly composed of microtubular arrays. Ciliate actin has been studied in several species, and it was established early on that this ciliate protein is very different from that of other eukaryotes. Similarly, the actin-binding proteins studied in ciliates display great differences with those of other eukaryotes. Consequently, ciliate actin has been considered as "unconventional," and this review focuses on molecular data leading to this conclusion.     

Sphingomyelinase cleavage of sphingomyelin in pure and mixed lipid membranes. Influence of the physical state of the sphingolipid

Sphingomyelin hydrolysis by sphingomyelinase is essential in regulating membrane levels of ceramide, a well-known metabolic signal. Since natural sphingomyelins have a gel-to-fluid transition temperature in the range of the physiological temperatures of mammals and birds, it is important to understand the influence of the physical state of the lipid on the enzyme activity. With that aim, large unilamellar vesicles consisting of pure egg sphingomyelin (gel-to-fluid crystalline transition temperature ca. 39 degrees C) were treated with sphingomyelinase in the temperature range 10-70 degrees C. The vesicles were also examined by differential scanning calorimetry (DSC). Shingomyelinase was active on pure sphingomyelin bilayers, leading to concomitant lipid hydrolysis, vesicle aggregation, and leakage of aqueous liposomal contents. Enzyme activity was found to be much higher when the substrate was in the fluid than when it was in the gel state. Sphingomyelinase activity was found to exhibit lag times, followed by bursts of activity. Lag times decreased markedly when the substrate went from the gel to the fluid state. When egg phosphatidylcholine, or egg phosphatidylethanolamine were included in the bilayer composition together with sphingomyelin, sphingomyelinase activity at 37 degrees C, that was negligible for the pure sphingolipid bilayers, was seen to increase with the proportion of glycerophospholipid, while the latency times became progressively shorter. A DSC study of the mixed-lipid vesicles revealed that both phosphatidylcholine and phosphatidyletanolamine decreased in a dose-dependent way the transition temperature of sphingomyelin. Thus, as those glycerophospholipids were added to the membrane composition, the proportion of sphingomyelin in the fluid state at 37 degrees C increased accordingly, in this way becoming amenable to rapid hydrolysis by the enzyme. Thus sphingomyelinase requires the substrate in bilayer form to be in the fluid state, irrespective of whether this is achieved through a thermotropic transition or by modulating bilayer composition.