Demographic stochasticity versus spatial variation in the competition between fast and slow dispersers

Dispersal is an important strategy that allows organisms to locate and exploit favorable habitats. The question arises: given competition in a spatially heterogeneous landscape, what is the optimal rate of dispersal? Continuous population models predict that a species with a lower dispersal rate always drives a competing species to extinction in the presence of spatial variation of resources. However, the introduction of intrinsic demographic stochasticity can reverse this conclusion. We present a simple model in which competition between the exploitation of resources and stochastic fluctuations leads to victory by either the faster or slower of two species depending on the environmental parameters. A simplified limiting case of the model, analyzed by closing the moment and correlation hierarchy, quantitatively predicts which species will win in the complete model under given parameters of spatial variation and average carrying capacity.     

Guidelines for indications for coronary revascularization in The Netherlands

In 1996 the Minister of Public Health, Welfare and Sports in The Netherlands published a 'Planning Decree Special Interventions in the Heart'. She requested from the professional organizations guidelines for the indications for interventions in the heart. A working group was formed with representatives from the Dutch professional organizations for cardiology and thoracic surgery, to address this issue for patients with coronary artery disease. The working group confirmed the need to discuss all patients who are considered for either elective or emergency revascularization during a multidisciplinary consultation in (or with) one of the specialized Dutch hospitals. During this meeting of the 'heart team', at least one interventional cardiologist and one thoracic surgeon should be present. There are three possible outcomes of the heart team's consultations for each patient: drug therapy only ('conservative management'), coronary surgery or catheter intervention. For each case, the team should indicate the expected benefit, the risk of the intervention, the urgency and the estimated waiting time. The guidelines presented in this paper address these issues for three patient categories: stable angina pectoris, unstable angina pectoris and acute myocardial infarction.     

Accumulation of protein-bound epidermal glucosylceramides in beta-glucocerebrosidase deficient type 2 Gaucher mice

The epidermal permeability barrier for water is essentially maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the main components of these membranes, derive in large part from hydrolysis of glucosylceramides mediated by the lysosomal enzyme beta-glucocerebrosidase. As analyzed in this work, the beta-glucocerebrosidase deficiency in type 2 Gaucher mice (RecNci I) resulted in an accumulation of all epidermal glucosylceramide species accompanied with a decrease of the related ceramides. However, the levels of one ceramide subtype, which possesses an alpha-hydroxypalmitic acid, was not altered in RecNci I mice suggesting that the beta-glucocerebrosidase pathway is not required for targeting of this lipid to interstices of the stratum corneum. Most importantly, omega-hydroxylated glucosylceramides which are protein-bound to the epidermal cornified cell envelope of the transgenic mice accumulated up to 35-fold whereas levels of related protein-bound ceramides and fatty acids were decreased to 10% of normal control. These data support the hypothesis that in wild-type epidermis omega-hydroxylated glucosylceramides are first transferred enzymatically from their linoleic esters to proteins of the epidermal cornified cell envelope and then catabolized to protein-bound ceramides and fatty acids, thus contributing at least in part to the formation of the lipid-bound envelope.     

Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion

Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency.     

An alpha-1,3-mannosyltransferase of Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus, distinguished by an elaborate polysaccharide capsule that is essential for its virulence. As part of an effort to understand the biosynthesis of this important structure, we initiated purification of an alpha-1,3-mannosyltransferase with appropriate specificity for a role in building the main capsule polysaccharide, glucuronoxylomannan. A pool of proteins that was 5,000-fold enriched in this activity included several polypeptides, which acted potentially as the catalytic protein. These were analyzed using sequence information and double-stranded RNA interference. Interference that targeted a sequence corresponding to part of a 46 kDa protein in the enriched fraction abolished the activity of interest and reduced the capsule on the affected cells. This gene was cloned and expressed in active form in Saccharomyces cerevisiae to confirm function, and was termed CMT1, for cryptococcal mannosyltransferase 1. CMT1 has no confirmed homologs in GenBank other than CAP59, a cryptococcal gene encoding a protein of unknown function that is required for capsule synthesis and virulence. The Cmt1p protein also co-purifies with a homolog of CAP64, a gene whose product has similarly been implicated in capsule synthesis and virulence. A strain disrupted in CMT1 was generated in C. neoformans; this had no effect on virulence in an animal model of cryptococcosis.     

Determinants of inhibition of transiently expressed voltage-gated calcium channels by omega-conotoxins GVIA and MVIIA

The Conus magus peptide toxin omega-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both omega-conotoxin GVIA and MVIIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EF-hand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (approximately 3 microm), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.     

RNA interference in the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus responsible for serious disease in immunocompromised individuals. This organism has recently been developed as an experimental system, with initiation of a genome project among other molecular advances. However, investigations of Cryptococcus are hampered by the technical difficulty of specific gene replacements. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, may help solve this problem. We have shown that expression of double-stranded RNA corresponding to portions of the cryptococcal CAP59 and ADE2 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Specific modulation of protein expression through introduction of double-stranded RNA thus operates in C. neoformans, which is the first demonstration of this technique in a fungal organism. Use of RNA interference in Cryptococcus should allow manipulation of mRNA levels for functional analysis of genes of interest and enable efficient exploration of genes discovered by genome sequencing.     

Sequence heterogeneity of the ferripyoverdine uptake (fpvA), but not the ferric uptake regulator (fur), genes among strains of the fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida

Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida are of importance to medicine, agriculture and biocycling. These microbes acquire ferric ion via the use of the siderophores pyochelin and the family known as the pyoverdines or pseudobactins. The ferric uptake regulator (fur) gene is responsible, at least in part, for the regulation of siderophore synthesis and uptake in P. aeruginosa.To determine whether the organisms contain single or multiple homologues of the siderophore-related genes fpvA (ferripyoverdine uptake) and fur, and whether these homologues displayed sequence heterogeneity, their chromosomal DNAs were probed with fur and fpvA sequences. As a representative of a non-fluorescent pseudomonad, the bacterium Burkholderia (Pseudomonas) cepacia was also examined.The pseudomonads all contained fpvA- and fur-like homologues, and heterogeneity was observed among the different species. The presence of two or more fpvA-like genes is indicated in all of the fluorescent pseudomonads surveyed. In contrast, B. cepacia DNA either did not hybridize to these probes, or did so only very weakly, suggesting that fur- and fpvA-like homologues are either absent or significantly different in B. cepacia compared to the fluorescent pseudomonads examined.     

Genetic ablation of caveolin-2 sensitizes mice to bleomycin-induced injury

Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1^−/−) and Cav-2-deficient mice (Cav-2^−/−) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2^−/− mice, but not Cav-1^−/− mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2^−/− mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2^−/− mice. Since Cav-1^−/− mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.