The high-osmolarity glycerol response pathway in the human fungal pathogen Candida glabrata strain ATCC 2001 lacks a signaling branch that operates in baker's yeast

The high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway mediates adaptation to high-osmolarity stress in the yeast Saccharomyces cerevisiae. Here we investigate the function of HOG in the human opportunistic fungal pathogen Candida glabrata. C. glabrata sho1Delta (Cgsho1Delta) deletion strains from the sequenced ATCC 2001 strain display severe growth defects under hyperosmotic conditions, a phenotype not observed for yeast sho1Delta mutants. However, deletion of CgSHO1 in other genetic backgrounds fails to cause osmostress hypersensitivity, whereas cells lacking the downstream MAP kinase Pbs2 remain osmosensitive. Notably, ATCC 2001 Cgsho1Delta cells also display methylglyoxal hypersensitivity, implying the inactivity of the Sln1 branch in ATCC 2001. Genomic sequencing of CgSSK2 in different C. glabrata backgrounds demonstrates that ATCC 2001 harbors a truncated and mutated Cgssk2-1 allele, the only orthologue of yeast SSK2/SSK22 genes. Thus, the osmophenotype of ATCC 2001 is caused by a point mutation in Cgssk2-1, which debilitates the second HOG pathway branch. Functional complementation experiments unequivocally demonstrate that HOG signaling in yeast and C. glabrata share similar functions in osmostress adaptation. In contrast to yeast, however, Cgsho1Delta mutants display hypersensitivity to weak organic acids such as sorbate and benzoate. Hence, CgSho1 is also implicated in modulating weak acid tolerance, suggesting that HOG signaling in C. glabrata mediates the response to multiple stress conditions.     

Role of ischemia and deformation in the onset of compression-induced deep tissue injury: MRI-based studies in a rat model.

A rat model was used to distinguish between the different factors that contribute to muscle tissue damage related to deep pressure ulcers that develop after compressive loading. The separate and combined effects of ischemia and deformation were studied. Loading was applied to the hindlimb of rats for 2 h. Muscle tissue was examined using MR imaging (MRI) and histology. An MR-compatible loading device allowed simultaneous loading and measurement of tissue status. Two separate loading protocols incorporated uniaxial loading, resulting in tissue compression and ischemic loading. Uniaxial loading was applied to the tibialis anterior by means of an indenter, and ischemic loading was accomplished with an inflatable tourniquet. Deformation of the muscle tissue during uniaxial loading was measured using MR tagging. Compression of the tissues for 2 h led to increased T2 values, which were correlated to necrotic regions in the tibialis anterior. Perfusion measurements, by means of contrast-enhanced MRI, indicated a large ischemic region during indentation. Pure ischemic loading for 2 h led to reversible tissue changes. From the MR-tagging experiments, local strain fields were calculated. A 4.5-mm deformation, corresponding to a surface pressure of 150 kPa, resulted in maximum shear strain up to 1.0. There was a good correlation between the location of damage and the location of high shear strain. It was concluded that the large deformations, in conjunction with ischemia, provided the main trigger for irreversible muscle damage.     

Functional HLA-DR T cell epitopes of CEA identified in patients with colorectal carcinoma immunized with the recombinant protein CEA

A baculovirus-produced recombinant CEA (rCEA) protein comprising the extracellular region was used for vaccination of CRC patients with or without GM-CSF as an adjuvant cytokine. Ten patients with a significant proliferative T cell response against rCEA were selected for T cell epitope mapping. Fifteen-aa-long overlapping peptides covering the entire aa sequence of the external domain of CEA were used in a proliferation assay. In six of the patients a repeatable T cell response against at least one peptide was demonstrated. For the first time, nine functional HLA-DR epitopes of CEA were defined. Two of the peptides were recognized by more than one patient, i.e., two and three patients, respectively. Those 15-mer peptides that induced a proliferative T cell response fitted to the actual HLA-DR type (SYFPEITHI). The affinity of the native peptides for the T cell receptor was in the low to intermediate range (scores 6–19). The 15-mer peptides also contained 9-mer peptide sequences that could be predicted to bind to the actual HLA-ABC genotypes (SYFPEITHI/BIMAS). Blocking experiments using monoclonal antibodies indicated that the proliferative T cell response was both MHC class I and II restricted. The defined HLA-DR T cell epitopes were spread over the entire CEA molecule, but a higher frequency was noted towards the C-terminal. Peptides with a dual specificity may form a basis for production of subunit cancer vaccines, but modifications should be done to increase the T cell affinity, thereby optimizing the antitumoral effects of the vaccine.Abbreviations aa amino acid - CRC colorectal carcinoma - GM-CSF granulocyte/monocyte colony stimulating factor - CEA carcino-embryonic antigen - BCP baculovirus control protein - MHC major histocompatibility complex - pp peptide - TAA tumor associated antigen     

Flow cytometric analysis of mitochondria from CA1 and CA3 regions of rat hippocampus reveals differences in permeability transition pore activation

Mitochondria are important in the pathophysiology of several neurodegenerative diseases, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes and release of apoptogenic proteins are involved in these processes. Following brain insults, cell death often occurs in discrete regions of the brain, such as the subregions of the hippocampus. To analyse mitochondrial structure and function in such subregions, only small amounts of mitochondria are available. We developed a protocol for flow cytometric analysis of very small samples of isolated brain mitochondria, and analysed mitochondrial swelling and formation of ROS in mitochondria from the CA1 and CA3 regions of the hippocampus. Calcium-induced mitochondrial swelling was measured, and fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), to measure membrane potential (tetramethylrhodamine-methyl-ester, 1,1',3,3,3',3'-hexamethylindodicarbocyanine-iodide) and to measure production of ROS (2',7'-dichlorodihydrofluorescein-diacetate). We found that formation of ROS and mitochondrial permeability transition pore activation were higher in mitochondria from the CA1 than from the CA3 region, and propose that differences in mitochondrial properties partly underlie the selective vulnerability of the CA1 region to brain insults. We also conclude that flow cytometry is a useful tool to analyse the role of mitochondria in cell death processes.     

Diversification in the tropical pacific: comparisons between marine and terrestrial systems and the importance of founder speciation

Patterns of distribution and processes of differentiation haveoften been contrasted between terrestrial and marine biotas.The islands of Oceania offer an excellent setting to explorethis contrast, because the geographic setting for terrestrialand shallow-water, benthic, marine organisms are the same: themyriad islands strewn across the vast Pacific. The size of speciesranges and the geographic distribution of endemism are two biogeographicattributes that are thought to differ markedly between terrestrialand marine biotas in the Pacific. While terrestrial speciesare frequently confined to single islands or archipelagoes throughoutOceania, marine species tend to have wide to very wide distributions,and are rarely restricted to single island groups except forthe most isolated archipelagoes. We explore the conditions underwhich species can reach an island by dispersal and differentiate.Genetic differentiation can occur either through founder speciationor vicariance; these processes are requisite ends of a continuum.We show that founder speciation is most likely when few propagulesenter the dispersal medium and survive well while they travelfar. We argue that conditions favorable to founder speciationare common in marine as well as terrestrial systems, and thatterrestrial-type, archipelagic-level endemism is likely commonin marine taxa. We give examples of marine groups that showarchipelagic level endemism on most Pacific island groups aswell as of terrestrial species that are widespread. Thus boththe patterns and processes of insular diversification are variable,and overlap more between land and sea than previously considered.     

Scale-up of stirring as foam disruption (SAFD) to industrial scale

Foam disruption by agitation—the stirring as foam disruption (SAFD) technique—was scaled up to pilot and production scale using Rushton turbines and an up-pumping hydrofoil impeller, the Scaba 3SHP1. The dominating mechanism behind SAFD—foam entrainment—was also demonstrated at production scale. The mechanistic model for SAFD defines a fictitious liquid velocity generated by the (upper) impeller near the dispersion surface, which is correlated with complete foam disruption. This model proved to be scalable, thus enabling the model to be used for the design of SAFD applications. Axial upward pumping impellers appeared to be more effective with respect to SAFD than Rushton turbines, as demonstrated by retrofitting a 12,000 l bioreactor, i.e. the triple Rushton configuration was compared with a mixed impeller configuration from Scaba with a 20% lower ungassed power draw. The retrofitted impeller configuration allowed 10% more broth without risking excessive foaming. In this way a substantial increase in the volumetric productivity of the bioreactor was achieved. Design recommendations for the application of SAFD are given in this paper. Using these recommendations for the design of a 30,000 l scale bioreactor, almost foamless Escherichia coli fermentations were realised. Electronic Publication     

Gates,GAVI, the glorious global funds and more: all you ever wanted to know

Global immunization programmes have achieved some remarkable successes. In 1977, Frank Fenner's Commission declared smallpox to have been eradicated by an 11-year-long intensive campaign. The Expanded Programme on Immunization encompassed six important childhood vaccines and reached over three-quarters of the world's children. Polio eradication has gone remarkably well, with only 10 out of 200 countries reporting residual cases. But amidst all the good news, there is also bad news. Coverage is variable; infrastructure is crumbling; and newer vaccines are not being incorporated in many country programmes. The Bill and Melinda Gates Foundation has introduced a new dynamic here. From their initial gift of $100 million in December 1998, their commitment to date is US$1.5 billion - and rising. At the centre is a Global Children's Vaccine Fund which permitted the launch, in January 2000, of the Global Alliance for Vaccines and Immunization. This is targeted to the 74 poorest countries of the world and is designed to improve vaccination infrastructure, to purchase newer vaccines and to support research and development. Even before we know how successful this programme will be, it has had its imitators. The Global Fund to Fight AIDS, TB and Malaria borrowed many concepts from GAVI. The Global Alliance for Improved Nutrition announced in May 2002 does so as well, and is heavily supported by Gates. Highly effective parasite control programmes antedate all this but will be much strengthened. However, we still face a sizeable budgetary gap both for research and for bringing the best advances to all people who need them.     

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.     

Linkage of a gene for familial hypobetalipoproteinemia to chromosome 3p21.1-22

Familial hypobetalipoproteinemia (FHBL) is an apparently autosomal dominant disorder of lipid metabolism characterized by less than fifth percentile age- and sex-specific levels of apolipoprotein beta (apobeta) and low-density lipoprotein-cholesterol. In a minority of cases, FHBL is due to truncation-producing mutations in the apobeta gene on chromosome 2p23-24. Previously, we reported on a four-generation FHBL kindred in which we had ruled out linkage of the trait to the apobeta gene. To locate other loci containing genes for low apobeta levels in the kindred, a genomewide search was conducted. Regions on 3p21.1-22 with two-point LOD scores >1.5 were identified. Additional markers were typed in the region of these signals. Two-point LOD scores in the region of D3S2407 increased to 3.35 at O = 0. GENEHUNTER confirmed this finding with an nonparametric multipoint LOD score of 7.5 (P=.0004). Additional model-free analyses were conducted with the square root of the apobeta level as the phenotype. Results from the Loki and SOLAR programs further confirmed linkage of FHBL to 3p21.1-22. Weaker linkage to a region near D19S916 was also indicated by Loki and SOLAR. Thus, a heretofore unidentified genetic susceptibility locus for FHBL may reside on chromosome 3.