Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers

Fibril formation of islet amyloid polypeptide (IAPP) is associated with cell death of the insulin-producing pancreatic beta-cells in patients with Type 2 Diabetes Mellitus. A likely cause for the cytotoxicity of human IAPP is that it destroys the barrier properties of the cell membrane. Here, we show by fluorescence confocal microscopy on lipid vesicles that the process of hIAPP amyloid formation is accompanied by a loss of barrier function, whereby lipids are extracted from the membrane and taken up in the forming amyloid deposits. No membrane interaction was observed when preformed fibrils were used. It is proposed that lipid uptake from the cell membrane is responsible for amyloid-induced membrane damage and that this represents a general mechanism underlying the cytotoxicity of amyloid forming proteins.     

Bacterial diversity and ecosystem function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by chemolithoautotrophic "Epsilonproteobacteria"

Filamentous microbial mats from three aphotic sulfidic springs in Lower Kane Cave, Wyoming, were assessed with regard to bacterial diversity, community structure, and ecosystem function using a 16S rDNA-based phylogenetic approach combined with elemental content and stable carbon isotope ratio analyses. The most prevalent mat morphotype consisted of white filament bundles, with low C:N ratios (3.5-5.4) and high sulfur content (16.1-51.2%). White filament bundles and two other mat morphotypes had organic carbon isotope values (mean delta13C=-34.7 per thousand, 1sigma=3.6) consistent with chemolithoautotrophic carbon fixation from a dissolved inorganic carbon reservoir (cave water, mean delta13C=-7.4 per thousand for two springs, n=8). Bacterial diversity was low overall in the clone libraries, and the most abundant taxonomic group was affiliated with the "Epsilonproteobacteria" (68%), with other bacterial sequences affiliated with Gammaproteobacteria (12.2%), Betaproteobacteria (11.7%), Deltaproteobacteria (0.8%), and the Acidobacterium (5.6%) and Bacteriodetes/Chlorobi (1.7%) divisions. Six distinct epsilonproteobacterial taxonomic groups were identified from the microbial mats. Epsilonproteobacterial and bacterial group abundances and community structure shifted from the spring orifices downstream, corresponding to changes in dissolved sulfide and oxygen concentrations and metabolic requirements of certain bacterial groups. Most of the clone sequences for epsilonproteobacterial groups were retrieved from areas with high sulfide and low oxygen concentrations, whereas Thiothrix spp. and Thiobacillus spp. had higher retrieved clone abundances where conditions of low sulfide and high oxygen concentrations were measured. Genetic and metabolic diversity among the "Epsilonproteobacteria" maximizes overall cave ecosystem function, and these organisms play a significant role in providing chemolithoautotrophic energy to the otherwise nutrient-poor cave habitat. Our results demonstrate that sulfur cycling supports subsurface ecosystems through chemolithoautotrophy and expand the evolutionary and ecological views of "Epsilonproteobacteria" in terrestrial habitats.     

The beta-adrenergic receptor in human lymphocytes: subclassification by the use of a new radio-ligand, (+/-)-125 Iodocyanopindolol

(±)?¹²⁵Iodocyanopindolol (ICYP), a new radio-ligand with high affinity and specificity to β-adrenoceptors was used to identify and characterized β-adrenergic receptors in human lymphocytes. Binding of ICYP was saturable with 1.56 ± 0.2 fmol ICYP specifically bound/10⁶ cells at maximal occupancy of the sites and of high affinity (K_D=57 ± 7.1pM, N=4. In contrast to ¹²⁵Iodohydroxybenzylpindolol ICYP-binding was not affected by phentolamine (up to 10^?4M) or serotin (up to 10^?5M). Analysis of inhibition of ICYP-binding via a pseudo-Scatchard-plot (“Hofstee-plot”) by β_1-selective (practocol, metaprolol) and β_2-selective (IPS 339, zinterol) adrenergic drugs resulted in linear plots suggesting the existence of a homogeneous population of β-adrenergic receptorsin human lymphocytes. From the resulting K_D-values for practolol (16.8 μM), metoprolol (4.11 μM), zinterol (0.08 μM) and IPS 339 (0.002 μM) is concluded that the β-adrenergic receptor present in human lymphocytes is of the β₂-subtype. According to its low non-specific binding and its high specificity to β-adrenergic receptors ICYP appears to be an ideal ligand for long-term studies on the regulation of β-adrenergic receptors of human lymphocytes.     

A pharmacokinetic model of filgrastim and pegfilgrastim application in normal mice and those with cyclophosphamide-induced granulocytopaenia

Objectives:  Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is widely used as treatment for granulocytopaenia during cytotoxic chemotherapy; however, optimal scheduling of this pharmaceutical is unknown. Biomathematical models can help to pre-select optimal application schedules but precise pharmacokinetic properties of the pharmaceuticals are required at first. In this study, we have aimed to construct a pharmacokinetic model of G-CSF derivatives filgrastim and pegfilgrastim in mice.
Methods:  Healthy CD-1 mice and those with cyclophosphamide-induced granulocytopaenia were studied after administration of filgrastim and pegfilgrastim in different dosing and timing schedules. Close meshed time series of granulocytes and G-CSF plasma concentrations were determined. An ordinary differential equations model of pharmacokinetics was constructed on the basis of known mechanisms of drug distribution and degradation.
Results:  Predictions of the model fit well with all experimental data for both filgrastim and pegfilgrastim. We obtained a unique parameter setting for all experimental scenarios. Differences in pharmacokinetics between filgrastim and pegfilgrastim can be explained by different estimates of model parameters rather than by different model mechanisms. Parameter estimates with respect to distribution and clearance of the drug derivatives are in agreement with qualitative experimental results.
Conclusion:  Dynamics of filgrastim and pegfilgrastim plasma levels can be explained by the same pharmacokinetic model but different model parameters. Beause of a strong clearance mechanism mediated by granulocytes, granulocytotic and granulocytopaenic conditions must be studied simultaneously to construct a reliable model. The pharmacokinetic model will be extended to a murine model of granulopoiesis under chemotherapy and G-CSF application.     

Managing future Gulf War Syndromes: international lessons and new models of care

After the 1991 Gulf War, veterans of the conflict from the United States, United Kingdom, Canada, Australia and other nations described chronic idiopathic symptoms that became popularly known as 'Gulf War Syndrome'. Nearly 15 years later, some 250 million dollars in United States medical research has failed to confirm a novel war-related syndrome and controversy over the existence and causes of idiopathic physical symptoms has persisted. Wartime exposures implicated as possible causes of subsequent symptoms include oil well fire smoke, infectious diseases, vaccines, chemical and biological warfare agents, depleted uranium munitions and post-traumatic stress disorder. Recent historical analyses have identified controversial idiopathic symptom syndromes associated with nearly every modern war, suggesting that war typically sets into motion interrelated physical, emotional and fiscal consequences for veterans and for society. We anticipate future controversial war syndromes and maintain that a population-based approach to care can mitigate their impact. This paper delineates essential features of the model, describes its public health and scientific underpinnings and details how several countries are trying to implement it. With troops returning from combat in Afghanistan, Iraq and elsewhere, the model is already getting put to the test.     

Terrorism, trauma, and mass casualty triage: how might we solve the latest mind-body problem?

The global war on terrorism has led to increased concern about the ability of the U.S. healthcare system to respond to casualties from a chemical, biological, or radiological agent attack. Relatively little attention, however, has focused on the potential, in the immediate aftermath of such an attack, for large numbers of casualties presenting to triage points with acute health anxiety and idiopathic physical symptoms. This sort of "mass idiopathic illness" is not a certain outcome of chemical, biological, or radiological attack. However, in the event that this phenomenon occurs, it could result in surges in demand for medical evaluations that may disrupt triage systems and endanger lives. Conversely, if continuous primary care is not available for such patients after initial triage, many may suffer with unrecognized physical and emotional injuries and illness. This report is the result of an expert planning initiative seeking to facilitate triage protocols that will address the possibility of mass idiopathic illness and bolster healthcare system surge capacity. The report reviews key triage assumptions and gaps in knowledge and offers a four-stage triage model for further discussion and research. Optimal triage approaches offer flexibility and should be based on empirical studies, critical incident modeling, lessons from simulation exercises, and case studies. In addition to staging, the proposed triage and longitudinal care model relies on early recognition of symptoms, development of a registry, and use of non-physician care management to facilitate later longitudinal followup and collaboration between primary care and psychiatry for the significant minority of patients who develop persistent idiopathic symptoms associated with reduced functional status.     

A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells.

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.     

LOCATION AND EXPRESSION OF FRUSTULINS IN THE PENNATE DIATOMS CYLINDROTHECA FUSIFORMIS, NAVICULA PELLICULOSA, AND NAVICULA SALINARUM (BACILLARIOPHYCEAE)

A detailed immunocytochemical and biochemical study of the location and expression of frustulins, a family of proteins associated with the frustules of diatoms, has been performed for Cylindrotheca fusiformis Reimann et Lewin, Navicula pelliculosa (Brébisson et Kützing) Hilse, and Navicula salinarum (Grunow) Husted. Immunocytochemistry revealed that frustulins, which share homologous epitopes but are different in size, were predominantly located in the organic casing. Based on timed immunolocalization experiments and Western blotting analysis of cell extracts obtained sequentially after repleting silicate to Si-synchronized cells, the continuous presence of the frustulins in the mature and parental organic casing of the examined species was observed. The frustulins of N. pelliculosa appeared as proteins similar to those of C. fusiformis, sharing identical epitopes. The extractions, however, yielded a markedly lower abundance of frustulins in N. pelliculosa. Peak concentrations of extracted frustulins appeared to be expressed just ahead of the silicification process in C. fusiformis, whereas the level of expression in N. pelliculosa increased along with maturation of the new valves. For N. salinarum, the presence of the frustulins could not be confirmed properly by Western blotting, most probably because of the small sample volumes, inefficient extraction, and a lower amount of homologous frustulins in the casing of this species. It is concluded that the frustulins of these species are not associated with the silicalemma of the newly formed silica deposition vesicles and therefore do not seem to be involved in the silicification process itself. Overall, the results imply a structural role of the frustulins in the casing of diatoms rather than a regulation of the silicification process.     

Assembly and disulfide rearrangement of recombinant surfactant protein A in vitro

The surfactant-associated protein, protein A, produced by transgenic Chinese hamster ovary cells exhibits a heterogeneous population of structures. Electron microscopy reveals lollipop-shaped monomers consisting of a collagenous triple helix and a globular domain as well as oligomers in which two, three or more protomers are connected by their collagenous stalks. Each protomer consists of three alpha-chains (36 kDa) but under non-reducing conditions few free alpha-chains are observed by SDS/PAGE. Instead gamma-components (three chains), gamma 2 (six chains) and higher components are observed which are derived from intra- and inter-protomer disulfide cross-linking. Complete reduction at low temperature dissociates the oligomers, but preserves the intact structure of monomers as demonstrated by electron microscopy and trypsin digestion. Circular dichroism revealed an unfolding of the collagen triple helices of fully reduced protein A at 26 degrees C and of the unreduced protein A around 41.5 degrees C. Reoxidation of the fully reduced protein A re-established mainly the disulfide bonds within the triple helix but not between monomers. Very few higher assembly forms were reformed even at high protein A concentrations. Cellular in vivo systems must possess an efficient assembly mechanism which cannot be simulated by an in vitro system.