Characterisation of the conformational changes in platelet factor 4 induced by polyanions: towards in vitro prediction of antigenicity.

Heparin-induced thrombocytopenia (HIT) is the most frequent drug-induced immune reaction affecting blood cells. Its antigen is formed when the chemokine platelet factor 4 (PF4) complexes with polyanions. By assessing polyanions of varying length and degree of sulfation using immunoassay and circular dichroism (CD)-spectroscopy, we show that PF4 structural changes resulting in antiparallel ß-sheet content >30% make PF4/polyanion complexes antigenic. Further, we found that polyphosphates (polyP-55) induce antigenic changes on PF4, whereas fondaparinux does not. We provide a model suggesting that conformational changes exposing antigens on PF4/polyanion complexes occur in the hairpin involving AA 32-38, which form together with C-terminal AA (66-70) of the adjacent PF4 monomer a continuous patch on the PF4 tetramer surface, explaining why only tetrameric PF4 molecules express "HIT antigens". The correlation of antibody binding in immunoassays with PF4 structural changes provides the intriguing possibility that CD-spectroscopy could become the first antibody-independent, in vitro method to predict potential immunogenicity of drugs. CD-spectroscopy could identify compounds during preclinical drug development that induce PF4 structural changes correlated with antigenicity. The clinical relevance can then be specifically addressed during clinical trials. Whether these findings can be transferred to other endogenous proteins requires further studies.

The adaptor protein FADD and the initiator caspase-8 mediate activation of NF-κB by TRAIL.

Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor-proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD's ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.

[Peripheral suprathreshold stimulation in preperimetric glaucoma]. / Uberschwellige periphere Stimulation bei präperimetrischen Glaukomen.

BACKGROUND AND PURPOSE: Early glaucomatous visual field defects can occur outside the central 30 degrees , which is usually examined in perimetric tests used for glaucoma diagnosis and screening. This study aimed to evaluate the diagnostic value of peripheral suprathreshold stimulation in open angle glaucoma before the development of reproducible visual field damage in standard 30 degrees automatic white-on-white perimetry. METHODS: A total of 352 eyes of 352 patients (ages 35-69 years; visual acuity 0.8 or better) from the Erlanger Glaucoma Registry were included in this study. They were divided into two groups: normal eyes and preperimetric glaucoma. All patients underwent a standardized glaucoma examination including Octopus 500EZ static perimetry (G1 program, all three phases); 95 eyes of 95 patients also received a 135-point suprathreshold test pattern of the Humphrey Field Analyzer (model 750i) for detecting peripheral visual field defects. Sensitivity and specificity were calculated for any single test point in phase 3 of the G1 test pattern and the Humphrey 135-point pattern. A score was calculated, and cluster analysis was performed. RESULTS: In 33 of 176 (18.8%) eyes with preperimetric glaucoma, the score was 3 or higher in phase 3 of the G1 program (normal eyes: 19 of 196; 9.7%). For both examination modalities, the highest sensitivity was found in test locations in the superior nasal midperiphery, corresponding to neuroretinal rim loss predominantly in the inferotemporal sector in early glaucomatous optic disc atrophy. CONCLUSION: Positive test results using suprathreshold stimulation in the midperiphery can be found in patients with preperimetric glaucoma at a significantly higher frequency than in normal subjects. Longitudinal studies will show whether such tests can be useful for predicting perimetric manifestation of the disease.

Phytophthora spp. Associated with Forest Soils in Eastern and North-Central U.S. Oak Ecosystems.

A survey of soils associated with oak species was conducted in 2003 and 2004 in Indiana, Illinois, Maryland, Michigan, Minnesota, Pennsylvania, Ohio, West Virginia, and Wisconsin to investigate the occurrence of Phytophthora spp. Soils taken from around the base of healthy and declining oak trees were flooded with H2O and Quercus robur leaflets were used as bait for Phytophthora spp. From 829 soil samples collected near trees, 21% were positive for Phytophthora spp., with 55% of the 125 sites surveyed yielding a Phytophthora sp. Phytophthora cinnamomi was the most frequently isolated species, representing 69.4% of the Phytophthora-infested sites surveyed. Other species, in decreasing order of isolation frequency were Phytophthora sp. 2, P. citricola, P. europaea, P. cambivora, P. quercina-like isolates, and Phytophthora sp. 1. No significant association was found between the presence of Phytophthora organisms and site characteristics such as latitude, elevation, soil pH, or the crown condition of the trees. However, in P. cinnamomi-infested sites, a significant association was found with the deteriorating crown status of Q. alba and the presence of P. cinnamomi. The absence of P. cinnamomi above the 40°N latitude range also was noteworthy.

[Conjunctival chemosis resistant to therapy]. / Therapierefraktäre Bindehautchemosis.

We saw a 62-year-old male with a conjunctival chemosis, which was resistant to local therapy. Excisional biopsy and histological work-up of the tissue were performed. Additional immunohistochemical staining using the Lyve-1 antibody (specific for lymphatic vascular endothelium) allowed the diagnosis of a lymphangioma of the conjunctiva to be made.

First Report of Phytophthora europaea in Oak Forests in the Eastern and North-Central United States.

In 2003 and 2004, soils in oak forest ecosystems in nine central and eastern states of the United States were surveyed for Phytophthora spp. Soil samples were collected around healthy and symptomatic trees. Symptoms included dieback of branches, gaps in lateral branch systems, yellowing of foliage, wilting and clustering of leaves, and the presence of epicormic shoots. Soil subsamples were collected in each of the four cardinal directions and at a distance of 1 to 2 m from the base of a tree. The four subsamples were bulked to produce a sample of approximately 2,000 ml. In the laboratory, each sample was mixed thoroughly and a single 250-g subsample was flooded with 500 ml of distilled water and baited with Quercus robur leaflets for 3 to 5 days at 17 to 20°C. Discolored leaflets were examined microscopically (×200) and those with sporangia typical of Phytophthora spp. were plated on PARPNH selective medium (1). Phytophthora europaea was recovered from soil samples collected from Q. alba in West Virginia, Q. rubra in Minnesota, West Virginia, and Wisconsin, Q. phellos in Ohio, and Q. velutina in Pennsylvania. Cultures were identified as P. europaea by their morphological, physiological, and molecular characteristics (4). Average dimensions of nine isolates were determined. Oogonia were 40 ± 3.9 µm in diameter and often had few bullet protuberances and tapered bases; oospores mostly filled the oogonia and averaged 36 ± 3.7 µm; sporangia dimensions averaged 42 ± 6.1 × 30 ± 4.1 µm with a length/width ratio of 1:4. Isolates produced larger oogonia and oospores but had similar sporangia length/width ratios comparable to the species description (4). Growth optimum (5.8 to 6.9 mm day-1) on V8 juice agar (V8A) occurred at 25°C. On potato dextrose agar, colonies produced dense, felt-like mycelia, often with a central mound of aerial hyphae. DNA also was extracted from eight representative isolates and the internal transcribed spacer (ITS) region of rDNA from each isolate was amplified and sequenced. ITS sequences were identical to those of P. europaea in the NCBI GenBank database (Accession No. DQ313222). Pathogenicity of six isolates (one from each site) was confirmed by wounding stems of 2-year-old Q. alba, Q. rubra, and Q. velutina seedlings and inoculating wounds with V8A plugs (6 mm) containing mycelia; V8A plugs without mycelia were used for controls. Two months after inoculation, P. europaea was reisolated on PARPNH medium from advancing lesions on all inoculated seedlings but was not isolated from control plants. Mean lesion lengths on seedlings inoculated with P. europaea were significantly greater (P < 0.05) than those on control plants; lesions averaged 0.46 cm on Q. alba, 1.38 cm on Q. rubra, and 1.01 cm on Q. velutina. Previously, P. europaea only was reported from oak trees and soil in forests of Austria, France, and Germany (1-4). These findings extend the current distribution of P. europaea and raise questions about its origin and role in the health of oak forests in eastern and north-central United States. Q. alba, Q. phellos, Q. rubra, and Q. velutina are new host associations for P. europaea. References: (1) Y. Balci and E. Halmschlager. For. Pathol. 33:157, 2003. (2) E. Hansen and C. Delatour. Ann. Sci. For. 56:539, 1999. (3) G. Hartmann and R. Blank. Forst Holz. 57:539, 2002. (4) T. Jung et al. Mycol. Res. 106:397, 2002.

Toxicogenomics, drug discovery, and the pathologist.

The field of toxicogenomics, which currently focuses on the application of large-scale differential gene expression (DGE) data to toxicology, is starting to influence drug discovery and development in the pharmaceutical industry. Toxicological pathologists, who play key roles in the development of therapeutic agents, have much to contribute to DGE studies, especially in the experimental design and interpretation phases. The intelligent application of DGE to drug discovery can reveal the potential for both desired (therapeutic) and undesired (toxic) responses. The pathologist's understanding of anatomic, physiologic, biochemical, immune, and other underlying factors that drive mechanisms of tissue responses to noxious agents turns a bewildering array of gene expression data into focused research programs. The latter process is critical for the successful application of DGE to toxicology. Pattern recognition is a useful first step, but mechanistically based DGE interpretation is where the long-term future of these new technologies lies. Pathologists trained to carry out such interpretations will become important members of the research teams needed to successfully apply these technologies to drug discovery and safety assessment. As a pathologist using DGE, you will need to learn to read DGE data in the same way you learned to read glass slides, patiently and with a desire to learn and, later, to teach. In return, you will gain a greater depth of understanding of cell and tissue function, both in health and disease.

31P-CP-MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE.

The binding of tetraphenylphosphonium (TPP+) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by 31P cross-polarisation-magic-angle spinning nuclear magnetic resonance spectroscopy (CP-MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP-MAS could selectively distinguish binding of TPP+ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.

Genomewide search for type 2 diabetes susceptibility genes in four American populations.

Type 2 diabetes is a serious, genetically influenced disease for which no fully effective treatments are available. Identification of biochemical or regulatory pathways involved in the disease syndrome could lead to innovative therapeutic interventions. One way to identify such pathways is the genetic analysis of families with multiple affected members where disease predisposing genes are likely to be segregating. We undertook a genomewide screen (389-395 microsatellite markers) in samples of 835 white, 591 Mexican American, 229 black, and 128 Japanese American individuals collected as part of the American Diabetes Association's GENNID study. Multipoint nonparametric linkage analyses were performed with diabetes, and diabetes or impaired glucose homeostasis (IH). Linkage to diabetes or IH was detected near markers D5S1404 (map position 77 cM, LOD = 2.80), D12S853 (map position 82 cM, LOD = 2.81) and GATA172D05 (X-chromosome map position 130 cM, LOD = 2.99) in whites, near marker D3S2432 (map position 51 cM, LOD = 3.91) in Mexican Americans, and near marker D10S1412 (map position 14 cM, LOD = 2.39) in African Americans mainly collected in phase 1 of the study. Further analyses showed evidence for interactions between the chromosome 5 locus and region on chromosome 12 containing the MODY 3 gene (map position 132 cM) and between the X-chromosome locus and region near D12S853 (map position 82 cM) in whites. Although these results were not replicated in samples collected in phase 2 of the GENNID study, the region on chromosome 12 was replicated in samples from whites described by Bektas et al. (1999).

Leaf area and net photosynthesis during development of Prunus serotina seedlings.

We used the plastochron index to study the relationship between plant age, leaf age and development, and net photosynthesis of black cherry (Prunus serotina Ehrh.) seedlings. Leaf area and net photosynthesis were measured on all leaves >/= 75 mm of plants ranging in age from 7 to 20 plastochrons. Effects of plant developmental stage on leaf area and net photosynthesis were evaluated for leaves of differing age (horizontal series), leaves on plants of constant age (vertical series), and leaves of constant age (oblique series). Regression techniques were used to estimate leaf area from leaf blade dimensions. The best equations for predicting leaf area had R(2) values of 0.991-0.992 and used linear or logarithmic functions of both leaf length and width. Suitable, but less precise, equations with R(2) values of 0.946-0.962 were developed from either leaf length or leaf width. Leaf area development in black cherry seedlings was similar to that in other indeterminate species. Leaves of young plants reached full expansion at a lower leaf plastochron age than leaves of older plants. Maximum net photosynthesis per unit leaf area occurred 2-3 plastochrons before full leaf expansion. There was strong ontogenetic drift in net photosynthesis with leaf age; net photosynthesis decreased as plant age increased in leaves of the same plastochron age. Plots of the oblique series were particularly useful in providing information about interaction effects.

The political economy of health care: colonial Namibia 1915-1961.

The political economy of health care services in colonial Namibia during 1915-1961 closely reflected the extreme racial and class imbalance of power in a conquest state. The colonial power allocated to the indigent nine-tenths, the poorest nine-tenths of the people, an average 43% of the health care budget between 1922 and 1954. The League of Nations mandate proved toothless in pressuring the Mandatory power to rectify this or other inequalities.

Conformation of the 18-23 loop region of bovine prothrombin in the absence and presence of a model Ca2+ ion. An energy minimization study.

The conformation of the cyclic peptide Ac-Cys-Leu-Gla-Gla-Pro-Cys-NHMe, representing the 18-23 disulfide loop of bovine prothrombin, was studied by energy minimization with the ECEPP (Empirical Conformational Energy Program for Peptides) algorithm. Parameters for charge and geometry for the gamma-carboxyglutamic acid (Gla) residue were obtained for inclusion in the ECEPP data set. Construction of the 18-23 cyclic peptide, for which no crystal structure is available, was carried out by using a scheme that took advantage of the constraints imposed by the requirement of disulfide ring closure and utilized known low-energy structures of single residues and dipeptides. Both cis and trans isomers about the Gla 21-Pro 22 peptide bond were considered. The lowest-energy conformation found for the isolated 18-23 cyclic peptide with arbitrary reduction of the charge on the Gla residues (to simulate hydration roughly) is a trans form, differing in energy by 11 kcal-mol-1 from the lowest-energy cis form. However, when the energy calculation includes one model Ca2+ ion, X2+, introduced at a fixed distance of 2.3 A from a single oxygen atom of either of the side-chain carboxyl groups of Gla with the C delta-O-X2+ bond angle fixed at one of three values, the lowest-energy cis conformation is about 1 kcal-mol-1 lower in energy than the lowest-energy trans conformation; i.e. the two structures have similar energies. In these structures, four oxygen atoms, two from each Gla side-chain, approach the model Ca2+ ion closely, in a manner similar to that seen in crystals of calcium alpha-ethylmalonate (Zell, A., Einspahr, H. & Bugg, C.E. (1985) Biochemistry 24, 533-537). It appears that the binding of Ca2+ to the 18-23 cyclic peptide may alter the equilibrium between cis and trans structures such that the fraction of cis isomers is greater in the presence of Ca2+.

A kinetic model describing the interaction of bovine prothrombin fragment 1 with calcium ions.

A kinetic model is derived for the interaction of bovine prothrombin fragment 1 with calcium ions. The model requires binding of a minimum of two calcium ions for induction of the observed biphasic fluorescence decrease as a function of time. The model is shown to be consistent with experimental kinetic and equilibrium data by fitting theoretical curves for the biphasic fluorescence change to the data through exact solution of the nonlinear differential rate equations derived from the model. The rate constants for the binding of these two required calcium ions are calculated from the solutions as best fit parameters. The thermodynamic equilibrium constants, K1 and K2, for the binding of these two calcium ions are calculated from ratios of the forward and reverse rate constants as 0.6 X 10(4) and 5.4 X 10(4), respectively. Thus, the model correctly predicts positively cooperative calcium ion binding for at least the two calcium ions required to induce fluorescence quenching.

Influence of intercellular agents on proliferation and gene activity of cultured human skin epithelium cells (NCTC 2544).

"Human skin epithelium cells" (NCTC strain 2544; HSEpicell) are established cells and grow to a monolayer the same way as epithelial cells. Addition of proliferating or antiproliferating substances results in a typical dose-dependent influence on the cell growth: steroids inhibit mitosis, polyamines stimulate proliferation, while prostaglandin E2, theophylline and papaverine reduce cell growth. Since the pattern of chromosomal nonhistone proteins indicates alterations of gene activity, DNA-binding proteins of HSE picells are analysed. Compared to native human skin fibroblasts (La Col 1115) there are only slight differences, in contrast to cancer cells. Therefore HSE picells may represent undifferentiated non-cancer cells. Hydrocortisone and theophylline inhibit cell proliferation by different mechanisms. As indicated by the pattern of DNA-binding proteins, both substances also act on HSEpicells in two different ways. As HSEpicells can be used for studying cell regulation, water-soluble extract and DNA-binding proteins of psoriatic scales as well as sera of psoriasis patients are tested in respect to any proliferating component. However, no influence on cell proliferation could be found.

Long-term tissue culture of epithelial-like cells from human skin (NCTC strain 2544). II. Viscosity changes after enzyme treatment.

Human skin epithelial-like cells (NCTC strain 2544) were grown in NCTC 135 medium. Neuraminidase and hyaluronidase were added to the growth medium. Cells were incubated 96 h at 36 degrees C. Growth rate and viscosity of cell suspensions were measured after forming single cells mechanically (mopping). With addition of neuraminidase and hyaluronidase, respectively, the growth rate remains unchanged. With neuraminidase a distinct raise in viscosity was achieved, whereas with hyaluronidase only a small effect was seen. The characteristic structure viscosity is maintained in all forms of the viscosity curves at different shear-rates.

Separation, isolation, and amino acid composition of the albumin-like fraction from psoriatic scales.

The albumin-like fraction of psoriatic scales was isolated either directly by slab gel electroporesis or after previous treatment with polyethylene glycol6000. After both isolation procedures the amino acid composition was determined. Both proteins were rich of glutamic acid, glycine, and alanine. On the other hand both contained very small amounts of cysteine and tyrosine. Only after the direct isolation procedure the amino-sugar galactosamine could be detected.