Evaluating the effects of hypoxic storage on platelet function and health using a novel storage system.

BACKGROUND: Thousands of units of whole blood (WB) and blood components are transfused daily to treat trauma patients. Improved methods for blood storage are critical to support trauma-related care. The Hemanext ONE® system offers a unique method for hypoxic storage of WB, with successfully demonstrated storage of clinically viable RBCs. This work evaluated the system for the storage of WB, focusing on platelet health and function. STUDY DESIGN AND METHODS: WB was collected from healthy donors and processed through the Hemanext ONE® system. Hemoglobin oxygen saturation (HbSO2) levels of WB were depleted to 10%, 20%, or 30% of total HbSO2 and then stored in PVC bags sealed in oxygen-impermeable bags (except for normoxic control) with samples collected on days 1, 7, and 14 post-processing. Flow cytometry assessed the activation and apoptosis of platelets. Clot dynamics were assessed based on aggregometry and thromboelastography assays, as well as thrombin generation using a calibrated-automated thrombogram method. RESULTS: Hypoxic storage conditions were maintained throughout the storage period. Hypoxia triggered increased lactate production, but pH changes were negligible compared to normoxic control. Storage at 10% HbSO2 had a significant impact on platelet function, resulting in increased activation and reduced clot formation and aggregation. These effects were less significant at 20% and 30% HbSO2. DISCUSSION: This study indicates that platelets are sensitive to hypoxic storage and suffer significant metabolic and functional deterioration when stored at or below 10% HbSO2.

CLIMATE CHANGE: The Causes of 20th Century Warming.

Global air surface temperatures increased by about 0.6 degrees C during the 20th century, but as Zwiers and Weaver discuss in their Perspective, the warming was not continuous. Two distinct periods of warming, from 1910 to 1945 and since 1976, were separated by a period of very gradual cooling. The authors highlight the work by Stott et al., who have performed the most comprehensive simulation of 20th century climate to date. The agreement between observed and simulated temperature variations strongly suggests that forcing from anthropogenic activities, moderated by variations in solar and volcanic forcing, has been the main driver of climate change during the past century.

Peptide-based analysis of amino acid sequences important to the biological activity of eosinophil granule major basic protein.

Synthetic peptides corresponding to amino acid sequences in eosinophil granule major basic protein (MBP) were evaluated for cytotoxic activity toward K562 cells and for ability to stimulate basophil mediator release. Results obtained using 14 peptides spanning the 117-amino acid sequence of MBP in overlapping fashion indicated that the activities mapped to peptide sequences near the amino and carboxy termini of MBP. The activity of these regions was confirmed using two peptides corresponding to MBP residues 18-45 and 89-117. A 20-h incubation with 5 microM peptide 18-45 or peptide 89-117 caused approximately the same levels (>60%) of cytotoxicity in K562 cells as 5 microM MBP. Similarly, a 30-min incubation with peptides 18-44 and 89-117 stimulated basophil histamine release in a concentration-dependent manner over the range of 5-20 microM. The level of release stimulated by 20 microM peptide 89-117 approached that stimulated by 2 microM MBP. A 20 microM concentration of peptide 89-117 also stimulated leukotriene C4 (LTC4) production by the basophils. Neither peptide 18-45 nor peptide 89-117 was cytotoxic for basophils under the experimental conditions for histamine and LTC4 release, as determined by 51Cr release. These results indicate that two MBP peptide sequences, including one (89-117) that contains a unique carbohydrate-binding region, share the biologic activities of MBP.

A 10-year review of research on chaplains and community-based clergy in 3 primary oncology nursing journals: 1990-1999.

A manual examination of 3 primary oncology nursing journals was conducted to identify quantitative studies about chaplains and community-based clergy that were published between 1990 and 1999. This systematic review identified 7 studies involving chaplains and/or clergy dealing with a range of issues. Although the rate at which such studies were published in the oncology nursing literature was relatively low (1 in 123 studies), this rate far exceeds the rate found in a similar review of psychology journals (1 in 600 studies). The nature of the 7 studies and the issues they addressed are discussed and the authors make recommendations for future collaborative efforts.

Marriage and family therapists and the clergy: a need for clinical collaboration, training, and research.

This article calls for greater collaboration between clergy and marriage and family therapists. It spells out the reasons for potential collaboration and suggests some specific ways it can occur. Marriage and family therapists acknowledge the highest rates of religious involvement of any mental health profession, placing them in a unique position to be involved in the continuing education of clergy. There is a clear need for research to understand how marriage and family therapists and clergy can more effectively work together. The dearth of research, training, and collaboration between the two vocations is all the more unfortunate given the clear evidence of the importance of religion in the personal lives of the clients we serve.

A review of research on religious and spiritual variables in two primary gerontological nursing journals: 1991 to 1997.

All articles published between 1991 and 1997 in the Journal of Gerontological Nursing and Geriatric Nursing were classified as qualitative research, quantitative research, or non-research. Of the 784 articles reviewed, 5.1% mentioned religion or spirituality. Research articles (7.7%) were more likely than non-research articles (2.8%) to address religion and spirituality. No statistical difference was found between the percentage of qualitative (10.7%) and quantitative (6.8%) studies addressing religious and spiritual factors. The percentage of quantitative studies including religious and spiritual variables was found to be higher than that found by systematic reviews of the research literature in various health professions.

The distressed family and wounded children.

This article addresses the issue of the need for clergy to be better informed regarding the assessment of child abuse and neglect. It provides a quick reference guide to assist clergy in assessing the risk factors in the abusive family with guidelines for the recognition of child maltreatment. In a society that is marked by unprecedented levels of child maltreatment reporting and an ever increasing shortage of mental health services, clergy are increasingly being confronted by situations involving potential child maltreatment that require expert crisis intervention skills. This article provides specific, concrete guidelines for clergy confronted with situations that call for the recognition of child maltreatment.

Has there been a failure to prepare and support parish-based clergy in their role as frontline community mental health workers: a review.

Addresses the issue that parish-based clergy, functioning as frontline community mental health workers, often do so with inadequate training and limited support from the mental health community. Claims that although a clergyperson is as likely to have a severely mentally distressed person seek her or his assistance as is a mental health specialist, there is inadequate research on the function of clergy in the mental health network or the psychological dynamics of religion. Suggests that clergy can serve most effectively in the mental health network as skilled facilitators, identifying the needs of persons, and connecting them to a larger circle of specialized helpers. Argues that the mental health and religious communities share many common values and goals and need to work together more effectively for the best interest of those they are called to serve.

Elderly suicide, mental health professionals, and the clergy: a need for clinical collaboration, training, and research.

This article addresses the need for improved clergy-mental health professional collaboration in the assessment and treatment of elderly suicide. Millions of older adults with personal problems seek the counsel of clergy. A recent Gallup survey found that elders are more willing to turn to their clergy than their medical doctor or a mental health specialist for help when a friend is contemplating suicide (Gallup Organization, 1992). Elder suicide prevention presents the mental health and religious communities with unique opportunities to work together in the best interests of those they serve.

Tryptophan sidechain dynamics in hydrophobic oligopeptides determined by use of 13C nuclear magnetic resonance spectroscopy.

Two oligopeptides, t-boc-LAWAL-OMe and t-boc-LALALW-OMe, were synthesized for the purpose of examining the sidechain dynamics of the tryptophan residue in hydrophobic environments by 13C nuclear magnetic resonance and fluorescence spectroscopy. In both peptides, the tryptophan sidechain was greater than 95% enriched with 13C at the C delta 1 position. Spin-lattice relaxation time (T1) and steady-state nuclear Overhauser effect (NOE) data were obtained at 50.3 and 75.4 MHz for both peptides in CD3OD, and at 75.4 MHz for t-boc-LALALW-OMe in lysolecithin-D2O micelles. We have adapted the model-free approach of G. Lipari and A. Szabo (1982, J. Am. Chem. Soc. 104:4546) to interpret the 13C-NMR data. Computer-generated curves based on experimental data obtained at a single frequency demonstrate relationships between an effective correlation time for tryptophan sidechain motion (tau e), a generalized order parameter (sigma) describing the extent of motional restriction, and an overall correlation time for the peptide (tau m). Assuming predominantly dipolar relaxation, least-squares fits of the dual frequency relaxation data provide values for these parameters for both peptides. The contribution of chemical shift anisotropy (CSA), however, is also explicitly assessed in the data analysis, and is shown to perturb the predicted sigma, tau e, and tau m values and to decrease chi(2) values observed in nonlinear least-squares analysis of the data. Because of uncertainty in the contribution of CSA to the relaxation of the indole ring 13C delta 1 atom, nonlinear least-squares analysis of the relaxation data were performed with and without inclusion of a CSA term in the appropriate relaxation equations. Neglecting CSA, an overall peptide correlation time of 0.69 ns is predicted for t-boc-LAWAL-OMe in CD3OD at 20 degrees C compared with 1.28 ns for t-boc-LALALW-OMe. Given these tau m values and taking into account the effect of measurement error in the T1 and NOE data, the internal dynamics of the tryptophan residue of t-boc-LAWAL-OMe in this isotropic environment are described by a range of tau e values from 70 to 112 ps and sigma values between 0.22 and 0.36. Similarly, for t-boc-LALALW-OMe, 68 less than or equal to tau e less than or equal to 93 ps and 0.09 less than or equal to sigma less than or equal to 0.17. The Ch-terminal position of the tryptophan residue in the hexapeptide may account for its lower order parameter. In lysolecithin micelles, the model-free approach applied tot-boc-LALALW-OMe predicts a Te between 0.87 and 1.08 ns, and an order parameter range of 0.72-0.80, assuming an average Tm of 14 ns (Saunders, L. 1966. Biochim. Biophys. Acta. 125:70) for a typical peptide-micelle complex. In this case, measurement of only two 13C relaxation parameters at a single frequency yields sufficient information, plotted in the form of a composite T1-NOE solution curve, to constrain the allowed values of the model-free motional parameters within a relatively narrow range. The predicted range of eV and Te values for the peptide-micelle complex demonstrate that both the rate and spatial mobility of the indole moiety are markedly restrained in the anisotropic micelle environment relative to free methanol solution. Steady-state fluorescence anisotropy measurements made on the peptides dissolved in methanol or with synthetic lysolecithins in water were used to calculate apparent order parameters for tryptophan motion; these values agree well with order parameters calculated from 13C NMR data. The reported results are relevant to the issue of protein dynamic events occurring on the picosecond time scale predicted by molecular dynamics simulations.

Posttraumatic stress, mental health professionals, and the clergy: a need for collaboration, training, and research.

This article addresses the need for improved clergy-mental health professional collaboration in the assessment and treatment of posttraumatic stress disorder (PTSD). Tens of millions of North Americans with personal problems seek the counsel of clergy. There is an absence of research on the function of clergy as helpers with the traumatized and on the psychological dynamics of religious coping among the traumatized. Psychological trauma presents the mental health and religious communities with unique opportunities to work together in the best interest of those they serve.

Research on religion and serious mental illness.

According to this review, religion plays a largely positive role in mental health; future research on severe mental disorders should include religious factors more directly.

Characterization of selectively 13C-labeled synthetic melittin and melittin analogues in isotropic solvents by circular dichroism, fluorescence, and NMR spectroscopy.

The spectroscopic and functional characterization of 13C-labeled synthetic melittin and three analogues is described. Selectively 13C-enriched tryptophan ( [13C delta 1]-L-Trp) and glycine ( [13C alpha]Gly) were incorporated into melittin and three analogues by de novo peptide synthesis. 13C-Labeled tryptophan was incorporated into melittin at position 19 and into single-tryptophan analogues of melittin at positions 17, 11, and 9, respectively. Each of the synthetic peptides contained 13C-labeled glycine at position 12 only. The peptides were characterized functionally in a cytolytic assay, and spectroscopically by CD, fluorescence, and NMR. The behavior of 13C-labeled synthetic melittin was, in all respects, indistinguishable from that of the naturally occurring peptide. All of the analogues were found to be efficient lytic agents and thus were functionally similar to the native peptide, yet no evidence was found for formation of a melittin-like tetramer by any of the analogues in aqueous media, although there was a propensity for apparently nonspecific peptide aggregation, especially for MLT-W9. Since the analogues did exhibit fractional helicities by CD comparable to or even greater than melittin itself in the presence of methanol, we infer that tetramer assembly requires not only the ability to form alpha-helix but also a very precise packing of amino acid side chains of the constituent monomers. The 13C chemical shift of the Gly-12 C alpha was found to be a sensitive marker for helix formation in all of the peptides. For melittin itself, 13C NMR spectra revealed a downfield shift of approximately 1.8 ppm for the Gly-12 13C alpha resonance of the tetramer relative to that observed for the free monomer in D2O. In mixed samples containing melittin monomer and tetramer, two discrete Gly-12 13C alpha peaks were observed simultaneously, suggestive of slow exchange between the two species. We conclude that melittin's ability to form a soluble tetramer is not a prerequisite for cytolytic activity, nor is cytolytic potential precisely correlated with the ability to form an amphiphilic helix.

Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents.

The dynamics in isotopic solvents of selectively 13C labeled synthetic melittin and three analogues have been investigated by using NMR and fluorescence techniques both separately and in combination. In conjunction with the "model-free" approach to interpretation of NMR relaxation data [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment T1, T2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique- and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). In particular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree very well with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of +/- 71 degrees about the beta-gamma bond with a correlation time of 159 +/- 24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of +/- 37 degrees, yet demonstrates an increased rate of motion with a correlation time of 56 +/- 8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferred from molecular dynamics simulations.

Degenerate recognition of alloantigenic peptides on a positive-selecting class I molecule.

The well-defined 2C T cell was used to investigate alloreactive degeneracy. A panel of class I molecules that are known ligands for the 2C TCR were sensitized with three known peptide ligands, p2Ca (LSPFPFDL), dEV-8 (EQYKFYSV), and SIYR-8 (SIYRYYGL). The peptide p2Ca was originally identified as the allopeptide seen in the Ld class I molecule by 2C T cells, 2C recognizes the dEV-8 peptide as the ligand in the Kbm3 class I molecule, and SIYR-8 was recently identified as a peptide ligand for 2C in the context of the Kb class I molecule. Strong recognition of all three Ag-presenting molecules occurred in the context of their respective allopeptides, but 2C recognized all three peptides to a measurable extent in the context of Kb. Molecular modeling of these Kb/peptide complexes revealed a high degree of similarity between dEV-8 and SIYR-8, but very little conformational similarity of either of these peptides with p2Ca. Furthermore, the structural changes in the mutant Kbm3 binding site resulted in generalized changes in the conformation of each of five bound peptides compared with those of the same peptides bound to Kb. The finding that degenerate recognition occurs on Kb, the restriction element responsible for selecting 2C T cells, suggests a unique relationship between a TCR and the Ag-presenting molecule that mediates its positive selection.

Inactivation of Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase by an enzyme-generated ethoxyacetylenic ketone in the presence of a thiol.

Replacement of the 21-methyl group of 20 beta-hydroxypregn-4-en-3-one with an ethoxyacetylene group yields a compound that is an excellent substrate (pH 7.4, Km = 2.3 microM, Vmax = 4.6 nmol min-1 micrograms-1) for the Streptomyces hydrogenans NAD(H)-dependent 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53). The enzyme-generated ethoxyacetylenic ketone product is a potent inactivator of the enzyme. Gel filtration chromatography of enzyme inactivated with radiolabeled steroid demonstrates that covalent modification of the enzyme has occurred. Both NAD and NADH retard the rate of inactivation, suggesting that only free enzyme is susceptible to covalent modification. Consequently, enzymatically formed ethoxyacetylenic ketone does not react with the enzyme while it is part of the ternary complex. Moreover, the kinetically preferred release of this reactive ketone prior to NADH release assures that enzyme inactivation occurs only when released ketone subsequently encounters free enzyme. Kinetic analysis of inactivations carried out with chemically prepared ethoxyacetylenic ketone and enzyme at pH 7.4 and 9.2 yields bimolecular rate constants for the inactivation process of 1.15 X 10(4) L mol-1 s-1 and 6.94 X 10(4) L mol-1 s-1, respectively. This bimolecular reaction is faster than the bimolecular reaction of the ethoxyacetylenic ketone with either glutathione, mercaptoethanol, or dithiothreitol. Thus, complete inactivation by ketone generated from 5 microM alcohol and 5 microM NAD occurs in 30 min at pH 7.4 in the presence of 1 mM glutathione.

The crystal structure of the GroES co-chaperonin at 2.8 A resolution.

The GroES heptamer forms a dome, approximately 75 A in diameter and 30 A high, with an 8 A orifice in the centre of its roof. The 'mobile loop' segment, previously identified as a GroEL binding determinant, is disordered in the crystal structure in six subunits; the single well-ordered copy extends from the bottom outer rim of the GroES dome, suggesting that the cavity within the dome is continuous with the polypeptide binding chamber of GroEL in the chaperonin complex.

Electron microscopy of thin-sectioned three-dimensional crystals of SecA protein from Escherichia coli: structure in projection at 40 A resolution.

SecA is a single-chain, membrane-associated polypeptide (102 kDa) which functions as an essential component of the protein export machinery of Escherichia coli. SecA has been crystallized from ammonium sulfate as small, three-dimensional bipyramidal crystals (0.1 x 0.1 x 0.05 mm). These crystals did not demonstrate detectable diffraction of X-rays from rotating anode sources. For study by electron microscopy, individual crystals were cross-linked in glutaraldehyde and OsO4 solutions, dehydrated, embedded in epoxy resin, and sectioned normal to crystallographic axial directions inferred from the external morphology of the crystals. Fourier transformation of processed images of untilted thin sections stained with uranyl acetate and lead citrate show reflections extending to 31 A resolution. Diffraction data and reconstructed images of the projected density of the unit cell contents indicate that the bipyramidal SecA crystals belong to orthorhombic space group C222(1) with unit cell dimensions a = 414 A, b = 381 A, and c = 243 A. Filtered images and density maps of mutually orthogonal projections of the unit cell contents are consistent with a three-dimensional model in which the asymmetric unit contains eight SecA monomers. The large unit cell dimensions and packing of protein monomers suggest that SecA is crystallizing as an oligomer of either dimers or tetramers.