Procoagulant activity, but not number, of microparticles increases with age and in individuals after a single venous thromboembolism.

The Calibrated Automated Thrombogram (CAT), a plate-based assay that measures thrombin generation and inhibition in plasma samples, is modified to measure the procoagulant activity of phospholipid associated with plasma microparticles (MP). The assay uses a tissue factor trigger without addition of 4 µM exogenous phospholipid (PL) used in the standard CAT. Calibrated with 4:1 posphatidylcholine- phosphatidylserine (PCPS) liposomes, the assay defines a median of 40 nM procoagulant phospholipid (PPL) equivalents in plasma containing MPs from 50 normal donors, with a range from 20 - 200 nM. Like the standard CAT, the modified assay detected no difference in plasma from 36 individuals with a history of a single episode of venous thromboembolism. However the male cases had double the PPL activity, as measured by rate of thrombin generation, of females; and there was a significant correlation among cases of increased thrombin generation with age. In contrast, there were no gender disparities or age correlations among control plasmas. The findings suggest that procoagulant activity of plasma microparticles, facilitated by a simplified, one-stage plate-based assay, offer a promising avenue of investigation of mechanisms and management of venous thromboembolic disorders.

Thermal stability of MHC class I-beta 2-microglobulin peptide complexes in the endoplasmic reticulum is determined by the peptide occupancy of the transporter associated with antigen processing complex.

Once MHC class I heavy chain binds beta(2)-microglobulin (beta(2)m) within the endoplasmic reticulum, an assembly complex comprising the class I heterodimer, TAP, TAPasin, calreticulin, and possibly Erp57 is formed before the binding of high affinity peptide. TAP-dependent delivery of high affinity peptide to in vitro translated K(b)beta(2)m complexes within microsomes (TAP(+)/TAPasin(+)) was studied to determine at which point peptide binding becomes resistant to thermal denaturation. It was determined that the thermal stability of K(b)-beta(2)m-peptide complexes depends on the timing of peptide binding to K(b)beta(2)m relative to TAP binding high affinity peptide. Premature exposure of the TAP complex to high affinity peptide before its association with class I heavy chain results in K(b)beta(2)m-peptide-TAP complexes that lose peptide upon exposure to elevated temperature after solubilization away from microsome-associated proteins. These findings suggest that the order in which class I heavy chain associates with endoplasmic reticulum-resident chaperones and peptide determines the stability of K(b)beta(2)m-peptide complexes.

Crystal structure and activity of human p23, a heat shock protein 90 co-chaperone.

p23 is a co-chaperone for the heat shock protein, hsp90. This protein binds hsp90 and participates in the folding of a number of cell regulatory proteins, but its activities are still unclear. We have solved a crystal structure of human p23 lacking 35 residues at the COOH terminus. The structure reveals a disulfide-linked dimer with each subunit containing eight beta-strands in a compact antiparallel beta-sandwich fold. In solution, however, p23 is primarily monomeric and the dimer appears to be a minor component. Conserved residues are clustered on one face of the monomer and define a putative surface region and binding pocket for interaction(s) with hsp90 or protein substrates. p23 contains a COOH-terminal tail that is apparently less structured and is unresolved in the crystal structure. This tail is not needed for the binding of p23 to hsp90 or to complexes with the progesterone receptor. However, the tail is necessary for optimum active chaperoning of the progesterone receptor, as well as the passive chaperoning activity of p23 in assays measuring inhibition of heat-induced protein aggregation.

The hsp90-related protein TRAP1 is a mitochondrial protein with distinct functional properties.

The hsp90 family of molecular chaperones was expanded recently due to the cloning of TRAP1 and hsp75 by yeast two-hybrid screens. Careful analysis of the human TRAP1 and hsp75 sequences revealed that they are identical, and we have cloned a similar protein from Drosophila. Immunofluorescence data show that human TRAP1 is localized to mitochondria. This mitochondrial localization is supported by the existence of mitochondrial localization sequences in the amino termini of both the human and Drosophila proteins. Due to the striking homology of TRAP1 to hsp90, we tested the ability of TRAP1 to function as an hsp90-like chaperone. TRAP1 did not form stable complexes with the classic hsp90 co-chaperones p23 and Hop (p60). Consistent with these observations, TRAP1 had no effect on the hsp90-dependent reconstitution of hormone binding to the progesterone receptor in vitro, nor could it substitute for hsp90 to promote maturation of the receptor to its hormone-binding state. However, TRAP1 is sufficiently conserved with hsp90 such that it bound ATP, and this binding was sensitive to the hsp90 inhibitor geldanamycin. In addition, TRAP1 exhibited ATPase activity that was inhibited by both geldanamycin and radicicol. Thus, TRAP1 has functions that are distinct from those of hsp90.

TAP association influences the conformation of nascent MHC class I molecules.

The influence of TAP-MHC class I interactions on peptide binding to the class I heavy chain is assessed during TAP-dependent assembly using Kb-specific Abs that recognize conformational changes induced by assembly with beta2-microglobulin (beta2m) and by peptide binding. A significant portion (45%) of Kb molecules in TAP+, RMA-derived microsomes are associated with the TAP complex as measured by coimmunoisolation of Kb using anti-TAP1 Abs, while only 20% of the Kb heavy chain molecules are isolated as Kbbeta2m complexes with the alpha-Kb-specific Abs, Y-3 or K-10-56. The amount of Kb isolated with Y-3 and K-10-56 increases in proportion to transport and binding of peptide to the Kb molecules within the RMA microsomes. In contrast, less than 5% of the Kb within TAP2-RMA-S microsomes associated with the remaining TAP1 subunit. However, greater than 60% of Kb heavy chain is isolated as K-10-56- and Y-3-reactive Kbbeta2m complexes. We propose that a TAP-MHC class I interaction serves to stabilize the MHC class I:beta2m complex in an immature conformation (Y-3 and K-10-56 nonreactive) prior to high affinity peptide binding, preventing the export of class I molecules complexed with low affinity peptide ligands from the ER.

Nursing law violations. A threat to competent and safe nursing practice.

Nurse Practice Act violations pose threats to consumers of nursing services and lead to disciplinary actions against nurses by boards of nursing. To analyze nursing law violations, the actions and decisions of boards of nursing, and evaluate trends in negligent and unsafe nursing practice, the authors reviewed nursing law violations as well as rates of recidivism among nurses who received actions against their nursing licenses in Kentucky. The authors discuss how their findings can assist nurse administrators in investigating nurse care givers before employment and in initiating safeguards against nurse violations that affect client safety.

Quantitation of free-living amoebae and bacterial populations in eyewash stations relative to flushing frequency.

This study investigated the concentration of amoebic and bacterial populations in eyewash station water relative to various flushing regimens. Amoebae concentrations averaged approximately 200 amoebae/100 mL in 13 of 15 stations positive for amoebae and consisted of Hartmannella and Acanthamoeba. Bacterial concentrations ranged from 10(0) to more than 10(5) colony forming units per mL. Amoebic concentrations differed notably between stations located in Buildings X and Y (p < 0.0001). Further study indicated that removal of diffusing screens did not substantially change (p > 0.05) the concentration of amoeba. Amoebic and bacterial concentrations temporarily decreased with the various flushing regimens tested. Lower amoebic concentrations were not sustained by a weekly 3-minute or a monthly 1-minute flushing regimen. However, weekly 3-minute flushes appeared to be more effective in maintaining lowered bacterial concentrations (p < 0.0001).

Protection of human health from mixtures of radionuclides and chemical in drinking water.

This study was undertaken to develop a common scale for evaluating health risks from contaminated drinking water. For different agents, many unrealistic models of risk have been used. By intent, regulatory toxicology depends on "data-sparse, model-intensive" analogies from exotic animal genetics and novel exposures (NCRP 1989). The question is, does a risk evaluation so derived have any predictive validity? Absence of data prevents answer because regulatory toxicology rationalizes in step-by-step logic, which we call absolute (i.e., predicts cases of disease in a population). Absolute models ensure safety, but do so at the cost of realism. In contrast, we make relative comparisons in the manner of horsepower or RBE from radiation biology. All pollutants are assumed to contribute to toxic injury. Next, relative potencies are linked to the most credible standards. Thus, experience is transferred from well-studied chemicals to the new chemical by "data-intensive, model-sparse" methods. This logos provides much relative precision. Then, pollutants are compared with: (1) common foodstuffs, (2) ambient radiation background, or (3) utility-pure drinking water. Finally, an assessment is made for a waste disposal area.

Interaction of factor Xa with heparin does not contribute to the inhibition of factor Xa by antithrombin III-heparin.

Factor Xa modified by reductive methylation (greater than 92%) loses the capacity to bind heparin as determined both by gel chromatography and by sedimentation equilibrium ultracentrifugation. The kinetic properties of methylated factor Xa differ, with respect to KM and Vmax for a synthetic tripeptide substrate and for antithrombin III inhibition rate constants, from those of the unmodified enzyme. The 10,000-fold rate enhancement elicited by the addition of heparin to the antithrombin III inhibition reaction, however, is the same. The observed second-order rate constants (k"obs) for antithrombin III inhibition of factor Xa and methylated factor Xa are 3000 and 340 M-1 s-1, respectively, whereas k"obs values for the inhibition of factor Xa or methylated factor Xa with antithrombin III-heparin are 4 X 10(7) and 3 X 10(6) M-1 s-1, respectively. These findings provide direct evidence that the interaction of factor Xa with heparin is not involved in the heparin-enhanced inhibition of this enzyme.

Literature-derived absorption coefficients for 39 chemicals via oral and inhalation routes of exposure.

Absorption refers to the amount of a chemical or substance that is able to cross biological membranes and be taken up by the blood for subsequent distribution to target tissues. The term absorption coefficient, as used here, is a numerical descriptor characterizing that fractional uptake by the blood and represents an approximation of the biological "dose" ultimately responsible for toxicity or other effects following exposure or chemical administration. Regulatory agencies utilize absorption coefficients in deriving acceptable daily intake values and health advisory indices, as well as in quantifying radiological risk. However, absorption coefficients do not exist for many chemicals due to a paucity of appropriate toxicological data. As a result, regulatory policy must often provide default options that assume, for example, 100% absorption by all routes to permit evaluation of "data-gap" chemicals. This paper attempts to improve the situation by providing a discrete source of route-specific absorption coefficients that are based on experimental data reported in the open literature. The estimates presented here are the result of an extensive investigation of three data bases (TOXLINE, HSDB, and CIS), many agency documents, and nearly 200 articles from 30 scientific journals. Acknowledging that absorption efficiency varies with dietary status, age, and several other situation-specific factors, the estimates presented here are intended to reflect absorption by the average adult human.

Formaldehyde in drinking water: comparative hazard evaluation and an approach to regulation.

Formaldehyde, a widely used industrial chemical to which humans are ubiquitously exposed, presented cause for concern when it was demonstrated to be carcinogenic in laboratory animals. Risk assessment protocols subsequently applied to formaldehyde are of questionable validity in light of the results of recent mechanistic investigations of biological responses to formaldehyde. Further, the hazard of ingested formaldehyde is not addressed in current assessment protocols. This paper addresses the potential human health risks accompanying low-level exposure to formaldehyde as a contaminant in drinking water. In this exposure scenario, noncarcinogenic risk from inhalation of formaldehyde from drinking water is evaluated through knowledge of the metabolism and biological effects of formaldehyde exposure. Noncarcinogenic risk from ingestion of formaldehyde in drinking water is evaluated from the perspective gained by comparison with dietary sources of formaldehyde. Carcinogenic risk to humans is evaluated in light of recent investigations into the mechanisms underlying biological responses to formaldehyde exposure. Finally, based on a comparison of ingestion of formaldehyde in drinking water with ingestion of naturally occurring formaldehyde in foods, a comparative hazard approach to formaldehyde regulation is offered as a supplement to the rigid evaluation protocols currently used.

Hazard evaluation for complex mixtures: relative comparisons to improve regulatory consistency.

The traditional "absolute decision-making" process used by federal regulatory agencies to derive permissible exposure concentrations for hazardous substances is initiated by an evaluation of the "weight-of-evidence" that a substance is a potential human carcinogen. Subsequent conservative procedures applied variably to noncarcinogens and carcinogens yield exposure limits for individual substances based on "data-sparse, model-intensive" techniques which may lack consistency and have difficulty directly addressing the hazards from complex mixtures. This paper describes how a "relative decision-making" technique applicable to complex mixtures can supplement the "absolute" approach currently used. Estimates obtained through this "data-intensive, model-sparse" technique may be evaluated by comparisons to estimates representing a range of hazards "generally regarded as safe" derived through analyses of chlorinated drinking water, cigarette smoke condensate, and other common human exposures. Comparisons are also used to evaluate the relative degree of consistency in risk estimates between 58 suspect human carcinogens analyzed by the U.S. Environmental Protection Agency Carcinogen Assessment Group and by the authors.

Isolation of a heparin-like anticoagulant from the plasma of a patient with metastatic bladder carcinoma.

A 73-year-old woman with metastatic transitional cell carcinoma of the bladder developed vaginal bleeding a few days after undergoing radical cystectomy. She had no other signs of mucocutaneous bleeding. Coagulation studies revealed a markedly prolonged thrombin time (greater than 600 seconds), a slightly prolonged reptilase time (20 seconds), and mildly elevated fibrinogen (4.39 g/L), and fibrin D-dimer (200 to 500 ng/mL) levels. Treatment of the patient's plasma in vitro with protamine or barium sulfate normalized the thrombin time. The anticoagulant activity corresponded to 0.15 heparin U/mL when measured by a thrombin time assay using normal plasma as substrate and standardized with porcine heparin. The anticoagulant was quantitatively bound to and subsequently eluted with 1 mol/L NaCl from quaternary aminoethyl (QAE) Sephadex, and then isolated by affinity chromatography on immobilized antithrombin III. The isolated anticoagulant was shown to be sensitive to heparinase digestion. Therefore, the inhibitor has functional and chemical properties similar to those of high-affinity heparin. Thus far, this is the only anticoagulant of this type isolated from the plasma of a patient bearing a tumor other than plasma cell myeloma.

Biotesting of wastewater: a comparative study using the Salmonella and CHO assay systems.

Means to assess the toxicity of wastewaters are essential to implementing the Federal Clean Water Act. Health risk assessment based on single chemicals is limited by the number of chemicals that can be identified and to those chemicals for which toxicity data are available. Long-term whole animal tests on large numbers of wastewater samples are not practical. In this study, two short-term tests, the Salmonella mutagenicity assay and the Chinese hamster ovary (CHO) cell assay for mutagenicity and cytotoxicity, were evaluated as potentially useful biomonitors of wastewaters. Standard assay protocols were modified to allow testing of up to 2.5 and 3.4 ml of unconcentrated water in the bacterial and mammalian cell tests, respectively. Cytotoxicity and mutagenicity were detected in some unconcentrated wastewater samples using these modifications. Data on eight wastewater samples, representing five different sites, indicated that the Salmonella test is the more sensitive indicator of mutagenic activity in those samples, whereas the CHO test is a sensitive indicator of the presence of cytotoxic components. Wastewater concentrates, prepared by adsorption onto XAD-2 and "blue cotton," were compared in the two bioassays. In a single concentrate, the two short-term tests detected distinctly different mutagens. Advantages of using the CHO-AS52 cell line instead of the CHO-K1BH4 line for detecting wastewater mutagens were indicated. This study illustrates the complementary use of multiple bioassays and concentration methods to detect and characterize toxic components in wastewater.

Chemical scoring by a rapid screening of hazard (RASH) method.

A rapid screening of hazard method (RASH) is presented for deriving relative potency estimates for hazardous substances. The method utilizes data from any available toxicological database such as the Registry of Toxic Effects of Chemical Substances (RTECS) or EPA's GENE-TOX database on genetic activity profiles. The method has been applied to derive relative potency values and permissible environmental concentrations for 278 chemicals. The derived values have been compared with recommendations of expert committees where possible, and substantial agreement is found.

Effects of fasting on the myocardial subcellular distribution and lipid distribution of terminal p-iodophenyl-substituted fatty acids in rats.

The myocardial lipid pool distribution and subcellular distribution of radiolabeled methyl-branched fatty acids in rats was evaluated under conditions of fasting (24 h) and feeding. With the unbranched iodophenyl fatty acid, fasting resulted in increased myocardial extraction and clearance time with a decrease in the incorporation into triglycerides and greater radioactivity in the mitochondrial fraction. With the monomethyl-branched analogue, the effects of fasting on lipid and subcellular distribution were minor except for a decrease in triglyceride incorporation. Like the unbranched analogue, the dimethyl-branched iodophenyl fatty acid showed increased myocardial extraction with fasting, however, this structurally-modified fatty acid showed increased rather than decreased incorporation into triglycerides.

Evaluation of the metabolism in rat hearts of two new radioiodinated 3-methyl-branched fatty acid myocardial imaging agents.

The biological fate of two new radioiodinated 3-methyl-branched fatty acids has been evaluated in rat hearts following intravenous administration. Methyl-branching was introduced in [15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) and 15-(p-iodophenyl)-3,3-dimethylpentadecanoic acid (DMIPP) to inhibit beta-oxidation. The goals of these studies were to correlate the effects of methyl-branching on the incorporation of these agents into the various fatty acid pools and subcellular distribution profiles, and to relate these data to the myocardial retention properties. The properties of BMIPP and DMIPP were compared with the 15-(p-iodophenyl)pentadecanoic acid straight-chain analogue (IPP). Differences in the heart retention of the analogues after intravenous administration in rats correlated with differences observed in subcellular distribution patterns. The dimethyl DMIPP analogue showed the longest retention and the highest association with the mitochondrial and microsomal fractions (34%, 38%) 30 min after injection. These data are in contrast to the rapid clearance of the straight-chain IPP analogue which showed much lower relative association with the mitochondria and microsomes (18%, 15%). The distribution patterns of each analogue in the various lipid pools appeared consistent with the expected capacity of the analogues to be metabolized by beta-oxidation. In contrast to the rapid oxidation of the straight-chain IPP analogue, the 3-monomethyl BMIPP analogue appeared to undergo slower oxidation and clearance, whereas the dimethyl-branched DMIPP analogue was apparently not catabolized by the myocardium. All three analogues showed some incorporation into triglycerides. The metabolism patterns of the branched analogues reported here may provide useful information in the description of the mechanisms by which BMIPP and DMIPP are retained in rat myocardium.

Binding of iron by factor IX. Possible role for beta-hydroxyaspartic acid.

In concentrations exceeding 1 mg/ml, bovine factor IX exhibits a pink color that arises from a broad absorption band with a lambda max = 500 nm. Analysis by x-ray fluorescence reveals the presence of iron but no other transition metals in the factor IX preparation. Quantitative analysis by atomic absorption spectroscopy indicates that 1 g atom of iron is bound tightly to 1 mol of factor IX. The iron is removed slowly (t1/2 = 3 h) by EDTA. In contrast, prothrombin binds no detectable iron, and factor X binds less than 0.2 g atom/mol. alpha-Hydroxybutyrate chelates Fe3+ with sufficient stability to preclude formation of [Fe(OH)3]n. It is proposed that factor IX binds iron with physiologically significant affinity and that the beta-hydroxyaspartate residue in factor IX is a chelator for the bound metal.

Myocardial imaging agents: synthesis, characterization, and evaluation of (Z)- and (Z,E)-18-[82Br]bromo-5-tellura-17-octadecenoic acids.

The effects of replacement of radioiodide with radiobromide on the biological properties of a model vinyl halide substituted tellurium fatty acid have been evaluated. The use of a facile radiobromination method involving the reaction of [82Br]Br2 with vinylmercuric bromide substrates has been investigated. Unexpectedly, both the cis- and trans-vinyl bromide products are formed as confirmed by chromatographic and spectral studies. With use of this technique, (E,Z)-1-[82Br]bromo-13-iodo-1-tridecene was prepared and used as the substrate to prepare (E,Z)-18-[82Br]bromo-5-tellura-17-octadecenoic acid (13-E,Z), which was evaluated in rats. Both [82Br]-13-E,Z and [82Br]-13-Z, prepared by an established procedure using N-chorosuccinimide-Br- reaction with the trans-boronovinyl substrates, showed tissue distribution properties similar to those of (E)-18-[125I]iodo-5-tellura-17-octadecenoic acid. These results demonstrate that replacement of I with Br and also the stereochemistry about the olefinic bond do not drastically affect heart uptake and retention.

Decreased immunoglobulin production by a human lymphoid cell line following melphalan treatment.

The effect of melphalan on immunoglobulin G (IgG) production by a human lymphoblastoid cell line (BF) was studied. The amount of secreted IgG and the percentage of cells containing cytoplasmic IgG were measured by immunoassay and cytofluorometry, respectively. Dose-response studied indicated that melphalan concentrations of 2 X 10(-8) M had no effect, while concentrations of 8 X 10(-7) M were totally toxic, after 72-hr exposures to the drug. Statistically significant, persistent, alterations in both synthesis and secretion of IgG by BF cells were observed following treatment for 72 hr with 4 X 10(-7) M melphalan, and there was an increase in population-doubling time from 24 to 72 hr in these drug-treated cells. The percentage of IgG-containing cells in melphalan-treated cultures was significantly decreased as compared to control cultures. IgG secretion was also decreased in these cultures, and the variation in IgG secretion as a function of cellular growth was significantly altered following melphalan treatment. Decreased IgG production following melphalan treatment may be related to altered cell cycle kinetics. Based on immunological analysis, there was no evident alteration in the IgG secreted by melphalan-treated cells, nor did melphalan treatment produce a cellular population lacking IgG entirely.