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PubMed1988

Structural requirements of lipid A species in activation of clotting enzymes from the horseshoe crab, and the human complement cascade.

The structure/activity relationship of lipid A, a bioactive center of endotoxic lipopolysaccharides, in the activation of the clotting enzyme cascade of a horseshoe crab amoebocyte lysate (Limulus activity) and the complement system in human serum, was examined using synthetic lipids A and related compounds. Regarding Limulus activity, a newly developed colorimetric method, which utilizes a mixture of recombined clotting factors and a chromogenic substance, was much more sensitive for detecting changes in the chemical structure of test compounds than the conventional gelation method using the amoebocyte whole lysate. (beta 1-6)-D-Glucosamine disaccharide bisphosphates, which had neither 3-hydroxyacyl nor 3-acyloxyacyl groups, and acylglucosamine phosphates, which in structure correspond or are analogous to the non-reducing or reducing moieties of lipids A and biosynthetic disaccharide lipid A precursors showed only negligible activity in the colorimetric tests, but they exhibited a distinct though much weaker gelation activity than the parent disaccharide molecules. The assay results obtained by the colorimetric Limulus test correlate better with the pyrogenicity of the test synthetic compounds than those given by the gelation method, although the dependence of pyrogenicity on chemical structure is greater. The presence of 3-hydroxyacyl groups on the bisphosphorylated (beta 1-6)-D-glucosamine disaccharide backbone is the prerequisite for effective activation of the clotting enzyme cascade of horseshoe crab amoebocyte lysate, while the presence of an adequate number (one or two) of 3-acyloxyacyl groups on the disaccharide bisphosphate backbone is needed for full pyrogenicity. Complement activation, on the other hand, showed structural requirements quite different from those for the colorimetric Limulus activity and the pyrogenicity. The disaccharide compounds that had only non-hydroxylated acyl groups, acylated glucosamine phosphates that had the structure of the non-reducing portion of lipids A and biosynthetic disaccharide precursors, which were scarcely active in the colorimetric Limulus test, caused complement activation comparable to or sometimes stronger than that of the parent disaccharide molecules. Acylglucosamine phosphates, corresponding in structure to the reducing moiety of disaccharide compounds, however, showed little activity.

Animals↗
PubMed1979

Adaptation, saturation, and physiological masking in single auditory-nerve fibers.

Results are reviewed concerning some effects, at a units's characteristic frequency, of a short-term conditioning stimulus on the responses to perstimulatory and poststimulatory test tones. A phenomenological equation is developed from the poststimulatory results and shown to be consistent with the perstimulatory results. According to the results and equation, the response to a test tone equals the unconditioned or unadapted response minus the decrement produced by adaptation to the conditioning tone. Furthermore, the decrement is proportional to the driven response to the conditioning tone and does not depend on sound intensity per se. The equation has a simple interpretation in terms of two processes in cascade--a static saturating nonlinearity followed by additive adaptation. Results are presented to show that this functional model is sufficient to account for the "physiological masking" produced by wide-band backgrounds. According to this interpretation, a sufficiently intense background produces saturation. Consequently, a superimposed test tone cause no change in response. In addition, when the onset of the background precedes the onset of the test tone, the total firing rate is reduced by adaptation. Evidence is reviewed concerning the possible correspondence between the variables in the model and intracellular events in the auditory periphery.

Acoustic Stimulation↗
PubMed1988

Extrinsic-pathway enzyme-linked coagulation assay (EP-ELCA). A clot-based alternative to prothrombin time for measurement of extrinsic pathway factors in plasma.

This solid-phase colorimetric microtiter-plate clotting assay is much more sensitive than standard clotting tests. In enzyme-linked coagulation assay (ELCA), enzyme-labeled fibrinogen and solid-phase fibrinogen are the substrate for thrombin generated in the clotting cascade. We used this assay to measure the factors of the extrinsic pathway by an extrinsic pathway-specific assay (EP-ELCA) and to determine the individual factors of the extrinsic pathway (VII, X, V, II) in plasmas of coumadin-treated and heparin-treated patients, with prothrombin time (PT) values used as a reference. In the ELCA method, samples and controls are incubated on the same plate, eliminating the requirement for pre-standardization of the substrate "plasma" before the factor assay is done. Concentrations of factors are determined by serially diluting sample and control plasmas to yield equivalent activity at given dilutions, a more direct approach for measuring specific factors than determining log concentrations vs log clotting time. Changes in the concentrations of clotting factors are seen before changes are apparent by PT. For coumadin-treated patients, all vitamin K-dependent factors were significantly (P less than or equal to 0.001) less than in normal controls, whereas factor V concentrations were normal, as expected. For patients treated with heparin, concentrations of factors X and VII were less than in normal controls (P less than 0.01) and results for EP-ELCA, II, and V assays were normal. This methodology can readily be automated.

Adult↗
PubMed1999

JSAP1, a novel jun N-terminal protein kinase (JNK)-binding protein that functions as a Scaffold factor in the JNK signaling pathway.

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.

Adaptor Proteins, Signal Transducing↗
PubMed1989

Detection of chemically induced renal injury: the cascade of degenerative morphological and functional changes that follow the primary nephrotoxic insult and evaluation of these changes by in-vitro methods.

A diversity of chemicals cause discrete lesions in the kidney by a number of different mechanisms, and similar types of chemicals may give rise to more than one target cell injury. Screening for these lesions in vivo may be unreliable if a single noninvasive or invasive criterion is used. Instead, evidence of nephrotoxicity must be based on an array of tests, applied over a period of time. These should include biochemical, pathological and histochemical tests conducted in tandem. A primary toxic injury to the kidney may give rise to recovery, to permanently altered functional reserve or to a clinically identifiable effect, such as acute or chronic renal failure or malignancy. These clinical effects occur as a result of a cascade of degenerative changes which are a consequence of a primary lesion but also affect other parts of the kidney. A number of factors can modulate the progression of the primary insult to the end-effect. In-vitro nephrotoxicity screening is also difficult, but a rational approach can be based on current understanding of how chemicals target for and damage cells in anatomically well-defined regions of the kidney. In-vitro techniques can provide answers to specific questions about the mechanisms by which chemicals damage these discrete cell types. It is essential that a number of different in-vitro systems be developed in parallel to address the mechanistic aspects and screening of nephrotoxicity properly. Data generated in vitro must be related to the situation in vivo and used to devise reliable noninvasive tests for assessing nephrotoxicity in man.

Cell-Free System↗
PubMedDate not supplied

Inhibitors of guanylate cyclase inhibit phototransduction in limulus ventral photoreceptors.

The second messenger systems involved in the final stages of the phototransduction cascade in Limulus photoreceptors remain unclear. Excised patches of transducing membrane contain cGMP-gated channels, suggesting the involvement of cGMP in the excitation process. To further explore this possibility, we tested the effects of inhibitors and agonists of guanylate cyclase. The active site cyclase inhibitors guanosine 5'-tetraphosphate and adenosine 5'-tetraphosphate produced a reversible reduction of the response to light without affecting resting membrane properties. The cyclase inhibitor Rp-GTPalphaS produced a similar reduction, but the effect was only slightly reversible. The reduction in the response produced by these inhibitors was robust, often producing over a 95% decrease in the amplitude of the light response. Previous work had shown that an end-product cyclase inhibitor, imidodiphosphate, also inhibited the response. The consistent results with four different guanylate cyclase inhibitors strongly support the involvement of this enzyme in the phototransduction cascade. To determine whether the guanylate cyclase involved is the NO-dependent soluble form, we applied inhibitors and activators of the nitric oxide synthase/guanylate cyclase pathway such as L-N5-(1-iminoethyl) ornithine, sodium nitroprusside, and carboxy-PTIO. None of these agents had any substantial effect on phototransduction. Taken together, these results support a role for a particulate guanylate cyclase in Limulus photoreceptor excitation.

Animals↗
PubMed1986

Immunobiological activities of synthetic lipid A analogs with low endotoxicity.

Synthetic lipid A analogs, beta(1-6)glucosamine disaccharide 1,4'-bisphosphates, which possesses four tetradecanoyl groups at the 2- and 2'-amino, and 3- and 3'-hydroxyl groups (LA-17-PP), and each two of the (R)-3-hydroxytetradecanoyl and tetradecanoyl groups at the 2- and 2'-amino and 3- and 3'-hydroxyl groups, respectively (LA-18-PP), were far less endotoxic than synthetic (506, LA-15-PP) and bacterial Escherichia coli type lipid A's; neither compound showed any detectable lethal toxicity in chicken embryos or preparatory activity for the local Shwartzman reaction in rabbits. Also both compounds were only weakly pyrogenic and comparably less lethally toxic in galactosamine-loaded mice than the reference synthetic and bacterial lipid A's and a synthetic counterpart to biosynthetic lipid A precursor Ia (406, LA-14-PP). Nevertheless, LA-17-PP and LA-18-PP exhibited definite in vivo immunoadjuvant activity in mice, and the ability to induce a possible tumor necrosis factor and alpha/beta interferon in Mycobacterium bovis BCG and Propionibacterium acnes-primed mice, respectively, although these activities were weaker than those of the reference lipid A's. 4'-Monophosphate analogs of the above two test compounds exhibited neither endotoxic nor beneficial activities, but they showed remarkable in vitro bioactivities comparable to those of the corresponding bisphosphate compounds; the ability to activate the human complement system and the clotting enzyme cascade of horseshoe crab amoebocyte lysate, stimulatory effects on guinea pig and murine peritoneal macrophages, and murine splenocytes.

Adjuvants, Immunologic↗
PubMed1983

Deposition of the terminal C5b-9 complement complex on erythrocytes by human red cell autoantibodies.

A radioassay for the detection of complement activating autoantibodies (autohaemolysins) in patients with autoimmune haemolytic anaemia (AIHA) is described. The method is based on immunoradiometric quantitation of the cytolytic C5b-9 complement complex following its antibody-dependent deposition on red blood cells (RBC). The use of affinity-purified, radiolabelled antibodies directed against the neoantigens of the C5b-9 complex ensured specificity of the test which proved more sensitive than conventional haemolytic assays and was not subject to disturbances by haemolytic sera. The results obtained in 70 patients with various forms of AIHA (warm type N = 45); cold type (N = 22); Donath-Landsteiner type (N = 3] support the prevailing assumption that autohaemolysins can trigger the complement cascade to completion. Since intact RBC from patients with detectable autohaemolysins carried C3/C4 components but never C5b-9, it is inferred that regulatory mechanisms operate in vivo at the level of C4b/C3b inactivation to arrest the cascade and rescue the autologous cells.

Adolescent↗
PubMedDate not supplied

Antibody, macrophages, dengue virus infection, shock, and hemorrhage: a pathogenetic cascade.

Dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in children is reliably associated with the presence of dengue antibody--actively or passively acquired--before the onset of illness. Limited observations by electron microscopy and fluorescent antibody testing and the recovery of virus from tissues obtained at autopsy show that dengue viruses are consistently associated with cells of mononuclear phagocyte lineage. In particular, virus is associated with Kupffer cells, pulmonary macrophages, and mononuclear cells in skin and blood. Endothelial cells fail to demonstrate necrosis or inflammatory changes. Since acute vascular permeability, shock, and hemorrhage occur late in illness, a plausible hypothesis is that phlogistic factors, resulting from interactions with elements of the immune response, are released from virus-infected mononuclear phagocytes. Such phenomena as generalized depression of mitotic activity of bone marrow cells, destruction of mature polymorphonuclear leukocytes, complement activation, and abnormal hemostasis may serve as markers of these phlogistic factors. It will be of interest to establish whether other viral hemorrhagic fevers involve the same target cells as in DHF/DSS and are mediated by similar effector mechanisms.

Age Factors↗
PubMed2003

The stress cascade and schizophrenia: etiology and onset.

Psychosocial stress is included in most etiologic models of schizophrenia, frequently as a precipitating factor for psychosis in vulnerable individuals. Nonetheless, the stress-diathesis model has not been tested prospectively in prodromal patients as a predictor of psychosis. The biological effects of stress are mediated by the hypothalamic-pituitary-adrenal (HPA) axis, which governs the release of steroids, including cortisol. The past few decades have witnessed an increased understanding of the neural effects of stress and cortisol, including both normal and abnormal diatheses. As few biological markers have been evaluated as risk factors for psychosis in prodromal patients, the HPA axis and its interaction with intervening life events are apt candidates for study. In this article, we review the HPA axis and its neural effects, present a model for how stress might precipitate psychosis in vulnerable individuals, review the empirical evidence of a link between stress and schizophrenia symptoms, and propose a research design and appropriate statistical models to test the stress-diathesis model for psychosis onset in prodromal patients.

Animals↗
PubMed2006

Cascade screening for familial hypercholesterolaemia: implications of a pilot study for national screening programmes.

OBJECTIVES: To determine what proportion of cases of heterozygous familial hypercholesterolaemia would be identified by cascade screening conducted by a specialist hospital clinic, and by how much this would increase the prevalence of diagnosed cases. SETTING: Hospital clinic serving a population of 605,900 in Oxfordshire, UK. METHODS: A specialist nurse obtained details of living first-degree relatives from 227 adult patients with heterozygous familial hypercholesterolaemia currently or previously attending Oxford lipid clinic after excluding 79 adults without relatives living in Oxfordshire and 48 children. Index cases were asked to invite relatives resident in Oxfordshire for testing. RESULTS: A total of 227 index cases had 1075 first-degree relatives, including 442 adults and 117 children aged < 18 years resident in Oxfordshire. We excluded 171 previously screened adults and 46 for other reasons. Among 225 eligible adult relatives, 28 responders (12%) planned to consult their general practitioner and 52 (23%) attended the clinic for testing. Parents of 113 children (97%) wanted them tested. The positive diagnostic rate was 29% (15/52) in adults and 32% (36/113) in children. Screening increased prevalence by 14.4%, from 0.58/1000 (95% confidence intervals [CI] 0.52-0.65) to 0.67/1000 (95% CI 0.60-0.73), representing 33.5% of predicted cases. CONCLUSIONS: Cascade screening conducted by a specialist hospital clinic within its population catchment area did not substantially increase the prevalence of diagnosed familial hypercholesterolaemia. To maximize response rates, clinic staff need to approach relatives directly. Validated age, sex and country-specific diagnostic criteria should be defined, possibly with access to DNA-based tests, to help resolve diagnostic uncertainty.

Adult↗
PubMed1997

Conservation of cholera toxin gene in a strain of cholera toxin non-producing Vibrio cholerae O1.

BT23, a Vibrio cholerae O1 E1 Tor isolate, possesses the cholera toxin (CT) gene as determined by PCR. However, CT was not detected in the culture medium by the reversed passive latex agglutination test, nor in the whole cell lysate as examined by Western blotting. The toxin-coregulated pilus (TCP) was not detected by Western blotting. This suggests the presence of defects in the regulatory cascade. toxR, toxS and toxT, members of the regulatory cascade, were examined by PCR. toxR and toxS were conserved but toxT was not. CT and TCP production was complemented by transformation of toxT. The lack of toxT was suspected to be the cause of the undetectable production of CT in strain BT23.

Bacterial Proteins↗
PubMed2005

Mensuration and cleaning of the jets in Andersen cascade impactors.

PURPOSE: Fifty-three Andersen Cascade Impactors (404 stages) have been investigated using an automated visual stage mensuration technique. A cleaning method was suggested for stages with jets smaller than nominal diameters. The impact of nonapproved jet diameters on result parameters from particle size analysis was evaluated theoretically. METHODS: The jet diameters were measured using the Andersen Visual Inspection Device. A stepwise cleaning procedure was performed to recover the jets of noncompliant stages, and after each step a new stage mensuration was performed. RESULTS: The result of this extensive investigation, including measurements of each jet, is compared to other studies, to tolerance limits applied at AstraZeneca Lund and also to limits used by the manufacturer. Sixteen of the investigated stages were outside applied tolerance limits due to too small average diameters. Insertion of a go gauge into every jet of the stages was the only technique of those tested that increased the jet diameters toward nominal dimensions. Moreover, the relative standard deviation of the jet diameters decreased considerably after use of go gauges. CONCLUSIONS: Stage mensuration is a valuable technique for detection of improper jet dimensions of the Andersen Cascade Impactor, and use of go gauges is an effective cleaning method especially for jets with small diameters. However, use of stop/go gauges as a periodical quality control test on a small number of randomly selected jets was a poorly discriminating test, as both compliant and noncompliant stages would most probably pass such a test.

Aerosols↗
PubMed1994

Flip side of synaptic plasticity: long-term depression mechanisms in the hippocampus.

There is growing interest in the phenomenon of long-term depression (LTD) of synaptic efficacy that, together with long-term potentiation (LTP), is a putative information storage mechanism in mammalian brain. In neural network models, multiple learning rules have been used for LTD induction. Similarly, in neurophysiological studies of hippocampal synaptic plasticity, a variety of activity patterns have been effective at inducing LTD, although experimental paradigms are still being optimized. In this review the authors summarize the major experimental paradigms and compare what is known about the mechanisms of LTD induction. Although all paradigms appear to initiate a cascade of events leading to an elevated level of Ca2+ postsynaptically, the extent to which these paradigms involve common expression mechanisms has not yet been tested. The authors discuss several critical experiments that would address this latter issue. Numerous questions about the properties and mechanisms of LTD(s) in the hippocampus remain to be answered, but it is clear that LTD has finally arrived, and will soon be attracting attention equal to its flip side, LTP.

Animals↗
PubMedDate not supplied

Dual-regulated expression technology: a new era in the adjustment of heterologous gene expression in mammalian cells.

BACKGROUND: On the basis of the compatible streptogramin- and tetracycline-responsive expression systems, a series of dual-regulated expression systems have been established for use in sophisticated biopharmaceutical manufacturing, advanced gene therapy, and tissue engineering. METHODS: Dual-regulated expression concepts enable streptogramin- and tetracycline-responsive control of two different (sets of) transgenes (multi-regulated multigene metabolic engineering), dual-autoregulated expression configurations for one-step chromosomal integration of two antibiotic-adjustable expression units, and artificial regulatory cascades for multi-level regulation of transgenes and optimized integration of molecular interventions into mammalian regulatory networks. RESULTS: This report describes the construction and testing of a family of dual-regulated expression vectors which are compatible with the pTRIDENT vector construction kit, and, in some cases, adapted for retroviral expression technology enabling straightforward transduction of difficult-to-transfect cell lines such as primary cells and stem cells. CONCLUSIONS: Dual-regulated expression technology will probably become of prime interest for a variety of therapeutic applications, including biopharmaceutical manufacturing, gene therapy, and tissue engineering.

Animals↗
PubMed2002

Assessment of mutagenicity and toxicity of different-size fractions of air particulates from La Plata, Argentina, and Leipzig, Germany.

Airborne particulates, especially fine particles and bound chemical compounds, are a potential mediator of adverse health effects. In this study an analysis was done of the concentration and size distribution of air particulate matter, the content of bound polycyclic aromatic hydrocarbons (PAHs), and the biological effects of organic extracts from different fractions of dust that had been influenced by urban and industrial emissions from the regions of La Plata, Argentina, and Leipzig, Germany, along with samples from control areas. Air particulate matter was sampled in summer and winter in each region using a high-volume sampler with a six-stage cascade impactor, classifying dust in six size fractions from 10-microm particles to those less than 0.49 microm in size. Organic extracts of dust were tested for mutagenicity (Ames test, Salmonella typhimurium TA98 strain, S9+) and cytotoxicity (Tetrahymena pyriformis test system, growth rate, cell respiration). The content of PAHs was analyzed by high-performance liquid chromatography (HPLC) with diode array and fluorescence detection. Mutagenic and cytotoxic effects were found to be associated with very fine (<0.49 microm) and fine (<1.5 microm) particle-bound compounds, corresponding to a higher content of PAHs. The mutagenic potency (revertants/m(3)) associated with particles less than 0.49 microm from urban areas of La Plata was about 1 order of magnitude higher than in particles in the range of 3.0 to 0.49 microm. Fine fractions from sites with an industrial burden were also found to exhibit high mutagenic potency. A similar tendency was observed in cytotoxicity tests with T. pyriformis. This cell system proved to be very sensitive to toxicants in tested dust fractions. The observed biological effects were found to be correlated significantly with concentrations of total PAH, carcinogenic PAHs, and benzo[a]pyrene.

Air Pollutants↗
PubMed1992

The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction.

The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2005

Consequences for Protaphorura armata (Collembola: Onychiuridae) following exposure to genetically modified Bacillus thuringiensis (Bt) maize and non-Bt maize.

Studies on the effect of genetically modified Bacillus thuringiensis (Bt) crops on true soil dwelling non-target arthropods are scarce. The objective of this study was to assess the influence of a 4-week exposure to two Bt maize varieties (Cry1Ab) Cascade and MEB307 on the collembolan Protaphorura armata. For comparison three non-Bt maize varieties, Rivaldo (isogenic to Cascade), Monumental (isogenic to MEB307) and DK242, and two control diets based on baker's yeast (uncontaminated and contaminated with Bt toxin Cry1Ab) were also tested. Due to a lower C:N ratio, individuals reared on yeast performed significantly better in all of the measured endpoints than those reared on maize. P. armata performed equally well when reared on two Bt and three non-Bt maize varieties. Although there were no negative effects of Bt maize in this experiment, we recommend future studies on Bt crops to focus on species interactions in long-term, multi-species experiments.

Animals↗