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Comparison between the DNA precipitation and alkali unwinding assays for detecting DNA strand breaks and cross-links.

A new DNA precipitation assay used together with the alkali unwinding assay may provide a rapid means of detecting DNA damage in addition to strand breaks based on the relative amount of damage measured by the two assays. X-rays, Adriamycin, 4-nitroquinoline-N-oxide, N-methyl-N'-nitrosoguanidine, bleomycin, RSU 1172, and five other drugs produced the same relative amount of strand breakage by using the DNA precipitation and alkali unwinding assays. However, strand breaks produced by the bifunctional alkylating agents bis(2-chloroethyl)nitrosourea, RSU 1069, and RSU 1131 were detected with greater efficiency by the DNA precipitation assay, while the unwinding assay measured more strand breaks than the precipitation assay after damage by the topoisomerase inhibitors VP-16 and VM-26 and the DNA-condensing agents acridine orange and pyronin Y. Based on the reported mechanisms of action of these drugs, and studies with known DNA cross-linking agents, it appears that in addition to DNA strand breaks, the alkali unwinding assay is more sensitive to interstrand than to DNA-protein cross-links, while the DNA precipitation assay can be used to detect both types of cross-links. While quantification of specific lesions is not possible with this approach, the concomitant use of these two assays may provide a rapid and simple method for screening genotoxic drugs for DNA damage, and may also help to differentiate between DNA lesions which include strand breaks, interstrand and protein cross-links, DNA-phosphate adducts, and DNA-drug precipitates.

Animals↗

The role of C1, C1-inactivator and C4 in modulating immune precipitation.

To clarify the mechanism of inhibition of immune precipitation by early components of the classical pathway of complement, aggregation of 125I-BSA-rabbit-anti-BSA antibody complexes was performed in the presence of purified C1, C1-inactivator (C1-In) and C4. C1 delayed the rate of immune precipitation in a concentration dependent manner. This phenomenon was not influenced by the presence of 0.3 mM p-nitrophenyl-p-guanidinobenzoate (NPGB) which inhibits C1 activation. The antiaggregational effect of C1 was reversed by 10 mM EDTA and by C1-In at a C1-In/C1 molar ratio of greater than or equal to 4/1. C1-In was not effective when the reaction was performed in the presence of NPGB. Thus, although the inhibitory effect of C1 on immune precipitation was not dependent upon C1 activation, the formation of C1 was required to observe the effect of C1-In. The addition of C4 to C1 did not modify the slow aggregation of complexes, even when a limiting concentration of C1 was used. C1-In and EDTA were both able to cause similar rapid precipitation of complexes prepared in the presence of C1 and C4, demonstrating that C4 did not play a significant role in delaying the precipitation reaction. However, soluble complexes prepared in the presence of C1 and C4 were specifically precipitated by the addition of excess anti-C4 antibody, attesting to the binding of C4 to immune complexes. These observations suggest that the processing of immune complexes in vivo may not be similar in different classical pathway complement deficiency states.

Animals↗

Prevention of immune precipitation by purified classical pathway complement components.

The role of the classical pathway of complement in the prevention of immune precipitation has been investigated using purified complement components and immune complexes (IC) consisting of rabbit anti-BSA and BSA. C1 reduced the rate of immune precipitation. As C1q, EDTA treated C1 or C1-inhibitor treated C1 were unable to retard the precipitation of IC, it was concluded that the intact C1 molecule was required for this function. Use of phenylmethylsulphonyl fluoride and benzamidine showed that the enzymatic site on C1 was not required for this activity. C4 and C2 did not affect immune precipitation significantly when C1 was present at the concentrations present in serum. When C3 was added to C1, C4 and C2 precipitation of IC did not occur. These data demonstrate that classical pathway activation alone is sufficient for the prevention of immune precipitation.

Antigen-Antibody Complex↗

Distribution of plasma fibronectin (cold-insoluble globulin) and components of the factor VIII complex after heparin-induced precipitation of plasma.

Factor VIII procoagulant (VIII:C) activity, factor VIII coagulant antigen (VIII:CAg), von Willebrand ristocetin cofactor (VIIIR:RC) activity, factor VIII-related antigen (VIIIR:Ag), and plasma fibronectin (CIg; cold-insoluble globulin) were measured in the heparin precipitable fraction (HPF) and heparin supernatant fraction (HS) of normal human plasma. Following heparin induced precipitation, most measurable VIII:C activity (77% +/- 24%) was recovered in the HS. Although there was little VIII:C activity (less than 1%) in the HPF, 20% +/- 6.5% VIII:CAg was present as well as CIg (81% +/- 5.6%). VIIIR:RC activity (72% +/- 12%), and VIIIR:Ag (34 +/- 5.2%). As assessed by Na dodecyl SO4 glyoxyl agarose electrophoresis, the multimeric forms of plasma VIIIR:Ag could be resolved into a series of bands. Larger multimers tended to precipitate with the HPF whereas the smaller multimers tended to remain supernatant. Plasma from a subject with congenital afibrinogenemia was also studied. Although the afibrinogenemic HPF contained CIg, neither VIIIR:RC activity nor VIIIR:Ag was precipitated. However, both were present in the HPF from afibrinogenemic plasma to which fibrinogen had been added, suggesting that they are incorporated in this precipitate because of an affinity for fibrinogen. The ability of heparin to induce precipitation of CIg while leaving most VIII:C activity in the supernatant plasma may be useful in the preparation of procoagulant-rich plasma subfractions, since VIII:C can subsequently be recovered in good yield by cryoprecipitation.

Afibrinogenemia↗

Purification of endo-polygalacturonase by affinity precipitation using alginate.

The precipitation of alginate by Ca2+ at pH 3.8 was found to occur concomitantly with the precipitation of endo-polygalacturonase from Aspergillus niger. Under optimum conditions, 92% of the enzyme activity was precipitated. The enzyme could be recovered from the precipitate by washing with 0.5 M NaCl/0.2 M Ca2+. All the precipitated endo-polygalacturonase activity could be recovered in this way. The enzyme thus obtained was purified 10-fold. A comparison of SDS/PAGE gel patterns of the crude preparation and enzyme purified by the affinity precipitation also showed a significant purification of the enzyme.

Alginates↗

Reference standards for quantification of skeletal alkaline phosphatase activity in serum by heat inactivation and lectin precipitation.

Putative standards of skeletal alkaline phosphatase (ALP) (from bone, bone cells, osteosarcoma cells, and Pagetic serum) and hepatic ALP (from cholestatic serum and bile) were used to compare three methods for quantifying skeletal ALP activity in serum: heat inactivation, precipitation with wheat germ agglutinin (WGA), and precipitation with concanavalin A (Con A). All the skeletal ALP standards were similarly sensitive to heat inactivation, as were the hepatic ALP standards. Heat inactivation separated skeletal from hepatic ALP by a 50% difference in remaining ALP activities (e.g., 23% and 74% remaining skeletal and hepatic ALP activities after 30 min at 52 degrees C). Differential precipitations with WGA and with Con A were less efficient at separating skeletal from hepatic ALP (maximum differences of < 30% remaining ALP activity). Although both types of hepatic ALP standard (cholestatic serum and bile) were precipitated with similar efficiencies by WGA and Con A, the skeletal ALP standards were not (e.g., at 2.7 g/L, WGA precipitated 78-86% of the ALP activity in Pagetic serum, but only 49% of the ALP activity in extracts of human bone). These data suggest that heat inactivation is preferable to precipitation with WGA or Con A for quantifying skeletal ALP activity in serum: it better separates skeletal from hepatic ALP activity and is not sensitive to glycosyl heterogeneity.

Alkaline Phosphatase↗

Reverse flux filtration: a new mode of therapy improving the efficacy of heparin-induced extracorporeal LDL precipitation in hyperlipidemic hemodialysis patients.

In the steady state after a run-in phase of 3 months, the acute effects of 3 modifications of weekly heparin-induced extracorporeal LDL precipitation (HELP) were studied in 5 ESRD and 2 non-uremic hypercholesterolemic coronary patients. In ESRD patients (n = 29 sessions), HELP reduced LDL-cholesterol (LDL-C) (56 +/- 7%) and fibrinogen (FIB) (54 +/- 10%) by a similar percentage as compared to non-uremic controls (60 +/- 4% and 61 +/- 3%, resp.; n = 5). In order to eliminate the need for extra HELP sessions in addition to the normal dialysis regimen, newly developed hardware was then used to perform combined synchronous HELP/HD (n = 12). However, premature precipitate filter plugging probably due to hyperfibrinogenemia in ESRD patients, accentuated by ultrafiltration (UF), decreased the corresponding reductions to 26 +/- 9% (LDL-C) and 34 +/- 11% (FIB). Therefore, the procedure was modified by reversing the filtrate flux through the precipitate filter membrane after 900 ml of treated plasma ("reverse flux filtration", RFF; n = 11). Thus, in RFF-HELP/HD the LDL/FIB/heparin coprecipitate was deposited on both filter membrane sides which caused a significant enhancement of the filter capacity and improved reductions to 46 +/- 14% for LDL-C and 51 +/- 15% for FIB. Elution of the precipitate from the precipitate filter after the sessions showed that RFF-HELP/HD had trapped 1733 +/- 238 mg LDL-C and 8108 +/- 1876 mg FIB in ESRD patients, while HELP eliminated 1890 +/- 333 mg LDL-C and only 3663 +/- 369 mg FIB in non-uremics. Filter precipitate recoveries (relative to the mass removed from the patient plasma pool) amounted to 97 +/- 18% for LDL-C and 158 +/- 67% for FIB in the ESRD group treated by RFF-HELP/HD vs. 70 and 76% in the non-uremic HELP group. Probably, passive transport of lipoproteins and FIB from the interstitium into the vascular space caused repletion of this compartment during HELP/HD where an UF induced solvent drag is effective. In summary, the new RFF-HELP/HD procedure effectively reduced LDL-C and FIB in ESRD patients who could not be adequately treated by the conventional HELP/HD system.

Blood Component Removal↗

Association of precipitating anti-adrenal anti-adrenal antibodies with moniliasis-polyendocrinopathy syndrome.

The association of precipitating anti-adrenal antibodies with different subgroups of idiopathic Addison's disease were studied. We had previously found these antibodies in patients with the moniliasis-polyendocrinopathy syndrome. Sera of 36 adult patients suffering from different froms of Addison's disease were examined for the presence of adrenal antibodies demonstrable either by immunofluorescence (IFL) or by gel diffusion. 3 of the 17 patients with tuberculous and 17 of 19 patients with idiopathic Addison's disease had IFL antibodies but only one had precipitating antibodies. There was one typical case of Schmidt's syndrome, and four additional cases with Addison's disease combined with diabetes or thyroiditis, who may later develop the syndrome. None of htese patients had precipitating anti-adrenal antibodies. The only patients with precipitating adrenal antibodies had the moniliasis-polyendocrinopathy syndrome. He was not typical as Addison' disease appeared unusually late and he did not have hypoparathyroidism. The presence of precipitating anti-adrenal antibodies in this patient, and the absence of these in other groups of Addison's disease, is further evidence for the association of precipitating antibodies with the moniliasis-polyendocrinopathy syndrome.

Addison Disease↗

Precipitants and additives for membrane crystallization of lysozyme.

Membrane crystallization is a newly developed crystallization technique that has proven to be superior in producing good crystal forms under operating conditions that are not appropriate to perform the crystallization process by other traditional techniques. In this work, static membrane crystallization was carried out on lysozyme, with hollow-fiber microporous hydrophobic membranes. Numerous precipitant and additive types and concentrations were employed in the crystallization processes in order to select the most appropriate precipitant and additive types and to find their corresponding concentration levels that can yield the best crystal forms. The crystallization processes were analyzed in two ways: firstly, by evaluation of the transmembrane fluxes obtained by using different precipitants and additives; secondly, by utilization of the images and results obtained from the micrography and IR spectra in comparisons and evaluations of the crystals formed under all kinds of conditions. Moreover, the size distributions of the crystals yielded under several typical crystallization conditions were analyzed, and turbidity and induction time periods obtained during typical crystallization experiments were also measured. Amongst the numerous precipitants and additives tested, the most appropriate precipitant type and additive were chosen and their concentrations were optimized. Good lysozyme crystals were obtained using a certain precipitant and additive. The obtained results from this work further support the advantages of utilizing the membrane crystallization technique for macromolecule crystallizations.

Biotechnology↗

Surface Precipitation of Co(II)(aq) on Al2O3

Surface precipitation is an important process in many areas of science and technology, including modeling contaminant segregation from groundwater to solid phases and dispersion of active phases on catalyst supports. XAFS, TEM, and XPS measurements of Co(II) sorbed on Al2O3 demonstrate that surface precipitates have formed from solutions that are undersaturated with respect to any known bulk solid phase. The precipitates have a structure similar to that of Co(OH)2(s), but are disordered and have a high concentration of Co vacancies. The data plus thermodynamic reasoning have been used to analyze the plausibility of various models for surface precipitation and to show that for Co(II)/Al2O3 it occurs by forming a double-hydroxide phase containing substrate-derived Al(III) ions. This idea was corroborated by mixing aqueous solutions of Al(III) and Co(II) at the pH and concentration of the sorption samples, forming a stable colloidal precipitate that is less soluble than either Al(OH)3 or Co(OH)2. The Co XAFS of this material was similar to that of the sorption samples. Successful quantitative models of metal ion transport in groundwater need to include the possibility of forming ternary and higher order precipitates that include ions derived from sparingly soluble solids. For catalyst impregnation, surface coprecipitation can prevent production of a well-dispersed precursor material.

Journal Article↗

Kinetics of the Formation and Dissolution of Ni Precipitates in a Gibbsite/Amorphous Silica Mixture.

There have been a number of studies that have examined metal precipitation reactions on an array of natural soil materials. While many of these investigations have focused on model single-component systems, recent research has appeared on metal precipitation on soils and clay fractions of soils. However, few studies have explored mixed model component systems, which may lead to a better understanding of metal reactions on soils and clay fractions. Furthermore, only a few studies have appeared on the stability of the metal surface precipitates. In light of this, we investigated Ni sorption and dissolution kinetics and mechanisms on a mixture of gibbsite and amorphous silica by combining macroscopic studies with X-ray absorption fine structure (XAFS) and diffuse reflectance spectroscopies (DRS), and high-resolution thermogravimetric analysis (HRTGA). Batch sorption experiments were conducted at pH 7.5 and at different reaction times to elucidate the sorption process and to study the role of residence time on metal precipitate stability. Spectroscopic and HRTGA investigations revealed alpha-Ni(OH)(2) precipitates formed on the gibbsite/silica mixture initially and over time evolved to a Ni phyllosilicate. The available Si source was derived from partial dissolution of the sorbent during Ni sorption. With increasing residence time, the precipitate phases drastically increased in stability, as shown by decreasing amounts of Ni release as effected by nitric acid (HNO(3)) and ethylenediaminetetraacetic acid (EDTA) treatments. This aging effect may be explained by the silicate-for-nitrate exchange during the first days of reaction and subsequently by silicate polymerization and partial grafting onto the hydroxide layers to form a phyllosilicate precursor phase (R. G. Ford, A. C. Scheinost, K. G. Scheckel, and D. L. Sparks, Environ. Sci. Technol. 33(18), 3140-3144, 1999). Copyright 2000 Academic Press.

Journal Article↗

Comparison of withdrawal precipitating properties of various morphine antagonists and partial agonists in relation to their stereospecific binding to brain homogenates.

In morphine-dependent rats the withdrawal precipitating properties of various morphine antagonists and partial agonists were studied by quantitatively evaluating a variety of different withdrawal signs. A comparison of the dose response curves of the various substances obtained for the different signs revealed marked differences in respect to the lowest effective doses (EDs) necessary to precipitate the withdrawal signs as well as in the maximum frequencies of the signs induced. The "pure" antagonist, naloxone, which was judged very potent according to the ED, precipitated the lowest levels of jumping, whereas certain partial agonists of the benzomorphane type, which were less potent according to the ED, induced very high levels of this sign. These latter compounds, however, failed to precipitate "complete" withdrawal, as evidenced by the nearly complete absence of some of the withdrawal signs. The jumping precipitating potency of the antagonists as judged from the ED was found to be highly correlated to the stereospecific binding of these substances to rat brain homogenate. On the other hand, the ability of the substances to precipitate high levels of jumping was seen to increase, at least within a certain range, with increasing degree of agonistic properties, as indicated by the ratio of stereospecific binding in the presence and absence of sodium.

Benzomorphans↗

Facial pain. IV. A prospective study of 1052 patients with a view of: precipitating factors, associated symptoms, objective psychiatric and neurological symptoms.

In a prospective material of 1052 patients the precipitating factors, associated symptoms, psychological and neurological deficits have been examined. Mastication and talking are the most frequently occurring precipitating factors, 76% as regards Neuralgia, with typical starting difficulties. As regards Non-neuralgiform Pain 24%, with precipitation late in the masticatory process. There were trigger zones in 50% of the cases of Typical Trigeminal Neuralgia and in 9% of the patients with Non-neuralgiform Pain. In a series of cases the jaw joint is perceived as a trigger zone. Cold precipitates pain in 48%-39%. Other precipitating factors are much more rare--psychological stress in 15% of the patients with Non-neuralgiform Pain, however. "Vegetative" associated symptoms were relatively frequent, lacrimation occurred in 31% of the cases of Typical Trigeminal Neuralgia and in 20% of the cases of Non-neuralgiform Pain. Rhinorrhea and salivation were less frequent. In terms of figures migrainoid associated symptoms had no connection with vegetative associated symptoms or with pain in the eye. In 11% of the patients pain occurred most frequently during the night and in 20% the frequency of pain was the same day and night. About 1/3 of the patients with Neuralgia experienced seasonal variations. Tenderness of foramina is a symptom of no significance. Very few patients had primary sensory loss. No eye or ear symptoms have been found which may be referred to as the patho-anatomical basis of the pain. About 1/3 of the patients with Non-neuralgiform Pain had psychological symptoms whereas hardly any patients with Neuralgia had them. MMPI test performed on a small matched material showed no difference between Neuralgia and Non-neuralgiform Pain. In material B an examination has been made of the jaw joint arthrosis symptoms. A restriction of the diagnosis of arthrosis has had the effect that it must be recognized that patients with facial pain do not have the high frequency of jaw joint diseases previously assumed. As was also the case in a series of normal material previously published, between 1, 5 and 1, 3 of the patients with Neuralgia had jaw joint arthrosis which was due to old age. This study has not revealed any connection between previous diseases, the onset of pain, the character and course of the pain, the character of the attack, the localization of pain, precipitating factors, associated symptoms and symptoms of loss on the one hand and the patho-anatomical substratum on the other.

Arousal↗

Spontaneous versus precipitated AUR: the same?

The differentiation between spontaneous and precipitated acute urinary retention was initially made by the PLESS study group. Acute urinary retention (AUR) occurring in the PLESS study, whether considered spontaneous or precipitated, was BPH-related as it occurred in a carefully selected population. It is known that AUR can be precipitated in patients without BPH by events such as anaesthesia and CVA. Therefore when assessing how to manage a patient with AUR, one should first consider whether or not the event is BPH-related. If BPH is thought to be present, then the management is likely to involve a trial without catheter following administration of an alpha-blocker. The presence of a factor that is considered to have precipitated or provoked the episode of AUR may influence the outcome in the longer term but there is little evidence that it alters the likelihood of a successful trial of voiding. Consequently spontaneous and precipitated AUR may be considered to be the same when they are BPH-related, as the approach to management need not be altered. Such differentiation is only likely to influence immediate management when AUR is truly precipitated in the absence of BPH.

Acute Disease↗

Effects of an increase in summer precipitation on leaf, soil, and ecosystem fluxes of CO2 and H2O in a sotol grassland in Big Bend National Park, Texas.

Global climate models predict that in the next century precipitation in desert regions of the USA will increase, which is anticipated to affect biosphere/atmosphere exchanges of both CO(2) and H(2)O. In a sotol grassland ecosystem in the Chihuahuan Desert at Big Bend National Park, we measured the response of leaf-level fluxes of CO(2) and H(2)O 1 day before and up to 7 days after three supplemental precipitation pulses in the summer (June, July, and August 2004). In addition, the responses of leaf, soil, and ecosystem fluxes of CO(2) and H(2)O to these precipitation pulses were also evaluated in September, 1 month after the final seasonal supplemental watering event. We found that plant carbon fixation responded positively to supplemental precipitation throughout the summer. Both shrubs and grasses in watered plots had increased rates of photosynthesis following pulses in June and July. In September, only grasses in watered plots had higher rates of photosynthesis than plants in the control plots. Soil respiration decreased in supplementally watered plots at the end of the summer. Due to these increased rates of photosynthesis in grasses and decreased rates of daytime soil respiration, watered ecosystems were a sink for carbon in September, assimilating on average 31 mmol CO(2) m(-2) s(-1) ground area day(-1). As a result of a 25% increase in summer precipitation, watered plots fixed eightfold more CO(2) during a 24-h period than control plots. In June and July, there were greater rates of transpiration for both grasses and shrubs in the watered plots. In September, similar rates of transpiration and soil water evaporation led to no observed treatment differences in ecosystem evapotranspiration, even though grasses transpired significantly more than shrubs. In summary, greater amounts of summer precipitation may lead to short-term increased carbon uptake by this sotol grassland ecosystem.

Asparagaceae↗

Nitrate sequestration by corticolous macrolichens during winter precipitation events.

Nitrogen is an essential nutrient in the biogeochemistry of forested ecosystems. The influence of canopy lichens on the winter biogeochemistry of nitrate in broadleaved deciduous forests is examined and it is hypothesized that nitrate sequestration will not differ between winter precipitation events. Rejection of this hypothesis would mean that meteorological conditions of winter precipitation events have a detectable influence on nitrate sequestration by canopy lichens and nitrate input to the forest floor. Canopy lichens of the genus Parmelia were found to influence winter nitrate stemflow inputs to forest soils differentially. Epiphytic lichens on an individual Carya glabra Mill. (pignut hickory) canopy tree, centrally located within the stand of an open deciduous forest, actively sequestered nitrate leached from the tree's woody frame, lowering aqueous stemflow inputs at the tree base. The quantities of nitrate sequestered by corticolous lichens during the 2 February 1999 mixed-precipitation event were significantly greater than those during all other precipitation events examined. Greater rates of nitrate uptake during the 2 February 1999 event may be attributed to (1) its intermediate rain intensity, which would have soaked the lichen thalli in a nutrient-rich bath, and (2) an air temperature range between -2 degrees C and 8 degrees C that would have increased viscosity and surface tension of stemflow drainage, thereby decreasing stemflow velocity and increasing the contact time of stemflow water on the lichen thalli. Other precipitation events were either too cold to promote metabolic activity by canopy lichens or too warm and intense for an optimal contact time of stemflow with lichen thalli, resulting in lower quantities of nitrate sequestered. Meteorological conditions of winter precipitation events have been documented to influence sequestration of nitrate by corticolous lichens and decrease aqueous stemflow inputs to the forest floor of broadleaved deciduous forests.

Ecosystem↗

Evaluation of polyethylene glycol's suitability for quantitative determination of non-precipitating antibody.

Polyethylene glycol (PEG) has been used extensively in the quantitative precipitin reaction and for the determination of soluble immune complexes. It has now been found that the optimum concentration of PEG for precipitation of non-precipitating antibodies in the quantitative precipitin reaction varies with the dilution of antisera. Immune precipitates obtained with PEG were examined for their protein content by polyacrylamide gel electrophoresis to detect non-specific precipitation of serum proteins. Determination of non-precipitating antibodies in sera of goats and pigs immunized with chicken egg-white lysozyme by the quantitative precipitin reaction in the presence of PEG, yielded lower values than obtained by immunoabsorbents. Care should be exercised in interpreting results when PEG is used to enhance precipitation.

Animals↗

Specific antibodies to Besnoitia besnoiti precipitated from serum of cattle by live parasites and by soluble antigen.

Specific precipitable antibodies of both IgG and IgM classes were detected in sera of cattle naturally infected with B. besnoiti. The amount of specific antibodies of the IgG class precipitated by soluble antigen was in the range of 17-50 micrograms/ml serum while that of the IgM class ranged between 5 and 24 micrograms/ml serum. Specific antibodies precipitated by live B. besnoiti parasites were in amounts of 10 to 22 micrograms/ml serum for IgG and 4 to 26 micrograms/ml serum for IgM. Different ratios of IgG/IgM were obtained by the two methods of precipitation. This might indicate that antibodies to B. besnoiti of the IgM class can be precipitated and detected in sera of naturally infected cattle in similar amounts either by live parasites or by solubilized antigen, whereas antibodies of the IgG class can be preferentially detected when solubilized antigen is used for precipitation.

Animals↗