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PubMed1983

Protein content of precipitates present in pancreatic juice of alcoholic subjects and patients with chronic calcifying pancreatitis.

Chronic calcifying pancreatitis is characterized by the formation of intraductal protein plugs or precipitates and calcified stones in ducts. Similar precipitates may be collected by endoscopic retrograde catheterization of the main pancreatic duct. They are present in the pancreatic juice of alcoholic subjects and patients with chronic calcifying pancreatitis. Protein analysis of these precipitates was performed to try to elucidate the mechanism of stone formation. Two protein fraction were separated by extraction of precipitates. One fraction was easily soluble in saline and contained a small amount of most of the proteins of pancreatic juice. The other fraction was soluble in citrate or ethylenediaminetetraacetate and contained a few proteins with close isoelectric points and identical molecular weight (13,500). These proteins showed immunological identity with the "stone protein" isolated from human pancreatic calculi. Our data demonstrate that the major citrate-soluble protein of precipitates in pancreatic juice is identical with "stone protein". They are strongly support the concept that this protein is the organic matrix of pancreatic stones. Different mechanisms are proposed to explain the phenomenon of protein precipitation that frequently occurs in alcoholic subjects and patients with chronic calcifying pancreatitis.

Alcoholism↗

PubMed1995

Comparison of benzodiazepine receptor ligands with partial agonistic, antagonistic or partial inverse agonistic properties in precipitating withdrawal in squirrel monkeys.

Benzodiazepine receptor (BZR) ligands previously characterized as differing in intrinsic efficacy were evaluated first for potency in antagonizing flunitrazepam-induced sleep in monkeys. Data from these experiments were used to define approximately equieffective doses for subsequent use in precipitating withdrawal in diazepam-treated monkeys. It was shown that partial agonists with intermediate intrinsic efficacy (bretazenil, Ro 41-7812) were relatively ineffective in precipitating withdrawal reactions in diazepam-treated squirrel monkeys. The potent and specific BZR antagonist flumazenil, which possesses weak intrinsic efficacy, was more effective in precipitating a withdrawal reaction in diazepam-treated monkeys. In contrast, the highest dose of the BZR antagonist ZK 93426 that could be administered failed to precipitate withdrawal under the same experimental conditions. Finally, the BZR partial inverse agonist sarmazenil was the most effective of these BZR ligands in eliciting a precipitated withdrawal reaction. Thus, the results of the present investigation strongly suggest that BZR ligands differing in intrinsic efficacy differentially precipitate withdrawal in squirrel monkeys treated chronically with diazepam.

Animals↗

PubMed1993

Intravenous flumazenil following acute and repeated exposure to lorazepam in healthy volunteers: antagonism and precipitated withdrawal.

The effects of i.v. administered flumazenil (3.0 mg) were studied in healthy male subjects who received pretreatment with p.o. placebo or lorazepam. The duration of placebo or lorazepam (3.0 mg single p.o. daily dose) pretreatment before a flumazenil or placebo injection was 1, 3, 7 or 14 days in four sequential groups of subjects. Initial administration of lorazepam produced a classic sedative profile of effects on various psychomotor/behavioral performance, observer-rated and subject-rated measures. Tolerance to repeated daily administration of lorazepam was suggested by a progressive diminution of performance disrupting effects. In subjects pretreated with placebo, flumazenil increased subject-ratings of dizziness over preinjection ratings. Flumazenil produced an immediate reversal of lorazepam effects in subjects who were not tolerant to lorazepam (1- and 3-day pretreatment groups). Flumazenil did not precipitate withdrawal symptoms in subjects who received a single administration of lorazepam. Precipitated withdrawal symptoms were evident after 3 and 7 days of lorazepam pretreatment, and there was a tendency toward precipitated withdrawal symptoms (that included one panic attack) after 14 days of lorazepam pretreatment. Precipitated withdrawal was characterized by an elevation in subject-rated symptoms including dizziness, tenseness, tachycardia, perceptual disturbance and sweating. Symptoms were maximal immediately after injection, usually mild in severity and usually resolved within 1 hr. There was no evidence of precipitated withdrawal on psychomotor/behavioral performance or observer ratings. The present study provides the strongest human experimental evidence to date that flumazenil can precipitate withdrawal symptoms after a history of repeated benzodiazepine exposure.

Adolescent↗

PubMed1998

[Precipitating factors in patients with repetitive exacerbation of chronic left heart failure].

The precipitating factors of repetitive exacerbation were investigated in 110 consecutive patients with chronic left heart failure admitted due to acute exacerbation more than twice to the medical emergency ward of National Cardiovascular Center from January, 1992 to December, 1996. The controls were 189 consecutive patients with chronic left heart failure admitted to the ward due to acute exacerbation only once during the same period. Excessive intake of water or sodium, overwork and infection were common precipitating factors in the first decompensation of left heart failure, but the former two factors became less common with repeated admission. Patient mistakes such as excessive intake of water or sodium, overwork and noncompliance with medications, and new onset arrhythmias were common precipitating factors in patients (n = 13) admitted to the ward more than four times. Infection was a common precipitating factor (63%) in patients with a time interval between readmission and the last discharge of longer than 2 years. Despite repeated admission, infection was a common precipitating factor in patients with valvular heart disease (n = 31), patient mistakes were common in heart disease with left ventricular hypertrophy (n = 20), and infection and new onset arrhythmias were common in dilated cardiomyopathy (n = 28) and old myocardial infarction (n = 31). Patient mistakes and new onset arrhythmias were the common factors that led to repetitive exacerbation of left heart failure, and precipitating factors were characterized by the etiology of left heart failure.

Aged↗

PubMed1998

Electron microscopic localization of acetylcholinesterase activity in the central nervous system: chemical basis of a catalytic activity of Hatchett's brown (cupric ferrocyanide) precipitate revealed by 3,3'-diaminobenzidine.

The chemical basis of the histochemical method for acetylcholinesterase described by Tago et al. was studied. The primary histochemical reaction of Tago's method is performed with diluted Karnovsky's medium, resulting in the in situ formation of Karnovsky's precipitate in minute quantities. The visualization of acetylcholinesterase activity is made in a secondary reaction where diaminobenzidine (DAB) is oxidized in the presence of hydrogen peroxide by the peroxidase-like activity of the histochemical precipitate. Since Karnovsky's precipitate is already known to be a mixture of Cu+3Fe (CN)6 and Cu++2Fe++(CN)6, the catalytic activity of these two complexes was studied in vitro comparatively to cupric ferricyanide (non-histochemical precipitate) and to copper and iron ions participating in the complexes. We showed that only the first two complexes intensely oxidized DAB with or without hydrogen peroxide. Thus, Tago's method is based on the same catalytic activity as Hanker's method (oxidation of DAB using non-diluted Karnovsky's medium without hydrogen peroxide). Since both complexes are able to oxidize DAB without hydrogen peroxide, we propose to describe this catalytic activity as oxidoreductase-like activity. Tago's method was modified for ultrastructural observations by avoiding metal intensification of the DAB precipitate. We obtained fine localization of DAB precipitate in the lumen of endoplasmic reticulum and Golgi apparatus of the neurons of substantia nigra.

3,3'-Diaminobenzidine↗

PubMed2004

Fractionation of protein, RNA, and plasmid DNA in centrifugal precipitation chromatography using cationic surfactant CTAB containing inorganic salts NaCl and NH(4)Cl.

Centrifugal precipitation chromatography (CPC) is a separation system that mainly employs a moving concentration gradient of precipitating agent along a channel and solutes of interest undergo repetitive precipitation-dissolution, fractionate at different locations, and elute out from the channel according to their solubility in the precipitating agent solution. We report here for the first time the use of a CPC system for fractionation of protein, RNA, and plasmid DNA in clarified lysate produced from bacterial culture. The cationic surfactant cetyltrimethylammonium bromide (CTAB) was initially used as a precipitating agent; however, all biomolecules showed no differential solubility in the moving concentration gradient of this surfactant and, as a result, no separation of protein, RNA, and plasmid DNA occurred. To overcome this problem, inorganic salts such as NaCl and NH(4)Cl were introduced into solution of CTAB. The protein and RNA were found to have higher solubility with the addition of these salts and separated from the plasmid DNA. Decreasing surface charge density of CTAB upon addition of NaCl and NH(4)Cl was believed to lead to lower surfactant complexation, and therefore caused differential solubility and fractionation of these biomolecules. Addition of CaCl(2) did not improve solubility and separation of RNA from plasmid DNA.

Centrifugation↗

PubMedDate not supplied

Affinity precipitation of proteins.

Affinity precipitation is being studied as a technique to be introduced at an early stage of downstream processing for the selective isolation of proteins. The technique utilizes a heterobifunctional ligand, which, in addition to having affinity for the target protein(s), possesses another function for controlling precipitation. The latter component is comprised of a polymer which can be made reversibly soluble and insoluble by altering a specific parameter such as pH or temperature. Different polymers of natural and synthetic origin have been used for this purpose. The soluble form of the ligand is used for the affinity binding step and precipitation is induced for obtaining separation of the affinity complex. Some of the polymers used in this laboratory include chitosan, alginate, Eudragit S-100 (copolymer of methacrylic acid and methyl methacrylate) and polyethyleneimine. Chitosan and alginate served as natural ligands for wheat germ agglutinin and pectinase, respectively. The aromatic dye Cibacron Blue 3GA coupled to Eudragit S 100 and polyethyleneimine way used for the affinity precipitation of some model enzymes such as lactate dehydrogenase and alcohol dehydrogenase. As prior removal of cell debris, etc., is essential for affinity precipitation, the possibility of integration of the technique with extraction in aqueous two-phase systems was also demonstrated.

Chemical Precipitation↗

PubMed2002

Fractional precipitation of plasmid DNA from lysate by CTAB.

Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA.

Cetrimonium↗

PubMed2005

Development of a novel adenovirus purification process utilizing selective precipitation of cellular DNA.

The use of recombinant adenoviral vectors for vaccination and gene therapy requires the development of purification processes that are cost-effective, scalable, and capable of robust host cell DNA clearance. An adenovirus purification process was developed which incorporates selective precipitation of host cell DNA, enabling a reduction in the use of costly nucleases and chromatographic resins while substantially improving DNA and protein clearance capabilities. In this work, three cationic detergents were evaluated for their capacity to selectively precipitate DNA from adenovirus-containing cell lysate. Parameters including pH, sodium chloride concentration, nonionic surfactant concentration, and cell density were investigated during development of the precipitation step. In a novel application, the cationic detergent domiphen bromide was found to have superior selectivity for host cell DNA. In addition, domiphen bromide-induced precipitation of adenovirus was shown to be reversible, which reduces the importance of mixing. Precipitation of DNA in the cell lysate coupled with primary clarification resulted in 3 logs of DNA clearance and improved impurity clearance in the subsequent ultrafiltration step. As a result, nuclease treatment and/or anion exchange chromatography can be eliminated, or included exclusively to improve process robustness.

Adenoviridae↗

PubMed2006

Kinetics and equilibria of lysozyme precipitation and crystallization in concentrated ammonium sulfate solutions.

The kinetics and thermodynamics of lysozyme precipitation in ammonium sulfate solutions at pH 4 and 8 and room temperature were studied. X-ray powder diffraction (XRD) was used to characterize the structure of lysozyme precipitates. It was found that, if sufficient time was allowed, microcrystals developed following an induction period after initial lysozyme precipitation, even up to ionic strengths of 8 m and at acidic pH, where lysozyme is refractory to crystallization in ammonium sulfate. The full set of precipitation and crystallization data allowed construction of a phase diagram of lysozyme, showing the ammonium sulfate dependence. It suggests that precipitation may reflect a frustrated metastable liquid-liquid phase separation, which would allow this process to be understood within the framework of the generic phase diagram for proteins. The results also demonstrate that XRD, more frequently used for characterizing inorganic and organic polycrystalline materials, is useful both in characterizing the presence of crystals in the dense phase and in verifying the crystal form of proteins.

Ammonium Sulfate↗

PubMed1983

In vitro method for detecting precipitation of parenteral formulations after injection.

Many injectable formulations currently on the market, including diazepam and alprazolam, utilize one or more cosolvents to solubilize the active constituents. On injections into an aqueous medium, some of these components tend to precipitate. A simple procedure is described for measuring the degree of precipitation that occurs when a solubilized drug is injected. This in vitro technique was used to show that alprazolam injection shows less precipitation than diazepam injection under all tested conditions, and that the precipitation observed with diazepam can be controlled by ensuring that the formulation is injected very slowly. This simple technique also can be used during preformulation development to evaluate the relative potential for precipitation of various formulations.

Alprazolam↗

PubMed2001

Inhibition of oligo(glutamine) precipitation by glutamine-containing peptides.

When a solution of glutamine is added to solid 1,1'-carbonyldiimidazole, a mixture of oligo(glutamine)s up to about the 11-mer is formed rapidly. On standing overnight, the solution deposits a precipitate that does not easily redissolve. We have studied the inhibition of this precipitation by glutamine-containing peptides. We find that the alternating peptides (arg.gln)(4) and (arg.gln)(8) are efficient inhibitors of precipitation while arg(5), (glu.gln)(4), and a nonalternating octapeptide of the same composition as (arg.gln)(4) do not inhibit precipitation even though all the arg-containing peptides readily adsorb to oligo(glutamine) precipitates. A possible structural basis for this difference is discussed.

Adsorption↗

PubMed2001

A new method for the isolation of histatins 1, 3, and 5 from parotid secretion using zinc precipitation.

Histatins, a family of small-molecular-weight, histidine-rich cationic salivary proteins, have been difficult to isolate in an efficient way by conventional procedures due to their anomalous interactions with chromatographic resins. In the present study we explored the possibility of developing a new isolation procedure based on recent observations that histatins associate with various metal ions, including zinc. Since solubility studies showed that histatin 5 forms precipitates with zinc under alkaline conditions, we investigated whether this characteristic could be exploited for the preparative isolation of histatins from salivary secretions. A fast and efficient two-step procedure was developed using zinc precipitation of histatins from human parotid secretion followed by final purification using reversed-phase high-performance liquid chromatography (HPLC). Analysis of zinc precipitates by Tricine-SDS-PAGE, cationic PAGE, HPLC, and mass spectrometry revealed the presence of the three major histatins, 1, 3, and 5, as well as statherin. The histatin yield obtained by the precipitation step was approximately 90%. Therefore, zinc precipitation of histatins from glandular salivary secretions is a novel, rapid, and effective means for the isolation of these proteins.

Chemical Precipitation↗

PubMed1977

The effect of iron, tin, aluminium, and chromium on fading, discoloration, and precipitation in berry and red beet juices.

The effect of iron(II), tin(II), aluminium(III), and chromium(III) on the properties of red whortleberry, blackcurrant, and red beet juices was followed during storage for 10 months at 5 degrees C. The colour and pH changes were studied, and the precipitates formed were weighed and their metal contents assayed. Of the metals tested, only tin caused bluish discoloration in the berry juices. In the case of iron, aluminium, and chromium the low pH prevented this type of discoloration. In berry juices, some increases in colour intensity took place with the lowering of pH value, whereas in red beet juice the opposite change occurred. The colour changes due to storage appear to take place irrespective of the metals. Precipitation is enhanced in red whortleberry juice only by tin, and in blackcurrant juice by tin and iron. In red beet juice, precipitation is increased by the lowering of pH resulting from metal salt addition. In general, increase in the amount of metal added increased the metal content of the precipitate. Iron showed the greatest tendency to become bound in the precipitate, particularly in blackcurrant and red beet juice. The suitabilities of the metals for canning purposes are considered. Chromium, in particular, has interesting possibilities in view of the low degree of colour change associated with it.

Aluminum↗

PubMed1985

In vitro precipitation of calcium phosphate under intracellular conditions: formation of brushite from an amorphous precursor in the absence of ATP.

Release of mitochondrial calcium has been shown to occur concomitant with mineral ion loading of matrix vesicles at the onset of mineralization in epiphyseal growth plate cartilage. Matrix vesicles contain amorphous calcium phosphate (ACP), a mineral form that usually results from rapid precipitation at high initial levels of Ca2+ and/or inorganic P (Pi). Since the cytosol of growth plate chondrocytes has been found to contain high levels of Pi, rapid release of mitochondrial Ca2+ into the cytosol may cause local precipitation of calcium phosphate and thus be coupled with matrix vesicle formation. Studies were carried out to determine the kinetics and nature of mineral formation that occur when small amounts of Ca2+ are added under various conditions to a Pi buffer composed of electrolytes matched in concentrations and pH to that of the cytosol of epiphyseal chondrocytes. Depending on the manner in which Ca2+ was added, ACP, dicalcium phosphate dihydrate (DCPD), or apatite (HA) first formed. In the presence of ATP, ACP was the only solid phase detected, being stable for at least 24 h. However, in its absence, ACP rapidly transformed into DCPD. Increasing the pH of the reaction buffer from 6.9 to 7.5 increased the amount of ACP initially formed, but DCPD was consistently found upon ACP transformation. Yet at pH 8.0, ACP persisted for at least 24 h. The amount of precipitate formed was proportional to the level of added Ca2+; precipitates formed when as little as 1.0 mmole was added per liter of buffer. Our findings support the possibility that rapid release of mitochondrial Ca2+ may cause localized intracellular precipitation of ACP. Since nascent ACP is known to stimulate membrane fusion and blebbing of vesicles, these findings may explain the presence of ACP in matrix vesicles. The rapid conversion of ACP to DCPD in the absence of ATP under these conditions may also explain the reported occurrence of DCPD in samples of early mineralizing tissue.

Adenosine Triphosphate↗

PubMed1987

Calcium phosphate precipitation in aqueous suspensions of phosphatidylserine-containing anionic liposomes.

Liposomes prepared from 6.3:1.8:0.9:1.0 molar mixtures of phosphatidylcholine, dicetyl phosphate, cholesterol, and phosphatidylserine, respectively, (PS(+) liposomes) were compared with similarly prepared liposomes without the phosphatidylserine (PS(-) liposomes) for their effect on calcium phosphate precipitate formation in aqueous solutions at pH 7.4 and 22 degrees C. The liposomes, encapsulated with 50 mM phosphate (PI), were suspended in buffered 2.2 mM CaCl2, 0 or 1.5 mM KH2PO4 solutions and made permeable to Ca2+ fluxes with the ionophore, X-537A. External solution Ca2+ losses were found to be small in both PS(+) and PS(-) liposome suspensions when no ionophore was added. Even with 1.5 mM PI in the external solution, these losses did not exceed 0.2 mM. However, inoculating both liposome preparations with X-537A resulted in rapid, appreciable losses in solution Ca2+. Previous studies showed that in PS(-) liposomes, these latter losses were due to calcium phosphate precipitation, with the precipitate confined to the interior of the liposomes when no external PI was present, but extending to outside the liposomes when the suspending medium was rendered metastable. In the present study, Ca2+ losses resulting from intraliposomally confined precipitation were found to be marginally greater in PS(+) liposomes due primarily to a larger volume of entrapped PI available for reaction in these liposomes. However, with the addition of PI to the external solution, the reverse was observed, i.e., considerably less Ca2+ was lost in PS(+) than in PS(-) suspensions, a result of markedly less X-537A-induced precipitate forming outside PS(+) liposomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium↗

PubMed1983

Affinity precipitation of dehydrogenases.

Affinity precipitation, a novel technique closely related to immunoprecipitation and affinity chromatography, has been evaluated in systems comprised of dehydrogenases and a bifunctional NAD derivative, Bis-NAD. Lactate dehydrogenase and glutamate dehydrogenase were easily precipitated whereas yeast alcohol dehydrogenase required the presence of salt to enhance the affinity precipitation. Liver alcohol dehydrogenase did not precipitate, probably because most of the affinity complexes formed were composed of only two enzyme molecules. Affinity precipitation was carried out on a preparative scale for the isolation of ox heart lactate dehydrogenase from a crude extract. The yield and purity of the enzyme and the general properties of the procedure are considered very satisfactory.

Affinity Labels↗

PubMed1987

The role of histidyl residues in zinc-induced precipitation of alpha 2-macroglobulin-proteinase complexes.

When alpha 2-macroglobulin (alpha 2M) is reacted with proteinases including trypsin, plasmin, alpha-thrombin, or with CH3NH2, each resulting alpha 2M derivative is precipitated by Zn2+ in a similar manner. By contrast, unreacted alpha 2M is not precipitated over the same Zn2+ concentration range. Zn2+-induced precipitation of alpha 2M-CH3NH2 or alpha 2M-trypsin is prevented by acylation of the protein employing the histidine-specific reagent diethylpyrocarbonate (DEP). The Zn2+-induced precipitation of alpha 2M-trypsin is prevented by acylation of the preformed alpha 2M-trypsin complex or by the reaction of acylated native alpha 2M with trypsin. Acylation of alpha 2M by treatment with DEP results in the modification of 13.5 histidyl residues per subunit of either native alpha 2M or alpha 2M-CH3NH2. Subsequent treatment with hydroxylamine reverses the modification of 10.5 histidyl residues per subunit in each protein preparation. These results indicate that histidyl residues are involved in the Zn2+-induced precipitation of alpha 2M-proteinase or alpha 2M-CH3NH2 complexes, and that these residues are accessible to extensive protein-metal interactions only after alpha 2M has undergone a major conformational change. These appear to be the same histidyl residues which, upon acylation by DEP, are responsible for recognition of alpha 2M-proteinase complexes by the acyl-low-density-lipoprotein cell surface receptor (S. V. Pizzo, P. A. Roche, S. R. Feldman, and S. L. Gonias (1986) Biochem. J. 238, 217-225).

Acylation↗