Saturated Ammonium Sulfate Precipitation Research Articles

Review on Desirable Microbial Phytases as a Poultry Feed Additive: Their Sources, Production, Enzymatic Evaluation, Market Size, and Regulation.

Poultry's digestive tract lacks hydrolytic phytase enzymes, which results in chelation of dietary minerals, vital amino acids, proteins, and carbohydrates, phytate-phosphate unavailability, and contamination of the environment due to phosphorus. Therefore, it is necessary to use exogenous microbial phytases as feed additive to chicken feed to catalyze the hydrolysis of dietary phytate. Potential sources of microbial isolates that produce desired phytases for chicken feed supplementation have been isolated from agricultural croplands. It is achievable to isolate phytase-producing bacteria isolates using both broth and agar phytase screening media. Potential substrates for submerged fermentation (SmF) for bacterial phytase production and solid-state fermentation (SSF) for fungal phytase production include rice and wheat bran. Following fermentation, saturated ammonium sulphate precipitation is typically used to partially purify microbial culture filtrate. The precipitate is then desalted. Measurements of the pH optimum and stability, temperature optimum and stability, metal ions stability, specificity and affinity to target substrate, proteolysis resistance, storage stability, and in vitro feed dephosphorylation are used to perform an enzymatic evaluation of phytase as an additive for poultry feed. The growth of the feed phytase market is primarily due to the expansion of chicken farms to meet the demand for meat and eggs from humans. The Food and Drug Administration in the USA and the European Food and Safety Authority are primarily in charge of putting rules pertaining to feed phytase use in chicken feed into effect. Conclusively, important components of the production of phytase additives for poultry feed include identifying a reliable source for potential microbe isolation, selecting an economical method of phytase production, thoroughly characterizing the biochemical properties of phytase, and comprehending the size and regulation of the current feed phytase market.

Cytotoxic activity of arginine deiminase purified from Lactobacillus sp

The goal of this research was to characterize ADI isolated from Lactobacillus sp. and test its anticancer activity in vitro. Because the arginine-degrading enzyme is a strong anticancer agent, it was isolated in two phases from a Lactobacillus sp. clinical isolate: 70% saturated ammonium sulphate precipitation followed by gel filtration chromatography (2x35cm). The most active fractions in enzyme activity (7.3 U/ ml), Purified enzyme had a special action activity of (48.6 U/mg) with nine folds of purgation and 61.4% enzyme recovery, The cytotoxic activity of purified arginine deiminase on PC3,A375 and WRL68 cells for 24 was examined. Purified arginine deiminase inhibited the proliferation of cancer cell lines PC3 and A375, with IC50 values of 68.64 g/ml and 136.3 g/ml, respectively. At a concentration of 6.25 ϻg/ml, 94.83% and 95.68% cell viability were observed of PC3 and A375 after treatment with purified arginine deiminase, respectively. However, cell viability reached to 42.52% and 48.77% using 400 ϻg/ml concentration purified arginine deiminase, respectively while ADI didn't show a significant effect on the viability of normal cell line.

Isolation and partial characterization of serum immunoglobuline in two species of sturgeons, Siberian sturgeon (Acipenser baerii) and Stellate sturgeon (Acipenser stellatus)

The immunoglobulins (Igs) were isolated and purified respectively from serum of Siberian sturgeon (Acipenser baerii) and Stellate sturgeon (Acipenser stellatus) by using 50% saturated ammonium sulfate precipitation. Analysis of the purified Ig of the two species of fishes on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions revealed two bands, heavy chains and light chains respectively. The molecular weight of the heavy chains of two fishes was about 70 kD and the molecular weight of the light chains was 27kD for Siberian sturgeon and 27.5 kD for Stellate sturgeon. The results suggested that the isolated and purified serum Immunoglobuline in two species of sturgeons, Siberian sturgeon (A. baerii) and Stellate sturgeon (A. stellatus) can be used to produce polyclonal and monoclonal antibodies against sturgeon Ig for developing diagnostic tests against a wide variety of pathogens.

Development of In-vitro Assays to Estimate Rabies Viral Protein in Vaccine Preparation

Vaccine against rabies is prepared by cell culture technology and these vaccines are free from many side effects when compared to nerve tissue vaccines. The vaccine production is a continuous process involving propagation of virus, harvesting, concentration, inactivation, purification and formulation with preservatives. The quantification of viral protein in the intermediate biological product is an in-process quality control test to reduce the product loss during various process of vaccine manufacturing. The conventional in-vivo & in-vitro tests employed for the quantification of rabies viral protein are time consuming, laborious and requires laboratory animals. In this study, we attempted to develop in-house serological methods such as sandwich ELISA, Dot Blot for the detection and quantification of rabies antigen in the intermediate biological material during vaccine preparation. The hyper immune sera was prepared by immunizing two animal models i.e. Guinea Pigs and rabbits with standard Rabies antigen. The sera samples were purified by saturated ammonium sulphate precipitation and further by G50 gel column. The anti-rabies antibody titre in the purified preparation was estimated using Rapid Fluorescent Focus Inhibition Test (RFFIT). National Reference Rabies Vaccine received from Central Drug Laboratory, Kasauli was used to prepare the local reference standard and it was included in the in-house serological methods to validate the assay. Our in-house tests are found to be simple, rapid and cost effective and require less time when compared to in-vivo animal challenge and cell culture based in-vivo tests.

Production and characterization of anti-Salmonella polyclonal antibodies as bio-recognition element for developing a microbial monitoring method

For the construction of the microbial monitoring method, anti-Salmonella polyclonal antibodies (pAbs) were produced from a rabbit and purified by saturated ammonium sulfate precipitation and protein A affinity column. The reactivity of anti-Salmonella pAbs was compared to that of commercial ones by using an indirect ELISA. The specificity of anti-Salmonella pAbs was investigated using 20 Salmonella serotypes and 20 non-Salmonella strains. A capturing ability of anti-Salmonella pAbs was investigated by exposing antibody-immobilized gold biosensor to different concentration of Salmonella mixture. Anti-Salmonella pAbs were successfully produced and purified with an antibody concentration of 2.0 mg/mL The reactivity of purified anti-Salmonella pAbs was greater than that of commercial one at all tested concentrations. All Salmonella serotypes, except S. Diarizonae, showed excellent binding efficiency with purified anti-Salmonella pAbs. Moreover, the purified anti-Salmonella pAbs showed excellent specificity against all non-Salmonella strains. The anti-Salmonella pAbs immobilized on the gold biosensor demonstrated the successful capturing capability against Salmonella with a dose-response manner. Therefore, the anti-Salmonella pAbs exhibited sufficient reactivity, specificity, as well as capturing capability against Salmonella to be considered as a bio-recognition element.

Production of Erythropoietin-Specific Polyclonal Antibodies.

Background Erythropoietin, as a principal hormone promotes red blood cell production in bone marrow. Varieties of erythropoietin biosimilar are being produced by recombinant DNA technology in cell cultures. The detection or quantifi cation of these molecules are being performed by diff erent methods which some of theme such as Western blot and enzymelinked immunosorbent assay (ELISA) require specifi c antibodies. High cost, inappropriate shipping (cold chain failures), reduced sensitivity and thus poor detection performance are common pitfalls of using commercial kits for performing immunological tests. Objectives To produce in-house polyclonal antibody against active pharmaceutical ingredient (API) of recombinant human erythropoietin (rh-EPO) was the aim of this study. Materials and Methods Two healthy female albino rabbits were injected four times in 14 days interval using rh-EPO API as antigen. The produced antibody was separated from plasma via either caprylic acid or saturated ammonium sulfate precipitation and the results were compared from each purification methodologies. The antibody was further purified by ion exchange chromatography. Acceptable purity and good immunogenicity were detected respectively by SDS-PAGE and western blot analysis. The purified antibody was compared with a commercial kit to determine rh-EPO concentration in diff erent steps of production batches via ELISA. Results The purity of antibodies after ion exchange chromatography, obtained from caprylic acid and ammonium sulfate precipitation were 97 and 80%, respectively. Conclusions As producing in house kits is one of the important challenges of bio- pharmaceutical manufacturers, a simple, cost- and time-effective, and easy to scale up strategy for making in-house polyclonal antibody was set up. Caprylic acid precipitation resulted higher purity than ammonium sulfate and fi nally purified antibody (97% purity) used as a capture antibody in sandwich ELISA test was able to detect erythropoietin antigen as sensitive (100%) and specifi c (100%) as commercial kits.

Preparation of monoclonal antibody of anti-feline calicivirus and establishment of double-antibody sandwich enzyme-linked immunosorbent assay detecting method.

This study aimed to prepare monoclonal antibody of feline calicivirus (FCV) and identify its basic biological characteristics. Saturated ammonium sulfate precipitation, combined differential centrifugation, and cesium chloride density gradient centrifugation were used for the purification of FCV. The purified FCV was used as antigen to immunize BALB/c mice. The hybridoma lines of anti-FCV monoclonal antibodies were established using cell fusion and hybridoma screening techniques. The subtypes of the monoclonal antibody were identified. The results showed that 3 strains of hybridoma cell lines stably secreted anti-FCV monoclonal antibody; they were named as D8, E5, and H4. The D8 and E5 were IgM subtype antibodies, and H4 was IgG2b subtype antibody. The monoclonal antibody obtained shared no cross-reactivity with feline parvovirus, canine parvovirus, and canine distemper virus. According to the different recognition sites of 2 monoclonal antibodies E5 and H4 to the FCV, they were used to coat microtiter plates and prepare 2 enzyme-labeled secondary antibodies to establish double-antibody sandwich enzyme-linked immunosorbent assay detecting method.

Production, Partial Purification and Characterization of an Extracellular Psychrotrophic Lipase from Pseudomonas Sp. ADT3

Psychrotrophic Pseudomonas ADT3 (NCBI GenBank Acc.no.JX914667) is capable of growth on lipid as the sole carbon source. In this paper, we report the purification and characterization of an extracellular lipase from psychrotroph, isolated from soil sample of Ny- Alesund, Svalbard, Arctic region. The Pseudomonas ADT3 isolate produces lipase enzyme in the extracellular minimal media with only 1% olive oil. The lipase was purified from the concentrated culture supernatant. The crude enzyme was partially purified by saturated ammonium sulphate precipitation followed by extensive dialysis. Enzyme activity was found to be induced 6-folds in presence of 1.2 mM lead ion but strongly inhibited by heavy metals Hg2+ as well as EDTA and β-mercaptoethanol. The purified lipase has activity at two pH optima of pH i.e. pH 3.5 and 8.5. Optimum temperature for lipase activity was recorded at 22cC. The purified active fraction of lipase exhibits specific activity of 527.8 U/mg. The Vmax and Km was 144.93 U/mg/min and 0.260 mM respectively determined using Lineweaver-Burk plot. Zymogram analysis revealed prominent lipase band at 13.9 kDa range in the 80% saturated ammonium sulphate purified enzyme fraction.

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

BackgroundSilkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.Methodology/Principal FindingsSilkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.Conclusions/SignificanceThe study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.

Characterization and biological activities of lectin isolated from <i>Pinellia ternata</i>

In this paper, Pinellia ternata lectin (PTL) purified from 95% saturated ammonium sulfate precipitation by applying mannose-Sepharose 4B affinity column chromatography was first described. The mannose-Sepharose 4B affinity adsorption gel has already been designed and prepared before the experiments according the combinative characterization between lectin and mannose. Mass spectrometry analysis result shows that PTL is a glycoprotein with the molecular weight of 12.165 kD. It was conclude that PTL had strong agglutination effects on mouse red blood cells, and the minimum reaction concentration was 25 μg/ml according the Hemagglutination. Results of automatic amino acid analysis indicated that PTL mainly contained 15 varieties of amino acids, of which the minimum content was cysteine and aspartate was the maximum. Cell experiment results suggested that PTL of low concentrations (0.004 mg/ml, 0.02 mg/ml and 0.1 mg/ml) promoted HeLa cell proliferation, but the effect weakened with the concentrations and treated-time increasing. However, the HeLa cells pro- liferation was intensively inhibited by higher PTL concentrations (0.5 mg/ml and 1 mg/ml), and the effect increased in a dose and time dependent manner.

Preliminary X-ray data analysis of crystalline hibiscus chlorotic ringspot virus

Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense monopartite single-stranded RNA virus that belongs to the Carmovirus genus of the Tombusviridae family, which includes carnation mottle virus (CarMV). The HCRSV virion has a 30 nm diameter icosahedral capsid with T = 3 quasi-symmetry containing 180 copies of a 38 kDa coat protein (CP) and encapsidates a full-length 3.9 kb genomic RNA. Authentic virus was harvested from infected host kenaf leaves and was purified by saturated ammonium sulfate precipitation, sucrose density-gradient centrifugation and anion-exchange chromatography. Virus crystals were grown in multiple conditions; one of the crystals diffracted to 3.2 A resolution and allowed the collection of a partial data set. The crystal belonged to space group R32, with unit-cell parameters a = b = 336.4, c = 798.5 A. Packing considerations and rotation-function analysis determined that there were three particles per unit cell, all of which have the same orientation and fixed positions, and resulted in tenfold noncrystallography symmetry for real-space averaging. The crystals used for the structure determination of southern bean mosaic virus (SBMV) have nearly identical characteristics. Together, these findings will greatly aid the high-resolution structure determination of HCRSV.

Isolation and characterization of a protective bacterial culture isolated from honey active against American Foulbrood disease

Bacterial strains isolated from US domestic honey were screened for antibacterial activity against Paenibacillus larvae ssp. larvae, the causative agent of American Foulbrood (AFB) in apiaries. A bacterial isolate (TH13) showing a high level of antimicrobial activity against P. larvae ssp. larvae ATCC 9545 was selected and identified as Paenibacillus polymyxa by 16S rRNA gene sequencing. The antimicrobial compound was purified by 80% saturated ammonium sulfate precipitation followed by CM-sepharose chromatography and reverse-phase HPLC. The molecular mass of the compound was determined to be 1168.78 Da by ESI-qTOF MS, matching that of polymyxin E1. The producer strain showed a broad range of antibacterial activity against gram-positive and -negative bacteria including P. larvae ssp. larvae ATCC 25747 and foodborne pathogens such as Bacillus cereus F4552 and Escherichia coli O157:H7 ATCC 43895. The selection of antibiotic-producing bacterial strains indigenous to honey as protective cultures against AFB may lessen the use of antibiotics in apiaries.

Proteomic analysis of Helicobacter pylori cellular proteins fractionated by ammonium sulfate precipitation

Among 1590 ORFs in the Helicobacter pylori genome, >250 have been identified as authentic genes by proteomic analysis. Low-abundance proteins need to be enriched to a minimal amount for MALDI-TOF analysis and salt precipitation has generally been used for protein enrichment. Here, a whole-cell extract of H. pylori strain 26695 was subjected to protein fractionation with stepwise concentrations of ammonium sulfate and the proteins were displayed by 2-DE. The protein spots were quantified using PDQUEST software and identified by peptide fingerprinting. The 2-DE profiles and intensities of individual protein spots differed among the protein fractions. Out of the 98 identified proteins, 61 were found in the stepwise ammonium sulfate fractions but not in the whole-cell extract. Out of these, 37 proteins, including KdsA, were found exclusively in a single fraction. In contrast, GroEL, UreA, UreB, TrxA, NapA, and FldA were ubiquitously present in all fractions. Iron-containing proteins such as NapA, SodB, CeuE, and Pfr were found predominantly in the 100% saturated ammonium sulfate precipitate. Additionally, 29 proteins were newly identified in this study. These data will facilitate the preparation of significant H. pylori proteins, as well as provide information about low-abundance proteins.

Construction and evaluation of anti-gastrin immunogen based on P64K protein

To construct two kinds of anti-gastrin immunogen based on P64K protein from Neisseria meningitids and to compare their immunogenic effect. G17P64K gene was cloned and ligated into pET28a plasmid, then transformed into BL21(DE3). After inoculation of LB medium and IPTG induction, the recombinant protein was solubly expressed at a high level. The purification of G17P64K fusion protein was similar to that of P64K. An initial step of purification consisting of 30% saturated ammonium sulfate precipitation was done. Additional fine optimizations included phenyl-sepharose, G200 Sephadex gel filtration and Q-sepharose anion exchanger chromatography. Highly purified protein was obtained and sequenced at the N-terminal amino acid residues. Polypeptide was synthesized by Fmoc solid phase chemical method and cross-linked to carrier protein P64K and DT mutant by MBS method and then the rabbit anti-gastrin 17 antibody was prepared by immunizing rabbit with cross-linked and fused protein. The titer and the activity in vitro of antibody were assessed. G17P64K gene and the recombinant bacteria were obtained. After four steps purification, protein sample that has the purity above 90% was achieved. At the 84(th) day after the first immunization, the titer of antibody against cross-linked protein reached 51,200. Evaluation of the antibody in vitro manifested that it had a high inhibitory activity on the growth of tumor cell SW480. The P64K-polypeptide cross-linked immunogen immunized rabbit and achieved a higher titer antibody against gastrin 17 than the G17P64K fusion protein immunogen, which could inhibit the growth of the tumor cell SW480.

Purification and characterization of a chitinase from Amycolatopsis orientalis with N-acetyllactosamine-repeating unit releasing activity

We report a novel enzyme from the culture filtrate of Amycolatopsis orientalis, that endoglycosidically releases an N-acetyllactosamine-repeating unit (Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAc, LN 2) from a synthetic chromogenic substrate Galβ1,4GlcNAcβ1,3Galβ1,4GlcNAcβ- pNP ( 1). The enzyme activity was purified by 80% saturated ammonium sulfate precipitation followed by gel filtration and affinity chromatography. The enzyme splits 1, Galβ1,4GlcNAcβ- pNP ( 2), GlcNAcβ1,3Galβ1,4GlcNAcβ- pNP ( 3), and GlcNAcβ1,4GlcNAcβ- pNP ( 4) into the corresponding oligosaccharides and p-nitrophenol. The catalytic efficiencies ( k cat/ K m) for compounds 1, 2, and 4 were 0.6, 0.05, and 13, respectively. Compound 4 acts as a fairly good substrate for the enzyme, and LN 2-releasing activity was inhibited by 4 and GlcNAcβ1,4GlcNAcβ1,4GlcNAcβ- pNP ( 7), indicating that this enzyme activity is derived from a kind of chitinase. The enzyme hydrolyzed 1 by a mechanism leading to retention of the anomeric configuration. This is the first report of a N-acetyllactosamine-repeating unit releasing enzyme.

Comparison of Rapid Physical Stability Tests

Our goal was to find a one-day physical stability test that would give an acceptable correlation with long-term chill haze. Over the course of several months, beers were tested from a number of breweries. The rapid methods examined were sensitive proteins, tannoids, saturated ammonium sulfate precipitation limit, alcohol chill, and simple flavanols tests. The Schneider PT-standard instrument was also tested. Results were compared with chill haze produced by long-term storage at 75°F (23.9°C). Only the alcohol chill test gave an acceptable correlation. The alcohol chill results from the Pfeuffer tannometer gave results that were not significantly different from those obtained when the test was run manually.

A biologically active lectin of enteroaggregative Escherichia coli

The pathogenesis of enteroaggregative Escherichia coli, a major contributor to paediatric diarrhoea, is still not clearly understood. A complex carbohydrate specific lectin was identified from the culture supernatant of an enteroaggregative E. coli strain. The lectin was purified to 660-fold by a combination of sequential saturated ammonium sulphate precipitation and gel filtration chromatography in the FPLC system. The homogeneity of the purified lectin was established by analytical isoelectrofocusing [pI 6.75]. Hemagglutination of rabbit erythrocytes by the purified lectin was best inhibited by fetuin. The N-terminal sequence of the 41.7 kDa subunit showed homology to the outermembrane porins and the 23.4 kDa subunit showed homology to a hypothetical protein of Yersinia pestis and secreted Hcp protein. This protein could induce extensive morphological changes in HEp-2 cells and significant amount of fluid accumulation in rabbit ileal loop. GM1 showed maximum binding to the lectin among all other gangliosides. This purified protein showed cross-reactivity to the binding subunit of cholera toxin in western immunoblot. The presence of this toxin in some of the clinical isolates of enteroaggregative E. coli was also observed. The structural and functional characteristics of the toxin revealed that it is a novel virulence determinant of aggregative E. coli.

Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography

A quantitative comparison was made on the fractionation of pepsin-digested horse antivenoms by ammonium sulfate (AS) fractional precipitation and ion-exchange chromatography on Q-Sepharose. In the precipitation process, pepsin digested horse anti- Naja kaouthia serum was precipitated by 30% saturated AS followed by 50% saturated AS. The recovery of antibody activity [as measured by an enzyme-linked immunosorbent assay (ELISA) against the cobra postsynaptic neurotoxin 3] from the 30–50% saturated AS precipitate was 53% with a 1.93-fold purification. For the chromatographic process, the behavior of the horse antitoxin antibody and its F(ab′) 2 fragments was first studied. The pepsin digested horse serum was then desalted on a Bio-gel P-2 column followed by chromatography on Q-Sepharose using a linear gradient (20 m M Tris-HCl, pH 8.0 containing 0.0 to 0.5 M NaCl). A peak containing primarily the F(ab′) 2 antibody could be obtained. This peak constituted 73% of the total antivenom activity with 2.08-fold purification. The total recovery of antibody activity by the chromatographic process was 90%. The yield of antibody activity was about 2-fold higher than that reported previously with other fractionation procedures. The implications of these results for the refining of horse therapeutic antivenoms are discussed.

Zymogen Activation in Pancreatic Endoproteolytic Preparations and Influence on Some Whey Protein Hydrolysate Characteristics

ABSTRACTA proteolytic preparation from porcine pancreas was isolated. Trypsin, chymotrypsin and elastase were characterized and their time‐dependent stability at 37°C was studied. The supematant of a 30% (w/v) saturated ammonium sulfate precipitation of a pancreatic extract (30S) was developed to pilot‐scale level. The influence of zymogen activation time on molecular characteristics of whey protein hydrolysates produced by 30S and the commercial pancreatic preparation Corolase PP were compared. Amino acid analysis and gel permeation chromatography were used to characterize lactalbumin hydrolysates produced. Physicochemical characteristics of whey protein hydrolysates could be altered by manipulation of zymogen activation conditions in pancreatic proteinase preparations to be used during subsequent protein hydrolysis.

Detection and partial purification of ischaemia-related neurotrophic activity in the periinfarcted brain tissue.

In the rat model of middle cerebral artery (MCA) occlusion, axons originating from the ipsilateral cortical and thalamic neurons are injured by ischaemia. The cortical neurons survive thereafter without retrograde degeneration, but thalamic neurons slowly die because of retrograde degeneration. The fate of these two neurons is remarkably different and may be related to neurotrophic activity induced by ischaemia. We detected ischaemia-related neurotrophic activity, and partially purified the factor. Tissue samples were obtained from the cortex adjacent to the infarction and contralateral corresponding site at 4, 8 and 12 days after occlusion of the MCA. They were homogenated with a culture medium and ultracentrifuged. The supernatant was obtained and used for neurotrophic assay. Foetal cortical neurons were obtained from 17 days rat embryo and cultured. Neurotrophic activity was assayed by applying tissue extract to the culture medium. Application of periischaemic cortical extract obtained at 8 and 12 days after ischaemia improved neuronal survival by 50% and 200% as compared to contralateral cortical extract, respectively. The activity was not detectable at 4 days after ischaemia. The neurotrophic activity disappeared by heating the extract at 90 degrees C for 10 min. We fractionated the extract by saturated ammonium sulphate precipitation, followed by gel-filtered with Superose 12 column. The neurotrophic activity was detected in the precipitation of 30 to 60% saturation fraction of ammonium sulphate. With gel-filtration we separated neurotrophic activity in several fractions, which included marker proteins of 8, 22 and 30 kilodaltons. The activities were only detected in the lesioned side but not in the contralateral side.(ABSTRACT TRUNCATED AT 250 WORDS)