Gel Permeation Column Research Articles

Identification of an endothelial-like type III NO synthase in LLC-PK1 kidney epithelial cells.

Porcine kidney tubular epithelial cells (LLC-PK1) produce nitric oxide or a related compound (e.g., a nitrosothiol) after stimulation with various agonists. We now report the identification and characterization of a constitutive, particulate nitric oxide (NO) synthase from LLC-PK1 cells. After partial purification on adenosine 2',5'-bisphosphate-Sepharose, the particulate NO synthase activity eluted anomalously from Superose 6 gel permeation columns near the total included volume, similar to that observed for the endothelial (type III) NO synthase. Substrate/cofactor requirements of the epithelial and endothelial NO synthases were identical, i.e., dependency on L-arginine, (6R)-5,6,7,8-tetrahydrobiopterin, FAD, calcium and calmodulin. The epithelial enzyme activity was inhibited by the arginine analogues, NG-methyl-L-arginine (100 microM) and NG-nitro-L-arginine (100 microM), as well as the calmodulin antagonists, trifluoperazine (100 microM) and calmidazolium (30 microM). Anti-type III (H32), but not anti-type I (brain, 6763-5) or anti-type II (macrophage, 8196) NO synthase antibodies, detected a single immunoreactive band in the LLC-PK1 particulate fraction of approximately 140 kDa by Western blot analysis. Finally, the presence of type III NO synthase mRNA in LLC-PK1 cells was demonstrated using the polymerase chain reaction. These data indicate that LLC-PK1 kidney epithelial cells contain type III NO synthase, which has been classically associated with the vascular endothelium.

COMPOSITIONAL ANALYSIS OF A VACUUM RESIDUE

ABSTRACT A sample of vacuum residue (VR) was evaluated for its physical and chemical properties. The VR was separated into sixteen fractions according to molecular size distribution on preparative gel permeation chromatography (GPC) columns. Elemental analysis including heteroatoms (N, S) and metal (Ni, V) was carried out on fractions 1 through 9. Molecular weight distribution and number average molecular weight (M¯n) were obtained by analytical GPC for fractions 1 through 11. Forty percent of the VR contains high molecular weight materials (i.e., materials with molecular weights ranging from 1170 to 3920) and 64.3% total vanadium, 62.5% total nickel, 34.1% total sulphur, and 41.6% total nitrogen.

Inversion of configuration around the seven-coordinated cobalt center induced by an interaction between sugars and tetrahedral oxoanions

Carbohydrates have a widespread Occurrence in nature and are indispensable compounds to living organisms.' Naturally rare carbohydrates have also proved to be involved in many glycoproteins, glycolipids, and antibiotics and play important roles in various biological processesS2 Further, in some enzymatic reactions of carbohydrates, alkali, alkaline earth, and some of transition metal ions were suggested to act in cooperation with sugars.3 In this regard, it is very important to investigate the interactions between sugars and metal ions including the stereochemical behavior of sugars in transition metal complexes. We have studied the synthesis and characterization of nickel(I1) and cobalt(II1) complexes containing N-glycosides derived from the reaction of sugars and diamine^.^ Here, we wish to report a synthesis of novel seven-coordinated cage-type cobalt complexes containing N-glycosides from mannose-type aldoses (D-mannose or L-rhamnose (=6-deoxy-~-mannose)) and tris(Zaminoethy1)amine (tren) and an unprecedented inversion of configuration around the metal center induced by an interaction between sugars and tetrahedral oxoanions. A methanolic solution containing D-mannose (D-Man) or L-rhamnose (L-Rha) (4 equiv) and tren (1 equiv) was incubated at 60 OC for 1 h. Then, CoXz.6HzO (X = C1-, B r ) (1 equiv) was added to the solution and the mixture was refluxed for 1 h. The resultant solution was purified on a Sephadex LH-20 gel permeation column. The wine-red major band was collected and concentrated, and an addition of ethanol gave microcrystals of [Co((aldose)3-tren)]Xz.nHzO (la, aldose = D-Man, X = C1-; lb, aldose = L-Rha, X = C1-; 2a, aldose = D-Man, X = B r ; 2b, aldose = L-Rha, X = B r ) , where (aldose)3-tren is tris(N-aldosyl2-aminoeth~l)amine.~ When CoSOcnHzO was used as a metal source, similar complexes formulated as [C~((aldose)~-tren)]S04.nH20 (3a, aldose = D-Man; 3b, aldose = L-Rha) were ~ b t a i n e d . ~ The electronic absorption (AB) spectra of 1-3 in solution showed a characteristic band around 20 X lo3 cm-I, corresponding to d-d transitions of Co(I1) ions. The transmittance spectra (Nujol mull) also showed a similar absorption indicating no significant structural change around the metal on dissolution. In the circular dichroism (CD) spectra, a large Cotton effect was observed at about 20 X 103 cm-l, suggesting the coordination of sugar moieties to the cobalt ion. Further, it should be noted that the sign of the Cotton effect of 1 and 2 containing halide

Enrichment of germanium-73 with the magnetic isotope effect

-(0.1 i 1.0) +( 12.3 2.0) O b(zGe) = ((ZGe/74Ge)/(ZGe/74Ge)0) - 1. 2) X le3 and 8.0 X le2 mol dm-3, respectively. The irradiation of the sample was carried out with a 500-W deep UV lamp. The details of the sample preparation and photolysis are described in the previous paper.7 For germanium isotope analysis after photolysis, the reaction products and starting compound (Ph3- MeGe) should be isolated from the micellar solution, but this isolation was very difficult. We isolated the products and starting compound by using a JAI-LC908 recycle high-performance liquid chromatograph (HPLC) with a gel permeation chromatography (GPC) column. The isotope ratios were measured using a VG- ELEMENT PQ-s2 inductively coupled plasma mass spectrometer (ICP-MS). Although the relative standard deviation (RSD) of the observed isotope ratio was <0.3% in the specifications of the commercially available ICP-MS, it was not so easy to realize this RSD value.

Comparison of Gel Permeation Chromatography, Sweep Codistillation, and Florisil Column Adsorption Chromatography as Sample Cleanup Techniques for the Determination of Organochlorine Pesticide Residues in Animal Fats

Methods using a commercial sweep codistillation apparatus, gel permeation chromatography (GPC), and Florisil column adsorption chromatography were compared as cleanup techniques for the determination of organochlorine pesticide residues in animal fats by Megabore capillary gas chromatography with electron capture detection. Animal fat that had been previously found to contain no detectable organochlorine residues was spiked with 17 organochlorine pesticides at levels ranging from 0.1 to 0.4 mg/kg and cleaned up by each of the 3 techniques. Recoveries obtained for all 3 methods were in the range 73-113%, with coefficients of variation between 1.1 and 11.2%. Equivalence of method performance was further demonstrated by performing replicate analyses of beef and sheep fat containing naturally incurred residues of heptachlor epoxide, dieldrin, p,p'-DDE, p,p'-DDD and p,p'-DDT. All 3 methods offer effective cleanup and acceptable recovery of organochlorine pesticides in animal fat. The sweep codistillation method has the advantages of low solvent and reagent use, simultaneous cleanup of 10 samples, and rapid turnaround, although thermal degradation of p,p'-DDT requires monitoring and control. GPC offers a high degree of automation but is a relatively slow sequential sample cleanup with high solvent use. Florisil column adsorption chromatography is a simple, proven technique but requires considerable solvent and reagent and has a low potential for automation.

Tubulin binding of two 1-deaza-7,8-dihydropteridines with different biological properties: Enantiomers NSC 613862 (S)-(-) and NSC 613863 (R)-(+)

Several fluorescence properties of two enantiomers, NSC 613862 (S)-(-) and NSC 613863 (R)-(+), have been compared. Even though the two isomers showed the same fluorescence behavior in solution in different solvents, drastic differences were observed after binding to purified calf brain tubulin. Binding measurements for the two compounds were performed both by fluorescence spectroscopy and by column gel permeation, a direct method of measurement. For both isomers, the binding was characterized by the presence of one high-affinity binding site with an apparent association constant of (3.2 +/- 0.5) x 10(6) M-1 and (4.1 +/- 0.9) x 10(6) M-1 for the R- and S-isomer, respectively, and by several low-affinity sites. Both isomers were also shown to induce GTPase activity in tubulin. The high-affinity binding site seems to be the same for the two isomers. Moreover, fluorescence competition experiments suggest at least a partial overlap of the colchicine and podophyllotoxin site. To explain the differences in fluorescence behavior after binding to tubulin, we hypothesize that the R-isomer is positioned differently in its binding locus as compared with the S-isomer.

Purification and Characterization of NAD Glycohydrolase from Rabbit Erythrocytes

The NAD glycohydrolase (NADase) was solubilized from intact erythrocytes with bacterial phosphatidylinositol-specific phospholipase C and purified to homogeneity by affinity chromatography on Cibacron blue-agarose. This purification procedure resulted in an ∼85-fold purification with an overall recovery of 75%. The purified NADase has a molecular weight of 65,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 63,000 as determined by gel permeation column chromatography at pH 7.0. Two hybridoma cell lines secreting antibodies against NADase were established and the antibodies recognized the purified enzyme as well as a 65-kDa band from the extracts of rabbit erythrocyte ghost. The enzyme displayed a K m of 43 μM for β-NAD, a V max of 23 μmol/min/mg, a broad pH optimum around pH 7.0, and p I of 5.0. Nicotinamide and isoniazid are inhibitors ( K i values, 2.5 and 3.5 mM, respectively) of the noncompetitive type. Adenosine diphosphoribose acts as a competitive inhibitor ( K i = 2.0 mM). Cibacron blue 3GA is a potent competitive inhibitor of NADase ( K i = 96 nM). The purified enzyme splits β-NAD, NADP, and nicotinamide hypoxanthine dinucleotide among the compounds tested and does not exhibit transglycosidase activity. Amino acid composition of the rabbit erythrocyte enzyme differed from that of NADases of other species, and the purified NADase contains 8% carbohydrate and a stoichiometric amount of inositol.

Formation of a cleavasome: enhancer DNA-2 stabilizes an active conformation of NaeI dimer.

Cleavage of DNA by NaeI-type restriction enzymes is stimulated by a DNA element with affinity for the activator site of the enzyme: a cleavage-enhancer DNA element. Measurements of the mobility of NaeI activity in comparison with protein standards on gel permeation columns and glycerol gradients demonstrated that NaeI, without enhancer, can form a 70,000 MW dimer. The dimer, however, is inactive: it could not cleave the "resistant" NaeI site in M13mp18 DNA in the absence of enhancer. In cleavage assays, enhancer stimulated either DNA nicking or DNA cleavage, depending upon NaeI concentration, and reduced the NaeI concentration required for the transition from nicking to cleavage activity. A gel mobility-shift assay of the interaction of NaeI with enhancer showed the formation of two complexes. Results using different sized DNAs and different percentage acrylamide gels for gel mobility-shift analysis implied that the two complexes were caused by NaeI monomer and dimer structures rather than one and two DNA binding. Dimer formation increased with the affinity of enhancer for NaeI. UV cross-linking "captured" the NaeI-enhancer complex; electrophoretic analysis of the cross-linked products showed NaeI dimer bound to enhancer. These results imply a model for cleavage enhancement in which enhancer binding stabilizes an active NaeI dimer conformation ("cleavasome") that cleaves both DNA strands before dissociating.

Regional Differences in Sulfated Oligosaccharides of Rat Gastrointestinal Mucin as Detected by Two-Dimensional Chromatography

Radiolabeled sulfated mucin was obtained from rat gastrointestinal tract tissue (corpus, antrum, duodenum, jejunum, ileum, or colon) incubated with [35S]sulfate in vitro by fractionation of extracts on Superose 6 followed by purification with CsCl density gradient centrifugation. Significant amounts of 35S-labeled mucin were obtained from each tissue and radioactivity differed according to region, in the order colon > duodenum > jejunum > corpus > antrum > ileum. Gel-permeation chromatography of 35S-labeled oligosaccharides liberated from each mucin by alkaline-borohydrate treatment indicated the 35S-labeled oligosaccharides of each mucin differ with respect to elution position. Further examination of fractions eluted from the gel-permeation column by thin-layer chromatography showed region-specific chromatograms for the sulfated oligosaccharides. Thus, not only the degree of sulfate incorporation but sulfated oligosaccharide structure differs according to the region in gastrointestinal mucin.

Germ cell-conditioned medium contains multiple factors that modulate the secretion of testins, clusterin, and transferrin by Sertoli cells.

In view of the evidence showing that germ cells regulate Sertoli cell (SC) function, the aim of this study was to examine if germ cell (GC)-conditioned media contained multiple biological factors that affect SC secretory functions. Total GC were isolated from adult rat testes. Pachytene spermatocytes (SPC) and early spermatids (SPT) were enriched to about 90% pure by centrifugal elutriation. GC, SPC, and SPT were cultured in serum-free medium for 20 hours with a viability greater than 95%. Conditioned media derived from these cells were fractionated by anion-exchange high-performance liquid chromatography (HPLC). An aliquot from each of these fractions was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and proteins were visualized by silver staining. The patterns of protein staining using media from GC, SPC, and SPT were similar. Bioassays of these column fractions on SC showed that the transferrin stimulatory, the testins inhibitory, and the clusterin inhibitory activities were eluted from the anion-exchange HPLC column in overlapping fractions. To determine whether these activities were confined to one or several molecules, further fractionations were performed. Eleven liters of GC-conditioned medium were fractionated by sequential HPLC using anion-exchange, gel permeation, and reversed-phase columns. Throughout the entire fractionation scheme, the HPLC fractions were bioassayed using primary SC-enriched cultures prepared from 20-day-old rats by incubating SC with aliquots of these fractions for 24 hours to monitor their effects on SC secretory function. The concentrations of transferrin, clusterin, and testins were quantified by specific radioimmunoassays. These studies showed that the transferrin stimulatory activity can be fractionated into four peaks (I, IIa, IIb, and IIc); clusterin inhibitory activity into three peaks (A, B, and C); and testins inhibitory activity into two peaks (1 and 2). Some of these bioactivities were eluted in overlapping fractions such as I and B, IIb and 1, and IIc and 2, whereas A, C, and IIa were not associated with any other assayed activities. In summary, additional GC modulators of SC function were identified for the first time, these include clusterin and testins inhibitors. The previously identified transferrin stimulatory activity was also resolved into multiple molecular forms.

Automated high-performance liquid chromatographic determination of hydroxylysylpyridinoline and lysylpyridinoline in urine using a column-switching method.

An on-line urine clean-up system was developed for the simultaneous determination of free and total pyridinoline, hydroxylysyl-pyridinoline (HP) and lysylpyridinoline (LP) by high-performance liquid chromatography (HPLC) using a column-switching technique. The method is based on a combination of gel permeation chromatography (GPC) and ion-pair reversed-phase HPLC. In the GPC column, pyridinoline is preseparated from endogenous urinary substances with 0.03 M heptafluorobutyric acid (HFBA) as the mobile phase. After column switching, the eluate fraction containing pyridinoline is further separated by ion-pair chromatography using an octadecylsilica (ODS) column with 0.03 M HFBA-acetonitrile (81:19) as the mobile phase. The detection limits were 36 and 44 pmol/ml for free and total HP, respectively, and 44 pmol/ml for both free and total LP at a signal-to-noise ratio of 3. The coefficients of variation for free and total pyridinoline were 1.5 and 3.5%, respectively. The determination of one sample including the clean-up is completed within 25 min. This system is precise and is useful for the determination of pyridinoline in large amounts of urine. The usefulness of pyridinoline as a biomedical marker for bone resorption was also examined.

A Multi-Dimensional Liquid Chromatography Method for Determination of Androgen Hormone Residues in Cattle Liver

Abstract A multidimensional HPLC method is described for the determination of trenbolone and 19-nortestosterone steroid hormone residues in cattle liver. Sample extracts are defatted by size exclusion chromatography. The evaporated residue is injected onto a high performance gel permeation column and the two fractions containing the steroids are diverted onto individual silica concentration columns, where the hormone residues are retained. The trapped hormones are then determined separately. Trenbolone held on one concentration column is eluted onto a cyano HPLC column and the steroid peak from this is transferred onto a final analytical silica HPLC column. After re-equilibration of the system the second concentration column is treated similarly for determination of 19-nortestosterone. Detection is by UV at 340 nm for trenbolone and 247 nm for 19-nortestosterone. Detection limits in cattle liver are 0.1 μg/kg for trenbolone and 0.3 μg/kg for 19-nortestosterone. For liver spiked at 2 μg/kg. recoveries of 59% and 39%, with CVs of 7% and 10% were obtained for trenbolone and 19-nortestosterone respectively. The low overall recovery for 19-nortestosterone was primarily attributable to losses during extraction.

Evaluation of gel permeation chromatographic techniques and diode-array UV detection for the characterization of biotechnological fermentation substrates and broths

Gel permeation chromatography (GPC) was used for the analysis and characterization of fermentation substrates and broths. Chemical or enzymatic hydrolysis of wood and waste waters from the pulp industry were used as the substrate in ethanol fermentation. Sample pretreatment was carried out by liquid-liquid extraction with dichloromethane and water. The two phases were subjected to analysis by organic and aqueous GPC using a polymer-based GPC column, a silica support and a GPC column with dichloromethane, water and water-miscible tetrahydrofuran as the mobile phases. respectively. Diode-array UV spectrophotometric detection (DAD) of substrates and broths was utilized for characterization complementary to the GPC separations. The DAD method will give UV absorption spectra for different samples providing unique fingerprints for the specific compounds present.

Fumarase a from Escherichia coli: purification and characterization as an iron-sulfur cluster containing enzyme.

It has been shown previously that Escherichia coli contains three fumarase genes designated fumA, fumB, and fumC. The gene products fumarases A, B, and C have been divided into two classes. Class I contains fumarases A and B, which have amino acid sequences that are 90% identical to each other, but have almost no similarity to the sequence of porcine fumarase. Class II contains fumarase C and porcine fumarase, which have amino acid sequences 60% identical to each other [Woods, S.A., Schwartzbach, S.D., & Guest, J.R. (1988) Biochim. Biophys. Acta 954, 14-26]. In this work it is shown that purified fumarase A contains a [4Fe-4S] cluster. This conclusion is based on the following observations. Fumarase A contains 4 Fe and 4 S2- per mole of protein monomer. (The mobility of fumarase A in native polyacrylamide gel electrophoresis and the elution volume on a gel permeation column indicate that it is a homodimer.) Its visible and circular dichroism spectra are characteristic of proteins containing an Fe-S cluster. Fumarase A can be reduced to an EPR active-state exhibiting a spectrum consisting of a rhombic spectrum at high fields (g-values = 2.03, 1.94, and 1.88) and a broad peak at g = 5.4. Upon addition of substrate, the high field signal shifts upfield (g-values = 2.035, 1.92, and 1.815) and increases in total spins by 8-fold, while the g = 5.4 signal disappears.(ABSTRACT TRUNCATED AT 250 WORDS)

New oligomeric alumoxanes

Acetooligoalumoxanes with molecular mass 407–467 were obtained upon the thermolysis of aqueous solutions of aluminum acetate at 210°C for 5 h. Gel permeation column chromatography was used to determine and isolate the oligomers obtained.

An evaluation of the HPLC—Gel chromatographic method for analyzing metallothioneins in aquatic organisms

The use of high-performance liquid chromatography to determine the concentrations of metal-binding proteins (MBP) in freshwater mussels has been evaluated. Initial use of dilute buffers ( e.g., 1Om M TRIS, pH 7 or 8), to minimize competition between the buffer and the metal-cytosolic ligand complexes, proved unacceptable; high losses of low molecular weight marker proteins occurred during chromatographic separation, presumably as a result of adsorption to the gel-permeation column packing. Losses were more dependent on salt than pH; satisfactory recoveries were obtained in the pH range 6–8 with 1Om M buffer solutions and 100m M added electrolyte (NACl; KCl). Competition experiments performed with commercial metallothionein (pre-labelled with 109Cd) and fresh mussel cytosol extract demonstrated that no appreciable metal exchange occurred during the 20-min pre-equilibration or the subsequent chromatographic separation step. With 203Hg-labelled metallothionein occasional losses were noted, however, appreciable loss of the radioisotope ranging from 50 to 58% did occur with 65Zn-labelled metallothionein during the chromatographic separation step. These results, particularly for Zn indicate that the recovery of metals after separation using this chromatographic method may vary, even for metals sharing similar chemical properties.

Characterization of the aryl hydrocarbon receptor in the human C-4II cervical squamous carcinoma cell line

Treatment of C-4II human cervical squamous carcinoma cells with 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) gave a concentration-dependent increase in ethoxyresorufin O-deethylase (EROD) activity. The EC 50 for this response was approximately 1 nM and the maximum induced activity was 27 pmol/min/mg protein. The molecular properties of the cytosolic and nuclear aryl hydrocarbon (Ah) receptor complex were determined by velocity sedimentation analysis, photoaffinity labeling, gel retardation using a consensus dioxin responsive element (DRE), and DNA-Sepharose, DRE-Sepharose and Sephaeryl S-300 gel permeation column chromatography. The apparent molecular masses of the cytosolic and nuclear photoaffinity-labeled Ah receptor complexes were 110kDa and differed from the corresponding values obtained for the Ah receptor from other animal species. In contrast, most of the other molecular properties of the Ah receptor were not significantly different from those previously reported for other species. The relative Ah-responsiveness of the C-4II cells was assessed by determining the ratio of the induced EROD activity/nuclear Ah receptor levels for a submaximal inducing dose of [ 3H]TCDD. The induced activity/binding ratio for the human C-4II cells was 0.77 and was at least one order of magnitude lower than the corresponding value for the Ah-responsive rat hepatoma H-4-II E cells.

Acetylene inhibition of Azotobacter vinelandii hydrogenase: acetylene binds tightly to the large subunit.

Acetylene is a slow-binding inhibitor of the Ni- and Fe-containing dimeric hydrogenase isolated from Azotobacter vinelandii. Acetylene was released from hydrogenase during the recovery from inhibition. This indicates that no transformation of acetylene to another compound occurred as a result of the interaction with hydrogenase. However, the release of C2H2 proceeds more rapidly than the recovery of activity, which indicates that release of C2H2 is not sufficient for recovery of activity. Acetylene binds tightly to native hydrogenase; hydrogenase and radioactivity coelute from a gel permeation column following inhibition with 14C2H2. Acetylene, or a derivative, remains bound to the large 65,000 MW subunit (and not to the small 35,000 MW subunit) of hydrogenase following denaturation as evidenced by SDS-PAGE and fluorography of 14C2H2-inhibited hydrogenase. This result suggests that C2H2, and by analogy H2, binds to and is activated by the large subunit of this dimeric hydrogenase. Radioactivity is lost from 14C2H2-inhibited protein during recovery. The inhibition is remarkably specific for C2H2: propyne, butyne, and ethylene are not inhibitors.

High-performance liquid chromatographic method to resolve and determine lipopolysaccharide sub-groups of Escherichia coli endotoxin in isolated perfused rat liver perfusate

A high-performance liquid chromatographic method was developed for resolving heterogeneous preparations of fluorescently labelled endotoxin derived from Escherichia coli (Serotype 0111:B4) into separate lipopolysaccharide sub-groups. The endotoxin was chromatographed on an analytical gel permeation column using a mobile phase of acetonitrile (20%, v/v) and 100 m M phosphate buffer (pH 7.75). Four fluorescent peaks were resolved, representing sub-groups of markedly different molecular sizes. Three of the four sub-groups contained the core polysaccharide 2-keto-3-deoxyoctonate, confirming that they contained lipopolysaccharide. Fluorescein isothiocyanate (FITC)-labelled endotoxins derived from Vibrio cholerae and Salmonella minnesota chromatographed using the same system eluted with distinctly different patterns of peaks from each other and from E. coli. Extraction of E. coli FITC—endotoxin from a buffer solution using a phenol—diethyl ether method and subsequent chromatography allowed the determination of three of the four fluorescent sub-groups over the concentration range 1–15 μg/ml.

High-performance liquid chromatographic determination of fatty acid binding proteins in rat liver with fluorescence detection.

A highly sensitive, simple and reproducible method for the quantitative determination of fatty acid binding protein in rat liver by post-column high-performance liquid chromatography with fluorescence detection is presented. Fatty acid binding protein in rat liver cytosols is separated by gel-permeation column chromatography, which is followed by fluorescence detection. The detection makes use of the fluorescence enhancement observed when a fluorescent fatty acid probe, dansylundecanoic acid, binds to fatty acid binding protein. The method is rapid, simple to perform and highly sensitive. The method was applied to the determination of fatty acid binding protein in liver from control and hypolipidaemic drug treated rats.