The effect of adrenalectomy on stress-induced c-fos mRNA expression in the rat brain

Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of postranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phase high-performance liquid chromatography (RP-HPLC) we have successfully separated various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea-Triton gel electrophoresis. Our results demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to traditional separation techniques and will be useful in studying the biological function of histone acetylation.     

The microheterogeneity of the mammalian H1(0) histone. Evidence for an age-dependent deamidation

Histone H1(0) is known to consist of two subfractions named H1(0)a and H1(0)b. The present work was performed with the aim of elucidating the nature of these two subfractions. By using reversed-phase high performance liquid chromatography in combination with hydrophilic interaction liquid chromatography, we fractionated human histone H1(0) into even four subfractions. Hydrophilic interaction liquid chromatographic analysis of the peptide fragments obtained after cleavage with cyanogen bromide and digestion with chymotrypsin suggested that the four H1(0) subfractions differ only in their small N-terminal end of the H1(0) molecule (30 residues). Edman degradation of the N-terminal H1(0) peptide fragments and mass spectra analysis have indicated that human histone H1(0) consists of intact histones H1(0) (named H1(0) Asn-3) and deamidated H1(0) forms (H1(0) Asp-3) having an aspartic acid residue at position 3 instead of asparagine. Moreover, both H1(0) Asn-3 and H1(0) Asp-3 are blocked (H1(0)a Asn-3, H1(0)a Asp-3) and unblocked (H1(0)b Asn-3, H1(0)b Asp-3) on their N terminus. Acid-urea gel electrophoretic analysis has shown that the histone subfraction, in the literature originally named H1(0)a, actually consists of a mixture of H1(0)a Asn-3 and H1(0)a Asp-3, whereas H1(0)b consists of H1(0)b Asn-3 and H1(0)b Asp-3. Furthermore, we found that hydrophilic interaction liquid chromatography separates rat and mouse histone H1(0) just like human H1(0) into four subfractions. Hydrophilic interaction liquid chromatographic analysis of brain and liver histone H1(0) from rats of different ages revealed an age-dependent increase of both the N-terminally acetylated and the deamidated forms of H1(0). In addition, we found that the relative proportions of the four forms of H1(0) histones differ from tissue to tissue.     

Microscale synthesis of phosphatidyl-[3H]choline from 1,2-diacylglycerol. Assessment of isomerization by reversed-phase high-performance liquid chromatography

The synthesis of rac-1-palmitoyl-2-oleoylglycero-3-phospho-[3H]choline of high specific activity was carried out on a microscale by making 7 mumol of rac-1-palmitoyl-2-oleoylglycerol react first with an equimolar amount of POCl3 and then of [3H]choline. After purification by thin-layer chromatography and normal-phase high-performance liquid chromatography and normal-phase high-performance liquid chromatography (HPLC), the yield of the synthesis of [3H]phosphatidylcholine (120 microCi/mumol) was 22%. rac-1-Palmitoyl-2-oleoylglycerol was purified before use by reversed-phase HPLC under conditions which were nonisomerizing and allowed the separation of 1,2- and 1,3-isomers of diacylglycerol. Ethanol, but not benzene, was shown to cause isomerization of long-chain diacylglycerol and, therefore, was not used for drying the substrate before reaction. A rapid and complete separation of 1,2- and 1,3-isomers of long-chain phosphatidylcholine was obtained by reversed-phase HPLC using 20 mM choline chloride in methanol/acetonitrile/water (50:50:1, by vol) isocratically as the mobile phase. Under these conditions, analysis of the synthesized rac-1-palmitoyl-2-oleoylglycero-3-phospho-[3H]choline showed a total absence of 1,3-isomer.     

F0F1-ATPase genes from an archaebacterium, Methanosarcina barkeri

The widely used high-performance liquid chromatography (HPLC) procedure to determine glutathione in biological samples utilizing iodoacetic acid as thiol quenching agent and 1-fluoro-2,4-dinitrobenzene for derivatization has been modified regarding tissue sample processing and storage of the working solutions. The modified procedure compared with the original method reduces artifactual oxidation in rat liver glutathione measurement (1.47+/-0.8% vs. 2.84+/-0.69%, respectively). In both HPLC procedures, an increase in artifactual oxidation was found in both standard glutathione solutions and hepatic samples when N-ethylmaleimide instead of iodoacetic acid was used for thiol trapping.     

Mitochondrial DNA mutations in multiple symmetric lipomatosis

We describe an analytical technique for measuring residues of imidacloprid, a relatively new and highly active insecticide, in water and soil using high-performance liquid chromatography (HPLC). All analyses were performed on reversed-phase HPLC with UV detection at 270 nm using a mobile phase of acetonitrile-water (20:80, v/v). Fortified water samples were extracted with either solid-phase extraction (SPE) or liquid-liquid extraction methods. A detection limit of 0.5 microgram/l was achieved using the SPE method. The imidacloprid residues in soils were extracted with acetonitrile-water (80:20, v/v), and the extract was then evaporated using a rotary evaporator. The concentrated extract was redissolved in 1 ml of acetonitrile-water (20:80, v/v) prior to analysis by reversed-phase HPLC. A detection limit of 5 micrograms/kg was obtained by this method which is suitable for analysis of environmental samples. Accuracy and precision at 10 and 25 micrograms/kg soil samples were 85 +/- 6% and 82 +/- 4%, respectively.     

Monitoring of tricyclic antidepressants in human serum and plasma by HPLC: characterization of a simple, laboratory developed method via external quality assessment

A reversed-phase high performance liquid chromatography (HPLC) method for the determination of plasma and serum levels of amitriptyline (AMI), nortriptyline (NORT), imipramine (IMI), desipramine (DESI), clomipramine (CLOMI), and norclomipramine (NCLOMI) is described. The assay is based upon single step liquid/liquid extraction of these compounds using hexane at pH 11 (recovery between 92 and 105%), a Nova-Pack C-18 HPLC cartridge column, a mobile phase composed of a phosphate buffer with 50% (v/v) acetonitrile and about 0.2% (v/v) diethylamine (final pH: 8) and solute detection at 242 nm. Using 1 ml of plasma or serum and econazole as internal standard, drug levels between 20 and 400 ng ml(-1) (about 60-1450 nM) were found to provide linear calibration graphs. For drug concentrations in the range of 70-120 ng ml(-1) (about 240-430 nM), intraday and interday imprecisions (n = 5) were determined to be < 6.0, and < 15%, respectively. Data reported include those gathered over a 3-year period during which this assay was employed for therapeutic drug monitoring and clinical toxicology. The performance of the laboratory developed assay was assessed via analysis of monthly samples provided by an external quality control scheme.     

Effect of combined administration of insulin-like growth factor and platelet-derived growth factor on the regeneration of transected and anastomosed sciatic nerve in rats

The development and application of membrane solid phase extraction (SPE) in 96-well microtiter plate format is described for the automated analysis of drugs in biological fluids. The small bed volume of the membrane allows elution of the analyte in a very small solvent volume, permitting direct HPLC injection and negating the need for the time consuming solvent evaporation step. A programmable liquid handling station (Quadra 96) was modified to automate all SPE steps. To avoid drying of the SPE bed and to enhance the analytical precision a novel protocol for performing the condition, load and wash steps in rapid succession was utilized. A block of 96 samples can now be extracted in 10 min., about 30 times faster than manual solvent extraction or single cartridge SPE methods. This processing speed complements the high-throughput speed of contemporary high performance liquid chromatography mass spectrometry (HPLC/MS) analysis. The quantitative analysis of a test analyte (Ziprasidone) in plasma demonstrates the utility and throughput of membrane SPE in combination with HPLC/MS. The results obtained with the current automated procedure compare favorably with those obtained using solvent and traditional solid phase extraction methods. The method has been used for the analysis of numerous drug prototypes in biological fluids to support drug discovery efforts.     

A validated method for the determination of paracetamol and its glucuronide and sulphate metabolites in the urine of HIV+/AIDS patients using wavelength-switching UV detection

Paracetamol is a safe drug which has been used as an in-vivo probe to determine phase II metabolism in a HIV+/AIDS population. Due to the biohazard nature of HIV-infected samples, a high performance liquid chromatography (HPLC) assay which offers minimal sample manipulation and maximal specificity was developed. This reverse-phase HPLC method uses wavelength-switching UV detection for the simultaneous determination of paracetamol and its glucuronide and sulfate metabolites in HIV-infected urine samples. The solvent systems involves a simple isocratic elution with a composition of 50 mM sodium acetate buffer, pH adjusted to 3.5; acetonitrile (96:4 v/v) modified with 0.35% trifluroacetic acid. The validated method is highly reproducible with an inter-assay variation of < 7%. This method also shows good precision and sensitivity, making it an ideal assay for phenotyping studies to determine the extent of glucurondiation and sulfation activities.     

Analysis of the Morgan-Elson chromogens by high-performance liquid chromatography

The Morgan-Elson method for quantitative N-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to yield a colored product. In the present investigation, the products formed in the first step of the procedure were analyzed by high-performance liquid chromatography (HPLC) on a reversed-phase (C18) column, which was eluted with a water-methanol gradient; the absorbance of the effluent was monitored at 229 nm. The profile generated from alkali-treated N-acetylglucosamine exhibited two major peaks, in a ratio of approximately 2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the peak area, was eluted in an intermediate position, and several smaller peaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's reagent, indicating that they were Morgan-Elson chromogens. The HPLC profile of alkali-treated N-acetylmannosamine was identical to that of the products generated from N-actylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the chromogens. N-Acetylgalactosamine yielded two major peaks, which were eluted in the same positions as the two major products formed from N-acetylglucosamine, but the intermediate peak seen in the N-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as approximately 25 ng of N-acetylglucosamine and may therefore be of value as an alternative to the complete Morgan-Elson procedure when only small amounts of sample are available for quantitative analysis.     

The cytoplasmic F-box binding protein SKP1 contains a novel pentasaccharide linked to hydroxyproline in Dictyostelium

SKP1 is involved in the ubiquitination of certain cell cycle and nutritional regulatory proteins for rapid turnover. SKP1 from Dictyostelium has been known to be modified by an oligosaccharide containing Fuc and Gal, which is unusual for a cytoplasmic or nuclear protein. To establish how it is glycosylated, SKP1 labeled with [3H]Fuc was purified to homogeneity and digested with endo-Lys-C. A single radioactive peptide was found after two-dimensional high performance liquid chromatography. Analysis in a quadrupole time-of-flight mass spectrometer revealed a predominant ion with a novel mass. Tandem mass spectrometry analysis yielded a set of daughter ions which identified the peptide and showed that it was modified at Pro-143. A second series of daughter ions showed that Pro-143 was hydroxylated and derivatized with a potentially linear pentasaccharide, Hex-->Hex-->Fuc-->Hex-->HexNAc-->(HyPro). The attachment site was confirmed by Edman degradation. Gas chromatography-mass spectrometry analysis of trimethylsilyl-derivatives of overexpressed SKP1 after methanolysis showed the HexNAc to be GlcNAc. Exoglycosidase digestions of the glycopeptide from normal SKP1 and from a fucosylation mutant, followed by matrix-assisted laser desorption time-of-flight mass spectrometry analysis, showed that the sugar chain consisted of D-Galpalpha1-->6-D-Galpalpha1-->L-Fucpalpha1-->2-D- Galpbeta1--> 3GlcNAc. Matrix-assisted laser-desorption time-of-flight mass spectrometry analysis of all SKP1 peptides resolved by reversed phase-high performance liquid chromatography showed that SKP1 was only partially hydroxylated at Pro-143 and that all hydroxylated SKP1 was completely glycosylated. Thus SKP1 is variably modified by an unusual linear pentasaccharide, suggesting the localization of a novel glycosylation pathway in the cytoplasm.     

Sensitive determination of a new antiarrhythmic agent, trecetilide, in plasma by high-performance liquid chromatography with fluorescence detection

Two high-performance liquid chromatography (HPLC) methods were developed for the determination of trecetilide in plasma samples. Differing only in the addition of a derivatization step and different detection wavelengths, the two methods encompassed a wide concentration range. In both methods, plasma samples (0.1 ml) with added internal standard were applied to solid-phase extraction discs containing a non-polar/strong cation mixed-phase, washed and eluted with an acetone-acetonitrile triethylamine mixture. The eluate was evaporated to dryness, and either reconstituted and directly injected onto an HPLC column or first derivatized with 1-naphthyl isocyanate before HPLC analysis. In both methods, the separation was performed isocratically on a cyano analytical column utilizing a mobile phase composed of acetonitrile-pH 7.9 phosphate buffer (70:30, v/v). The column effluent was monitored by fluorescence detection at 290/345 nm (with derivatization) or 235/320 nm (without derivatization). The limits of detection and quantitation of the assay were 0.57 and 1.9 ng/ml, respectively, when derivatization was used, or 4.3 and 14 ng/ml, respectively, without derivatization.     

Interaction of histone H1 with cis-platinum modified DNA

Cis-diamminedichloroplatinum(II) (cis-DDP) is known as an effective anticancer drug. Its therapeutic effect is supposed to be a consequence of the covalent binding to DNA. A number of cellular proteins were found to bind selectively to DNA modified by cis-DDP (but not by its isomer trans-DDP). Here we present our observations on interaction of the linker histone H1 with cis- and trans-DDP modified DNA fragments. The results afford new experimental information about the preferential binding of histone H1 to cis-DDP-distorted DNAs versus trans-DDP modified ones.     

Infarct tolerance against temporary focal ischemia following spreading depression in rat brain

A sensitive and versatile analytical method utilizing high-performance liquid chromatography (HPLC) and precolumn derivatization of 1H-4-substituted imidazole compounds is described. A HPLC method using 4-dimethylaminoazobenzene-4'-sulfonyl chloride (dabsyl chloride) and ultraviolet (UV) detection was developed for the analysis of histamine (HA) H3-selective compounds in human plasma, rat plasma, or homogenized rat cortical tissue. The average intra- and inter-assay variability, over a range of 10 to 0.01 microg/ml, was determined to be acceptable. The lower limit of detection for the dabsylated ligands was estimated to be <1.0 ng/ml while the lower limit of quantitation (LLOQ) was determined to be 10 ng/ml of conjugate. This assay has demonstrated it's suitability for the sensitive quantitation of several structurally diverse 1H-4-substituted imidazole HA H3-receptor antagonists in biological matrices for pharmacokinetic and biodistribution studies.     

The mouse histone H1 genes: gene organization and differential regulation

There are six mouse histone H1 genes present in the histone gene cluster on mouse chromosome 13. These genes encode five histone H1 variants expressed in somatic cells, H1a to H1e, and the testis-specific H1t histone. Two of the genes that have not been assigned previously to the five somatic H1 subtypes have been identified as encoding the H1b and H1d subtypes. Three of the H1 genes, H1a, H1c and H1t, are present on an 80 kb segment of DNA that contains nine core histone genes. Two others, H1d and H1e, are present in a second patch, while the H1b gene is at least 500 kb away in a patch containing 14 core histone genes. The histone H1 genes are differentially expressed. All five genes for the somatic histone H1 proteins are expressed in exponentially growing cells. However, the levels of H1a, H1b and H1d mRNAs are greatly reduced in cells that are terminally differentiated or arrested in G0, while the H1c and H1e mRNAs continue to be expressed. In addition to the major RNA that ends at the stem-loop, the H1c gene expresses a longer, polyadenylated mRNA in differentiated cells, although in varying amounts. None of the other histone H1 genes encodes detectable amounts of polyadenylated mRNAs.     

Solid-phase extraction with end-capped C2 columns for the routine measurement of racemic citalopram and metabolites in plasma by high-performance liquid chromatography

An assay based on solid-phase extraction followed by high-performance liquid chromatography (HPLC) was developed for the measurement of citalopram and its main metabolites desmethylcitalopram and didesmethylcitalopram. The best extraction procedure was performed with end-capped C2 column utilising secondary silanol interactions to obtain clean extract. The HPLC analysis was done on a phenyl column with a mobile phase without any amine additives. Fluorescence detection gave a limit of detection of 0.8 nmol/l plasma for the compounds analysed.     

The in situ labeling of histone H3 in chromatin by a fluorescent probe

A sulfhydryl-specific fluorescent probe, N-3-pyrene maleimide, has been shown to label with high efficiency the sulfhydryl groups of histone H3 in nonsheared chromatin. The probe labels chromatin preparations obtained by mild homogenization or nuclease treatment of rat liver and mouse thymocyte, but not chick erythrocyte nuclei. Mononucleosomes from all nuclear preparations are labeled by the probe. The reaction is inhibited by prior reaction of the chromatin with N-ethyl maleimide. The reaction kinetics show fast and slow components representing reactions with cysteinyl sulfhydryl groups and lysyl epsilon-amino groups, respectively. Dissociation of the chromatin by urea (6 M) or sodium dodecyl sulfate (SDS) increases the fluorescence intensity (2-3 fold) and is maximal at approx. 0.01-0.02% (w/v) SDS. Histones extracted from the labelled chromatin show that approx. 80-90% of the label is associated with the histone fraction and column chromatography of this fraction shows that the label is primarily associated with histone H3. Labelling of the isolated histone fractions shows significant labelling only of histone H3. The intrinsic fluorescence of tryptophan is quenched by the labelled histone H3, but not by iodide, suggesting that non-histone (tryptophan-containing) proteins lie in close proximity to the labelled histone H3 but are not immediately accessible to external solvent. The labelled chromatin exhibits fluorescence anistropy, the anistropy parameter being 0.19 +/- 0.003 for chromatin, 0.05 +/- 0.01 for mononucleosomes and 0.0 for isolated histone H3. This demonstrates the restriction placed on the label's mobility by the chromatin fiber. The formation of a superhelix at 60-100 mM NaCl has been monitored with the probe. An increase in fluorescence intensity at 80 mM NaCl is observed with intact chromatin (but not H-1 depleted chromatin) followed by dissociation of the octamer in 1.50-2.0 M salt accompanied by a large increase in labelling.     

Separation of 1-phenyl-3-methyl-5-pyrazolone derivatives of monosaccharides by capillary electrochromatography

1-Phenyl-3-methyl-5-pyrazolone (PMP) derivatives of component monosaccharides in glycoproteins (fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine) and epimeric aldopentoses (arabinose, lyxose, ribose and xylose) were well separated from each other by capillary electrochromatography on a Hypersil ODS column with a mixture of 50 mM N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) buffer, pH 6.0 to approximately 6.3, and acetonitrile (2.2:1 v/v) as eluent. The elution of these compounds showed relatively strong dependence on the pH and concentration of the buffer salts contained in the eluent, as compared to the elution by pressure-driven high-performance liquid chromatography (HPLC) on the same stationary phase, but separation of PMP-monosaccharides was better than that by HPLC. Retention times of PMP-monosaccharides were highly reproducible with a relative standard deviation (RSD) of approximately 0.6%, and quantification with an RSD less than 5% could be achieved using 3-O-methylglucose as an internal standard.     

Identification and characterization of a calreticulin-binding nuclear protein as histone (H1), an autoantigen in systemic lupus erythematosus

Our objective was to identify nuclear calreticulin-binding protein(s) and investigate whether there is a correlation between presence of autoantibodies against calreticulin and calreticulin-binding protein(s) in the sera of patients suffering from systemic lupus erythematosus (SLE). The ligand overlay procedure using digoxigenin-labelled calreticulin was used to identify a calreticulin-binding protein in the nuclear fraction of bovine brain. Fractionation of the nuclear components was used to localize the major positive calreticulin-binding protein. The protein was partially purified using hydroxylapatitie chromatography and subjected to NH2-amino acid sequence analysis. Immunoblots using the sera of SLE patients were then carried out on calreticulin and the calreticulin-binding protein. The calreticulin-binding protein present in the nucleoplasm was identified as histone H1. Approximately 62% (26/42) patients with SLE had IgG antibodies directed against H1 whereas the sera of healthy individuals did not react with the antigen; 36% of patients with SLE had both anti-calreticulin and anti-histone H1 antibodies. Phosphorylation of the latter protein did not alter its immunoreactivity. These findings demonstrate that the concomitant presence of autoantibodies directed against both calreticulin and histone H1 occurs frequently in patients with SLE and may help shed some light on the mechanisms which bring about the autoimmune response.     

A sensitive procedure for the quantitation of free and N-(2-hydroxypropyl) methacrylamide polymer-bound doxorubicin (PK1) and some of its metabolites, 13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in human plasma and urine by reversed-phase HPLC with fluorimetric detection

A high-performance liquid chromatographic assay has been developed and validated for the determination in plasma and urine of doxorubicin (DXR) and some of its metabolites released in vivo from an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer containing DXR linked through its aminosugar moiety to the polymer via an oligopeptide spacer (PK1). The method also allows measurement of the DXR still bound to the polymer. Following addition of two internal standards, the free compounds were extracted twice with isopropanol-chloroform (25:75, v/v). The first extraction was performed at physiological pH and the second after buffering at pH 8.4, in order to extract the aglycones and the glycosides, respectively. Determination of total DXR (polymer-bound plus free DXR) was performed, after quantitative acid hydrolysis to release doxorubicinone from free or polymer-bound DXR, by extraction with the same solvent mixture at pH 7.4. In both cases the organic phase was evaporated to dryness; the compounds were then separated by reversed-phase high-performance liquid chromatography (HPLC) under isocratic conditions and quantitated by fluorimetric detection. In the chromatograms all the analytes appeared to be separated at the baseline and no interference from blank human plasma and urine was observed. The suitability of the method for in vivo samples was checked by the analysis of plasma and urine samples obtained from a cancer patient who had received a single intravenous dose of the test compound.     

Chromosome mapping of rat histone genes H1fv, H1d, H1t, Th2a and Th2b

Chromosome assignment of the rat histone genes H1t, H1d (H1.4), H1fv (H10), Th2a and Th2b is described. The testicularly expressed histone genes H1t, Th2a and Th2b could be assigned to rat chromosome (RNO) 17 by PCR analysis of somatic cell hybrid DNAs. The H1d gene was mapped to RNO17p12-->p11 by FISH. These genes might form a histone gene cluster homologous to that found on HSA6p21.3 in humans and MMU13A2-3 in mice. The rat histone H1fv gene was assigned to RNO7 by PCR. This result allows the inclusion of rat H1fv to an established conserved group of syntenic genes in rat, mouse and human on chromosomes RNO7, MMU15 and HSA22, respectively.