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PubMed1999

Determination of sparfloxacin in plasma and urine by a simple and rapid liquid chromatographic method.

A simple, specific, sensitive, and rapid method has been developed and validated for the determination of sparfloxacin in human plasma and urine. The assay consisted of reversed-phase HPLC with ultraviolet detection. Plasma proteins were efficiently removed by precipitation with perchloric acid after the addition of grepafloxacin as an internal standard. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a C18 reversed-phase column. The quantification limit was 0.025 mg/L in plasma and 0.5 mg/L in urine. The coefficients of variation (CV) were less than 10% for intra-day and inter-day analyses. The recovery of sparfloxacin added to plasma and urine ranged from 96.7% to 97.9%. The method has been successfully applied to pharmacokinetic studies.

Anti-Infective Agents↗

PubMed2004

In vivo proton magnetic resonance spectroscopy of human spinal mass lesions.

OBJECTIVE: To observe metabolic differences between spinal tumor and other diseases in human spinal mass lesions, in vivo 1H magnetic resonance spectroscopy (MRS) was attempted to obtain metabolic signals in patients with various spinal mass lesions. METHODS: 1H nuclear magnetic resonance (NMR) spectra were obtained from 14 patients before surgery using a receive-only surface coil on a 1.5 T clinical magnetic resonance imaging (MRI) unit. MRS findings were compared with the histopathologic results from biopsy. In addition, tumor spectra were compared with the spectra of other benign diseases including disc herniation, which can mimic spinal cord tumor. In vitro 1H-NMR spectra were also collected from perchloric acid extracts of some spinal tumors. RESULTS: Typical water resonance line widths were in the 6- to 10-Hz range, but the metabolic signals observed were sufficiently resolved to be assigned from comparison with the 1H spectra of brain tissue. Choline was detected in all tumor spectra (n = 6) except ependymoma, whereas it was absent in other benign diseases including disc herniation (mimicking spinal cord tumors), dermoid cyst, tuberculosis, and non-multiple sclerosis myelitis. Spectral patterns of meningiomas, schwannomas, metastasis from renal cell carcinoma, and ependymomas in the spinal cord were similar to those of central nervous system (CNS) tumors. It was not possible to observe distinctive metabolic differences between benign diseases owing to relatively larger line broadening of some signals compared with that in CNS tissue. CONCLUSIONS: It appeared that acquisition of in vivo 1H-NMR signals was possible in human spinal mass lesions on a 1.5 T clinical MRI unit. Detection of choline only in the spinal tumors may indicate that there is some potential in using in vivo 1H-MRS to distinguish spinal tumors from disc herniation mimicking spinal cord tumors, non-multiple sclerosis myelitis, and dermoid cysts. On the basis of our NMR findings, however, it was not possible to distinguish between benign diseases.

Biomarkers, Tumor↗

PubMed2004

Identification of salt-sensitive genes in the kidneys of Dahl rats.

OBJECTIVE: Inherited differences in renal function underlie the effect of high salt diets on blood pressure in Dahl rats. We probed the kidneys of inbred Dahl SS/Jr and SR/Jr for anonymous and candidate genes whose expression was regulated by dietary sodium. METHODS: mRNA quantitation of both candidate genes implicated in sodium excretion and anonymous gene products found by differential hybridization in the kidneys of salt-resistant (SR) and salt sensitive (SS) inbred Dahl rats on high and low salt diets for 21 days. RESULTS: Differential screening revealed a cDNA clone (H1) that showed increased dietary salt-dependent expression only in SS rats. Sequencing of the H1 cDNA showed it was the Dahl rat homologue to a perchloric acid soluble protein expressed in liver and kidney. Among candidate genes, transcript levels of arginosuccinate synthetase (AS) and arginosuccinate lyase (AL) were higher in SS on low salt diets, and AS mRNA increased in response to a high salt diet in SR. Renal mRNA for the ANP-A and the vasopressin type II receptors did not differ by strain or dietary conditions. CONCLUSIONS: Three new salt-sensitive genes were detected in the kidneys of inbred Dahl rats. Two genes encode enzymes in the biosynthesis of L-arginine. The upregulation of these genes by dietary salt indicates increased demand and biosynthesis of L-arginine in Dahl SS rats. A third gene encodes a small acid-soluble protein thought to influence the transcription/translation of numerous genes. Further studies will be needed to determine the nature of the association of these genes with salt-sensitivity and blood pressure.

Animals↗

PubMed1980

Production and initial characterization of rat interferon.

Rat interferon of relatively high specific activity (about 10(6) units/mg protein) was produced in embryonic rat cells treated with Newcastle disease virus at a high m.o.i. The cells were cultured in serum-free medium and the interferon was precipitated and concentrated with 0.02 M-zinc acetate or with ammonium sulphate at 85% saturation. With both methods the increase in interferon activity was greater than the concentration factor. The rat interferon activity was stable on treatment with 0.15 M-perchloric acid or three cycles of freezing and thawing, but incubation at 37 degrees C for 1 h resulted in a 50% loss in activity. It had no cross activity in human or mouse cells. The sensitivity of different types of rat cells for rat interferon differed widely and was dependent on the challenge virus. Human interferons had no detectable antiviral activity on rat cells and did not block the activity of rat interferon.

Animals↗

PubMed1985

Characterization of proteins in membrane vesicles from scrapie-infected hamster brain.

Previous studies have shown that the scrapie agent is highly membrane-associated. We examined the protein composition of gradient fractions enriched for large membrane vesicles prepared from scrapie-infected and uninfected hamster brain using various methods to extract membrane proteins. We also examined proteins in detergent-extracted membrane vesicles fractionated on CsCl gradients. No qualitative differences in protein composition were seen comparing scrapie-infected and uninfected samples by one-dimensional gel electrophoresis. Extraction of proteins from membrane vesicles by phenol, pyridine, perchloric acid or lithium diiodosalicylate also failed to reveal any unique proteins in scrapie-infected hamster brain. Attempts to solubilize hydrophobic proteins (proteolipids) from CsCl gradient fractions into organic solvents were unsuccessful. These findings indicate that any hydrophobic protein associated with the scrapie agent is not a proteolipid, and that the ability of solvents to reduce scrapie infectivity is not a result of extraction of a proteolipid.

Animals↗

PubMed1990

31P NMR studies on the effect of phosphite on Phytophthora palmivora.

31P NMR spectra were obtained from perchloric acid (PCA) and KOH extracts of Phytophthora palmivora mycelium. Signals indicating the presence of large amounts of short-chain polyphosphate were observed in the spectra of PCA extracts of mycelia grown under both low (0.1 mM) and high (10 mM) phosphate conditions. The mean chain length of polyphosphate was calculated from the relative areas of signals arising from terminal and internal P nuclei in the polyphosphate chain. The small amount of polyphosphate evident in the KOH extract had an average chain length similar to PCA-soluble polyphosphate. 32P tracer studies indicated that phosphorus in the PCA fraction accounted for between 50 and 60% of total phosphorus, the bulk of the remainder being divided between the lipid and KOH extracts. The presence of the fungicide phosphorous acid markedly reduced the average chain length of acid-soluble polyphosphate. This reduction occurred both under low-phosphate conditions, in which treatment with phosphorous acid retards growth, and under high-phosphate conditions, in which no significant growth retardation is observed. Treatment with phosphorous acid perturbed phosphorus distribution and lipid composition under low-phosphate conditions.

Chytridiomycota↗

PubMed1979

Measurement of ribulose 1,5-bisphosphate from spinach chloroplasts.

A technique has been developed for the rapid and simple measurement of ribulose 1,5-bisphosphate from isolated spinach chloroplasts. The endogenous ribulose bisphosphate was detected enzymically using (14)CO(2) and ribulose bisphosphate carboxylase/oxygenase released from the chloroplasts. Ribulose 5-phosphate kinase was inhibited with 0.4 to 0.6 millimolar 2,6-dichlorophenol-indophenol and 4 micromolar carbonyl cyanide m-chlorophenylhydrazone. Phosphoenolpyruvate carboxylase activity was low with washed chloroplasts and its labeled product, [(14)C]oxalacetate, was destroyed by heating with 1.0 n HCl at 90 C. The assay method was linear from 0.05 to 0.87 nanomoles ribulose bisphosphate per milliliter. The latter value was determined with chloroplast material having 44 micrograms of chlorophyll per milliliter. This technique was simple and direct, used less chloroplast material, yet provided results comparable to a previously described enzymic technique in which ribulose bisphosphate was determined after the precipitation of chloroplast proteins by perchloric acid.

Journal Article↗

PubMed1980

Enzymic determination of metabolites in the subcellular compartments of spinach protoplasts.

A method for determining the subcellular metabolite levels in spinach protoplasts is described. The protoplasts are disrupted by centrifugation through a nylon net, releasing intact chloroplasts which pass through a layer of silicone oil into perchloric acid while the remaining cytoplasmic components remain over the oil and are simultaneously quenched as acid is centrifuged into them. Cross-contamination is measured and corrected for using ribulose 1,5-bisphosphate as a chloroplastic marker and phosphoenolpyruvate carboxylase as a cytoplasmic marker. A method for separation of intact protoplasts from the medium by silicone oil centrifugation is described, which allows a correction to be made for the effect of free chloroplasts and broken protoplasts. Methods for inhibiting chloroplast photosynthesis, without inhibiting protoplasts, are presented. It is demonstrated that ribulose 1,5-bisphosphate, ATP, ADP, AMP, inorganic phosphate, hexose phosphate, triose phosphate, fructose 1,6-bisphosphate, and 3-phosphoglycerate can be reliably recovered in the subcellular fractions isolated from protoplasts, and measured by enzymic substrate analysis.

Journal Article↗

PubMed1989

In VivoP NMR Spectroscopic Studies of Soybean Bradyrhizobium Symbiosis: I. Optimization of Parameters.

(31)P NMR spectroscopy was used to study in vivo the symbiotic state established between soybean (Glycine max [L.] Merr. cv Williams) and Bradyrhizobium japonicum (USDA 110 and 138). Different experimental conditions were used to maintain perfused, respiring detached or attached nodules in an NMR magnet. The pH of the perfusion medium affected the cytoplasmic pH and the resolution of the spectra. The internal Pi content and distribution were assessed as a function of nodule age and green-house growth conditions and the rate of glucose and 2-deoxyglucose uptake into nodules in split and intact states. The major metabolites (glucose-6-P, fructose-1,6-diP, P-choline, Pi, NTP, UDP-glc, and NAD) were readily identified from (31)P NMR spectra of perchloric acid extracts of nodules with the exception of one unknown phosphorus metabolite. Nodules stressed by glucose deprivation demonstrated movement of Pi between the vacuole and cytoplasmic compartments not previously observed in (31)P NMR studies.

Journal Article↗

PubMed1990

Isolation and characterization of a novel nuclear protein from pollen mother cells of lily.

Pollen mother cells of the lily (Lilium speciosum) were found to have a histone-H1-like protein (PMCP) not detected in other tissues. The PMCP appears from the late S-G(2) period of premeiosis and is present in mature pollen. PMCP and H1 were extracted from pollen mother cells with 5% perchloric acid and isolated by reverse-phase high-performance liquid chromatography. The amino acid composition of PMCP differs from that of somatic H1. However, PMCP is similar to H1t in mammalian testis with regard to amino acid composition.

Journal Article↗

PubMed2000

2,2'-Bipyridinium bis(perchlorate)

The title compound, [H(2)bipy](ClO(4))(2) or C(10)H(10)N(2)(2+). 2ClO(4)(-), was obtained at the interface between an organic (2, 2'-bipyridine in methanol) and an aqueous phase (perchloric acid in water). The compound crystallizes in space group P-1 and comprises discrete diprotonated trans-bipyridinium cations, [H(2)bipy](2+), and ClO(4)(-) anions. The cations and anions are connected through N-H.O and C-H.O hydrogen bonds [distances N.O 2.817 (4) and 2.852 (4) A, and C.O 3.225 (6)-3.412 (5)A]. The C-C bond distance between the two rings is 1.452 (5) A. The bipyridinium cation has a trans conformation and the N-C-C-N torsion angle is 152.0 (3) degrees.

Journal Article↗

PubMed1989

1H NMR spectroscopy: an approach to evaluation of diseased skin in vivo.

1H nuclear magnetic resonance (NMR) spectroscopy was tested for its applicability in evaluating diseased skin. In order to explore potential spectral markers characteristic of diseased tissue, perchloric acid (PCA) extracts of psoriasis and malignant melanoma tissues were compared with normal skin, and changes in melanoma after heat treatment were monitored. In psoriatic plaque extract, the spectral peak intensity ratios of Glu: Ser, creatine: Gly, and taurine: Ala were approximately three-fold compared with symptom-free or normal skin, whereas Val: Leu/Ile was one-half the normal skin ratio. In melanoma extracts, the phosphorylcholine (PC)/glycerophosphorylcholine (GPC): Ala, Glu: Ser, and lactate: Ala ratios were five-, three-, and two-fold higher, respectively, than normal skin and the Val: Leu/Ile ratio was two-thirds of normal skin. With heat treatment, PC/GPC: Ala and Glu: Ser ratios decreased, whereas lactate: Ala and Val: Leu/Ile ratios increased three-fold and one-third, respectively. Results indicate that 1H NMR spectroscopy is a sufficiently sensitive technique to distinguish normal from diseased skin. The main attraction of this technique lies in the possibility of non-invasive study of various skin diseases, malignant transformation of benign tumors, and responses to treatment. Several methodologic problems remain to be resolved before a meaningful interpretation of in vivo observations becomes feasible. Correlation of in vivo and in vitro findings is an essential step toward this goal.

Adult↗

PubMed1984

Proliferative response and renewal of hepatic function following cocaine administration in mice.

Experiments were undertaken to investigate the hepatic, temporal and spatial sequence of events following a single injection of cocaine, a known hepatotoxin. Centrilobular necrosis was induced in male mice (DBA/2Ha) 24 hr post-injection (PI). The time course of hepatic damage was monitored by assaying microsomal cytochrome P450 content, the activity of microsomal FAD-containing monooxygenase (FAD-M) and by determining the levels of serum glutamic pyruvic transaminase (SGPT). Kinetics of the onset of DNA synthesis were determined by autoradiography of thin liver sections and the incorporation of 3H-methyl thymidine into perchloric-acid-precipitable material. There was no increase in the labelling index (LI) and thymidine (TdR) incorporation in the first 24 hr PI. The LI rose to 14.6% and TdR incorporation showed a 5-fold increase over control values 48 hr PI. Both indices declined slightly at 72 hr PI and returned to control values by 96 hr PI. In contrast, the cytochrome P450 content declined by 69%, the FAD-M activity dropped by 40% and the SGPT levels showed an 18-fold increase at 24 hr PI, coincident with cytological signs of necrosis. Although the patterns of recovery differed between these selected enzymes, normal values were attained by 96 hr PI. These results demonstrate that cell damage and hepatic dysfunction precede the onset of DNA synthesis and subsequent proliferation.

Alanine Transaminase↗

PubMed1983

Granular cell myoblastoma: an immunoperoxidase study using a variety of antisera to human carcinoembryonic antigen.

Immunoperoxidase staining using five antisera to human carcinoembryonic antigen (CEA), including a mouse monoclonal antibody, was performed to investigate the expression of CEA reactivity in ten cases of granular cell myoblastoma. The granular cells were negative with four of the antisera although control sections of CEA producing colon carcinoma were positive. The single positive antiserum gave intense granular cytoplasmic staining of all tumour cells in the ten specimens studied. This reactivity was abolished after absorption of the antiserum with a perchloric acid extract of human lung to remove cross-reacting antibodies against non-specific cross-reacting antigen (NCA); a procedure which did not affect the staining of colon carcinoma specimens. The results indicate that the granular cells do not contain CEA but express a related antigen and that care in the choice of primary antiserum is important if the immunocytochemical detection of this antigen is to be used as a diagnostic aid.

Animals↗

PubMed1976

Carcinoembryonic antigen and cystic fibrosis protein in blood from cystic fibrosis homozygotes and heterozygote carriers.

Carcinoembryonic antigen (CEA) activity was measured by radioimmunoassay in blood from cystic fibrosis (CF) homozygotes, heterozygote carriers of CF, normal healthy controls, and other patient controls with carcinomas involving gastrointestinal organs. All samples were also screened by electrofocusing for cystic fibrosis protein (CFP), a metabolic marker previously shown to be associated with the CF gene. Significantly increased levels of CEA activity were found in all CFP-positive groups; however, with one exception all patient controls with marked increases in CEA activity were CFP-negative. Immunodiffusion of perchloric acid extracts of CEA-like material from heterozygote carrier blood indicated that the CEA-like material, which was elevated in homozygotes and heterozygotes for CF, showed only partial identity with two separate CEA preparations obtained from colon carcinomas and was not identical to either A, B, or O(H) blood group substances. This glycoprotein material did, however, react with three different anti-CEA antisera. Our finding of an abnormally increased glycoprotein in cystic fibrosis, taken together with previous reports demonstrating abnormalities in the carbohydrate portion of glycoproteins found in various exocrine secretions in CF, further suggests that the primary defect in this disease is manifested partly as a defect in glycoprotein metabolism. This defect may result from an abnormality in one or more of the glycosyltransferases, possibly caused by a more primary defect in polyamine metabolism.

Adolescent↗

PubMed1977

Phosphorylation in vitro and in vivo of ribosomal proteins from Saccharomyces cerevisia.

Crude ribosomes from Saccharomyces cerevisiae cultures were phosphorylated in vitro when incubated in the presence of [gamma-32P]ATP. Analysis of the ribosomal proteins with two-dimensional electrophoresis revealed that of the 29 proteins identified in the small subunit, only protein S6 was phosphorylated. Of the 37 proteins identified in the large subunit, one was highly phosphorylated (L3) and two only slightly phosphorylated (L11 and L14). The protein kinase activity associated with the ribosomes was extracted with 1 M KCl and was not dependent on adenosine 3':5'-monophosphate; it preferentially phosphorylated casein and phosvitin, but was less active on histones. Structural ribosomal proteins were also phosphorylated in vivo when the yeast cultures were incubated with [32P]orthophosphate; the radioactivity resistant to hydrolysis by hot perchloric acid was incorporated into the proteins of the two subunits. Radioactive phosphoserine was found by subjecting hydrolysates of ribosomal proteins to high-voltage electrophoresis. After two-dimensional electrophoresis, one poorly phosphorylated protein (S10) was identified in the small subunit. In the large subunit, one protein (L3) was highly labelled, and two proteins (L11 and L24) only slightly labelled.

Amino Acids↗

PubMed1980

Cross-linking of histone H1 in chromatin.

Arrays of neighbouring histone H1 molecules are present in rat liver chromatin, and can be cross-linked to each other by bisimidoesters. The product, poly(H1), can be extracted from cross-linked chromatin in high yield with 5% perchloric acid and thus distinguished from cross-linked oligomers of the core histones. On analysis in dodecyl sulphate/polyacrylamide gels poly(H1) gives a striking pattern of alternating strong and weak bands which have been shown, by the use of cleavable cross-linked reagents and two-dimensional gel electrophoresis, to arise respectively from H1 homopolymers and H1 polymers linked to the nucleosome core histones. H1-H1 proximities as measured by cross-linking exist both at low ionic strength where the nucleosome filament is extended, and at higher ionic strengths at which it is folded into a 30-nm-diameter fibre, probably in the form of a solenoid, although some additional H1-H1 contacts seem to occur in the folded form. A similar pattern of H1-H1 cross-linking is observed for nucleosome oligomers too short to form a complete turn of a solenoid. The lack of any strong dependence of the H1 cross-linking pattern on ionic strength, and the results for short oligomers, suggest that in a solenoid the major H1-H1 interactions are lateral (i.e. they occur between neighbouring nucleosomes along a turn) although additional vertical contacts (between successive turns) are not excluded. For a short nucleosome oligomer containing n nucleosomes, the number of H1 molecules that may be cross-linked into an array at low ionic strength (approximately 15 mM) is n, consistent with the presence of one H1 per nucleosome. H1-H1 cross-linking occurs only within the chromatin framework; when cross-linking is carried out in 0.5 M NaCl so that H1 is dissociated from chromatin, no poly(H1) is formed. When H1 which has been dissociated in 0.5 M NaCl is allowed to reassociate by gradual lowering of the ionic strength, the poly(H1) is identical with that in native chromatin, suggesting faithful rebinding of H1 molecules. H1-H1 proximities do not seem to be mediated by the presence of bound high-mobility-group non-histone proteins because the poly(H1) pattern persists when cross-linking is carried out at 365 mM ionic strength, when these high-mobility-group proteins are dissociated from chromatin. The H1 cross-linking pattern described here could prove to be a useful assay for the native arrangement of H1 molecules in reconstituted chromatin and for changes in H1-H1 contacts that might result from cell-cycle modifications (e.g phosphorylation) of H1. It may not prove a good assay for higher-order structure because of the relative insensitivity to ionic strength of the poly(H1) pattern as studied here. A detailed analysis will be necessary to determine whether there are any subtle differences in the interactions between H1 molecules in the folded and extended nucleosome filament.

Animals↗

PubMed1982

C1 proteins: a class of high-mobility-group non-histone chromosomal proteins from the fruit fly Ceratitis capitata.

1. CM-cellulose chromatography of a fraction soluble in 5% perchloric acid from Ceratitis capitata chromatin yields three proteins, C1a1, C1a2 and C1b, which have been purified to electrophoretical homogeneity. 2. C1a1, C1a2 and C1b analyse like high mobility group (HMG) non-histone chromosomal proteins, although they do not exactly correspond with those from vertebrates. It is proposed that C1 proteins, as well as Drosophila D1 [Rodríguez Alfageme et al. (1980) Chromosoma, 78, 1-31] are representative of a class of insect-specific HMG proteins. Tryptic fingerprints show that C1a1 and C1a2 are very similar, but C1b is a somewhat distinct protein. Circular dichroism studies have shown that these preparations do not appreciably fold on increasing ionic strength. 3. The interactions between DNA and C1 proteins have been studied. These proteins precipitate DNA in 0.15 M NaCl, 0.015 M sodium citrate and the precipitation curves are cooperative. Soluble complexes between C1 proteins and DNA could be prepared in low ionic strength media and their thermal denaturation profiles obtained. C1 proteins do not destabilize DNA under the conditions used to prepare the complexes but the three proteins stabilize DNA to a different degree. From these studies it has been concluded that the association constant of C1b to DNA is smaller than that of C1a1 and C1a2.

Animals↗