Rapid, simple and sensitive high-performance liquid chromatographic method for detection and determination of acyclovir in human plasma and its use in bioavailability studies

A rapid, simple and sensitive reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the measurement of acyclovir concentrations in human plasma and its use in bioavailability studies is evaluated. Unchanged acyclovir has been quantified without the introduction of an internal standard using the present method. Human plasma proteins were selectively precipitated by the addition of 7% perchloric acid to spiked plasma samples or to the plasma samples obtained after acyclovir administration to human volunteers and the mixture was spun at 1000 g for 10 min. The supernatant was directly injected into a Novaflex C₁₈ column and detected at 254 nm. The mobile phase consisted of octane sulfonic acid buffer (pH 2.5) and methanol (92:08). The limit of quantitation for acyclovir in plasma was 20 ng/ml, which enabled the determination of the area under the curve (AUC) more precisely, that is, it is much closer to its extrapolated value. The present method has been successfully applied to samples from bioavailability studies.     

Behavioral and physiological effects of chronic 2,450-MHz microwave irradiation of the rat at 0.5 mW/cm2

Adult male Long-Evans rats were intermittently exposed to 2450 MHz CW microwaves at an average power density of 0.5 mW/cm2 for 90 days. The resulting SAR was 0.14 W/kg (range 0.11 to 0.18 W/kg). The animals were exposed 7 h/day, 7 days/wk, for a total of 630 h in a monopole-above-ground radiation chamber while housed in Plexiglas holding cages. Daily measures of body mass and food and water intake indicated no statistically significant effects of microwave exposure. Monthly assessment of reactivity to electric footshock, levels of cholinesterase and sulfhydryl groups in blood, and 17-ketosteroids in urine revealed no reliable differences between 14 sham-exposed and 14 microwave-exposed rats. After the 90 days of exposure, seven rats, randomly chosen from each group, were assessed for open-field behavior, shuttlebox performance, and schedule-controlled (IRT schedule) lever pressing for food pellets. Statistically significant differences between microwave-exposed and sham-exposed rats were observed in shuttlebox performances and lever pressing. Post mortem measures of mass of several organs and microscopic examination of adrenal tissue revealed no differences between the two groups of animals.     

Isolation and sequencing of a cDNA clone encoding lysosomal membrane glycoprotein mouse LAMP-1. Sequence similarity to proteins bearing onco-differentiation antigens

We have isolated and sequenced a cDNA clone encoding the mouse LAMP-1 (mLAMP-1) major lysosomal membrane glycoprotein. The deduced protein sequence, which included the NH2-terminal portion of the mLAMP-1 molecule, consisted of 382 amino acids (Mr 41,509). The predicted structure of this protein included an NH2-terminal intralumenal domain consisting of two homology units of approximately 160 residues each separated by a proline-rich hinge region. Each homology unit contained four cysteine residues with two intercysteine intervals of 36-38 residues and one of 68 or 76 residues. The molecule also contained 20 asparagine-linked glycosylation sites within residues 1-287, a membrane-spanning region from residues 347 to 370, and a carboxyl-terminal cytoplasmic domain of 12 residues. The biochemical properties and amino acid sequence of mLAMP-1 were highly similar to those of two other molecules that have been studied as cell surface onco-differentiation antigens: a highly sialylated polylactosaminoglycan-containing glycoprotein isolated from human chronic myelogenous leukemia cells (Viitala, J., Carlsson, S. R., Siebert, P. D., and Fukuda, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, in press) and the mouse gp130 (P2B) glycoprotein, in which an increase in beta 1-6 branching of asparagine-linked oligosaccharides has been correlated with metastatic potential in certain tumor cells (Dennis, J.W., Laferte, S., Waghorne, C., Breitman, M.L., and Kerbel, R.S. (1987) Science 236, 582-585).     

Molecular basis for the isozymes of bovine glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase exists as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis, isoelectric focusing, and ion-exchange chromatography. GPI from bovine heart was purified to homogeneity and each of the isozymes resolved. Four of the five isozymes were characterized with regard to their physical, chemical, and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118,000) and subunit (59,000) molecular weights but had different apparent pI values of 7.2, 7.0, 6.8, and 6.6. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes in either reaction direction. Structural analyses showed that the amino termini were blocked and the carboxyl terminal sequences were -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes at pH extremes, at high ionic strength, in the presence of denaturants, or upon exposure to proteases. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release of ammonia along with the spontaneous shift to more acidic isozymes indicates deamidation as the molecular basis for the formation of the acidic isozymes both in vivo and in vitro. The change in the peptide fragmentation patterns following cleavage by hydroxylamine further suggests that deamidation of specific Asn-Gly bonds accounts for the structural basis of the isozymes.     

In vitro deamidation of human triosephosphate isomerase

The effects of pH, temperature, buffer ion, ionic strength, protein concentration, and substrate on the rates of specific, spontaneous deamidations of Asn-15 and Asn-71 of human triosephosphate isomerase were examined. Elevated temperature and pH facilitate the deamidations, and the deamidation rate is dependent on the specific buffer ions indicating a general base catalysis mechanism. The presence of substrate also enhances the rates of deamidation. The effect of substrate may be related to conformational changes in the catalytic center which are known to cause changes in the subunit-subunit contact sites where Asn-15 and Asn-71 are located. The enhanced deamidation in the presence of substrate may, in part, account for the more rapid rate of deamidation observed in vivo.     

Microwave radiation absorption in the rat: frequency-dependent SAR distribution in body and tail

Experiments were conducted using twin-well calorimetry to determine the averaged whole-body specific absorption rate (SAR) for rat carcasses exposed to 360, 700, 915, and 2,450 MHz CW radiation in an anechoic chamber. All exposures were done with the long axis of the rat in an E-polarization. Additional experiments were conducted using a fiber optical temperature probe to determine local SAR in the brain, esophagus, colon, rectum, and tail during microwave exposure. The whole-body averaged SAR for the radiation frequencies examined follows a nonmonotonic function with 700 MHz as the resonant frequency. This result agrees with previous analytical estimates. Local SARs within the body and tail are nonuniform with significant frequency-specific hotspots in the colon, rectum, and tail.     

Metal-ion catalysis of aldose-ketose isomerizations in acidic solutions

A comparative study has been made of the ability of metal ions to catalyze the Lobry de Bruyn-Alberda van Ekenstein transformation. An analytical enzymic assay system, which is highly sensitive and selective, was developed to monitor the rates of isomerization of hexose and triose phosphates. The metal ions catalyze the isomerization of hexose phosphates by virtue of their Lewis acid character with the first transition series being most effective. The metal ions also appear to have no ability to direct the course of the reactions. Amino acids also catalyze the isomerization apparently by virtue of the free carboxylate anion.     

Ribonucleic Acid and protein synthesis in chick embryo cells infected with fowl plague virus

Fowl plague virus comprised four major protein components and several minor ones, two strains of the virus giving similar results. One of the components was identified as the nucleocapsid protein. Synthesis of the virion proteins could readily be detected in infected cells 3 hr after infection. The two subcellular fractions associated with viral ribonucleic acid (RNA) polymerase activity (nuclei and ribosomal pellet) were associated with the protein of the nucleocapsid and a second virion protein of unidentified function. Measurement of viral RNA and protein synthesis in cells infected with preparations of ultraviolet irradiated virus showed that the capacity to synthesise the RNA and protein species of highest molecular weight was lost most quickly, suggesting that the pieces of viral RNA function independently.