Enantiospecific quantification of hexobarbital and its metabolites in biological fluids by gas chromatography/electron capture negative ion chemical ionization mass spectrometry.

A highly sensitive and specific assay based on gas chromatography/electron capture negative ion chemical ionization mass spectrometry has been developed for the analysis of the enantiomers of hexobarbital and its major metabolites in human urine and plasma. S-(+)-(5-2H3)hexobarbital and R-(-)-(5-2H3)hexobarbital were synthesized for clinical studies along with (+/-)-(1,5-2H6)hexobarbital and the deuterated major metabolites for use as internal and reference standards. Hexobarbital enantiomers and their metabolites were analyzed after pentafluorobenzyl and trimethylsilyl derivatization, following solid-phase extraction from plasma and urine. Intense negative ion spectra were observed for all of the derivatives. The base peak in the spectra corresponded to the M-pentafluorobenzyl anion [M-PFB]- except for 1,5-dimethylbarbituric acid, where M-. was the most abundant ion. The applicability of the method was demonstrated by following the plasma concentration-time profiles and urinary excretion in a male extensive metabolizer of mephenytoin who was given a pseudoracemic oral dose of hexobarbital containing equal 50 mg amounts of S-(+)-2(H0)hexobarbital and R-(-)-(2H3)hexobarbital. Marked stereoselective disposition was observed, with the R-(-)-enantiomer being more efficiently metabolized, primarily by alicyclic oxidation and ring cleavage.     

Induction of erythropoietin responsiveness in murine hematopoietic cells by the gag-myb-ets-containing ME26 virus.

ME26 virus, which was generated by inserting the coding region of the acute avian leukemia-inducing virus E26 into a murine retrovirus vector, encodes a 135-kDa gag-myb-ets fusion protein. Amphotropic murine leukemia virus pseudotypes of ME26 virus induce a high incidence of erythroleukemia 2 to 4 months after injection into newborn NFS/N mice. Spleen cells from the majority of these mice proliferate to high levels in the presence of the erythroid hormone erythropoietin (Epo) and can easily be established as permanent Epo-dependent cell lines. The cell lines contain multiple copies of ME26 viral DNA and express viral message and protein. An Epo receptor mRNA of normal size can be detected in these cells, and binding studies reveal a single class of lower-affinity Epo receptor with an affinity for Epo that is in the range of that previously reported for erythroid cells. The ME26 virus-induced Epo-dependent cell lines, however, appear more immature than previously described erythroid cell lines and more closely resemble early hematopoietic precursor cells, suggesting that the virus may be activating the Epo receptor in hematopoietic cells that do not normally express it. Consistent with this idea, we are able to infect an interleukin-3-dependent myeloid cell line, FDC-P2, with ME26 virus and convert it to Epo dependence. The ME26 virus-infected FDC-P2 cells, even before growth on Epo, showed a large increase in the amount of Epo receptor mRNA. However, no ME26 viral integrations can be detected adjacent to the Epo receptor gene, indicating that the virus is not activating the Epo receptor gene by promoter/enhancer insertion. Our results are more consistent with the hypothesis that the gag-myb-ets-encoded viral fusion protein, which is known to bind DNA, is directly or indirectly activating the expression of the Epo receptor gene in these cells.     

Differences in lipogenesis in tissues of control and gold-thioglucose obese mice after an isocaloric meal

Lipogenesis was measured in 2 and 5 week gold-thioglucose (GTG) obese mice after a single meal of 0.5 g of standard chow. Compared to control mice the rate of lipogenesis in GTG obese mice, was 4-fold higher in liver and 10-fold higher in white adipose tissue (WAT). In brown adipose tissue (BAT) of GTG-injected mice the lipogenic rate was only 50% of that of controls. These results indicate that the increased lipid synthesis observed in GTG-injected mice is not due solely to hyperphagia and that some other stimuli, such as increased basal insulin levels and/or decreased thermogenesis and insulin resistance in BAT, contribute to the high rates of fat synthesis in this animal model of obesity.     

Biochemistry and ultrastructure of iridescent virus type 29

Physical and chemical parameters of iridescent virus type 29, isolated from the mealworm, Tenebrio molitor, have been analyzed. The icosahedral capsid is 130–135 nm in diameter and is surrounded by a fringe of coarse filaments. The virus has a buoyant density in CsCl of 1.31 g cm^?3 and contains 20 to 25 structural proteins as analyzed by isoelectric focusing and SDS-polyacrylamide gel electrophoresis. The DNA has a buoyant density in CsCl of 1.6874 g cm^?3 indicating a G + C content of approximately 28%. The lipid components of this virus differ from those of the host cell; the virus contains about 80% cardiolipin and 20% phosphatidyl choline.     

The radiolysis of tryptophan and leucine with32Pβ-radiation

Summary We have extended earlier experiments on the radiolysis of DL-tryptophan using³²P-radiation to longer reaction times, observing complete destruction of the tryptophan by secondary, non-radiolytic processes. We have also undertaken the irradiation of DL-leucine with³²P's at -196°, achieving radiolyses to the extents of ca. 20–30%, but observing no concomittant asymmetric bias. The implications of these observations are discussed with regard to the Vester-Ulbricht mechanism for the origin of optical activity.     

Synthesis and repair of RNA-containing supercoiled plasmid ColE1 DNA in Escherichia coli

Supercoiled plasmid molecules sensitive to nicking by RNase or alkali have been shown to accumulate during replication of colicinogenic factor E1 (ColE1) in Escherichia coli in the presence of chloramphenicol. The possibility that this sensitivity is due to the covalent integration of RNA molecules during the synthesis of plasmid DNA is supported by the demonstration that (a) strands of supercoiled ColE1 newly replicated in the presence of chloramphenicol exhibit sensitivity to RNase and alkali treatment, while (b) RNase- and alkali-resistant circular strands of plasmid DNA synthesized either before or after the addition of chloramphenicol remain resistant during subsequent replication of the plasmid in the presence of chloramphenicol. Furthermore, newly made plasmid DNA strands cannot act as templates for further rounds of replication if they possess an RNA segment. The existence of a repair mechanism for the removal of the RNA segment from supercoiled ColE1 DNA molecules was demonstrated by pulse-chase experiments. It was observed that the proportion of RNase-sensitive molecules is considerably higher in pulse-labeled as compared to continuously labeled ColE1 DNA synthesized in the presence of chloramphenicol, and the proportion of pulse-labeled ColE1 DNA that is RNase sensitive is greatly reduced during a chase period. Removal of the RNA segment is also carried out effectively at the restrictive temperature in temperature-sensitive DNA polymerase I mutants. In a survey of other bacterial mutants defective in the repair of damaged DNA, a substantial increase in the rate of accumulation of RNase-and alkali-sensitive supercoiled ColE1 DNA in the presence of chloramphenicol was observed in recBC and uvrA mutants in comparison with the wild-type strains.     

Piriform Syndrome

Among a variety of deep muscle trigger points, the piriform muscle trigger point is selected for individual scrutiny. This seems fully justified by the great potential for confusing this entity with discogenic disease and consequently having unnecessary surgical procedures carried out.The diagnosis can be made from findings on simple physical diagnostic tests and an appropriate history. Low back and hip pain with pain radiating down the back of the leg should suggest piriform syndrome as part of the differential diagnosis. This is especially true if a female patient has complaint of dyspareunia.Pain and weakness on resisted abduction-external rotation of the thigh is a sign of piriform syndrome. This is confirmed by tenderness and reproduction of the patient''s complaints by digital pressure over the belly of the piriform muscle, completing the diagnostic criteria.Local injection of the muscle belly is curative. There are no laboratory or x-ray findings leading to a diagnosis.     

The action of chelating agents on human liver aldehyde dehydrogenase.

Human liver aldehyde dehydrogenase was inhibited by aromatic chelating agents. However, structurally related compounds with much lower metal-complexing ability displayed affinities for enzyme essentially equal to those of their respective chelating analogues. Inhibition was competitive with respect to the coenzyme. It is suggested that hydrophobic interactions between the inhibitors and the coenzyme-binding site of the enzyme are responsible for the observed effects on activity.     

A comparison of immunomagnetic and surface adhesion immunofluorescent techniques for the rapid detection of Listeria monocytogenes and Listeria innocua in meat

an immunomagnetic immunofluorescent method was investigated for the rapid detection of Listeria monocytogenes and Listeria innouca . This technique involved enrichment of the suspect sample at 30°C overnight. Listeria monocytogenes cells were isolated from the enriched sample using immunomagnetic separation and Listeria were subsequently visualized using an immunofluorescent microscopy technique. This technique was used in the detection of Listeria cells from pure culture, inoculated beef mince samples and naturally contaminated retail beef mince samples. A detection level of approximately 1×10³ cfu ml⁻¹ was achieved. When compared with traditional detection methods no false negatives or positives were recorded for L. monocytogenes or L. innocua . The immunomagnetic immunofluorescent technique had a detection level similar to a previously described surface adhesion immunofluorescent technique. Isolation of the Listeria cells by surface adhesion involved dipping a membrane attached to a microscope slide into the enriched sample for 10 min. This was quicker and simpler to perform than the immunomagnetic separation technique which took 2 h to carry out.