Determination of 8-oxoguanine in human plasma and urine by high-performance liquid chromatography with electrochemical detection

A highly sensitive and selective method for determining 8-oxoguanine in plasma and urine was developed by high-performance liquid chromatography with electrochemical detection. The compound was separated by gradient elution on a C₁₈ reversed-phase column with a mobile phase of acetonitrile and 0.1 M sodium acetate, pH 5.2. 8-Hydroxy-2′-deoxyguanosine was used as internal standard. 8-Oxoguanine was detected electrochemically by setting the potential to +300 mV vs. Pd reference. The sensitivity of the assay was 22 ng/ml with a signal-to-noise ratio of 7:1. The within-day relative standard deviations for 8-oxoguanine quality control samples with concentrations of 3340, 1340 and 84 ng/ml were 3.6, 4.3 and 5.7% for plasma, and 4.1, 4.6 and 6.2% for urine, respectively. The day-to-day relative standard deviations for the same samples were 3.8, 6.8 and 7.1% for plasma, and 3.9, 7.0 and 7.9% for urine, respectively. The method is designed to study the pharmacokinetics and metabolic fate of O⁶-benzylguanine in a phase I clinical trial. Previously, O⁶-benzyl-8-oxoguanine was identified as the primary metabolite of O⁶-benzylguanine in humans. We now demonstrate that 8-oxoguanine is a further metabolite of O⁶-benzylguanine.     

1-Thioglycerol: inhibitor of glycerol kinase activity in vitro and in situ

The infantile form of glycerol kinase (GK) deficiency (McKusick No. 30703) (1) is characterized by adrenal cortical insufficiency, adrenal hypoplasia and developmental delay. The underlying biochemical mechanism(s) responsible for the observed clinical presentations are undetermined. Pursuant to our examination of the molecular pathogenesis of this enzyme deficiency, we have endeavored to develop a model for this disorder. 1-thioglycerol (1-TG) was investigated as a potential GK inhibitor in adrenal gland, an organ consistently affected, and in cultured fibroblasts, available from affected individuals. In 105,000 g bovine adrenal supernatant the Ki for 1-TG was 1.9 mM. In human fibroblast 105,000 g supernatant, the Ki for 1-TG was 3.4 mM. In both tissues the inhibition was purely competitive with respect to glycerol. Using incorporation of [14C(U)]-glycerol into protein as an index of GK activity in situ in human skin fibroblasts, GK deficient fibroblasts incorporate less than 10% of that observed in normal fibroblasts. Addition of 1-TG to normal fibroblasts resulted in inhibited incorporation rates. The specificity of these effects in situ was examined. Our findings indicate that 1-TG may be a suitable inhibitor of GK activity for the development of a model for glycerol kinase deficiency.     

Autoradiographic localization of subcomponents of the macromolecular GABA receptor complex

The autoradiographic localization of subcomponents of the gamma-aminobutyric acid (GABA) receptor-chloride ionophore complex has provided insight into the distribution of this macromolecular system. GABA inhibits neurons by preferentially increasing the permeability of the affected membrane to chloride ions. This inhibition can be modified by the presence of other substances which bind to the GABA receptor complex. Autoradiographic localization of specific receptor subtypes associated with this complex has been accomplished in the central nervous system. This type of analysis has been performed on high and low affinity GABAA, benzodiazepine (BZ; both BZ1 and BZ2) and convulsant sites. These receptor sites are situated in distinct brain regions and co-exist in several areas. Other receptor subtypes, which may be influenced by the presence of GABA, can be analyzed for comparison in order to define regions of the brain where GABA may be exerting independent effects (i.e., those not associated with chloride channels). Microscopic localization of receptor sites indicates specific areas to investigate in further studies concerning the characterization of subcomponents of the macromolecular GABA complex associated with chloride ion channels.     

Direct DNA transfer using electric discharge particle acceleration (ACCELL™ technology)

Direct DNA transfer methods based on particle bombardment have revolutionized plant genetic engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been engineered using variations of this technology. In many cases variety-independent and efficient transformation methods have been developed enabling application of molecular biology techniques to crop improvement. The focus of this article is the development and performance of electric discharge particle bombardment (ACCELL™) technology. Unique advantages of this methodology compared to alternative propulsion technologies are discussed in terms of the range of species and genotypes that have been engineered, and the high transformation frequencies for major agronomic crops that enabled the technology to move from the R&D phase to commercialization. Creation of transgenic soybeans, cotton, and rice will be used as examples to illustrate the development of variety-independent and efficient gene transfer methods for most of the major agronomic crops. To our knowledge, no other gene transfer method based on particle bombardment has resulted in variety-independent and practical generation of large numbers of independently-derived crop plants. ACCELL™ technology is currently being utilized for the routine transfer of valuable genes into elite germplasm of soybean, cotton, bean, rice, corn, peanut and woody species.     

Increased efficiency of substrate utilization by exposure of the thermotolerant yeast strain,Kluyveromyces marxianus IMB3 to electric-field stimulation

Summary In order to enhance cellobiose utilization and conversion of substrate to ethanol by the thermotolerant yeast strain, Kluyveromyces marxianus IMB3, the organism was exposed to short, intense electric pulses. When cells were treated with pulses measuring 0.25kV for 10mS, in the presence of cellobiose, ethanol production was found to increase by almost 40% above that found in fermentations containing non-treated cells. When the extracellular culture filtrate was assayed for -glucosidase activity no significant difference in levels was detected between treated and control systems. Increasing the voltage of the pulses resulted in a decrease in ethanol production.     

Identification of a developmentally regulated keratan sulfate proteoglycan that inhibits cell adhesion and neurite outgrowth.

Monoclonal antibodies have been used to identify a 320 kd keratan sulfate proteoglycan that is primarily expressed in the embryonic chick nervous system. Immunohistochemical localization of the proteoglycan shows that it is expressed by putative midline barrier structures in the developing chick central nervous system. When added to laminin or neural cell adhesion molecule that has been adsorbed onto nitrocellulose-coated dishes, the proteoglycan abolishes cell attachment and neurite outgrowth on these adhesive substrata. This effect can be reversed by keratanase treatment and incubation with a monoclonal antibody that recognizes the keratan sulfate chains of the proteoglycan. These data suggest that this neural keratan sulfate proteoglycan plays an important role in the modulation of neuronal cell adhesion during embryonic brain development.     

TGFβ alters growth and differentiation related gene expression in proliferating osteoblasts in vitro,preventing development of the mature bone phenotype

This study examines the mechanism by which TGF-β1, an important mediator of cell growth and differentiation, blocks the differentiation of normal rat diploid fetal osteoblasts in vitro. We have established that the inability for pre-osteoblasts to differentiate is associated with changes in the expression of cell growth, matrix forming, and bone related genes. These include histone, jun B, c-fos, collagen, fibronectin, osteocalcin, alkaline phosphatase, and osteopontin. Morphologically, the TGF-β1-treated osteoblasts exhibit an elongated, spread shape as opposed to the characteristic cuboidal appearance during the early stages of growth. This is followed by a decrease in the number of bone nodules formed and the amount of calcium deposition. These effects on differentiation can occur without dramatic changes in cell growth if TGF-β1 is given for a short time early in the proliferative phase. However, continuous exposure to TGF-β1 leads to a bifunctional growth response from a negative effect during the proliferative phase to a positive growth effect during the later matrix maturation and mineralization phases of the osteoblast developmental sequence. Extracellular matrix genes, fibronectin, osteopontin and α1(I) collagen, are altered in their expression pattern which may provide an aberrant matrix environment for mineralization and osteoblast maturation and potentiate the TGF-β1 response throughout the course of osteoblast differentiation. The initiation of a TGF-β1 effect on cell growth and differentiation is restricted to the proliferative phase of the culture before the cells express the mature osteoblastic phenotype. Second passage cells that are accelerated to differentiate by the addition of dexamethasone or by seeding cultures at a high density are refractory to TGF-β1. These in vitro results indicate that TGF-β1 exerts irreversible effects at a specific stage of osteoblast phenotype development resulting in a potent inhibition of osteoblast differentiation at concentrations from 0.1 ng/ml. © 1994 Wiley-Liss, Inc.     

DXS28 (C7) maps centromeric to DXS68 (L1-4) and DXS67 (B24) by deletion analysis

Complex glycerol kinase deficiency (CGKD) is a contiguous gene syndrome consisting of glycerol kinase deficiency together with Duchenne muscular dystrophy (DMD), congenital adrenal hypoplasia, and/or Aland Island eye disease. Deletion mapping of genomic DNA from patients with CGKD was carried out and allowed definitive ordering of loci DXS28 (C7), DXS68 (L1-4), and DXS67 (B24). Most reports have placed DXS68 centromeric to DXS28 and DXS67 on the basis of the initial mapping of the Iowa patient 3, but others have presented evidence consistent with the placement of DXS28 telomeric to DXS68 and DXS67. Through the use of DNA from CGKD patients with a variety of genomic deletions, this controversy is resolved and the order Xcen...DMD-DXS28-DXS68-DXS67...pter is definitively demonstrated.