Urine GABA levels in ovarian cancer patients: elevated GABA in malignancy.

The association of malignancy with elevated diamine oxidase (DAO), an enzyme producing gamma-aminobutyric acid (GABA) is well documented. In ovarian cancer, increased DAO occurs in the malignant tissues and plasma. Since higher DAO levels cause GABA accumulation, elevated GABA may occur in ovarian cancer and be reflected in urine. We tested this hypothesis and found that half the ovarian cancer patients had a clearly elevated urine GABA, a result that is in agreement with previous reports on DAO and malignancy. The published findings on DAO, GABA and malignancy suggest that elevated GABA is associated with cancer. This proposal could lead to a GABA urine monitor or new directions in cancer treatment or research.

A bacterial high-affinity GABA binding protein: isolation and characterization.

A gamma-aminobutyric acid (GABA) binding protein (GBP) was isolated from a bacterial mutant which has high-affinity GABA binding characteristics comparable with the GABA(A) brain receptor in mammals. The GBP was partially purified and characterized and was shown to be a periplasmic protein of approximately 42,000 molecular weight. To determine the molecular weight, a bacterial GABA binding assay was used with SDS-PAGE. This procedure did not require large amounts or complete purification of protein and may be useful as a simple method in estimating the molecular weight of other bacterial binding proteins.

Patterns of c-fos and c-jun proto-oncogene expression, apoptosis, and proliferation in rat pleural mesothelial cells exposed to erionite or asbestos fibers.

Erionite, a naturally occurring fibrous zeolite, is associated with the development of nonmalignant and malignant lung diseases and is more carcinogenic than asbestos fibers in man and rodent inhalation models of disease. To investigate the possible molecular mechanisms of erionite-induced toxicity and carcinogenesis and whether cationic content of erionite fibers was important, we examined c-fos and c-jun mRNA levels, activator protein-1 (AP-1) binding to DNA, and changes in cell proliferation and apoptosis in rat pleural mesothelial (RPM) cells exposed to different cation-substituted erionite fibers or crocidolite asbestos at various concentrations (1, 5, or 10 microg/cm2 dish) at time periods from 8 to 48 h after addition of minerals. c-fos mRNA levels in cells exposed to equal weight concentrations of various erionites and crocidolite fibers were increased comparably. When compared to other fibers, Na-erionite caused significantly increased levels of c-jun mRNA at lower mass concentrations (1 and 5 microg/cm2) than crocidolite asbestos, but comparable AP-1 binding to DNA. In comparison to untreated controls, numbers of RPM cells incorporating 5'-bromodeoxyuridine (BrdU) were increased dramatically after exposure to asbestos or Na-erionite at 5 and 10 microg/cm2. Significant dose-dependent increases in apoptosis were observed with asbestos at all time points, whereas erionites failed to induce apoptosis at 8 or 24 h, with minimal induction at higher concentrations than asbestos at 48 h. These data suggest that erionite increases the balance between cell proliferation (and/or abnormal DNA repair) and apoptosis, a normal mechanism of elimination of transformed or proliferating cells.

Mineral properties and their contributions to particle toxicity.

It has been recognized since at least as early as the mid-1500s that inhaled minerals (i.e., inorganic particles) can pose a risk. Extensive research has focused on the biological mechanisms responsible for asbestos- and silica-induced diseases, but much less attention has been paid to the mineralogical properties and geochemical mechanisms that might influence a mineral's biological activity. Several important mineralogical characteristics control a mineral's reactivity in geochemical reactions and are likely to determine its biological reactivity. In addition to the traditionally considered variables of particle size and shape, mineralogical characteristics such as dissolution behavior, ion exchange, sorptive properties, and the nature of the mineral surface (e.g., surface reactivity) play important roles in determining the toxicity and carcinogenicity of a particle. Ultimately, a mineral's species (which provides direct information on a mineral's structure and composition) is probably one of the most significant yet most neglected factors that must be considered in studies of toxicity and carcinogenicity.

Bacterial assay for quantitative measurement of nanomolar concentrations of a metabolite.

A mutant of Pseudomonas fluorescens was used to develop a radioligand, competitive binding assay to quantitatively measure gamma-aminobutyric acid. The highly reliable and reproducible assay was sensitive (nM detection), rapid, and easy to perform. Nonspecific activity and scatter were insignificant. Radiolabel was irreversibly fixed by the cells in an energy-dependent reaction. The finding that a bacterium was effective in quantitatively detecting nanomolar amounts of a metabolite suggests that other bacteria or their mutants might be used in competitive binding assays to detect and quantify amino acids or other substances occurring in trace amounts.

Determination of gamma-aminobutyric acid levels in human cerebrospinal fluid using Pseudomonas.

The principle objective was to demonstrate the efficacy of a bacterial, radioligand, competitive binding method in determining gamma-aminobutyric acid (GABA) levels in human cerebrospinal fluid (CSF). In a double blind study, CSF GABA concentrations were measured by the bacterial method using a mutant of Pseudomonas fluorescens and by a standard radioligand competitive binding assay using rat brain membranes. Linear regression analysis demonstrated a highly significant correlation (r = 0.84; P < 0.001) between the two methods. In an ancillary study, a similar correlation (r = 0.90; P < 0.001) was found between the bacterial method and HPLC, another standard procedure in determining CSF GABA levels. Furthermore, the mean CSF GABA measurements found with the bacterial method were in agreement with those reported in the literature using the brain membrane method or HPLC. The bacterial method was highly reproducible and reliable with a detection to 5 nM GABA. It was specific for GABA and was not affected by other naturally occurring compounds which are found in higher concentrations in CSF than GABA or which were suspected to interfere with the neurotransmitter in a binding assay. The bacterial radioligand method was shown to be an accurate, sensitive, and rapid assay for CSF GABA.

Mineralogical characteristics of silica polymorphs in relation to their biological activities.

Numerous aspects of silica polymorphs can affect their biological activities, including periodic structures, compositional variations, dissolution characteristics, surface properties, and particle size and shape. For an understanding of mineral-induced pathogenesis from a mechanistic perspective, the links between these properties and biochemical processes must be elucidated. This paper presents some strategies for designing assays to evaluate these properties.

High-resolution and analytical transmission electron microscopy of mineral disorder and reactions.

Crystal defects and chemical reactions occurring at scales beyond the resolution of light microscopes have major effects on the chemical and physical properties of rocks and minerals. High-resolution imaging, diffraction, and chemical analysis in the transmission electron microscope have become important methods for exploring mineral defect structures and reaction mechanisms and for studying the distribution of phases resulting from reactions. These techniques have shown that structural disorder is common in some rock-forming minerals but rare in others. They have also established mechanisms by which many reactions occur at the atomic cluster scale. These data thus provide an atomistic basis for understanding the kinetics of geological reactions. Furthermore, apparent major-element, minor-element, and trace-element chemistry of minerals can be influenced by submicroscopic inclusions or intergrowths, which commonly form as products of solid-state reactions.

Alcohol as a nutrient: interactions between ethanol and carbohydrate.

This study examined metabolic interactions between two nutrients--ethanol and carbohydrate. Both nutrients are metabolized by a common pathway to fatty acids from acetyl-coenzyme A by lipogenic enzymes. The effects of ethanol and carbohydrate on the induction of lipogenic enzymes in livers of rats were examined using two types of base diets differing in carbohydrate and lipid content and using isocaloric substitutions of ethanol, carbohydrate, and fat. Three nonlipogenic enzymes were used for comparison. Isocaloric substitution of both fat and carbohydrate for ethanol was necessary to show the specific effects of alcohol on the activity of lipogenic or nonlipogenic enzymes. Carbohydrate, and not ethanol, induced lipogenic enzymes. Ethanol specifically reduced the activity of lactate dehydrogenase and malic enzyme, but did not affect those of alcohol dehydrogenase or glycerol 3-phosphate dehydrogenase. Ethanol interacted with carbohydrate to increase the activity of ATP citrate lyase. In addition, we studied the effects of ethanol and different kinds of carbohydrates on the growth of rats and on the morphology of their livers and intestines. Ethanol significantly decreased growth characteristics (weight gain, growth rate, and caloric efficiency). Fructose, either as a monosaccharide or in sucrose, decreased this alcohol effect. Sucrose was better than glucose in lowering lipid accumulation in livers of rats. Fragility of intestinal villi was found with an alcohol, low carbohydrate diet, but was not present in alcohol diets with a higher level of carbohydrate. In contrast to carbohydrate, ethanol lacked some characteristics of a nutrient, namely, it did not induce some enzymes involved in its metabolism and did not promote optimum growth.

gamma-Aminobutyric acid uptake by a bacterial system with neurotransmitter binding characteristics.

gamma-Aminobutyric acid (GABA), an amino acid, has been found in every class of living organisms. In higher organisms, GABA is a neurotransmitter and binds with high affinity and specificity to GABA receptors on neurons in a sodium-independent reaction that is saturable. The role of GABA in organisms lacking nervous tissue is not known. This report describes, in a strain of Pseudomonas fluorescens, a GABA uptake system with binding characteristics like those of the GABA (type A) brain receptor. The binding was saturable and specific for GABA, was sodium-independent, was of high affinity (Km = 65 nM), and was inhibited competitively by muscimol, a potent GABA analogue. The bacterial GABA system included a homogeneous binding site, and no cooperative interaction was found between sites. To our knowledge, such a system for GABA, or other neurotransmitters, in a bacterium has not been reported.

Accumulation of protoporphyrin-IX by the chlorophyll-less y-y mutant of Chlamydomonas reinhardtii.

A pigment accumulating in a Mendelian mutant (y-y) of Chlamydomonas reinhardtii, which has essentially no chlorophyll and lacks inner chloroplast membranes in the light and dark, was isolated and characterized. It was identified as protoporphyrin-IX (PROTO) by spectral analysis using two different methods of extraction and fractionation. The amount of PROTO was estimated to be 10(7) molecules per cell. Since PROTO was the only intermediate of chlorophyll biosynthesis that accumulated, we conclude the y-y lesion in the pathway is after PROTO.