Species differences in the metabolism of benzoic acid by isolated hepatocytes and kidney tubule fragments

The metabolism of benzoic acid to hippuric acid and benzoyl glucuronide has been studied in viable hepatocytes and renal tubule fragments from two species of omnivores (rat, hamster) and two species of carnivores (ferret, dog). Hippuric acid formation was detected in hepatocytes and tubules from omnivores but was not detectable in hepatocytes from the two carnivore species. High levels of hippuric acid were produced in the tubules from the carnivores. A small amount of glucuronidation occured in hepatocytes of all these species tested and in the carnivores this reaction was the predominant pathway of benzoic acid metabolism. These results indicated that the marked species differences in patterns of benzoic acid conjugation are related to differences in the ability of liver and kidney cells to carry out glycine and glucuronic acid conjugation.     

Adult human retinal cells in culture. Identification of cell types and expression of differentiated properties

A method for culturing adult mammalian retinal neurons in serum-free N2 medium supplemented with nerve growth factor (NGF) is described. Identification of neurons in cultures of dispersed human retina was based upon morphology, immunocytochemical localization of bound tetanus toxin, and autoradiographic localization of 3H-neurotransmitter candidates (gamma-aminobutyric acid, glycine, dopamine) accumulated by high-affinity uptake mechanisms. Neurons would not attach to glass or plastic substrates, consequently the present studies were performed using neurons plated upon a feeder layer. Serum was required for the initial phase of attachment. The feeder layer was derived from retinal cells that had been plated on glass or plastic in the presence of serum and had later been passaged. Since these cells exhibited glial fibrillary acidic protein (GFAP) immunoreactivity, they were tentatively identified as being glial in origin. Under these conditions, neuron- and glia-specific properties were retained up to 28 days. The presence of interstitial retinol-binding protein (IRBP) in medium of cultures of neuronal cells on feeder layers was demonstrated by an immunoblot technique using rabbit antibovine IRBP antibodies. No IRBP was detected in medium in which the feeder layers alone had been cultured. IRBP biosynthesis was demonstrated by incubation of the cultures with [35S]methionine. Immunoprecipitable [35S]IRBP was detected only in medium from cultures containing neurons; cells of the feeder layer did not synthesize and secrete this glycoprotein. These findings are consistent with the hypothesis that IRBP, a 135K constituent of the interphotoreceptor matrix, is synthesized in vivo by a neuronal cell, specifically, the photoreceptors.     

The two-step model of bacterial UV mutagenesis

Recent results are discussed which have led to a two-step model for UV mutagenesis in excision-deficient Escherichia coli. After exposure to UV, the replication fork is assumed to continue until immediately before certain photoproducts where it stops and leaves a gap which cannot be dealt with by recombination repair. In the first (misincorporation) step, bases (a proportion of which are ‘wrong’) are postulated to be inserted opposite the photoproduct under the direct influence of the recA gene product. These misincorporated bases can be revealed as mutations by delayed photoreversal in umuD, C and lexA (ind⁻) bacteria. Their level is determined by the particular allele of recA that is present (recA441 > recA⁺ > recA430) and their rate of formation by the amount of recA protein in the cell and the degree of enrichment of the medium. No other protein needs to be synthesized for this step to occur. The second (bypass) step requires induced levels of the products of the umuD and C genes which are postulated to facilitate continued DNA synthesis on the priming end opposite the photoproduct. In principle, further errors could be made at this stage which might appear as ‘hitch-hiking’ rather than ‘targeted’ mutations.     

Aryl mono-oxygencise activity in hepatic microsomes isolated by isoelectric precipitation

The activity of the aryl mono-oxygenase (AMO) system has been compared in hepatic microsomes isolated by precipitation at pH 5.4 and by conventional centrifugation. “Acid” microsomes were shown to contain comparable levels of AMO activity, when measured on a per gram of liver basis, in control and induced rat liver and in human liver. Acid microsomes from control rat liver possess extra nonmicrosomal protein that appears to be cytoplasmic in origin.