Search

The subcellular distribution of the first three enzymes in the ommochrome eye pigment pathway were studied by means of differential centrifugation. Tryptophan pyrrolase and kynurenine formamidase are found in the soluble fraction of the homogenate. A relatively small amount of tryptophan pyrrolase, 10 to 15% of the total, is sedimentable. The head region of pupae contains approximately 15% of the total activity of the animal. The subcellular distribution of the activity of the head is not appreciably different from the distribution of activity in the rest of the animal. Therefore no evidence suggests that the enzyme exists in a particulate form specifically in the head. Kynurenine hydroxylase is judged to be a mitochondrial enzyme on the basis of its co-sedimentation with particles containing cytochrome oxidase and by the demonstration of mitochondria in the sedimented fractions containing kynurenine hydroxylase activity. Kynurenine hydroxylase was judged not to be present in pigment granules since developmental and genetic changes in pigment granule morphology do not affect the sedimentation of kynurenine hydroxylase containing particles. These results on the three enzymes lead us to conclude that they are not organized into a single multi-enzyme complex as has been previously suggested. A consideration of the six ommochrome deficient mutants indicates that four of these can be explained by existing data. A hypothesis which does not involve enzyme complexes is presented to account for the remaining two.

The administration of chloroquine or hydroxychloroquine to albino and pigmented (hooded) rats at a daily dosage of 40 mg/kg produces a rapid rise in tissue concentrations of the drugs during the first month of medication, but comparatively little further rise when the medications are continued for two additional months. The mean tissue concentration of chloroquine at 1 month is about 100 mg/kg, compared to about 30 mg/kg for hydroxychloroquine. However, rats degrade the latter compound more extensively, so that their total mean tissue content of 4-aminoquinoline bases is greater than 30 mg/kg at that time. The order of increasing tissue concentration for both drugs is almost invariably: muscle, eye, heart, kidney, liver, lung, and spleen. In the pigmented rats, the order is: muscle, heart, kidney, liver, lung, spleen, and eye. When medication is discontinued, the concentrations of both drugs in the tissues decrease very rapidly, except in the eyes of the pigmented rats. Long-continued exposure of the eyes of the pigmented rats to high concentrations (approaching 0.1%) of chloroquine or hydroxychloroquine and their degradation products does not produce any detectable ocular histopathology.

The study of genetic regulatory mechanisms operating in plants and animals is of paramount importance in contemporary biology. A precise understanding of the mechanisms that underlie normal cellular differentiation is a prerequisite for understanding neoplastic transformation and genetic disease. At present, we are not aware of a single assay system that can give answers to all questions we are already able to pose. Studies of RNA synthesis are valuable because they provide a direct measurement of transcriptional activity. But these studies remain incomplete until we succeed in unraveling the metabolic roles of the molecules whose synthesis we study. In this respect, the study of enzyme synthesis represents a better defined assay system, although the interpretation of observed fluctuations in synthetic rates is made difficult by the many steps that intervene between the genes and their finished protein products. We propose that a combination of protein biosynthetic and cytogenetic analysis is a promising assay system for further investigation.

In vitro synthesis of RNA by nuclei isolated from various tissues has been observed in many laboratories (1-3). Several investigators have realized the usefulness of isolated nuclei for studying the regulation of RNA synthesis. The effects of nuclear proteins (4) and various hormones, (5-7) for example, on the incorporation of various precursors into RNA have been analyzed recently. However, if the full potential of isolated nuclei as an assay system for studying the regulation of RNA synthesis is to be realized, then it is necessary that the characterization of the synthesized RNA be more complete. In the present studies, we have used the technique of DNA-RNA hybridization in an attempt to characterize the RNA synthesized in vitro by thymus nuclei. We have shown that the isolated thymus nucleus is capable of synthesizing a population of RNA molecules that is specific to thymus. The intracellular distribution of RNA species was also investigated and differences were found between nuclear RNA and cytoplasmic RNA. The isolated nucleus was shown to synthesize RNA similar to that found in the nucleus and in the cytoplasm.