No abstract

RECENT studies have indicated that L-phenylalanine inhibits antibody response to diphtheria toxoid in rats and rabbits ( RYAN and CARVER, 1964). It was suggested, as an explanation for the inhibition, that excess phenylalanine caused a disturbance in the free amino acids of the animals with a resultant decrease in antibody (protein) synthesis. It is also conceivable that chronic alterations in the amino acids of the central nervous system induced by phenylalanine could also give rise to changes in protein synthesis. Since an excess or deficiency of an amino acid might exert an effect on the distribution of other amino acids, a quantitative study of the amino acids in rat brain and liver was conducted to assess the influence of phenylalanine on the concentration of the other amino acids. METHODS L-Phenylalanine (Nutritional Biochemical) was dissolved in dilute HC1-saline, neutralized with NaOH and brought to volume. All injections were made intraperitoneally in a volume not exceeding 2.0 ml. Sprague-Dawley male rats weighing between 150-175 g were used. The control animals received saline; the experimental groups were injected with 100 mg L-phenylalanine. At 15, 30, and 120 min after injection, the rats were placed under light ether anaesthesia, a midline incision was made, the heart was exposed and blood was removed by cardiac puncture. The hepatic vein and spinal cord were clamped with haemostats, and saline was perfused through the heart for 5 sec to free the brain of blood (CHIIUGOS, GREENGARD and UDENFRIEND, 1960). The animals were sacrificed, the brains and livers were quickly removed and blotted with filter paper, and the free amino acids were isolated with minor modifications according to TALLAN, MOORE and STEIN (1956) and quantitated by ion-exchange chromatography (SPACKMAN, STEIN and MOORE, 1958). RESULTS 45

Disaggregation of polyribosomal structures has been demonstrated in fetal rat brains following treatment of the maternal animal with para‐chloro‐d,l‐phenylalanine (used primarily to inhibit maternal phenylalanine hydroxylase, EC 1.14.3.1) and with phenylalanine (used to raise the level of circulating phenylalanine in maternal and fetal plasma). Since highly disaggregated polyribosomal systems cannot support normal levels of protein synthesis in vitro, it has been postulated that the composition of the free amino acid pool(s) plays a regulatory role in protein synthesis through the intermediary effect of polyribosomal aggregation‐disaggregation. We believe that a possibly prolonged period of disaggregation of neuronal polyribosomes might disrupt neuronal protein synthesis sufficiently in utero to produce the mental insufficiency observed in the offspring of untreated maternal phenylketonurics.

terized by marked degeneration and necrosis of monolayer cultures. Multiple small foci of degeneration and necrosis were observed as early as 16 hours post inoculation. These focal areas enlarged until most cells were affected by 72 hours after exposure to the virus. ' Early cellular changes consisted of rounding of cells with condensation of the cytoplasm. These changes were often accompanied by vacuolization or ballooning of the cytoplasm and shrinkage of the nucleus. Next, necrosis, as evidenced by pyknosis of 'khe nucleus and lysis of the cell, occurred 'rapidly. Many of the pyknotic nuclei as-* sumed a rod or horseshoe shape. Neither intranuclear nor intracytoplasmic inclusions were observed.Discussion. Duck hepatitis virus had been propagated in cell cultures derived from chicken embryos but cytopathic changes were not observed(2-4). CPE was first noticed in our experiments during the 8th passage, of DHV in DEK cell cultures. Cytopathic effects occurred consistently from the 16th through the 26th passage in this system and usually appeared 24 to 36 hours after inoculation with virus.Cell culture fluid containing infectious virus produced gross lesions of DHV in chicken embryos(5). Duck serum, known to contain antibodies against DHV, inhibited the production of CPE in DEK cell cultures and prevented occurrence of lesions in chicken embryos,In other experiments, ducks inoculated with 2nd egg passage origin DHV exhibited gross and microscopic lesions of duck hepa-titis (6). A liver suspension obtained from one of the affected birds was inoculated into DEK cell cultures. Focal areas of cytopathic changes similar to those described above have been sporadically observed during 5 passages of the liver material in DEK cell cultures. Fluid from the 25th DEK cell passage containing infectious virus and showing CPE failed to produce cytopathic changes in 2 passages in chicken embryo kidney cell cultures prepared in the same manner and with the same nutrient medium used for preparation of DEK cultures. These findings would suggest that the host cell is of prime importance in determining the nature and degree of cytopathic changes resulting from infection by DHV.Summary. The cytopathic effect of duck hepatitis virus on duck embryo kidney cell cultures was characterized by marked degeneration and necrosis of the cells of the monolayers. Cytopathic effects occurred consistently through several viral passages and were usually apparent within 24 to 36 hours after inoculation. Inclusion bodies were not observed in the infected cells.

Antibody synthesis in response to the injection of diphtheria toxoid into rabbits and rats was used to study the effects of an excess of the amino acid L-phenylalanine on protein synthesis. Injections of phenylalanine produced a marked inhibition of antibody synthesis in both the rat and the rabbit. The dosage of phenylalanine used caused an increase over the normal concentration of phenylalanine in the plasma and spleen of rats, but did not cause a loss of weight or serum protein changes in the treated animals.