Tyrosinaemia type I--de novo mutation in liver tissue suppressing an inborn splicing defect.

Many patients with tyrosinaemia type 1 have a mosaic pattern of fumarylacetoacetase (FAH) immunopositive or immunonegative nodules in liver tissue. This phenomenon has been explained by a spontaneous reversion of the mutation in one allele to a normal genotype, but only a few nodules have been examined. We now report on a Norwegian patient, compound heterozygous for the mutations IVS12g(+5)-->a and G(1009-->)A, with liver mosaicism, but with an immunopositive nodule in which both primary mutations were intact. In the immunopositive hepatocytes of this nodule, genetic analyses showed a new mutation, C(1061-->)A, 6 bp upstream of the primary mutation IVS12g(+5)-->a in the FAH gene. The splicing defect caused by the primary mutation is most likely suppressed by the new mutation due to improvement of the splicing site. In the same liver we demonstrate another nodule of regenerating immunopositive tissue due to reversion of one of the primary mutations to a normal genotype. Together with the original cells this makes a triple mosaicism of hepatocytes with one, two or three point mutations in the FAH gene.

Role of compensatory growth in lactation: a stair-step nutrient regimen modulates differentiation and lactation of bovine mammary gland.

Twenty Holstein heifers were assigned to either a control or test (stair-step compensatory; food restriction, followed by refeeding) growth group. The stair-step growth model was designed to induce distinctive compensatory allometric mammary development during three different hormonal states, coinciding with prepubertal, pubertal, and late gestational stages. Mammary tissues obtained by biopsy from pregnant and lactating cows were used for acini culture and chemical composition analysis. Test mammary tissues from late gestation heifers contained less (P = .067) fat than control counterparts (731 vs. 628 mg/g). DNA, RNA, and protein contents in test mammary tissue from late gestation cows were higher (P = .001 to .088) compared to control tissue. Milk protein secretion of test acini in culture was increased more than 20% over that of the control acini. Lactating mammary acinar cells in culture from test cows exhibited a 14% increase in amino acid uptake over that of the control. RNA dot-blot hybridization analysis revealed that alpha s1- and beta-casein mRNA accumulation in acini from test tissue was increased (P = .027 to .042) as much as 40-50%. During both food restriction and refeeding, concentrations of plasma growth hormone were elevated. Food restriction decreased levels of plasma insulin, whereas levels of insulin were elevated during refeeding. The long-term influence of compensatory growth upon subsequent lactation performance was also evaluated. Milk production data derived from previous two sequential growth trials showed that cows from test groups produced approximately 10% more (P = .001) milk compared to control counterparts. Results reinforce our postulation that compensatory growth induced by nutritional modulation regulates the differentiation and functional activity of the mammary gland.

Casein gene expression in bovine mammary gland.

The objectives were to examine the rate of synthesis of casein mRNA transcripts in bovine mammary tissue at different hormonal states and to study the effects of hormonal stimuli (insulin, hydrocortisone, and prolactin) on the accumulation of casein mRNA and on the rate of protein secretion by epithelial cells from bovine mammary tissues. Total cytoplasmic RNA was extracted from mammary tissues of cows obtained by biopsy (8 mo pregnant) and upon slaughter (lactating). The relative specific activities of cytoplasmic mRNA for alpha s1-, alpha s2-, beta-, and kappa-casein were about 3.2, 4.6, 3.3, and 4.5-fold higher in tissues of lactating cows than in those of 8 mo pregnant cows. Mammary alveolar epithelial cells retained hormone-inducible milk protein gene expression for total milk protein gene expression for total milk protein secretion and for alpha s1- and beta-casein messages. Prolactin, even in the absence of insulin and hydrocortisone, induced significant amounts of milk protein mRNA. Hydrocortisone in the presence of prolactin amplified the lactogenic effects on mammary epithelium. Maximal induction of beta-casein mRNA and protein secretion occurred when all three hormones were present.

Toxicity of 1-phenylcyclohexene and its interaction with phencyclidine.

The toxicity of 1-phenylcyclohexene (PC), a pyrolysis product of phencyclidine (PCP), and its interaction with PCP were evaluated. The ip LD50 of PC in Swiss male mice was 22 mmol/kg. Treatment of mice with PC at 2.2 mmol/kg/day, ip, for up to 7 days increased the liver/body weight ratio, which returned to normal within 7 days after PC withdrawal. Increases of 32% in serum glutamic-oxalacetic transaminase (SGOT) and 94% in serum glutamic-pyruvic transaminase (SGPT) were observed within 4 hr following the initial (Day 1) dose of PC. Smaller increases in the SGOT activity continued following Day 2 and 3 PC administrations. The SGPT activity remained elevated after these treatments. Activities of both enzymes, however, returned to normal within 24 hr following daily PC injections. No pathologic changes were observed in liver, brain, spleen, kidneys, and lungs with light microscopy. PC treatment for 4 days at 2.2 or 4.4 mmol/kg produced proliferation along with dilatation and fragmentation of the endoplasmic reticulum in liver. Scattering of ribosomes in the cytoplasm and dilatation of rough-surfaced cisternae were prominent at the higher dosage. Pretreatment of animals for 4 days with PC (1.1, 2.2, and 4.4 mmol/kg, ip) decreased pentobarbital- (60 mg/kg) induced sleeping time by 27, 64, and 80% and lowered PCP- (16.4 mumol/kg) stimulated locomotor activity by 18, 28, and 41%, respectively. Pretreatment of animals with PC for 1 hr inhibited (ED50: 2.3 mmol/kg) the PCP-induced locomotion. These results indicate that the PC treatment during a 7-day period produces some undesirable effects on liver function, which are reversible on its discontinuation. However, PC also weakens toxic effects of PCP.

The role of hepatic microsomal enzymes in the modulation of phencyclidine-induced toxicity.

The LD50 of phencyclidine (PCP, 234 mumol/kg, i.p.) in male Swiss mice decreased by 62% in animals pretreated with 2-diethylamino-2,2-diphenylvalerate hydrochloride (SKF-525A, 40 mg/kg), and increased by 74% and 20% in animals pretreated with sodium phenobarbital (75 mg/kg), and 3-methylcholanthrene (70 mg/kg), respectively, No Significant change in the LD50 was observed with cysteine or diethylmaleate pretreatment. The treatment with PCP at 179 mumol/kg/day i.p. for 7 days resulted in body weight decrement in the first 2 days and gradual increment thereafter. The increase was only 33% of the control group. The food intake was also lower in the PCP treated group of animals. PCP withdrawal led to an increase in food intake as well as body weight at a normal rate. The ratio of liver weight to body weight was not significantly higher than that of control during the treatment period. The administration of PCP for 7 days did not alter the activities of liver function enzyme markers. However, within 12 h of the initial PCP treatment a 85% increase in activity of serum glutamicoxalacetic transaminase was observed. Later the enzyme activity reached close to normal levels. No liver lesions at the light microscopic level were observed. Treatment of mice for 4 days with PCP (179 mumol/kg) caused no significant change in pentobarbital sleeping time.