Impaired development of mammary glands in scorbutic rats unable to synthesize ascorbic acid

The effects of ascorbic acid (AsA)-deficiency on the development of mammary glands were investigated using mutant rats (osteogenic disorder syndrome rats; ODS rats) with hereditary inability to synthesize AsA. Female ODS rats of 21 days old were castrated and divided into two groups. One group was given AsA in their drinking water, and the other was not. All the rats received a daily injection of oestradiol-17 beta and progesterone (EP) from day 28 to day 49 of age. After EP treatment, the concentrations of AsA in the mammary glands of rats not given AsA were less than one tenth of those of rats given AsA and the contents of hydroxyproline in the mammary glands of the former rats were about half of those in the latter. Furthermore, the concentration of serum prolactin in rats not given AsA was reduced to about one third of that in rats given AsA. After EP treatment, whole mounts of mammary glands showed that in rats not given AsA the development of ducts was impaired and there was extensive accumulation of endbuds. Consistent with this finding, EP injections did not increase the area of parenchyma in the mammary glands of rats not given AsA, whereas they increased it about 2-fold in rats given AsA. Moreover, after EP treatment the amount of alpha-lactalbumin was significantly less in the mammary parenchyma of rats not given AsA than in that of rats given AsA. On the other hand, AsA deficiency did not impair the response of the mammary cells to insulin or prolactin in terms of DNA synthesis and alpha-lactalbumin production. These findings indicate that AsA deficiency impaired the development of mammary glands. This effect may be partly attributable to a defect in collagen synthesis in the mammary glands and a decrease in the concentration of serum prolactin.     

Screening for prolonged incubation of HTLV-I infection in British and Jamaican relatives of British patients with tropical spastic paraparesis.

OBJECTIVE--To compare the prevalence of antibody to and proviral DNA of the retrovirus HTLV-I in relatives of 11 British patients with tropical spastic paraparesis who had migrated from Jamaica before they developed symptoms, and to examine factors possibly related to transmission of HTLV-I. DESIGN--Migrant, family study. Antibody state was determined by several methods and confirmed by western blotting; the polymerase chain reaction was used to detect proviral DNA. SETTING--Britain and Jamaica. SUBJECTS--All available first degree relatives: those born and still resident in Jamaica (group 1); those born in Jamaica who migrated to Britain (group 2); and index patients'' children who were born and resident in Britain (group 3). All had been breast fed and none had had blood transfusions. RESULTS--Of the 66 living relatives, 60 were traced. Seroprevalence among those born in Jamaica (irrespective of current residence) was 22% (10/46; 95% confidence limits 9 to 34%) compared with zero among British born offspring (0/14) and was higher in group 2 at 33% (7/21; 12 to 55%) than in group 1 at 12% (3/25; 0 to 25%). (Patients in group 1 had the greatest mean age.) Proviral DNA was not detected in any subject negative for HTLV-I antibody, making prolonged viral incubation in those negative for the antibody unlikely. CONCLUSION--In this sample factors related to place of birth and early residence were more important in transmission of HTLV-I than maternal or age effects. In areas with a low to moderate prevalence policies of preventing mothers who are carriers of the virus from breast feeding would be premature.     

Molecular genetics of polyamine synthesis in eukaryotic cells

The polyamines putrescine, spermidine and spermine are important cellular constituents involved in the regulation of cell growth and differentiation. Their intracellular levels are regulated by a multitude of mechanisms affecting their synthesis, degradation, uptake and excretion. As a result of the application of molecular biology techniques, some of these mechanisms are presently being unravelled, and are providing a basis for the rational development of novel agents effective against proliferative disorders and various parasitic diseases.     

The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification.

The amplified DNA of HC50474, a Chinese hamster fibroblast cell line selected in three steps for high resistance to coformycin, consists chiefly of 150 copies of a large inverted duplication including the adenylate deaminase gene. Most if not all of these units are more than 2 x 120 kb long. The inverted duplication was first detected in the cells recovered from the second selection step, at the same chromosomal location as the first step amplified units. Its formation and amplification appear to be coupled since the second step cell line already contained 40 copies of this novel structure. Reamplification of the inverted duplication occurred at the third step of selection concomitant with the loss of amplified DNA acquired during the first step. The head-to-head junction has been formed by recombination within a recombinational hotspot described previously [Hyrien, O., Debatisse, M., Buttin, G. and Robert de Saint Vincent, B. (1987) EMBO J., 6, 2401-2408]. Sequences at the joint and in the corresponding wild-type region reveal that the crossover sites, one of which occurs in the putative promoter region of B2 repeat, are located at the top of significant stem-loop structures and that patchy homologies between the parental molecules on one side of the breakpoints allow alignment of these crossover sites. We present a model which explains the formation and amplification of this and other large inverted duplications by errors in DNA replication.     

Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells.

H-35 rat hepatoma cells were labelled with [32P]orthophosphate and their insulin receptors isolated on wheat germ agglutinin (WGA)-agarose and anti-(insulin receptor) serum. The incubation of these cells with 10 mM-H2O2 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the beta subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H2O2-treated H-35 cells and the purified fractions incubated with [gamma-32P]ATP and Mn2+. Phosphorylation of the beta subunit of insulin receptors obtained from H2O2-treated cells was 150% of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H2O2-treated cells, as measured by phosphorylation of src-related synthetic peptide, was increased about 4-fold over control cells. These data suggest that in intact cell systems, H2O2 may increase the insulin receptor kinase activity by inducing phosphorylation of the beta subunit of insulin receptor.     

Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes.

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.     

Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15.

It has been previously reported that both the cysteinyl-endo-oligopeptidase A and the metalloendopeptidase EC 3.4.24.15 are able to generate enkephalin from a number of enkephalin-containing peptides, including dynorphin A1-8. The present study shows that only endo-oligopeptidase A is able to generate [Leu5]enkephalin and [Met5]enkephalin from dynorphin A1-8 and from metorphamide respectively. It is also shown that endo-oligopeptidase A neither hydrolyses the specific EC 3.4.24.15 substrate alpha-N-benzoyl-Gly-Ala-Ala-Phe p-aminobenzoate, nor is inhibited by the specific EC 3.4.24.15 inhibitor N-[1(RS)-carboxy-2-phenylethyl]-alpha-Ala-Ala-Phe p-aminobenzoate.