Effects of inhibitors of ornithine and S-adenosylmethionine decarboxylases on L6 myoblast proliferation

The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.     

The preparation of bile acid amides and oxazolines, II, the synthesis of the amides and oxazolines of ursodeoxycholic acid, deoxycholic acid, hyodeoxycholic acid and cholic acid

Bile acid amides and oxazolines were synthesized by a sequence of steps involving the reaction of the free bile acid with formic acid to yield the formyloxy derivative, preparation of the formyloxy acid chloride, condensation of the acid chloride with 2-amino-2-methyl-1-propanol to give the amide and, finally, cyclization of the amide with thionyl chloride to give the oxazoline. The oxazolines were characterized by physical constants, thin layer and gas-liquid chromatography and identified by elemental analysis and gas-liquid chromatography-mass spectrometry. Some of the bile acid oxazoline derivatives alter the activity of bacterial 7-dehydroxylases $\text{in vitro}$, and inhibit the growth of certain anaerobic bacteria in pure culture.     

Different repetition frequencies of a 120 base-pair DNA-element and its arrangement in Chironomus thummi thummi and Chironomus thummi piger

The repetition frequency of a highly repetitive DNA sequence has been measured in the genomes of Ch. thummi thummi and Ch. th. piger. This sequence is known to be involved in the evolutionary duplication of defined chromosomal segments leading to a significant increase in the genome size of Ch. th. thummi. Reassociation of this highly repetitive DNA sequence which has a repeat length of 120 base-pairs, with total Ch. th. thummi and Ch. th. piger DNA has shown that the repetition frequency in the Ch. th. thummi DNA is 5.5 fold higher than in Ch. th. piger. In both genomes a 120 base-pair sequence is present as tandemly repeated sequence as shown by Southern analysis.     

Structure of the rat prolactin gene

The organization and sequence of the rat preprolactin gene has been investigated. Analysis of two different plasmids containing pituitary cDNA inserts has provided the complete 681-nucleotide coding sequence of preprolactin as well as 17 nucleotides preceding the initiation codon and 90 nucleotides following the termination codon. Digestion of rat chromosomal DNA with the restriction endonuclease Eco RI followed by size fractionation and hybridization to a labeled prolactin cDNA probe has demonstrated that prolactin genomic sequences are located on 6.0-, 3.9-, and 2.9-kilobase fragments. The 6.0- and 3.9-kilobase fragments were isolated from a library of cloned rat DNA fragments. The sequence of more than 1800 nucleotides of the cloned DNA has been determined. The sequenced region contains coding regions of 180 and 189 nucleotides which specify the COOH-terminal 123 amino acids of the 227-amino-acid sequence of rat preprolactin. These coding regions are separated by an intervening sequence of 597 nucleotides. At least one other large intervening sequence separates this region from the region coding for the NH2-terminal portion of preprolactin. Hybridization experiments suggested that the intervening sequences of the rat prolactin gene contain DNA sequences which are repeated elsewhere in the rat genome.     

Localization and characteristics of rat liver mitochondrial aldehyde dehydrogenases.

Two NAD-dependent aldehyde dehydrogenase enzymes from rat liver mitochondria have been partially purified and characterized. One enzyme (enzyme I) has molecular weight of 320,000 and has a broad substrate specificity which includes formaldehyde; NADP is not a cofactor for this enzyme. This enzyme has K_m values for most aldehydes in the micromolar range. The isoelectric point was found to be 6.06. A second enzyme (enzyme II) has a molecular weight of 67,000, a K_m value for most aldehydes in the millimolar range but no activity toward formaldehyde. NADP does serve as a coenzyme, however. The isoelectric point is 6.64 for this enzyme. By utilization of the different substrate properties of these two enzymes it was possible to demonstrate a time-dependent release from digitonin-treated liver mitochondria. The high K_m, low molecular weight enzyme (enzyme II) is apparently in the intermembrane space while the low K_m, high molecular weight enzyme (enzyme I) is in the mitochondrial matrix and is most likely responsible for oxidation of acetaldehyde formed from ethanol.     

Characteristics of the induction of aldehyde dehydrogenase in rat liver

The activity of rat liver supernatant aldehyde dehydrogenase is increased by phenobarbital treatment in one selected strain (RR) but not in another strain (rr) of animals derived from randomally bred populations (Deitrich, Collins, and Erwin (1972) J. Biol. Chem., 247, 7232). Before 14 days of age, increased enzyme activity after phenobarbital treatment is minimal but between 30 and 60 days of age there is a maximal increase in activity after phenobarbital treatment. Using animals of this age, it was shown that both cycloheximide and actinomycin D block this response to phenobarbital. Phenobarbital treatment decreases heat stability of crude preparations of the enzyme from RR rats, but increases heat stability of the enzyme from rr animals.     

A comparison of two timed-mating strategies for breeding pigtail monkeys. (Macaca nemestrina)

Pigtail monkeys (Macaca nemestrina) were time-mated using female perineal “sex skin” tumescence cyclicity as an indicator of ovulation time. The goal of these matings was production of infants of known gestational age for an investigation using a nonhuman primate model to study causes, correlates, and consequences of premature birth. Two breeding strategies were employed. The first involved allowing breeders constant access to one another for 72 hours at the time ovulation was predicted to occur from previous data on cyclicity of perineal tumescence. The second method limited exposure to two hours daily until perineal detumescence occurred. The second strategy has been considerably more effective than the first.