Effects of antihypertensive drugs on hepatic heme biosynthesis,and evaluation of ferrochelatase inhibitors to simplify testing of drugs for heme pathway induction

Effects of a series of antihypertensive drugs on the activity of δ-aminolevulinate synthase and on the formation of porphyrins and cytochrome P-450 were examined in the 18-day-old chick embryo liver in ovo. Hydralazine, pargyline, phenoxybenzamine, clonidine, and spironolactone were found to induce δ-aminolevulinate synthase in this system. These drugs therfore have the potential to precipitate clinical expression in human hereditary hepatic porphyrias and should be avoided or used with caution in patients with these disorders. Differential effects of these and other drugs were observed in the avian liver, in that δ-aminolevulinate synthase was more commonly induced thatn were porphyrins and cytochrome -450; the synthase was usually highest 6–12 h after injection, whereas porphyrins and cytochrome P-450 were highest at 24 h. Furthermore marked porphyrin accumulation was not seen with many drugs that induce σ-aminolevulinate synthase and cytochrome P-450 but was more characteristic of compounds that reduced the metabolism of protoporphyrin to heme, such as 1,4-dihydro-3,5-dicarbethoxycollidne (DDC) and high dose of hydralazine. A sensitive and convenient method to test for capacity to induce heme biosynthesis was adapted for use in the chick embryo liver. This employed a relatively small “priming” dose (0.25 mg) of DDC given with a drug being tested and a fluorometric assay of porphyrins in a liver homogenate obtained at 24 h. This simple method should facilitate screening for those drugs which induce the synthesis of δ-aminolevulinate synthase and/or cytochrome P-450 and are potentially dangerous to patients with hereditary hepatic porphyria.     

Bimodal activation of SMC ATPase by intra- and inter-molecular interactions.

Structural maintenance of chromosomes (SMC) proteins play fundamental roles in higher-order chromosome dynamics from bacteria to humans. It has been proposed that the Bacillus subtilis SMC (BsSMC) homodimer is composed of two anti-parallel coiled-coil arms, each having an ATP-binding domain at its distal end. It remains totally unknown, however, how the two-armed structure supports ATP-dependent actions of BsSMC. By constructing a number of mutant derivatives including 'single-armed' BsSMC, we show here that the central hinge domain provides a structural flexibility that allows opening and closing of the two arms. This unique structure brings about bimodal regulation of the SMC ATPase cycle. Closing the arm can trigger ATP hydrolysis by allowing an end-end interaction within a dimer (intramolecular mode). When bound to DNA, ATP promotes a dimer-dimer interaction, which in turn activates their DNA-dependent ATPase activity (intermolecular mode). Our results reveal a novel mechanism of ATPase regulation and provide mechanistic insights into how eukaryotic SMC protein complexes could mediate diverse chromosomal functions, such as chromosome condensation and sister chromatid cohesion.     

The Tc3 Family of Transposable Genetic Elements in Caenorhabditis Elegans

We describe genetic and molecular properties of Tc3, a family of transposable elements in Caenorhabditis elegans. About 15 Tc3 elements are present in the genomes of several different wild-type varieties of C. elegans, but Tc3 transposition and excision are not detected in these strains. Tc3 transposition and excision occur at high frequencies, however, in strain TR679, a mutant identified because of its highly active Tc1 elements. In TR679, Tc3 is responsible for several spontaneous mutations affecting the unc-22 gene. Tc3-induced mutations are unstable, and revertants result from precise or nearly precise excision of Tc3. Although Tc3 is very active in TR679, it is not detectably active in several other mutator mutants, all of which exhibit high levels of Tc1 activity. Tc3 is 2.5 kilobases long, and except for sequences near its inverted repeat termini, it is unrelated to Tc1. The termini of Tc3 are inverted repeats of at least 70 base pairs; the terminal 8 nucleotides of Tc3 are identical to 8 of the terminal 9 nucleotides of Tc1.     

A radiation-reduced hybrid cell line containing 5 Mb/17 cM of human DNA from 9q34.

Disease gene loci for tuberous sclerosis (TSC1), idiopathic torsion dystonia (DYT1), and nail-patella syndrome (NPS1) have been mapped by genetic linkage analysis to human chromosome 9q band 34. To create a resource for physical mapping and manipulation of this region of the genome, we have created a radiation-reduced hybrid cell line containing DNA from human 9q34 as its only human component. This cell line, E6B, has been characterized by Southern blot and PCR analysis using a panel of 9q markers and fluorescent in situ hybridization. We estimate that it contains 5 Mb of human DNA, equal to 17 cM of genetic distance, extending from AK1 to ABO on 9q34.     

Effect of relaxin on facilitation of parturition in dairy heifers

Purified pig relaxin (3000 U/mg) was injected i.m. into pregnant Holstein dairy heifers on Day 276 or 277 to determine its effect on parturition and sequential measurements of the pelvic area, cervical dilatation, and peripheral blood-plasma concentrations of progesterone and relaxin. Treatments included phosphate-buffer saline (2 ml, Group C, N = 7), relaxin once (1 mg, Group 1R, N = 7), and twice (2 mg, 12 h apart; Group 2R, N = 7). Intervals (mean +/- s.e.) between the first injection of relaxin or PBS and calving were 64 +/- 17, 80 +/- 19 and 125 +/- 34 h for Groups 2R, 1R and C, respectively. The calving intervals were reduced in Groups 2R (P less than 0.01) and 1R (P less than 0.05) compared with Group C. The incidence of dystocia was 29% (2 of 7) in Group 2R and 43% (3 of 7) in Group 1R compared with 57% (4 of 7) in Group C (P less than 0.01). Body weights and ratios of males to females of the calves were similar (P greater than 0.05) between groups. Progesterone plasma concentrations decreased (P less than 0.01) earlier in Groups 1R and 2R compared with Group C, and this acute decrease began within 6 h of treatment. At 24 h after relaxin or PBS injection, progesterone concentrations were 2.7 +/- 1.1 ng/ml for Group 2R, 3.5 +/- 0.9 ng/ml for Group 1R, and 6.0 +/- 0.1 ng/ml for Group C. Relaxin reached peak blood-plasma levels of 19 +/- 2.2 ng/ml 1 h after injection of relaxin, but remained unchanged, 0.3 +/- 0.01 ng/ml, in Group C. Pelvic area was increased 26%, 22% and 14% and cervical dilatation was increased 109%, 76% and 53% 48 h after injection in Groups 2R, 1R and C, respectively, but these responses were similar among groups at the time of parturition. We conclude that two i.m. injections of relaxin facilitated earlier calving, acutely decreased progesterone secretion, increased cervical dilatation and pelvic area expansion, and decreased the incidence of dystocia in dairy heifers.     

Early indicators of bladder carcinogenesis produced by non-genotoxic agents

There are several early indicators of non-genotoxic bladder tumorigenicity. The non-invasive indications are polydipsia, diuresis, changes in urine pH and urinary cation concentrations, especially Na and Ca. The indicators requiring invasive techniques are increased bladder weight and increased cell replication assessed by DNA labeling or histologically as epithelial hyperplasia. SEM has been used to characterize bladder surface changes, and a reduction of bladder tissue Ca has been implicated in one mechanism leading to bladder cancer. Wherever multiple species have been tested, the non-genotoxic bladder carcinogens have induced bladder responses only in rats. This is true whether the criterion was complete carcinogenesis, promotion or short-term indicators. It is also evident that the response can vary greatly within rat strains and is dependent upon the diet being fed. These variables make the relevance of the results obtained in the rat bladder of questionable significance to man. In relation to chronic studies it is clear that as the male rat ages it loses the capacity to concentrate urine, probably because of the endemic, age-progressive loss of functional renal tissue. It is also clear that the bladder grows to accommodate the increase in urine output. Thus it is likely that any agent or treatment that causes bladder damage may be associated with increased neoplasia expression in aged male rats. No other species shows the degree of spontaneous nephrosis seen in the male rat, a condition which is both rat strain- and diet-dependent. Finally, it should be recognized that while there are some early indicators of bladder tumorigenesis that can be useful as warning signs, each compound is likely to yield unique responses when its mechanism is studied in detail. To facilitate discussion of the parameters that have been identified as early indicators of bladder tumorigenesis associated with non-genotoxic agents, the proposed mechanisms of cancer development, the information which led to these proposals and a critique of the mechanisms have been presented.