Mercaptan and dicarboxylate inhibitors of hamster dihydroorotase

In mammals, dihydroorotase is part of a trifunctional protein, dihydroorotate synthetase, which catalyzes the first three reactions of de novo pyrimidine biosynthesis. Dihydroorotase catalyzes the formation of a peptide-like bond between the terminal ureido nitrogen and the beta-carboxyl group of N-carbamyl-L-aspartate to yield heterocyclic L-dihydroorotate. A variety of evidence suggests that dihydroorotase may have a catalytic mechanism similar to that of a zinc protease [Christopherson, R. I., & Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370]. Tight-binding inhibitors of the zinc proteases, carboxypeptidase A, thermolysin, and angiotensin-converting enzyme have been synthesized that combine structural features of the substrates with a thiol or carboxyl group in an appropriate position to coordinate a zinc atom bound at the catalytic site. We have synthesized (4R)-2-oxo-6-thioxohexahydropyrimidine-4-carboxylate (L-6-thiodihydroorotate) and have found that this analogue is a potent competitive inhibitor of dihydroorotase with a dissociation constant (Ki) in the presence of excess Zn2+ ion of 0.17 +/- 0.02 microM at pH 7.4. The potency of inhibition by L-6-thiodihydroorotate in the presence of divalent metal ions decreases in the order Zn2+ greater than Ca2+ greater than Co2+ greater than Mn2+ greater than Ni2+; L-6-thiodihydroorotate alone is less inhibitory and has a Ki of 0.85 +/- 0.14 microM. 6-Thioorotate has a Ki of 82 +/- 8 microM which decreases to 3.8 +/- 1.4 microM in the presence of Zn2+. Zn2+ alone is a moderate inhibitor of dihydroorotase and does not enhance the potency of other inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular forms of acetylcholinesterases in Alzheimer's disease

In this study, we examined 26 cases of Alzheimer's disease (AD) and 14 age-matched controls. In Brodmann area 21 cerebral cortex of the AD cases, there was no change in soluble G1 and G4 acetylcholinesterase (AChE) (EC 3.1.1.7), a significant 40% decrease in membrane-associated G4 AChE, significant 342 and 406% increases in A12 and A8 AChE, and a significant 71% decrease in choline acetyltransferase (ChAT) (EC 2.3.1.6). Our working hypothesis to account for these changes postulates that soluble globular forms are unchanged because they are primarily associated with intrinsic cortical neurons that are relatively unaffected by AD, that ChAT and membrane-associated G4 AChE decrease because they are primarily associated with incoming axons of cholinergic neurons that are abnormal in AD, and that asymmetric forms of AChE increase because of an acrylamide-type impairment of fast axonal transport in diseased incoming cholinergic axons. In the nucleus basalis of Meynert (nbM) of the 26 AD cases, there was a significant 61% decrease in the number of cholinergic neurons, an insignificant 23% decrease in nbM ChAT, a significant 298% increase in nbM ChAT per cholinergic neuron, and a significant 7% increase in the area of cholinergic perikarya. To account for the increased ChAT in cholinergic neurons and the enlargement of cholinergic perikarya, we propose that slow axonal transport may be impaired in nbM cholinergic neurons in AD.     

Citrate-cleavage enzyme, `malic'' enzyme and certain dehydrogenases in embryonic and growing chicks

The activities of several enzymes possibly implicated in lipogenesis were measured in the soluble fraction of homogenates of liver and adipose tissue of embryonic and growing chicks. The activities of adipose-tissue enzymes showed little or no change. The activities of hepatic hexose monophosphate-shunt dehydrogenases, malate dehydrogenase, 3-phosphoglyceraldehyde dehydrogenase and NAD-linked α-glycerophosphate dehydrogenase also showed little or no change. Isocitrate dehydrogenase activity in liver rose to a peak on the day of hatching and fell to half the peak value during the next 12 days, where it remained to 26 days after hatching. The activities of `malic' enzyme and citrate-cleavage enzyme showed very low stable values in embryonic liver and remarkable rises during the early part of the post-hatching period. An 85-fold increase in the activity of `malic' enzyme activity was completed in 7 days and a 15-fold increase in that of citrate-cleavage enzyme in 5 days. The activities then attained were maintained up to 26 days after hatching. 2. The increases in the activities of hepatic citrate-cleavage enzyme and `malic' enzyme occurred simultaneously with a marked increase in lipogenesis, suggesting a relationship of these enzymes to lipogenesis in chick liver. By contrast, activity of the hexose monophosphate-shunt dehydrogenases does not appear to be thus associated.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Regulation of the activity and synthesis of malic enzyme in 3T3-L1 cells

Malic enzyme activity in differentiated 3T3-L1 cells was about 20-fold greater than activity in undifferentiated cells. A new steady-state level was achieved about 8 days after initiating differentiation of confluent cultures with a 2-day exposure to dexamethasone, isobutylmethylxanthine, and insulin. This increase in enzyme activity resulted from an increase in the mass of malic enzyme as detected by immunotitration of enzyme activity with goat antiserum directed against purified rat liver malic enzyme. Malic enzyme synthesis was undetectable in undifferentiated cells and increased to about 0.2% of soluble protein in differentiated cells, suggesting that the increase in enzyme mass was due primarily to an increase in enzyme synthesis. Thyroid hormone, a potent stimulator of malic enzyme activity in hepatocytes in culture and in liver and adipose tissue in intact animals, decreased or increased malic enzyme activity in differentiating 3T3-L1 cells by about 40% when it was removed or added to the medium, respectively. Insulin, another physiologically important regulator of malic enzyme activity in vivo, had no effect on the initial rate of accumulation of malic enzyme activity in the differentiating cells and caused a 30 to 40% decrease in the final level of enzyme activity in the fully differentiated cells. Cyclic AMP, a potent inhibitor of malic enzyme synthesis in hepatocytes in culture, inhibited this process in 3T3-L1 cells by 30%. Malic enzyme is like several other enzymes in that the large increase in its concentration which accompanies differentiation of 3T3-L1 cells is due to increased synthesis of enzyme protein. However, the hormonal modulation of malic enzyme characteristic of liver and adipose tissue in intact animals does not appear to occur in differentiated 3T3-L1 cells, suggesting that differentiated 3T3-L1 cells may not be an appropriate model system in which to study the hormonal modulation of malic enzyme that occurs in liver and adipose tissue of intact animals.     

Nutritional and hormonal regulation of the translatable levels of malic enzyme and albumin mRNAs in avian liver cells in vivo and in culture

Relative synthesis of malic enzyme is stimulated 25-to 100-fold by feeding neonatal ducklings or by incubating embryonic chick hepatocytes in culture with triiodothyronine. Synthesis of the enzyme is almost completely blocked when fed birds are starved or when triiodothyronine-treated hepatocytes in culture are also treated with glucagon. Cytoplasmic poly(A)+ RNA was isolated from livers of intact ducklings or hepatocytes in culture treated as described above and translated in an mRNA-dependent rabbit reticulocyte lysate. The identity of malic enzyme synthesized in the cell-free system was confirmed by virtue of its antigenicity, subunit molecular weight, and proteolytic peptide pattern. Translatable levels of malic enzyme mRNA paralleled changes in relative synthesis of malic enzyme in vivo and in hepatocytes in culture. Translatable levels od albumin mRNA were either unaffected or changed in a direction opposite to that of malic enzyme mRNA. Thus, both nutritional and hormonal regulation of malic enzyme synthesis involves regulation of cytoplasmic translatable malic enzyme mRNA levels. The hepatocyte culture system is ideally suited for future studies on the regulation of malic enzyme mRNA synthesis and/or degradation by thyroid hormone and glucagon.     

Bacteriophage biocontrol of plant pathogens: fact or fiction?

Bacterial resistance due to the misuse of antibiotics has become a global issue and alternative methods are being developed that might decrease the use of antimicrobials in agricultural settings. Bacteriophage therapy represents a novel way to control the growth of plant-based bacterial pathogens. Although this method shows promise, a recent paper by Gill and Abedon has shown that the complex bacteriophage-host interactions in the plant environment must be investigated further.