Poly(ADP-Ribose) Synthetase Activation: An Early Indicator of Neurotoxic DNA Damage

Abstract: DNA damage activates a nuclear enzyme poly(ADP-ribose) synthetase (PARS) that facilitates DNA repair by adding multiple ADP-ribose groups to nuclear proteins such as histones and PARS itself. N -Methyl- d -aspartate neurotoxicity may involve DNA damage excessively activating PARS to deplete its substrate NAD, as PARS inhibitors prevent this toxicity. We now show that PARS is rapidly and markedly activated in PC12 cells following treatment with neurotoxic agents, including the amyloid β-protein, hydrogen peroxide, N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its active metabolite N -methyl-4-phenylpyridine (MPP⁺). With MPP⁺, PARS activity is increased fivefold in 1 h and 20-fold by 3 h. By contrast, direct measurement of DNA damage by the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay shows no significant increase by 3 h and less than fourfold by 24 h. These findings indicate that PARS activity can provide a simple, sensitive, and early index of DNA damage following neurotoxic insults.     

A Genetic Analysis of the 63e-64a Genomic Region of Drosophila Melanogaster: Identification of Mutations in a Replication Factor C Subunit

We have performed and F(2) genetic screen to identify lethal mutations within the 63E-64A genomic region. We have isolated 122 mutations in 20 different complementation groups. Of these groups, 16 are represented by multiple alleles. We have also established that the Rop and Ras2 genes are located within the 63E-64A genomic domain at 64A10,11. We have sequenced 10.2 kb of DNA surrounding this gene pair and find that in addition to Rop and Ras2 there is another gene located within this DNA sequence. The gene product, which we have named Rfc40, shows 68% identity to the 40-kDa subunit of replication factor C. We find that the members of one complementation group (13 alleles) derived from our screen correspond to mutations in the Rop gene, whereas the members of another (five alleles) correspond to mutations in the Rfc40 gene. In addition we have isolated 11 new mutant alleles of the disembodied gene.     

Exocrine pancreatic secretion in response to a new CCK-analog, CCK33 and caerulein in dogs

We studied the relative molar potencies of a newly synthetized cholecystokinin nonapeptide [Thr28,Nle31]CCK[25-33], natural porcine CCK33 and synthetic caerulein in conscious dogs with chronic gastric and pancreatic fistulas. Peptides were dissolved in albumin-containing solutions to prevent loss from solution. The three peptides were found to be equipotent on a molar basis in stimulating exocrine pancreatic secretion. As [Thr28,Nle31]CCK9 is a peptide less susceptible to oxidation than other forms of CCK, it is an interesting analog with many uses for medical and biological research.     

Migration of smolts of Atlantic salmon (Salmo salar L.) and sea trout (Salmo trutta L.) in a chalkstream

Synopsis Migration of smolts in the River Piddle, Dorset, was studied over three years in relation to factors that could influence downstream movement. The river originates mostly from groundwater springs, resulting in stable flows and low turbidity except in very rainy weither. Fish were intercepted at the tidal limit in a fixed trap-net, and measurements of water temperature, discharge, turbidity, barometric pressure, rainfall and solar radiation taken nearby. Slightly increased turbidity and discharge following heavy rain initiated major movements during two nights of the total of 55 days studied. At other times large-scale movements rock place during sunny warm afternoons. Both solar radiation and water temperature were correlated with intensity and timing of movement. The pattern of migration is different from that reported on other rivers, reflecting the relatively stable flow regime of the chalkstream.     

Biosynthesis of prostaglandins and thromboxane B2 by fetal lung homogenates

The conversion of arachidonic acid to prostaglandins (PG's) and thromboxane B₂ (TXB₂) was investigated in homogenates from fetal and adult bovine and rabbit lungs. Adult bovine lungs were very active in converting arachidonic acid (100 μg/g tissue) to both PGE₂ (10.7 μg/g tissue) and TXB₂ (6.2 μ/g tissue). Smaller amounts of PGF_2α (0.9 μ/g) and 6-oxoPGF_1α were formed. Homogenates from fetal calf lungs during the third trimester of pregnancy were quite active in converting arachidonic acid to PGE₂, but formed very little TXB₂, PGF_2α or 6-oxoPGF_1α. Homogenates from rabbit lungs converted arachidonic acid (100 μg/g) mainly to PGE₂, both before and after birth. The amount of PGE₂ formed increased during gestation to a maximum of about 6 μg/g tissue at 28 days of gestation. It then decreased to a minimum (1.5 μg/g) which was observed 8 days after birth, followed by an increase to about 4 μg/g in older rabbits.     

Membrane mediated link between ion transport and metabolism in human red cells

When 10^?6 M oubain is added to human red cells that have been incubated without glucose for two hours, there is a significant shift in the ³¹P nuclear magnetic resonances of both phosphate groups of cellular 2,3-diphosphoglycerate, which is not found in control cells incubated with glucose. This means that an effect induced by ouabain on the outside of the red cell membrane is transmitted through the membrane to alter the environment of an intracellular metabolite. Experiments with glycolytic cycle inhibitors have indicated that the intracellular ligand responsible for the resonance shifts is monophosphoglycerate mutase which requires 2,3-diphosphoglycerate as a cofactor for the reaction it catalyzes. To account for this finding a hypothesis is presented that the (Na⁺ + K⁺)-ATPase in human red cells is linked to monophosphoglycerate mutase through the agency of phosphoglycerate kinase. Evidence is presented for the existence of phosphoglycerate kinase/monophosphoglycerate mutase in solution. It is shown that this complex can interact with the cytoplasmic face of (Na⁺ + K⁺)-ATPase at the outside surface of inside out red cell vesicles, and that this interaction is inhibited when 10^?6 M ouabain is contained within the vesicle. Neither monophosphoglycerate mutase nor phosphoglycerate kinase is significantly bound to the inside surface of the intact human red cell, but glyceraldehyde 3-phosphate dehydrogenase is; it is shown that this enzyme also interacts with the cytoplasmic face of the (Na⁺ + K⁺)-ATPase and that the interaction is inhibited by 10^?6 M ouabain.     

Enzymic activity and conformational properties of native and crosslinked glucose oxidase.

Glucose oxidase has been modified by reacting it with glutardialdehyde. The products obtained are water soluble and merely intramolecularly crosslinked; they exhibit high enzymic activity and good stability towards denaturing agents. Comparative circular dichroism studies have been performed with the native and crosslinked enzymes, as well as with the corresponding apoenzymes. The results suggest that the FAD coenzyme is not a gross structural determinant of glucose oxidase.     

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.