Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta

Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM.     

Optimisation of total urinary aldosterone estimation: comparison with other laboratory methods for assessment of mineralocorticoid status

We have demonstrated that conventional methods for measuring total urinary aldosterone (TUA) may markedly and inconsistently underestimate aldosterone output, since under the conditions usually employed (pH 1.0), the hydrolysis of aldosterone conjugates in urine is incomplete. The use of more acidic hydrolysis conditions (pH 0.2) overcomes this problem. However free aldosterone may be damaged at this pH. Therefore to accurately measure TUA output, it is necessary to isolate the undamaged aldosterone chromatographically and to correct for procedural losses based on the recovery of aldosterone tracer added to the urine prior to hydrolysis. We compared a number of laboratory estimates of aldosterone status (including urinary free aldosterone) with the 24-h urinary sodium output in normal subjects, since this provides a good bioassay of aldosterone. Sodium output correlated best with "optimised" 24 h TUA, i.e. hydrolysed at pH 0.2, (r = -0.589, P less than 0.001), and with plasma aldosterone (r = -0.504, P less than 0.005). Both aldosterone in random urine specimens and plasma renin activity correlated poorly with 24-h sodium output. Therefore, while the measurement of optimised TUA excretion provides the best index of aldosterone activity, assay of aldosterone in random specimens of plasma, which is more convenient for patient and laboratory, may be adequate for many clinical purposes.     

Cytochemical changes in hepatocytes of rats with endotoxemia or sepsis: localization of fibronectin, calcium, and enzymes

Bacterial lipopolysaccharide (LPS) is known to be implicated in the pathogenesis of endotoxemia and septic shock. The liver is the first vital organ to exhibit pathological alterations in shock. The present studies include immunoelectron microscopic localization of tissue fibronectin and cytochemical localization of calcium and enzymes in hepatocytes of animals with LPS-induced endotoxemia or cecal ligation-induced septic shock. The results showed increased staining of fibronectin in the basal (perisinusoidal) surfaces and in the cisternae of rough endoplasmic reticulum and the Golgi complex of hepatocytes in rats with endotoxemia or septic shock. Intracellular calcium content was significantly increased in the LPS-treated or septic rats. Calcium pyroantimonate precipitate was deposited predominantly on the outer surfaces of the RER of hepatocytes. In addition, diminution or depletion of glycogen, reduction of catalase-containing peroxisomes, increase of G-6-Pase activity, and depletion of cytochrome c oxidase in many mitochondria were also observed in hepatocytes of experimental animals. The overall results suggest that LPS stimulates: (a) hepatic synthesis and secretion of fibronectin; (b) uptake of calcium by hepatocytes; and (c) G-6-Pase activity. LPS treatment also leads to reduced numbers of peroxisomes and depletion of cytochrome c oxidase.     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.