Inhibition of Escherichia coli DNA polymerase-I by the anti-cancer drug cis-diaminedichloroplatinum(II): what roles do polymerases play in cis-platin-induced cytotoxicity?

Activities of Escherichia coli DNA polymerase-I were examined in the presence of the anti-tumor drug cis-diaminedichloroplatinum(II) and its inactive geometric isomer trans-diaminedichloroplatinum(II). The trans-isomer did not inhibit the enzyme activity. The anti-tumor drug, on the other hand, retarded the enzyme in its ability to extend the primer strand of DNA. Two alternative mechanisms of inhibition, covalent binding of cis-diaminedichloroplatinum(II) to the polymerase and to the template DNA, were explored. Selective preincubations of the platinum drug with the polymerase and DNA reveal that the inhibition is primarily due to covalent binding to the enzyme. The rates of inhibition were found to be first order in enzyme and zeroth order in platinum in the concentration range 0.05-3.0 mM. A mechanism that deals with the formation of an initial platinum-polymerase-I complex with a binding constant > 10(5) M(-1) followed by a further reaction to form an inhibitory complex is consistent with the kinetic data. The rate limiting first order rate constant for the formation of the inhibitory complex is comparable to that observed for the thiol coordination of peptides containing cysteine residues. Analyses of known structures and functions of catalytic domains of various polymerases point to the direction that the inhibition is perhaps due to the distortion of the DNA binding domain of the enzyme due to platinum coordination.     

Protein kinase A inhibitors enhance radiation-induced apoptosis

In addition to a role for de novo protein synthesis in apoptosis we have previously shown that activation of a protein phosphatase or loss of activity of a kinase is also important in radiation-induced apoptosis in human cells [Baxter, and Lavin (1992): J Immunol 148:149–1954]. We show here that some inhibitors of protein kinases exacerbate radiation-induced apoptosis in the human cell line BM13674. The specific protein kinase A inhibitor isoquinoline sulfonamide (20 μM) gave rise to significantly increased levels of apoptosis at 2–6 h postirradiation compared to values after radiation exposure only. The same concentration of isoquinolinesulfonamide, which was effective in increasing apoptosis, reduced activity markedly. A 66% inhibition of cyclic AMP-dependent protein kinase A activity occurred in unirradiated cells at this concentration of H89 and activity was reduced to 58% in irradiated cells. Calphostin C, a specific inhibitor of protein kinase C, at a concentration of 0.1 μM, which caused 68% inhibition of enzyme activity in irradiated cells, failed to enhance the level of radiation-induced apoptosis. Other kinase inhibitors did not lead to an additional increase in apoptosis over and above that observed after irradiation. The results obtained here provide further support for an important role for modification of existing proteins during radiation-induced apoptosis.     

Goldston syndrome: report of a case.

Cerebrohepatorenal malformation is a rare familial disorder characterized by typical renal lesions combined with Dandy-Walker malformation, and congenital hepatic fibrosis. In this case report, a male premie with the diagnosis of cerebrorenal syndrome or so called Goldston syndrome is presented. Besides the rarity of this syndrome, this case is the second reported patient diagnosed prenatally.     

Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations AFP antifreeze protein - CD circular dichroism - DTT dithiothreitol - HPLC high pressure liquid chromatography - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

Activation of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Purified antifreeze proteins (AFPs) from the larvae of the beetle Dendroides canadensis do not produce the high levels of antifreeze activity seen in the hemolymph of overwintering larvae, even when the purified AFPs are assayed at very high concentrations. However, addition of certain proteins or agar (at concentrations sufficiently low that the gel state does not result) to the Dendroides AFP resulted in a 2–3-fold increase in activity. A 70-kDa protein with AFP-activating capabilities was purified from Dendroides larvae. Addition of this endogenous activator protein to a 4 mg·ml^-1 solution of AFP increased the activity of the AFPs to values comparable to those of the hemolymph of overwintering larvae. Data derived from a modified immunoblot technique demonstrate that the activators bind to the AFP, or vice versa. Formation of this association must allow the AFP to block ice crystal growth by binding to the surface of potential seed crystals in the normal fashion. However, because the AFP-activator complex is much larger than the AFP alone, the complex probably blocks a greater surface area of the crystal and is thus a more efficient antifreeze.Abbreviations AFP antifreeze protein - BSA bovine serum albumine - DEAE diethylaminoethyl - Ig immunoglubolin - LPIN lipoprotein ice nucleator - PIN protein ice nucleator - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - TH thermal hysteresis     

Isolation of a cDNA clone for the alpha subunit of the human GABA-A receptor

A cDNA clone of an alpha subunit of the human GABA-A receptor has been isolated. The human clone (pCLL800) contains 1055 nucleotides in an open reading frame and 260 nucleotides in the 5' non-coding region. The 351 amino acid sequence of this human alpha subunit shows 97% homology with its bovine counterpart. Hybridization of pCLL800 to Northern blots shows a 3.9/4.3 Kb RNA doublet in human cortex, rat whole brain, cortex, hippocampus, midbrain, olfactory bulb and cerebellum. Developmental studies show that the levels of the rat alpha mRNA increase between one and three weeks of age in a manner similar to the development of the benzodiazepine binding sites.     

An Examination of the Involvement of Phospholipases A2 and C in the α-Adrenergic and γ-Aminobutyric Acid Receptor Modulation of Cyclic AMP Accumulation in Rat Brain Slices

Experiments were undertaken to define the role of two calcium-associated enzyme systems in modulating transmitter-stimulated production of cyclic nucleotides in rat brain. Cyclic AMP (cAMP) accumulation was examined in cerebral cortical slices using a prelabeling technique. The enhancement of isoproterenol-stimulated cAMP production by alpha-adrenergic and gamma-aminobutyric acid-B (GABAB) agonists was reduced by exposing the tissue to EGTA, a chelator of divalent cations, or quinacrine, a nonselective inhibitor of phospholipase A2. Likewise, chronic (2 weeks) administration of corticosterone decreased the alpha-adrenergic and GABAB receptor modulation of second messenger production. Neither cyclooxygenase nor lipoxygenase inhibitors selectively influenced the facilitating response of alpha-adrenergic and GABAB agonists. Other experiments revealed that although norepinephrine and 6-fluoronorepinephrine stimulated inositol phosphate (IP) production in cerebral cortical slices with potencies equal to those displayed in the cyclic nucleotide assay, selective alpha 1-adrenergic agonists were less efficacious on IP formation and were without effect in the cAMP assay. Conversely, a selective alpha 2-adrenergic receptor agonist facilitated the cAMP response to a beta-adrenergic agonist without affecting IP formation. The rank orders of potency of a series of alpha-adrenergic antagonists suggest that IP accumulation is mediated solely by alpha 1-adrenergic receptors, whereas the augmentation of cAMP accumulation is regulated by a mixed population of alpha-adrenergic sites. The results suggest that the alpha-adrenergic and GABAB receptor-mediated enhancement of isoproterenol-stimulated cAMP formation appears to be more closely associated with phospholipase A2 than phospholipase C and may be mediated by arachidonate or some other fatty acid.     

Desipramine Binding: Relationship to Central and Sympathetic Noradrenergic Activity

We examined the effects of treatments affecting norepinephrine release on the number of norepinephrine reuptake recognition sites as reflected by desipramine binding. To do this, we used manipulations having similar presynaptic but contrasting postsynaptic effects. Presynaptic inhibition by 6-hydroxydopamine lesion or by clonidine, and postsynaptic receptor stimulation by isoproterenol, reduced desipramine binding. Presynaptic stimulation by d-amphetamine and postsynaptic receptor blockade by prazosin increased desipramine binding. Similar effects and binding properties were seen in cerebral cortex, heart, and soleus muscle. After unilateral noradrenergic lesions, reduction in desipramine binding correlated with reduction in norepinephrine uptake. These results show that norepinephrine reuptake appears to be regulated by transmitter release regardless of effects on postsynaptic transmission, and that this regulation is analogous in the central and sympathetic nervous systems.     

Further studies on the involvement of the circadian system in photoperiodic control of antifreeze protein production in the beetle Dendroides canadensis

The role of circadian rhythmicity in the photoperiodic time measuring processes regulating antifreeze protein production in the beetle Dendroides canadensis was further investigated. Using “T” experiments larvae were exposed to environmental light cycle periods close to the period length of the endogenous circadian oscillator. The following light cycles were employed: light/dark 8/13, 8/14, 8/16, 8/18 and 8/19 corresponding to period lengths of 21, 22, 24, 26 and 27 h. Larvae maintained in cycles equal to or less than 24 h displayed a characteristic short-day response, showing significantly (P < 0.01) greater antifreeze protein activity than did those measured on the day of collection in late summer. In contrast, a long-day response was observed in larvae maintained under a 26- or 27-h light cycle in that antifreeze protein activity did not differ from that measured on the initial collection date.

The role of photoperiod and temperature in influencing the photoperiodic timing processes were examined with a series of resonance experiments. The first group consisted of a 24, 36, 48, 60 or 72-h light cycle, each with an 8-h photophase at temperatures of 20 or 17°C. Rhythmic increases in antifreeze protein levels at intervals of 24 h occurred under both temperatures. However, the lower temperature displaced the resonance curve in the vertical direction (i.e. increasing % population response) and reduced the difference between peaks and troughs on the resonance curve. Resonance experiments incorporating a 14-h photophase resulted in low antifreeze protein activity under all conditions except a 36-h light cycle in which a 67% induction was observed.

Eight hour resonance experiments were also conducted with D. canadensis collected in early spring to determine whether the circadian system participates in the photoperiodic timing processes influencing the spring termination of antifreeze protein production. Positive resonance results were obtained in that only larvae maintained in cycles of 36 and 60 h displayed significantly (P < 0.01) lower antifreeze activity when compared to animals on the initial collection date.

The combined results emphasize the involvement of the circadian system in the photoperiodic control of antifreeze protein production by D. canadensis during the fall and spring. Furthermore, the induction of antifreeze protein production is a function of light cycle and its waveform (photoperiod). Temperature appears to modify the photoperiodic response in some manner involving the photoperiodic time measuring processes. It is concluded that the photoperiodic response of antifreeze protein production by D. canadensis is dependent upon the entrainment of the circadian system by the light cycle.     

Chronic Electroconvulsive Seizures Down–Regulate Expression of the Immediate-Early Genes c-fos and c-jun in Rat Cerebral Cortex

Acute seizures and other stimuli that increase neuronal activity cause a rapid induction of the immediate-early genes c-fos and c-jun, also referred to as nuclear proto-oncogenes, in the nervous system. In the present study, rats were administered one or more electroconvulsive seizures (ECS) and the responsiveness of c-fos and c-jun to an acute, "test" seizure was examined. Four hours after a single ECS, the induction of c-fos mRNA by a test seizure was blocked, in agreement with earlier findings, but by 18 h the levels of c-fos mRNA could be reinduced by the test seizure, suggesting that 1 day is sufficient to "reset" the responsiveness of this system. However, it was found that chronic, daily ECS treatments resulted in a time-dependent decrease in the expression of c-fos mRNA in response to a test seizure administered 18 h after the last daily ECS; this effect was maximal after 8-10 days of treatment, at which time the induction of c-fos mRNA by the test seizure was blocked dramatically. Chronic ECS also blocked the induction of c-jun in response to an acute, test seizure. The effect of chronic ECS on levels of Fos protein was also investigated. It was found that basal levels of Fos protein were reduced after chronic (10 days) ECS and were not induced by a test seizure. Because levels of Fos protein remain elevated 4 h after a single seizure this finding suggests that the mechanisms by which acute (4 h) and chronic (8-10 days) ECS block the induction of c-fos may differ.