Processing enzyme ribonuclease E specifically cleaves RNA I. An inhibitor of primer formation in plasmid DNA synthesis

When the RNA processing enzyme RNAase E is inactivated in an Escherichia coli strain carrying derivatives of the colicin E1 plasmid, a small RNA, about 100 nucleotides long, accumulates. Structural analysis of this RNA showed that it is RNA I, the RNA that inhibits plasmid DNA synthesis. RNA I is a specific substrate for RNAase E and the cleavage takes place between the fifth and sixth nucleotides from the 5' end of the molecule. This is only the second natural RNA substrate that has been found, so far, for the RNA processing enzyme ribonuclease E, the other being a precursor for 5 S ribosomal RNA. It is remarkable that nine nucleotides around the cleavage sites are identical in both substrates: (Formula: see text). Therefore, we suggest that at least part of the interaction between RNAase E and its substrate is controlled by these nine nucleotides.     

Thermodynamics of the reaction of ferric myoglobin from Aplysia limacina with azide and fluoride. Dependence of enthalpy changes on pH

The effect of pH on the enthalpy changes for binding of azide and fluoride to ferric myoglobin from Aplysia limacina, which lacks the distal histidine, has been investigated. Over the whole pH range explored (3.8 to 9.5), -delta H degrees values for the formation of the hemoprotein-ligand complexes are: (1) much greater than the variations in -delta G degrees; (2) always negative; and (3) show a dependence upon pH characterized by a maximum for azide and a minimum for fluoride binding, centered at pH 4.55 (identical to pHch). This value agrees well with that expected from the linear correlation between pHch and the simple function "Lys+Arg-Glu-Asp-2" proposed by Beetlestone and others. Data reported here greatly extend the pH range for which the linear correlation between the net charge of the macromolecule and pHch has been found to hold, and indicate unequivocally that the pH dependence of -delta H degree for the binding of anionic ligands does not uniquely require the presence of the histidyl residue at the distal position.     

A plasmodial alpha-tubulin cDNA from Physarum polycephalum. Nucleotide sequence and comparative analysis

As the first step towards correlating structure and function of tubulin in the slime mold Physarum polycephalum we have elucidated the nucleotide sequence of a cDNA that appears to code for all but the last 25 to 30 C-terminal amino acids of a plasmodial alpha-tubulin. Differences in amino acid sequence from those of other alpha-tubulins are distributed fairly evenly throughout the sequence, although a relatively extensive conserved region is found in position 396 to 426 near the C terminus. A small region in position 298 to 307 contains a cluster of amino acid residues unique to Physarum alpha-tubulin. The sequence is 70% homologous to two yeast alpha-tubulins and about 83% homologous to five animal alpha-tubulins. A comparison of the homologies of all the known alpha-tubulins indicates that a large decrease in the accepted point mutation rate has occurred during the evolution of the metazoa, suggesting a major functional specialization of microtubules.     

Folding of thermolysin fragments. Identification of the minimum size of a carboxyl-terminal fragment that can fold into a stable native-like structure

The COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin.     

An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda

We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.     

Nitrobenzylthioinosine binding in brain: an interspecies study

The binding of the potent adenosine uptake inhibitor [3H]nitrobenzylthioinosine ( [3H]NBI) to cerebral cortical membrane preparations from human, dog, guinea pig, rat, and mouse was investigated. Reversible, specific, saturable, high affinity binding was found in all five species with similar kinetic parameters. (Kd = 0.16-0.44 nM; Bmax = 128-196 fmol/mg prot.). Dilazep, hexobendine, and dipyridamole were all high potency inhibitors of [3H]NBI binding in human, dog, guinea pig and mouse preparations but not in rat. These compounds showed a competitive inhibition of [3H]NBI binding indicating that they are acting at the same site. Discrepancies seen in the rat appear to be a unique, species related anomaly. The dihydropyridine calcium antagonists also inhibited binding with lower potency than the adenosine uptake blockers. This inhibition was most potent in dog and human and suggests a relationship between the calcium channel and the adenosine uptake site.     

CSF distribution of morphine, methadone and sucrose after intrathecal injection

The lumbar to cisternal CSF distribution of morphine and methadone were compared to C-14 sucrose, a standard marker of CSF bulk flow, after lumbar subarachnoid injections in a sheep preparation. Morphine appeared and peaked simultaneously with C-14 sucrose in cisternal CSF at 90 to 190 minutes. The mean peak cisternal CSF morphine concentrations were sustained for 30-40 minutes, and averaged 148 ng/ml, representing 0.3% of the administered dose. Methadone was not detectable in cisternal CSF up to 240-300 minutes after lumbar subarachnoid administration. The C-14 sucrose/morphine ratio was increased an average of 6.7 times in cisternal CSF as compared to the ratio of the two compounds injected into the lumbar subarachnoid space. These studies demonstrate that morphine, a hydrophilic opioid, given intrathecally moves rostrally and appears in cisternal CSF by bulk flow. Furthermore the rostral redistribution of morphine is associated with the clearance of morphine from CSF. Methadone, a lipophilic opioid, appears to be completely cleared from CSF before it reaches the cisterna magna. These pharmacokinetic studies support a contribution of supraspinal sites to the analgesic and adverse effects produced by morphine given by spinal routes of administration. In contrast methadone appears to exert its effects predominantly at spinal sites.     

The effects of the acute administration of buprenorphine hydrochloride on the release of anterior pituitary hormones in the rat: evidence for the involvement of multiple opiate receptors

Multiple opiate receptor agonists and antagonists have been found to produce different patterns of anterior pituitary hormone release. The present studies examined the pattern of anterior pituitary hormone release produced by buprenorphine. The effects of the kappa agonist ethylketocyclazocine on thyroid stimulating hormone release were also examined. Following buprenorphine, serum levels of corticosterone and luteinizing hormone were not changed while growth hormone release was stimulated in a dose-dependent manner. Prolactin release was stimulated after the lowest dose of buprenorphine while the highest dose induced a fall in serum prolactin. Similar biphasic effects on thyroid stimulating hormone were seen after either buprenorphine or ethylketocyclazocine. The results provide support for the role of multiple opiate receptors in opiate-induced changes in anterior pituitary hormone release.     

Inhibition of growth of C6 astrocytoma cells by inhibitors of calmodulin

We evaluated the effect of several classes of calmodulin inhibitors on the activity of calmodulin prepared from C6 astrocytoma cells and studied the activity of these drugs as inhibitors of the growth of C6 cells in tissue culture. There was a good correlation between the activity of the drugs as inhibitors of calmodulin and their activity as inhibitors of cell growth. The most potent compounds were calmidazolium and melittin as compared to the phenothiazines, trifluoperazine, chlorpromazine, chlorpromazine-sulfoxide or the diphenylbutylpiperidine, pimozide. The mechanism by which the inhibition of calmodulin leads to the death of cells could not be attributed entirely to inhibition of the calmodulin-sensitive cyclic nucleotide phosphodiesterase. Calmodulin is a heat stable, calcium-binding protein involved in numerous biological processes. Recent evidence indicates that calcium and calmodulin may be important for cellular proliferation. For example, this protein changes in concentration during the cell cycle; is involved in the disassembly of the mitotic apparatus; is increased in concentration in rapidly growing hepatomas and in transformed fibroblasts. Weiss and co-workers demonstrated that phenothiazines and structurally similar drugs are capable of binding to and inhibiting the activity of calmodulin. It has been recently observed that certain drugs that inhibit the activity of calmodulin also inhibit the growth of malignant cells in vitro and in vivo. In these studies, however, there was no direct correlation of the effect of the drugs on the calmodulin from the cell type under investigation with cytotoxicity. To learn more about the relationship between a drug's ability to inhibit calmodulin and its antiproliferative activity, we correlated the effect of drugs on the activity of calmodulin prepared from the C6 astrocytoma cell line with their effect on cellular proliferation. Since many inhibitors of calmodulin readily cross the blood-brain barrier and since no acceptable treatment for malignancies of the central nervous system exist, we chose this cell line as a model for elucidating the potential antineoplastic effects of calmodulin inhibitors.