Prostaglandin I2 is less relaxant than prostaglandin E2 on the lamb ductus arteriosus.

Prostaglandin (PG) I2 and its stable metabolite, 6-keto-PGF1alpha, were tested on the isolated ductus arteriosus from mature fetal lambs. PGI2 relaxed the ductus in high doses (threshold 10(-6)M) and its activity disappeared on standing at room temperature for 30 minutes. 6-keto-PGF1alpha was inactive at all doses. By contrast, PGE2 produced a dose-dependent relaxation over a range between 10(-10) and 10(-6)M. These findings confirm that PGE2 is the most potent ductal relaxant among the known derivatives of arachidonic acid. PGE2 probably maintains ductus patency in the fetus and, together with PGE1, remains the compound of choice in the management of newborns requiring a viable ductus for survival.     

Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B.

We have constructed three different truncated versions of diphtheria toxin (a 535-amino-acid polypeptide) which correspond to the N-terminal 290, 377, and 485 amino acids of the toxin. These lengths include one, three, and all four of the putative membrane-spanning sequences of the toxin which are thought to play a role in the translocation of fragment A into cells. Each of these three genes has been modified at its 3' end to code for a C-terminal cysteine (to allow for disulfide linkage of a targeting ligand) or a gene fusion with alpha-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (ptox) with the lambda pR promoter in an effort to overexpress these proteins. The truncated genes are expressed in Escherichia coli from both the tox promoter in a constitutive fashion and from the pR promoter by using the heat-inducible cI857 repressor. The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated Mr on Western blots, and which have ADP-ribosyltransferase activity. Constitutive synthesis from ptox leads to severe proteolytic degradation even in a protease-deficient strain. High-level expression from the pR promoter in the same lon htpR strain allows the full-length polypeptides to accumulate but also stops the growth of the cells. It appears that removal of as few as 50 amino acids from the C-terminus of diphtheria toxin alters its conformation, making it a target for proteases and causing overexpression lethality in the host cells.     

Evidence for a role of prostaglandin I2 and thromboxane A2 in the ductus venosus of the lamb

The prostaglandin (PG) endoperoxide, PGH2, and the thromboxane (TX) A2 analog, 9,11-epithio-11,12-methano-TXA2, were tested in vitro on the ductus venosus sphincter from fetal (premature and mature) and neonatal (1-day-old) lambs. PGH2 relaxed the indomethacin-contracted fetal ductus in a dose-dependent manner and its action was reduced after treatment with 15-hydroperoxyarachidonic acid. In contrast, reduced glutathione did not affect the PGH2 relaxation in the indomethacin-treated ductus, nor did it modify the response of the untreated ductus to constrictor stimuli. Unlike PGH2, the stable 9 alpha,11 alpha-epoxymethano-PGH2 analog contracted the vessel. Similarly, the TXA2 analog was a contractile agent, its action exceeding that of the PGH2 analog in potency and efficacy. The TXA2 analog was active on preparations from both premature (minimum 117 days gestation) and mature lambs, but a maximal effect was attained during the perinatal period. These results confirm the existence of a PG-mediated relaxing mechanism in the ductus venosus and suggest that the active compound is PGI2. This mechanism is likely responsible for keeping the ductus patent in the fetus. TXA2, formed within the liver parenchyma, is well suited for playing a role in postnatal closure of the vessel.     

A peroxide/ascorbate-inducible catalase from Haemophilus influenzae is homologous to the Escherichia coli katE gene product.

Bacterial catalases are induced by exposure to peroxide (e.g., Escherichia coli katG) or entry into stationary phase (e.g., E. coli katE). To study regulatory systems in Haemophilus influenzae, we complemented an E. coli rpoS mutant, which is unable to induce katE in stationary phase, with a plasmid library of H. influenzae Rd- chromosomal DNA. Nineteen complementing clones with a catalase-positive phenotype were obtained and characterized after screening about 10(5) transformants. All carried the same structural gene for an H. influenzae catalase. The DNA sequence of this gene, called hktE, encodes a 508-amino-acid polypeptide with strong homology to eukaryotic catalases and E. coli katE. However, hktE is regulated like E. coli katG, with catalase activity increasing 10-fold and hktE mRNA levels increasing 4-fold upon exposure to ascorbic acid, which serves to generate hydrogen peroxide. Mutations in the known global regulatory genes of H. influenzae--crp, cya, and sxy--do not affect the inducibility of hktE. The hktE gene maps to a 225-kb segment of the H. influenzae chromosome in a region encoding resistance to spectinomycin.     

Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.     

Daily dosing of rifapentine cures tuberculosis in three months or less in the murine model

Background

Availability of an ultra-short-course drug regimen capable of curing patients with tuberculosis in 2 to 3 mo would significantly improve global control efforts. Because immediate prospects for novel treatment-shortening drugs remain uncertain, we examined whether better use of existing drugs could shorten the duration of treatment. Rifapentine is a long-lived rifamycin derivative currently recommended only in once-weekly continuation-phase regimens. Moxifloxacin is an 8-methoxyfluoroquinolone currently used in second-line regimens.

Methods and Findings

Using a well-established mouse model with a high bacterial burden and human-equivalent drug dosing, we compared the efficacy of rifapentine- and moxifloxacin-containing regimens with that of the standard daily short-course regimen based on rifampin, isoniazid, and pyrazinamide. Bactericidal activity was assessed by lung colony-forming unit counts, and sterilizing activity was assessed by the proportion of mice with culture-positive relapse after 2, 3, 4, and 6 mo of treatment. Here, we demonstrate that replacing rifampin with rifapentine and isoniazid with moxifloxacin dramatically increased the activity of the standard daily regimen. After just 2 mo of treatment, mice receiving rifapentine- and moxifloxacin-containing regimens were found to have negative lung cultures, while those given the standard regimen still harbored 3.17 log₁₀ colony-forming units in the lungs (p < 0.01). No relapse was observed after just 3 mo of treatment with daily and thrice-weekly administered rifapentine- and moxifloxacin-containing regimens, whereas the standard daily regimen required 6 mo to prevent relapse in all mice.

Conclusions

Rifapentine should no longer be viewed solely as a rifamycin for once-weekly administration. Our results suggest that treatment regimens based on daily and thrice-weekly administration of rifapentine and moxifloxacin may permit shortening the current 6 mo duration of treatment to 3 mo or less. Such regimens warrant urgent clinical investigation.     

Pre- and postjunctional alpha(2)-adrenergic receptors in fetal and adult ovine cerebral arteries

In ovine cerebral arteries, adrenergic-mediated vasoconstrictor responses differ significantly with developmental age. We tested the hypothesis that, in part, these differences are a consequence of altered alpha(2)-adrenergic receptor (alpha(2)-AR) density and/or affinity. In fetal (approximately 140 days) and adult sheep, we measured alpha(2)-AR density and affinity with the antagonist [(3)H]idazoxan in main branch cerebral arteries and other vessels. We also quantified contractile responses in middle cerebral artery (MCA) to norepinephrine (NE) or phenylephrine in the presence of the alpha(2)-AR antagonists yohimbine and idazoxan and contractile responses to the alpha(2)-AR agonists clonidine and UK-14304. In fetal and adult cerebral artery homogenates, alpha(2)-AR density was 201 +/- 18 and 52 +/- 6 fmol/mg protein, respectively (P < 0.01); however, antagonist affinity values did not differ. In fetal, but not adult, MCA, 10(-7) M yohimbine significantly decreased the pD(2) for NE-induced tension in the presence of 3 x 10(-5) M cocaine, 10(-5) M deoxycorticosterone, and 10(-6) M tetrodotoxin. In fetal, but not adult, MCA, UK-14304 induced a significant decrease in pD(2) for the phenylephrine dose-response relation. In addition, stimulation-evoked fractional NE release was significantly greater in fetal than in adult cerebral arteries. In the presence of 10(-6) M idazoxan to block alpha(2)-AR-mediated inhibition of prejunctional NE release, the fractional NE release was significantly increased in both age groups. We conclude that in fetal and adult ovine cerebral arteries, alpha(2)-AR appear to be chiefly prejunctional. Nonetheless, the fetal cerebral arteries appear to have a significant component of postjunctional alpha(2)-AR.