Influence of ion gradients on the transbilayer distribution of dibucaine in large unilamellar vesicles

The uptake of dibucaine into large unilamellar vesicles in response to proton gradients (delta pH; inside acidic) or membrane potentials (delta psi; inside negative) has been investigated. Dibucaine uptake in response to delta pH proceeds rapidly in a manner consistent with permeation of the neutral (deprotonated) form of the drug, reaching a Henderson-Hasselbach equilibrium where [dibucaine]in/[dibucaine]out = [H+]in/[H+]out and where the absolute amount of drug accumulated is sensitive to the buffering capacity of the interior environment. Under appropriate conditions, high absolute interior concentrations of the drug can be achieved (approximately 120 mM) in combination with high trapping efficiencies (in excess of 90%). Dibucaine uptake in response to delta psi proceeds more than an order of magnitude more slowly and cannot be directly attributed to uptake in response to the delta pH induced by delta psi. This induced delta pH is too small (less than or equal to 1.5 pH units) to account for the transmembrane dibucaine concentration gradients achieved and does not come to electrochemical equilibrium with delta psi. Results supporting the possibility that the charged (protonated) form of dibucaine can be accumulated in response to delta psi were obtained by employing a permanently positively charged dibucaine analogue (N-methyldibucaine). Further, the results suggest that delta psi-dependent uptake may depend on formation of a precipitate of the drug in the vesicle interior. The uptake of dibucaine into vesicles in response to ion gradients is of direct utility in drug delivery and controlled release applications and is related to processes of drug sequestration by cells and organelles in vivo.     

Increase in the formation of leukotriene B4 and other lipoxygenase products in peritoneal macrophages of adrenalectomized rats

The effect of adrenalectomy on the formation of cyclooxygenase and lipoxygenase products by activated peritoneal rat macrophages was determined. After isolation, the cells were incubated with [1-14C]arachidonic acid and the calcium ionophore A23187 and the metabolites isolated by HPLC chromatography. The main components formed in the controls are 6-keto-prostaglandin F1 alpha, thromboxane B2 and 12-HETE. One peak represents 5,12-di-HETE. Smaller amounts of prostaglandin F2 alpha, prostaglandin E2, prostaglandin D2, leukotriene B4 and 15-HETE are also present. After adrenalectomy, a considerable increase occurs in the amounts of leukotriene B4, 15-HETE and 12-HETE. The increase in the prostaglandins is smaller. The compounds formed from endogenous arachidonic acid are also determined. In the cells of the controls, 6-keto-prostaglandin F1 alpha and thromboxane B2 are produced in higher amounts than leukotriene B4. After adrenalectomy, the formation of leukotriene B4 is much more increased than that of 6-keto-prostaglandin F1 alpha. These effects are most probably related to a diminished amount or inactivation of lipocortin, a glucocorticosteroid-induced peptide with phospholipase A2 inhibitory activity in adrenalectomized animals.     

Interactive roles of progesterone, prostaglandins, and collagenase in the ovulatory mechanism of the ewe

Interrelationships between production of progesterone (P4), prostaglandin (PG) E2 and PGF2 alpha, and collagenase by periovulatory ovine follicles and their possible involvements in the ovulatory process were investigated. Follicles were isolated from ovaries at intervals (0 to 24 h) after the initiation of the preovulatory surge of luteinizing hormone (LH). Progesterone and PGs within follicles were determined by radioimmunoassay. Digestion of radioactive collagen during coincubation with tissue homogenates was used to assess the production of a bioactive follicular collagenase(s). Follicular accumulation of PGs and P4 increased at 12 and 16 h, respectively, after the onset of the surge of LH; PGE2 then decreased at 20 h. Collagenolytic activity of follicular tissue increased at 20 h and was maximal at 24 h (during the time of follicular rupture). An inhibitor of synthesis of P4 (isoxazol) or PGs (indomethacin) was injected into the follicular antrum at 8 h. Isoxazol did not prevent the initial rise in PGs, but inhibited synthesis of PGF2 alpha at 16 h and therafter. Isoxazol negated the decline in PGE2 and increase in collagenolysis. Indomethacin did not influence synthesis of P4; however, it suppressed collagenolytic activity of follicular tissue. Ovaries with treated follicles were left in situ and observed for an ovulation point at 30 h. Isoxazol or indomethacin was a potent inhibitor of ovulation. The blockade of ovulation by isoxazol was reversed by systemic administration of P4 or PGF2 alpha, but not by PGE2. Reversal of the blockade by indomethacin was accomplished with PGE2 or PGF2 alpha. Collagenolytic activity of follicular tissue was likewise restored by such treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolactin modulates steroidogenesis by rat granulosa cells: I. Effects on progesterone

Either testosterone or follicle-stimulating hormone (FSH) stimulates progesterone secretion by granulosa cells from rats but the combination of the two hormones increases progesterone production in a synergistic manner. We have investigated the effects of graded doses of prolactin (0, 0.02, 0.2, 2, or 10 micrograms/ml) alone or in combination with testosterone (0.5 microM), FSH (300 ng/ml), or FSH + testosterone on progesterone secretion by granulosa cells at two stages of differentiation. Relatively undifferentiated granulosa cells from immature, diethylstilbestrol-treated, hypophysectomized (HPX) rats were cultured in defined (serum-free) medium for 3 days. More highly differentiated granulosa cells were obtained on the morning of proestrus from the preovulatory follicles of 30-day-old rats induced to undergo an estrous cycle by injection with 4 IU pregnant mare's serum gonadotropin; these cells were cultured in medium containing 10% fetal bovine serum. Prolactin alone did not enhance the negligible secretion of progesterone by cells from HPX rats, but increased progesterone secretion by cells from proestrous rats. Prolactin significantly enhanced the stimulatory effects of testosterone or FSH alone on cells from both HPX and proestrous rats. When cultures containing both FSH + testosterone were treated with prolactin, progesterone secretion by cells from proestrous rats was significantly enhanced, whereas secretion by cells from HPX rats was significantly depressed. Therefore when cells from HPX rats were cultured with both FSH and testosterone, the direction of the effect of prolactin was reversed from that observed with prolactin + FSH or testosterone alone, and from that observed when cells from proestrous rats were cultured with prolactin + FSH + testosterone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Classification of Histoplasma capsulatum isolates by restriction fragment polymorphisms

Twenty isolates of the dimorphic, pathogenic fungus Histoplasma capsulatum were divided into three classes based on comparisons of restriction enzyme digests of their mitochondrial DNA and rDNA. The majority of isolates, including most North American strains and the African H. capsulatum var. duboisii variants, belong to class 2. Isolates from Central America and South America make up class 3. The attenuated Downs strain is the only member of class 1.     

Role of progesterone in regulating uteroovarian venous concentrations of PGF2 α and PGE2 during the estrous cycle and early pregnancy in ewes

The role of progesterone in regulation of uteroovarian venous concentrations of prostaglandins F₂ α (PGF₂α) and E₂ (PGE₂) during days 13 to 16 of the ovine estrous cycle or early pregancy was examined. At estrus, ewes were either mated to a fertile ram or unmated. On day 12 postesturus, ewes were laparotomized and a catheter was inserted into a uteroovarian vein. Six mated and 7 unmated ewes received no further treatment. Fifteen mated and 13 unmated ewes were ovariectomized on day 12 and of these, 7 mated and 5 unmated ewes were given 10 mg progesteron sc and an intravaginal pessary containing 30 mg of progesterone. Uteroovarian venous samples were collected every 15 min for 3 h on days 13 to 16 postestrus. Mating resulted in higher mean daily concentrations of PGE₂ in the uterovarian vein than in unmated ewes. Ovariectomy prevented the rise in PGE₂ with day in mated ewes but had no effect in unmated ewes. Progesterone treatment restored PGE₂ in ovariectomized, mated ewes with intact embros. Mating had no effect on mean daily concentrations of PGF₂α or the patterns of the natural logarithm (ln) of the invariance of PGF₂α. Ovariectomy resulted in higher mean concentrations and ln invariances of PGF₂α on day 13 and lower mean concentrations and ln invariances of PGF₂α on days 15 and 16. Replacement with progesterone prevented these changes in patters of mean concentrations and ln variances of PGF₂α following ovariectomy. It is concluded that progesterone regulates the release of PGF₂α from the uterus, maintaining high concentrations while also preventing the occurrence of the final peaks of PGF₂α which are seen with falling concentrations of progesterone. This occurs in both pregnant and non-pregnant ewes. Progesterone is also needed to maintain increasing concentrations of PGE₂ in mated ewes.     

Characterization of Bacteria by Particle Beam Mass Spectrometry

A technique is described for detecting and characterizing bacteria on a single-particle basis by mass spectrometry. The method involves generation of a particle beam of single whole cells which are rapidly volatilized and ionized in vacuum in the ion source of a quadrupole mass spectrometer. The particle beam can be generated, with minimal sample handling, from a naturally occurring aerosol or from a solution of bacteria that can be dispersed as an aerosol. The mass spectrum is generated by successively measuring the average intensities of different mass peaks. The average intensity is obtained by measuring the ion intensity distribution at the particular mass (m/e) for ion pulses from more than 1,000 bacteria particles. Bacillus cereus, Bacillus subtilis, and Pseudomonas putida samples were analyzed to test the capability of the instrument for differentiating among species of bacteria. Significant ion-intensity information was produced over the m/e range of 50 to 300, an improvement over previous pyrolysis-mass spectrometry results. The complex mass spectra contained a few unique peaks which could be used for the differentiation of the bacteria. A statistical analysis of the variations in peak intensities among the three bacteria provided a quantitative measure of the reproducibility of the instrument and its ability to differentiate among bacteria. The technique could lead to a new rapid method for the analysis of microorganisms and could be used for the detection of airborne pathogens on a continuous, real-time basis.     

Mapping parathyroid hormone, β-globin,insulin, and LDH-A genes within the human chromosome 11 short arm by spot blotting sorted chromosomes

Summary Rearranged human chromosomes carrying segments of chromosome 11 were separated from the normal chromosome 11 by high-resolution chromosome sorting. Sorted chromosomes were tested with parathyroid hormone, -globin, insulin, and LDH-A gene-specific probes to determine the genes carried by each chromosome segment. Based on the gene content and karyotypes of these abnormal chromosomes, the parathyroid hormone, -globin, insulin, and LDH-A genes and the unique restriction fragment ADJ-762 are all located on the terminal band of the short arm of human chromosome 11 (band 11p15), with LDH-A proximal to the other loci.