Biomedical subjects
Publications and source records attributed to M Manning.
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There is a growing awareness of vegetation's role as a source of potentially reactive hydrocarbons that may serve as photochemical oxidant precursors. This study assessed the influence of light and temperature, independently, on monoterpene emissions from slash pine (Pinus elliottii Engelm.). Plants were preconditioned in a growth chamber, then transferred to an environmentally controlled gas exchange chamber. Samples of the chamber atmosphere were collected; the monoterpenes were concentrated cryogenically and measured by gas chromatography. Five monoterpenes (alpha-pinene, beta-pinene, myrcene, limonene, and beta-phellandrene) were present in the vapor phase surrounding the plants in sufficient quantity for reliable measurement. Light did not directly influence monoterpene emission rates since the emissions were similar in both the dark and at various light intensities. Monoterpene emission rates increased exponentially with temperature (i. e. emissions depend on temperature in a log-linear manner). The summed emissions of the five monoterpenes ranged from 3 to 21 micrograms C per gram dry weight per hour as temperature was increased from 20 to 46 C. Initially, emission rates from heat-stressed needles were similar to healthy needles, but rates decreased 11% per day. Daily carbon loss through monoterpene emissions accounted for approximately 0.4% of the carbon fixed during photosynthesis.
We have previously shown that the substitution of 8-ornithine and 2-O-methyltyrosine alone and in combination in [1-deaminopenicillamine] oxytocin (dPOT) brought about enhancements in antagonistic potencies to responses to oxytocin in vivo. To explore the effects of these substitutions in analogs of dPOT containing larger alkyl substitutents on the beta carbon at position 1 and on the tyrosine residue at position two, the following six analogs were synthesized: [1-(beta-mercapto-beta, beta-diethylpropionic acid), 8-ornithine] vasotocin (1, dEt2OVT); (1-beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 8-ornithine] vasotocin (2, d(CH2)5OVT): [1-beta-mercapto-beta, beta-diethylpropionic acid), 2-O-methyltyrosine, 8-ornithine]vasotocin [3, dEt2 Tyr(Me)OVT]; [1-(beta-mercapto-beta, beta-diethylpropionic acid), 2-O-ethyltyrosine, 8-ornithine]vasotocin [4, dEt2 Tyr(Et)OVT]; [1-beta-mercapto-beta', beta-cyclopentamethylenepropionic acid), 2-O-methyltyrosine, 8-ornithine]vasotocin [5, d(CH2)5 Tyr(me)OVT]: [1-beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-O-methyltyrosine, 8-ornithine]vasotocin [6,d(CH2)5 Tyr(Et)OVT]. The required protected intermediates were synthesized by a combination of solid-phase synthesis and by individual 8 + 1 couplings in solution. All six analogs antagonize the actions of oxytocin on the rat uterus in the absence of Mg2+, in the presence of 0.5 mM Mg2+ and in situ. They also antagonize milk ejection responses to oxytocin, and the vasopressor responses to arginine vasopressin, and all have very low antidiuretic activities. With pA2 values of 7.35 +/- 0.08 and 7.37 +/- 0.17, respectively, compounds 3 and 5 are the two most potent in vivo antagonists of oxytocin reported to date.
The extent of the macromolecular change accompanying yeast to mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis of the cytoplasmic proteins of the two growth forms after antibody cross-absorption experiments. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium (LSM). The two strains selected for this study were strain 4918, which produces pure mycelial (M) cultures in LSM at 37 degrees C (designated 4918-37M) and yeasts (Y) at 24 degrees C (4918-24Y), and strain 2252, which produces yeasts exclusively at both 24 and 37 degrees C in LSM (2252-24Y and 2252-37Y). The proteins of both strains were labeled at both temperatures with [35S]sulfate, and cytoplasmic fractions were prepared by mechanical disruption and ultracentrifugation. Rabbits were immunized with the 4918-24Y and 4918-37M cytoplasmic fractions to produce anti-yeast-phase and anti-mycelial-phase hyperimmune sera. Each radiolabeled cytoplasmic fraction was absorbed with anti-mycelial-phase immunoglobulin, anti-yeast immunoglobulin, and immunoglobulin from normal rabbit serum. Staphyloccal protein A was used to remove immune complexes. The labeled, nonabsorbed proteins were also analyzed by two-dimensional electrophoresis. Highly reproducible protein spot patterns were obtained which defined hundreds of proteins in each extract. The specificity of the immunoglobulin hundreds of proteins in each extract. The specificity of the immunoglobulin preparations was extremely broad, and as many as 168 cytoplasmic antigens were detected. Eighty-three antigens were recognized in the mycelial-phase extract only by the anti-mycelial-phase immunoglobulin. However, comparative analysis revealed that all of these proteins were present in at least one other extract. Therefore, none of them was unique to the mycelial morphology. Eleven antigens were detected in the 2252-37Y extract that were not present in the extracts from strain 4918, which indicates that proteins obtained from different strains may express similar antigenic determinants, but differ in their physiochemical properties.
A low-sulfate synthetic medium was developed in which pure cultures of yeast- and mycelial-phase Candida albicans could be cultivated for investigations of the molecular biology of dimorphism. The medium contained ammonium ions, phosphate buffer, salts, glucose, and biotin. Morphogenesis was found to be dependent upon the strain of C. albicans. Of six strains tested in the low-sulfate medium at 37 degrees C, three formed mixed cultures of yeasts, true mycelium and pseudomycelium, two formed pure cultures of true mycelium, and one maintained yeast growth. All six strains produced pure cultures of yeasts at 24 degrees C. The buffering capacity of the medium maintained the pH at 6.9 even at high-density cell growth. The low concentration of sulfate and the absence of amino acids in the medium provided conditions in which to radiolabel cellular constituents with [35S]sulfate. For molecular investigations, the use of two strains is suggested, one forming yeasts and one forming true mycelium in low-sulfate medium at 37 degrees C, thus providing controls for both strain variation and for molecular changes induced by environmental change but unrelated to morphogenesis.
The extent of change in cytoplasmic proteins which accompanies yeast-to-mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium. The two strains selected for this study were strain 4918, which produces pure mycelial cultures in low-sulfate medium at 37 degrees C and yeast cells at 24 degrees C, and strain 2252, which produces yeast cells exclusively at both 24 and 37 degrees C in low-sulfate medium. The proteins of both strains were labeled at both temperatures with [35S]sulfate, cytoplasmic fractions were prepared by mechanical disruption and ultracentrifugation, and the labeled proteins were analyzed by two-dimensional electrophoresis. Highly reproducible protein spot patterns were obtained which defined hundreds of proteins in each extract. Ten protein spots were identified on the two-dimensional gels of the 4918 mycelial-phase extract which were not present in the 4918 yeast-phase extract. These proteins appeared to be modifications of preexisting yeast-phase proteins rather than proteins synthesized de novo in the mycelial cells because 5 were absorbed by rabbit anti-yeast-phase immunoglobulin and each of the 10 was also present in extracts of strain 2252 grown at 24 and 37 degrees C, indicating that they were neither unique to filamentous cells nor sufficient for induction or maintenance of the mycelial morphology. Thirty-three proteins were identified in the 4918 yeast-phase extract which were not present in the 4918 mycelial-phase extract. Pulse-chase experiments revealed the synthesis of new proteins during yeast-to-mycelial conversion, but none of these was unique to mycelial cells. No differences in the major cytoplasmic proteins of any of the yeast- or mycelial-phase extracts were identified. This finding suggests that the major structural proteins of the cytoplasm are not extensively modified and argues instead that proteins unique to either phase may serve a regulatory function.
Several new synthetic analogs of the oxytocin antagonist [1-deaminopenicillamine]oxytocin have been prepared and tested for their abilities to inhibit responses to oxytocin by the isolated rat uterus in the absence and presence of Mg++, by the rat uterus in situ, and by the rat mammary gland in situ. Substituting 2-O-methyltyrosine in [1-deaminopenicillamine]oxytocin strikingly enhances antagonism of all uterin responses, and [1-deaminopenicillamine, 2-O-methyltyrosine]oxytocin and its 4-threonine analog are also potent inhibitors of the milk ejection response. Substituting 2-phenylalanine in [1-deaminopenicillamine]oxytocin also enhances antagonistic activities in all uterine assays, but [1-deaminopenicillamine, 2-phenylalanine]oxytocin retains agonistic activity on milk ejection assays. From these studies we can conclude that changes in the 1-position (1-deaminopenicillamine substitution) and the 2-position (2-O-methyltyrosine or 2-phenylalanine substitution) can have additive effects on antagonistic activities. Substitution of an 8-ornithine also enhances inhibitory potency in vivo, and this effect may also be additive to those of the substitutions in 1- and 2-positions. These findings provide many clues that may lead to the design of even more effective antagonists; several of the analogs reported here appear to the most effective antagonists of oxytocin in vivo yet reported and may be useful agents in further studies on the physiological functions of endogenous oxytocin.
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Substitution of the amino acid glycine for 7-proline in oxytocin produces an analog, [7-glycine]oxygocin, having markedly reduced antidiuretic and vasopressor activities but retaining considerable natriuretic activity. We administered this analog alone and with distally acting diuretic agents to test the hypothesis that oxytocin and its analogs decrease proximal tubular fluid reabsorption. If [7-glycine]oxytocin shares a common mechanism of action with any of these agents, the response to combined treatment should be less than additive, otherwise, the saluretic effect of both agents should be maintained. Subcutaneous administration of [7-glycine]oxytocin, 10 micrograms/kg, to conscious fluid-loaded rats produced a natriuresis, diuresis and kaliuresis. Hydrochlorothiazide and [7-glycine]oxytocin administered together to the same rats had a greater than additive effect on urine volume and sodium excretion. Furosemide and [7-glycine]oxytocin had an additive effect on urine volume, sodium and chloride excretion. Triamterene blocked the kaliuresis caused by [7-glycine]oxytocin, although the two agents administered together had a greater than additive effect on sodium excretion. These results indicate that [7-glycine]oxytocin does not share a common natriuretic mechanism or site of action with the above diuretics. Our data are consistent with the hypothesis that oxytocin and its analogs may increase urinary sodium excretion by decreasing net proximal tubular fluid reabsorption.
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As part of a program in which we are attempting to synthesize in vivo antagonists of oxytocin, the following four analogues were synthesized and tested for antagonistic activities in rat uterus and rat vasopressor assay systems: [-(beta-mercapto-beta,beta-diethylpropionic acid), 4-threonine]oxytocin (1, dEt2TOT), [1-beta-mercapto-beta,beta-cyclopenta-methylenepropionic acid), 4-threonine]oxytocin [2, d(CH2)5TOT], [1-deaminopenicillamine,2-O-methyltyrosine]oxytocin [3, dPTyr(Me)OT], and [1-deaminopenicillamine,2-O-methyltyrosine,4-threonine]oxytocin [4, dPTyr(Me)TOT]. The required protected intermediates were synthesized by a combination of solid-phase peptide synthesis and by individual 8 + 1 couplings in solution. All four analogues antagonize the actions of oxytocin on the rat uterus (a) in the absence of Mg2+, (b) in the presence of 0.5 mM Mg2+, and (c) in situ. They exhibit, respectively, the following pA2 values in each of the assay systems a-c: (1) (a) 7.72 +/- 0.11, (b) 7.36 +/- 0.09, (c) 6.47 +/- 0.11; (2) (a) 7.91 +/- 0.13, (b) 7.81 +/- 0.09, (c) 6.94 +/- 0.11; (3) (a) 7.76 +/- 0.12, (b) 7.80 +/- 0.12, (c) 6.86 +/- 0.12; (4) (a) 7.64 +/- 0.14, (b) 7.79 +/- 0.09, (c) 6.84 +/- 0.10. They have the following antivasopressor pA2 values: (1) 6.30 +/- 0.13; (2) 5.86 +/- 0.03; (3) 7.59 +/- 0.05; (4) 7.32 +/- 0.04. Compounds 2-4 are among the most potent in vivo antagonists of oxytocin reported to date.
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[1-Deaminopenicillamine,4-threonine]oxytocin was prepared in duplicate from S-benzyl-3-mercapto-3,3-dimethylpropanoyl-Tyr(Bzl)-Ile-Thr(Bzl)-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 (I) by removal of the Bzl-protecting groups with Na-NH3, followed by cyclization of the resulting disulfhydryl compound with K3Fo(CN)6. The analogue was purified by desalting on Sephadex G-15 in 50% acetic acid and gel filtration of Sephadex G-15. The protected peptide I was synthesized (a) by the solid-phase method and (b) by a combination of solid-phase synthesis and an [8 + 1] coupling in solution. The analogue has no detectable agonist activity in rat vasopressor or isolated rat uterus assays. It has an antivasopressor pA2 of 6.67 +/- 0.09. It is a potent inhibitor of the in vitro oxytocic response to oxytocin and has a pA2 value of 7.46 +/- 0.04. (Material from the repeat synthesis has a pA2 value of 7.59 +/- 0.08.) Thus the substitution of threonine for glutamine in the antagonist [1-deaminopenicilliamine]oxytocin (pA2, 7.14 +/- 0.05) has effected a twofold increase in inhibitory potency. [1-deaminopenicillamine,4-threonine]oxytocin is one of the most potent inhibitors of oxytocin known to date.
As part of a program in which we are attempting the design and synthesis of an antagonist of the antidiuretic response to arginine-vasopressin (AVP) [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),4-valine,8-D-arginine]vasopressin [d(CH2)5VDAVP] was synthesized and assayed for antidiuretic, vasopressor, and oxytocic activities. The required protected intermediate was synthesized by a combination of solid-phase synthesis and an [8 + 1] coupling in solution. d(CH2)5VDAVP has an antidiuretic potency of 0.10 +/- 0.02 unit/mg, less than 1/10000 that of its parent [deamino,4-valine,8-D-arginine]vasopressin (dVDAVP). d(CH2)5VDAVP is a specific antagonist of the vasopressor responses to AVP. It has an antivasopressor pA2 value of 7.68 +/- 0.05 when tested against AVP. It is also an antagonist of the in vitro oxytocic response to oxytocin (pA2 value = 6.62 +/- 0.07). With its negligible antidiuretic activity, absence of oxytocic activity, and its potent and specific ability to antagonize the vasopressor effects of AVP, d(CH2)5VDAVP is one of the most potent and selective vasopressor antagonists reported to date. It should thus be a useful tool with which to probe the possible role(s) that AVP may play in cardiovascular regulation under normal and pathological conditions.
As part of a program in which we are attempting to design and synthesize antagonists of the vasopressor response to arginine-vasopressin (AVP), [1-deaminopenicillamine]arginine-vasopressin (dPAVP), [2-O-methyl)tyrosine]-arginine-vasopressin [Tyr(Me)AVP], and [1-deaminopenicillamine,2-(O-methyl)tyrosine]arginine-vasopressin [dPTyr(Me)AVP] were synthesized by the solid-phase method and assayed for vasopressor, antidiuretic, and oxytocic activities. Tyr(Me)AVP has a vasopressor potency of 9.7 +/- 0.5 units/mg and an antidiuretic potency of 386 +/- 36 units/mg. These values are 2.5 and 120%, respectively, of the corresponding potencies of AVP. The analogue is an antagonist of the in vitro response to oxytocin (pA2 = 7.44 +/- 0.12). dPAVP has an antivasopressor pA2 of 7.45 +/- 0.11. Its antidiuretic potency is 42.2 +/- 2 units/mg, 2.5% that of its parent, 1-[deamino]arginine-vasopressin (dAVP). It is an antagonist of the in vitro response to oxytocin (pA2 value = 6.93 +/- 0.10). dPTyr(Me)AVP has an antivasopressor pA2 of 7.96 +/- 0.05 and an antidiuretic potency of 3.5 +/- 0.5 units/mg. It is also an antagonist of the in vitro oxytocic response to oxytocin (pA2 value = 7.61 +/- 0.14). It is thus one of the most potent vasopressor antagonists reported to date.