Biomedical subjects

S W May

Publications and source records attributed to S W May.

At least 19 recordsLinked to original sources

PubMed2001

Speciation and identification of organoselenium metabolites in human urine using inductively coupled plasma mass spectrometry and tandem mass spectrometry.

A method for speciation and identification of organoselenium metabolites found in human urine samples using high performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS) and tandem mass spectrometry (MS/MS) is described. Reversed-phase chromatographic separation was used for sample fractionation with the ICP-MS functioning as an element-selective detector, and six distinct selenium-containing species were detected in a human urine sample. Fractions were then collected and analyzed using a triple quadrupole mass spectrometer with electrospray ionization and collision-induced dissociation to obtain structural information. The first two fractions were identified specifically as selenomethionine and selenocystamine, estimated to be present at approximately 11 and 40 ppb, respectively. To the best of our knowledge, this is the first time these two metabolites have been positively identified in human urine.

Chromatography, High Pressure Liquid↗

PubMed2001

High-performance liquid chromatographic enantiomeric separation of an enzyme inhibitor which possesses both a chiral center and tautomeric moieties.

The enantiomeric separation using high-performance liquid chromatography (HPLC) on chiral stationary phases (CSPs) of a chiral compound which exists in solution in several tautomeric forms is described. 2,4-Dioxo-5-acetamido-6-phenylhexanoic acid is the most potent inhibitor known for peptidylamidoglycolate lyase (PGL, EC 4.3.2.5), an enzyme which plays an essential role in carboxyl-terminal amidation of many biological peptides. Synthesis of this inhibitor entails an alkaline hydrolysis step, under which condition the compound is racemized; thus, HPLC with a CSP was employed to obtain the individual enantiomers of this inhibitor. Since 2,4-dioxo-5-acetamido-6-phenylhexanoic acid exists in solution in several tautomeric forms, the strategy of first converting this compound from its multiple enol forms into a single diketo tautomer, which was then applied to various CSPs, was employed. Successful preparative scale enantiomeric separation of this compound was achieved using a Chiralpak AD CSP. Enantiomeric separation was also accomplished on a D-penicillamine column, but this CSP was found to be less satisfactory for preparative purposes.

Acetamides↗

PubMed2001

Evaluation of cardiovascular parameters of a selenium-based antihypertensive using pulsed Doppler ultrasound.

The pharmacology of selenium is of much interest because selenium deficiency has been linked to cardiovascular diseases, cancer, and arthritis, and selenoenzymes are critical cellular antioxidants. We have previously reported that phenyl-2-aminoethylselenide (PAESe) and its derivatives represent a novel class of selenium-based antihypertensive agents that exhibit unique biochemical and pharmacologic properties. We now report on experiments designed to probe the hemodynamic mechanism of action of these compounds in spontaneously hypertensive rats (SHR). A noninvasive pulsed Doppler ultrasound probe was used to measure peak blood flow velocity in the aortic arch from the right second intercostal space. PAESe was found to increase peak aortic blood flow velocity (+44%), heart rate (+16%), and blood flow acceleration (+105%), while decreasing left ventricular ejection time (LVET) (-37%) concomitant with a decrease in mean arterial pressure (-54%). These results were compared with the known vasodilator hydralazine, which had similar effects on mean arterial pressure (MAP) and peak velocity but caused an increase in LVET (+42%) and a decrease in heart rate (-18%). Taken together, our results suggest that PAESe decreases blood pressure via a decrease in peripheral resistance, which overcomes the initial increase in heart rate and acceleration to give a net decrease in MAP.

Animals↗

PubMed2000

Kinetic and stereochemical studies on novel inactivators of C-terminal amidation.

C-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymic action by peptidylglycine alpha-mono-oxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). Here we introduce novel compounds in which an olefinic functionality is incorporated into peptide analogues as the most potent turnover-dependent inactivators of PAM. Kinetic parameters for PAM inactivation by 4-oxo-5-acetamido-6-phenyl-hex-2-enoic acid and 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid were obtained by using both the conventional dilution assay method and the more complex progress curve method. The results obtained from the progress curve method establish that these compounds exhibit the kinetic characteristics of pure competitive inactivators (i.e. no ESI complex forms during inactivation). On the basis of k(inact)/K(i) values, 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid is almost two orders of magnitude more potent than benzoylacrylate, a chemically analogous olefinic inactivator that lacks the peptide moiety. Stereochemical studies established that PAM inactivation by 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid is stereospecific with respect to the moiety at the P(2) position, which is consistent with previous results with substrates and reversible inhibitors. In contrast, 2, 4-dioxo-5-acetamido-6-phenylhexanoic acid, which is a competitive inhibitor with respect to ascorbate, exhibits a low degree of stereospecificity in binding to the ascorbate sites of both PAM and dopamine-beta-hydroxylase.

Acetamides↗

PubMed2000

Reaction of phenylaminoethyl selenides with peroxynitrite and hydrogen peroxide.

Peroxynitrite, a reactive cytotoxic species generated by the reaction of superoxide with nitric oxide, rapidly oxidizes phenylaminoethyl selenide (PAESe) and its para-substituted derivatives with second-order rate constants ranging from 900 to 3000 M(-1) s(-1) at neutral pH (pH 7.0) and 25 degrees C. These values are approximately 3 x 10(4) times greater than the corresponding rate constants for the reactions of selenides with hydrogen peroxide. The peroxynitrite reaction was also studied at alkaline pH. HPLC analysis confirms that both the peroxynitrite and hydrogen peroxide reactions produced the corresponding phenylaminoethyl selenoxide (PAESeO) as the sole selenium-containing product, with a stoichiometry of 1 mol of PAESe oxidized per 1 mol of PAESeO formed per 1 mol of oxidant reacted. The influence of para-substituents on the rate constants was investigated using Hammett plots; in both cases the data are consistent with an S(N)2-type mechanism, wherein the selenium atom acts as the nucleophile. Our results provide further evidence that organoselenium compounds may play a protective role in the defense against the many reactive oxidizing species produced in cellular metabolism.

Antihypertensive Agents↗

PubMed2000

Liquid chromatography/electrospray mass spectrometry of organoselenium compounds with postcolumn crown ether complexation.

Postcolumn addition is an effective means of alleviating or solving ionization-related problems in liquid chromatography/electrospray ionization mass spectrometry (LC/ESIMS). In the current study, initial attempts to develop a direct LC/ESIMS method for an organoselenium compound, 4-hydroxyphenyl 2-methyl-2-aminoethyl selenide (HOMePAESe), were unsuccessful because of extensive fragmentation which occurred even under the mildest in-source collision-induced dissociation (CID) conditions. To reduce the extent of compound fragmentation, a crown ether, 18-crown-6, was added postcolumn to the system, forming a complex with HOMePAESe, which survived the electrospray ionization process with reduced fragmentation and hence improved sensitivity for the major ions. The general applicability of this crown ether complexation approach to clinical samples was demonstrated by the analysis of HOMePAESe in human urine, using a structural analogue, 4-fluorophenyl 2-aminoethyl selenide (FPAESe) as an internal standard. The limit of detection for HOMePAESe, based on a signal-to-noise ratio of 3:1, was estimated to be 5 pg/microL in urine. The potential application of this approach to the general analysis of other amine-containing compounds was also evaluated.

Chromatography, Liquid↗

PubMed2000

Vascular and endothelial actions of inhibitors of substance P amidation.

Formation of mature active neuropeptides such as substance P (SP) from their glycine extended precursors entails alpha-amidation of peptide precursors by the sequential enzymatic action of peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL). We reported that these two enzymes that can produce mature active neuropeptides are present in cultured bovine aortic endothelial cells (BAECs). We hypothesize that alpha-amidation of peptides occurs in endothelial cells and that these peptides are critically involved in the overall regulation of cardiovascular function. In this study, this hypothesis was tested using specific amidation inhibitors to determine their effects on the actions of SP and its glycine-extended precursor (SP-Gly). We have found that SP and SP-Gly are equipotent in stimulating nitric oxide (NO) release by BAECs. At 10(-5) M, the specific inhibitors of PAM (4-phenyl-3-butenoic acid; PBA) and PGL (5-acetamido-2,4-diketo-6-phenyl-hexanoic acid and its methyl ester) reduced NO basal release by 40, 34, and 45%, respectively. They also reduced the production of NO induced by SP-Gly by 63, 68, and 69%, respectively, but had no effect on NO production in response to either SP or acetylcholine. SP and SP-Gly also were equipotent in relaxing rat aortic segments. The vasorelaxation to SP-Gly was endothelium dependent and inhibited by the NOS antagonist L-nitroarginine methyl ester (L-NAME), but it was not affected by inhibition of prostaglandin synthesis. Inhibitors of both PAM and PGL significantly reduced the vasorelaxing actions of SP-Gly, whereas responses to SP were not affected. A cumulative infusion of PBA into the femoral artery of rabbits, at final concentrations of 2.4, 24, and 240 microM for 20 min each, increased the vascular resistance (VR), indicating the tonic production of vasodilating amidated peptide(s). This effect was maximum at 60 min after infusion (20.5 +/- 4.7 vs. 8.2 +/- 0.7 mm Hg/ml/min; p < 0.05). These results suggest that endothelial cells can produce mature SP from its SP-Gly precursor and that a product of peptide alpha-amidation tonically stimulates endothelial cell NO release to control vascular tone.

Amidine-Lyases↗

PubMed1999

Kinetic and inhibition studies on substrate channelling in the bifunctional enzyme catalysing C-terminal amidation.

A series of experiments has been conducted to investigate the possibility that substrate channelling might occur in the bifunctional forms of enzymes carrying out C-terminal amidation, a post-translational modification essential to the biological activity of many neuropeptides. C-terminal amidation entails sequential action by peptidylglycine mono-oxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5), with the mono-oxygenase catalysing conversion of a glycine-extended pro-peptide into the corresponding alpha-hydroxyglycine derivative, which is then converted by the lyase into amidated peptide plus glyoxylate. Since the mono-oxygenase and lyase reactions exhibit tandem reaction stereospecificities, channelling of the alpha-hydroxy intermediate might occur, as is the case for some other multifunctional enzymes. Selective inhibition of the mono-oxygenase domain by competitive ester inhibitors, as well as mechanism-based mono-oxygenase inactivation by the novel olefinic inhibitor 5-acetamido-4-oxo-6-phenylhex-2-enoate (N-acetylphenylalanyl acrylate), has little to no effect on the kinetic parameters of the lyase domain of the AE from Xenopus laevis. Similarly, inhibition of the lyase domain by the potent dioxo inhibitor 2,4-dioxo-5-acetamido-6-phenylhexanoate has little effect on the activity of the monooxygenase domain in the bifunctional enzyme. A series of experiments on intermediate accumulation and conversion were also carried out, along with kinetic investigations of the reactivities of the monofunctional and bifunctional forms of PAM and PGL towards substrates and inhibitors. Taken together, the results demonstrate the kinetic independence of the mono-oxygenase and lyase domains, and provide no evidence for substrate channelling between these domains in the bifunctional amidating enzyme.

Acrylates↗

PubMed1999

Applications of oxidoreductases.

Oxidoreductases comprise the large class of enzymes that catalyze biological oxidation/reduction reactions. Because many chemical and biochemical transformations involve oxidation/reduction processes, developing practical biocatalytic applications of oxidoreductases has long been an important goal in biotechnology. During the past year, significant progress has been made in the development of oxidoreductase-based diagnostic tests and improved biosensors, in the design of innovative systems for regeneration of essential coenzymes, in the construction bioreactors for biodegradation of pollutants and for biomass processing, and in the development of oxidoreductase-based approaches for synthesis of polymers and oxyfunctionalized organic substrates.

Biodegradation, Environmental↗

PubMed1998

Determination of the absolute configuration of alpha-hydroxyglycine derivatives by enzymatic conversion and chiral high-performance liquid chromatography.

We describe an approach for facile determination of the absolute configuration of enantiomerically chromatographed racemates by combining enzymatic conversion and chiral chromatography. The method involves initial rapid development of chiral HPLC methods using polar organic eluents with polysaccharide chiral phases. We present here evidence for using the stereospecific peptidylamidoglycolate lyase (PGL, E.C. 4.3.2.5) to determine the absolute configuration of alpha-hydroxyglycine derivatives. The racemic solute was incubated with PGL, lyophilized and then enantiomerically chromatographed using the CHIRALPAK AD column. Based on the specificity of the enzyme reaction, the unreacted enantiomer was assigned the absolute configuration R.

Amidine-Lyases↗

PubMed1998

Pharmacological evaluation of 1-(carboxymethyl)-3,5-diphenyl-2-methylbenzene, a novel arylacetic acid with potential anti-inflammatory properties.

OBJECTIVE AND DESIGN: 1-(Carboxymethyl)-3,5-diphenyl-2-methylbenzene (CDB), a novel arylacetic acid, was evaluated in vivo for its ability to inhibit acute and chronic inflammation as well as acute pain. MATERIALS AND METHODS: The effects of CDB were evaluated using the following assays: 1) acute inflammation induced by the injection of carrageenan, bradykinin and serotonin into the subplantar region of the hind paw of rats; 2) chronic inflammation produced by the injection of Mycobacterium butyricum into the base of the tail of rats; 3) acute pain induced by the i.p. injection of phenyl-p-quinone into mice resulting in the production of writhes; 4) cyclooxygenase (COX) activity, including COX-1 and COX-2, evaluated using whole blood; and 5) activity of peptidylglycine alpha-monooxygenase (PAM) isolated from Xenopus laevis skin. RESULTS: CDB (10 to 100mg/kg s.c.) produced a dose-dependent inhibition of carrageenan edema (ED50 of 41 mg/ kg at 3 h) which continued for up to 12 h. Using a therapeutic dosing regimen, this compound inhibited hind paw inflammation (>70%) and arthogram scores in rats with adjuvant-induced arthritis. This compound also possessed significant analgesic activity in mice (70% inhibition with 50mg/kg). CDB, however, lacked inhibitory activity on bradykinin and serotonin-induced edema. In addition, CDB significantly inhibited COX-I activity (IC50 approximately = 17 microM) while having only a weak inhibitory activity on both COX-2 and PAM activity. CONCLUSIONS: CDB is an effective anti-inflammatory/analgesic agent whose mechanism of action appears to be associated with inhibition of COX-1 activity.

Acetates↗

PubMed1998

Selenium-based antihypertensives. Rationale and potential.

Selenium, long recognised as an important 'dietary antioxidant', is now known to be an essential component of the active sites of a number of enzymes, including the glutathione peroxidase selenoenzyme family which scavenge hydroperoxides to prevent cellular damage. Dietary selenium deficiency has been linked to diseases as diverse as cancer, heart disease, arthritis and AIDS, and epidemiological evidence is now emerging for the beneficial effects of selenium supplementation. Thus, the pharmacology, biology and biochemistry of selenium metabolism have become subjects of considerable interest, which are spurring efforts to develop synthetic selenium-containing compounds as potential therapeutic agents. Phenylaminoalkyl selenides were developed in the authors' laboratories as novel, selenium-based pharmacological agents. We demonstrated that these compounds exhibited dose-dependent antihypertensive activity in spontaneously hypertensive rats. Biochemical studies established that as a consequence of the redox properties of their selenium moieties, these phenylaminoalkyl selenides possessed the remarkable property of propagating a cycle of turnover-dependent local depletion of reduced ascorbate when processed by the key enzyme of catecholamine metabolism, dopamine-beta-monooxygenase. On the basis of inductively coupled plasma/mass spectroscopic analyses, corroborated by operant behaviour and locomotor activity investigations, an orally-active phenylaminoalkyl selenide with restricted CNS permeability was successfully developed. To our knowledge, this compound--4-hydroxy-alpha-methyl-phenyl-2-aminoethyl selenide--is the first orally active, selenium-based anti-hypertensive compound ever reported. In the future, we anticipate more widespread efforts to incorporate selenium into rationally designed pharmaceutical agents, with the goal of developing novel compounds which may be of therapeutic benefit toward a variety of human diseases.

Animals↗

PubMed1997

Pyruvate-extended amino acid derivatives as highly potent inhibitors of carboxyl-terminal peptide amidation.

Carboxyl-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymatic action by peptidylglycine monooxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). The monooxygenase, PAM, first catalyzes conversion of a glycine-extended pro-peptide to the corresponding alpha-hydroxyglycine derivative, and the lyase, PGL, then catalyzes breakdown of this alpha-hydroxyglycine derivative to the amidated peptide plus glyoxylate. We now introduce the first potent inhibitors for peptidylamidoglycolate lyase. These inhibitors, which can be viewed as pyruvate-extended N-acetyl amino acids, constitute a novel class of compounds. They were designed to resemble likely transient species along the reaction pathway of PGL catalysis. A general synthetic procedure for preparation of pyruvate-extended N-acetyl amino acids or peptides is described. Since these compounds possess the 2,4-dioxo-carboxylate moiety, their solution tautomerization was investigated using both NMR and high performance liquid chromatography analyses. The results establish that freshly prepared solutions of N-Ac-Phe-pyruvate consist predominantly of the enol tautomer, which then slowly tautomerizes to the diketo form when left standing for several days in an aqueous medium; upon acidification, formation of the hydrate tautomer occurs. Kinetic experiments established that these novel compounds are highly potent, pure competitive inhibitors of PGL. Kinetic experiments with the ascorbate-dependent copper monooxygenases, PAM and dopamine-beta-monooxygenase, established that these compounds also bind competitively with respect to ascorbate; however, pyruvate-extended N-acyl-amino acid derivatives possessing hydrophobic side chains are much more potent inhibitors of PGL than of PAM. Selective targeting of N-Ac-Phe-pyruvate so as to inhibit the lyase, but not the monooxygenase, domain was demonstrated with the bifunctional amidating enzyme of Xenopus laevis. The availability of potent inhibitors of PGL should facilitate studies regarding the possible biological role of alpha-hydroxyglycine-extended peptides.

Amides↗

PubMed1997

Amidative peptide processing and vascular function.

Substance P (SP), an amidated peptide present in many sensory nerves, is known to affect cardiovascular function, and exogenously supplied SP has been shown to activate nitric oxide synthase (NOS) in endothelial cells. We now report that SP-Gly, the glycine-extended biosynthetic precursor of SP (which is enzymatically processed to the mature amidated SP), causes relaxation of rat aortic strips with an efficacy and potency comparable to that of SP itself. Pretreatment of the aortic strips with 4-phenyl-3-butenoic acid (PBA), an irreversible amidating enzyme inactivator, results in marked inhibition of the vasodilation activity induced by SP-Gly but not of that induced by SP itself. Isolated endothelial cell basal NOS activity is also decreased by pretreatment with PBA, with no evidence of cell death or direct action of PBA on NOS activity. Both bifunctional and monofunctional forms of amidating enzymes are present in endothelial cells, as evidenced by affinity chromatography and Western blot analysis. These results provide evidence for a link between amidative peptide processing, NOS activation in endothelial cells, and vasodilation and suggest that a product of amidative processing provides intrinsic basal activation of NOS in endothelial cells.

Amino Acid Sequence↗

PubMed1997

Antiinflammatory and analgesic activity of an inhibitor of neuropeptide amidation.

4-Phenyl-3-butenoic acid (PBA) has been shown in vitro to be a turnover-dependent inactivator of peptidylglycine alpha-monooxygenase (PAM), the rate-limiting enzyme involved in the formation of amidated neuropeptides from their glycine-extended precursors. In the studies reported herein, we have shown that PBA produces a dose-dependent (50-500 mg/kg s.c.) inhibition of serum PAM activity in normal rats without affecting peptidylamidoglycolate lyase activity. Because amidated neuropeptides such as substance P and calcitonin gene-related peptide are involved in acute inflammation, we evaluated the effects of PBA on carrageenan-induced inflammation in rats. The acute administration of PBA (s.c. or i.p.) produced a dose-related inhibition of edema with maximum inhibition (67%) observed at 2 hr postphlogistic agent. In addition, the continuous administration of PBA to animals over a 7-day period using osmotic pumps not only inhibited hind paw swelling induced by carrageenan but also inhibited serum PAM activity and reduced tissue levels of substance P in hind paws. These results demonstrate for the first time a correlation between the antiinflammatory activity produced by an inhibitor of peptide amidation with its ability to inhibit serum PAM activity and lower endogenous tissue levels of substance P. Moreover, these results confirm our contention that PAM is an excellent pharmacological target for controlling the acute inflammatory response. We also demonstrate the ability of PBA to inhibit phenyl-p-quinone and acetylcholine-induced writhing in mice without affecting the spinally mediated tail immersion assay in rats. Because this analgesic effect was extremely rapid (within 15 min), PBA may be producing this effect by a mechanism other than peptide amidation.

Amidine-Lyases↗

PubMed1997

An orally active selenium-based antihypertensive agent with restricted CNS permeability.

We report here the first orally active, selenium-based antihypertensive agent, and we demonstrate its restricted CNS permeability using inductively coupled plasma/mass spectroscopy (ICP/MS) and operant behavioral analysis. The biochemistry and pharmacology of selenium are subjects of intense current interest. As a consequence of the redox chemistry of the selenium moiety, phenylaminoalkyl selenides possess the remarkable characteristic of propagating a cycle of turnover-dependent local depletion of reduced ascorbate when processed by the key enzyme of catecholamine metabolism, dopamine-beta-monooxygenase. ICP/MS analysis was used to determine the pharmacokinetic parameters for selenide compounds after i.v. administration to anesthetized rats. Analysis of the data using a two-compartment pharmacokinetic model established very rapid initial clearance and a short beta-elimination half-life from blood. We developed an oxidative procedure for digestion and processing of tissue samples in order to obtain ICP/MS data on the tissue distributions of Se-containing metabolites after the administration of selenide compounds. The results establish that aromatic ring hydroxylation of the selenides results in a marked reduction in brain levels of Se-containing metabolites. The comparative effects of selenide compounds on locomotor activity and operant behavior were then investigated, and the results fully corroborate the ICP/MS analytical results. The novel compound, 4-hydroxy-alpha-methyl-phenyl-2-aminoethyl selenide, exhibits both restricted CNS permeability and oral antihypertensive activity in spontaneously hypertensive rats. This compound is the first orally active selenium-based antihypertensive agent ever reported, and it possesses properties that are highly desirable in pharmacological agents being developed for treatment of chronic diseases such as hypertension.

Administration, Oral↗

PubMed1996

Mechanism-based inactivation of dopamine beta-monooxygenase in adrenal chromaffin cells.

Dopamine beta-monoxygenase (DBM, E.C. 1.14.17.1) is an attractive target point for possible modulation of adrenergic activity, and a variety of DBM-targeted pseudosubstrates and inhibitors have been developed in this laboratory and other laboratories. We now demonstrate the efficacy of a DBM-targeted mechanism-based inactivator, as well as enzymatic processing of two alternate DBM substrates, within functional adrenal chromaffin cells. When cultured adrenal medullary chromaffin cells were incubated with the mechanism-based inactivator 1-(4'-hydroxyphenyl)-1-(aminomethyl)-ethene (HOPAME), vesicular DBM activity was markedly decreased. Similarly, the alternate substrates 4'-hydroxyphenyl-2-aminoethyl sulfide and 4'-hydroxyphenyl-2-aminopropyl selenide each undergo uptake and DBM-catalyzed oxygenation within these cells. The simultaneous action of both the mechanism-based inactivator and an alternate substrate within functional chromaffin cells was also demonstrated. These results provide support for a direct mechanistic link between the enzymological properties of DBM-targeted adrenergic agents and their in-vivo pharmacological activities.

Adrenal Medulla↗

PubMed1996

Virulence of feline Chlamydia psittaci in mice is not a function of the major outer membrane protein (MOMP).

The virulent strain of feline Chlamydia psittaci, the Cello strain, produces conjunctivitis and upper respiratory disease in cats. This same strain produces a lethal disease in mice when inoculated intraperitoneally (i.p.). In this study we have shown that the Baker strain of feline C. psittaci is attenuated in the mouse model system. Intraperitoneal inoculation of mice with the Baker strain produced no disease but did stimulate an immune response that protected the mice from subsequent produced i.p. inoculation with the virulent Cello strain. To determine if the difference between these two strains was in the major outer membrane protein (MOMP), the omp1 gene which codes for this protein was sequenced for both the Baker and Cello strains. The MOMP was chosen to study because in Chlamydia trachomatis this protein has been shown to contain neutralizing epitopes and has been shown to play a role in cell attachment. These functions make it a likely structural component capable of mutating and causing altered cell tropism and virulence. The DNA sequence of the omp1 was determined by amplifying the gene with PCR, cloning the PCR product into the pCR-II cloning vector and determining the DNA sequence of the inserted gene using primers to sites in the plasmid vector. From the DNA sequence, the deduced amino acid sequence of MOMP was determined for both the attenuated Baker and the virulent Cello strains of feline C. Psittaci. The results indicated that the omp1 gene of both strains contained 1179 base pairs which coded for a protein 392 amino acids. The DNA sequences of the omp1 gene of the two strains differed by only two base pairs which resulted in two amino acid changes in the MOMP. The Baker strain had a serine instead of a tryptophan at amino acid 7 and a tyrosine instead of an aspartic acid at amino acid 125 of the uncleaved protein. Neither amino acid change was in an area of the MOMP which could logically account for the difference in biological activity. Amino acid 7 was in the leader sequence which is cleaved from the authentic MOMP and is not present in the infectious elementary body. Amino acid 125 was in a conserved hydrophobic area of one of the constant regions of the protein. A change at this location was not likely surface exposed and thus could not affect cell adhesion, tissue tropism or neutralizing epitopes. Therefore, the differences in the primary structure of the MOMP from the Baker and Cello strains of feline C. psittaci could not account for the attenuation of the Baker strain for mice. The molecular basis of their difference is yet to be determined.

Amino Acid Sequence↗