The influence of phosphodiesterase inhibitor, rolipram, on hemodynamics in lipopolysaccharide-treated rats.

Administration of bacterial endotoxin (lipopolysaccharide, LPS) intravenously has been noted to produce a shock state, which is characterized by hypotension and multi-organ system failure. The aim of the present investigation was to (a) examine the influence of rolipram on hemodynamics, plasma levels of tumor necrosis factor-alpha (TNF-alpha) levels, and production of inducible nitric oxide synthase (iNOS) in the lungs, ex vivo, in LPS-treated rats, and (b) determine the cardiovascular effects of a selective alpha1-adrenoceptor agonist, methoxamine, in the absence or presence of rolipram in rats treated with LPS. Blood pressure, cardiac index, heart rate and arterial resistance were assessed in Long-Evans rats anesthetized with thiobutabarbital. Administration of LPS to animals resulted in a significant reduction in cardiac index over time. The administration of LPS to rats resulted in a substantial rise in the plasma levels of TNF-alpha. Furthermore, the injection of LPS resulted in a significant increase in the iNOS activity in the lungs. Pre-treatment with rolipram prevented the decline in cardiac index in animals that received LPS. Infusion of methoxamine into animals injected with rolipram and pre-treated with LPS did not result in significant changes in cardiac index. Pre-treatment with rolipram or dexamethasone in animals injected with LPS significantly prevented the rise in TNF-alpha when compared to the respective values in vehicle-treated animals. Our present observations support the view that the cardiac index can be maintained in animals treated with LPS independent of iNOS inhibition.

Site-specific deoxynucleotide substitutions in yeast U6 snRNA block splicing of pre-mRNA in vitro.

We have identified 2'-hydroxyl groups of the U6 phosphate-ribose backbone which are required for reconstitution of splicing activity in U6-depleted yeast extract. To screen the 2'-hydroxyls of yeast U6 at nucleotides 39-88, spanning the conserved central domain, synthetic U6 RNAs were constructed with deoxyribonucleotides incorporated site specifically. Only four individual deoxynucleotide substitutions blocked splicing activity: dA51 (in the ACAGAG sequence), dA62 (next to the AGC triad), and dU70 and dC72 (both in the loop of the 3' intramolecular stem-loop). Native gel analysis revealed that these deoxy-substituted U6 RNAs were competent for assembly of spliceosomes. Interestingly, a 2'-O-methyl substituent at A51, A62, U70 or C72 did not inhibit splicing activity, indicating that the essential 2'-OH groups at these positions in U6 act as hydrogen bond acceptors or neutral coordinated ligands. The requisite 2'-hydroxyls at A62, U70 and C72 show both similarities and differences relative to the positions of essential 2'-hydroxyls of catalytic domain V of group II ribozymes. The identification of the essential 2'-hydroxyls at positions 62, 70 and 72 corroborates that the 3' intramolecular stem-loop in U6 plays an important role in pre-mRNA splicing.

Reactions between tunichrome Mm-1, a tunicate blood pigment, and vanadium ions in acidic and neutral media.

Tunichromes are yellow, polyphenolic tripeptides prevalent in blood cells of tunicates (suborders phlebobranchia and stolidobranchia). Spectrophotometric studies of reactions between tunichrome Mm-1 and VV or VIV ions were conducted in vitro in various media to crudely approximate cellular conditions: deionized water, aqueous methanol, and aqueous buffers at pH 2 and 7. Catechol was used in parallel studies for comparison to tunichrome and was found to be a good model for tunichrome reactivity. For VIV in pH 7 buffer, both catechol and Mm-1 formed complexes with VIV ions, and no redox products were found. For VV in pH 2 buffer, both catechol and Mm-1 were oxidized by VV ions. Room temperature EPR qualitatively showed that Mm-1 in pH 2 buffer reduced VV ions to free VIV ions. For VV in pH 7 buffer, Mm-1 was oxidized by VV ions and formed VIV complexes. At higher concentrations, the VIV complexes were observed by low temperature EPR [Grant, K. B. (1994) Dissertation, Columbia University; Grant, K. B., et al. (1996) J. Inorg. Biochem. (manuscript in preparation)]. Using a colorimetric assay for VIII, we found that reactions between Mm-1 and VV or VIV ions in pH 7 buffer clearly did not generate appreciable quantities of VIII products. Thus, the colorimetric VIII assay resolved the issue of VIII product formation raised in EPR studies of Mm-1 [cf. Ryan, D. E., et al. (1992) Biochemistry 35, 8651-8661]. Overall, the results provide insights into tunichrome-vanadium chemistry and identify conditions which promote complexation and/or redox reactions in vitro.

Reactions between vanadium ions and biogenic reductants of tunicates: spectroscopic probing for complexation and redox products in vitro.

Several species of marine tunicates store oxygen-sensitive VIII in blood cells. A sensitive colorimetric VIII assay was used to survey the leading candidates for the native reducing agent of vanadate in tunicates (i.e., An-type tunichromes, glutathione, NADPH, and H2S) in reactions with VV or VIV ions under anaerobic, aqueous conditions at acidic or neutral pH. Except for the case of An-1 and VV ions in pH 7 buffer, the assay results for the biogenic reducing agents clearly showed that appreciable quantities of VIII products were not generated under the conditions tested. Therefore, the assay results place new limits on hypothetical mechanisms of VIII formation in vivo. For reactions between An-1 and VV ions in pH 7 buffer, low levels of VIII products could not be ruled out because of an interfering peak in the colorimetric assays. For similar reactions between VV ions and An-1, or an An-1,2 mixture, in mildly to moderately basic media, the product mixtures precipitated as greenish black solids. Analyses of the precipitated V/An mixtures using vanadium K-edge X-ray absorption spectroscopy (XAS) showed that the major products were tris(catecholate)-type VIV complexes (65 +/- 6%) and bis(catecholate)-type VIVO complexes (20 +/- 4%). XAS analysis of the V/An-1 product mixture also provided evidence of a minor VIII component (9 +/- 5% of total V), notable for possible relevance to tunicate biochemistry. The combined results of XAS studies, spectrophotometric studies [Ryan, D. E., et al. (1996) Biochemistry 35, 8640-8650], and EPR studies [Grant, K. B., et al. (1996) J. Inorg. Biochem. (manuscript in preparation)] consistently establish that reactions between tunichromes (Mm-1 or An-1) and VV ions generate predominantly VIV-tunichrome complexes in neutral to moderately basic aqueous media.

The effects of intra-articular deposition of betamethasone in the goat temporomandibular joint.

PURPOSE: This study evaluated the response of the articular surfaces of the normal goat temporomandibular joint (TMJ) to intra-articular injections of betamethasone suspension. METHODS: Thirty female goats were divided into four experimental groups of seven each and one control group of two animals. The design resulted in 24 joints receiving from one to nine weekly injections of betamethasone suspension, 0.085 mg/kg. The 24 contralateral joints received an identical array of saline injections. In the remaining 12 joints, four were unilaterally injected with saline, four were unilateral uninjected joints, and four were bilateral uninjected joints. All of the joints were inspected grossly and histologically assessed for any intra-articular changes associated with corticosteroid injections. RESULTS: Comparative examination of the gross and histologic features of the injected joints and the eight normal joints showed no significant adverse effects on nondiseased TMJs. CONCLUSION: Intra-articular injection of betamethasone suspension at the dosage and delivery rate used had no harmful effects on the TMJs of nondiseased adult female goats. Because of possible differences between species, these results should not be taken as an indication that betamethasone suspension will not have adverse effects in the human TMJ. These findings support the need for further investigations into the physical and biochemical responses of normal and diseased articular fibrocartilage to different glucocorticosteroids at various concentrations and delivery rates using the goat and other animal models.

Insertion of a structural domain of interleukin (IL)-1 beta confers agonist activity to the IL-1 receptor antagonist. Implications for IL-1 bioactivity.

We showed previously that replacement of Lys-145 in the IL-1 receptor antagonist (IL-1ra) with Asp resulted in an analog (IL-1ra K145D) with partial agonist activity. To identify additional amino acids that affect IL-1 bioactivity, we created second site mutations in IL-1ra K145D. Substitutions of single amino acids surrounding position 145 were made; none of these substitutions increased the bioactivity of IL-1ra K145D. However, the insertion of the beta-bulge (QGEESN) of IL-1 beta at the corresponding region of IL-1ra K145D resulted in a 3-4-fold augmentation of bioactivity. An additional increase in agonist activity was observed when the beta-bulge was co-expressed with a second substitution (His-54 --> Pro) in IL-1ra K145D. We also show that the bioactivity of both IL-1ra K145D and the triple mutant IL-1ra K145D/H54P/QGEESN is dependent on interaction with the newly cloned IL-1 receptor accessory protein.

Monoclonal antibodies of differentiating specificities as probes of cytochrome P450h (2C11).

A panel of 30 monoclonal antibodies against rat hepatic microsomal cytochrome P450h (2C11) has been produced, purified, and characterized. A broad range of reactivities was observed when 13 purified rat cytochrome P450 isozymes were tested for epitope relatedness in a noncompetitive enzyme-linked immunosorbent assay or on immunoblots. Several antibodies were antigen-specific, others reacted with additional members of the 2C subfamily, and other monoclonal antibodies recognized cytochromes P450 from the 2E, 2B, 2A, and 1A subfamilies. Cytochromes P450p (3A1) and P4501 (3A2) did not react with any of the antibodies. A minimum of seven spatially distinct epitopes on cytochrome P450h were defined by the panel of antibodies. Immunoblot analysis of rat microsomes illustrated the male specificity of cytochrome P450h expression which extended to extrahepatic tissues including kidney and lung. A survey of various species by immunoblot analysis with several antibodies revealed little if any epitope relatedness among microsomal proteins from rats, mice, rabbits, hamsters, squirrel monkeys, guinea pigs, or humans. All of the antibodies were screened as potential inhibitors of cytochrome P450h-mediated testosterone hydroxylation in a reconstituted system. Although most of the antibodies were noninhibitory, greater than 70% inhibition of 2 alpha- and 16 alpha-hydroxylation of testosterone was observed with selected antibodies. These inhibitory antibodies gave similar results when benzphetamine N-demethylation was evaluated in the reconstituted system. The inhibitory antibodies were then used to assess the role of cytochrome P450h in microsomal benzphetamine N-demethylation, since this isozyme exhibits high catalytic activity for this substrate. Only 20-25% inhibition of benzphetamine metabolism was attained in microsomal preparations from adult male rats, and the antibodies did not influence the microsomal catalytic activity of immature males or females or adult females. Thus, despite the high level of expression of cytochrome P450h in microsomes from adult male rats and the high catalytic activity of the purified protein for benzphetamine, this isozyme contributes only a small portion of the metabolism of this substrate in microsomes.

Regulation of two members of the steroid-inducible cytochrome P450 subfamily (3A) in rats.

Monoclonal antibodies have been successfully isolated which are isozyme-specific for cytochrome P450p (3A1) or P4501 (3A2), two members of the steroid-inducible cytochrome P450 subfamily exhibiting 89% amino acid sequence homology, and these antibodies show less than 5% cross-reaction with 11 other cytochromes P450 (P450a-P450k). A library of 28 purified monoclonal antibodies was established and characterized as to epitope specificity. Appropriate antibodies were selected and utilized to investigate the regulation of expressed cytochrome P450p and P4501 proteins as a function of age, sex, and treatment of rats with various inducing agents. Cytochrome P450p is not detectable in hepatic microsomes from untreated immature or adult male and female rats. Following dexamethasone treatment, expression of cytochrome P450p is observed in all groups with the levels reaching 30-37% of total microsomal cytochrome P450. Administration of other inducers such as pregnenolone 16 alpha-carbonitrile also yield enhanced levels of cytochrome P450p. Measurable amounts of constitutive cytochrome P4501 were detected in hepatic microsomes from immature and adult males as well as immature females but not in adult females. Cytochrome P4501 expression is inducible by dexamethasone in immature rats of both sexes and adult males, although dexamethasone is more effective as an inducer of cytochrome P450p than cytochrome P4501. Hence, not only is cytochrome P4501 protein expressed in immature animals of both sexes, it is also inducible in both sexes. These studies show that constitutive expression and induction of steroid-inducible cytochrome P450s may vary as a function of age.

Temporomandibular disorders.

Temporomandibular disorders include a variety of intracapsular and extracapsular disorders that may or may not be related to each other and the diagnosis of which can be complicated. The causes of these disorders have not been identified scientifically and instead rely on anecdotal clinical experience. Preliminary epidemiologic studies have begun to identify predisposing, initiating, or perpetuating factors. Recent advances in imaging techniques have created opportunities for a more rational approach to the treatment of these disorders. Nonsurgical therapies are used to manage most of these disorders. Refinements in surgical procedures have increased the predictability of results for patients who have followed nonsurgical treatments. Arthroscopy has added to the macroscopic and microscopic knowledge of intracapsular disorders and offers a less invasive approach to surgical management. With proper diagnosis and treatment selection, quality of life of the majority of patients can be improved.

Novel stereoselectivity of rat liver cytochrome(s) P450 toward enantiomers of the trans-1,2-dihydrodiol of triphenylene.

Metabolism of 3H-labeled (+)-(S,S)- and (-)-(R,R)-1,2-dihydrodiols of triphenylene by rat liver microsomes and 11 purified isozymes of cytochrome P450 in a reconstituted monooxygenase system has been examined. Although both enantiomers were metabolized at comparable rates, the distribution of metabolites between phenolic dihydrodiols and bay-region, 1,2-diol 3,4-epoxide diastereomers varied substantially with the different systems. Treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) caused a slight reduction or less than a twofold increase, respectively, in the rate of total metabolism (per nanomole of cytochrome P450) of the enantiomeric dihydrodiols compared to microsomes from control rats. Among the 11 isozymes of cytochrome P450 tested, only cytochromes P450c (P450IA1) and P450d (P450IA2) had significant catalytic activity. With either enantiomer of triphenylene 1,2-dihydrodiol, both purified cytochrome P450c (P450IA1) and liver microsomes from MC-treated rats formed diol epoxides and phenolic dihydrodiols in approximately equal amounts. Purifed cytochrome P450d (P450IA2), however, formed bay-region diol epoxides and phenolic dihydrodiols in an 80:20 ratio. Interestingly, liver microsomes from control or PB-treated rats produced only diol epoxides and little or no phenolic dihydrodiols. The diol epoxide diastereomers differ in that the epoxide oxygen is either cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 1-hydroxyl group. With either purified cytochromes P450 (isozymes c or d) or liver microsomes from MC-treated rats, diol epoxide-2 is favored over diol epoxide-1 by at least 4:1 when the (-)-enantiomer is the substrate, while diol epoxide-1 is favored by at least 5:1 when the (+)- enantiomer is the substrate. In contrast, with liver microsomes from control or PB-treated rats, formation of diol epoxide-1 relative to diol epoxide-2 was favored by at least 2:1 regardless of the substrate enantiomer metabolized. This is the first instance where the ratio of diol epoxide-1/diol epoxide-2 metabolites is independent of the dihydrodiol enantiomer metabolized. Experiments with antibodies indicate that a large percentage of the metabolism by microsomes from control and PB-treated rats is catalyzed by cytochrome P450p (P450IIIA1), resulting in the altered stereoselectivity of these microsomes compared to that of the liver microsomes from MC-treated rats.

Trigeminal nerve neurofibroma: case report.

A case of left trigeminal nerve neurofibroma is presented. The location of the lesion required a multidisciplinary surgical effort for total excision. A vertical ramus osteotomy allowed access to the infratemporal space and base of skull. The frequency and clinical features of trigeminal nerve tumors are discussed.

Retention of antimony in skin biopsies of leishmaniasis patients after treatment with N-methylglucamine antimoniate.

Leishmaniasis patients were treated with N-methylglucamine antimoniate by intravenous injections of 10 or 20 mg of Sb per kilogram of body weight per day for 10 or 20 days. Digests of skin biopsies taken from the site of lesion before and after treatment were analyzed for antimony by instrumental neutron activation (INAA). The detection limit of the assay was 20 ng, and no Sb could be measured in digests of samples (less than 2.45 mg) taken before treatment. Biopsies taken after injections of Sb showed concentrations in the range of 8.32 to 70.68 ng/g skin. We discuss the usefulness of INAA in the study of Sb in small samples of tissues.

Induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by a series of 4'-substituted-2,3,4,5-tetrachlorobiphenyls.

We have shown previously that 2,3,4,5-tetrachlorobiphenyl is ineffective as an inducer of rat liver microsomal cytochrome P-450. Addition of a single para-chloro substituent in the otherwise unsubstituted phenyl ring, to give 2,3,4,4',5-pentachlorobiphenyl, produces a potent cytochrome P-450 inducer with both phenobarbital- and 3-methylcholanthrene-type characteristics. In the present study, 2,3,4,5-tetrachlorobiphenyl was substituted in the para(4') position with 12 other functional groups. The 4'-X-C12H5Cl4 derivatives were tested as inducers of cytochromes P-450a--P-450e and epoxide hydrolase, by immunochemical analysis of liver microsomes prepared 4 days after a single treatment (500 mumol/kg) of 1-month-old male Long Evans rats. When the para' substituent was a halogen (F, Cl, Br or I), the derivative induced both cytochromes P-450b and P-450e, and cytochromes P-450c and P-450d, which are the major phenobarbital- and 3-methylcholanthrene-inducible isozymes, respectively. A similar type of induction was observed with a second group of derivatives substituted with CN, NO2 or CF3. However, a derivative containing CH3CO--(which is also a meta-directing, ring-activating substituent) failed to induce cytochromes P-450a-P-450e at the dosage and time tested. Members of a third group of derivatives, which contained an ortho/para-directing, ring-activating substituent) were either ineffective inducers (OH, CH3, CH3O--), or were inducers of cytochromes P-450c and P-450d (isopropyl or t-butyl). Hence, 4'-substitution with a bulky lipophilic substituent conferred 3-methylcholanthrene- but not phenobarbital-type characteristics on 2,3,4,5-tetrachlorobiphenyl. Some of the derivatives tested, namely those substituted with Cl, Br, I and CF3, were remarkably effective inducers of cytochrome P-450a, causing a 10-11-fold induction of this isozyme. Data on the induction of cytochrome P-450c were analyzed by multiparameter linear regression in an attempt to correlate the biological activity of the 4'-X-C12H5Cl4 derivatives with the physiochemical properties of the various substituents. From these results, and those reported recently, we propose that binding of the 4'-X-C12H5Cl4 derivatives to the rat cytosolic Ah receptor is favored by increasing the electronegativity, lipophilicity and hydrogen bonding characteristics of the 4' substituent, whereas enzyme induction (both in vivo and in cultured rat hepatoma cells) is also governed by a fourth characteristic, the STERIMOL factor, which gives a measure of the width of the substituent.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of androstenedione formation from testosterone and epitestosterone catalyzed by purified cytochrome P-450b.

A purified rat hepatic monooxygenase system containing cytochrome P-450b oxidizes testosterone to androstenedione and 16 alpha- and 16 beta-hydroxytestosterone at approximately equal rates. The metabolism of epitestosterone by the same system is characterized by a marked stereoselectivity in favor of 16 beta-hydroxylation (4- to 5-fold relative to 16 alpha-hydroxylation), formation of 15 alpha-hydroxyepitestosterone, and a rate of androstenedione formation which is three to five times higher than that observed with testosterone. Apparent Km values for 16 alpha- and 16 beta-hydroxylation and androstenedione formation are 20-30 microM with either substrate. Mass spectral analysis of the androstenedione formed from [16,16-2H2]testosterone and [16,16-2H2] epitestosterone indicates essentially complete retention of deuterium, thereby ruling out a mechanism of androstenedione formation via C-16 hydroxylation followed by loss of water and rearrangement. Mass spectral analysis of the C-16 hydroxylation products from incubations of testosterone or epitestosterone in 18O2 shows essentially complete incorporation of 18O (greater than 95%). Androstenedione formed from testosterone is enriched in 18O only 2-fold (5-8%) over background, while the androstenedione formed from epitestosterone shows 84% enrichment. Kinetic experiments utilizing [17-2H]testosterone and [17-2H]epitestosterone as substrates indicate that cleavage of the C-17 carbon-hydrogen bond is involved in a rate-limiting step in the formation of androstenedione from both substrates. Taken together, our results indicate that androstenedione formation from epitestosterone proceeds exclusively through the gem-diol pathway, while androstenedione formation from testosterone may proceed through a combination of gem-diol and dual hydrogen abstraction pathways.