Reactive granulomatous dermatitis as a clinically relevant and unifying term: a retrospective review of clinical features, associated systemic diseases, histopathology and treatment for a series of 65 patients at Mayo Clinic.

BACKGROUND: Reactive granulomatous dermatitis (RGD) is an umbrella term used to describe interstitial granulomatous dermatitis (IGD), palisaded neutrophilic and granulomatous dermatitis (PNGD), and interstitial granulomatous drug eruption (IGDR). OBJECTIVE: The aim of this study was to describe systemic associations of RGD, explore possible associations between histopathologic findings and systemic RGD associations and determine clinical relevance of RGD subtypes. METHODS: We retrospectively studied clinical and histopathologic characteristics of patients with RGD from 1990 through 2020. RESULTS: Of 65 patients with RGD (41 women, 24 men; median age at diagnosis, 62 years), 37 had IGD, 26 had PNGD, and 2 had IGDR. Fifty patients (76.9%) had an associated systemic condition; rheumatologic conditions were identified for 34 (52.3%) patients. The associated systemic condition occurred before RGD in approximately 75% of patients. Statistical analyses did not show significant associations between specific subtypes of RGD and systemic diseases or treatment response, and specific histopathologic findings were not predictive of an associated systemic disease. CONCLUSIONS: Although most patients with RGD had an associated systemic condition, subtypes of RGD did not correlate with systemic associations, lending support to the use of the umbrella term RGD.

Leukocytoclastic vasculitis in children: clinical characteristics, subtypes, causes and direct immunofluorescence findings of 56 biopsy-confirmed cases.

BACKGROUND: Leukocytoclastic vasculitis (LCV) in children is a complex group of conditions. OBJECTIVES: This study presents the demographics, clinical features, direct immunofluorescence (DIF) results and suspected aetiologies of 56 biopsy-confirmed cases of leukocytoclastic vasculitis in children. METHODS: Retrospective review of 56 children seen at Mayo Clinic in Rochester, Minnesota, from 1993 to 2013 with clinical features and cutaneous biopsy consistent with LCV. RESULTS: Twenty-seven (48%) cases were found to be due to IgA vasculitis (Henoch-Schonlein purpura). The remaining cases were found to be due to cutaneous small-vessel vasculitis (n = 19, 34%), urticarial vasculitis (n = 5, 9%), ANCA-associated vasculitis (n = 4, 7%) and acute haemorrhagic oedema of infancy (n = 1, 2%). IgA vasculitis was found to be associated with abdominal pain (P = 0.008), whereas the non-IgA vasculitis group was associated with headache (P = 0.052). Children with IgA vasculitis had palpable purpura (P = <0.001), petechia (P = 0.057), vesicles (P = 0.009) and involvement of the buttock (P = 0.004) more frequently than the non-IgA vasculitis group. On DIF, perivascular IgA was positive in IgA vasculitis compared to non-IgA vasculitis cases (P = <0.001), the other conjugates were similar between the two groups. CONCLUSION: The most common subtype of biopsy-confirmed LCV in children is IgA vasculitis. Clinical features, exam characteristics and DIF results can be helpful in determining the subtype of cutaneous vasculitis in children.

Henoch-Schönlein purpura and systemic disease in children: retrospective study of clinical findings, histopathology and direct immunofluorescence in 34 paediatric patients.

BACKGROUND: Henoch-Schönlein purpura (HSP), an IgA-mediated small vessel vasculitis, is the most common form of vasculitis in children. HSP is commonly associated with systemic involvement of the gastrointestinal tract, joints and kidneys. Renal involvement is the main cause of morbidity and mortality in HSP. OBJECTIVES: To characterize the clinical, histopathological and direct immunofluorescence (DIF) findings, and to correlate the findings with systemic disease in 34 children with HSP seen at our institution. METHODS: This was a retrospective review of paediatric patients with HSP and with available biopsy specimens seen at our institution between 1993 and 2013. RESULTS: Thirty-four paediatric patients were identified (mean age 10·7 years). Renal involvement was found in 17 (50%) patients, gastrointestinal tract involvement in 22 (65%) and joint involvement in 23 (68%). Renal involvement was significantly associated with papillary dermal oedema on histopathology (P < 0·01) and the presence of perivascular C3 on DIF (P = 0·01). The presence of lesions above the waist was significantly associated with gastrointestinal involvement (P = 0·03), as was the presence of clinically apparent oedema (P = 0·01). CONCLUSIONS: This study suggests that in children with HSP, microscopic dermal oedema and C3 on DIF may be predictive of renal involvement. Patients with clinically apparent oedema and lesions above the waist are more likely to have gastrointestinal involvement.

[Computer model of 3D structure of cytochrome P450 2B4]. / Komp'iuternaia model' trekhmernoi struktury tsitokhroma P450 2B4.

Cytochromes P450 (CYPs) play an important role in the oxidative metabolism of xenobiotics. Three-dimensional structures of CYPs are needed to study structure-function relationships in their molecules and interaction with partner proteins. Experimental determination of eucaryotic CYPs 3D structures is difficult because of hydrophobic membrane anchors and surface hydrophobic regions that prevent their crystallization. Replacement of surface hydrophobic amino acids by hydrophilic residues without any changes in protein structure and function can help to solve this problem. Such modification can be proposed using the analysis of 3D model of protein. In this work computer aided 3D structure of microsomal P450 2B4 (CYP2B4) was modeled for the further prediction of surface mutations for hydrophilization of the protein surface. The model of 3D structure of CYP2B4 was constructed by homology with CYP2C5 Model optimization was made by energy minimization and molecular dynamics simulation of protein molecule in water environment. The model was verified by using special statistic software and by comparison with the experimental data on the substrate recognition site, redox-partner binding sites and chemical modification of the protein surface.

A major CYP2D6 autoepitope in autoimmune hepatitis type 2 and chronic hepatitis C is a three-dimensional structure homologous to other cytochrome P450 autoantigens.

Liver-kidney microsomal antibodies type 1 (LKM) are a diagnostic marker for autoimmune hepatitis type 2 (AIH-2), however, LKM autoantibodies are also detected in a small percentage of patients with chronic hepatitis C. The major target of LKM antibodies as evidenced by indirect immunofluorescence is cytochrome P4502D6 (CYP2D6). Anti-CYP2D6 titers of 62 LKM positive sera, 196 sera of patients with hepatic and rheumatic diseases and 33 sera of healthy blood donors (BD) were determined by an in vitro transcription/in vitro translation assay (ITT). Twenty five out of 26 AIH-2 sera and 33/36 LKM positive hepatitis C virus (HCV) sera were anti-CYP2D6 positive by ITT and antibody titers were similar in both patient groups. Epitope mapping experiments were performed by a series of truncated CYP2D6 proteins and by single epitopes of 257-269, 321-351, 373-389 and 410-419 amino acid (aa) expressed as DHFR-fusion proteins in Escherichia coli. The major linear epitope consists of 257-269 aa. This epitope is recognized with a significantly higher prevalence (64%) in AIH-2 than in LKM sera from patients with chronic hepatitis C (24%) (p < 0.001). None of the other autoepitopes showed significant differences in the prevalence of recognition by sera from both patient groups. Minor binding sites consisted of 321-351 aa, which was recognized by less than 20% of LKM sera and in the C-terminal region of 350-494 aa, which was recognized by less than 5% of LKM sera Our study revealed an epitope of 321-379 an on CYP2D6, which was shown to be conformation dependent. It was recognized by the vast majority of LKM sera, specifically by 76% of sera from HCV positive LKM patients and also by 76% of sera from patients with AIH-2. This epitope is homologous to three-dimensional epitopes detected by autoantibodies directed against hepatic cytochromes P450s in drug induced hepatitis and to an autoepitope on CYP21B associated with adrenal failure.

Identification of human CYP2C19 residues that confer S-mephenytoin 4'-hydroxylation activity to CYP2C9.

CYP2C19 is selective for the 4'-hydroxylation of S-mephenytoin while the highly similar CYP2C9 has little activity toward this substrate. To identify critical amino acids determining the specificity of human CYP2C19 for S-mephenytoin 4'-hydroxylation, we constructed chimeras by replacing portions of CYP2C9 containing various proposed substrate recognition sites (SRSs) with those of CYP2C19 and mutating individual residues by site-directed mutagenesis. Only a chimera containing regions encompassing SRSs 1--4 was active (30% of wild-type CYP2C19), indicating that multiple regions are necessary to confer specificity for S-mephenytoin. Mutagenesis studies identified six residues in three topological components of the proteins required to convert CYP2C9 to an S-mephenytoin 4'-hydroxylase (6% of the activity of wild-type CYP2C19). Of these, only the I99H difference located in SRS 1 between helices B and C reflects a change in a side chain that is predicted to be in the substrate-binding cavity formed above the heme prosthetic group. Two additional substitutions, S220P and P221T residing between helices F and G but not in close proximity to the substrate binding site together with five differences in the N-terminal portion of helix I conferred S-mephenytoin 4'-hydroxylation activity with a K(M) similar to that of CYP2C19 but a 3-fold lower K(cat). Three residues in helix I, S286N, V292A, and F295L, were essential for S-mephenytoin 4'-hydroxylation activity. On the basis of the structure of the closely related enzyme CYP2C5, these residues are unlikely to directly contact the substrate during catalysis but are positioned to influence the packing of substrate binding site residues and likely substrate access channels in the enzyme.

Identification of peroxisome proliferator-responsive human genes by elevated expression of the peroxisome proliferator-activated receptor alpha in HepG2 cells.

In mice and other sensitive species, PPARalpha mediates the induction of mitochondrial, microsomal, and peroxisomal fatty acid oxidation, peroxisome proliferation, liver enlargement, and tumors by peroxisome proliferators. In order to identify PPARalpha-responsive human genes, HepG2 cells were engineered to express PPARalpha at concentrations similar to mouse liver. This resulted in the dramatic induction of mRNAs encoding the mitochondrial HMG-CoA synthase and increases in fatty acyl-CoA synthetase (3-8-fold) and carnitine palmitoyl-CoA transferase IA (2-4-fold) mRNAs that were dependent on PPARalpha expression and enhanced by exposure to the PPARalpha agonist Wy14643. A PPAR response element was identified in the proximal promoter of the human HMG-CoA synthase gene that is functional in its native context. These data suggest that humans retain a capacity for PPARalpha regulation of mitochondrial fatty acid oxidation and ketogenesis. Human liver is refractory to peroxisome proliferation, and increased expression of mRNAs for the peroxisomal fatty acyl-CoA oxidase, bifunctional enzyme, or thiolase, which accompanies peroxisome proliferation in responsive species, was not evident following Wy14643 treatment of cells expressing elevated levels of PPARalpha. Additionally, no significant differences were seen for the expression of apolipoprotein AI, AII, or CIII; medium chain acyl-CoA dehydrogenase; or stearoyl-CoA desaturase mRNAs.

Amino acid residues critical for differential inhibition of CYP2B4, CYP2B5, and CYP2B1 by phenylimidazoles.

The molecular basis for reversible inhibition of rabbit CYP2B4 and CYP2B5 and rat CYP2B1 by phenylimidazoles was assessed with active-site mutants and new three-dimensional models based on the crystal structure of CYP2C5. 4-Phenylimidazole was 17- to 32-fold more potent toward CYP2B4 and CYP2B1 than CYP2B5. The 3D models, along with site-directed mutagenesis data, revealed the importance of residue 114 for sensitivity to inhibition of all three CYP2B enzymes. Besides Ile 114, Val 367 was also found to be critical for inhibition of CYP2B4 and CYP2B1. The most interesting new insights were obtained from analysis of the CYP2B5 model and the CYP2B5 active-site mutants. Simultaneous substitution of residues 114, 294, 363, and 367 with the corresponding residues of CYP2B4 decreased the IC(50) value for inhibition by 4-phenylimidazole 12-fold. Docking 4-phenylimidazole into the models of CYP2B5 mutants demonstrated that the inhibitor-binding site is strongly influenced by residue-residue interactions, especially between residues 114 and 294. A chlorine substitution at position 4 of the phenyl moiety of 4- and 1-phenylimidazole resulted in IC(50) values 95- and 130-fold lower for CYP2B4 than for CYP2B5, respectively, suggesting that these compounds are selective inhibitors of CYP2B4. Overall, the study revealed that differences in the determinants of inhibition between CYP2B4 and CYP2B5 are caused not only by single residue inhibitor contacts but also by residue-residue interactions. This new generation of CYP2B models may provide valuable information for the design of selective inhibitors of human CYP2B6 and for the development of drugs that avoid drug interactions due to P450 inhibition.

A novel pH2 control on the expression of flagella in the hyperthermophilic strictly hydrogenotrophic methanarchaeaon Methanococcus jannaschii.

The methanarchaeon, Methanococcus jannaschii, a hyperthermophilic, autotrophic, and strictly hydrogenotrophic inhabitant of submarine hydrothermal vents, was cultivated in a reactor at two hydrogen partial pressure (p(H(2))) values, 178 kPa (high) and 650 Pa (ultralow), and the cells were subjected to a comparative proteome analysis. From these studies, it was discovered that, when p(H(2)) was high and the cell density was low (a combination representing a hydrogen-excess condition), the cells possessed very low or undetectable levels of four flagella-related polypeptides (FlaB2, FlaB3, FlaD, and FlaE); electron microscopic examination showed that most of these cells were devoid of flagella. Flagella synthesis occurred when hydrogen became limiting either at high cell density under high p(H(2)) or at low cell density under low p(H(2)). The results from a p(H(2))-shift experiment corroborated the above observations. The p(H(2))-dependent changes in the levels of two methanogenic enzymes (MTD and HMDX) were as expected, and thus they served as internal controls. To our knowledge, this is the first example for the regulation of expression of flagella by hydrogen in any domain of life and for a control of any kind on flagella synthesis in the archaea. Our work also provides the only known example for each of the following: (i) the pure culture cultivation of a methanogen at an ultralow, near ecologically relevant p(H(2)); (ii) experimental functional genomics for M. jannaschii; and (iii) the use of proteomics with M. jannaschii.

Microsomal cytochrome P450 2C5: comparison to microbial P450s and unique features.

Although microsomal P450s represent the majority of P450s, only microbial P450s have been amenable to crystal structure solution. We have recently solved the first crystal structure of a microsomal P450, 2C5, a progesterone hydroxylase from rabbit. We discuss the features of the protein in common with existing structures of microbial P450s and limitations of homology modeling mammalian P450s based on the microbial structures. Unique features involving membrane, substrate and cytochrome P450 reductase interactions are also discussed.

Insights into the HER-2 receptor tyrosine kinase mechanism and substrate specificity using a transient kinetic analysis.

The HER-2/erbB-2/c-neu proto-oncogene encodes for an EGF receptor-like protein which has been implicated in the pathogenesis of several human malignancies. Although much has been learned about the physiological significance of this receptor tyrosine kinase, its catalytic mechanism remains poorly understood. We have expressed, purified, and characterized two recombinant proteins corresponding to a full-length (HCD) and truncated (HKD) construct of the HER-2 intracellular tyrosine kinase domain and have identified an optimal substrate (GGMEDIYFEFMGGKKK; HER2Peptide) through screening of a degenerate peptide library. We have conducted a transient kinetic analysis of the HER-2 proteins (HCD and HKD) to illuminate mechanistic details of the HER-2 pathway. In particular, stopped-flow fluorescence studies with mant (N-methylanthraniloyl)-nucleotide derivatives provided direct measurements of the association and dissociation rate constants for these nucleotide interactions with the HER-2 recombinant proteins, thereby enabling the determination of nucleotide K(d) values. Moreover, the actual step of chemical catalysis was isolated using rapid chemical quench techniques and shown to occur approximately 3-fold faster than the steady-state rate which corresponds to product release. Evidence is also provided that suggests a conformational change that is partially rate-limiting at least in HCD. Furthermore, the role that the phosphorylation state of the protein may play on catalysis was examined. Studies carried out with pre-phosphorylated recombinant HER-2 proteins suggest that while autophosphorylation is not a prerequisite for enzymatic activity, this protein modification actually directly affects the catalytic mechanism by enhancing the rate of ADP release and that of the rate-limiting step. While a pre-steady-state kinetic analysis has been carried out on the catalytic subunit of cAMP-dependent serine/threonine kinase, to our knowledge, this study represents the first reported transient kinetic investigation of a receptor tyrosine kinase. This work serves as a basis for comparison of these two important protein kinase families and in this report we highlight these similarities and differences.

Rabbit pregnane X receptor is activated by rifampicin.

Reverse transcriptase-polymerase chain reaction was used to amplify a partial cDNA from rabbit lung mRNA that shared 77% protein sequence identity with the mouse pregnane X receptor (PXR). Rapid amplification of cDNA ends from a rabbit kidney lambdaZAP expression library resulted in the isolation of overlapping cDNAs spanning the complete coding sequence. The deduced amino acid sequence of 411 residues exhibited 79% overall amino acid identity with human PXR and 77% identity with mouse PXR. Based on this protein sequence relationship and a similar degree of conservation exhibited by the mouse and human PXR orthologs, the cDNA appears to encode the rabbit PXR ortholog. 5'-rapid amplification of cDNA ends performed on an adaptor-ligated cDNA library from rabbit liver revealed the presence of an alternate mRNA, which differed at the 5'-terminus. RNase protection assays indicated that the alternate mRNA was expressed at >50-fold lower levels in rabbit kidney and liver. Rifampicin treatment of CV-1 cells cotransfected with a rabbit PXR expression plasmid and a luciferase reporter construct containing two copies of the DR3 enhancer from CYP3A23 produced a 6-fold induction of luciferase activity. In contrast, rat PXR was not responsive to this antibiotic under the same conditions. Pregnenolone 16alpha-carbonitrile was an efficacious activator of rat PXR, but failed to significantly activate rabbit PXR at equivalent concentrations. These results indicate that the ligand activation profile of rabbit PXR is distinct from rat PXR and more closely resembles that of human PXR. The rabbit PXR activation profile is consistent with the cytochrome P450 (P450) 3A6 induction profile in rabbits.

Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity.

Microsomal cytochrome P450s participate in xenobiotic detoxification, procarcinogen activation, and steroid hormone synthesis. The first structure of a mammalian microsomal P450 suggests that the association of P450s with the endoplasmic reticulum involves a hydrophobic surface of the protein formed by noncontiguous portions of the polypeptide chain. This interaction places the entrance of the putative substrate access channel in or near the membrane and orients the face of the protein proximal to the heme cofactor perpendicular to the plane of the membrane for interaction with the P450 reductase. This structure offers a template for modeling other mammalian P450s and should aid drug discovery and the prediction of drug-drug interactions.

CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes.

Tolbutamide is a sulfonylurea-type oral hypoglycemic agent whose action is terminated by hydroxylation of the tolylsulfonyl methyl moiety catalyzed by cytochrome P-450 (CYP) enzymes of the human CYP2C subfamily. Although most studies have implicated CYP2C9 as the exclusive catalyst of hepatic tolbutamide hydroxylation in humans, there is evidence that other CYP2C enzymes (e.g., CYP2C19) may also participate. To that end, we used an immunochemical approach to assess the role of individual CYP2Cs in microsomal tolbutamide metabolism. Polyclonal antibodies were raised to CYP2C9 purified from human liver, and were then back-adsorbed against recombinant CYP2C19 coupled to a solid-phase support. Western blotting revealed that the absorbed anti-human CYP2C9 preparation reacted with only recombinant CYP2C9 and the corresponding native protein in hepatic microsomes, and no longer recognized CYP2C19 and CYP2C8. Monospecific anti-CYP2C9 not only retained the ability to inhibit CYP2C9-catalyzed reactions, as evidenced by its marked (90%) inhibition of diclofenac 4'-hydroxylation by purified CYP2C9 and by human liver microsomes, but also exhibited metabolic specificity, as indicated by its negligible (<15%) inhibitory effect on S-mephenytoin 4'-hydroxylation by purified CYP2C19 or hepatic microsomes containing CYP2C19. Monospecific anti-CYP2C9 was also found to inhibit rates of tolbutamide hydroxylation by 93 +/- 4 and 78 +/- 6% in CYP2C19-deficient and CYP2C19-containing human liver microsomes, respectively. Taken together, our results indicate that both CYP2C9 and CYP2C19 are involved in tolbutamide hydroxylation by human liver microsomes, and that CYP2C19 underlies at least 14 to 22% of tolbutamide metabolism. Although expression of CYP2C19 in human liver is less than that of CYP2C9, it may play an important role in tolbutamide disposition in subjects expressing either high levels of CYP2C19 or a catalytically deficient CYP2C9 enzyme.

Engineering microsomal cytochrome P450 2C5 to be a soluble, monomeric enzyme. Mutations that alter aggregation, phospholipid dependence of catalysis, and membrane binding.

Deletion of the N-terminal membrane-spanning domain from microsomal P450s 2C5 and 2C3 generates the enzymes, 2C5dH and 2C3dH, that exhibit a salt-dependent association with membranes indicating that they retain a monofacial membrane interaction domain. The two proteins are tetramers and dimers, respectively, in high salt buffers, and only 2C5dH requires phospholipids to reconstitute fully the catalytic activity of the enzyme. Amino acid residues derived from P450 2C3dH between residues 201 and 210 were substituted for the corresponding residues in P450 2C5 to identify those that would diminish the membrane interaction, the phospholipid dependence of catalysis, and aggregation of 2C5dH. Each of four substitutions, N202H, I207L, S209G, and S210T, diminished the aggregation of P450 2C5dH and produced a monomeric enzyme. The N202H and I207L mutations also diminished the stimulation of catalytic activity by phospholipid and reduced the binding of P450 2C5dH to phospholipid vesicles. The modified enzymes exhibit rates of progesterone 21-hydroxylation that are similar to that of P450 2C5dH. These conditionally membrane-bound P450s with improved solubility in high salt buffers are suitable for crystallization and structural determination by x-ray diffraction studies.

Reactor-scale cultivation of the hyperthermophilic methanarchaeon Methanococcus jannaschii to high cell densities.

For the hyperthermophilic and barophilic methanarchaeon Methanococcus jannaschii, we have developed a medium and protocols for reactor-scale cultivation that improved the final cell yield per liter from approximately 0.5 to approximately 7.5 g of packed wet cells ( approximately 1.8 g dry cell mass) under autotrophic growth conditions and to approximately 8.5 g of packed wet cells ( approximately 2 g dry cell mass) with yeast extract (2 g liter(-1)) and tryptone (2 g liter(-1)) as medium supplements. For growth in a sealed bottle it was necessary to add Se to the medium, and a level of 2 microM for added Se gave the highest final cell yield. In a reactor M. jannaschii grew without added Se in the medium; it is plausible that the cells received Se as a contaminant from the reactor vessel and the H(2)S supply. But, for the optimal performance of a reactor culture, an addition of Se to a final concentration of 50 to 100 microM was needed. Also, cell growth in a reactor culture was inhibited at much higher Se concentrations. These observations and the data from previous work with methanogen cell extracts (B. C. McBride and R. S. Wolfe, Biochemistry 10:4312-4317, 1971) suggested that from a continuously sparged reactor culture Se was lost in the exhaust gas as volatile selenides, and this loss raised the apparent required level of and tolerance for Se. In spite of having a proteinaceous cell wall, M. jannaschii withstood an impeller tip speed of 235.5 cms(-1), which was optimal for achieving high cell density and also was the higher limit for the tolerated shear rate. The organism secreted one or more acidic compounds, which lowered pH in cultures without pH control; this secretion continued even after cessation of growth.

Structure-function of cytochromes P450 and flavin-containing monooxygenases: implications for drug metabolism.

This article is a report on a symposium held at Experimental Biology '98 in San Francisco, California. Recent developments in site-directed mutagenesis, computer-modeling, and mechanistic analysis of cytochromes P450 and flavin-containing monooxygenases are described. A unifying theme is the elaboration of general approaches for understanding and predicting the function of individual forms of these enzymes. A related goal is the production of soluble forms of mammalian cytochromes P450 for X-ray crystallography.

The value of acoustic rhinometry in assessing nasal responses to cat exposure.

BACKGROUND: Acoustic rhinometry (AR) uses sonar principles to map the anatomy of the nasal cavity and has been used in other studies to assess acute airway responses to allergen exposure. OBJECTIVE: The purpose of this study was to evaluate the utility of AR in assessing acute airway responses to cat allergen exposure by using a well-characterized cat exposure model. METHODS: Thirty subjects with a history of cat-induced rhinitis and a positive skin prick test response to cat allergen underwent an environmental cat challenge. Of these 30 subjects, 10 also had repeat challenges at lower levels of antigen to determine whether there was a dose response. Five subjects with negative skin test responses to cat were recruited as control subjects. During the 1-hour cat exposure, upper and lower respiratory symptoms were scored every 5 minutes, and spirometry and AR were obtained every 15 minutes. RESULTS: Although 29 of 30 subjects had changes in AR measurements, no correlations were detected between upper respiratory symptom scores and any of the changes observed in AR. In comparing the baseline challenges with lower antigen level challenges, upper respiratory symptom scores differed significantly (P = .002), whereas AR responses were nearly identical. Subjects without cat allergy did exhibit less response by AR (P = .05 to .13), but the greatest differences remained in the upper respiratory symptoms scores (P < .0001). CONCLUSION: We conclude that although AR does provide an objective measure of nasal response to allergen exposure, it has significant limitations. These are evidenced by the lack of correlation with symptoms, the inability to measure a dose response, and the changes noted even among the control subjects.

Identification of amino acid substitutions that confer a high affinity for sulfaphenazole binding and a high catalytic efficiency for warfarin metabolism to P450 2C19.

Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, KS, of <1 microM. P450 2C19 is not affected by SFZ at concentrations up to 100 microM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227-338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (KS approximately 4 microM) that was not seen when a shorter fragment of 2C9 was substituted (227-282). However, replacement of amino acids 283-338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a KS < 10 microM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (KS < 5 microM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower KM for (S)-warfarin metabolism (<10 microM) typical of 2C9.