Development of a Stability-Indicating Purity Method for Ubrogepant through Stress Degradation Analysis, Extraction, and Characterization of Unidentified Degradation Products Using Flash Chromatography, NMR, IR, and LC-MS.

BACKGROUND: Ubrogepant is a prescription medication used to prevent migraine headaches. It is currently available in tablet form. OBJECTIVE: The goal of this work is to investigate the drug degradation profile of Ubrogepant, as well as to isolate and characterize undiscovered Ubrogepant degradation products by utilizing LC-MS, NMR, and IR spectroscopic analytical techniques. Furthermore, to develop a high resolution, sensitive, stability-indicating analytical approach for detecting and quantifying Ubrogepant degradation products in its pharmaceutical formulation. METHOD: To identify and quantify the degradation products of Ubrogepant in pharmaceutical products, a novel gradient reverse-phase HPLC (RP-HPLC) technique with a PDA detector was developed by utilizing a C18 stationary phase column. The eluent comprised a mixture of acetonitrile and water with 0.1% v/v ortho-phosphoric acid. To establish the intrinsic stability of Ubrogepant pharmaceutical product, it was stress tested under various degrading circumstances, including water, alkaline, acid hydrolysis, photolytic, oxidative, and thermal. Flash chromatography was used to isolate the two major degradants, and the structures were determined using NMR (1H , 13C, DEPT 135), IR, and LC-MS methods. RESULTS: The Ubrogepant medication was relatively more degradable in alkaline and acidic circumstances, and two unique degradation products were discovered. Based on spectroscopic and chromatographic evidence, it was conclusively demonstrated that these unique compounds were Ubrogepant hydrolysis products. All degradation products have been separated with a resolution greater than 2.0. The peak purity data shown that the Ubrogepant peak in all of the stress samples examined was pure. Under all stress environments, Ubrogepant achieved a minimum mass balance of 95%. The validated developed approach was sensitive enough to quantify Ubrogepant degradation products at 0.03% of the Ubrogepant test concentration. CONCLUSIONS: The proposed method was found to be stability-indicating since it fits all of the regulatory authorities' typical requirements. This method is highly efficient for detecting and quantifying impurities in Ubrogepant drug substances and drug products in QC laboratories. HIGHLIGHTS: Two new degradation products of Ubrogepant were successfully extracted and characterized using NMR, IR, and LC-MS spectroscopic methods. The proposed HPLC method can accurately quantify the degradation products of Ubrogepant in pharmaceutical products and is sensitive enough to detect degradation products of Ubrogepant as low as 0.17 µg/mL.

A Comprehensive Review of Thrombocytopenia With a Spotlight on Intensive Care Patients.

Thrombocytopenia is a common entity seen in ICU patients and is associated with increased morbidity such as bleeding and transfusions, and mortality in ICU patients. Various mechanisms such as decreased platelet production, sequestration, destruction, consumption, and sometimes a combination of these factors contribute to thrombocytopenia. An understanding of the mechanism is essential to diagnose the cause of thrombocytopenia and to help provide appropriate management. The management strategies are aimed at treating the underlying disorder, such as platelet transfusion to treat complications like bleeding. Several studies have aimed to provide the threshold for platelet transfusions in various clinical settings and recommend a conservative approach in the appropriate scenario. In this review, we discuss various pathophysiological mechanisms of thrombocytopenia and the diverse scenarios of thrombocytopenia encountered in the ICU setting to shed light on the varied thresholds for platelet transfusion, alternative agents to platelet transfusion, and future directions for the implementation of thromboelastography (TEG) in multiple clinical scenarios to assist in the administration of appropriate blood products to correct coagulopathy.

From Chest Wall Resection to Medical Management: The Continued Saga of Parapneumonic Effusion Management and Future Directions.

Pleural space infections have been described since the time of Hippocrates and to this day remains a significant pathology. Every year in the USA approximately there are one million hospital admissions for pneumonia with 20%-40% associated with some form of pleural space infections leading to pleural effusions with increased morbidity and mortality. Often, management of these effusions mandate combination of medical treatment and surgical drainage with debridement and decortication. There has been a lot of ongoing research regarding the safety and efficacy of intrapleural fibrinolytics in the management of complicated pleural effusions and empyema. Till this day, areas of debate and controversies exist among clinicians treating pleural space infection. Empyema is historically considered a surgical disease. There have been societies and guidelines for the management of infected parapneumonic effusions with antibiotics and chest tube drainage as an initial empiric treatment modality. With the advances in the use of Intrapleural fibrinolytics and minimally invasive procedures such as video-assisted thoracoscopic surgery (VATS), empyema a surgical disease is now more favoring medical management. Surgical option, such as open thoracotomy, is reserved for patients who failed conservative management and chronic empyema. The aim of this comprehensive review is to shed light on the evolution of various management strategies from the era of Hippocrates to current day practice and how there continues to be a paradigm shift in treating empyema as a surgical condition to a medical disease.

Comparative appraisal of ghee and common vegetable oils for spectral characteristics in FT-MIR reflectance spectroscopy.

FT-MIR spectra of ghee (anhydrous milk fat) and common vegetable oils were acquired using HATR in 4000-650 cm-1 region. The differences in absorbance by carbon-hydrogen (C-H) stretch in fatty acid chain at 3.48 µm and absorbance by carbonyl (C-O) stretch of ester linkage at 5.7 µm in ghee and that in vegetable oils were studied. The clear differences in the spectra of ghee and that of the vegetable oils were noticed in fingerprint region, which can be very well utilized to develop FT-MIR spectroscopy as a promising tool to detect presence of common vegetable oils mixed in the ghee as an adulterant.

Iliac vein stenosis is an underdiagnosed cause of pelvic venous insufficiency.

BACKGROUND: Reflux in the ovarian veins, with or without an obstructive venous outflow component, is reported to be the primary cause of pelvic venous insufficiency (PVI). The degree to which venous outflow obstruction plays a role in PVI is currently ill-defined. METHODS: We retrospectively reviewed the charts of 227 women with PVI who presented to the Center for Vascular Medicine from January 2012 to September 2015. Assessments and interventions consisted of an evaluation for other causes of chronic pelvic pain by a gynecologist; preintervention and postintervention visual analog scale (VAS) pain score; complete venous duplex ultrasound examination; and Clinical, Etiology, Anatomy, and Pathophysiology classification. All patients underwent diagnostic venography of their pelvic and left ovarian veins as well as intravascular ultrasound of their iliac veins. Patients were treated in one of six ways: ovarian vein embolization (OVE) alone (chemical ± coils), OVE with staged iliac vein stenting, OVE with simultaneous iliac vein stenting, iliac vein stenting alone, OVE with venoplasty, and venoplasty alone. RESULTS: Of the 227 women treated, the average age and number of pregnancies was 46.4 ± 10.4 years and 3.36 ± 1.99, respectively. Treatment distribution was the following: OVE, n = 39; OVE with staged stenting, n = 94; OVE with simultaneous stenting, n = 33; stenting alone, n = 50; OVE with venoplasty, n = 8; and venoplasty alone, n = 3. Seven patients in the OVE and stenting groups (staged) and one patient in the OVE + venoplasty group required a second embolization of the left ovarian vein. Eighty percent (181/227) of patients demonstrated an iliac stenosis >50% by intravascular ultrasound. Average VAS scores for the entire cohort before and after intervention were 8.45 ± 1.11 and 1.86 ± 1.61 (P ≤ .001). In the staged group, only 9 of 94 patients reported a decrease in the VAS score with OVE alone. VAS score decreased from 8.6 ± 0.89 before OVE to 7.97 ± 2.10 after OVE. After the planned staged stenting, VAS score decreased to 1.33 ± 2.33 (P ≤ .001). Similarly, in the simultaneous group, preintervention scores were 8.63 ± 1.07 and decreased to 2.36 ± 2.67 after OVE + stenting (P ≤ .001). CONCLUSIONS: The majority of patients in our series (80%) demonstrated a significant iliac vein stenosis. These observations indicate that the incidence of iliac vein outflow obstruction in PVI is greater than previously reported. In patients with combined ovarian vein reflux and iliac vein outflow obstruction, our data suggest that pelvic venous outflow lesions should be treated first and that ovarian vein reflux should be treated only if symptoms persist. In women with an outflow lesion, ovarian vein reflux, and a large pelvic reservoir, we recommend simultaneous treatment.

Chronic kidney disease after orthotopic liver transplantation in recipients receiving tacrolimus.

INTRODUCTION: Chronic kidney disease (CKD) is common in liver transplant recipients receiving calcineurin inhibitors. METHOD AND POPULATION: The goals of this case-control study were to identify risk factors associated with CKD and its effect on mortality in 294 liver transplant recipients receiving calcineurin inhibition with tacrolimus. RESULTS: Hepatitis C virus (HCV) was the most common indication (42%) for transplantation. CKD 4 and 5 (estimated glomerular filtration rate (eGFR) of <=29 ml/min/1.73 m2) developed in 10.8% of recipients during a mean follow-up of 52 months. The incidence density of CKD was 2.56 per 100 patient-years. End-stage renal disease developed in 2.7%. By univariate analysis, CKD patients were older (mean±sd, 57±10 vs. 51±11, p<0.05) with hypertension (56 vs. 32%, p<0.05), had lower preoperative hematocrit (31±6 vs. 34±5, p<0.05), alanine aminotransferase (median (95% confidence limit) 46 (34­80) vs. 68 (56­77), <0.05) and eGFR (56±28 vs. 91±35 ml/ min/1.73 m2, p<0.05), had higher preoperative prothrombin time (16.1 (14.6­17.2) vs. 14.8 (14.5­15.1) seconds, p<0.05), and required more perioperative renal replacement therapy (RRT) (41% vs. 6.5%, p<0.05) compared to controls. Perioperative need for RRT (hazard ratio (95% CI) 2.72 (1.05­7.03)) and lower preoperative eGFR: 60­89 (4.08 (1.23­13.5)), 30­59 (4.26 (1.18­15.36)), and<=29 (5.91 ((1.28­27.19)) vs. eGFR>=90 ml/min/1.73 m2 were independently associated with development of CKD adjusting for important covariates. The development of CKD (2.36 (1.22­4.59)) was independently associated with late mortality with an attributable risk of 12.8%. CONCLUSION: Data demonstrate that CKD is an important clinical event associated with increased risk for death after primary liver transplantation.

Compression neuropathy of common peroneal nerve caused by an extraneural ganglion: a report of two cases.

Peroneal nerve entrapment is most common in the popliteal fossa, but is rarely caused by a ganglion. Although ganglionic cysts are very common lesions, they seldom cause serious complications. Ganglionic cysts developing in the sheath of a peripheral nerve or joint capsule may cause compression neuropathy. We report on two cases of compression neuropathy of the common peroneal nerve caused by an extraneural ganglion and its evaluation with magnetic resonance imaging (MRI) and ultrasonography. The differential diagnosis should involve L5 root pathology, a posttraumatic intraneural hemorrhage, a nerve compression near the tendinous arch located at the fibular insertion of the peroneal longus muscle, and a nerve-sheath tumor. The combination of MRI and ultrasonography is useful for the accurate diagnosis of this condition, and it should be treated by microsurgical exploration as soon as possible.

Identification and characterization of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from the Gram-positive pathogen Streptococcus pneumoniae.

The UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from a Gram-positive pathogen, Streptococcus pneumoniae, was identified and characterized. The enzyme from S. pneumoniae shows 31% identity with the MurB protein from Escherichia coli, and contains the catalytic residues, substrate-binding residues and FAD-binding motif identified previously in the E. coli protein. The gene was cloned into the pET28a+ expression vector, and the 34.5 kDa protein that it encodes was overexpressed in E. coli strain BL21(DE3) to 30% of total cell protein. The majority of the protein was found to be insoluble. A variety of methods were used to increase the amount of soluble protein to 10%. This was then purified to near homogeneity in a two-step process. The absorption spectrum of the purified protein indicated it to be a flavoprotein, like its E. coli homologue, with a characteristic absorption at 463 nm. The enzyme was shown to be active, reducing UDP-N-acetylglucosamine enolpyruvate with the concomitant oxidation of NADPH, and was characterized kinetically with respect to its two substrates. The enzyme showed properties similar to those of its E. coli counterpart, being activated by univalent cations and being subject to substrate inhibition. The characterization of an important cell wall biosynthesis enzyme from a Gram-positive pathogen provides a good starting point for the discovery of antibacterial agents against MurB.

Structure-function aspects and inhibitor design of type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3).

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) type 5 has been cloned from human prostate and is identical to type 2 3alpha-HSD and is a member of the aldo-keto reductase (AKR) superfamily; it is formally AKR1C3. In vitro the homogeneous recombinant enzyme expressed in Escherichia coli functions as a 3-keto-, 17-keto- and 20-ketosteroid reductase and as a 3alpha-, 17beta- and 20alpha-hydroxysteroid oxidase. The enzyme will reduce 5alpha-DHT, Delta(4)-androstene-3,17-dione, estrone and progesterone to produce 3alpha-androstanediol, testosterone, 17beta-estradiol and 20alpha-hydroxprogesterone, respectively. It will also oxidize 3alpha-androstanediol, testosterone, 17beta-estradiol and 20alpha-hydroxyprogesterone to produce 5alpha-androstane-3,17-dione, Delta(4)-androstene-3,17-dione, and progesterone, respectively. Many of these properties are shared by the related AKR1C1, AKR1C2 and AKR1C4 isoforms. RT-PCR shows that AKR1C3 is dominantly expressed in the human prostate and mammary gland. Examination of k(cat)/K(m) for these reactions indicates that as a reductase it prefers 5alpha-dihydrotestosterone and 5alpha-androstane-3,17-dione as substrates to Delta(4)-androstene-3,17-dione, suggesting that in the prostate it favors the formation of inactive androgens. Its concerted reductase activity may, however, lead to a pro-estrogenic state in the breast since it will convert estrone to 17beta-estradiol; convert Delta(4)-androstene-3,17-dione to testosterone (which can be aromatized to 17beta-estradiol); and it will reduce progesterone to its inactive metabolite 20alpha-hydroxyprogesterone. Drawing on detailed structure-function analysis of the related rat 3alpha-HSD (AKR1C9), which shares 69% sequence identity with AKR1C3, it is predicted that AKR1C3 catalyzes an ordered bi bi mechanism, that the rate determining step is k(chem), and that an oxyanion prevails in the transition state. Based on these relationships steroidal-based inhibitors that compete with the steroid product would be desirable since they would act as uncompetitive inhibitors. With regards to transition state analogs steroid carboxylates and pyrazoles may be preferred while 3alpha, 17beta or 20alpha-spiro-oxiranes may act as mechanism-based inactivators.

Beta-lactamase-inhibitor combinations in the 21st century: current agents and new developments.

Combinations of beta-lactams and beta-lactamase inhibitors have become one of the most successful antibacterial strategies in our global battle against bacterial infections. The success of these agents is particularly emphasized by the continued efficacy of Augmenting (amoxicillin and clavulanate) after nearly 20 years of clinical use. The clinical situation now dictates that second-generation beta-lactamase inhibitors capable of encompassing both class A and class C beta-lactamases would combat emerging resistance and provide a vital addition to our armory of hospital antibiotics. This realization has generated a renewed interest in beta-lactamase inhibitors and improved the prospects for the delivery of such agents in the future.

Adjuvant therapy of bullous pemphigoid with mycophenolate mofetil: old drug, new use.

Bullous Pemphigoid (BP) is an autoimmune subepidermal blistering disease appearing predominantly in the elderly. The disease is primarily treated with systemic corticosteroids. However, the treatment can be associated with significant morbidity. Adjuvant corticosteroid sparing therapy can also be associated with significant morbidity. In this study a case of BP which was difficult to control with systemic steroids was successfully treated with mycophenolate mofetil as adjuvant therapy. Mycophenolate mofetil used previously in transplantation, has recently been shown to be useful in autoimmune blistering disorders. Further study to confirm this significant finding and to determine if the long term prognosis of BP can be altered by the drug, is required.

Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones.

The kinetic parameters, steroid substrate specificity and identities of reaction products were determined for four homogeneous recombinant human 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) isoforms of the aldo-keto reductase (AKR) superfamily. The enzymes correspond to type 1 3alpha-HSD (AKR1C4), type 2 3alpha(17beta)-HSD (AKR1C3), type 3 3alpha-HSD (AKR1C2) and 20alpha(3alpha)-HSD (AKR1C1), and share at least 84% amino acid sequence identity. All enzymes acted as NAD(P)(H)-dependent 3-, 17- and 20-ketosteroid reductases and as 3alpha-, 17beta- and 20alpha-hydroxysteroid oxidases. The functional plasticity of these isoforms highlights their ability to modulate the levels of active androgens, oestrogens and progestins. Salient features were that AKR1C4 was the most catalytically efficient, with k(cat)/K(m) values for substrates that exceeded those obtained with other isoforms by 10-30-fold. In the reduction direction, all isoforms inactivated 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one; 5alpha-DHT) to yield 5alpha-androstane-3alpha,17beta-diol (3alpha-androstanediol). However, only AKR1C3 reduced Delta(4)-androstene-3,17-dione to produce significant amounts of testosterone. All isoforms reduced oestrone to 17beta-oestradiol, and progesterone to 20alpha-hydroxy-pregn-4-ene-3,20-dione (20alpha-hydroxyprogesterone). In the oxidation direction, only AKR1C2 converted 3alpha-androstanediol to the active hormone 5alpha-DHT. AKR1C3 and AKR1C4 oxidized testosterone to Delta(4)-androstene-3,17-dione. All isoforms oxidized 17beta-oestradiol to oestrone, and 20alpha-hydroxyprogesterone to progesterone. Discrete tissue distribution of these AKR1C enzymes was observed using isoform-specific reverse transcriptase-PCR. AKR1C4 was virtually liver-specific and its high k(cat)/K(m) allows this enzyme to form 5alpha/5beta-tetrahydrosteroids robustly. AKR1C3 was most prominent in the prostate and mammary glands. The ability of AKR1C3 to interconvert testosterone with Delta(4)-androstene-3,17-dione, but to inactivate 5alpha-DHT, is consistent with this enzyme eliminating active androgens from the prostate. In the mammary gland, AKR1C3 will convert Delta(4)-androstene-3,17-dione to testosterone (a substrate aromatizable to 17beta-oestradiol), oestrone to 17beta-oestradiol, and progesterone to 20alpha-hydroxyprogesterone, and this concerted reductive activity may yield a pro-oesterogenic state. AKR1C3 is also the dominant form in the uterus and is responsible for the synthesis of 3alpha-androstanediol which has been implicated as a parturition hormone. The major isoforms in the brain, capable of synthesizing anxiolytic steroids, are AKR1C1 and AKR1C2. These studies are in stark contrast with those in rat where only a single AKR with positional- and stereo-specificity for 3alpha-hydroxysteroids exists.

Mutation of nicotinamide pocket residues in rat liver 3 alpha-hydroxysteroid dehydrogenase reveals different modes of cofactor binding.

Rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), an aldo-keto reductase, binds NADP(+) in an extended anti-conformation across an (alpha/beta)(8)-barrel. The orientation of the nicotinamide ring, which permits stereospecific transfer of the 4-pro-R hydride from NAD(P)H to substrate, is achieved by hydrogen bonds formed between the C3-carboxamide of the nicotinamide ring and Ser 166, Asn 167, and Gln 190 and by pi-stacking between this ring and Tyr 216. These residues were mutated to yield S166A, N167A, Q190A, and Y216S. In these mutants, K(d)(NADP(H)) increased by 2-11-fold but without a significant change in K(d)(NAD(H)). Steady-state kinetic parameters showed that K(m)(NADP)()+ increased 13-151-fold, and this was accompanied by comparable decreases in k(cat)/K(m)(NADP)()+. By contrast, K(m)(NAD)()+ increased 4-8-fold, but changes in k(cat)/K(m)(NAD)()+ were more dramatic and ranged from 23- to 930-fold. Corresponding changes in binding energies indicated that each residue contributed equally to the binding of NADP(H) in the ground and transition states. However, the same residues stabilized the binding of NAD(H) only in the transition state. These observations suggest that different modes of binding exist for NADP(H) and NAD(H). Importantly, these modes were revealed by mutating residues in the nicotinamide pocket indicating that direct interactions with the 2'-phosphate in the adenine mononucleotide is not the sole determinant of cofactor preference. The single mutations were unable to invert or racemize the stereochemistry of hydride transfer even though the nicotinamide pocket can accommodate both anti- and syn-conformers once the necessary hydrogen bonds are eliminated. When 4-pro-R-[(3)H]NADH was used to monitor incorporation into [(14)C]-5alpha-dihydrotestosterone, a decrease in the (3)H:(14)C ratio was observed in the mutants relative to wild-type enzyme reflecting a pronounced primary kinetic isotope effect. This observation coupled with the change in the binding energy for NAD(P)(H) in the transition state suggests that these mutants have altered the reaction trajectory for hydride transfer.

The arginine 276 anchor for NADP(H) dictates fluorescence kinetic transients in 3 alpha-hydroxysteroid dehydrogenase, a representative aldo-keto reductase.

Fluorescence stopped-flow studies were conducted with recombinant rat liver 3 alpha-HSD, an aldo-keto reductase (AKR) that plays critical roles in steroid hormone inactivation, to characterize the binding of nicotinamide cofactor, the first step in the kinetic mechanism. Binding of NADP(H) involved two events: the fast formation of a loose complex (E.NADP(H)), followed by a conformational change in enzyme structure leading to a tightly bound complex (E.NADP(H)), which was observed as a fluorescence kinetic transient. Binding of NAD(H) was not characterized by a similar kinetic transient, implying a difference in the mode of binding of the two cofactors. Unlike previously characterized AKRs, the rates associated with the formation and decay of E.NADP(H) and E.NADP(H) were much faster than kcat for the oxidoreduction of various substrates, indicating that binding and release of cofactor is not rate-limiting overall in 3 alpha-HSD. Mutation of Arg 276, a highly conserved residue in AKRs that forms a salt bridge with the adenosine 2'-phosphate of NADP(H), resulted in large changes in Km and Kd for NADP(H) that were not observed with NAD(H). The loss in free energy associated with the increase in Kd for NADP(H) is consistent with the elimination of an electrostatic link. Importantly, this mutation abolished the kinetic transient associated with NADPH binding. Thus, anchoring of the adenosine 2'-phosphate of NADPH by Arg 276 appears to be obligatory for the fluorescence kinetic transients to be observed. The removal of Trp 86, a residue involved in fluorescence energy transfer with NAD(P)H, also abolished the kinetic transient, but mutation of Trp 227, a residue on a mobile loop associated with cofactor binding, did not. It is concluded that in 3 alpha-HSD, the time dependence of the change in Trp 86 fluorescence is due to cofactor anchoring, and thus, Trp 86 is a distal reporter of this event. Further, the loop movement that accompanies cofactor binding is spectrally silent.

Retention of NADPH-linked quinone reductase activity in an aldo-keto reductase following mutation of the catalytic tyrosine.

Aldo-keto reductases (AKR) are monomeric oxidoreductases that retain a conserved catalytic tetrad (Tyr, Lys, Asp, and His) at their active sites in which the Tyr acts as a general acid-base catalyst. In rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD, AKR1C9), a well-characterized AKR, the catalytic tyrosine is Tyr 55. This enzyme displays a high catalytic efficiency for a common AKR substrate 9,10-phenanthrenequinone (9,10-PQ). Surprisingly, Y55F and Y55S mutants of 3alpha-HSD reduced 9,10-PQ with high kcat values. This is the first report whereby the invariant catalytic tyrosine of an AKR has been mutated with retention of kcat values similar to wild-type enzyme. The Y55F and Y55S mutants displayed narrow substrate specificity and reduced select aromatic quinones and alpha-dicarbonyls. kcat versus pH profiles for steroid oxidoreduction catalyzed by wild-type 3alpha-HSD exhibited a single ionizable group with a pK= 7.0-7.5, which has been assigned to Tyr 55. This group was not evident in the kcat versus pH profiles for 9, 10-PQ reduction catalyzed by either wild-type or the Tyr 55 mutant enzymes, indicating that the protonation state of Tyr 55 is unimportant for 9,10-PQ turnover. Instead, wild-type and the active-site mutants Y55F, Y55S, H117A, D50N, K84R, and K84M showed the presence of a new titratable group with a pKb = 8.3-9.9. Thus, the group being titrated is not part of the tetrad. All the mutants decreased kcat/Km considerably more than they decreased kcat. Thus, the K84R mutant demonstrated a 30-fold decrease in the pH-independent value of kcat but 2200-fold decrease in the pH-independent value of kcat/Km. This suggests that all the tetrad residues influence quinone binding and that Lys 84 plays a dominant role in maintaining proper substrate orientation. Using wild-type enzyme, the energy of activation (Ea) for 9,10-PQ reduction was approximately 11 kcal/mol less than steroid oxidoreduction. The Ea for 9,10-PQ reduction was unchanged in the Tyr 55 mutants, suggesting that the reaction proceeds through the same low-energy barrier in the wild-type enzyme and these mutants. The retention of quinone reductase activity in this AKR in the absence of Tyr 55 with kcat versus pH rate profiles and activation energies identical to wild-type enzyme suggests that quinone reduction occurs via a mechanism that differs from 3-ketosteroid reduction. In this mechanism, the electron donor (NADPH) and acceptor (o-quinone) are bound in close proximity, which permits hydride transfer without formal protonation of the acceptor carbonyl by Tyr 55. This represents a rare example where one enzyme can catalyze the same chemical reaction (carbonyl reduction) by either acid catalysis or by a propinquity effect and where these two mechanisms can be discriminated by site-directed mutagenesis.

Analyses of the NRAMP1 and IFN-gammaR1 genes in women with Mycobacterium avium-intracellulare pulmonary disease.

Mycobacterium avium-intracellulare (MAI) pulmonary disease causes substantial morbidity in a population of older, HIV-negative women without preexisting lung disease. The cause for disease susceptibility in these patients is unknown, although their relative phenotypic homogeneity suggests the existence of a common, subtle immune deficiency. An investigation was undertaken to determine if these patients have a defect in their natural resistance-associated macrophage protein (NRAMP1) or interferon gamma receptor 1 (IFN-gammaR1) genes. A point mutation in murine nramp, an autosomal recessive gene controlling resistance to intracellular organisms, correlates with overwhelming Mycobacterium bovis infection in mice. The corresponding region in human NRAMP1, two coding polymorphisms and one promoter NRAMP1 polymorphism, as well as two IFN-gammaR1 polymorphisms, were analyzed to determine if an allele was present to correlate with disease. Genomic DNA was purified from eight women with MAI pulmonary disease and four controls. Regions of interest were amplified by PCR; three sites were analyzed by restriction fragment length polymorphisms, and three were analyzed using denaturing high-performance liquid chromatography. The NRAMP1 promoter polymorphism of 18 additional random controls was analyzed by microsatellite sizing. No allelism was found in NRAMP1 corresponding to the murine mutation, or in the two coding regions. In the NRAMP1 promoter microsatellite, 3 of 8 patients were heterozygous for a dinucleotide sequence insertion, as were 10 of 22 controls. None of the patients had either of the two known IFN-gammaR1 mutations. In conclusion, in women with MAI pulmonary disease, there is no evidence for a genetic defect in NRAMP1 or IFN-gammaR1 to correlate with disease.

Spectroscopic and kinetic characterization of the recombinant cytochrome c reductase fragment of nitrate reductase. Identification of the rate-limiting catalytic step.

The recombinant NADH-cytochrome c reductase fragment of spinach NADH-nitrate reductase (EC 1.6.6.1), consisting of the contiguous heme-containing cytochrome b domain and flavin-containing NADH-cytochrome b reductase fragment, has been characterized spectroscopically and kinetically. Reductive titration with sodium dithionite indicates heme reduction takes place prior to flavin reduction, which correlates well with the reduction potentials for enzyme-bound heme (15 mV) and FAD (-280 mV). Reductive titration with NADH also indicates that the reduced enzyme forms a charge-transfer complex with NAD+. The circular dichroism spectrum of the oxidized fragment is primarily due to the flavin, whereas the ferrous heme dominates the circular dichroism spectrum of reduced enzyme. Three kinetic phases are observed in the course of the reaction of the enzyme with NADH, each with a distinct spectral signature. The fast phase represents flavin reduction, concomitant with the formation of a charge-transfer complex between reduced flavin and NAD+, and exhibits hyperbolic dependence on NADH concentration with a Kd of 3 microM and a limiting rate constant of 560 s-1. Electron transfer from reduced flavin to heme with a rate constant of 12 s-1 is the intermediate phase, which is rate-limited by breakdown of the charge-transfer complex between NAD+ and reduced flavin. The slow phase is dismutation of a pair of molecules of two-electron reduced enzyme (generated at the end of the second phase of the reaction) to give one molecule each of one- and three- electron reduced enzyme, with a second order rate constant of 2 x 10(6) M-1 s-1. In the presence of excess NADH, this dismutation reaction is followed by the rapid reaction of the one-electron reduced enzyme with a second equivalent of NADH to generate fully reduced enzyme. On the basis of this work, it appears that dissociation of NAD+ from the reduced flavin site rate limits electron transfer to the cytochrome and likely represents the overall rate-limiting step of catalysis.

Purification of xanthine dehydrogenase and sulfite oxidase from chicken liver.

Xanthine dehydrogenase and sulfite oxidase from chicken liver are oxomolybdenum enzymes which catalyze the oxidation of xanthine to uric acid and sulfite to sulfate, respectively. Independent purification protocols have been previously described for both enzymes. Here we describe a procedure by which xanthine dehydrogenase and sulfite oxidase are purified simultaneously from the same batch of fresh chicken liver. Also, unlike the protocols described earlier, this procedure avoids the use of acetone extraction as well as a heat step, thus minimizing damage to the molybdenum centers of the enzymes.

Idiopathic hypersensitivity vasculitis: clinicopathologic correlation of 61 cases.

BACKGROUND: Little is known of the course and prognosis of hypersensitivity vasculitis (HV). METHODS: Sixty-one cases of idiopathic HV were biopsied and followed-up for 5 years. RESULTS: There were two predominant patterns of presentation: palpable purpura (PP) and vasculitic ulcers (VU). The former was self-limiting, whereas the prognosis of the latter was poor. Findings on histopathology correlate well in PP patients having acute episodes, whereas in patients with VU there is correlation with duration and recurrence. CONCLUSION: A biopsy of a fresh lesion is recommended to help in determining the outcome.

Chronic discoid lupus erythematosus: an immunopathological and electron microscopic study.

We studied 100 cases of chronic cutaneous discoid lupus erythematosus (DLE) to evaluate the diagnostic sensitivity of immunoreactant deposition and its possible role in basement membrane thickening. Histopathology was diagnostic in 71% of cases. Sixty-two percent (41/66) of lesions with thickened and 50% (17/34) with normal basement membrane had immunoreactant deposition. Ultrastructural study of 6 cases (3 with and 3 without immunoreactant deposition) with thickened basement membrane all showed reduplication of the lamina densa. Thickening of the basement membrane appears to be contributed mainly by reduplication of the lamina densa rather than by immunoreactant deposition. The direct immunofluorescence (DIF) test of lesional skin was positive in 58% of patients and was independent of duration of lesion, age and sex. Light microscopy has greater diagnostic sensitivity in confirming DLE lesions than DIF. Direct salt split skin technique did not increase DIF sensitivity. Scalp lesions showed the highest frequency (83%) of immunoreactant deposition. As C1q was the commonest immunoreactant found in our study, we suggest that it should be routinely used when DIF is employed in the evaluation of DLE. DIF is especially helpful in confirming cicatricial alopecia due to DLE.