ABSTRACT
Innate lymphocyte populations, such as innate lymphoid cells (ILCs), γδ T cells, invariant natural killer T (iNK T) cells and mucosal-associated invariant T (MAIT) cells are emerging as important effectors of innate immunity and are involved in various inflammatory and autoimmune diseases. The aim of this study was to assess the frequencies and absolute numbers of innate lymphocytes as well as conventional lymphocytes and monocytes in peripheral blood from a cohort of anti-neutrophil cytoplasm autoantibody (ANCA)-associated vasculitis (AAV) patients. Thirty-eight AAV patients and 24 healthy and disease controls were included in the study. Patients with AAV were sampled both with and without immunosuppressive treatment, and in the setting of both active disease and remission. The frequencies of MAIT and ILC2 cells were significantly lower in patients with AAV and in the disease control group compared to healthy controls. These reductions in the AAV patients remained during remission. B cell count and frequencies were significantly lower in AAV in remission compared to patients with active disease and disease controls. Despite the strong T helper type 2 (Th) preponderance of eosinophilic granulomatosis with polyangiitis, we did not observe increased ILC2 frequency in this cohort of patients. The frequencies of other cell types were similar in all groups studied. Reductions in circulating ILC2 and MAIT cells reported previously in patients with AAV are not specific for AAV, but are more likely to be due to non-specific manifestations of renal impairment and chronic illness. Reduction in B cell numbers in AAV patients experiencing remission is probably therapy-related.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , B-Lymphocytes/immunology , Kidney/pathology , Lymphocyte Subsets/immunology , Mucosal-Associated Invariant T Cells/immunology , Natural Killer T-Cells/immunology , T-Lymphocytes/immunology , Adult , Aged , Aged, 80 and over , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/drug therapy , Cohort Studies , Female , Humans , Immunity, Innate , Immunosuppression Therapy , Lymphocyte Count , Male , Microcirculation , Middle Aged , Receptors, Antigen, T-Cell, gamma-delta/metabolismABSTRACT
BACKGROUND AND PURPOSE: Although the amino acid sequences of rat and human 5-hydroxytryptamine (5-HT) and noradrenaline (NA) transporters (i.e. SERT and NET) are highly homologous, species differences exist in the inhibitory effects of drugs acting at these transporters. Therefore, comparison of the potencies of drugs acting at SERT and NET in native human and rat neocortex may serve to more accurately predict their clinical profile. EXPERIMENTAL APPROACH: Synaptosomes prepared from fresh human and rat neocortical tissues were used for [(3)H]-5-HT and [(3)H]-NA saturation and competition uptake experiments. The drugs tested included NA reuptake inhibitors (desipramine, atomoxetine and (S,S)-reboxetine), 5-HT reuptake blockers (citalopram, fluoxetine and fluvoxamine) and dual 5-HT/NA reuptake inhibitors (duloxetine and milnacipran). KEY RESULTS: In saturation experiments on synaptosomal [(3)H]-5-HT and [(3)H]-NA uptake, the dissociation constants did not indicate species differences although a smaller density of both SERT and NET was observed in human tissues. In competition experiments with the various drugs, marked species differences in their potencies were observed, especially at SERT. The rank order of selectivity ratios (SERT/NET) in human neocortex was as follows: citalopram >or= duloxetine = fluvoxamine >or= fluoxetine > milnacipran > desipramine = atomoxetine > (S,S)-reboxetine. Significant species differences in these ratios were observed for duloxetine, atomoxetine and desipramine. CONCLUSIONS AND IMPLICATIONS: This study provides the first compilation of drug potency at native human neocortical SERT and NET. The significant species differences (viz., human vs. rat) in drug potency suggest that the general use of rodent data should be limited to predict clinical efficacy or profile.
Subject(s)
Adrenergic Uptake Inhibitors/pharmacology , Norepinephrine Plasma Membrane Transport Proteins/antagonists & inhibitors , Selective Serotonin Reuptake Inhibitors/pharmacology , Serotonin Plasma Membrane Transport Proteins/drug effects , Adolescent , Adult , Aged , Animals , Child , Female , Humans , Male , Middle Aged , Neocortex/metabolism , Norepinephrine Plasma Membrane Transport Proteins/metabolism , Rats , Rats, Wistar , Serotonin Plasma Membrane Transport Proteins/metabolism , Species Specificity , Synaptosomes/drug effects , Synaptosomes/metabolism , Young AdultABSTRACT
Anticonvulsant, analgesic, and anxiolytic effects have been observed both in preclinical and clinical studies with gabapentin (GBP) and pregabalin (PGB). These drugs appear to act by binding to the alpha(2)delta subunit of voltage-sensitive Ca(2+) channels (VSCC), resulting in the inhibition of neurotransmitter release. In this study, we examined the effects of GBP and PGB (mostly 100 microM, corresponding to relatively high preclinical/clinical plasma levels) on the release of neurotransmitters in human neocortical slices. These slices were prelabeled with (3)H-dopamine ((3)H-DA), (3)H-choline (to release (3)H-acetylcholine ((3)H-ACh)), (3)H-noradrenaline ((3)H-NA), and (3)H-serotonin ((3)H-5-HT), and stimulated twice in superfusion experiments by elevation of extracellular K(+) in the presence and absence of GBP and PGB. The alpha(2)delta ligands produced significant inhibitions of K(+)-evoked (3)H-ACh, (3)H-NA, and (3)H-5-HT release between 22% and 56% without affecting (3)H-DA release. Neither drug reduced (3)H-NA release in the presence of L: -isoleucine, a putative alpha(2)delta antagonist. Interestingly, this antagonism did not occur using the enantiomer, D: -isoleucine. These results suggest that GBP and PGB are not general inhibitors of VSCC and neurotransmitter release. Such alpha(2)delta ligands appear to be selective modulators of the release of certain, but not all, neurotransmitters. This differential modulation of neurotransmission presumably contributes to their clinical profile.
Subject(s)
Amines/pharmacology , Analgesics/pharmacology , Anticonvulsants/pharmacology , Cyclohexanecarboxylic Acids/pharmacology , gamma-Aminobutyric Acid/analogs & derivatives , Acetylcholine/metabolism , Adolescent , Adult , Aged , Calcium Channels/metabolism , Child , Dopamine/metabolism , Female , Gabapentin , Humans , In Vitro Techniques , Isoleucine/pharmacology , Ligands , Male , Middle Aged , Neocortex/drug effects , Neocortex/metabolism , Norepinephrine/metabolism , Potassium/pharmacology , Pregabalin , Serotonin/metabolism , Stereoisomerism , gamma-Aminobutyric Acid/pharmacologyABSTRACT
alpha-plutonium's volume-corrected polycrystal elastic moduli were measured between 18 K and the upper limit of its occurrence, near 400 K. The two independent moduli for a polycrystal-bulk and shear-behave smoothly, indicating no phase transition. Both moduli show the same 50% increase on cooling, an order of magnitude larger than in other metals. The Debye temperature obtained from low-temperature elastic moduli, 207 K, significantly exceeds most previous estimates. The Gruneisen parameter gamma=5.3, obtained from the temperature dependence of the bulk modulus, is intermediate among previous estimates using other approaches, alpha-plutonium's Poisson ratio nu is low: 0.18, nearly temperature independent, and its small decrease on warming opposes usual behavior. The high gamma, large but equal bulk modulus and shear modulus fractional stiffening on cooling, and near-temperature-invariant nu are attributed to a single mechanism: 5-f electron localization-delocalization.
ABSTRACT
Tranylcypromine (TCP), an amphetamine, is a reversible inhibitor of copper-containing amine oxidases. We have solved the structure of the complex of TCP with the amine oxidase from E. coli (ECAO) and shown that only the (+)-enantiomer of TCP binds. Kinetic studies on 2-phenylethylamine and TCP binding to wild-type ECAO and mutational variants fully support the model in which binding of the protonated amine is the first step in the catalytic cycle. Hydrazines are irreversible inhibitors of copper-containing amine oxidases. Binding of hydrazines leads to an adduct ("Adduct 1") with a chromophore at 430 nm which converts at higher pH to another adduct ("Adduct 2") with a chromophore at 520 nm. We have determined the structures of Adduct 1 and 2 for 2-hydrazinopyridine reacted with ECAO. It has been found that Adduct 1 corresponds to the hydrazone and azo tautomers whilst Adduct 2 corresponds to the azo tautomer coordinated to the active site copper. The implications of these results in developing more specific drugs are discussed.
Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Amphetamines/chemistry , Catalytic Domain/drug effects , Hydrazines/chemistry , Tranylcypromine/chemistry , Amine Oxidase (Copper-Containing)/drug effects , Amine Oxidase (Copper-Containing)/metabolism , Amphetamines/metabolism , Amphetamines/pharmacology , Binding Sites/drug effects , Binding Sites/physiology , Catalytic Domain/physiology , Copper/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Hydrazines/metabolism , Hydrazines/pharmacology , Isomerism , Molecular Conformation , Molecular Structure , Monoamine Oxidase Inhibitors/chemistry , Monoamine Oxidase Inhibitors/metabolism , Monoamine Oxidase Inhibitors/pharmacology , Protein Binding/drug effects , Protein Binding/physiology , Pyridones/chemistry , Pyridones/metabolism , Pyridones/pharmacology , Tranylcypromine/metabolism , Tranylcypromine/pharmacologyABSTRACT
Galactose oxidase (GO; EC 1.1.3.9) is a monomeric 68 kDa enzyme that contains a single copper and an amino acid-derived cofactor. The mechanism of this radical enzyme has been widely studied by structural, spectroscopic, kinetic and mutational approaches and there is a reasonable understanding of the catalytic mechanism and activation by oxidation to generate the radical cofactor that resides on Tyr-272, one of the copper ligands. Biogenesis of this cofactor involves the post-translational, autocatalytic formation of a thioether cross-link between the active-site residues Cys-228 and Tyr-272. This process is closely linked to a peptide bond cleavage event that releases the N-terminal 17-amino-acid pro-peptide. We have shown using pro-enzyme purified in copper-free conditions that mature oxidized GO can be formed by an autocatalytic process upon addition of copper and oxygen. Structural comparison of pro-GO (GO with the prosequence present) with mature GO reveals overall structural similarity, but with some regions showing significant local differences in main chain position and some active-site-residue side chains differing significantly from their mature enzyme positions. These structural effects of the pro-peptide suggest that it may act as an intramolecular chaperone to provide an open active-site structure conducive to copper binding and chemistry associated with cofactor formation. Various models can be proposed to account for the formation of the thioether bond and oxidation to the radical state; however, the mechanism of prosequence cleavage remains unclear.
Subject(s)
Galactose Oxidase/metabolism , Binding Sites , Coenzymes/metabolism , Copper/analysis , Enzyme Precursors/metabolism , Fusarium/enzymology , Galactose Oxidase/chemistry , Galactose Oxidase/genetics , Oxidation-Reduction , Protein Processing, Post-TranslationalABSTRACT
We previously reported that 3-pyrroline and 3-phenyl-3-pyrroline effect a time-dependent inactivation of the copper-containing quinone-dependent amine oxidase from bovine plasma (BPAO) (Lee et al. J. Am. Chem. Soc. 1996, 118, 7241-7242). Quinone cofactor model studies suggested a mechanism involving stoichiometric turnover to a stable pyrrolylated cofactor. Full details of the model studies are now reported along with data on the inhibition of BPAO by a family of 3-aryl-3-pyrrolines (aryl = substituted phenyl, 1-naphthyl, 2-naphthyl), with the 4-methoxy-3-nitrophenyl analogue being the most potent. At the same time, the parent 3-phenyl analogue is a pure substrate for the flavin-dependent mitochondrial monoamine oxidase B from bovine liver. Spectroscopic studies (including resonance Raman) on BPAO inactivated by the 4-methoxy-3-nitrophenyl analogue are consistent with covalent derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The distinction of a class of compounds acting as an inactivator of one amine oxidase family and a pure substrate of another amine oxidase family represents a unique lead to the development of selective inhibitors of the mammalian copper-containing amine oxidases.
Subject(s)
Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Monoamine Oxidase/metabolism , Pyrroles/pharmacology , Amine Oxidase (Copper-Containing)/metabolism , Animals , Cattle , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Kinetics , Mitochondria, Liver/enzymology , Monoamine Oxidase/chemistry , Pyrroles/chemistry , Pyrroles/metabolism , Pyrrolidines/chemistry , Pyrrolidines/metabolism , Spectrophotometry , Spectrum Analysis, Raman , Substrate SpecificityABSTRACT
BACKGROUND: Female genital tract tuberculosis (TB) is a common cause of infertility in developing countries. It is a paucibacillary form of the disease of which smears and cultures are usually negative. CASE: We were able to use polymerase chain reaction (PCR) amplification of Mycobacterium tuberculosis DNA to support a clinical and histologic diagnosis of a typical case of culture negative female genital tract TB. CONCLUSION: PCR may be a useful adjunct to diagnostic efforts in gynecologic tuberculosis.
Subject(s)
DNA, Bacterial/genetics , Mycobacterium tuberculosis/genetics , Polymerase Chain Reaction , Tuberculosis, Female Genital/diagnosis , Tuberculosis, Female Genital/genetics , Adult , Diagnosis, Differential , False Negative Reactions , Female , Humans , Mycobacterium tuberculosis/pathogenicity , Peritoneum/microbiologyABSTRACT
BACKGROUND: Data from multiple clinical, epidemiologic, and in vitro studies are conflicting regarding the effect of estrogen replacement therapy (ERT) on airway function in postmenopausal women with asthma. OBJECTIVE: To determine the impact of withdrawal of estrogen administration in postmenopausal, asthmatic women. METHODS: Twenty asthmatic women who were postmenopausal for at least 2 years and undergoing ERT were recruited for this prospective crossover study. Subjects continued taking baseline estrogen for 28 days, stopped taking estrogen for 28 days, and then resumed taking the medication for 14 days. Objective measurements were obtained by recording daily peak flows in the morning and evening and formal spirometry at days 14, 28, 42, 56, and 70. Compliance was measured by evaluating serum estradiol levels at days 28 and 56. Daily use of short-acting beta-agonist bronchodilators was also recorded. RESULTS: Differences in estradiol levels indicated compliance with the medication regimen. The combined day 14 and 28 (taking estrogen) mean percent predicted forced expiratory volume in 1 second (FEV(1)) was 77% compared with the combined day 42 and 56 (not taking estrogen) mean FEV(1) of 78% and the day 70 (taking estrogen again) FEV(1) of 76% (P>.05). Average peak flow measurements were 295.5 L/min for the duration of ERT, 293.9 L/min while not undergoing ERT, and 291.8 L/min when ERT was restarted for the final 2 weeks of the study (P>.05). Use of short-acting beta-agonist bronchodilators did not differ between study periods. CONCLUSION: These data indicate that neither the discontinuation nor reinitiation of ERT in postmenopausal, asthmatic women has any effect on objective measures of airway obstruction.
Subject(s)
Asthma/physiopathology , Estrogen Replacement Therapy , Postmenopause , Asthma/drug therapy , Bronchodilator Agents/therapeutic use , Cross-Over Studies , Female , Humans , Middle Aged , Peak Expiratory Flow Rate , Prospective Studies , SpirometryABSTRACT
Galactose oxidase (EC ) is a monomeric enzyme that contains a single copper ion and catalyses the stereospecific oxidation of primary alcohols to their corresponding aldehydes. The protein contains an unusual covalent thioether bond between a tyrosine, which acts as a radical center during the two-electron reaction, and a cysteine. The enzyme is produced in a precursor form lacking the thioether bond and also possessing an additional 17-aa pro-sequence at the N terminus. Previous work has shown that the aerobic addition of Cu(2+) to the precursor is sufficient to generate fully processed mature enzyme. The structure of the precursor protein has been determined to 1.4 A, revealing the location of the pro-sequence and identifying structural differences between the precursor and the mature protein. Structural alignment of the precursor and mature forms of galactose oxidase shows that five regions of main chain and some key residues of the active site differ significantly between the two forms. The precursor structure provides a starting point for modeling the chemistry of thioether bond formation and pro-sequence cleavage.
Subject(s)
Enzyme Precursors/chemistry , Galactose Oxidase/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Molecular Sequence Data , Protein Conformation , Protein Processing, Post-TranslationalABSTRACT
OBJECTIVE: Transforming growth factor-beta(1) (TGF-beta(1)) strongly inhibits the proliferation and differentiation of primitive CD34(+)CD38(-) hematopoietic cells. In contrast, Flt3 ligand (FL) is a positive effector of CD34(+)CD38(-/low) cell proliferation. Because apoptosis plays a critical role in hematopoietic development, TGF-beta(1) and FL were analyzed as possible modulators of apoptosis. Specifically, this report examined expression of apoptotic promoters Bax and Bad and apoptotic inhibitors Bcl-2 and Bcl-x (all members of the Bcl-2 protein family). Protein levels were determined in fresh and cultured CD34(+)CD38(+) cells and CD34(+)CD38(-/low) cells with and without treatment with TGF-beta(1) and FL. MATERIALS AND METHODS: Cells fractions were purified by sorting CD34(+)-enriched mononuclear cells from mobilized peripheral blood. Expression of Bcl-2, Bcl-x, Bax, and Bad and the extent of apoptosis were determined by flow cytometric analysis of freshly isolated cells and cells cultured with TGF-beta(1) and FL effectors. RESULTS: TGF-beta(1) reduced CD34(+)CD38(+) cell expansion and arrested cell division. Inhibition of growth was not accompanied by an increase in apoptosis. In CD34(+)CD38(-)(/low) cells, serum TGF-beta(1) and added TGF-beta(1) inhibited cell growth and significantly increased apoptotic cell death. Freshly isolated CD34(+)CD38(+) and CD34(+)CD38(-/low) cells expressed Bcl-2 at similar low levels. However, after 3 days, Bcl-2 expression was markedly higher in cultured CD34(+)CD38(+) cells. TGF-beta(1) significantly increased Bax expression in both fractions after 3 days cultivation (p = 0.0034). Thus, addition of TGF beta-1 further reduced the already low Bcl-2:Bax ratio in CD34(+)CD38(-/low) cells. CONCLUSIONS: Compared to CD34(+)CD38(+) cells, CD34(+)CD38(-/low) cells were slow to up-regulate expression of Bcl-2 during ex vivo culture. TGF-beta(1) up-regulated Bax expression by both CD34(+)CD38(+) and CD34(+)CD38(-)(/low) cells and promoted apoptosis in the latter fraction. This suggests that the preferential induction of apoptosis in primitive cells by TGF-beta(1) may be due to its further reduction of the Bcl-2:Bax ratio.
Subject(s)
Antigens, CD34/blood , Antigens, CD , Antigens, Differentiation/blood , Apoptosis/drug effects , Hematopoietic Stem Cells/drug effects , NAD+ Nucleosidase/blood , Proto-Oncogene Proteins c-bcl-2/metabolism , Transforming Growth Factor beta/pharmacology , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Carrier Proteins/drug effects , Carrier Proteins/metabolism , Cell Division/drug effects , Flow Cytometry , Hematopoietic Stem Cells/immunology , Hematopoietic Stem Cells/metabolism , Humans , Membrane Glycoproteins , Membrane Proteins/metabolism , Membrane Proteins/pharmacology , Proto-Oncogene Proteins/drug effects , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/drug effects , Transforming Growth Factor beta1 , bcl-2-Associated X Protein , bcl-Associated Death Protein , bcl-X ProteinABSTRACT
The copper-containing amine oxidase from Arthrobacter globiformis has been expressed and purified as a fusion protein with a C-terminal Strep-tag II peptide. This tag facilitates the rapid purification of the enzyme on a large scale using the StrepTactin POROS medium. For example, we have demonstrated that 50 mg of protein can be obtained in 2 days from 2 L of Escherichia coli. The purified fusion protein displays turnover and spectroscopic properties that are essentially identical to those of the wild-type enzyme. Given the location of the C-terminus in four amine oxidase crystal structures, this strategy should be quite general for the rapid purification of amine oxidases from multiple sources.
Subject(s)
Amine Oxidase (Copper-Containing)/genetics , Arthrobacter/enzymology , Oligopeptides , Amine Oxidase (Copper-Containing)/isolation & purification , Amine Oxidase (Copper-Containing)/metabolism , Circular Dichroism , Cloning, Molecular , Escherichia coli/genetics , Kinetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismABSTRACT
Nitrous oxide reductase (N(2)OR) from Pseudomonas stutzeri, a dimeric enzyme with a canonical metal ion content of at least six Cu ions per subunit, contains two types of multinuclear copper sites: Cu(A) and Cu(Z). An electron-transfer role for the dinuclear Cu(A) site is indicated based on its similarity to the Cu(A) site in cytochrome c oxidase (CcO), a dicysteinate-bridged, mixed-valence cluster. The Cu(Z) site is the catalytic site, which had long been thought to have novel spectroscopic properties. However, the low-energy electronic transitions and resonance Raman features attributable to Cu(Z) have been difficult to reconcile with a lack of conserved cysteine residues in standard alignments of N(2)OR sequences, other than those associated with the Cu(A) site. Recent evidence indicates that nitrous oxide reductase contains acid-labile sulfide and that this sulfide is a constituent of the Cu(Z) site (Rasmussen, T.; Berks, B. C.; Sanders-Loehr, J.; Dooley, D. M.; Zumft, W. G.; Thomson, A. J. Biochemistry 2000, 39, 12753-12756). We have used resonance Raman (RR) spectroscopy to selectively probe the Cu(A) and Cu(Z) sites of N(2)OR in three oxidation states (oxidized, semireduced, and reduced) as well as Cu(A)-only and Cu(Z)-only variants. The Cu(A) (mixed-valence, also designated as A(mv)) RR spectrum exhibits 10 vibrational modes between 220 and 410 cm(-1), with >1-cm(-1) (34)S isotope shifts that sum to -16.6 cm(-1). Many of these modes are also sensitive to (65)Cu and (15)N(His) and, thus, can be assigned to coupling of the Cu-S stretch, nu(Cu-S), with cysteine and histidine vibrations of the Cu(2)Cys(2)His(2) core. The RR spectrum of the Cu(Z) site (Z(ox)) reveals a novel Cu-sulfur chromophore with four S isotope-sensitive modes at 293, 347, 352, and 408 cm(-1), with a total (34)S shift of -19.9 cm(-)(1). The magnitude of the S isotope shifts and wide spread of perturbed frequencies are similar to those observed in Cu(A) and therefore suggest a sulfur-bridged cluster in Z(ox). The Z(ox) site has its nu(Cu-S)-containing modes at higher energy and exhibits less mixing with ligand deformations, compared to Cu(A). Reduction by dithionite produces a mixed-valence Cu(Z) site (Z(mv)) with six S isotope-sensitive RR modes between 282 and 382 cm(-1) and a total (34)S-shift of -16.9 cm(-1). The observation of a nearly identical RR spectrum in the C622D variant of N(2)OR, which lacks one of the conserved Cu(A) Cys residues, establishes that Cu-S vibrations observed in this variant arise from the Z(mv) site. Furthermore, none of the features assigned to Cu(Z) are detected in a second variant that contains only Cu(A). Therefore the resonance Raman spectra reported here provide compelling evidence for a unique Cu-S cluster in the catalytic site of nitrous oxide reductase.
Subject(s)
Oxidoreductases/chemistry , Bacterial Proteins/chemistry , Catalytic Domain , Copper/chemistry , Histidine/chemistry , Ligands , Models, Molecular , Oxidation-Reduction , Pseudomonas/enzymology , Soil Microbiology , Spectrum Analysis, Raman , Sulfur/chemistryABSTRACT
To elucidate the mechanism of action of the anticonvulsant gabapentin (GBP), we compared its effects on K+-evoked [3H]-noradrenaline ([3H]-NA) release from rat hippocampal and human neocortical slices with those of the KATP channel opener pinacidil and the Na+ channel blockers phenytoin, carbamazepine and lamotrigine. Rat hippocampal and human neocortical slices were loaded with [3H]-NA and superfused. [3H]-NA release was evoked by increasing the extracellular [K+] from 3 to 15 mM. GBP decreased [3H]-NA release from rat hippocampal with a pIC50 of 5.59 and a maximum inhibition of 44%. Concentration-dependent inhibition was also seen in human neocortical slices (39% inhibition with 100 microM GBP). These inhibitory effects were antagonized by the KATP channel antagonist glibenclamide, yielding a pA2 of 7.50 in the rat. The KATP channel opener pinacidil (10 microM), like GBP, decreased [3H]-NA release from rat hippocampal slices by 27% and this effect was also antagonized by glibenclamide. In human neocortical slices the inhibition by pinacidil (10 microM) was 31%. Although phenytoin (10 microM), carbamazepine (100 microM) and lamotrigine (10 microM) also decreased [3H]-NA release (by 25%, 57% and 22%, respectively), glibenclamide did not antagonize the effects of these classical Na+ channel blockers. These findings suggest that GBP inhibits K+-evoked [3H]-NA release through activation of KATP channels. To establish whether the KATP channels under investigation were located on noradrenergic nerve terminals or on other neuronal elements, the effects of GBP were compared in the absence and in the presence of tetrodotoxin (TTX 0.32 microM) throughout superfusion. Since the functional elimination of the perikarya of interneurons by TTX reduced the inhibitory effect of GBP, the KATP channels mediating the effect of GBP may be located on nerve terminals, probably on both noradrenergic and glutamatergic nerve endings.
Subject(s)
Acetates/pharmacology , Amines , Anticonvulsants/pharmacology , Cyclohexanecarboxylic Acids , Norepinephrine/metabolism , Potassium Channels/metabolism , Potassium Chloride/pharmacology , gamma-Aminobutyric Acid , Animals , Dose-Response Relationship, Drug , Gabapentin , Glyburide/pharmacology , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Hypoglycemic Agents/pharmacology , In Vitro Techniques , Male , Neocortex/drug effects , Neocortex/metabolism , Pinacidil/pharmacology , Rats , Rats, Wistar , Retrospective Studies , Vasodilator Agents/pharmacologyABSTRACT
BACKGROUND: Group F streptococci are gram-positive cocci typically isolated from wound infections and abscesses. Bacteremia with group F streptococcus is uncommon, and the lower gynecologic tract has not been reported as a source. We report a case of a Bartholin's abscess leading to group F streptococcal bacteremia. CASE: A 31-year-old female noted fever and rigors 30 min after manipulation of a 3-day-old vulvar abscess. An empty Bartholin's gland abscess was found on examination, and blood cultures grew beta-hemolytic group F streptococci. The patient was treated with ampicillin/sulbactam, symptoms improved, and follow-up blood cultures revealed no growth. CONCLUSION: Group F streptococci are known to inhabit various body sites and have a predilection for forming abscesses; however, bacteremia is infrequent. They have occasionally been identified in true infections of the genitourinary tract but only very rarely in Bartholin's abscesses. This case of group F streptococcal bacteremia following self-drainage of a Bartholin's abscess constitutes the first such description in the medical literature.
Subject(s)
Abscess/complications , Bacteremia/microbiology , Bartholin's Glands/microbiology , Streptococcal Infections/complications , Abscess/drug therapy , Abscess/microbiology , Adult , Ampicillin/therapeutic use , Bacteremia/drug therapy , Drug Therapy, Combination/therapeutic use , Female , Humans , Streptococcal Infections/drug therapy , Streptococcal Infections/microbiology , Streptococcus/isolation & purification , Sulbactam/therapeutic useABSTRACT
NosL, one of the accessory proteins of the nos (nitrous oxide reductase) gene cluster, has been heterologously expressed, purified, and characterized. NosL is a monomeric protein of 18,540 MW that specifically and stoichiometrically binds Cu(I). The copper ion in NosL is ligated by a Cys residue, and one Met and one His are thought to serve as the other ligands. While it is possible to oxidize Cu(I)-NosL with ferricyanide, the Cu(II) ion thus formed appears to dissociate from the protein. The function of Cu(I)NosL is not yet known, but the data indicate that NosL does not act as an electron transfer partner to nitrous oxide reductase. NosL is encoded on the same transcript as three other gene products (NosD, NosF, and NosY). These have been shown to be required for assembly of the active site in nitrous oxide reductase, which is thought to be a copper cluster. Accordingly, it is possible that NosL is a copper chaperone involved in metallocenter assembly.
Subject(s)
Alcaligenes/chemistry , Bacterial Outer Membrane Proteins/chemistry , Copper/chemistry , Lipoproteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Binding Sites , Escherichia coli , Gene Expression Regulation , Lipoproteins/genetics , Lipoproteins/metabolism , Metalloproteins/chemistry , Metalloproteins/genetics , Metalloproteins/metabolism , Molecular Chaperones , Molecular Structure , Oxidoreductases , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, X-Ray EmissionABSTRACT
Lysyl oxidase from Pichia pastoris has been successfully overexpressed. EPR and resonance Raman experiments have shown that copper and TPQ are present, respectively. Lysyl oxidase from P. pastoris has a similar substrate specificity to the mammalian enzyme (both have been shown to oxidize peptidyl lysine residues) and is 30% identical to the human kidney diamine oxidase (the highest of any non-mammalian source). This enzyme also has a relatively broad substrate specificity compared to other amine oxidases. Molecular modeling data suggest that the substrate channel in lysyl oxidase from P. pastoris permits greater active site access than observed in structurally-characterized amine oxidases. This larger channel may account for the diversity of substrates that are turned over by this enzyme.
Subject(s)
Copper/chemistry , Pichia/enzymology , Protein-Lysine 6-Oxidase/chemistry , Amino Acid Sequence , Animals , Binding Sites , Circular Dichroism , Cloning, Molecular , Crystallography, X-Ray , Electron Spin Resonance Spectroscopy , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein-Lysine 6-Oxidase/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Spectrum Analysis, RamanABSTRACT
Incubation of bovine plasma amine oxidase (BPAO) with benzylamine and various p-substituted analogues results in a time-dependent inactivation that is attributable to buildup of the H(2)O(2)-turnover product on the basis of protection afforded by coincubation with catalase. The mechanism of inactivation is distinct from that effected by H(2)O(2) itself, which requires higher concentrations. Solution studies using models for the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor reveal a loss of catalytic activity arising from oxidation of the dihydrobenzoxazole tautomer of the product Schiff base, that competes with hydrolytic release of benzaldehyde product. The resulting stable benzoxazole exhibits a characteristic absorption depending on the nature of the benzylamine p-substituent. For benzylamine itself, the model benzoxazole absorbs at 313 nm, in an area of strong absorption by the enzyme, whereas for 4-nitrobenzylamine, the absorption of the model benzoxazole is sufficiently red-shifted (at 365 nm) to be discerned above the background enzyme absorption. Inactivation of BPAO by 4-nitrobenzylamine is accompanied by loss of the resting TPQ anion absorption at 480 nm concomitant with generation of a new absorption near 360 nm. Resonance Raman spectra of the inactivated enzyme show a close correspondence with those for the model 4-nitrobenzylamine-derived benzoxazole. Substrate-dependent inactivation is also observed for the other two mammalian enzymes examined, equine plasma amine oxidase and human kidney amine oxidase. Catalase provides complete protection in these instances as well. Benzoxazole formation may constitute a common mechanism of inactivation of quinone-dependent amine oxidases by normal substrates in vitro if the product H(2)O(2) is permitted to accumulate. More importantly, the results suggest that the benzoxazole inactivation pathway may be important physiologically and may have influenced the distribution of amine oxidases and catalase in cells.