Suspect 'baggage canine heartworm' case: canine heartworm disease in a dog from Sydney, New South Wales.

Locally acquired canine heartworm (Dirofilaria immitis) infection in the temperate southern climate zones of Australia is currently rare. We report a case of locally acquired canine heartworm from Sydney, New South Wales in a 12-year-old Fox Terrier × Jack Russell female that presented with coughing and breathing difficulties. Absence of heartworm prevention and no travel outside Sydney was noted. Blood sample was D. immitis positive using antigen test, but negative on Modified Knott's testing. PCR confirmed the presence of D. immitis DNA in circulating blood. Echocardiographic examination revealed multiple parallel echogenic lines separated by a hypoechoic region ('tram-tracks') in the right pulmonary artery. The patient was treated and clinical condition gradually improved over the following 12 months. Antigen test remained positive for D. immitis at ~7 months and became negative at ~15 months after the start of the treatment. The most plausible scenario is importation of infected mosquito(s) in the luggage arriving from Queensland, Australia, common holiday destination for many Sydney-siders. We consider this a case of 'baggage canine heartworm'. Canine heartworm in dogs who did not travel should be considered in the differential diagnosis and D. immitis antigen test coupled with Modified Knott's test or PCR must be considered.

The discovery of tetrahydrofluorenones as a new class of estrogen receptor beta-subtype selective ligands.

Synthesis and derivatization of a series of substituted tetrahydrofluorenone analogs giving potent, ERbeta subtype selective ligands are described. Several analogs possessing ERbeta binding affinities comparable to 17beta-estradiol but with greater than 75-fold selectivity over ERalpha are reported.

The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1.

BACKGROUND: Granzyme B, one of the most abundant granzymes in cytotoxic T-lymphocyte (CTL) granules, and members of the caspase (cysteine aspartyl proteinases) family have a unique cleavage specificity for aspartic acid in P1 and play critical roles in the biochemical events that culminate in cell death. RESULTS: We have determined the three-dimensional structure of the complex of the human granzyme B with a potent tetrapeptide aldehyde inhibitor. The Asp-specific S1 subsite of human granzyme B is significantly larger and less charged than the corresponding Asp-specific site in the apoptosis-promoting caspases, and also larger than the corresponding subsite in rat granzyme B. CONCLUSIONS: The above differences account for the variation in substrate specificity among granzyme B, other serine proteases and the caspases, and enable the design of specific inhibitors that can probe the physiological functions of these proteins and the disease states with which they are associated.

Both coactivator LXXLL motif-dependent and -independent interactions are required for peroxisome proliferator-activated receptor gamma (PPARgamma) function.

Nuclear receptor activation is dependent on recruitment of coactivators, including CREB-binding protein (CBP/p300) and steroid receptor coactivator-1 (SRC-1). A three-dimensional NMR approach was used to probe the coactivator binding interface in the peroxisome proliferator-activated receptor gamma (PPARgamma) ligand binding domain (LBD). In the presence of a CBP peptide, peaks corresponding to 20 residues in helices 3, 4, 5, and 12 of the LBD were attenuated. Alanine mutants revealed that K301A, V315A, Y320A, L468A, and E471A were required for binding of both CBP and SRC-1 and for cell-based transcription. Several additional amino acids in helix 4 of the PPARgammaLBD were defective with respect to CBP recruitment, but retained relatively normal SRC-1 recruitment. Thus these amino acid residues may be important determinants of specificity for nuclear receptor LBD interactions with discrete coactivator molecules.

Positions of His-64 and a bound water in human carbonic anhydrase II upon binding three structurally related inhibitors.

The 3-dimensional structure of human carbonic anhydrase II (HCAII; EC 4.2.1.1) complexed with 3 structurally related inhibitors, 1a, 1b, and 1c, has been determined by X-ray crystallographic methods. The 3 inhibitors (1a = C8H12N2O4S3) vary only in the length of the substituent on the 4-amino group: 1a, proton; 1b, methyl; and 1c, ethyl. The binding constants (Ki's) for 1a, 1b, and 1c to HCAII are 1.52, 1.88, and 0.37 nM, respectively. These structures were solved to learn if any structural cause could be found for the difference in binding. In the complex with inhibitors 1a and 1b, electron density can be observed for His-64 and a bound water molecule in the native positions. When inhibitor 1c is bound, the side chain attached to the 4-amino group is positioned so that His-64 can only occupy the alternate position and the bound water is absent. While a variety of factors contribute to the observed binding constants, the major reason 1c binds tighter to HCAII than does 1a or 1b appears to be entropy: the increase in entropy when the bound water molecule is released contributes to the increase in binding and overcomes the small penalty for putting the His-64 side chain in a higher energy state.

FK-506-binding protein: three-dimensional structure of the complex with the antagonist L-685,818.

L-685,818 differs only slightly in structure from the immunosuppressive drug FK-506, and both compounds bind with comparable affinity to the 12-kDa FK-506-binding protein (FKBP12), the major intracellular receptor for the drug. Despite these similarities, L-685,818 is a potent antagonist of both the immunosuppressive and toxic effects of the drug. Here, we present a structural analysis of this problem. Although FK-506 and L-685,818 differ greatly in pharmacology, we have found that the three-dimensional structures of their complexes with FKBP12 are essentially identical. Approximately half of each ligand is in contact with the receptor protein, and half is exposed to solvent; the exposed region includes the two sites where the compounds differ. These results indicate that the profound differences in the pharmacology of these two compounds are not caused by any difference in their interaction with FKBP12. Rather, these effects arise because relatively minor changes in the exposed part of a bound ligand have a strong effect on how FKBP12-ligand complexes interact with calcineurin, their putative intracellular target. In addition, FK-506 complexes with FKBP12 proteins from several species all inhibit mammalian calcineurin. Analysis of the three-dimensional structure of the complex with respect to residues conserved among these proteins suggests a small number of surface residues near the bound ligands that may play a critical role in interactions between the protein-drug complex and calcineurin.

Site-directed mutagenesis of glutathione S-transferase YaYa. Important roles of tyrosine 9 and aspartic acid 101 in catalysis.

The roles of tyrosine 9 and aspartic acid 101 in the catalytic mechanism of rat glutathione S-transferase YaYa were studied by site-directed mutagenesis. Replacement of tyrosine 9 with phenylalanine (Y9F), threonine (Y9T), histidine (Y9H), or valine (Y9V) resulted in mutant enzymes with less than 5% catalytic activity of the wild type enzymes. Kinetic studies with purified Y9F and Y9T mutants demonstrated poor catalytic efficiencies which were largely due to a drastic decrease in kcat. The estimated pK alpha values of the sulfhydryl group of glutathione bound to Y9F and Y9T mutant enzymes were 8.5 to 8.7, similar to the chemical reaction, in contrast to the estimated pK alpha value of 6.7 to 6.8 for the glutathione enzyme complex of wild type glutathione S-transferase. These results indicate that tyrosine 9 is directly responsible for the lowering of the pKa of the sulfhydryl group of glutathione, presumably due to the stabilization of the thiolate anion through hydrogen bonding with the hydroxyl group of tyrosine. To examine the role of aspartic acid in the binding of glutathione to YaYa, 4 conserved aspartic acid residues at positions 61, 93, 101, and 157 were changed to glutamic acid and asparagine. All mutant enzymes retained either full or partial activity except D157N, which was virtually inactive. Kinetic studies with four mutant enzymes (D93E, D93N, D101E, and D101N) indicate that only D101N exhibited a 5-fold increase in Km toward glutathione. Also, the binding of this mutant to the affinity column was greatly reduced. These results demonstrate that aspartic acid 101 plays an important role in glutathione interaction to YaYa. The role of aspartic acid 157 in catalysis remains to be determined.

Synthesis and antiviral activity of a series of HIV-1 protease inhibitors with functionality tethered to the P1 or P1' phenyl substituents: X-ray crystal structure assisted design.

By tethering of a polar hydrophilic group to the P1 or P1' substituent of a Phe-based hydroxyethylene isostere, the antiviral potency of a series of HIV protease inhibitors was improved. The optimum enhancement of anti-HIV activity was observed with the 4-morpholinylethoxy substituent. The substituent effect is consistent with a model derived from inhibitor docked in the crystal structure of the native enzyme. An X-ray crystal structure of the inhibited enzyme determined to 2.25 A verifies the modeling predictions.

New isomeric classes of topically active ocular hypotensive carbonic anhydrase inhibitors: 5-substituted thieno[2,3-b]thiophene-2-sulfonamides and 5-substituted thieno[3,2-b]thiophene-2-sulfonamides.

A series of 5-substituted thieno[2,3-b]- and thieno[3,2-b)- and thieno[3,2-b)thiophene-2-sulfonamides was prepared and evaluated for topical ocular hypotensive activity in glaucoma models. The 5-substituents were varied to maximize both inhibitory potency against carbonic anhydrase and water solubility. At the same time, these substituents were varied in order to obtain compounds with the appropriate pKa to minimize pigment binding in the iris. All of these variables were optimized in the best compound, 5-[[(methoxyethyl)[(methoxyethyl)ethyl] amino]methyl]thieno[2,3-b]thiophene-2-sulfonamide hydrochloride (55).

Crystallographic analysis of a complex between human immunodeficiency virus type 1 protease and acetyl-pepstatin at 2.0-A resolution.

The mode of binding of acetyl-pepstatin to the protease from the human immunodeficiency virus type 1 (HIV-1) has been determined by x-ray diffraction analysis. Crystals of an acetyl-pepstatin-HIV-1 protease complex were obtained in space group P2(1)2(1)2 (unit cell dimensions a = 58.39 A, b = 86.70 A, c = 46.27 A) by precipitation with sodium chloride. The structure was phased by molecular replacement methods, and a model for the structure was refined using diffraction data to 2.0 A resolution (R = 0.176 for 12901 reflections with I greater than sigma (I); deviation of bond distances from ideal values = 0.018 A; 172 solvent molecules included). The structure of the protein in the complex has been compared with the structure of the enzyme without the ligand. A core of 44 amino acids in each monomer, including residues in the active site and residues at the dimer interface, remains unchanged on binding of the inhibitor (root mean square deviation of alpha carbon positions = 0.39 A). The remaining 55 residues in each monomer undergo substantial rearrangement, with the most dramatic changes occurring at residues 44-57 (these residues comprise the so-called flaps of the enzyme). The flaps interact with one another and with the inhibitor so as to largely preserve the 2-fold symmetry of the protein. The inhibitor is bound in two approximately symmetric orientations. In both orientations the peptidyl backbone of the inhibitor is extended; a network of hydrogen bonds is formed between the inhibitor and the main body of the protein as well as between the inhibitor and the flaps. Hydrophobic side chains of residues in the body of the protein form partial binding sites for the side chains of the inhibitor; hydrophobic side chains of residues in the flaps complete these binding sites.

A role for the aspartyl protease from the human immunodeficiency virus type 1 (HIV-1) in the orchestration of virus assembly.

Functional HIV-1 protease (PR) is required for the maturation of viral proteins, for the appearance of characteristic structural features in the virion (as determined by electron microscopy), and for the final assembly of mature virus. Most importantly, HIV-1 PR activity is required for the development of infectivity. Still largely undefined, however, is the timing and control of protease action in this assembly process. Based on the three-dimensional structure of HIV-1 PR2,3 and experimental data reported in the literature, we propose a comprehensive virus assembly model that highlights the role of HIV-1 PR, suggests further experiments to verify the validity of the model, and poses specific questions relevant to the ultimate exploitation of HIV-1 protease as a therapeutic target.

Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1.

The crystal structure of the protease of the human immunodeficiency virus type (HIV-1), which releases structural proteins and enzymes from viral polyprotein products, has been determined to 3 A resolution. Large regions of the protease dimer, including the active site, have structural homology to the family of microbial aspartyl proteases. The structure suggests a mechanism for the autoproteolytic release of protease and a role in the control of virus maturation.

Crystallization of the aspartylprotease from the human immunodeficiency virus, HIV-1.

The aspartylprotease of the human immunodeficiency virus HIV-1 (NY5) has been crystallized in a form suitable for x-ray diffraction analysis. The crystals are tetragonal bipyramids and produce an x-ray diffraction pattern that exhibits the symmetry associated with space group P4(1)2(1)2 (or its enantiomorph, P4(3)2(1)2). The unit cell parameters are a = b = 50.3 A, c = 106.8 A, alpha = beta = gamma = 90 degrees; measurable diffraction intensities are observed to a resolution of 2.5 A. Density measurements indicate one molecule of 9,400 daltons/asymmetric unit. The symmetry of this space group could accommodate the proposed active dimer species of the protease if the 2-fold axis were coincident with one of the crystallographic 2-fold axes.

Structure of human neutrophil elastase in complex with a peptide chloromethyl ketone inhibitor at 1.84-A resolution.

Human neutrophil elastase (HNE) has been implicated as a major contributor to tissue destruction in various disease states, including emphysema. The structure of HNE, at neutral pH, in complex with methoxysuccinyl-Ala-Ala-Pro-Ala chloromethyl ketone (MSACK), has been solved and refined to an R factor of 16.4% at 1.84-A resolution. Results are consistent with the currently accepted mechanism of peptide chloromethyl ketone inhibition of serine proteases, in that MSACK cross-links the catalytic residues His-57 and Ser-195. The structure of the HNE-MSACK complex is compared with that of porcine pancreatic elastase in complex with L-647,957, a beta-lactam inhibitor of both elastases. The distribution of positively charged residues on HNE is highly asymmetric and may play a role in its specific association with the underlying negatively charged proteoglycan matrix of the neutrophil granules in which the enzyme is stored.

Crystallization of human neutrophil elastase.

Human neutrophil elastase was inactivated by methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane. The modified enzyme was crystallized from 40 mM ammonium phosphate, pH 7.0 in the hexagonal space group P6(3) with unit cell parameters a = 74.53 A, b = 74.53 A, c = 70.88 A, alpha = beta = 90 degrees, gamma = 120 degrees. These crystals were resistant to radiation damage and diffracted beyond 1.84-A resolution. The asymmetric unit contained one 25,000-dalton monomer of human neutrophil elastase. Crystals were also grown from the enzyme modified with the analogous iodinated inactivator, p-iodoanilinosuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane. These crystals proved to be isomorphous with those of methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane-modified human neutrophil elastase, and served as a single-site, heavy atom derivative for solving the tertiary structure of the enzyme.

Fluorescence characterization of the low pH-induced change in diphtheria toxin conformation: effect of salt.

We have examined the effect of pH on diphtheria toxin conformation using intrinsic protein fluorescence and a new fluorescence quenching method. In aqueous solutions, fluorescence indicates toxin conformation undergoes a drastic change at low pH. This conformational change is closely associated with a switch from a hydrophilic conformation to a hydrophobic one, as judged by quenching-detected detergent binding. In the absence of NaC1 these changes occur around pH 4-4.5. However, in 150 mM NaCl the conformational change occurs in the pH 5-5.5 range, close to the pH the toxin is expected to encounter in endosomes and lysosomes. Therefore, the conformational change observed at low pH is likely to be physiologically significant.

Amine fluorescamine compounds inhibit oxidative phosphorylation in rat liver mitochondria.

The reaction of fluorescamine with ammonia, benzylamine, o,p-dimethylbenzylamine, 2-phenylethylamine, p-aminobenzoic acid, and the mycosamine-containing macrolide antibiotic, amphotericin B, yield compounds which induce significant effects on mitochondrial activities. From their effects on energy-yielding processes which lead to transmembranous proton movements, the compounds may be divided into three classes. While all modifiers significantly inhibit proton movement induced by both ATP hydrolysis and electron transfer in mitochondria, their influence on the primary energy yielding steps are quite different. Class I modifiers, e.g., the compound made from amphotericin B, inhibit electron transfer but have no effect on the Pi release associated with ATP hydrolysis. Class II modifiers, e.g., the compound made from benzylamine, inhibit respiration but stimulate Pi release. Class III modifiers, e.g., the compound made from p-aminobenzoic acid, on the other hand, only slightly increase Pi release but have no effect on redox reactions. These and other effects of the modifiers are taken to mean that the proton movements and their associated energy-yielding processes are only linked indirectly. The effects of the modifiers on State 3 mitochondrial activities were also investigated. Although all the modifiers decrease the rates of both State 3 respiration and its coupled ATP synthesis, the efficiency of energy conversion measured by the P/O ratio remains unaltered.

Oxygen-exchange studies on the pathways for magnesium adenosine 5'-triphosphate hydrolysis by actomyosin.

At an intermediate stage in the hydrolysis of magnesium adenosine 5'-phosphate (MgATP) by myosin or actomyosin, there is an exchange of oxygen between water and the P gamma group of enzyme-bound nucleotide. Starting with [P gamma-18O]ATP as substrate, the exchange is revealed in the [18O]Pi species that are ultimately released as product into the reaction medium. An analysis of the distribution of these labeled Pi species, which contain 3, 2, 1, or none of the 18O atoms originally on the P gamma of ATP, is used to probe intermediate stages of the hydrolytic mechanism. In recent years, studies of this kind by several groups have shown that more than one pathway of hydrolysis operates. The work reported here demonstrates that two of these pathways are spurious; one is a "nonexchanging MgATPase" that is present in fresh myosin preparations; the other is an induced slow exchange that develops in myosin during storage (-20 degrees C) and subsequent aging (4 degrees C). However, after correction for these artifacts, two normal pathways for actomyosin hydrolysis remain. These normal pathways differ in the mode of interaction between actin and myosin in the course of hydrolysis; one is the Lymn-Taylor pathway where oxygen exchange occurs at a stage when actin and myosin are dissociated; the other is a pathway in which actin and myosin are associated during oxygen exchange. Each of these two pathways contributes an equal amount of Pi to the product pool. Thus, on average, each myosin head uses each of these pathways half the time. The findings suggest, e.g., that during contraction, myosin can dissociate from the actin filament only during every other cycle of MgATP hydrolysis or that only half the heads, at any one time, can exchange oxygen while free of the actin filament.