Loop diuretics and in vitro relaxation of human fetal and newborn mouse airways.

This study was designed to test the hypotheses that furosemide directly causes relaxation in human fetal airway and that delivery of loop diuretics to either the adventitial or epithelial surface of newborn mouse airway results in equivalent relaxation. Isometric tension changes were measured in human fetal (11-16 wk) trachea and mainstem bronchus rings exposed to furosemide (300 microM) or saline after acetylcholine or leukotriene D(4) constriction. Significant decreases in isometric tension to furosemide were demonstrated after constriction with acetylcholine or leukotriene D(4). To examine the site of effect and mimic aerosolized and systemic administration, furosemide (3-300 microM) and bumetanide (0.3-30 microM) were applied separately to epithelial and adventitial surfaces of newborn mouse airways. No differences in airway diameter changes to epithelial or adventitial furosemide or bumetanide were observed, but a 10-fold difference in potency was found. In summary, human fetal airway relaxed to furosemide when constricted with either neurotransmitter or inflammatory mediator in vitro. Further, no differences in relaxation to equimolar epithelial and adventitial furosemide were observed in isolated newborn mouse airway. Taken together, this provides evidence that furosemide has a direct, nonepithelial-dependent effect on airway smooth muscle tone.

The mechanism of aubstrate eecognition of pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as determined by X-ray crystallography and site-directed mutagenesis.

Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O.H-N:Inh and Gln71:N-H.OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. The k(cat) value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (k(cat)/K(m)) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an "induced fit" mechanism.

Differential effects of inhaled nitric oxide and hyperoxia on pulmonary dysfunction in newborn guinea pigs.

This study tested the hypothesis that inhaled nitric oxide (NO) and combined NO and hyperoxia will result in less pulmonary dysfunction and delay onset of respiratory signs compared with hyperoxia-exposed newborn guinea pigs (GPs). GPs were exposed to room air (n = 14), 95% O(2) (n = 36), 20 parts per million (ppm) NO (n = 14), or combined 20 ppm NO and 95% O(2) (NO/O(2), n = 13) for up to 5 days. Data evaluated included latency interval for onset of respiratory distress, pressure volume curves, lung histology, and bronchoalveolar lavage (BAL) polymorphonuclear cells (PMNs), proteolytic activity, and total protein. NO-exposed GPs did not develop respiratory distress and had no evidence of pulmonary dysfunction. O(2)-exposed GPs developed respiratory distress after 1-5 days (median 4.0) vs. 3-5 days (median 5.0) for NO/O(2) exposure (P < 0.05). BAL from O(2)-exposed GPs showed increased PMNs compared with NO/O(2)-exposed GPs. O(2)- and NO/O(2)-exposed GPs had comparable reduced lung volumes, lung histology, and increased BAL proteinase activity and total protein. In summary 1) O(2) exposure resulted in multiple measures of pulmonary dysfunction in newborn GPs, 2) 5-day exposure to NO produced no noticeable respiratory effects and pulmonary dysfunction, and 3) short-term exposure (

Crystal structure of a plant ribonuclease, RNase LE.

Ribonuclease LE (RNase LE) from cultured tomato (Lycopersicon esculentum) cells is a member of the RNase T(2) family showing broad base specificity. The crystal structure of RNase LE has been determined at 1.65 A resolution. The structure consists of seven alpha-helices and seven beta-strands, belonging to an alpha+beta type structure. Comparison of the structure of RNase LE with that of RNase Rh, a microbial RNase belonging to the RNase T(2) family, reveals that while the overall folding topologies are similar to each other, major insertions and deletions are found at the N-terminal regions. The structural comparison, an amino acid sequence alignment of the RNase T(2) enzymes, and comparison of the disulfide-bonding pattern of these enzymes show that the structure of RNase LE shown here is the basic framework of the animal/plant subfamily of RNase T(2) enzymes (including a self-incompatibility protein called S-RNase), and the structure of RNase Rh is that of the fungal subfamily of RNase T(2) enzymes (including RNase T(2)). Subsequently, we superposed the active-site of the RNase LE with that of RNase Rh and found that (1) His39, Trp42, His92, Glu93, Lys96, and His97 of RNase LE coincided exactly with His46, Trp49, His104, Glu105, Lys108, and His109, respectively, of RNase Rh, and (2) two conserved water molecules were found at the putative P(1) sites of both enzymes. These facts suggest that plant RNase LE has a very similar hydrolysis mechanism to that of fungal RNase Rh, and almost all the RNase T(2) enzymes widely distributed in various species share a common catalytic mechanism. A cluster of hydrophobic residues was found on the active-site face of the RNase LE molecule and two large hydrophobic pockets exist. These hydrophobic pockets appear to be base binding sites mainly by hydrophobic interactions and are responsible for the base non-specificity of RNase LE.

m-hydroxy benzoylecgonine recovery in fetal guinea pigs.

Recently, meta-hydroxybenzoylecgonine (m-OH BE) was identified by gas chromatography-mass spectroscopy during quantitative analysis for cocaine. Identification of m-OH BE in addition to the routinely identified benzoylecgonine by gas chromatography-mass spectroscopy confirmatory assays may increase detection of cocaine-exposed infants and decrease false negative results. However, it is not known whether m-OH BE is derived directly from benzoylecgonine or from hydroxylated cocaine, or whether this metabolite is produced in the fetus or transferred across the placenta from the maternal circulation. We quantitated the recovery of cocaine, benzoylecgonine, and m-OH BE from amniotic fluid, fetal meconium, fetal intestine, and maternal urine for up to 4 days after single dose administration of either cocaine or benzoylecgonine to pregnant time-bred guinea pigs. m-OH BE was recovered from meconium after maternal injections of cocaine and benzoylecgonine. There was no significant detection of m-OH BE from amniotic fluid or intestine and minimal recovery from maternal urine after either cocaine or benzoylecgonine administration. Detection of m-OH BE in meconium increased the identification of in utero exposed guinea pigs, and the greatest yield of m-OH BE from meconium occurred later than that observed for cocaine or benzoylecgonine.

Crystal structure of prolyl aminopeptidase from Serratia marcescens.

Prolyl aminopeptidase from Serratia marcescens specifically catalyzes the removal of N-terminal proline residues from peptides. We have solved its three-dimensional structure at 2.3 A resolution by the multiple isomorphous replacement method. The enzyme consists of two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet and six helices. The smaller domain consists of six helices. The catalytic triad (Ser113, His296, and Asp268) is located near the large cavity at the interface between the two domains. Cys271, which is sensitive to SH reagents, is located near the catalytic residues, in spite of the fact that the enzyme is a serine peptidase. The specific residues which make up the hydrophobic pocket line the smaller domain, and the specificity of the exo-type enzyme originates from this smaller domain, which blocks the N-terminal of P1 proline.

Cross-desensitization to furosemide and salbutamol in isolated neonatal guinea pig airways.

Airway hyperresponsiveness in neonatal chronic lung disease is treated with both furosemide, a diurectic that inhibits the Na-K-2Cl cotrasporter, and salbutamol, a beta2-adrenoceptor agonist. Tachyphylaxis to both drugs in vitro has been described. This study was conducted to determine if the relaxation response in newborn guinea pig airways to furosemide and salbutamol can be cross-desensitized in vitro. Tracheal ring segments from 4- to 7-day-old guinea pigs were suspended in HEPES buffer for measurement of isometric tension. Segments were pre-treated with either furosemide (300 microM, 1 h) or salbutamol (10 microM, 30 min). After constriction with 3 microM acetylcholine, relaxation response to salbutamol or furosemide, respectively, was measured. Pretreatment with furosemide diminished relaxation response to salbutamol [87 +/- 3% (n = 11) vs. 117 +/- 8% (n = 10), p < 0.05], as compared to saline-treated controls. In addition, pretreatment with salbutamol diminished relaxation response to furosemide [53 +/- 2% (n = 11) vs. saline-treated (83 +/- 7%, n = 7, p < 0.05) and DMSO-treated controls (69 +/- 5%, n = 5, p < 0.05)]. Measurements of 86Rb uptake, cyclic AMP levels and responses in the presence of charybdotoxin make it unlikely that Na-K-2Cl cotransporter activity, stimulation of cAMP, or opening of maxi-K+ channels are mechanisms involved in the cross-desensitization to furosemide and salbutamol in vitro.

Synthesis of oligonucleotides containing the oxidatively modified kappaB site: unique binding affinity to the NFkappaB p50 homodimer.

Synthesis of oligonucleotide 26-mers including single 5-formyl-2'-deoxyuridine (1) or 5-formyl-2'-O-methyluridine (2) in place of thymidine at the kappaB site has been accomplished. One of the 26-mers with 1 was critically discriminated by the NFkappaB p50 homodimer in binding.

Structural analysis and molecular model of a self-incompatibility RNase from wild tomato.

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.

Amiloride-induced contraction of isolated guinea pig, mouse, and human fetal airways.

Nebulized amiloride has been proposed as therapy in cystic fibrosis to block Na+ hyperabsorption in airway epithelium and prevent dehydration of secretions. Patients with cystic fibrosis often have reaction airways. Bovine and canine trachea relax to amiloride in vitro, suggesting another benefit as a bronchodilator, whereas guinea pig trachea, a useful model of human airways, does not. We hypothesized that human airways would respond like guinea pig airways. Airway ring segments from guinea pigs, mice, and human fetuses were constricted with the concentration of acetylcholine producing 50-75% maximum contraction. Subsequent changes in isometric tension to cumulative additions of amiloride (10(-8)-10(-4) M) were measured. Guinea pig airways contracted 29 +/- 5%, mouse airways contracted 23 +/- 6%, and human fetal airways contracted 30 +/- 8%. Contraction to amiloride was mimicked by dimethylamiloride, a more selective inhibitor of the Na+/H+ antiporter, and was attenuated by protein kinase C (PKC) inhibition with GF109203X and staurosporine. The present study indicates that amiloride-induced airway contraction in guinea pigs and mice closely parallels the response in isolated human airways and that the mechanism may involve the Na+/H+ antiporter and PKC.

Identification of regions in which positive selection may operate in S-RNase of Rosaceae: implication for S-allele-specific recognition sites in S-RNase.

A stylar S-RNase is associated with gametophytic self-incompatibility in the Rosaceae, Solanaceae, and Scrophulariaceae. This S-RNase is responsible for S-allele-specific recognition in the self-incompatible reaction, but how it functions in specific discrimination is not clear. Window analysis of the numbers of synonymous (dS) and non-synonymous (dN) substitutions in rosaceous S-RNases detected four regions with an excess of dN over dS in which positive selection may operate (PS regions). The topology of the secondary structure of the S-RNases predicted by the PHD method is very similar to that of fungal RNase Rh whose tertiary structure is known. When the sequences of S-RNases are aligned with the sequence of RNase Rh based on the predicted secondary structures, the four PS regions correspond to two surface sites on the tertiary structure of RNase Rh. These findings suggest that in S-RNases the PS regions also form two sites and are candidates for the recognition sites for S-allele-specific discrimination.

Furosemide-induced airway relaxation in guinea pigs: relation to Na-K-2Cl cotransporter function.

This study tested the hypothesis that airway relaxation to furosemide is mediated via the Na-K-2Cl cotransporter. If this mechanism exists in airway smooth muscle like in vascular smooth muscle, changes in airway relaxation should be associated with changes in Na-K-2Cl cotransporter function, and both should be substrate dependent. Tracheal rings from newborn guinea pigs were bathed in standard (STD) or varying low Cl- concentration ([Cl-]) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Isometric relaxation to 300 microM furosemide or 10(-8) to 10(-5) M salbutamol was measured. Airway segments were incubated with rubidium-86 (86Rb) in STD or varying low [Cl-] HEPES, with and without 300 microM furosemide or 25 microM salbutamol. Furosemide was unable to reduce 86Rb uptake at 10 mM [Cl-], although relaxation was still observed in 10 mM [Cl-]. Salbutamol did not affect 86Rb uptake. This study demonstrated that there is a furosemide-sensitive Na-K-2Cl cotransporter in newborn guinea pig trachea. However, the effect of furosemide on cotransporter function did not always directly correspond to differences in relaxation, suggesting that the Na-K-2Cl cotransporter may play a major, but not exclusive, role in furosemide-induced airway relaxation.

Heliquinomycin, a new inhibitor of DNA helicase, produced by Streptomyces sp. MJ929-SF2 II. Structure determination of heliquinomycin.

The structure of heliquinomycin which was isolated from the culture broth of Streptomyces sp. MJ929-SF2 was studied by NMR spectroscopies, X-ray crystallographic analysis and degradation experiments. Heliquinomycin is the first member of glycosylated rubromycins and griseorhodins group antibiotics.

Stereocontrolled cyclization at the anomeric position of 6-(hydroxyalkyl)uridines using hypoiodite reaction.

Hypoiodite reaction of 6-(hydroxyalkyl)-2',3'-O-isopropylideneuridines (1-3) was found to yield a new class of spiro nucleosides having an anomeric orthoester structure. It appeared that the 6-hydroxyalkyl substituent and the 2',3'-O-isopropylidene group are working cooperatively to control the anomeric stereochemistry (beta/alpha = 35/1-2/1) of the cyclization.

Tachyphylaxis to furosemide in isolated airways of guinea pigs.

This in vitro study was conducted to determine whether tachyphylaxis of guinea pig airway to furosemide occurs under conditions that produce tachyphylaxis to the beta 2-adrenoceptor agonist, salbutamol. Isometric tension was measured in tracheal rings bathed in HEPES buffer from 4-6 d newborn guinea pigs of either sex, and 6 wk old males. Paired rings were first incubated with furosemide, 30 or 300 microM, or control for 60 min, washed, then constricted with 3 microM acetylcholine. At stable contraction, relaxation to furosemide (30 microM-1 mM) was measured. For comparison, similar experiments were performed with 10 microM salbutamol incubation for 30 min. 86Rb uptake, a marker for K+ transport and Na-K-Cl cotransport activity, was also measured in these airway segments. Pre-exposure to these airway relaxants did not affect contractile force generation by acetylcholine. Tracheal desensitization to both salbutamol and furosemide was observed. Partial recovery of furosemide induced relaxation was seen one hour after desensitization. Pre-exposure to 300 microM furosemide did not inhibit the decrease in 86Rb uptake normally observed with furosemide. In summary, we found that: 1) tachyphylaxis of guinea pig airway relaxation occurred with both salbutamol and furosemide under similar experimental conditions; however 2) inhibition of 86Rb uptake by furosemide was not affected by prior exposure. Taken together, these results suggest that furosemide induced airway relaxation could be affected by repeated or prolonged exposure, but this response may not be associated with changes in furosemide-sensitive Na-K-Cl cotransporter activity.

The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 A resolution.

The three-dimensional structure of ribonuclease Rh (RNase Rh), a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.0 A resolution. The overall structure of RNase Rh is completely different from those of other previously studied RNases, such as RNase A from bovine pancreas and RNase T1 from Aspergillus oryzae. In the structure of RNase Rh, two histidine residues (His46 and His109) and one glutamic acid residue (Glu105), which were predicted to be critical to the activity from the chemical modification and mutagenesis experiments, are found to be located close together, constructing the active site. The indole ring of Trp49 plays an important role in preserving the active site structure by its stacking interactions with the imidazole ring of His 109, and by hydrogen bonding with the carboxyl group of Glu105. There exists a hydrophobic pocket around the active site, which contains the aromatic side-chain of Trp49 and Tyr57. The results of mutagenesis studies suggest that this pocket is the base binding site of the substrate.

Derivatives of tetrodecamycin.

The derivatives of tetrodecamycin (1), being introduced acyl, carbamoyl and alkyl groups at 14-hydroxyl group and modified at exo-methylene group, were synthesized and evaluated on their antibacterial activities. Although 14-O-substituted tetrodecamycins (3 approximately 19) showed weak activity against Pasteurella piscicida, they were more active against Gram-positive bacteria than 1. Among them, 15 showed approximately 10-fold higher activity than 1. The derivatives (20 approximately 23) modified at 4 or 5 positions had moderate antibacterial activity. The absolute structure of 4(R),5-dibromotetrodecamycin (23) was determined by X-ray crystallographic analysis.

Tetrodecamycin and dihydrotetrodecamycin, new antimicrobial antibiotics against Pasteurella piscicida produced by Streptomyces nashvillensis MJ885-mF8. II. Structure determination.

Novel antimicrobial antibiotics against Pasteurella piscicida, tetrodecamycin (1) and weakly active dihydrotetrodecamycin (2) were isolated from a culture broth of Streptomyces nashvillensis MJ885-mF8. The planar structure of 1 was determined to be 2-acyl-4-ylidene tetronic acid alkyl ether containing decaline ring by various NMR spectral data of 1 and its acetyl derivative (3). The structure of 2 was elucidated by comparison with the spectral data of 1 and confirmed by catalytic reduction of 1 into 2. The X-ray crystallography of 2 showed the relative stereochemistry. Their absolute configurations were determined by using modified Mosher's method.