Role of interleukin-5 and substance P in development of airway hyperreactivity to histamine in guinea-pigs

In this study, we examined the mechanism by which bronchoalveolar lavage (BAL) cells induced hyperreactivity of the trachea in vitro. As both interleukin-5 (IL-5) and substance P (SP) appeared to be involved, the effect of these mediators was examined in vivo. Tracheae were incubated with BAL cells from ovalbumin or saline challenged animals, and from naive animals, in the absence or presence of either IL-5, SP, or both. In addition, the effect of intra-airway application of IL-5, SP, both, or vehicle on tracheal hyperreactivity was examined. Incubation of tracheae with BAL cells from ovalbumin challenged animals induced an increase (30 +/- 10%) in the maximal response to histamine. The hyperreactivity could be completely inhibited by co-incubation with 5-lipoxygenase inhibitor, AA861. The hyperreactivity could be mimicked by incubation of tracheae with BAL cells from naive animals in the presence of IL-5 and SP. After in vivo administration of either IL-5 or SP, maximal responses to histamine were increased and amounted to 105 +/- 35 and 101 +/- 37%, respectively. Administration of IL-5 but not SP induced a significant increase in the number of eosinophils (67 +/- 22%) and eosinophil peroxidase (EPO) activity (94 +/- 33%) in BAL cells. The simultaneous administration of IL-5 and SP did not potentiate the hyperreactivity and eosinophilia observed with IL-5 alone. These data suggest that IL-5 is important in the recruitment of eosinophils, whereas both IL-5 and substance P are involved in the induction of airway hyperreactivity.     

5-(5-硝基-2-吡啶偶氮)-2,4-二氨基甲苯分光光度法同时测定铜和钴

基于Cu ,Co 与5 (5 硝基 2 吡啶偶氮) 2,4 二氨基甲苯(简称5 NO2 PADAT)在弱酸性介质中均可发生灵敏的显色反应,但铜络合物可被EDTA或强酸分解,而钴络合物不被分解的特点,利用差减法建立了铜、钴同时测定的新方法。在10mL溶液中,0～7 0μg铜、0～3 6μg钴符合比尔定律,表观摩尔吸光系数分别为ε519Co=1 48×105。方Cu=6 70×104,ε586法用于镁合金及锌铝合金中铜、钴的同时测定,结果满意。     

Definition, distribution, and use of a conserved Bovidae retroposon element sequence motif

Based on a data-base search, the sequences of 32 Bovidae retroposon elements have been compared. Two conserved areas are identified, and one of the corresponding sequences of the derived bovine consensus was used to design oligonucleotides as primer molecules for random DNA amplification of Bovidae DNA. Such a primer binding site should occur on average every 10,000 bp in the bovine genome, as suggested by a survey of published sequences. This estimate about the distribution of these possible primer binding sites was experimentally substantiated by mapping four of these primer binding sites within 40 kb of contiguous bovine DNA, carrying the heretofore undescribed bovine lactoferrin gene. Furthermore, these conserved, ubiquitous sequence motifs prove to be useful for mapping of bovine DNA.     

Characterization of a highly conserved FAD-binding site in human monoamine oxidase B

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the apoenzyme at Cys397. Our previous studies identified two noncovalent flavin-binding regions in MAO B (residues 6-34 and 39-46) (Kwan, S.-W., Lewis, D. A., Zhou, B. P., and Abell, C. W. (1995) Arch. Biochem. Biophys. 316, 385-391; Zhou, B. P., Lewis, D. A., Kwan, S.-W., Kirksey, T. J., and Abell, C. W. (1995) Biochemistry 34, 9526-9531). In these regions, Glu34 and Tyr44 were found to be required for the initial binding of FAD. By comparing sequences with enzymes in the oxidoreductase family, we now have found an additional FAD-binding site in MAO B (residues 222-227), which is highly conserved across species (human, bovine, and rat). This conserved sequence contains adjacent glycine and aspartate residues (Gly226 and Asp227). Based on the x-ray crystal structures of several oxidoreductases (Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142; Van Driessche, G., Kol, M., Chen, Z.-W., Mathews, F. S., Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Van Beeumen, J. J. (1996) Protein Sci. 5, 1753-1764), the Gly residue at the end of a beta-strand facilitates a sharp turn and extends the beta-carbonyl group of Asp to interact with the 3'-hydroxyl group of the ribityl chain of FAD. To assess the hypothesis that Gly226 and Asp227 are involved in FAD binding in MAO B, site-specific mutants that encode substitutions at these positions were prepared and expressed in mammalian COS-7 cells. Our results indicate that Gly226 and the beta-carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent binding of FAD. Our studies also reveal that mutagenesis at Glu34 and Tyr44 not only interferes with covalent flavinylation and catalytic activity of MAO B, but also with noncovalent binding of FAD. Based on these collective results, we propose that the coupling of FAD to the MAO B apoenzyme is a multistep process.     

Role of histamine in the regulation of coronary circulation

AIMS/BACKGROUND: An objective method for detecting hemifield and quadrantic visual field defects has been developed using steady state visual evoked cortical potentials (VECPs), an adaptive noise canceller (ANC), and Hotelling's t2 statistic. The purpose of this study was to determine the sensitivity and specificity of the technique. METHODS: Nine subjects (mean age 44 years) were investigated with field loss due to a variety of causes including both anterior and posterior visual pathway lesions. Dynamic perimetry was performed by means of a Goldmann or Tübingen perimeter. VECP recordings were made from each visual field quadrant (23 degrees X 23 degrees) by means of a steady state reversing checkerboard (7.7 rev/s). The central 5 degrees of the visual field and the vertical and horizontal meridians were masked during these measurements. Recordings were made from three electrode sites, positioned over the visual cortex, relative to a mid frontal electrode. Each recording lasted 2 minutes, during which time fixation was monitored. The data from each recording were divided into 4 second segments, and the amplitude and phase of the VECP signal measured using the ANC. Hotelling's t2 statistic was applied to determine the probability of signal detection. Receiver operating characteristic curves were used to find the optimum signal detection threshold for identification of the visual field defects. RESULTS: The results of the study confirmed patterns of subjective visual field loss. The technique had a sensitivity and a specificity of 81% and 85%, respectively, for detecting 'non-seeing' areas in the inferior visual field, and 82% and 89%, respectively, for detecting 'non-seeing' areas in the superior visual field. CONCLUSION: These results demonstrate that the technique is of potential clinical value to ophthalmologists and neurologists when subjective perimetry is not possible.     

Interaction of a G-protein beta-subunit with a conserved sequence in Ste20/PAK family protein kinases

Serine/threonine protein kinases of the Ste20/PAK family have been implicated in the signalling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades. In the yeast Saccharomyces cerevisiae, Ste20 is involved in transmitting the mating-pheromone signal from the betagamma-subunits (encoded by the STE4 and STE18 genes, respectively) of a heterotrimeric G protein to a downstream MAP kinase cascade. We have identified a binding site for the G-protein beta-subunit (Gbeta) in the non-catalytic carboxy-terminal regions of Ste20 and its mammalian homologues, the p21-activated protein kinases (PAKs). Association of Gbeta with this site in Ste20 was regulated by binding of pheromone to the receptor. Mutations in Gbeta and Ste20 that prevented this association blocked activation of the MAP kinase cascade. Considering the high degree of structural and functional conservation of Ste20/PAK family members and G-protein subunits, our results provide a possible model for a role of these kinases in Gbetagamma-mediated signal transduction in organisms ranging from yeast to mammals.     

A conserved sequence in tRNA and rRNA promoters of Lactococcus lactis

A tRNA operon (trnA) from Lactococcus lactis consisting of seven tRNA genes and a 5S rRNA gene was cloned and sequenced. Promoter-fusion of the trnA promoter to a promoter-less beta-galactosidase gene of Leuconostoc mesenteroides resulted in high levels of beta-galactosidase activity in L. lactis. Searching for sequences with similarity to the sequence of the promoter region revealed a consensus sequence of promoters preceeding rRNA operons and tRNA operons from Lactococcus species including a not previously described conserved sequence (AGTT).     

The amino-terminal sequence of MUC5B contains conserved multifunctional D domains: implications for tissue-specific mucin functions

Otoacoustic emissions have historically been measured with an acoustical probe assembly hermetically sealed in the ear canal, imposing in most cases a limited stimulus bandwidth. A physically open recording system should afford the possibility of a greater stimulus bandwidth but the change in acoustical load may affect the magnitude of otoacoustic emissions obtained. Here it is reported that the authors have measured in the guinea pig transient-evoked otoacoustic emissions extending in frequency to 20 kHz and cubic distortion tone otoacoustic emissions for f2 = 4737 and 8096 Hz with a physically open sound system. To address the effect of acoustical load provided by a physically open versus hermetically sealed system, the authors compared the amplitude of electrically evoked otoacoustic emissions recorded from a guinea pig in each case. The change in acoustical load in the ear canal introduced by the change in recording setup did not appear to make a substantial difference to the magnitude of otoacoustic emissions measured. A physically open recording system provides a good alternative to traditional acoustical probe assemblies sealed in the ear canal for the laboratory measurement of acoustically evoked otoacoustic emissions, with the advantage of permitting a greater stimulus bandwidth.     

2,4-diamino-5-(3,5-dimethoxy-4-substituted)-benzyl-pyrimidines as ligands in affinity chromatography of bacterial dihydrofolate reductases

Inhibitors of the trimethoprim-type, bearing terminal amino, hydroxyl or carboxyl groups in position 4 of the benzene ring as well as methotrexate were coupled to either CH-Sepharose 4B, epoxy-activated Sepharose 6B or AH-Sepharose 4B, respectively. In contrast to the methotrexate-affinity gel, trimethoprim-like ligands retained bacterial but not mammalian dihydrofolate reductases. The affinity gels prepared with these ligands could be used for effective purification of bacterial dihydrofolate reductases (EC 1.5.1.3), but differed in their affinity for this enzyme.     

Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase

Mutations in the COX17 gene of Saccharomyces cerevisiae cause a respiratory deficiency due to a block in the production of a functional cytochrome oxidase complex. Because cox17 mutants are able to express both the mitochondrially and nuclearly encoded subunits of cytochrome oxidase, the Cox17p most likely affects some late posttranslational step of the assembly pathway. A fragment of yeast nuclear DNA capable of complementing the mutation has been cloned by transformation of the cox17 mutant with a library of genomic DNA. Subcloning and sequencing of the COX17 gene revealed that it codes for a cysteine-rich protein with a molecular weight of 8,057. Unlike other previously described accessory factors involved in cytochrome oxidase assembly, all of which are components of mitochondria, Cox17p is a cytoplasmic protein. The cytoplasmic location of Cox17p suggested that it might have a function in delivery of a prosthetic group to the holoenzyme. A requirement of Cox17p in providing the copper prosthetic group of cytochrome oxidase is supported by the finding that a cox17 null mutant is rescued by the addition of copper to the growth medium. Evidence is presented indicating that Cox17p is not involved in general copper metabolism in yeast but rather has a more specific function in the delivery of copper to mitochondria.     

The PC motif: a novel and evolutionarily conserved sequence involved in interaction between p40phox and p67phox, SH3 domain-containing cytosolic factors of the phagocyte NADPH oxidase

The superoxide-generating NADPH oxidase, dormant in resting phagocytes, is activated during phagocytosis following assembly of the membrane-integrated protein cytochrome b558 and cytosolic factors. Among the latter are the three proteins containing Src homology 3 (SH3) domains, p67phox, p47phox and p40phox. While the first two factors are indispensable for the activity, p40phox is tightly associated with p67phox in resting cells and is suggested to have some modulatory role. Here we describe a systematic analysis of the interaction between p40phox and p67phox using the yeast two-hybrid system and in vitro binding assays with recombinant proteins. Both methods unequivocally showed that the minimum requirements for stable interaction are the C-terminal region of p40phox and the region between the two SH3 domains of p67phox. This interaction is maintained even in the presence of anionic amphiphiles used for the activation of the NADPH oxidase, raising a possibility that it mediates constitutive association of the two factors in both resting and activated cells. The C-terminal region of p40phox responsible for the interaction contains a characteristic stretch of amino acids designated as the PC motif, that also exists in other signal-transducing proteins from yeast to human. Intensive site-directed mutagenesis to the motif in p40phox revealed that it plays a critical role in the binding to p67phox. Thus the PC motif appears to represent a novel module for protein-protein interaction used in a variety of signaling pathways.     

New substrates of papain, based on the conserved sequence of natural inhibitors of the cystatin family

A discontinuous borate/formate buffer system is presented for horizontal polyacrylamide gel electrophoresis of DNA fragments. The resolution potential of the system could be altered by changing the total monomer concentration (5-9%T), the concentration of the crosslinker piperazine diacrylamide (2-5%CPDA), as well as the concentration of formate in the gel (40-120 mM), the leading ion of the buffer system. The separation of DNA fragments would be improved by increasing the migration distance from 22 to 28 cm. This discontinuous polyacrylamide gel electrophoresis system proved highly reproducible.     

Competitive inhibition of swine kidney copper amine oxidase by drugs: amiloride, clonidine, and gabexate mesylate

Carotid artery injuries from seat belt shoulder straps have been reported involving primarily the internal carotid artery. This paper describes a case of common carotid artery disruption and tracheal transection after a shoulder strap seat belt injury. A successful method for management of this rare injury is presented. Repair involved autogenous arterial replacement in a contaminated field using an interposition graft of superficial femoral artery.     

Upregulation of xanthine oxidase by lipopolysaccharide, interleukin-1, and hypoxia. Role in acute lung injury

LPS and selected cytokines upregulate xanthine dehydrogenase/xanthine oxidase (XDH/XO) in cellular systems. However, the effect of these factors on in vivo XDH/XO expression, and their contribution to lung injury, are poorly understood. Rats were exposed to normoxia or hypoxia for 24 h after treatment with LPS (1 mg/kg) and IL-1beta (100 microg/kg) or sterile saline. Lungs were then harvested for measurement of XDH/XO enzymatic activity and gene expression, and pulmonary edema was assessed by measurement of the wet/dry lung weight ratio (W/D). Although treatment with LPS + IL-1beta or hypoxia independently produced a 2-fold elevation (p < 0. 05 versus exposure to normoxia and treatment with saline) in lung XDH/XO activity and mRNA, the combination of LPS + IL-1beta and hypoxia caused a 4- and 3.5-fold increase in these values, respectively. XDH/XO protein expression was increased 2-fold by hypoxia alone and 1.3-fold by treatment with LPS + IL-1beta alone or combination treatment. Compared with normoxic lungs, W/D was significantly increased by exposure to hypoxia, LPS + IL-1beta, or combination treatment. This increase was prevented by treatment of the animals with tungsten, which abrogated lung XDH/XO activity. In conclusion, LPS, IL-1beta, and hypoxia significantly upregulate lung XDH/XO expression in vivo. The present data support a role for this enzyme in the pathogenesis of acute lung injury.     

Role of the striatum, cerebellum, and frontal lobes in the learning of a visuomotor sequence

This study was designed to examine the role of the striatum, cerebellum, and frontal lobes in the implicit learning of a visuomotor sequence. The performance of patients with idiopathic Parkinson's disease (PD), with damage to the cerebellum, or with a circumscribed lesion to the frontal lobes was thus compared to that of separate groups of matched normal control subjects on an adapted version of the Repeated Sequence Test. This paradigm consists of a visual reaction-time task with a fixed embedded sequence of finger movements to be performed based on presentation of visual stimuli. Subjects received four blocks of trials (i.e., 40 presentations of a 10-item sequence) per day over 6 training days. Following the last experimental session, subjects were also given two tests measuring their declarative knowledge of the sequence. Only PD patients with a bilateral striatal-dysfunction or patients with lesions to the cerebellum failed to improve their performance in the last three training sessions, hence suggesting an impairment late in the acquisition process. Further analyses revealed that such impairment was mainly implicit in nature, and that it could not be ascribed to a general decline in cognitive functioning, to mood disturbances, or to the severity of the motor symptoms. By contrast, the level of declarative knowledge of the sequence did not differ between the three clinical groups and their respective groups of normal subjects. These findings suggest that, unlike declarative memory, the incremental acquisition of a new visuomotor skill depends upon the integrity of both the striatum and the cerebellum, but not of the frontal lobes.     

Role of xanthine oxidase and eicosanoids in development of pancreatic ischemia-reperfusion injury

The implication of different eicosanoids and oxygen free radicals in the development of pancreatic injury after an ischemia-reperfusion process has been evaluated. For this purpose we have compared the effect of allopurinol and indomethacin administration on the pancreatic levels of eicosanoids in a rat model of pancreatic ischemia-reperfusion. After 60 min of pancreatic ischemia and 2 h of reperfusion, significant increases in 6-keto-PGF1 alpha, PGE2, and LTB4 in pancreas tissue were detected. Allopurinol before the ischemic period reduced 6-keto-PGF1 alpha, PGE2, and LTB4 levels to the range of basal values, while prior indomethacin treatment significantly reduced 6-keto-PGF1 alpha and PGE2 levels, with LTB4 remaining unmodified. Increased postischemic plasma lipases were also significantly reduced by allopurinol to the range of sham-operated animals whereas indomethacin did not modify these levels. The data suggest a role for lipoxygenase metabolites in the development of pancreatic injury and the importance of the enzyme xanthine oxidase as an inductor of eicosanoid biosynthesis.     

Long-term effects of portacaval anastomosis on the 5-hydroxytryptamine, histamine, and catecholamine neurotransmitter systems in rat brain

Portacaval anastomosis (PCA) in the rat is used as a model for portal systemic encephalopathy. Changes in the serotonergic, histaminergic, and catecholaminergic neurotransmitter systems are often found shortly after PCA. We have examined the long-term effects of PCA on the aminergic systems in brains of male Wistar rats, which 8 months previously had been subjected to PCA. Precursors, amines, and metabolites were assayed by HPLC. Eight months after PCA, the catecholamine levels were unchanged in all brain regions. In contrast, tryptophan was evenly increased throughout the brain. The accumulation of 5-hydroxytryptophan after decarboxylase inhibition (NSD-1015; 100 mg/kg i.p.) and the endogenous levels of 5-hydroxyindoleacetic acid were significantly higher in PCA rats, particularly in the hypothalamus and midbrain, whereas 5-hydroxytryptamine concentrations were unchanged. Histamine levels were elevated throughout the brain with the greatest increase found in the hypothalamus and in the striatum. tele-Methylhistamine levels were significantly elevated in cortex and hypothalamus. We conclude that 8 months after PCA, catecholaminergic systems had reestablished their homeostasis, whereas serotonergic and histaminergic systems still show profound disturbances in their function. With histamine, this is reflected as an increase in the amounts of both transmitter and metabolite; serotonergic neurons respond by increasing only the level of the metabolite.