Effects of biogenic phosphates on protease-induced protein cleavage and functioning of plasminogen activators

We showed, using the method of lysis of fibrin plates and five substrate proteins in a thin layer of agar gel, that inorganic orthophosphate (0.001-0.06 M) enhances by 50-250% the activatory functions of streptokinase, urokinase, and tissue plasminogen activator and, in general, by 1.2-12.0 times enhances protein lysis by trypsin, alpha-chymotrypsin, subtilisin, papain, bacterial metalloprotease, and even pepsin at a concentration < 4 mM. At higher concentrations, phosphate sharply inhibited pepsin activity and inhibited by 40-50% gelatin lysis by papain and gelatin (at a peak concentration) and casein lysis by metalloprotease. Inorganic pyrophosphate ions at concentrations of 10(-8)-10(-1) M enhanced the cleavage of a number of proteins by serine proteases and, at concentrations of 10(-5) -10(-3) M, the activities of pepsin, plasminogen tissue activator, and streptokinase by 100 and 40%, respectively. The pyrophosphate concentrations of > 10(-3) and >10(-4) M inhibited pepsin- and metalloprotease-induced lysis of virtually all proteins. ATP increased casein lysis by serine proteases, metalloprotease, and pepsin by 20-60% at concentration of 10(-3) M and by 30-260% at 10(-2) M concentration. At concentrations of 10-2 M, it inhibited the cleavage of some proteins by trypsin, chymotrypsin, papain, and metalloprotease by 20-100%, and, at concentrations of 10(-3) M, lysis of albumin with pepsin and other proteins (except for fibrinogen) by metalloprotease. A GTP concentration of 10(-7)-10(-2) M increased protein degradation by serine proteases, papain, and gelatin lysis by pepsin by 20-90%, whereas albumin lysis was inhibited by 40-70%. The presence of 10(-6)-10(-5) M GTP led to a slightly increased degradation of hemoglobin and casein by bacterial metalloprotease, while 10(-3) M GTP induced a drop in the activity of the metalloprotease by 20-50%. ADP could enhance gelatin lysis by trypsin, casein lysis by pepsin and papain, and inhibited metalloprotease activity by 20-100% (at 10(-3) M). Peculiarities of the effects of AMP and GD(M)P on gelatin lysis were found.     

Synthesis of enantiopure 2-aryl-2-methoxypropionic acids and determination of their absolute configurations by X-ray crystallography

Racemic 2-aryl-2-methoxypropionic acids were enantioresolved by the use of (S)-(-)-phenylalaninol 4. For instance, racemic 2-methoxy-2-phenylpropionic acid (+/-)-7 was condensed with phenylalaninol (S)-(-)-4 yielding a diastereomeric mixture of amides, which was easily separated by HPLC on silica gel affording the first-eluted amide (-)-13a and the second-eluted amide (+)-13b: alpha = 3.19, Rs = 3.49. The absolute configuration of amide (-)-13a was determined to be (R;S) by X-ray crystallography by reference to the S configuration of the phenylalaninol moiety. Amide (R;S)-(-)-13a was converted to oxazoline (R;S)-(-)-14a, from which enantiopure 2-methoxy-2-phenylpropionic acid (R)-(-)-7 was recovered. Other 2-aryl-2-methoxypropionic acids, (R)-(-)-8, (R)-(-)-9, (R)-(+)-10, (R)-(-)-11, and (R)-(-)-12, were similarly prepared in enantiopure forms with the use of phenylalaninol (S)-(-)-4, and their absolute configurations were clearly determined by X-ray crystallography or by chemical correlation.     

Monohydroperoxides Formed by Autoxidation and Photosensitized Oxidation of Methyl Eicosapentaenoate

Methyl eicosapentaenoate (methyl 5,8,11,14,17-eicosapentaenoate) was subjected to autoxidation and methylene blue sensitized photooxidation. Methyl eicosapentaenoate monohydroperoxides, the primary products of the autoxidation and photosensitized oxidation, were isolated by silica gel column chromatography, and characterized by ultraviolet, infrared and nuclear magnetic resonance spectra. The isomeric composition of the monohydroperoxides were determined by gas chromatography-mass spectrometry as follows: the 5-, 8-, 9-, 11-, 12-, 14-, 15- and 18-isomers (autoxidation), and the 5-, 6-, 8-, 9-, 11-, 12-, 14-, 15-, 17- and 18-isomers (photosensitized oxidation). Methyl eicosapentaenoate was readily oxidized both by autoxidation and by photosensitized oxidation.     

猪链球菌2型FBPS的纤连蛋白结合部位的初步确定

根据猪链球菌2型江苏分离株HA9801的fbps基因序列,设计合成不同的引物,用含全长fbps的pMD-T-FBPS质粒为模板,通过PCR技术,扩增不同片段fbps,并按正确的阅读框架定向克隆到表达载体pET-32a( ),构建分别表达全长7~82、7~165和87~320氨基酸FBPS的重组表达质粒pFBPS、pFBPS(7~82)、pFBPS(7~165)和pFBPS(87~320);将重组质粒转化大肠杆菌BL21(DE)株,经IPTG诱导,表达rFBPS(7~82)、rFBPS(7~165)、rFBPS(87~320)和rFBPS(全长),分子量分别为29、34、42及83kD的融合蛋白。配基亲和Western blot试验表明,表达的融合蛋白除rFBPS(7~82)外,均可与人纤连蛋白(Fn)结合,由此可以推断SS2的纤连蛋白/血纤蛋白原结合蛋白(FBPS)N端87~165氨基酸区域为具有结合活性的线性部位。     

The effects of somatostatin on the arachidonate cascade of platelets

Somatostatin (10(-9) M) significantly elevated the synthesis of thromboxane B2 in rat platelets. The transformation of arachidonic acid to active lipoxygenase metabolites was suppressed by somatostatin (10(-9) and 10(-8) M). The ratio of the lipoxygenase/cyclooxygenase products was significantly reduced by the polypeptide (10(-9) and 10(-8) M) in rat platelets. Higher concentrations (10(-7), 10(-6) and 10(-5) M) of somatostatin did not modify the lipoxygenase pathway of the platelets. The synthesis of the vasoconstrictor - proaggregatory cyclooxygenase products was stimulated by the polypeptide (10(-9) and 10(-8) M), while the formation of vasodilatator - antiaggregatory cyclooxygenase metabolites was induced by higher concentrations of somatostatin (10(-7) and 10(-6) M). Somatostatin might act on the deacylation process of phospholipids, reducing the free arachidonic acid substrate level, resulting in a lower lipoxygenation rate in the platelets, which could be responsible for the increased formation of thromboxane. The contradictory results reported by others concerning the action of somatostatin on the platelet function might be explained by our results that the effect of somatostatin depends on the applied dose.     

Existence of two types of postjunctional alpha-adrenoceptors in the isolated canine internal carotid artery

The pharmacological characteristics of postjunctional alpha-adrenoceptors in isolated canine internal carotid arteries were investigated by the use of selective agonists and antagonists for alpha 1- and alpha 2-adrenoceptors. Norepinephrine, phenylephrine, and xylazine caused concentration-dependent contractions in the helical strips. The contraction induced by 10(-4)M xylazine was significantly smaller than that produced by 10(-4)M norepinephrine or 10(-4)M phenylephrine. The contraction induced by 10(-4)M phenylephrine was almost the same value as that induced by 10(-4)M norepinephrine. Phentolamine (10(-8) and 10(-7)M) caused a parallel shift to the right of the concentration-response curve to norepinephrine. The contractile responses to low concentrations of norepinephrine were significantly suppressed by pretreatment with an alpha 2-antagonist such as yohimbine (10(-9) and 10(-8)M) or DG5128 (10(-7) and 10(-6)M). On the other hand, the responses to higher concentrations of norepinephrine were mainly reduced by low concentrations of an alpha 1-antagonist, prazosin (3 x 10(-10) and 3 x 10(-9)M). These results suggest that both alpha 1- and alpha 2-adrenoceptors are located on the plasma membrane of smooth muscle cells in canine internal carotid arteries and that the norepinephrine-induced contractions at low and high concentrations are mainly mediated by activation of alpha 2- and alpha 1-adrenoceptors, respectively.     

Determination of amino acid sequence responsible for suppression of bone resorption by serum calcium-decreasing factor (caldecrin).

We previously reported on the serum calcium-decreasing activity of recombinant protein factor referred to as caldecrin [Tomomura et al. (1995) J. Biol. Chem. 270, 30315-30321]. To address the mechanism of this serum calcium-decreasing activity, we investigated the effect of rat caldecrin on osteoclastic bone-resorbing activity. Wild-type caldecrin suppressed resorption pit formation by osteoclast on a dentine slice in a dose-dependent manner. The suppressive effect on the bone resorption was not affected by treatment of caldecrin with phenylmethyl sulfonyl fluoride or by use of protease-deficient mutant caldecrins. Recombinant procaldecrin (-13-239), and its fragments (-13-125), (1-111), (1-46), (47-111), and (126-239) were expressed as His-tagged thioredoxin fusion proteins and investigated for their ability to suppress bone resorption. The proform (-13-239) and fragment (-13-125) did not affect the suppressive activity, whereas fragments (1-111) and (126-239) did suppress the bone resorption. The bone-resorbing activity was also suppressed by fragment (47-111), not by fragment (1-46). Overlapping fragments (47-62), (47-79), (47-98), (56-111), (71-111), and (85-111) were compared for their suppressive activity. The fragments (47-62) and (85-111) did not affect the activity, but the other fragments suppressed the bone resorption. A synthetic peptide having the (71-79) sequence suppressed the bone resorption. These results suggest that amino acid sequence corresponding to rat caldecrin (aa 71-79) is responsible for the suppression of bone resorption by caldecrin.     

Semisynthesis of carboxy-terminal fragments of thermolysin

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205-316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu302 residue into fragments 205-302 and 303-316. Upon incubation for 2-5 days of fragment 205-302 with a 5-fold excess of peptide 303-316, prepared by solid phase synthesis, with V8-protease in 0.1 M ammonium acetate, pH 6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to approximately 90% (based on fragment 205-302). The same procedure was used to prepare also the thermolysin fragments 205-315 and 205-311 by enzymatic coupling of fragment 205-302 to peptide 303-315 or 303-311, these last prepared by proteolytic digestion of the synthetic peptide 303-316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205-316 of thermolysin at the level of its helical segment encompassing residues 301-312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.     

Properties of adenylate cyclase from plasma membranes of the rabbit myometrium in different functional states

Adenylate cyclase of plasma membranes from the nonpregnant rabbit myometrium shows the maximum activity at pH 7.7-7.9, is characterized by apparent Km for ATP amounting to 0.38 +/- 0.09 mM, V--125 +/- 34.4 pmol min/mg protein, is activated at most by 15-20 mM Mg2+ and F-. Adenylate cyclase of plasma membranes from the pregnant rabbit myometrium is characterized by apparent Km for ATP amounting to 0.74 +/- 0.06 mM, V--77.3 +/- 6.0 pmol/min/mg protein, is activated at most by 5-10 mM Mg2+ and 10-15 mM F-; the pH optimum for the adenylate cyclase in this functional state is 7.3. Adenylate cyclase in the state of labour is characterized by apparent Km for ATP amounting to 0.46 +/- 0.11 mM, V--34.8 +/- 4.6 pmol/min/mg protein, is activated at most by 10-15 mM Mg2+ and F-, shows the same activity at pH 7.3-8.5. Adenylate cyclase of myometrium in three investigated states is activated by 2 mM EGTA; 10(-7) M Ca2+ decreases activation caused by EGTA; higher concentrations of Ca2+ decrease the basal activity of the enzyme.     

Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclizations of geranyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene

The conversion of geranyl pyrophosphate to (+)-alpha-pinene and to (-)-beta-pinene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)- and to (+)-(3S)-linalyl pyrophosphate, respectively, and the subsequent cyclization of the anti, endo-conformer of these bound intermediates by mirror-image sequences which should result in the net retention of configuration at C1 of the geranyl precursor. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with (+)-pinene cyclase and with (-)-pinene cyclase from common sage (Salvia officinalis) gave labeled (+)-alpha- and (-)-beta-pinene of unchanged 3H/14C ratio in all cases, and the (+)- and (-)-olefins were stereoselectively converted to (+)- and (-)-borneol, respectively, which were oxidized to the corresponding (+)- and (-)-isomers of camphor, again without change in isotope ratio. The location of the tritium was determined in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogens of these derived ketones. The results indicated that the configuration at C1 of the substrate was retained in the enzymatic transformations to the (+)- and (-)-pinenes, which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate, transoid to cisoid rotation, and anti, endo-cyclization of the latter. The absolute stereochemical elements of the antipodal reaction sequences were confirmed by the selective enzymatic conversions of (3R)- and (3S)-1Z-[1-3H]linalyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene, respectively, and by the location of the tritium in the derived camphors as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal pinenes.     

Identification of insulin intermediates and sites of cleavage of native insulin by insulin protease from human fibroblasts

We have studied the time sequence degradation of native insulin by insulin protease from human fibroblast using multiple steps involving purification of the products by high performance liquid chromatography, determination of peak composition by amino acid sequence analysis, and confirmation of structure by mass spectrometry and thus elucidated the sites of cleavage of insulin by human insulin protease. We observed that as early as 0.5 min of incubation, three major new peptide peaks, intact insulin, and four smaller peptide peaks can be detected. The major peptides are portions of the insulin molecule, with the amino ends of the A and B chains or the carboxyl ends of the A and B chains still connected by disulfide bonds. Peptide peak I is A1-13-B1-9. Peptide peak II is A1-14-B1-9. Peptide peak III is A14-21-B14-30. The smaller peptide peaks are A14-21-B17-30, A15-21-B14-30, A15-21-B10-30, and A14-21-B10-30. The major peptide bond cleavage sites therefore consist of A13-14, A14-15, B9-10, B13-14, and B10-17. With longer incubation times, peptide peak II appears to lose the A14 tyrosine to form peptide peak I. This peptide I, which is the amino end of the A and B chains, is not further degraded even after 1.5 h of incubation. With longer incubation times, the peptides containing the carboxyl ends of the A and B chains are further degraded to form products from cleavage at the A18-19, B14-15, B25-26, and a small amount of A19-20, B10-11, and B24-25 cleavage and the emergence of 2-5-amino acid peptide chains, tyrosine, alanine, histidine, and leucine-tyrosine. We conclude, based on the three-dimensional structure of insulin, that human insulin protease recognizes the alpha-helical regions around leucine-tyrosine bonds and that final degradation steps to small peptides do not require lysosomal involvement.     

Kinetics of oxidation of aliphatic and aromatic thiols by myeloperoxidase compounds I and II

Myeloperoxidase (MPO) is the most abundant protein in neutrophils and plays a central role in microbial killing and inflammatory tissue damage. Because most of the non-steroidal anti-inflammatory drugs and other drugs contain a thiol group, it is necessary to understand how these substrates are oxidized by MPO. We have performed transient kinetic measurements to study the oxidation of 14 aliphatic and aromatic mono- and dithiols by the MPO intermediates, Compound I (k3) and Compound II (k4), using sequential mixing stopped-flow techniques. The one-electron reduction of Compound I by aromatic thiols (e.g. methimidazole, 2-mercaptopurine and 6-mercaptopurine) varied by less than a factor of seven (between 1.39 +/- 0.12 x 10(5) M(-1) s(-1) and 9.16 +/- 1.63 x 10(5) M(-1) s(-1)), whereas reduction by aliphatic thiols was demonstrated to depend on their overall net charge and hydrophobic character and not on the percentage of thiol deprotonation or redox potential. Cysteamine, cysteine methyl ester, cysteine ethyl ester and alpha-lipoic acid showed k3 values comparable to aromatic thiols, whereas a free carboxy group (e.g. cysteine, N-acetylcysteine, glutathione) diminished k3 dramatically. The one-electron reduction of Compound II was far more constrained by the nature of the substrate. Reduction by methimidazole, 2-mercaptopurine and 6-mercaptopurine showed second-order rate constants (k4) of 1.33 +/- 0.08 x 10(5) M(-1) s(-1), 5.25 +/- 0.07 x 10(5) M(-1) s(-1) and 3.03 +/- 0.07 x 10(3) M(-1) s(-1). Even at high concentrations cysteine, penicillamine and glutathione could not reduce Compound II, whereas cysteamine (4.27 +/- 0.05 x 10(3) M(-1) s(-1)), cysteine methyl ester (8.14 +/- 0.08 x 10(3) M(-1) s(-1)), cysteine ethyl ester (3.76 +/- 0.17 x 10(3) M(-1) s(-1)) and alpha-lipoic acid (4.78 +/- 0.07 x 10(4) M(-1) s(-1)) were demonstrated to reduce Compound II and thus could be expected to be oxidized by MPO without co-substrates.     

Covalent structure of collagen: amino acid sequence of five consecutive CNBr peptides from type III collagen of human liver

Type III collagen was solubilized from human liver by limited pepsin digestion and purified by differential salt precipitation and carboxymethylcellulose chromatography. Digestion with cyanogen bromide yielded the nine distinct peptides previously described and an additional tripeptide not recognized in earlier studies. Five of these peptides, alpha1 (III)-CB1, 2, 4, 8, and 10, were further purified by molecular sieve and/or ion exchange chromatography. They contained 12, 40, 149, 125 and 3 amino acid residues, respectively. The amino acid sequence of these peptides was determined by automated Edman degradation of tryptic (before and after maleylation), chymotryptic, thermolytic or hydroxylamine-derived peptide fragments as well as the intact peptides. The alignment of these five peptides within the collagen chain is deduced to be 1-8-10-2-4 by homology with known alpha1 (I) sequences. The known CNBr peptide alignment of the NH2-terminal portion of type III collagen so far would, therefore, be alpha1 (III)-CB3-7-6-1-8-10-2-4 and correspond to the homologous region of alpha1 (I)-CB0-1-2-4-5-8-3 or residues 11-567 of the alpha1 (III) collagen chain.     

不同树种混交林及其纯林对土壤理化性质影响的研究

对针阔混交林土壤理化性质的研究表明,针阔混交林比针叶树纯林对土壤的改良作用要好,它使土壤总孔隙度增加２—１９％,水分含量增加６—３１％,枯枝落叶年凋落量增加２—２００％;土壤养分含量全Ｎ、ＮＨ４－Ｎ、代换性Ｃａ、代换性Ｍｇ和腐殖质含量分别增加４５—７５％、３３—８２％、５５—８５％、４４—８４％和３７—４６％．     

Biotechnological production of l(+)-lactic acid from wood hydrolyzate by batch fermentation of Enterococcus faecalis

The inhibition of lactic acid fermentation by wood hydrolyzate was decreased (approx. 20%) by adaptation of Enterococcus faecalis RKY1 to wood hydrolyzate-based medium whereby lactic acid productivity and cell growth were enhanced by 0.5 g l(-1) h(-1) and 2.1 g l(-1), respectively. When the diluted or concentrated wood hydrolyzate (equivalent to 25-100 g glucose l(-1)) was supplemented with 15 g yeast extract l(-1), 24-93 g lactic acid l(-1) was produced at a rate between 1.7 g l(-1) h(-1) and 3.2 g l(-1) h(-1).     

Comparative Induction of Nitrate and Nitrite Uptake and Reduction Systems by Ambient Nitrate and Nitrite in Intact Roots of Barley (Hordeum vulgare L.) Seedlings

The induction by ambient NO3- and NO2- of the NO3- and NO2- uptake and reduction systems in roots of 8-d-old intact barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with concentrations of NaNO3 or NaNO2 ranging from 0.25 to 1000 [mu]M. Uptake was determined by measuring the depletion of either NO3- or NO2- from uptake solutions. Enzyme activities were assayed in vitro using cell-free extracts. Uptake and reduction systems for both NO3- and NO2- were induced by either ion. The Km values for NO3- and NO2- uptake induced by NO2- were similar to those for uptake induced by NO3-. Induction of both the uptake and reduction systems was detected well before any NO3- or NO2- was found in the roots. At lower substrate concentrations of both NO3- and NO2- (5-10 [mu]M), the durations of the lag periods preceding induction were similar. Induction of uptake, as a function of concentration, proceeded linearly and similarly for both ions up to about 10 [mu]M. Then, while induction by NO3- continued to increase more slowly, induction by NO2- sharply decreased between 10 and 1000 [mu]M, apparently due to NO2- toxicity. In contrast, induction of NO3- reductase (NR) and NO2- reductase (NiR) by NO2- did not decrease above 10 [mu]M but rather continued to increase up to a substrate concentration of 1000 [mu]M. NO3- was a more effective inducer of NR than was NO2-; however, both ions equally induced NiR. Cycloheximide inhibited the induction of both uptake systems as well as NR and NiR activities whether induced by NO3- or NO2-. The results indicate that in situ NO3- and NO2- induce both uptake and reduction systems, and the accumulation of the substrates per se is not obligatory.     

Residues of 14-3-3 zeta required for activation of exoenzyme S of Pseudomonas aeruginosa.

Exoenzyme S (ExoS) is a mono-ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. ExoS requires a eukaryotic factor, the 14-3-3 protein, for enzymatic activity. Here, two aspects of the activation of the ADP-ribosyltransferase activity of ExoS by 14-3-3 proteins are examined. Initial studies showed that several isoforms of 14-3-3, including beta, zeta, eta, sigma, and tau, activated ExoS with similar efficiency. This implicates a conserved structure in 14-3-3 that contributes to the interaction between 14-3-3 and ExoS. One candidate structure is the conserved amphipathic groove that mediates the 14-3-3/Raf-1 interaction. The next series of experiments examined the role of individual amino acids of the amphipathic groove of 14-3-3 zeta in ExoS activation and showed that ExoS activation required the basic residues lining the amphipathic groove of 14-3-3 zeta without extensive involvement of the hydrophobic residues. Strikingly, mutations of Val-176 of 14-3-3 zeta that disrupted its interaction with Raf-1 did not affect the binding and activation of ExoS by 14-3-3. Thus, ExoS selectively employs residues in the Raf-binding groove for its association with 14-3-3 proteins.     

Role of LPS and receptor subtypes in the uptake of TNF by the murine lung

Tumor necrosis factor alpha (TNF) is an important mediator in lung injury. The kinetics of TNF uptake by the lung are not completely understood. In this study, we evaluated the role that lipopolysaccharide (LPS) and the two types of TNF receptor (p55 and p75) play in the uptake of circulating murine TNF by the murine lung. TNF radioactively labeled with 125I (I-mTNF) was administered intravenously (2 x 10(6) cpm/mouse) to mice with both receptors (wild-type) or to mice missing one (p55-/- or p75-/-) or both (p55-/- and p75-/-) TNF receptors. Blood to lung non-reversible sequestration (Ki) and reversible uptake (Vi) were measured with multiple-time regression analysis. Uptake by lung of I-mTNF in wild-type mice had reversible and non-reversible components. This uptake was decreased by intratracheal, but not by intravenous, LPS, suggesting modulation by local, rather than systemic, inflammation. The p75-/- deficient mice retained the Ki (saturable, non-reversible) component of TNF uptake, whereas p55-/- deficient mice retained the Vi (saturable, reversible) component of TNF uptake. Both Ki and Vi components of TNF uptake were absent in the lungs of p55-/- p75-/- deficient mice. These studies show that local inflammation inhibits the uptake of circulating I-mTNF by lung and that uptake consists of two distinguishable compartments: reversible uptake mediated by the p75 receptor and non-reversible sequestration mediated by the p55 receptor.     

Effects of diethylcarbamazine on the motility of Angiostrongylus cantonensis and Dirofilaria immitis

Effects of piperazine derivatives, especially of diethylcarbamazine (DEC) on adult Angiostrongylus cantonensis and Dirofilaria immitis were examined. Piperazine (3 X 10(-5)-10(-4) M) paralyzed A. cantonensis and the action was antagonized by picrotoxin. 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) (10(-5)-10(-4) M) caused contraction but little effect was produced by strychnine. An inhibitory effect on untreated preparations was caused by lower concentrations (3 X 10(-6)-10(-5) M) of diethylcarbamazine citrate (DEC) and also on the preparations contracted by eserine. A stimulatory effect was also seen when higher concentrations (10(-4)-3 X 10(-4) M) of this drug were applied to both preparations. The inhibitory action of DEC was antagonized by gabergic antagonists such as picrotoxin and bicuculline, but not by alpha-adrenergic antagonists like dibenamine and phentolamine. When the worm preparation was paralyzed by strychnine or hexylresorcinol (inhibitors of the release of acetylcholine in this worm), the stimulatory effect of DEC was blocked, but pyrantel (a nicotinic cholinergic agonist) contracted the paralyzed preparation. However, the effect of DEC on D. immitis (10(-7)-3 X 10(-4) M) was inhibitory, and this action was also antagonized by picrotoxin. These results suggest that the DEC inhibitory and stimulatory action is through the gabergic and cholinergic mechanisms in adult A. cantonensis and D. immitis.     

Chemical control of tracheal vascular resistance in dogs

With anesthetized dogs we have measured upper tracheal vascular resistance on both sides of the trachea simultaneously by perfusing the cranial tracheal arteries and measuring inflow pressures at constant flows. The ratio of pressure to flow gave vascular resistance (Rtv). Lung airflow, blood pressure (BP), heart rate, and pressure in a cervical tracheal balloon (Ptr) were also measured. In paralyzed dogs, systemic hypoxia due to artificial ventilation with 10% O2-90% N2 increased Rtv by +8.1 +/- 1.0% (SE), Ptr by +76 +/- 22.8%, and BP by +18.9 +/- 24%. After bilateral cervical vagosympathectomy the increases in Rtv and BP were present (+8.8 +/- 0.9 and +22.3 +/- 0.3%, respectively). After carotid body denervation Rtv, Ptr, and BP increased (+6.4 +/- 1.3, +58.6 +/- 31.6, and +14.6 +/- 3.3%, respectively). After vagotomy Rtv and BP increased (+14.1 +/- 1.7 and +22.4 +/- 10.1%, respectively). Tracheal perfusion with hypoxic blood caused a small vasodilation (-2.2 +/- 1.1%). Systemic hypercapnia due to artificial ventilation with 8% CO2-92% air increased Rtv by +16.7 +/- 3.8%, Ptr by +67 +/- 2.0%, and BP by +12.9 +/- 9.9%. Tracheal perfusion with hypercapnic blood caused a small vasodilation (-2.5 +/- 1.2%). Stimulation of the carotid body chemoreceptors with KCN caused a small increase in Rtv (+1.2 +/- 0.5%) and increases in Ptr (+49.8 +/- 13.6%) and BP (+11.1 +/- 2.1%). Systemic hypoxia and hypercapnia caused tracheal vasoconstriction mainly by an action on the central nervous system.