Scoring protein-protein docked structures based on the balance and tightness of binding.

One main issue in protein-protein docking is to filter or score the putative docked structures. Unlike many popular scoring functions that are based on geometric and energetic complementarity, we present a set of scoring functions that are based on the consideration of local balance and tightness of binding of the docked structures. These scoring functions include the force and moment acting on one component (ligand) imposed by the other (receptor) and the second order spatial derivatives of protein-protein interaction potential. The scoring functions were applied to the docked structures of 19 test targets including enzyme/inhibitor, antibody/antigen and other classes of protein complexes. The results indicate that these scoring functions are also discriminative for the near-native conformation. For some cases, such as antibody/antigen, they show more discriminative efficiency than some other scoring functions, such as desolvation free energy (deltaG(des)) based on pairwise atom-atom contact energy (ACE). The correlation analyses between present scoring functions and the energetic functions also show that there is no clear correlation between them; therefore, the present scoring functions are not essentially the same as energy functions.

Localization of Barrington's nucleus in the pontine dorsolateral tegmentum of the rabbit.

The localization of Barrington's nucleus in the dorsolateral pons of the rabbit and its projections to the sacral spinal cord were examined by using retrograde and anterograde labeling methods combined with immunohistochemistry. After injection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) or a fluorescence tracer, tetramethylrhodamine-dextran amine (TMR), into the sacral spinal cord segments, a cluster of neurons labeled with WGA-HRP or TMR were seen in the pontine dorsolateral tegmentum. To identify whether the retrogradely labeled neurons were situated within the locus coeruleus, the sections containing TMR-labeled neurons through the pons were incubated with anti-tyrosine hydroxylase (TH) antibody and observed under epifluorescence microscope. It was shown that the cluster of TMR-labeled neurons in the dorsolateral tegmentum were surrounded by TH-positive neurons, but they were negatively immunostained with TH-like immunoreactivity. In anterograde experiment, injection of WGA-HRP into the dorsolateral tegmentum resulted in many anterogradely labeled nerve fibers and terminals in the sacral spinal cord, including the sacral parasympathetic nucleus. The present results suggest that the cluster of neurons in the dorsolateral tegmentum of the rabbit may correspond to Barrington's nucleus revealed in the rat and cat, and thus may be involved in micturtion reflex of the rabbit.

Neurokinin B receptor (NK3)-containing neurons in the paraventricular and supraoptic nuclei of the rat hypothalamus synthesize vasopressin and express Fos following intravenous injection of hypertonic saline.

Subnuclear localization of neurokinin B receptor (NK3) in the paraventricular and supraoptic nuclei of the hypothalamus was immunohistochemically investigated in the rat. In the paraventricular hypothalamic nucleus, intense neurokinin B receptor-like immunoreactivity was found in the posterior magnocellular part, moderate to weak neurokinin B receptor-like immunoreactivity was seen in the other parts. In the supraoptic nucleus, intense neurokinin B receptor-like immunoreactivity was distributed in its principal part, and a few neurons with neurokinin B receptor-like immunoreactivity were found in the retrochiasmatic part. Co-localization of neurokinin B receptor-like immunoreactivity with vasopressin-like immunoreactivity was examined through serial adjacent sections. Neurons with both neurokinin B receptor-like immunoreactivity and vasopressin-like immunoreactivity were primarily found in the supraoptic nucleus and posterior magnocellular part of the pavaventricular nucleus. A small number of neurons with neurokinin B receptor-like immunoreactivity and vasopressin-like immunoreactivity were also seen in the circular nucleus and the region surrounding blood vessel in the anterior hypothalamus. Many neurokinin B receptor-containing neurons in the paraventricular and supraoptic nuclei, as well as in circular nucleus and the region surrounding the blood vessel, expressed Fos-like immunoreactivity after intravenous injection of hypertonic saline. The present study demonstrated that a large proportion of neurokinin B receptor-like immunoreactive neurons in the paraventricular hypothalamic and supraoptic nuclei contained vasopressin-like immunoreactivity, and expressed Fos-like immunoreactivity after intravenous administration of hypertonic saline. The results suggest that neurokinin B receptor in the two nuclei may be involved in modulation of the release of vasopressin when the internal environment is disturbed.

Co-localization of mu-opioid receptor-like immunoreactivity with substance P-LI, calcitonin gene-related peptide-LI and nitric oxide synthase-LI in vagal and glossopharyngeal afferent neurons of the rat.

Co-localization of mu-opioid receptor (MOR)-like immunoreactivity (-LI) with substance P (SP)-LI, calcitonin gene-related peptide (CGRP)-LI and nitric oxide synthase (NOS)-LI in the nodose, petrosal and jugular ganglia was examined in the rat by a double immunofluorescence histochemical method. About 0.6%, 41% and 95% of neurons with MOR-LI, respectively, in the nodose, petrosal and jugular ganglia showed SP-LI; about 2%, 51% and 66% of MOR-like immunoreactive neurons displayed CGRP-LI in the nodose, petrosal and jugular ganglia, respectively. In addition, about 59% of MOR-like immunoreactive neurons in the nodose ganglia displayed NOS-LI, whereas no NOS-LI was detected in the petrosal or jugular ganglion. These data provide evidence for co-localization of MOR-LI with SP-LI, CGRP-LI and NOS-LI in the vagal and glossopharyngeal afferent neurons, and suggest that MOR may regulate the release of SP, CGRP and nitric oxide from the visceral primary afferent terminals in the nucleus of the solitary tract of the rat.

Substance P receptor-expressing neurons in the medullary and spinal dorsal horns projecting to the nucleus of the solitary tract in the rat.

By using substance P receptor (SPR) immunofluorescence histochemistry combined with fluorescent retrograde labeling, we examined the distribution of the trigeminal and spinal neurons with SPR-like immunoreactivity (-LI) projecting to the nucleus of the solitary tract in the rat. After injection of Fluoro-Gold (FG) into the nucleus of the solitary tract, FG-labeled neurons showing SPR-LI were mainly seen in lamina I of the medullary and spinal dorsal horns, lamina V and the lateral spinal nucleus of the spinal cord. The present results suggest that the trigeminal and spinal neurons with SPR-LI, especially those in lamina I may be involved in the transmission of somatic and/or visceral nociceptive information from the medullary and spinal dorsal horns to the nucleus of the solitary tract.

Direct projections from the periaqueductal gray to pontine micturition center neurons projecting to the lumbosacral cord segments: an electron microscopic study in the rat.

Direct projections from the periaqueductal gray (PAG) to the pontine micturition center neurons directly projecting to the lumbosacral cord segments were observed electron microscopically in the rat by a double labeling method. Biotinylated dextran amine (BDA) was injected into the PAG and horseradish peroxidase (HRP) was injected into the lumbosacral cord segments. After injection of BDA into the ventrolateral part of the PAG, many BDA-labeled axons were seen light microscopically in Barrington's nucleus; a moderate number of them were found in the pontine tegmental region just ventral to Barrington's nucleus (D-region [Ding, Y-Q., Takada, M., Tokuno, H. and Mizuno, N., J. Comp. Neurol., 357 (1996) 318-330]). On the other hand, after injection of BDA into the dorsolateral part of the PAG, only a few BDA-labeled axons were seen in Barrington's nucleus or the D-region. BDA-labeled axon terminals were electron microscopically confirmed to be in synaptic contact with HRP-labeled dendrites and somata in Barrington's nucleus and the D-region. The results indicate that the ventrolateral part of the PAG is implicated in regulation of the micturition reflex.

[Preventive effect of quinacrine on the development of heat injury by stabilizing membrane phospholipids metabolism and beta-adrenoceptor].

Previous studies have demonstrated that the activation of phospholipase A2 (PLA2) during heat stress would caused disorder of membrane phospholipids metabolism, accompanied by a decrease in phosphatidylcholine and phosphatidyl-serine and an increase in arachidonic acid. In heat stressed rats, a down-regulation of beta-adrenergic receptor in lung tissue was observed due to activation of PLA2. In the present work, it was demonstrated that pretreatment of rats with PLA2 inhibitor, quinacrine (20 mg/kg, I.P.), 1 h before heat stress could block these membrane lipid alterations, and the tolerance of rats to heat exposure was enhanced. The effect of quinacrine on the thermotolerance of marching soldiers with 15 kg of load under hot environment was also investigated. The results indicated that in the soldiers taken orally 200 mg quinacrine 1 h before marching, their heart rate, body temperature, accumulation of blood lactic acid and the changes of index of heart function were significantly improved as compared to the control group at the end of 3 h marching. According to these data, it can be concluded that quinacrine is a useful drug to prevent derangement of beta-adrenoceptor and membrane phospholipids metabolism in the development of heat stress. So as to prevent heat injury, and to improve tolerance to heat stress.

[Type and content of adrenoceptors in toad heart].

The type and content of adrenoceptors in heart of normothermic and cold-acclimatized toad were studied by radioligand binding assay at different testing temperatures. The Bmax and Kd values of membrane from normal toad heart binding to 3H-DHA at 37 degrees C were respectively 55.11 +/- 6.22 fmol/mg protein and 2.15 +/- 0.42 nmol/L for the whole heart, 55.80 +/- 7.03 and 2.65 +/- 0.37 for sino-atrium and 54.27 +/- 3.06 and 1.84 +/- 0.14 for ventricle. While the binding to 3H-DHE was very low and nonspecific. When membrane from cold-acclimatized toad at 5-8 degrees C for 10 days was examined for its binding to 3H-DHA or 3H-DHE at 10 degrees C, the values of Bmax and Kd were as same as those of normothermic toad examined at 37 degrees C. The present results suggest that the toad heart is lacking of alpha-adrenoceptor, and the type and content of adrenoceptors are not influenced by temperature.

Changes of pulmonary glucocorticoid receptor and phospholipase A2 in sheep with acute lung injury after high dose endotoxin infusion.

In a sheep model of acute lung injury induced by an Escherichia coli endotoxin (5 micrograms/kg) with chronic lung lymph fistula (n = 15), we measured the changes in glucocorticoid receptor (GCR) binding capacity in lung tissue by means of radioligand binding assay. The content of cortisol and the activity of phospholipase A2 (PLA2) were also measured. The results showed that the maximal binding capacity (Bmax) of GCR in lung cytoplasma decreased continuously 2 h (113 +/- 3 versus 66 +/- 2 fmol/mg protein, p < 0.01), 4 h (105 +/- 6 versus 52 +/- 3 fmol/mg protein, p < 0.01), and 6 h (105 +/- 5 versus 37 +/- 2 fmol/mg protein, p < 0.01) after endotoxin infusion. Its affinity decreased markedly (p < 0.05) at 6 h after the infusion. The contents of cortisol in plasma elevated at 0.5 h and remained at a high level until 4 h after the infusion. PLA2 activity rose from 97 +/- 25 to 188 +/- 12 U (p < 0.05), 99 +/- 13 to 285 +/- 25 U (p < 0.01), and 106 +/- 14 to 354 +/- 32 U (p < 0.01) at 2, 4, and 6 h after endotoxin infusion, respectively. There was a negative correlation between the Bmax of GCR and PLA2 activity (r = -0.87, p < 0.01). The findings indicate that there was a secondary GCR abnormality and a higher PLA2 activity during endotoxin-induced lung injury. The glucocorticoid hypofunction caused by reduced GCR binding capacity may accelerate the pathologic response of acute lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)

[Changes of adrenoceptors and glucocorticoid receptor in the lungs of rats with experimental respiratory distress syndrome].

With the radioligand binding assay, the maximal binding capacity (Bmax) and affinity (Kd) of alpha, beta-adrenoceptors(alpha AR,beta AR) in the lung membrane and glucocorticoid receptor (GCR) in the lung cytoplasma of rats with experimental respiratory distress syndrome (RDS) induced by oleic acid have been measured. The results demonstrated that the content of alpha AR in rat lungs increased continuously during the experiment, the Bmax at 1st, 4th and 6th hour after oleic acid injection were 139 +/- 40, 127 +/- 12, 116 +/- 25 fmol/mg protein, significantly higher than normal value (83 +/- 7, n = 8-10, P < 0.01). Meanwhile, the content of beta AR and GCR decreased continuously, the Bmax at the same time were 364 +/- 18, 307 +/- 55, 240 +/- 66 and 146 +/- 28, 153 +/- 37, 150 +/- 32 fmol/mg protein respectively, significantly lower than their normal value (490 +/- 61, 227 +/- 14 fmol/mg protein, n = 6-10, P < 0.01). The results indicate that the changes of these receptors may be of significance in the pathogenesis of ARDS.

[Relationship between the changes in beta-adrenoceptor content and the metabolism of phospholipids in lung tissue from acute heat stressed rats].

To explore the relationship between the change of beta-adrenoceptor and the metabolism of phospholipids in lung tissue from acute heat stressed rats, the Bmax of beta-adrenoceptors, the activity of phospholipase A2 (PLA2), the content of phosphatidylcholine (PC) and phosphatidylserine (PS), and membrane fluidity in lung tissue of normal and heat stressed rats were investigated. The relevant parameter values mentioned above were 479 +/- 94 fmol/mg protein, 78.5 +/- 8.2 U, 53.5 +/- 1.7 mg/g.wet. w. and 425.1 +/- 68.1 micrograms/g.wet. w. respectively. Whereas in the heat stressed rats with rectal temperature raised to 42 degrees C for 15 min, the Bmax of beta-adrenoceptor was decreased by 43% (P less than 0.01), the activity of PLA2 increased by 83% (P less than 0.01), the contents of PC and PS decreased by 50% and 47% (P less than 0.01) respectively. A lower membrane fluidity in lung tissue for heat stressed rats was also demonstrated. The results suggest that the decreased binding sites of beta-adrenoceptor in lung tissue of rat during hyperthermia may be contributed to the activation of PLA2, which then accelerated the catabolism of phospholipids such as PC and PS in the cell plasma membrane, with a consequent alteration of membrane fluidity.

Distribution and characteristics of placental ANP receptors in normal and hypertensive pregnancy.

The ANP (atrial natriuretic peptide) receptor binding site was studied in human placentas of normal and hypertensive pregnancy. The results showed there were specific high affinity ANP receptors in the nonbrush border (fetal side), and their affinity to ANP was higher than that in the microvillous membrane (meternal side). The ANP receptor affinity in the nonbrush border and microvillous membrane of normal pregnancy was higher than that of hypertensive pregnancy. Though the weight of placentas of hypertensive pregnancy was lower than that of normal pregnancy, high ANP concentrations in the placental tissues, umbilical and maternal blood were found in hypertensive pregnancy. It is believed that the distribution of ANP receptors in the placentas is related to hemodynamics, maternal exchange and fluid and electrolyte balance. The decrease of ANP receptors and lowering of affinity in hypertensive pregnancy may influence the the target cell effect of ANP, especially in the fetal side. This may be related to the pathogenesis of hypertensive pregnancy.

[Study on molecular evolution for both variable segments of immunoglobulin heavy chain and T cell receptor].

In order to explain difference and similarity in producing antibody diversity between immunoglobulin (Ig) and T cell receptor (TCR), authors compared both codon substitution and concerted evolution rate between the variable segment of Ig heavy (Ig VH) and that of TCR (TCR V). The protein sequences of TCR V alpha (including 8 gene segments from mouse and 3 from human), TCR V beta (including 11 from mouse and one from human) and T cell V gamma (including 2 from mouse and 4 from human) were compiled, as well as the protein sequences of Ig VH (3 from human, 11 from mouse, 3 from caiman and one from shark) were collected. It is shown that: (1) the nucleotide substitution of TCR V segment is 2.4 times as large as that of Ig VH in coding region; (2) as for concerted evolution, gene duplicate rates in TCR V and Ig VH are 1.7 X 10(-8) and 1.6 X 10(-8)/gene/year, respectively. The number of TCR V(V alpha equals to 100 and V beta equals to 30) is less than the one of Ig VH (VH equals to 300), for TCR V is subject to negative selection of major histocompatibility complex according to the neutral theory. We discussed that is somatic mutation or DNA rearrangement the main force in producing antibody diversity and are there pseudogenes in TCR V or not.

A human embryonic lung fibroblast with a high density of muscarinic acetylcholine receptors.

Binding studies with the radiolabeled muscarinic antagonists dexetimide, quinuclidinyl benzilate and N-methylscopolamine showed that the human embryonic lung fibroblast CCL137 possesses approximately 2 X 10(5) muscarinic receptors/cell, i.e. 2.1 pmol/mg membrane protein. These receptors showed a marked stereoselectivity towards dexetimide and levetimide and only low affinity for another antagonist, pirenzepine. The muscarinic agonist carbamylcholine inhibited forskolin-stimulated adenylate cyclase and induced phosphatidylinositide turnover in the intact cells. Both effects were inhibited by the muscarinic antagonist atropine. Affinity labeling with tritiated propylbenzylcholine mustard revealed a protein of 72 kDa. Finally, down-regulation of the membrane receptors following prolonged treatment with the agonist carbamylcholine was assessed by means of the hydrophilic antagonist N-methylscopolamine.