Recurrent facial taste neurons of sea catfish Plotosus japonicus: morphology and organization in the ganglion.

This study investigated the morphology of the recurrent facial taste neurons and their organization in the recurrent ganglion of the sea catfish Plotosus japonicus. The recurrent ganglion is independent of the anterior ganglion, which consists of trigeminal, facial and anterior lateral line neurons that send peripheral fibres to the head region. The recurrent taste neurons are round or oval and bipolar, with thick peripheral and thin central fibres, and completely wrapped by membranous layers of satellite cells. Two peripheral nerve branches coursing to the trunk or pectoral fin originate from the recurrent ganglion. The results presented here show that the trunk and pectoral-fin neurons are independently distributed to form various sizes of groups, and the groups are intermingled throughout the ganglion. No distinct topographical relationship of the two nerve branches occurs in the ganglion. Centrally, the trunk and pectoral-fin branches project somatotopically in the anterolateral and intermediate medial regions of the trunk tail lobule of the facial lobe, respectively.

Membrane proteins in four acts: function precedes structure determination.

Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) ß-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to OmpF established the role of OmpF as an import channel for colicin nuclease cytotoxins. (IV) α-Synuclein, associated with the etiology of Parkinson's Disease, is an example of a protein, which is soluble and disordered in solution, but which can assume an ordered predominantly α-helical conformation upon binding to membranes. When subjected in its membrane-bound form to a trans-membrane electrical potential, α-synuclein can form voltage-gated ion channels. Summary of methods to assay functions/activities: (i) sensitive spectrophotometric assay to measure electron transfer activities; (ii) hydrophobic chromatography to deplete lipids, allowing reconstitution with specific lipids for studies on lipid-protein interactions; (iii) microbiological screen to assay high affinity binding of colicin receptor domains to Escherichia coli outer membrane receptors; (iv) electrophysiology/channel analysis (a) to select channel-occluding ligands for co-crystallization with ion channels of OmpF, and (b) to provide a unique description of voltage-gated ion channels of α-synuclein.

Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A.

The high resolution three-dimensional x-ray structure of the metal sites of bovine heart cytochrome c oxidase is reported. Cytochrome c oxidase is the largest membrane protein yet crystallized and analyzed at atomic resolution. Electron density distribution of the oxidized bovine cytochrome c oxidase at 2.8 A resolution indicates a dinuclear copper center with an unexpected structure similar to a [2Fe-2S]-type iron-sulfur center. Previously predicted zinc and magnesium sites have been located, the former bound by a nuclear encoded subunit on the matrix side of the membrane, and the latter situated between heme a3 and CuA, at the interface of subunits I and II. The O2 binding site contains heme a3 iron and copper atoms (CuB) with an interatomic distance of 4.5 A; there is no detectable bridging ligand between iron and copper atoms in spite of a strong antiferromagnetic coupling between them. A hydrogen bond is present between a hydroxyl group of the hydroxyfarnesylethyl side chain of heme a3 and an OH of a tyrosine. The tyrosine phenol plane is immediately adjacent and perpendicular to an imidazole group bonded to CuB, suggesting a possible role in intramolecular electron transfer or conformational control, the latter of which could induce the redox-coupled proton pumping. A phenyl group located halfway between a pyrrole plane of the heme a3 and an imidazole plane liganded to the other heme (heme a) could also influence electron transfer or conformational control.

The whole structure of the 13-subunit oxidized cytochrome c oxidase at 2.8 A.

The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified.

Redox-coupled crystal structural changes in bovine heart cytochrome c oxidase.

Crystal structures of bovine heart cytochrome c oxidase in the fully oxidized, fully reduced, azide-bound, and carbon monoxide-bound states were determined at 2.30, 2.35, 2.9, and 2.8 angstrom resolution, respectively. An aspartate residue apart from the O2 reduction site exchanges its effective accessibility to the matrix aqueous phase for one to the cytosolic phase concomitantly with a significant decrease in the pK of its carboxyl group, on reduction of the metal sites. The movement indicates the aspartate as the proton pumping site. A tyrosine acidified by a covalently linked imidazole nitrogen is a possible proton donor for the O2 reduction by the enzyme.

Structure of the cytochrome b6f complex: quinone analogue inhibitors as ligands of heme cn.

A native structure of the cytochrome b(6)f complex with improved resolution was obtained from crystals of the complex grown in the presence of divalent cadmium. Two Cd(2+) binding sites with different occupancy were determined: (i) a higher affinity site, Cd1, which bridges His143 of cytochrome f and the acidic residue, Glu75, of cyt b(6); in addition, Cd1 is coordinated by 1-2 H(2)O or 1-2 Cl(-); (ii) a second site, Cd2, of lower affinity for which three identified ligands are Asp58 (subunit IV), Glu3 (PetG subunit) and Glu4 (PetM subunit). Binding sites of quinone analogue inhibitors were sought to map the pathway of transfer of the lipophilic quinone across the b(6)f complex and to define the function of the novel heme c(n). Two sites were found for the chromone ring of the tridecyl-stigmatellin (TDS) quinone analogue inhibitor, one near the p-side [2Fe-2S] cluster. A second TDS site was found on the n-side of the complex facing the quinone exchange cavity as an axial ligand of heme c(n). A similar binding site proximal to heme c(n) was found for the n-side inhibitor, NQNO. Binding of these inhibitors required their addition to the complex before lipid used to facilitate crystallization. The similar binding of NQNO and TDS as axial ligands to heme c(n) implies that this heme utilizes plastoquinone as a natural ligand, thus defining an electron transfer complex consisting of hemes b(n), c(n), and PQ, and the pathway of n-side reduction of the PQ pool. The NQNO binding site explains several effects associated with its inhibitory action: the negative shift in heme c(n) midpoint potential, the increased amplitude of light-induced heme b(n) reduction, and an altered EPR spectrum attributed to interaction between hemes c(n) and b(n). A decreased extent of heme c(n) reduction by reduced ferredoxin in the presence of NQNO allows observation of the heme c(n) Soret band in a chemical difference spectrum.

Structure-function of the cytochrome b6f complex.

The structure and function of the cytochrome b6f complex is considered in the context of recent crystal structures of the complex as an eight subunit, 220 kDa symmetric dimeric complex obtained from the thermophilic cyanobacterium, Mastigocladus laminosus, and the green alga, Chlamydomonas reinhardtii. A major problem confronted in crystallization of the cyanobacterial complex, proteolysis of three of the subunits, is discussed along with initial efforts to identify the protease. The evolution of these cytochrome complexes is illustrated by conservation of the hydrophobic heme-binding transmembrane domain of the cyt b polypeptide between b6f and bc1 complexes, and the rubredoxin-like membrane proximal domain of the Rieske [2Fe-2S] protein. Pathways of coupled electron and proton transfer are discussed in the framework of a modified Q cycle, in which the heme c(n), not found in the bc1 complex, but electronically tightly coupled to the heme b(n) of the b6f complex, is included. Crystal structures of the cyanobacterial complex with the quinone analogue inhibitors, NQNO or tridecyl-stigmatellin, show the latter to be ligands of heme c(n), implicating heme c(n) as an n-side plastoquinone reductase. Existing questions include (a) the details of the shuttle of: (i) the [2Fe-2S] protein between the membrane-bound PQH2 electron/H+ donor and the cytochrome f acceptor to complete the p-side electron transfer circuit; (ii) PQ/PQH2 between n- and p-sides of the complex across the intermonomer quinone exchange cavity, through the narrow portal connecting the cavity with the p-side [2Fe-2S] niche; (b) the role of the n-side of the b6f complex and heme c(n) in regulation of the relative rates of noncyclic and cyclic electron transfer. The likely presence of cyclic electron transport in the b6f complex, and of heme c(n) in the firmicute bc complex suggests the concept that hemes b(n)-c(n) define a branch point in bc complexes that can support electron transport pathways that differ in detail from the Q cycle supported by the bc1 complex.

In meso structure of the cobalamin transporter, BtuB, at 1.95 A resolution.

Crystals of the apo form of the vitamin B12 and colicin receptor, BtuB, that diffract to 1.95 A have been grown by the membrane-based in meso technique. The structure of the protein differs in several details from that of its counterpart grown by the more traditional, detergent-based (in surfo) method. Some of these differences include (i) the five N-terminal residues are resolved in meso, (ii) residues 57-62 in the hatch domain and residues 574-581 in loop 21-22 are disordered in meso and are ordered in surfo, (iii) residues 278-287 in loop 7-8 are resolved in meso, (iv) residues 324-331 in loop 9-10, 396-411 in loop 13-14, 442-458 in loop 15-16 and 526-541 in loop 19-20 have large differences in position between the two crystal forms, as have residues 86-96 in the hatch domain, and (v) the conformation of residues 6 and 7 in the Ton box (considered critical to signal transduction and substrate transport) are entirely different in the two structures. Importantly, the in meso orientation of residues 6 and 7 is similar to that of the vitamin B12-charged state. These data suggest that the "substrate-induced" 180 degrees -rotation of residues 6 and 7 reported in the literature may not be a unique signalling event. The extent to which these findings agree with structural, dynamic and functional insights gleaned from site-directed spin labelling and electron paramagnetic resonance measurements is evaluated. Packing in in meso grown crystals is dense and layered, consistent with the current model for crystallogenesis of membrane proteins in lipidic mesophases. Layered packing has been used to locate the transmembrane hydrophobic surface of the protein. Generally, this is consistent with tryptophan, tyrosine, lipid and CalphaB-factor distributions in the protein, and with predictions based on transfer free energy calculations.

Review of global regulations concerning biowaivers for immediate release solid oral dosage forms.

The regulations with respect to biowaivers for immediate release (IR) solid oral dosage forms in the USA, the EU, Japan and from the World Health Organization (WHO) are summarized and compared. Two case studies are presented, one from our own files and one from the open literature, showing the similarities and the differences among the qualification requirements of the four systems. The regulatory experience gained up to now is reviewed and expected future trends are discussed.

Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases.

BACKGROUND: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics. To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp. strain KNK712. RESULTS: The crystal structure of DCase has been determined to 1.7 A resolution. The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold. The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases. CONCLUSIONS: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171. Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46. DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes.

Efficient radical trapping at the surface and inside the phospholipid membrane is responsible for highly potent antiperoxidative activity of the carotenoid astaxanthin.

The effects of the carotenoids beta-carotene and astaxanthin on the peroxidation of liposomes induced by ADP and Fe(2+) were examined. Both compounds inhibited production of lipid peroxides, astaxanthin being about 2-fold more effective than beta-carotene. The difference in the modes of destruction of the conjugated polyene chain between beta-carotene and astaxanthin suggested that the conjugated polyene moiety and terminal ring moieties of the more potent astaxanthin trapped radicals in the membrane and both at the membrane surface and in the membrane, respectively, whereas only the conjugated polyene chain of beta-carotene was responsible for radical trapping near the membrane surface and in the interior of the membrane. The efficient antioxidant activity of astaxanthin is suggested to be due to the unique structure of the terminal ring moiety.

Effects of ethyleneglycol chain length of dodecyl polyethyleneglycol monoether on the crystallization of bovine heart cytochrome c oxidase.

Tetragonal crystals that diffracted X-rays up to 5 A resolution were obtained from bovine heart cytochrome c oxidase isolated and solubilized with dodecyl octaethyleneglycol monoether, CH3(CH2)11O(CH2CH2O)8H. Comparison of observed structure factors between data sets each obtained from a different native crystal gave correlation coefficients of 0.92, 0.84 and 0.57 at 10 A, 7 A and 6 A resolution, respectively. The space group and the cell dimensions of the crystal are I4(1) or I4(3) and a = b = 253 A, c = 507 A, respectively. The perfection and stability of the tetragonal crystals are significantly higher than those of the hexagonal crystals of the protein stabilized with Brij-35, CH3(CH2)11O(CH2CH2O)23H (whose details are reported elsewhere). Examination of the effect of ethyleneglycol chain length on the crystallization revealed that only dodecyl polyethyleneglycol monoethers with eight and seven units were appropriate for producing this type of crystal, indicating an optimum size of the detergent for crystallization of the membrane protein.

The 1.55 A resolution structure of Nicotiana alata S(F11)-RNase associated with gametophytic self-incompatibility.

The crystal structure of Nicotiana alata (ornamental tobacco) S(F11)-RNase, an S-allelic glycoprotein associated with gametophytic self-incompatibility, was determined by X-ray diffraction at 1.55 A resolution. The protein has a tertiary structure typical of members of the RNase T(2) family as it consists of a variant of the (alpha+beta) fold and has eight helices and seven strands. A heptasaccharide moiety is also present, and amino acid residues that serve as the catalytic acid and base can be assigned to His32 and His91, respectively. Two "hypervariable" regions, known as HVa and HVb, are the proposed sites of S-allele discrimination during the self-incompatibility reaction, and in the S(F11)-RNase these are well separated from the active site. HVa and HVb are composed of a long, positively charged loop followed by a part of an alpha-helix and short, negatively charged alpha-helix, respectively. The S(F11)-RNase structure shows both regions are readily accessible to the solvent and hence could participate in the process of self/non-self discrimination between the S-RNase and an unknown pollen S-gene product(s) upon pollination.

Suppression of the luteinizing hormone releasing effect of luteinizing hormone releasing hormone by arginine-vasotocin.

The concentrations of 17-oxosteroids in the spermatic venous blood of anaesthetized dogs were used as an index of LH release to assess the effects of arginine-vasotocin on the response of the canine pituitary gland to exogenous luteinizing hormone releasing hormone (LH-RH). When injected into the carotid artery, arginine-vasotocin (1.0 microgram/kg body wt) caused no significant alterations in the testicular output of 17-oxosteroids. The administration of LH-RH (5 microgram/kg body wt, a standard dose) into the carotid artery produced typical stimulation of testicular 17-oxosteroid secretion. Administration of arginine-vasotocin (0.01, 0.1 or 1.0 microgram/kg body wt) into the carotid artery 3 h before the administration of a standard dose of LH-RH inhibited the testicular secretion of 17-oxosteroids normally induced by LH-RH. However, pretreatment with arginine-vasotocin (1.0 microgram/kg body wt) did not affect the testicular response to i.v. administration of human chorionic gonadotrophin (5 i.u./kg body wt). These results indicate that in the dog, arginine-vasotocin inhibits the LH-RH-induced release of LH by acting acting directly on the anterior pituitary gland.

Suppression of the luteinizing hormone releasing effect of luteinizing hormone releasing hormone by arginine-vasotocin in immature male dogs.

Arginine-vasotocin (0.1 or 10 ng/kg body wt) was administered into the carotid artery of anaesthetized immature male dogs 3 h before the administration of a standard dose of luteinizing hormone releasing hormone (LH-RH, 5 microgram/kg body wt) into the same vessel. The rate of secretion of 17-oxosteroids by the testes in vivo served as an index of luteinizing hormone (LH) secretion. The administration of LH-RH into the carotid artery of control dogs which had been injected with isotonic saline caused a slight but definite increase in the secretion of testicular 17-oxosteroids. This effect of LH-RH on the testicular secretion of steroids was markedly reduced by pretreatment with arginine-vasotocin. However, the testicular response to the i.v. administration of human chorionic gonadotrophin (5 i.u./kg body wt) was unaffected by pretreatment with arginine-vasotocin (10 ng/kg body wt). These results indicate that in immature male dogs, arginine-vasotocin is able to inhibit the action of LH-RH by acting directly on the anterior pituitary gland.

Inhibition by melatonin of the pituitary response to luteinizing hormone releasing hormone in vivo.

The rate of secretion of 17-oxosteroids by the testes of anaesthetized dogs in vivo was used as an index of LH secretion. Intracarotid injection of luteinizing hormone releasing hormone (LH-RH, 1, 5 or 10 microgram/kg body wt) resulted in an increase in the testicular 17-oxosteroid secretion which was roughly proportional to the dose administered and which reached a maximum 60 min after the injection. Testicular output of 17-oxosteroids was unaffected by administration of melatonin (10 or 100 microgram/kg body wt) into the carotid artery. When LH-RH (5 microgram/kg) was injected into the carotid artery 3 h after intracarotid injection of melatonin (10 or 100 microgram/kg), the testicular response to LH-RH was considerably diminished. Pretreatment with melatonin (100 microgram/kg) did not alter the testicular response to human chorionic gonadotrophin (20 i.u./kg body wt) given i.v. Is is concluded that melatonin may act directly on the anterior pituitary gland in dogs to inhibit the LH-RH-induced release of LH.

Action of prostaglandin E2 on the release of catecholamines from the canine adrenal gland and its interaction with acetylcholine.

The effect of prostaglandin E2 (PGE2) on the secretion of adrenaline and noradrenaline by the adrenal gland and the interaction between PGE2 and acetylcholine in the adrenal medulla were examined in anaesthetized dogs. In splanchnicotomized dogs, i.v. injection of PGE2 failed to induce any secretion of catecholamines from the adrenal gland, whereas administration of PGE2 into the lumboadrenal artery resulted in a slight, approximately dose-dependent increase in catecholamine secretion within 2 min of the injection. This effect of PGE2 was unaffected by i.v. administration of atropine. Intravenous administration of acetylcholine 1 min after the administration of PGE2 into the lumboadrenal artery of splanchnicotomized atropine-treated dogs had a markedly greater effect on adrenal catecholamine secretion; the resultant output was about twice that evoked by acetylcholine in the absence of PGE2. The effect was more than additive, since the response to acetylcholine was at least one order of magnitude greater than that to PGE2. This indicates that PGE2 and acetycholine may act synergistically in the adrenal medulla.

Reverse redistribution of Tc-99m-tetrofosmin in patients with acute myocardial infarction.

We examined reverse redistribution (RR) of Tc-99m-tetrofosmin after a single injection in patients with acute myocardial infarction (AMI). Tc-99m-tetrofosmin myocardial SPECT was performed in 28 patients with AMI 10-14 days after the onset. Myocardial images were obtained 30 min and 180 min after the injection of 740 MBq of Tc-99m-tetrofosmin. The left ventricular wall was divided into 9 segments. Regional myocardial uptakes of Tc-99m-tetrofosmin were scored by 4-point scoring (0 = normal, 1 = mildly reduced, 2 = moderately reduced, and 3 = defect). RR was defined as an increase of more than 1 in the regional score in images at 180 min. RR of Tc-99m-tetrofosmin was observed in 17 of 20 patients with direct PTCA and 3 of 8 patients without reperfused therapy. RR was observed in 61 of all 252 segments. Coronary angiography performed 1 month later revealed that the infarct-related artery was patent in 19 of 20 patients (95%) with RR and in 3 of 8 patients (37.5%) with persistent defects (PD) (p < 0.05). In segment-by-segment analysis, the incidence of regional wall motion abnormality I month later was reduced in regions with RR compared to those with PD (p < 0.0001). In conclusion, RR of Tc-99m-tetrofosmin was frequently observed in patients with successful direct PTCA. As the segments with RR showed signs of preserved function 1 month later, this phenomenon may reflect a salvaged myocardium in AMI.