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Analogs of opioid pentapeptide [D-Ala2,Leu5]enkephalin were prepared using two kinds of N-methylation reactions, namely quaternization and amide-methylation. Quaternization reaction with CH3I-KHCO3 in methanol was applied to the deprotected N-terminal group of the pentapeptide derivatives affording trimethylammonium group-containing analogs. [Me3+Tyr1,D-Ala2,Leu5]enkephalin and its amide were found to show opioid activity on guinea pig ileium assay only slightly lower than the parent unmethylated peptides. Application of amide-methylation reaction using CH3I-Ag2O in DMF to the protected pentapeptide yielded a pentamethyl derivative in which all of the five N atoms were methylated. Deprotection of the derivative gave pentamethyl analogs of [D-Ala2,Leu5]enkephalin, which showed no significant activity on the guinea pig ileum assay and opiate-receptor binding assay.

Brain injury-derived neurotrophic peptide (BINP) is a synthetic 13-mer peptide that supports neuronal survival and protects hippocampal neurons in primary cultures from cell death caused by glutamate. We have developed a monoclonal antibody named mAb 6A22 against the 40-kDa BINP-binding protein, p40BBP. mAb 6A22 inhibits binding between BINP and rat brain synaptosomes and abolishes the protective effect of BINP. The antigen of mAb 6A22 should be the BINP-binding protein that mediates the neuroprotective action of BINP. Using an expression cloning approach with mAb 6A22, we isolated a cDNA encoding a novel receptor protein that shows binding activity of BINP. COS7 cells transfected with the cloned cDNA show binding of BINP and cell surfaces that are stained by 6A22. The mRNA for p40BBP is specific for the rat brain and is increased after birth. From immunohistochemical studies using mAb 6A22, p40BBP increased after kainic acid treatment in rat hippocampal neurons.

Brain injury-derived neurotrophic peptide is a 13-amino acid peptide derived from a 15,000 mol. wt neurotrophic factor released from sites of mechanical injury in neonatal rat brain. This peptide promotes survival of septal cholinergic neurons and mesencephalic dopaminergic neurons, and protects hippocampal neurons from glutamate-induced neurotoxicity. In this study, we have developed a monoclonal antibody against a brain injury-derived neurotrophic peptide-binding protein by immunizing mice with septal synaptosomes from five-week-old rat brain. Monoclonal antibodies were screened for inhibition of the binding of a 125 I-labeled analogue of brain injury-derived neurotrophic peptide to rat brain synaptosomes. The monoclonal antibody 6A22 suppressed the biological activity of brain injury-derived neurotrophic peptide and abolished the protective effect of the neurotrophic peptide against glutamate-induced neurotoxicity. This monoclonal antibody recognized a 40,000 mol. wt brain injury-derived neurotrophic peptide-binding protein, which was also identified by cross-linking experiments. Immunohistochemical studies showed that the 6A22 antibody bound to the cell surfaces of a subpopulation (about 60%) of hippocampal neurons in culture. These results are consistent with the possibility that the 40,000 mol. wt protein belongs to brain injury-derived neurotrophic peptide receptors.

The paper investigates agglomerate formation in the world biggest pressurized fluidized bed combustor at Karita Power Station, Japan. It was found that significant amount of agglomerates was formed at an increased boiler load bringing about a significant decrease of bed density. The agglomerates had a glassy-like molten surface and consisted of a mixture of ash from char fines and limestone particles. The main components found in the agglomerate cross-section were Ca, Al and Si oxides, all quite uniformly distributed. Agglomerate formation occurred particularly when the unit was fired with a porous Blair Athol coal that was found to produce a porous char. Firing the boiler with other coal (Nanton) that produced less porous char prevented the formation of agglomerates and enabled to maintain stable operation of the combustor. A model was developed to calculate the horizontal distribution of char surface temperature in the PFBC based on a quasi-steady heat balance for a burning char by taking into consideration the distribution of volatiles above fuel feeding nozzles, as well as char porosity. In order to take into consideration the effect of porosity on combustion of a porous char a completely new expression to estimate the reaction rate was proposed. The calculation results indicated that the agglomerates were mainly formed due to the combustion of highly porous Blair Athol chars in the poorly fluidized areas in the bed, where air-to-fuel ratio became larger. The combustion rate of less porous Nanton char was much slower then that of the Blair Athol. Accordingly, combustion temperature of its char was lower bringing about no formation of agglomerates.Copyright © 2003 by ASME

Signal transduction by second messengers has long been hypothesized to be involved in the circadian system in the suprachiasmatic nucleus (SCN) of the mammalian brain. The present study reports that the concentration of adenosine-3',5'-monophosphate (cyclic AMP or cAMP) in the SCN region exhibited a circadian fluctuation with two peaks during a day under constant conditions. While a sharp peak at around the end of the subjective night was observed only in the SCN region, the other peak in the late subjective day was also found in the anterior hypothalamic area. The distinct cAMP peak at the late subjective night contrasts with the daytime peaks in electrical and metabolic activity rhythms of the SCN.

Brain injury-derived neurotrophic peptide is the fragmental 13-mer peptide of the novel neurotrophic factor which was extracted and purified from Sponge Gelform made of gelatin implanted at the mechanically-induced injury site in neonatal rat brains. Brain injury-derived neurotrophic peptide supports survival of septal cholinergic and mesencephalic dopaminergic neurons in culture, and rescues hippocampal neurons in culture from glutamate neurotoxicity. Here we studied the binding characteristics of brain injury-derived neurotrophic peptide to synaptosomes from normal adult rat brains and neurons in culture from neonatal rat brains. [ 125 I]Asp-[Tyr 11 ]-brain injury-derived neurotrophic peptide binding to rat brain synaptosomes was specific and saturable. Equilibrium binding studies revealed that [ 125 I]Asp-[Tyr 11 ]-brain injury-derived neurotrophic peptide bound to 1.1 pmol/mg protein with a K d (dissociation constant) of 0.17 μM in hippocampal synaptosomes and to 2.0 pmol/mg protein with a K d of 0.38 μM in septal synaptosomes. [ 125 I]Asp-[Tyr 11 ]-brain injury-derived neurotrophic peptide could bind to a subpopulation of hippocampal neurons in culture from embryonic rat brains. Affinity cross-linking with the car☐yl-reactive cross-linking reagent I-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-HCl and [ 125 I]Asp-[Tyr 11 ]-brain injury-derived neurotrophic peptide produced radiolabeled bands corresponding to 100,000, 50,000 and 40,000 mol. wt molecules on hippocampal neurons in culture. These results suggest that the 13-mer sequence of brain injury-derived neurotrophic peptide plays a crucial role in expressing the neurotrophic properties of the factor.

Neuronal survival is mediated by several kinds of proteins. Among these, neurotrophic factors play important roles in the nervous system by supporting neuronal activity and survival. It has been suggested recently that certain factors promote neuronal survival in the case of brain injury. To examine this possibility, we purified a novel neurotrophic factor from Gelfoam that was implanted at the site of injury caused in neonatal rats. During amino acid sequence analysis, we found that a fragmental peptide of this neurotrophic protein consisting of 13 amino acids showed neurotrophic activity. This 13-mer peptide promoted survival of septal cholinergic and mesencephalic dopaminergic neurons in culture and rescued hippocampal neurons from injury caused by glutamate in culture. This peptide rescued neurons from cell death caused by glutamate, even when added 4.5 h after glutamate exposure.

We identified the possible endogenous factor effective to modulate the binding of [ 3 H]-labeled excitatory amino acid agonists and antagonists in the 100,000 × g supernatant of Triton X-100 (0.01%)-treated cell membranes from frog spinal cords. The factor inhibited the binding of [ 3 H]glutamate to Triton X-100-treated cell membranes, to which the binding capacity of [ 3 H]glutamate increased much more than that to intact cell membranes. The binding capacities of [ 3 H]AMPA (an AMPA type agonist) and [ 3 H]CPP (an NMDA type antagonist) to cell membranes remained low by Triton treatment, but they were enhanced significantly by the addition of the factor. The effect of the factor on the [ 3 H]kainate binding was hardly observable. The factor may provide key information on receptor structures and the classification of receptor types concerning excitatory amino acids in the mammalian central nervous system.

The effect of the thermal fluctuation on the orientation distribution pattern of globular protein molecules in a two-dimensional lattice was investigated by the method of computer simulation. A set of interaction parameters was assigned to interaction sites on each molecule and the interaction energy between two molecules was given by the product of the parameters of facing sites. Orientation fluctuation was assumed to take place with the probability proportional to the Boltzmann factor. Patterns having different degrees of order appeared with the change of temperature. The entropy and other thermodynamic quantities of these patterns were calculated, and gradual and transitional changes of the pattern were discussed in comparison with the case of simple atoms or molecules.

Triton treatment of synaptic membranes from the frog spinal cord enhanced the specific binding of [3H]glutamic acid compared with non-treated fresh and frozen ones, but not that of [3H]kainic acid. Acromelic acid A specifically inhibited the binding of [3H]kainic acid, and was approximately 100 times more potent than kainic acid. Acromelic acid A and excitatory amino acids caused a depolarization in the ventral root of the frog spinal cord in a dose-dependent manner, and the effect of acromelic acid A was much superior to that of kainic acid or domoic acid. Acromelic acid A is one of the most potent kainic acid agonist at the frog spinal cord.

Movement is one of the fundamental properties of living cells. It is expressed in such diverse cellular activities in animal and plant cells as cytoplasmic streaming, amoeboid movement, phagocytosis, morphogenetic movement, cytokinesis, mitosis, secretory processes and probably the regulation of the topological distribution of membrane proteins as well.Recently proteins which closely resemble the major contractile proteins of muscle, actin and myosin, have found in most types of eukaryotic cells and even in prokaryotic cells. The universal occurence of these proteins suggests that they provide the basis of a general mechanism for producing cellular movement, of which muscle contraction is a specialized example.In general the nonmuscle actins (cytoplasmic actins) resemble the muscle actin, and this similarity was mainly emphasized in early researches. However, more extensive biochemical studies have increasingly revealed some differences, especially in microheterogenity among actins. Another significant focus of recent researches concerns the ability of nonmuscle actins to form supramolecular assemblies or higher aggregates corresponding to the microfilament bundles or stress fibers seen in microscopy. These studies suggest that actin may play a cytoskeletal as well as a contractile role in nonmuscle cells.In this review we have focused our attension to emphasize recent advances in the biochemical understanding of the nonmuscle actin. We discuss the properties of the purified actin and the morphological distribution of actin filaments and its properties. Most of the papers discussed here were published after 1974.

We considered the possible relationship between the serum levels of progesterone and estradiol-17 beta, which were controlled to that of several stages in normal pregnant rats, and the Ca2+ sensitivity, defined as the pCa required for half maximum activation of force production, of chemically skinned uterine muscle fibers from ovariectomized rats. The Ca2+ sensitivity was negligibly influenced by the levels of these hormones.

1. 1. The complex flagella of Rhizobium lupini H13-3 differ from plain bacterial flagella in the fine structure of their filaments dominated by conspicuous helical bands, in their fragility and their resistance against heat decomposition. To elucidate the basis of these differences, the composition of complex filaments and their subunits was analysed. 2. 2. Isolated complex flagella containing the filament and hook portions were purified by differential centrifugation. Hooks were separated by ultracentrifugation after acid degradation of filaments at pH 2. The complex filaments consist of 43 000 dalton monomers (cx-flagellin), the hooks are composed of 41 000 dalton subunits. 3. 3. Amino acid analysis of cx-flagellin indicated the presence of approx. 417 amino acid residues. These comprise 47% hydrophobic residues and 21% Asp and Glu (or amides), but no Cys, His, Pro and Trp. No carbohydrate, phosphate or lipid moieties have been detected. Fingerprint analysis after tryptic digestion yields approx. 36 peptides, about half of them clustered in the neutral region. A comparison with the composition of various known flagellins from plain flagella indicates a 7% higher content of hydrophobic amino acid residues in complex filaments; this is largely compensated for by the higher content of Glu and Asp (presumably as Gln and Asn) in plain filaments. 4. 4. Immunodiffusion and immunoelectrophoresis of cx-flagellin yield single precipitin bands indicating homogeneity. In contrast, isoelectric focusing lead to three close-running bands around pH 4.7. When isolated, the two major bands again produced an “isoelectric spectrum” suggesting that it reflects an allomorphism of cx-flagellin. 5. 5. Self-assembly experiments with cx-flagellin lead to coiled fibres including helical regions, but not to intact filaments. The products resemble heat-denatured complex filaments and may represent intermediates between monomers and complete polymers.

Effects of TI233, a calmodulin antagonist, on transmitter release were studied using a clonal pheochromocytoma cell line (PC12h). TI233, at a concentration of 30 microM, completely suppressed the release of preloaded [3H]NE and [3H]DA. The 50% suppression dose was around 3 microM. TI233 did not inhibit the [3H]NE release evoked by the calcium ionophore A23187. Electrophysiological examinations using a clonal neuroblastoma x glioma hybrid cell line (NG108-15) revealed that TI233 blocked the voltage-sensitive calcium channel of the membrane in the same concentration range. Thus it was suggested that TI233 inhibited transmitter release from neuronal cells by blocking the entry of calcium to the cytoplasm.

This paper describes the binding properties of [3H]peptidergic opioid ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [3H]-[D-Ala2-, Met5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl2 and 100 mM NaCl at 0 degree C (on ice) for 3 h. With this assay condition, the significant binding of following [3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [3H]-[D-Ala2, MePhe4, Gly-ol5]enkephalin (mu-type), [3H]-[D-Ala2, D-Leu5]enkephalin (delta-type) and [3H]dynorphin1-9 (kappa-type). The number of binding sites in solubilized preparations for each [3H]ligand corresponded to 40-50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [3H]ligand. Apparent dissociation constants between solubilized receptors and [3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycodeoxycholate and NaCl and the dilution process.

The effects of putative calmodul n antagonists on the binding of [3H]- opioid ligands to rat brain membranes were examined. Chlorpromazine, trifluoperazine, N-(6-aminohexyl)-5-chloro-1 -naphthalenesulfonamide (W-7) and N2-dansyl-L-arginine-4-t-butylpiperidine amide (Tl 233, No. 233) inhibited the binding of [3H] [D-Ala2, Met5]enkephalinamide ([3H]DALAMID) in a dose- dependent manner, and this inhibition was not necessarily specific for Ca2+. The order of the inhibitory potency on the binding of [3H] DALAMID was chlorpromazine, W-7, TI 233 and trifluoperazine. From the Scatchard plots, the effects of these compounds on the binding of [3FI]DALAMID were biphasic. Low concentrations of calmodulin antagonists caused a small but significant increase in the maximal binding (Bmax), but upon a further increase of the concentration, a significant decrease in Bmax was observable; the apparent dissociation constant for the ligand- receptor complexes increased with the increase in the concentration of calmodulin antagonists. TI 233 (1–30×10−6 M) inhibited, somewhat in a ligand selective manner, the binding of [3H]opioid ligands that have been thought to be selective for opioid receptor types. Calmodulin antagonists caused to increase in the fluorescence anisotropy obtained from 1,6-diphenyl-1,3,5-hexatriene-labeled membrane fractions from rat brains, indicating that these compounds were effective for the decrease in the membrane fluidity. The present results indicate that putative calmodulin antagonists can affect the binding of the opioid ligands to rat brain membranes, probably through a mechanism other than one involving calmodulin- related processes.

The effects of Ca2+ and calmodulin on contraction of saponin-treated (chemically skinned) uterine smooth muscle fibers of pregnant rats were examined. Ca2+ sensitivity, defined as the pCa required for half maximum activation of force production, was found to change with the progress of pregnancy; low in the early and middle stages and high in the later stages of pregnancy. The overall change of Ca2+ sensitivity was about pCa 1.5 during the period of pregnancy. The effect of calmodulin on contraction was also found to be dependent on the stages of pregnancy. Calmodulin was effective on the augmentation of the tension rather than the change in Ca2+ sensitivity, and this augmentation was large in the early and middle stages of pregnancy. The amount of calmodulin, which eluted out of uterine muscle cells during saponin treatment, was large in the early and middle stages of pregnancy. The results indicate that the contractile response of the uterine muscle cells during the period of pregnancy seems to be controlled by both the changes in Ca2+ sensitivity and in the amount of free calmodulin in uterine muscle cells.

A flow birefringent study suggests that the mean length of bacterial flagella of Salmonella (SJ-25) reconstituted in vitro does not exceed some critical length, say about 6 μm. Since monomers remaining in solution can polymerize upon addition of new seeds, the self-limiting mechanism of polymerization seems to be intrinsic.

In order to see the relationship between the monomer structure and the polymer structure of protein, stable patterns of the orientation distribution of globular molecules in a two-dimensional lattice were investigated by a computer. A set of interaction parameters was given to interaction sites distributed on the surface of the molecule and the energy was assumed to be given by the sum of products of parameters belonging to the facing sites of neighbouring molecules. Patterns having different degrees of order were obtained under various sets of interaction parameters. The response of the pattern to the environmental condition was also simulated and the gradual and transitional changes of the pattern were found.

Bacterial flagella are organelles of locomotion. Since their thickness is less than 200 A, it is impossible to observe individual intact flagella by light microscopy. In order to estimate the flexural rigidity of flagella in vitro, reconstituted flagella of Salmonella were studied by quasielastic scattering of laser light. The main findings were as follows. The bacterial flagellum is very stiff. Its flexural rigidity is of the order of 10−15 dyncm2 for straight-type flagella. Among mutant strains, the normal-type flagellum seems to be the most stiff. Curly- and straight-type flagella follow in that order. The mean length of reconstituted flagella is nearly equal irrespective of widely varied weight ratios of flagellin to seed at the reconstitution, provided that the ratio exceeds some critical value. This suggests that there exists some self-control mechanism in the polymerization process of flagellin.

Quaternization of primary amino groups of derivatives of [D-Ala2, Leu5]enkephalin and gramicidin S with Mel–KHCO3 in MeOH afforded new biologically active analogues possessing trimethylammonium group(s).

Effects of taurine(TAU) on the frog spinal cord was examined and compared with those of GABA. TAU(0.001 - 1 mM) caused concentration-dependent inhibitory actions on the ventral root potentials induced by stimulation of the dorsal root (DR-VRP). GABA also inhibited the potentials, but it needed about a hundred times higher concentrations than TAU did. Membrane conductance of the motoneuron was increased by both TAU and GABA to the same degree, and the hyperpolarization of motoneuron induced by both amino acids was accompanied by the same level of increase in the extracellular K-activity. Picrotoxinin and anisatin (10 μM) reduced GABA-induced depolarizations in the primary afferent terminal, while these agents also caused significant inhibition of TAU-induced depolarization. Strychnine (10 μM) and dendrobine (30 μM) showed no GABA-antagonizing action, but markedly antagonized the action of TAU. The presynaptic inhibition on the first spike potential in the DR-VRR was markedly blocked by these antagonists. The blocking actions of latter two agents were stronger than those of former two. Strychnine and dendrobine abolished the early components of the DR-DRP, while rather augmented the later components. On the other hand picrotoxinin and anisatin reduced the size of DR-DRP. These results suggest that the postsynaptic actions of GABA and TAU are almost equipotent, but the presynaptic action of TAU is more effective than that of GABA. The effects of antagonists indicate that early component of the DR-DRP may be mediated by TAU or its related compound, and the later component may be caused by GABAergic synapses. Thus the major neurotransmitter for the presynaptic inhibition in the frog spinal cord may not be GABA.