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This investigation ascertains whether, in (smooth muscle) myosin, certain residues engage in functional interactions with their actin conjugates in an actomyosin complex. Such interactions have been postulated from putting together crystallographic models of the two proteins [Rayment, I., Rypniewski, W. R., Schmidt-Bäse, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G., and Holden, H. M. (1993) Science 261, 50-58]. Here, in several instances, we ask whether mutation of a particular residue significantly impairs a function, and find that the answers are largely rationalized by the original postulation. Additionally, a novel element emerges from our investigation. To assess function, we test the wild type and mutant systems as they perform in the steady state of ATP degradation. In doing so, we assume, as usual, that degradation proceeds from an early stage in which the complex forms (and is described by parameter K(app)) to a later stage during which the product leaves the complex (and is described by parameter V(max)). Interestingly, certain defects induced by the mutations are associated with changes in K(app), and other defects are associated with changes in V(max), suggesting that our procedure at least roughly distinguishes between events according to the time in the degradation at which they occur. In this framework, we suggest that (1) in the actin-myosin association phase, cationic residues Lys-576 and Lys-578 interact with anionic residues of the so-called second actin, and (2) in the product leaving phase, hydrophobic residues Trp-546, Phe-547, and Pro-548, as well as the Thr-532/Asn-533/Pro-534/Pro-535 sequence, sever connections with the so-called first actin. The role of Glu-473 is also examined.

To investigate immunohistochemically the spatial localization of putative odour receptor proteins, we synthesized a polypeptide which corresponds to a region of a putative odour receptor protein, I3, and raised an antibody to the peptide. In rat olfactory epithelium, the antibody specifically recognized cilia of a small subset of olfactory receptor neurons, suggesting that the odour receptor protein is localized selectively in the cilia. Olfactory receptor neurons having immunoreactive cilia were distributed sparsely throughout the epithelium. This suggests that receptor neurons expressing a similar odour receptor protein are probably distributed similarly in the epithelium.

Publisher Summary Isolated stress fibers are useful for studying their properties and functions, but they are also useful as a nonmuscle contraction model system for investigating the regulatory mechanism for the actomyosin-based contractility in nonmuscle cells. This chapter discusses the isolation of stress fibers en masse from cultured cells. It also describes the way to observe contraction of stress fibers. The isolation procedure is simple and provides stress fibers that are pure enough for biochemical as well as structural and other studies. The contraction of stress fibers is best observed by using those still attached to the substrate surface. The contraction is an actomyosin-based, ATP-driven contraction. Rho kinase is involved in stress fiber contraction. Both RhoA and Rho kinase are present in isolated stress fibers. The chapter explains that the stress fiber model can contract in the absence of Ca 2+ and that this contraction is inhibited by a Rho kinase inhibitor. In this case, the myosin regulatory light chain is phosphorylated, not by myosin light chain kinase, but by Rho kinase. The studies presented in the chapter indicate that stress fiber contraction is regulated by two independent systems: one by the Ca 2+ - dependent myosin light chain kinase system and the other by the Ca 2+ - independent Rho kinase system.

The involvement of tetrodotoxin-sensitive Na+ channels and receptor-operated nonspecific Ca2+ channels, and the effects of short-chain fatty acids, on growth hormone (GH) release induced by GH-releasing hormone (GHRH) were investigated in cultured and freshly isolated caprine anterior pituitary cells. In 3-d cultured cells in Dulbecco's modified Eagle's medium, an increase in GH release induced by GHRH (10 nmol/l) was moderately, but significantly, reduced by a voltage-sensitive Na+ channel antagonist tetrodotoxin (1 micromol). The GHRH-induced GH increase, which was not affected by a simultaneous addition of a receptor-operated nonspecific Ca2+ channel antagonist tetramethrine (0.1 mmol/l), was significantly reduced by a voltage-sensitive L-type Ca2+ channel antagonist nifedipine (1 micromol/l). Propionate and butyrate at 10 mmol/l, however, not only suppressed basal GH release but also significantly reduced the GH increase induced by 10 nmol/l of GHRH. The inhibitory action of these acids was also reproduced by an addition of beta-hydroxy butyrate (10 mmol/l) and octanoate (10 mmol/l). In freshly isolated and perifused cells, butyrate (10 mmol/l) as well as somatostatin (100 nmol/l) significantly reduced the GH increase induced by GHRH. From these findings we conclude that tetrodotoxin-sensitive Na+ channels and voltage-dependent L-type Ca2+ channels are involved in the cellular mechanism for GHRH-induced GH release, and that short-chain fatty acids such as propionate and butyrate have a direct action on somatotrophs to reduce basal and GHRH-induced GH release, in caprine somatotrophs.

For analyzing the mechanism of energy transduction in the “motor” protein, myosin, it is opportune both to model the structural change in the hydrolytic transition, ATP (myosin-bound) + H 2 O → ADP⋅P i (myosin-bound) and to check the plausibility of the model by appropriate site-directed mutations in the functional system. Here, we made a series of mutations to investigate the role of the salt-bridge between Glu-470 and Arg-247 (of chicken smooth muscle myosin) that has been inferred from crystallography to be a central feature of the transition [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M., & Rayment, I. (1995) Biochemistry 34, 8960–8972]. Our results suggest that whether in the normal, or in the inverted, direction an intact salt-bridge is necessary for ATP hydrolysis, but when the salt-bridge is in the inverted direction it does not support actin activation. Normally, fluorescence changes result from adding nucleotides to myosin; these signals are reported by Trp-512 (of chicken smooth muscle myosin). Our results also suggest that structural impairments in the 470–247 region interfere with the transmission of these signals to the responsive Trp.

From their crystallographic comparisons, Fisher et al. (Biochemistry 34, 8960-8972, 1995) have proposed that in an important transition of myosin heads (M), MATP → M ADP Pi, an interdomain rotation occurs in Gly468 (of chicken smooth muscle myosin) and that the rotated state is stabilized by newly-formed interdomain contacts including the salt link between Glu470 and Arg247 (of chicken smooth muscle myosin). Here, we have studied the effects of Gly468, Glu470, and Arg247 mutations on the hydrolysis of ATP. The G468A HMM did not show a significant ATPase activity, a stoichiometric initial phosphate burst, and tryptophan fluorescence enhancement attributed to bound ADP Pi. The E470A HMM also did not show a significant ATPase activity and the phosphate burst, but the mutant gave tryptophan response attributed to bound ATP. The E470R/R247E HMM exhibited an ATPase activity and the phosphate burst which were comparable to those of the wild-type HMM, whereas neither the E470R HMM nor the R247E HMM showed such a significant ATPase activity and burst. We thus propose that both an unhindered rotation and a salt link that stabilizes the rotated state are necessary for ATP hydrolysis.

It has been proposed from crystallographic comparisons [Fisher, A. J., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M.,Rayment, I. (1995) Biochemistry 34, 8960-8972] that in one of the important transitions of myosin head (M), M x ATP --M x ADP x Pi, a rotation occurs in Gly468 (of chicken smooth muscle myosin). We find that mutation of this Gly to Ala does block the transition. Searching for proton acceptors in ATPase catalysis, we also find that mutation of a candidate (Glu470 of chicken smooth muscle myosin) blocks the transition. Interpretations of both findings are examined.

Stress fibers (SFs) are present along the apical (apical SF) and the basal (basal SF) portions of cultured cells. We have recently shown that apical SFs are anchored to the apical plasma membrane (PM) in a manner similar to how basal SFs are attached to focal adhesion sites. We propose calling such apical SF–membrane attachment sites “apical plaques.” To study the macromolecular composition of the apical plaque and the focal adhesion in endothelial cells (ECs) in situ, we examined by confocal laser scanning and fluorescence microscopy guinea pig aortae stained with various antibodies against focal adhesion–associated proteins. Basal SFs oriented parallel to the blood flow direction were mainly located in the upstream half of the cell. Thin apical SFs were also observed. Spotty staining patterns were observed in the basal and the apical portions of cells stained with anti-vinculin, anti-talin, anti-paxillin, or anti-fibronectin receptor, indicating the presence of focal adhesions and apical plaques in ECs in situ. Although fibronectin receptors were present in the apical plaque, fibronectin was not detected on the apical cell surface. Our data suggest that the molecules responsible for the SF-PM association are the same between in vitro and in situ cells. Our results appear to support a hypothesis that the SF system is involved in sensing and/or signal transduction of fluid mechanical forces.

The ends of stress fibers in the basal portion of cells (basal stress fibers) are anchored to focal adhesions, and stress fibers in the apical part of cells (apical stress fibers) are attached to the apical membrane, forming a structure (the apical plaque; KATOH, K. et al. (1995). Cell Motil. Cytoskel., 31: 177-195) resembling the focal adhesion. In addition to these two sites, stress fibers also make lateral contact with the plasma membrane but little is known about the molecular composition of this type of stress fiber-membrane interaction sites. Several actin-membrane association types are known, each employing a different set of proteins, and the focal adhesion and the erythrocyte membrane skeleton are the best characterized systems. We investigated by immunofluorescence microscopy if there is any morphological basis for the involvement of the erythrocyte membrane proteins in the stress fiber-plasma membrane association sites in cultured human fibroblasts. Our results indicated that fodrin (nonerythrocyte type spectrin) and ankyrin were generally associated with the plasma membrane, but that they were clearly excluded from the focal adhesion, the apical plaque and the stress fiber. Thus, it appears that the spectrin and the integrin based actin-membrane association systems are mutually excluded in the fibroblast membrane undercoat. Protein localization at the lateral stress fiber-membrane association site was also studied. Our data indicated that, while talin, vinculin, paxillin, fibronectin receptor and integrin beta 1 were present at the three stress fiber-membrane association sites, vitronectin receptor and integrin alpha v were absent from the apical plaque and the lateral association site. While the plasma membrane at the focal adhesion adheres tightly to a solid substrate, the cell surface of the apical plaque is free. Although the lateral association site faces the substrate, the molecular composition of this site is similar to that of the apical plaque.

In order to investigate the cellular mechanisms involved in amylase release in response to stimulation with short-chain fatty acids, changes in intracellular calcium concentration ([Ca2+]i), membrane current and amylase release were measured in pancreatic acinar cells of sheep. Both octanoate and acetylcholine raised [Ca2+]i in acinar cells in a concentration-dependent manner. The rise in [Ca2+]i in response to the stimulation with octanoate (10 mmol.l-1) was reduced in a medium without CaCl2, but was markedly enhanced by reintroduction of CaCl2 into the medium up to 2.56 mmol.l-1. Perfusion of the cells with a medium containing octanoate (5 mmol.l-1) or acetylcholine (0.5 mumol.l-1) immediately raised inward current across the cell membrane at a holding-membrane potential of -30 mV. The inward current became greater as the holding potential became more negative. The equilibrium potential was 1.8 mV and 3.9 mV for octanoate and acetylcholine, respectively, being consistent with that for Cl-. Although intracellular application of octanoate through a patch-clamp pipette also raised inward current after several minutes in some cells (4 out of 12), this possibility was significantly smaller than that for extracellular application. In other cells, even though the intracellular application of octanoate did not cause an increase in current, it always caused responses immediately after introduction of the fatty acid into the medium. Stimulation with fatty acid as well as acetylcholine raised amylase release in a concentration-dependent manner in cells dispersed from tissue segments with crude collagenase and trypsin inhibitor. Without trypsin inhibitor, crude collagenase significantly and selectively reduced the octanoate (10 mmol.l-1)-induced amylase release. Dispersion with crude collagenase and trypsin significantly reduced both responses induced by octanoate and acetylcholine (5.5 mumol.l-1). We conclude that fatty acids and acetylcholine increase [Ca2+]i, which consequently evokes a rise in transmembrane ion (Cl-) conductance and amylase release, and that trypsin-sensitive protein(s) in the cell membrane are involved in secretory processes activated by stimulation with fatty acids in ovine pancreatic acinar cells.

In the course of myosin-catalyzed ATP hydrolysis, certain amino acid residues in myosin interact with counterparts in actin to produce the relational changes that underlie muscle contraction; some of these interactions are ionic, but the stronger interactions are hydrophobic. In an effort to identify myosin residues participating in hydrophobic interactions, myosin (from smooth muscle) fragments with mutations at suspected sites were engineered and compared with wild-type fragments. It was found that the ATPase of doubly mutated (Trp546Ser and Phe547His) fragments was minimally activated by actin and did not decorate actin well to form the regular arrowhead pattern characteristic of myosin binding to actin filaments. Thus, we suggest that Trp546 and Phe547 are important participants in the hydrophobic actin-myosin interaction.

Human fibroblasts stained with fluorescently labeled phalloidin revealed many stress fibers within the apical cytoplasm in addition to those located along the basal plasma membrane and associated with focal adhesions. The staining patterns of these apical stress fibers with fluorescent phalloidin, anti-alpha-actinin, and antimyosin were identical to those of the basal stress fibers, suggesting the same macromolecular organization for both types of stress fibers. There were two types of apical stress fibers that clearly interacted with the apical plasma membrane, those extending between the basal and the apical plasma membrane and those having both ends on the basal membrane forming arches whose top interacted with the apical plasma membrane. By electron microscopy, we observed that apical stress fibers were associated with the apical plasma membrane via electron-dense plaques reminiscent of the focal adhesion. Since several proteins have been specifically localized to the focal adhesion site, we examined whether they were also present at the apical stress fiber-membrane association site by using immunocytochemical methods and image reconstruction techniques. We found that vinculin, talin, paxillin, a fibronectin receptor protein, several integrin subunits including beta 1, fibronectin, and proteins with phosphorylated tyrosine were also components of the apical plaque. These observations indicate that apical stress fibers are attached to the plasma membrane by using principally the same molecular assembly as the focal adhesion associated with the basal stress fiber. We suggest that the complex molecular organization of the focal adhesion is not demanded by cell adhesion, but rather it is needed for anchoring stress fibers to the plasma membrane. Apical plaques did not stain with the anti-integrin alpha v subunit or anti-focal adhesion associated kinase (FAK), although these antibodies stained focal adhesions. These results suggest that the apical stress fiber-membrane contact has some important functions different from those of the focal adhesion.

Most species living in temperate zones adapt their physiology and behavior to seasonal changes in the environment by using the photoperiod as a primary cue. The mechanisms underlying photoperiodic regulation of stress-related functions are not well understood. In this study, we analyzed the effects of photoperiod on the hypothalamic-pituitary-adrenal axis in photoperiod-sensitive Fischer 344 rats. We first examined how photoperiod affects diurnal variations in plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone. ACTH levels did not exhibit diurnal variations under long- and short-day conditions. On the other hand, corticosterone levels exhibited a clear rhythm under short-day condition with a peak during dark phase. This peak was not observed under long-day condition in which a significant rhythm was not detected. To analyze the mechanisms responsible for the photoperiodic regulation of corticosterone rhythms, ACTH was intraperitoneally injected at the onset of the light or dark phase in dexamethasone-treated rats maintained under long- and short-day conditions. ACTH induced higher corticosterone levels in rats examined at dark onset under short-day condition than those maintained under long-day condition. Next, we asked whether melatonin signals are involved in photoperiodic regulation of corticosterone rhythms, and rats were intraperitoneally injected with melatonin at late afternoon under long-day condition for 3 weeks. However, melatonin injections did not affect the corticosterone rhythms. In addition, photoperiodic changes in the amplitude of corticosterone rhythms were also observed in melatonin-deficient C57BL/6J mice, in which expression profiles of several clock genes and steroidgenesis genes in adrenal gland were modified by the photoperiod. Our data suggest that photoperiod regulates corticosterone rhythms by altered adrenal sensitivity through melatonin-independent mechanisms that may involve the adrenal clock.

1. Recordings of extracellular spike responses were made from single mitral/tufted cells in the ventromedial region of the main olfactory bulb of urethan-chloralose-anesthetized rabbits. Using periodic artificial inhalations, the olfactory epithelium was stimulated with series of aromatic and aliphatic compounds systematically varying in molecular conformation. 2. Analysis of response specificity of single mitral/tufted cells for alkylbenzenes indicated that the length of the hydrocarbon side chain attached to the benzene ring plays a role in determining the specificity of excitatory spike responses. 3. For a panel of isomeric (ortho-, meta-, and para-positions) disubstituted benzenes, single mitral/tufted cells tended to be activated selectively by one or two specific structural isomer(s). For a panel that contained both alkylbenzenes and disubstituted benzenes, single mitral/tufted cells were activated by subsets of odor molecules having similar conformations. These observations suggest that the overall conformation of the aromatic compounds plays an important role in determining tuning specificity of individual mitral/tufted cells. 4. For a panel of monosubstituted benzenes with various functional groups, single mitral/tufted cells in the ventromedial region tended to be activated not only by molecules having a hydrocarbon side chain (alkylbenzenes), but also by those having a methoxy group (--O--CH3), a bromine (--Br), or a chlorine (--Cl). However, most of the neurons were not activated by those having an amino group (--NH2), a hydroxy group (--OH), nor a carboxyl group (--COOH). 5. Examination with an expanded panel of stimulus odor molecules that included both aromatic and aliphatic compounds indicated that single mitral/tufted cells show excitatory spike responses to a range of odor molecules (molecular receptive range) having similar conformations. Different mitral/tufted cells in the ventromedial region typically showed different molecular receptive ranges. 6. In mitral/tufted cells with relatively high spontaneous discharges, single neurons in the ventromedial region showed inhibitory responses to subsets of odor molecules in addition to the excitatory response to other subsets of odor molecules. The odor molecules that caused inhibitory responses in single mitral/tufted cells showed molecular conformations resembling each other. 7. The present results together with previous studies indicate that determination of the molecular receptive range properties (both excitatory extent and inhibitory extent) of single mitral/tufted cells is a useful method for characterizing individual bulbar neurons. These results further support the hypothesis that conformational parameters of ligand odor molecules play a key role in sensory processing in the main olfactory bulb.

Monoclonal antibodies R4B12 and R1D1 label the same subsets of rabbit primary olfactory axons. In the present study, we characterized the R4B12 antigens using immunohistochemical, immunoelectron-microscopic, and biochemical techniques. The R4B12 antigens are expressed on the surface membrane of a subset of primary olfactory axons. Western blot analysis revealed the existence of two forms (115,000 and 90,000 mol. wt) of the R4B12 antigens with different membrane-anchoring structures. Of the two forms, the smaller antigen (90,000 mol. wt) is anchored to the plasma membrane via a phosphatidylinositol linkage and expressed exclusively by the olfactory system. When the rabbit olfactory epithelium was stimulated by odors for 2–8 h in situ , the R4B12 immunoreactivity disappeared from the primary olfactory axons in the glomeruli of the olfactory bulb. These results suggest that the cell surface antigens R4B12 expressed by subsets of primary olfactory axons undergo stimulus-dependent changes by odor stimulation and may be involved in plasticity of olfactory sensory neurons.

Various specimen preparations for thin-section electron microscopy were tested to better preserve and visualize actin filaments in the cortex of the early embryos of Drosophila melanogaster. When embryos were treated with phalloidin prior to fixation, many actin filaments were observed in their cortex comparable to the staining with fluorescently labeled phalloidin in light microscopy. Then we used various fixatives containing phalloidin. As far as we examined, actin filaments were best preserved in the specimen fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium phosphate buffer or in 0.1 M PIPES buffer (1 mM EGTA and 1 mM MgCl2) containing 10 microM phalloidin and 0.1% saponin. When embryos were glycerinated and then treated with tropomyosin before fixation, actin filaments were well visualized as thicker, uniform-sized filaments, though the number of filaments decreased probably owing to glycerination. This suggests that, like heavy meromyosin and its subfragment-1, this protein may protect actin filaments from being disrupted by chemical fixation. Using these improved fixation procedures, we successfully examined the distribution of actin filaments in the Drosophila embryo cortex during cellularization. These methods may be applicable to stabilize labile actin filaments in other types of cells.

Publisher Summary This chapter reviews results of research conducted on control mechanisms in the ruminant exocrine pancreas. It focuses on the significance of short-chain fatty acids (SCFA) in exocrine pancreatic regulation because they appear to be a new and unique candidate among postprandial humoral factors that regulate the exocrine pancreas of ruminants. The term “secretion” has a wide range of meanings and originally implies at least four steps: ingestion, synthesis, transportation and storage, and extrusion. The chapter describes the relationships of the nervous and systemic circulatory processes. For the regulation of exocrine pancreatic secretion in ruminants, intestinal and humoral phases are more important than cephalic and gastric phases. The role of SCFA as one of the factors affecting the exocrine pancreas is nutritionally important for some animal species such as ruminants that have a persistent digesta flow into the duodenum due to their large forestomachs. Thus, continuous digestion in the small intestine requires these animals to secrete pancreatic juice relatively continuously, associated with slow changes in the duodenal flow rate.

AVP (0.18mg/animal) was intravenously injected in dairy cattle to evaluate the potential of AVP as a releaser of ACTH and cortisol. AVP administration immediately increased serum ACTH concentration from 151.9±96.9pg/ml to the peak valne of 880.8±269.5pg/ml at 10min post injection (p

The three-dimensional organization of cytoskeletal components in the early Drosophila melanogaster embryos during cellularization was examined by confocal laser scanning microscopy of the whole embryos and thin section electron microscopy.For confocal microscopy, Drosophila embryos at 2-3 hr after egg-laying were dechorionated and fixed with 8% paraformaldehyde-1% picric acid in 0.1M phosphate buffer (pH 7.2). Embryos were first blocked with normal goat serum, incubated with monoclonal antibody raised against Drosophila embryo α-tubulin for 4 hr, and then were incubated with FITC-conjugated anti-mouse IgG for 2 hr. Some embryos were stained with rhodamine-labeled phailoidin for F-actin visualization. After staining, the whole embryos were mounted on slide glass with an appropriate spacer and examined under the confocal microscope (Bio-Rad, Lasersharp MRC-500). For electron microscopy, dechorionated embryos were fixed with 1/2 Karnovsky's fixative followed by OsO4 fixation. To better preserve actin filaments, embryos were fixed with the same 1/2 Karnovsky's fixative containing 10 μM phailoidin and 0.1% saponin in 0.1M phosphate buffer, pH 7.2. Such fixed embryos were dehydrated and then embedded in Epoxy resin. Thin sections were cut and examined under a Hitachi H-800 type electron microscope.

A specific method for the determination of two types of urokinase was developed, involving high-performance liquid chromatography (HPLC) with radioisotope detection using [3H]diisopropyl fluorophosphate (DFP) as a pre-labelling reagent. The serine moiety located in the active site of urokinase was reacted selectively with [3H]DFP to form [3H]DFP-urokinase adduct. Two types of urokinase were determined by monitoring the radioactivity of each peak separated by means of HPLC. The total radioactivity of the two peaks was in good agreement with the urokinase activity obtained by a modified fibrin plate method.

A new equation for the excess Gibbs energy similar to the UNIQUAC equation is presented. The equation is found to give better data reduction results for those binary systems where an alcohol is one of the components. Successful prediction of ternary vapor-liquid equilibria using only binary parameters is demonstrated. To obtain improved calculated results for ternary liquid-liquid equilibria, a total of six parameters (two per binary) are not sufficient and one additional parameter C is usually necessary, as shown in illustrative examples. C must be the same for three binary systems constituting a ternary system.

Liquid—liquid equilibrium data were obtained for two ternary systems: acetonitrile— ethanol—cyclohexane at 40°C, and acetonitrile—2-propanol—cyclohexane at 50°C. Binary vapor—Liquid equilibrium data were measured for acetonitrile—2-propanol at 50°C. The binary parameters of the Zeta and effective Zeta equations were evaluated from equilibrium data for binary pairs. The parameters obtained were used to predict the ternary liquid—liquid equilibrium data for six systems involving the present systems and the ternary vapor—liquid equilibrium data for one completely miscible system and two partially miscible systems without adding any ternary parameter. A heterogeneous area calculated by the Zeta equation is in general too large and does not decrease appreciably with increasing values of the third parameter ζ of the Zeta equation. However, the effective Zeta equation works much better than the original Zeta equation in data reduction.

The number of damaging incidents due to electrostatic discharges (ESD) on digital electronic equipment has been increasing with the increased use of integrated semiconductor elements with lower operation power. We proposed the idea that by visualizing the static electricity which caused ESD for realtime recognition and tracking, preventive measures could be applied before ESD occur. This paper proposes a measuring principle in which the electric potentials induced on an array of small square electrodes placed in parallel in the vicinity of a charged body are proportional to the amount of the charge of the body. A prototype based on this principle was built and attempts were made at visualization and tracking for the static electricity distribution of a charged body. As a result, we confirmed that we could visualize charge distributions of a charged body directly below the electrode array using the electric potential data from a finite number of measuring electrodes. Furthermore, we could observe a two-dimensional charge distribution of a charged body using an acrylic sheet.

Laser acupuncture is recognized as the biostimulation therapy with a low energy laser, while the mechanism has not been clear. However, in the pulsed laser acupuncture, the thermal stress caused by the photo-acoustic effect is thought to be the dominant factor of the biostimulation.This paper proposes a method of calculating the spatial distribution of the thermal stress inside a human skin tissue using Hu's model for analyzing the photo-acoustic wave in the liquid. Numerical calculations show that the shape of the thermal stress depends on the absorption coefficient of the laser, that is, the stress due to the CO2 laser occurs sharply compared with the case of the YAG laser.

The effects of the cholinergic and adrenergic nerve stimulations on amylase release from the segment isolated from the rat parotid gland were investigated, employing the combined techniques of electrical field stimulation (FS) and tyramine application with automated fluorescence method for measuring amylase. The maximum amylase release in response to FS for a short period (1 min) was attained at 16 Hz frequency, 2 ms pulse width and 8 V strength stimulation in the absence of any autonomic antagonist. Periodic short-lasting FS using these parameters at intervals of about 15 min could reproduce similar sizes of amylase release for about 2 h. Continuous long-lasting FS (3 V, 2 ms, 16 Hz) caused a transient sharp increase in amylase release followed by a sustained one. The FS-evoked amylase release was completely abolished by tetrodotoxin (TTX) (10(-7) g/ml) and markedly reduced by atropine (7 X 10(-6) M) or by propranolol (10(-5) M), while it was scarcely affected by hexamethonium (3 X 10(-4) M) and phentolamine (10(-5) M). The maximum stimulus frequency of short-lasting FS for amylase release in the presence of propranolol was similar to that (16 Hz) in the control, but it was higher (32 Hz) in the presence of atropine. Reduced response in amylase release to FS by propranolol was completely restored by the superimposed addition of dibutyryl cyclic AMP (10(-4) M). Application of tyramine (5 X 10(-4) M) evoked amylase release in the presence of atropine, which was blocked mostly by propranolol and partly by phentolamine, while tyramine was ineffective in the tissue segment from rats pretreated with 6-hydroxydopamine. From these results the following were suggested; that the intrinsic cholinergic neurotransmitter activates a muscarinic receptor of the acinar cell, while the adrenergic neurotransmitter stimulates mainly a beta-adrenergic and partly an alpha-adrenergic receptor, resulting in amylase release.

The effect of cholinergic neural excitation by field stimulation on the tension of the contractile system (myoepithelial cells) was investigated in superfused segments of rat salivary glands (sublingual, submaxillary, and parotid). Electric field stimulation evoked gland tissue contraction of a few mg, which was blocked by addition of TTX or atropine. The order of the magnitude of the contraction per wet tissue weight was sublingual greater than submaxillary greater than parotid. This correlates with the order of the distribution of myoepithelial cells described in morphological investigations. Cholinergic stimulants induced contraction in all salivary glands, but adrenergic stimulants hardly evoked contraction in the sublingual gland, whereas those drugs evoked a considerable amount of contraction in the submaxillary gland. These findings seem to suggest that the contraction may originate in the myoepithelial cells. The omission of Na from the superfusing solution quickly abolished the contraction evoked by field stimulation but not the contraction induced by ACh. The omission of Ca from the solution reduced the contraction evoked not only by field stimulation but also by ACh and by high K. The results suggest that extracellular Ca is important for the initiation of myoepithelial contraction.

Experimental liquid—liquid equilibrium data are reported for the systems acetonitrile—acetone—cyclohexane at 318.15 K and acetonitrile—methyl acetate—cyclohexane at 313.15 K. Two modified forms of the NRTL equation proposed by Renon are presented by substituting local surface fractions for local mole fractions and further by including Guggenheim's combinatorial entropy for athermal mixtures whose molecules differ in size and shape. The resultant equations involve three adjustable parameters and are extended to multicomponent systems without adding ternary (or higher) parameters. Calculated results of vapor—liquid and liquid—liquid equilibria for typical binary and ternary mixtures are presented.

It is well known that the early diagnosis of maxillary cancer (MC) is very difficult on the basis of its clinical appearance and symptoms, since nasal obstraction, bloody discharge, toothache and loosening of the teeth are common in the early stage.A new technique was devised for the isolation and charactrization of maxillary cancer associated antigens (MCAA), since elucidation of MCAA has been one of the major achievements of tumor immunology.Frozen sections of MC 10μm thick were applied directly to PAG-plates for isoelectric focusing. Saline extract of tumor tissues and sera were treated in the same manner.Specific components at around pH 6.1 were determined on PAG-plates and collected by chromatofocusing. Anti-MCAA rabbit serum was obtained by immunization with the components around pH 6.1 and anti-MCAA IgG serum was purified by protein A sepharose CL-4B.The purpose of this study was to demonstrate the location of MCAA in tumor sections and determine its approximate molecular weight. Tissues of 10 out of 11 (91%) cases of MC were stained positively by indirect immunofluorescence and immunoperoxidase techniques. This definitely positive staining was demonstrated on the cell membranes of MC, while in 8 control cases (normal maxillary sinus membrane, paratine tonsil, parotid gland and membranes from patients with chronic paranasal sinusitis) the staining was definitively negative.The molecular weight of MCAA was investigated by SDS-PAG and Immune-Blotting. The former showed two components (170k-225k), and the latter detected two components (120k-136k) and one minor component of 76k.

For the investigation of a possible mechanism causing the bio-effect of low energy laser beam, this paper describes the influences of the laser beam irradiationon protein conformation (spatial structure of a protein) relating to biological activation. Measurements of the myoglobin conformation are made by using the circular dichroism (CD) spectra, and the variation of the conformation is examined with respect to the irradiation time. The results show that the low energy laserbeam could promote the time fluctuation of the conformation content and might result in affecting the bio-effect.

Urokinae activity was determined by capillary-tube isotachophoresis using N-a-acetyl- l -lysine methyl ester as a synthetic substrate. The resulting N-a-acetyl- l -lysine was separated and detected isotachophoretically using methanolic potassium hydroxide solution adjusted to pH 5.32 by adding a-ketoglutaric acid as a leading electrolyte and methanolic betaine hydrochloride solution as a terminating electrolyte. The enzymatic reaction was stopped by addition of tannic acid and the resulting supernatant solution was injected into an isotachophoretic analyser. Linearity was observed at urokinase activities in the range 1–30 I.U. The urokinase activities in commercial products determined by the method were in good agreement with those determined by Walton's modified plate method. The coefficient of variation of the method was less than 3.4%.

体産鶏(白色レグホーン種)の血漿グルコース,インスリン,グルカゴンおよび遊離脂肪酸濃度(FFA)に対する24時間寒冷暴露の影響を検討した.環境温度を20°Cから0°Cに低下させると,血漿グルコース濃度は204±1.4mg/dlから徐々にかつ有意に減少し,16時間後に最低値(176±3.7mg/dl)を示した.血漿インスリン濃度は,寒冷暴露前値は3.10ng/mlであったが,12時間後に急激に有意な増加(最大値11721ng/ml)を示した.血漿グルカゴン濃度は,寒冷暴露でやや減少する傾向がみられ,12時間後に最低値を示したものの有意な変動ではなかった.また,FFAにも有意な変動は認められなかった.インスリン抗体を用いた免疫組織学的な観察では,寒冷に暴露した鶏の膵島細胞におけるインスリン濃度の減少が認められた.以上の結果から,鶏を寒冷に暴露すると血漿グルコース濃度が低下し,その原因にはインスリンの急激な膵分泌増加が関与するものと考えられる.

1. The stimulus-secretion coupling in the pancreatic exocrine responses to i.v. injection of sodium butyrate was investigated in guinea pigs in vivo and in vitro. 2. Intravenous single injection of sodium butyrate (12.5-100 mumol/100 g body wt) caused an increase in fluid and amylase secretion in a dose-dependent manner. The responses evoked by sodium butyrate (100 mumol/100 g body wt) were not affected by prior injection of atropine (0.14 mumol/100 g body wt) or hexamethonium (4 mumol/100 g body wt). 3. The chloride concentration in secreted fluid increased slightly with an increase in flow rate in response to sodium butyrate, but decreased in response to secretin. 4. The amylase release from the pancreatic segments evoked by sodium butyrate (10(-6)-10(-2) M) increased dose-dependently. The responses were potentiated in the presence of secretin (1 C.H.R.u./ml), but were suppressed in the presence of acetylcholine (10(-6) M) or in a Ca-free solution containing EGTA (10(-4) M). 5. These results suggest that the secretory effects in response to i.v. injection of sodium butyrate probably arise from direct action on the acinar cells, and that an increase in cellular calcium concentration might be an important step in the secretion process, in guinea pig exocrine pancreas.