A K+-selective,three-state channel from fragmented sarcoplasmic reticulum of frog leg muscle

Summary Sarcoplasmic reticulum (SR) vesicles from frog leg muscle were fused with a planar phospholipid bilayer by a method described previously for rabbit SR. As a result of the fusion, K⁺-selective conduction channels are inserted into the bilayer. Unlike the two-state rabbit channel, the frog channel displays three states: a nonconducting (closed) state and two conducting states and . In 0.1m K⁺ the single-channel conductances are 50 and 150 pS for and , respectively. The probabilities of appearearance of the three states are voltage-dependent, and transitions between the closed and states proceed through the state. Both open states follow a quantitatively identical selectivity sequence in channel conductance: K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺>Cs⁺. Both open states are blocked by Cs⁺ asymmetrically in a voltage-dependent manner. The zero-voltage dissociation constant for blocking is the same for both open states, but the voltage-dependences of the Cs⁺ block for the two states differ in a way suggesting that the Cs⁺ blocking site is located more deeply inside the membrane in the than in the state.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

The muscle ryanodine receptor and its intrinsic Ca2+ channel activity

In skeletal and cardiac muscle, contraction is initiated by the rapid release of Ca²⁺ ions from the intracellular membrane system, sarcoplasmic reticulum. Rapid-mixing vesicle ion flux and planar lipid bilayer-single-channel measurements have shown that Ca²⁺ release is mediated by a high-conductance, ligand-gated Ca²⁺ channel. Using the Ca²⁺ release-specific probe ryanodine, a 30 S protein complex composed of four polypeptides ofM _r 400,000 has been isolated. Reconstitution of the purified skeletal and cardiac muscle 30 S complexes into planar lipid bilayers induced single Ca²⁺ channel currents with conductance and gating kinetics similar to those of native Ca²⁺ release channels. Electron microscopy revealed structural similarity with the protein bridges (feet) that span the transverse-tubule-sarcoplasmic reticulum junction. These results suggest that striated muscle contains an intracellular Ca²⁺ release channel that is identical with the ryanodine receptor and the transverse-tubule-sarcoplasmic reticulum spanning feet structures.     

Anti-diabetic activity of <Emphasis Type="Italic">β</Emphasis>-glucans and their enzymatically hydrolyzed oligosaccharides from <Emphasis Type="Italic">Agaricus blazei</Emphasis>

-Glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of -glucans were 30–50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing -glucans with an endo--(16)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though -glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of -glucans with respect to anti-diabetic activity.     

Effects of polymyxin B on the sarcoplasmic reticulum membrane

Polymyxin B, a cyclic peptide antibiotic, inhibits Ca²⁺-ATPase, p-nitrophenyl phosphatase and phosphorylase kinase activities associated with rabbit skeletal muscle sarcoplasmic reticulum membranes; 50% inhibition is induced by 100 M, 130M and 550 M of polymyxin respectively. The fluorescence intensity of fluorescein isothiocyanate-labeled Ca²⁺-ATPase, decreases in the presence of polymyxin (50% of the total decrease at 70 M polymyxin). On the other hand, the polypeptide inhibits calmodulin-dependent endogenous phosphorylation of 60 kDa, 20 kDa and 14 kDa membrane proteins, while an increase of calmodulin-dependent phosphorylation is observed in 132 kDa and 86 kDa proteins.     

Polarities in Mammalian Evolution Seen Through Homologs of the Inner Cell Mass

Embryogenetic pathways differ markedly among monotremes, marsupials, and placentals, and their analysis provides information of fundamental importance to recognition of mammalian evolutionary directions. The cap of cuboidal cells of the marsupial late unilaminar blastocyst, generally known as the embryonic area, probably is induced to form (prior to origin of Hensen's node) by signals from earliest hypoblastic cells (anterior visceral endoderm). The thickened cap is a medullary plate of sauropsid terminology because it includes epiblastic cells presumptive to neurectoderm (including neural crest), Hensen's node, primitive streak, and gut endoderm. The remainder of the definitive embryo (i.e., parts of epidermal origin, including ectodermal placodes) derives from squamous ectoderm (surrounding the medullary plate) of the blastocyst's ill-named trophoblastic area. Amniotic ectoderm develops farther distally within the trophoblastic area. The autapomorphic inner cell mass (ICM) of placental mammals is homologous to medullary plate of the marsupial blastocyst plus morphologically undefined, proximal parts of surrounding ectoderm (of the trophoblastic area). Considerations of early cell lineages in marsupials are greatly affected by recognition that the boundary between future embryonic and extra-embryonic tissues does not match the margin of the medullary plate (i.e., embryonic area). Marsupials and monotremes largely conform to sauropsid early embryogenesis, but placentals express, at earliest developmental stages, innovations unique within Amniota that are linked to early establishment of the brain. Neonatal marsupials and hatchling monotremes are extremely altricial and closely comparable anatomically/physiologically; they share a temporal pattern in combining early morphogenesis of craniofacial features (related to suckling) with deferral of telencephalic completion into postnatal/posthatching life. Placentals contrast greatly in establishing the central nervous system prior to rudiments of the cranial skeleton and associated musculature, and they complete essentials of forebrain development before birth. Comparative evidence from transitory periderm suggests that primordial eutherians had extremely altricial hatchlings or newborns, whichever was the mode of early development. Details remain unknown about the origin of the unique specialization of ICM plus encapsulating trophoblast from the more generalized blastula of ancestral synapsids.     

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

Summary ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic -cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 m and as high as 500 m, with maximal activation between 10 and 100 m ADP in mouse -cell membrane patches. At concentrations greater than 500 m, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of -cell membrane potential, increasing ADP in the 10 and 100 m range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize -cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the ATP/ADP ratio, and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 m range as suggested by biochemical studies.We would like to thank Mr. Louis Stamps for expert technical assistance and Dr. Wil Fujimoto and Ms. Jeanette Teague for generously providing HIT cells obtained from Dr. Robert Santerre at Eli Lilly. We would also like to thank Dr. Michel Vivaudou for providing the program ALEX. Support was provided by the NIH and the Department of Veterans Affairs.     

A morphometric analysis of regional differences in myotomal muscle ultrastructure in the juvenile eel (Anguilla anguilla L.)

Summary Subpopulations of fast and slow fibres within the trunk musculature of elvers were examined using morphometric analysis of electron micrographs. Fibre regions were characterised by their histochemical staining characteristics, and individual fibres located using a coordinate mapping system utilising morphological features as reference points. Percentages of fibre volume occupied by mitochondria, myofibrils, sarcoplasmic reticulum (S.R.), and T-system were determined in each of the fibre groups, along a transect from the skin to the vertebral column (fibres 1–14, respectively).The fine structure of slow (red) fibres (1–2 fibres deep) is relatively homogeneous throughout its range, giving mean values for mitochondria, 21.4%; myofibrils, 61.0%; S.R., 2.10%; T-system, 0.31%. The fibres are relatively small (204 m²) and the mitochondrial cristae poorly developed.In contrast, there is a marked heterogeneity in the ultrastructure of fast (white) fibres, dependent on both position and size. The moderately small (333 m²) superficial fast fibres (3–4 fibres deep) have a significantly higher mitochondrial content (7.6%) than the larger deep fibres (1.2%) (6–12 fibres deep, 775 m²). The mean fractional volumes occupied by myofibrils, S.R., and T-system in the deep fibres are: 80.4%, 5.95%, and 0.38%, respectively. Fibres < 100 m² constitute up to 5% of the fast muscle and have a significantly higher mitochondrial volume (4.3%), more glycogen granules, and a slightly lower volume of S.R. (5.57%) than larger fibres.It is suggested that metabolic subpopulations of fast fibres correspond to different stages of fibre growth. The relatively poorly developed S.R. of eel fast muscle is thought to be correlated with the low frequency, high amplitude nature of the propagated waveform found in anguilliform locomotion.     

Effect of androgens on histochemical fibre type

Summary The temporal muscles of the guinea pig show a sexual differentiation reflected in their histochemical enzyme pattern. Using histochemical methods for mitochondrial (SDH, -GPDH), and glycolytic enzymes (phosphorylase, LDH) it could be shown, that in adult animals the male muscle is a white muscle with marked activity of glycolytic enzymes, the female muscle a red muscle displaying high activity of mitochondrial enzymes. This differential enzyme pattern can be converted by the application of testosterone to the female type during the postnatal development. The male sex hormone thus affects the histochemical enzyme pattern of the muscle, converting the red, female into a white, male muscle in the female guinea pig.     

In vitro comparison of ceftazidime,aztreonam, cefpirome,gentamicin, imipenem,enoxacin, and ticarcillin plus clavulanic acid against 108 strains ofPseudomonas maltophilia

Pseudomonas maltophilia is an uncommon cause of hospital-acquired infection and is resistant to most of the antimicrobial agents used in the treatment of gram-negative infections. Susceptibility of 108 isolates ofP. maltophilia to ceftazidime, aztreonam, defpirome, gentamicin, imipenem, enoxacin, and ticarcillin plus clavulanic acid was determined by an agar dilution method. The isolates were in general resistant to the antibiotics. Imipenem and cefpirome were not active at clinically achievable levels. Of the isolates, 20% were susceptible to 16 g/ml ceftazidime, 53% were susceptible to 4 g/ml enoxacin, 10% were susceptible to 4 g/ml gentamicin, and 25% were susceptible to 64 g/ml ticarcillin plus 2 g/ml clavulanic acid.     

Hexahydroindanone derivatives of steroids formed by Rhodococcus equi

Summary A mutant strain of Rhodococcus equi accumulates three metabolites from the androst-4-ene-3,17-dione or from its degradation intermediate, 3a-H-4(3'-propionic acid)-7a-methylhexahydro-1,5-indanedione (MEPHIP). These three metabolites are: 3a-H-4a(3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone--lactone (HIL); 3a-H-4(3'-trans acrylic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (^2'-5-hydroxy-MEPHIP); and 3a-H-4(3'-hydroxy-3'-propionic acid)-5-hydroxy-7a-methylhexahydro-1-indanone (3'-hydroxy-HIL). The behaviour of this mutant allows us to propose a pathway for degradation of the intermediates, methylperhydroindanone propionates. However, during this degradation, the side-chain propionate was eliminated by a-oxidation mechanism. Offprint requests to: A. Miclo     

Frequency response characteristics of a multi-loop representation of the segmental muscle stretch reflex

This paper continues the investigation of a three-loop representation of the segmental muscle stretch reflex system introduced in a preceding communication. Frequency response characteristics were computed for open-loop conditions, control and disturbance signal inputs under a variety of conditions: (i) in parallel and in series peripheral arrangements of muscle compartments, (ii) various patterns of central connectivity, (iii) various recruitment levels of motor units, (iv) various overall reflex gains, (v) absence or presence of muscle spindle accleration sensitivity. These computations disclosed a number of mechanisms by which the nervous system might improve system stability and behaviour. These mechanisms are discussed with regard to physiological data.     

Human lymphokine preparations which generate tumoricidal properties of human monocytes in vitro may be distinct from gamma interferon

Summary The purpose of these studies was to determine whether stimulated human lymphocytes produce lymphokines distinct from IFN, that can activate human blood monocytes to lyse tumor cells. We undertook this investigation because of the controversy concerning whether MAF and IFN are the same molecule. Crude lymphokine preparations prepared from normal human mononuclear cells incubated with Con A and rich in MAF activity also contained 1000 U/ml IFN as measured by the virus neutralization assay. However, the induction of tumoridical activity in monocytes by the lymphokine preparation could be dissociated from the IFN activity, based on the following data: (1) Heat treatment (100 °C for 2 min) removed the antiviral activity of the lymphokine yet did not diminish its MAF-like activity when measured in a 72 h cytotoxicity assay against ¹²⁵I IUdR-labeled human A375 melanoma cells. (2) Likewise, treatment of this lymphokine preparation with a twofold excess of anti-IFN antibody neutralized antiviral activity but once again had no effect on its ability to activate monocyte tumoricidal function. In contrast, both heat treatment and anti-IFN antibody abolished monocyte activation by equivalent units of human recombinant IFN. Taken together, these data suggest that there is a molecule(s) distinct from IFN which can activate human monocytes for tumoricidal function. Furthermore, this dissociation of MAF and IFN activity was dependent on the use of a long-term (72 h) assay, since activation of tumoricidal activity in an 18–24 h assay appeared to be attributable solely to IFN.     

Polymorphism of the HLA DR1 haplotype in the Israeli population investigated at the serological,cellular, and genomic levels

In the present report, we used serological, cellular, and restriction fragment length polymorphism (RFLP) to investigate the DR1 haplotype in the Israeli population. We describe an Israeli homozygous typing cell (HTC), HLA-DwLVA, which defines a new lymphocyte-activating determinant associated with Bw65, DR1 and distinct from Dwl. The parents of this donor, non-Ashkenazi Algerian Jews, are first cousins and share HLA-Cw8, Bw65, BfS, DR1, DQw1, DPw4. No specificity could be assigned to HLA-DwLVA using the 91 Ninth Workshop HTCs. Two families and forty unrelated DR1 individuals were studied with DwLVA and a panel of DR1/Dw1 HTCs. HLA-DwLVA showed segregation as a single determinant within families. This new specificity was present in 24 out of 40 (60%) unrelated DR1 individuals, indicating that in the Israeli population DwLVA is the main lymphocyte-defined determinant associated with the serologically defined DRI specificity, in contrast to non-Jewish Caucasoids where DR1 is significantly associated with Dw1. The vast majority of DwLVA-positive carriers were also Bw65 carriers, indicating that Bw65, DR1, DwLVA may represent a typical allele combination in the Israeli population. The RFLP analysis established the correlation of certain RFLPs with Dw1 and DwLVA. In addition, we describe a cluster of RFLPs that may correspond to a new Dw subtype associated with DR1, for which no serological and cellular reagents have been described so far.     

Histochemistry of some acid hydrolases in striated muscles of the rat

Summary The distribution of acid phosphatase, -n-acetylglucosaminidase, -glucuronidase, and acid -galactosidase was studied in mm. extensor digitorum longus, soleus, and diaphragm of rats. Using the technic of semipermeable membranes activities of these enzymes were demonstrated beside cells of the interstitial tissue in muscle fibers themselves as well. Acid phosphatase displayed the highest activity which appeared in many small dots dispersed in the fiber. The activity of acid phosphatase was about 1.2 x higher in the m. soleus than in the m. extensor digitorum longus. In the latter muscle a somewhat higher activity was often found in muscle fibers displaying a higher staining for NADH tetrazolium reductase. The activity of -n-acetylglucosaminidase was slightly lower, that of -glucuronidase very weak but still discernible. The activity of acid -galactosidase was not ascertained in the majority of fibers. The ratio of activities measured in an area of the same size in cells of the interstitial tissue and in muscle fibers amounted in average to 2.6: 1 in the case of acid phosphatase, 2.5:1 in the case of -n-acetylglucosaminidase, 5.7: 1 in the case of -glucuronidase, and 44.3:1 in the case of acid -galactosidase. The importance of the histochemical technic in studies concerned with acid hydrolases in striated muscle fibers in normal and pathological conditions is pointed out.     

The fine structure of red and white myotomal muscle fibres of the coalfish (Gadus virens)

Summary The fine structure of the red and white myotomal muscles of a marine teleost, the coalfish Gadus virens, has been examined and ultrastructural measurements and analyses carried out. The sarcomere lengths of the red and white fibres were found to be 1.60 minimum, 1.82 maximum and 1.70 minimum, 1.85 maximum, respectively. No significant difference was found between the red and white fibres in their percentage of sarcoplasmic reticulum and T system. Both were found to have regularly occurring triads at the Z disk level, to have distinctive M lines and to be multiply innervated. Ultrastructurally the two fibres can be distinguished by the thicker Z line and more abundant mitochondria of the red fibre, and by the ribbon-shaped peripheral myofibrils of the white fibres. The structure of the fibres in these two types of muscle is discussed in relation to their possible role in swimming.This work was supported by a research grant from the National Environmental Research Council.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.