Mg2+ Induces Conformational Changes in the Catalytic Subunit of Phosphorylase Kinase, Whether by Itself or as Part of the Holoenzyme Complex

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition ()₄. Previous studies have revealed that the activity of its catalytic subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb 79) has been generated against isolated subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the -calmodulin binary complex, and the free subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb 79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the subunit containing the epitope for mAb 79. The activating ligand Mg²⁺ also stimulated the binding of the PhK holoenzyme to immobilized mAb 79, as well as the binding of mAb 79 to immobilized subunit. Thus, Mg²⁺ increases the accessibility of the mAb 79 epitope in both the isolated subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg²⁺ on the quaternary structure of the PhK holoenzyme are directly mediated by the subunit.     

Integrins: cell adhesives and modulators of cell function

Summary Integrins encompass a family of cell-surface molecules which play a crucial role in cell-cell and cell-extracellular matrix interaction. Of these heterodimeric transmembrane glycoproteins (consisting of an and chain) as yet at least 20 different types have been described, all with a different pattern of reactivity with extracellular matrix components. In this review the cell and tissue distribution of the integrins is discussed, with special emphasis on immunohistochemical localization of the ₁ integrins and the ₆₄ integrin. The ₁ integrins comprise a subfamily in which eight chains combine with one (the ₁) chain. The ₂₁, ₃₁ and ₆₁ and the ₆₄ integrins are expressed on a wide variety of epithelia on the basolateral surface or exclusively on the basal surface facing the basement membrane (e.g. ₆₁ and ₆₄). Leucocyte integrins, which share a common ₂ chain, occur almost exclusively on white blood cells and their precursors. The vitronectin receptors, which share a common _v chain, occur in a wide variety of cell types. Integrins play a major role in the interaction of the cell with the extracellular matrix in order to create and maintain tissue architecture. It has become clear, however, that through integrin-ligand interaction cell function is also modulated. Furthermore, in pathological conditions integrins play a role of some significance. Integrins mediate leucocyte traffic in developing inflammatory processes and function in neoplastic growth when it comes to invasion and metastasis.     

Freeze-fracture analysis of junctional complexes in the nephron of the garter snake,Thamnophis sirtalis

Summary Zonulae occludentes are shown by freeze-fracture to be pleomorphic along the garter snake nephron. In the neck and proximal segments the occluding junctions are moderately complex with frequent discontinuities in their junctional fibrils. Junctional depth and complexity are maximal in the distal and collecting segments and discontinuities in fibrils are absent. Comparison of these results with similar observations on other tissues indicates that the zonulae occludentes in the neck and proximal segments are intermediate to leaky and that they may be very tight in the distal and collecting segments. These findings suggest that in the garter snake nephron transepithelial flow of fluid may occur primarily by passive diffusion through the zonulae occludentes in the neck and proximal segments and by cell-mediated osmotic flow in the distal and collecting segments. Gap junctions occur only in the proximal tubule and are probably involved in low resistance, intercellular movement of ions.Supported by the National Research Council of Canada. The authors wish to acknowledge the generous provision of freeze-fracture facilities by Dr. M.W. Brightman, NINDS, National Institutes of Health, Bethesda, Maryland, U.S.A.     

Ultrastructure of the new neuromuscular junctions formed during reinnervation of rat soleus muscle by a “foreign” nerve

Summary In rats the fast fibular nerve was transposed to the slow soleus muscle outside the original innervation band. Formation of new neuromuscular junctions was induced by cutting the soleus nerve after different periods of time. The morphological maturation of these junctions was studied by electron microscopy.New neuromuscular junctions do not form when the original innervation is left intact.Three to five days after denervation, vesicle-laden terminal boutons contact muscle fibers with only the basal lamina of the latter intervening. Three weeks after denervation, most boutons are larger and postsynaptic folds are present, although younger stages are also seen. Sixteen weeks after denervation, the neuromuscular junctions appear mature. This corresponds well with electrophysiological findings in the same material.The fully developed neuromuscular junctions sixteen weeks after denervation possess postsynaptic folds similar to those of normal fast muscle fibers. This suggests that the fast fibular nerve rather than the slow soleus muscle fibers determines the morphology of the postsynaptic folds.Possible trophic neuromuscular interactions are discussed.The authors are indebted to Mrs. Jorunn Line Vaaland, Miss Bjørg Riber, and Miss Berit Branil for technical assistance. Dr. T. Lømo and Dr. C. Slater have contributed constructive criticism and advice     

Quantitative analysis of type IV collagen alpha chains in the basement membrane of human urogenital epithelium

Type IV collagen is a major component of the basement membrane (BM), which consists of six genetically distinct (IV) chains. In this study the expression of these six (IV) chains was demonstrated immunohistochemically. In addition, the 2(IV) and 5(IV) chains were analysed quantitatively by confocal laser scanning microscopy in human urogenital epithelial BM. The 1/2(IV) and 5/6(IV) chains were immunoreactive in the epithelial BM, whereas, 3/4(IV) chains were not. The quantitative analysis revealed that the amount of 2(IV) and 5(IV) chains differed in each urogenital epithelial BM. The content of 5(IV) chains in the epithelial BM of the bladder was differentially high, and that of the foreskin was differentially low. It is concluded that the elasticity of epithelial BM of the bladder may be structurally related to the high content of 5/6(IV) chains.     

Structure of the parathyroid glands,as revealed by different methods of fixation

Summary Stereology and semi-automatic image analysis were used with the aim of comparing the structure of parathyroid glands from untreated adult Mongolian gerbils fixed by immersion with those fixed by perfusion. Subclassification of the chief cells based upon the staining affinity or electron density of the cytoplasm was readily performed only in glands fixed by immersion, and so-called atrophic cells were observed only in these glands. The atrophic cells were often surrounded by light chief cells. In glands fixed by perfusion, light chief cells were only rarely encountered. A significant difference between glands fixed by immersion and those fixed by perfusion was found only with regard to the form of cells and nuclei, those fixed by perfusion being more spherical. When comparing individual cells within glands fixed by immersion, light chief cells were more spherical and had a significantly larger nuclear and cellular size, and a lower mitochondrial volume density than the intermediate/dark chief cells. Otherwise there were no significant differences in any of the parameters investigated. These data indicate that occurrence of socalled light chief cells and atrophic cells is a result of improper fixation. The results of this study do not favour the concept of a functional cycle with a simultaneous occurrence of active and inactive cells within parathyroid glands.     

Fine structure of the stretch receptor in the bursa copulatrix of the butterfly,Pieris rapae crucivora

Summary A pair of multipolar stretch-receptive neurons were found in the bursa copulatrix of the female cabbage white butterfly, Pieris rapae crucivora. The cell body of each neuron, about 10 m in diameter, lies on the edge of the muscular region in the antero-lateral wall of the corpus bursae. No special accessory structure, such as a receptor muscle, is associated with the neuron. The several dendrites extend radially into the muscle layer. The dendrites are ensheathed except for their terminal tips, and, on their course, they anchor repeatedly on the epithelial cells or the muscle fibers in such a manner that their basement membranes fuse together. While the ensheathed dendrite is usually 0.1–0.2 m in diameter, it often forms 1–2 m varicosities especially at anchor sites, so that it looks like a varicose, or beaded, chain. The varicosities contain a number of mitochondria, but only microtubules are found in the fine interconnecting parts of the dendrite. The naked dendritic tips terminate in the basement membrane of the epithelial cell. The varicosities, as well as naked tips, seem to be important for stimulus transduction in the sensory cell of this type.     

Ultrastructural duality of extrafusal fibers in a slow (tonic) skeletal muscle

Summary Ultrastructural and stereological assessment of the mature avian anterior latissimus dorsi (ALD) muscle showed that it contains two kinds of extrafusal fibers. This fine structural dichotomy of fiber types in the ALD correlated well with their previously reported histochemical duality. Distinct differences occur in sarcomere banding, myofibrillar area, sarcotubular and mitochondrial density, and in morphology of motor-nerve terminals. Both myofiber types in this muscle were interpreted as representing varieties of slow or tonic muscle fibers.Both fibers contain myofibrils that, despite differences in cross-sectional area, were large, irregular, and ribbon-shaped, typical of the Felderstruktur appearance of true slow fibers. Whereas the majority of fibers (type-1) are devoid of well-defined M-bands, the minor fiber population (type-2) exhibit prominent M-bands in the center of each sarcomere. In addition, type-1 tonic fibers contain a significantly lower mitochondrial and sarcotubular volume than the tonic fibers of type-2. While both fiber types exhibit motor-nerve terminals that are small, smooth and punctate in appearance, those on the type2 fibers often had a number of shallow postjunctional folds. Whether or not these two classes of extrafusal fiber in this muscle represent two separate and distinct types of motor units remains to be determined functionally.Supported by grants from the Medical Research Council and the Muscular Dystrophy Association of Canada. The author gratefully acknowledges the excellent technical assistance of Susan L. Shinn     

Type-VI collagen in the human iris and ciliary body

Summary The distribution of type-VI collagen in the human iris and ciliary body was investigated by means of immunohistochemical techniques and compared with that of type-IV collagen, fibronectin and laminin. As has been described for other tissues, type-VI collagen surrounds type-I and-III collagen fibers. The aggregated from of type-IV collagen (the long-spacing or curly collagen), which has already been described in the trabecular meshwork and sclera, was also observed at the ciliary muscle tips surrounding the anterior elastic tendons of this muscle. In addition, staining for type-VI collagen was seen directly adjacent to the basement membranes of the ciliary muscle cells, the iris muscles, the uveal vascular endothelia and nerves, but not adjacent to the epithelial basement membranes. The staining did not form a discrete line like the immunoreaction for type-IV collagen, but bundles of marked fibrils extended into the surrounding connective tissue. We assume that type-VI collagen similar to type-VII collagen forms part of an anchoring system for these tissues. As type-VII collagen has been described only in connection with epithelial basement membranes, both type-VI and type-VII collagens may represent anchoring fibrils, however for different tissue components.     

Architecture of the media of the arterial vessels in the dog brain: A scanning electron-microscopic study

Summary The architecture of the media of arterial vessels in dog brain was investigated using scanning electron microscopy. The arrangement and shape of the circularly-oriented smooth muscle cells varied with vessel diameter: The arteries (>100 m in diameter) had 4–10 layers of spindle-shaped smooth muscle cells; the muscular arterioles (30–100 m), 2–3 layers of spindle-shaped smooth muscle cells; the terminal arterioles (10–30 m), a compact layer of spindle-shaped smooth muscle cells with more dominant nodular or rod-like processes and thin lateral processes; and the precapillary arterioles (5–15 m), a less compact layer of branched smooth muscle cells.Longitudinally-oriented muscles were observed in the medio-adventitial border. The distribution and arrangement of these muscles varied with vessel size: in the large arteries (> 300 m in diameter), at the branching sites only; in the small arteries (100–300 m), at both the branching and non-branching sites; in the muscular arterioles, at both the branching and non-branching sites in a reticular arrangement with some muscle cells having an asteroid appearance; in the terminal aterioles, only asteroid-like muscle cells were found at the branching and non-branching sites.     

Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells

Summary Selective biotinylation of the apical or basolateral domains of confluent MDCK monolayers grown on polycarbonate filters with a water soluble biotin analog, sulfo-NHS-biotin, was employed to reveal strikingly distinct patterns of endogenous peripheral and integral membrane proteins. Peripheral proteins were found to be approximately fivefold more abundant with this procedure than integral membrane proteins, both on the apical and on the basolateral surface. The distinct apical and basal patterns were shown to depend upon the integrity of the monolayer; when the tight junctions were disrupted by preincubation in calcium-depleted medium, the patterns appeared practically indistinguishable. Two-dimensional gel electrophoresis demonstrated that only a very small percentage of the biotinylated proteins were found in similar amounts on both apical and basolateral domains. These results indicate that the sorting mechanisms that segregate apical and basolateral epithelial proteins are very strict. The simple procedure described here has clear advantages over other methods available to label apical and basal epithelial surface domains, namely, higher accessibility of the biotin probe to the basolateral membrane, possibility of purifying biotinylated proteins via immobilized streptavidin and minimal exposure of the researcher to isotopes. It should be very useful in characterizing the apical and basolateral protein compositions of other epithelial cells and in studies on the development of epithelial cell polarity.     

Concluding remarks

On the basis of symposium contributions onChlorella, Hibbertia, Eucalyptus, Ambrosia and on numerical approaches some fundamental problems of (bio)systematics, evolution, and taxonomic categories are discussed: Methods available for analysing affinities; conflicting evidence from phenetic, biochemical, cytogenetic and other analyses; further classification problems in cases of intermediacy, etc. While sibs of various levels and their natural hierarchy often can be objectively defined, this appears impossible for particular taxonomic levels itself (e. g. species). A single objective taxonomic system of organisms is unrealistic. Certain guiding lines for relative and practicable concepts of species and genus are proposed.Presented at the symposium Speciation and the Species Concept during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

Intercellular junctions in nerve-free hydra

Summary Epithelial cells of nerve-free hydra contain septate and gap junctions. In thin sections the gap junctions are characterized by a gap of 3–4 nm. Freeze-fracture demonstrates the presence of septate junctions and two further types of structures: (i) the E-type or inverted gap junctions with particles in an en plaque conformation appearing as a raised plateau on the E-face or as a depression on the P-face; (ii) structures morphologically similar to gap junctions in rat liver, containing particles on the P-face and corresponding pits on the E-face, both having hexagonal packing with a lattice constant of 8 nm. We propose that these structures are also gap junctions.     

β-Alanine metabolism and high salinity stress in the sea anemone,Bunodosoma cavernata

Summary During high salinity stress, -alanine accumulates to high levels in the sea anemone,Bunodosoma cavernata. Following a salinity increase from 26 to 40 -alanine increased 28-fold from 1.5 to 41.9 moles/g dry weight. Both whole animal studies and experiments with cell free homogenates indicate that under high salinity conditions an increase in the rate of -alanine synthesis from aspartic acid as well as a decrease in the rate of -alanine oxidation are responsible for the observed accumulation of -alanine. The rate of aspartic acid decarboxylation to -alanine is about 3 times greater in anemones acclimated to 40 than for those in normal salinity water (26). -alanine oxidation to CO₂ and acetyl-CoA proceeds 2.5 to 3 times slower in high salinity adaptedB. cavernata than in those acclimated to normal salinity. There is always a rapid degradation of uracil to -alanine, but this does not change with salinity.Abbreviations CASF cold acid soluble fraction - FAA free amino acids - MES 2(N-morpholino) ethane sulfonic acid - NPS ninhydrin positive substances - PCA perchloric acid - TCA trichloroacetic acid     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

Electrical and mechanical responses of chromatophore muscle fibers of the squid,Loligo opalescens,to nerve stimulation and drugs

Summary The muscle fibers of brown and red chromatophores in the skin of the squid, Loligo opalescens, respond to motor nerve stimulation with non-propagating excitatory postsynaptic potentials (e.p.s.p.'s) of fluctuating amplitude. Depending on the strength of stimulation several size classes of e.p.s.p.'s are found, indicating polyneuronal innervation. Facilitation and summation are minimal even though the reversal potential of the e.p.s.p.'s is close to zero.Acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) have no effect on membrane characteristics of the muscle fiber, but ACh greatly augments the spontaneous quantal release of transmitter [increase in the frequency of miniature postsynaptic potentials (m.p.s.p.'s)] and thereby causes tonic contraction (miniature tetanus). 5-HT reduces the frequency of miniature potentials and abolishes tonic contraction. Inhibition of cholinesterase by eserine does not affect the amplitude or time course of e.p.s.p.'s and of m.p.s.p.'s. High concentrations of cholinergic blocking agents (atropine, banthine) reduce the postsynaptic effects of nerve stimulation in some cases. The natural transmitter substance of the motoneurones may not be ACh. The action of 5-HT appears to be intracellular.Neighboring muscle fibers are electrically coupled through low resistance pathways. These are most likely provided by the close junctions that form part of the myo-muscular junctions. The specific membrane resistance of the regular muscle fiber membrane was found to range from 1,056 to 1,320 Ohm×cm², that of the close junctions ranges from 12.8 to 22.6 Ohm×cm². The area occupied by close junctions is small, however, and only 10% of the current injected into one cell passes into the next. Some of the e.p.s.p.'s observed in a given muscle fiber most likely represent the electrotonic spread of the e.p.s.p.'s of the neighbor fibers. Of the six classes of e.p.s.p.'s observed in some muscle fibers, only two may originate in these fibers themselves.Chromatophores in aged preparations often exhibit pulsations. These are caused by spike potentials arising within muscle fibers whose membranes have become electrically excitable. Each spike is preceded by a generator depolarization. The electrical coupling of neighboring muscle cells permits conduction of the spike potentials throughout the set of muscle fibers of a pulsating chromatophore. Altered conditions within such preparations also lead to tonic contractions and contractures that are not necessarily accompanied by electrical activity. Several arguments are presented in support of the hypothesis that the tonic condition of nerve terminals (characterized by enhanced spontaneous transmitter release) and of muscle fibers (characterized by inability to relax) is due to an abnormal condition of intracellular calcium (lack of Ca-binding by sarcoplasmic reticulum or other storage sites).No evidence could be found for an inhibitory innervation of the chromatophore muscles. The nerve-induced relaxation of tonically contracted muscle fibers is caused by the action of motoneurones.Preliminary experiments on muscle fibers of the anterior byssus retractor muscle of Mytilus support the hypothesis that the tonic behavior (catch) of other molluscan muscles is due to mechanisms similar to those found in the chromatophore muscles.This investigation was supported by Public Health Service Grant No. NB 04145 from the National Institute of Neurological Diseases and Blindness. We are grateful to the director of the Friday Harbor Laboratories, Prof. R. L. Fernald for providing space and facilities for this investigation.Supported by a Training Grant GM 1194 from the National Institute of General Medical Sciences.     

The α and γ subunits of 7S nerve growth factor are present in excess concentrations in the mouse submandibular gland

Summary The 7S nerve growth factor molecule, found in the mouse submandibular gland, is comprised of three distinct protein subunits named , and -NGF. In this paper, radioimmunoassays specific for each subunit were used to measure the concentrations of these subunits in homogenates of mouse submandibular gland. It was determined that there were excess concentrations of both the and subunits, more than enough to bind all of the -NGF in the gland to form 7S-NGF. The radioimmunoassay data was confirmed by gel filtration experiments. In the gel filtration experiments, the excess and subunits eluted at positions which would indicate that these excess subunits were free and not bound in the 7S-NGF complex. The identity of the excess and subunits was substantiated by ion exchange chromatography, isoelectric focusing polyacrylamide gels and immunoblotting experiments. In conclusion, there are considerable quantities of and subunits present in the submandibular gland which are not bound to -NGE The functional significance of these excess concentrations of the and subunits is not known.     

A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes

A novel approach was used to compare male and female recombination rates in wheat. Doubled haploid lines were developed from an F₁ using two distinct approaches: the anther-culture technique and the Hordeum bulbosum system, from which sets of lines were developed from male and female meioses, respectively. The genotype of the lines was established at RFLP and isozyme markers polymorphic on chromosomes of homoeologous groups 6 and 7, and male and female linkage maps were calculated using this information. The markers in one segment of chromosome 6B exhibited disturbed segregation frequencies in the anther-culture population. The male and female maps differed significantly in recombination frequency between some markers on two chromosomes, and these were consistent in direction within chromosomes and inconsistent in direction between chromosomes. In two of the four chromosomes studied the male map was much longer than the female map. These results suggest that significant differences may exist in male and female recombination frequencies in bread wheat which are specific to certain chromosomal segments but are inconsistent in direction between chromosomes. Other factors, such as environmental influences, may also be important in creating differences.     

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.     

Starch hydrolysing enzymes in the developing barley grain

Summary The enzymes -amylase (-1, 4-glucan 4-glucanohydrolase, 3.2.1.1), -amylase (-1,4-glucan maltohydrolase, 3.2.1.2) and phosphorylase (-1,4-glucan: orthophosphate glucosyltransferase, 2.4.1.1) were assayed in whole grains of barley throughout the maturation period. -amylase and phosphorylase had peaks of activity between 25 and 30 days after anthesis. On the other hand the activity of -amylase in both the available and latent forms reached a maximum value at 35 days after anthesis which did not decrease thereafter. -amylase activity was also assayed throughout development in the endosperm, aleurone, testa pericarp and embryo. Latent -amylase reached a constant maximum value in endosperm at 35 days but available -amylase reached a peak of activity at 25 days and then declined to zero at 45 days. Only latent -amylase was associated with the aleurone layer and activity rose to a maximum value at 35 days. The testa pericarp had mainly latent -amylase whose activity fell from an early maximum at 21 days to zero at 35 days. No hydrolytic activity was associated with the embryo. The phosphorylase activity was low and mainly associated with the endosperm fraction.