Insulin resistance in denervated skeletal muscle. Inability of insulin to stimulate dephosphorylation of glycogen synthase in denervated rat epitrochlearis muscles

We have investigated the effects of insulin and motor denervation on the phosphorylation of glycogen synthase in skeletal muscle. Rat epitrochlearis muscles were denervated in vivo 3 days before the contralateral and denervated muscles were incubated in vitro with 32Pi to label sites in glycogen synthase. The 32P-labeled synthase was rapidly immunoprecipitated from extracts under conditions which prevented changes in the phosphorylation state of the enzyme. When 32P-labeled synthase from contralateral muscles was cleaved with CNBr, essentially all of the 32P was recovered in two fragments, denoted CB-1 and CB-2. Incubating these muscles with insulin decreased the 32P content of each fragment by approximately 25%, indicating that the hormone stimulated dephosphorylation of at least two sites. Peptide mapping by reverse phase high performance liquid chromatography was performed to resolve phosphorylation sites more completely. The results suggest that the enzyme was phosphorylated in sites 1a, 1b, 2, 3(a+b+c), and 5. Insulin stimulated dephosphorylation of sites in peptides presumed to contain sites 1b, 2, and 3(a+b+c). Synthase from denervated muscles appeared to contain the same amount of phosphate as enzyme from contralateral muscles, and denervation did not detectably affect the distribution of 32P within the subunit. However, denervation abolished the effect of insulin on decreasing the 32P content of synthase. The results indicate that the insulin resistance induced by denervation involves a loss in the ability of insulin to stimulate dephosphorylation of glycogen synthase.     

Catalytic site of rabbit glycogen synthase isozymes. Identification of an active site lysine close to the amino terminus of the subunit

Rabbit skeletal muscle glycogen synthase was inhibited by pyridoxal 5'-phosphate and irreversibly inactivated after sodium borohydride reduction of the enzyme-pyridoxal-P complex. The irreversible inactivation by pyridoxal-P was opposed by the presence of the substrate UDP-glucose. With [3H]pyridoxal-P, covalent incorporation of 3H label into the enzyme could be monitored. UDP-glucose protected against 3H incorporation, whereas glucose-6-P was ineffective. Peptide mapping of tryptic digests indicated that two distinct peptides were specifically modified by pyridoxal-P. One of these peptides contained the NH2-terminal sequence of the glycogen synthase subunit. Chymotrypsin cleavage of this peptide resulted in a single-labeled fragment with the sequence: Glu-Val-Ala-Asn-(Pyridoxal-P-Lys)-Val-Gly-Gly-Ile-Tyr. This sequence is identical to that previously reported (Tagaya, M., Nakano, K., and Fukui, T. (1985) J. Biol. Chem. 260. 6670-6676) for a peptide specifically modified by a substrate analogue and inferred to form part of the active site of the enzyme. Sequence analysis revealed that the modified lysine was located at residue 38 from the NH2 terminus of the rabbit muscle glycogen synthase subunit. An analogous tryptic peptide obtained from the rabbit liver isozyme displayed a high degree of sequence homology in the vicinity of the modified lysine. We propose that the extreme NH2 terminus of the glycogen synthase subunit forms part of the catalytic site, in close proximity to one of the phosphorylated regions of the enzyme (site 2, serine 7). In addition, the work extends the known NH2-terminal amino acid sequences of both the liver and muscle glycogen synthase isozymes.     

Suberized bundle sheaths in grasses (Poaceae) of different photosynthetic types. II. Apoplastic permeability

Summary The relative hydraulic conductivities of major and minor longitudinal veins, and the apoplastic permeability of the bundle sheaths surrounding all longitudinal and transverse veins were investigated in representatives of the C₃, C₄/NAD-ME, C₄/NAD-ME/PCK intermediate, C₄/PCK and C₄/NADP-ME photosynthetic types. Using the Hagen-Poiseuille equation and measurements of tracheary element diameters, the number of elements in each vein type and the numbers of each vein type, we calculated that 87–99% of the water flow in a longitudinal direction would be expected to occur in the major veins. The permeability of the mestome sheaths and parenchymatous bundle sheaths surrounding the veins was tested using the negatively-charged, fluorescent dye, trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS). This dye proved nontoxic to plant tissue at a concentration of 0.5%, according to a deplasmolysis test with onion epidermal strips. The PTS concentration achieved in the tested grass leaves was about 0.035%, well below the toxic limit. When a solution of PTS was fed to the leaves by means of a basal cut, the dye moved into the veins of all orders. From there, it moved outward into the surrounding tissues, indicating that the sheaths surrounding the veins of all orders in all species tested were permeable. Therefore, contrary to previous predictions based on structural observations and some tracer studies, bundle sheaths with suberized cell walls do not function as endodermal layers.     

Allometric shape change and heterochrony in the freeliving coral Trachyphyllia bilobata (Duncan)

Regression analysis has been used to study the relationship between age, size, shape, and surface area in two ancestral-descendant populations of the Neogene Caribbean coral Trachyphyllia bilobata. Analyses of the relationship between size and age show that the relationship is isometric and that little difference occurs between populations in mean corallite length or height and in their rates of growth. Onset of columella growth is significantly earlier, however, in the descendant population. Studies of the relationship between size and shape show that growth is allometric, with shape change occurring in both corallum elongation and pinching of the corallite wall during ontogeny. In the descendant population, pinching and elongation initiate earlier in the ontogeny of the coral. These results suggest that the evolutionary development of the meandroid form in freeliving corals has been accomplished by heterochrony, involving a complex set of disassociated peramorphic changes in ontogeny accompanied by paedomorphic changes in astogeny. Further analyses show that the observed heterochronic changes serve to decrease corallum surface area which may in turn enhance sediment removal and nutrition in unstable habitats.     

Immature maize spikelets develop and produce pollen in culture

Immature maize spikelets have been successfully grown in vitro. Culture conditions were refined to maximize development of normal pollen grains. Kinetin was not required for normal development, in contrast to the absolute requirement for this plant growth regulator for in vitro tassel development. Development occured in all stages sampled, from premeiosis to postvacuolation, and there was no lag in progression through the various stages of development as compared to greenhouse-grown material. Cultured spikelets produced pollen that appeared morphologically normal, accumulated starch and had the normal two sperm nuclei and single vegetative nucleus.     

Interactions between freshwater snails and tadpoles: competition and facilitation

Summary Freshwater snails and anuran tadpoles have been suggested to have their highest population densities in ponds of intermediate size where abiotic disturbance (e.g. desiccation) is low and large predators absent. Both snails and tadpoles feed on periphytic algae and, thus, there should be a large potential for competitive interactions to occur between these two distantly related taxa. In a field experiment we examined the relative strength of competition between two closely related snail species, Lymnaea stagnalis and L. peregra, and between L. stagnalis and tadpoles of the common frog, Rana temporaria. Snail growth and egg production and tadpole size at and time to metamorphosis were determined. Effects on the common food source, periphyton, were monitored with the aid of artificial substrates. Periphyton dry weight was dramatically reduced in the presence of snails and/or tadpoles. There were no competitive effects on growth or egg production of the two snail species when they were coexisting. Mortality of L. peregra was high (95%) after reproduction, but independent of treatment. Growth of L. stagnalis was reduced only at the highest tadpole densities, whereas egg production was reduced both by intraspecific competition and by competition with tadpoles. Differences in egg production were retained after tadpole metamorphosis. Tadpole larval period increased, weight of metamorphosing frogs decreased and growth rate was reduced as a function of increasing tadpole density. However, contrary to expectation, snails had a positive effect on tadpole larval period, weight and growth rate. Further, in experimental containers without snails there was a dense growth of the filamentous green alga Cladophora sp. We suggest that the facilitative effects of snails on tadpoles are due to an indirect mutualistic mechanism, involving competition between food sources of different quality (microalgae and Cladophora sp.) and tadpoles being competitively dominant over snails for the preferred food source (microalgae). In the presence of tadpoles snails will be forced to feed on low-quality Cladophora, increasing nutrient turnover rates, which results in enhanced productivity of microalgae, increasing tadpole food resources. Thus, tadpoles have a negative effect on snails through resource depression, while snails facilitate tadpole growth through an indirect enhancement of food availability.     

Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.