Glyconeogenic and glycogenic enzymes in chronically active and normal skeletal muscle

The chronically active (pseudomyotonic) gastrocnemius muscle in the C57B16J dy2J/dy2J mouse contains both elevated lactate and glycogen as well as fibers that have high amounts of glycogen and enhanced glyconeogenic activity. In the present study we analyze the activities of some key glyconeogenic enzymes to assess the causes of elevated muscle glycogen and to determine the pathway for glycogen synthesis from lactate. Glycogen synthase, malate dehydrogenase, phosphoenolpyruvate carboxykinase, and malic enzyme were all elevated in homogenates of the chronically active muscle. Activities of glycogen phosphorylase and fructose 1,6-bisphosphatase were decreased in whole muscle homogenates. Histochemistry demonstrated that the high-glycogen fibers were typically fast-twitch glycolytic fibers that had high glycogen synthase, glycogen phosphorylase, and malic enzyme activities. Malate dehydrogenase activity followed succinate dehydrogenase activity and did not correlate to high-glycogen fibers. Thus the high-glycogen fibers have an elevated enzymatic capacity for glycogen synthesis from lactate, and the pathway may involve use of the pyruvate kinase bypass enzymes.     

Augmentation of NK activity and/or macrophage-mediated cytotoxicity in the liver by biological response modifiers including human recombinant interleukin 2

Summary Administration of several biological response modifiers (BRMs) to mice strongly augmented natural killer (NK) activity of leukocytes isolated from the liver. This augmentation of NK activity was induced by two synthetic molecules (MVE-2 and poly ICLC), by two BRMs of bacterial origin (formalin-fixed Propionibacterium acnes: P. acnes and a streptococcal cell wall preparation designated OK-432), as well as a single injection of human recombinant interleukin-2 (hrIL 2). All of these BRMs augmented NK activity in the liver to a greater degree than in the spleen. In addition, adherent leukocytes (>90% macrophages) isolated from the liver following P. acnes administration also exhibited augmented macrophage-mediated cytotoxicity. This cytotoxicity was characterized as macrophage mediated and distinguished from NK activity, on the basis of adherence purification, kinetics of cytotoxicity, and target cell selectivity. The results demonstrate that a variety of BRMs induce augmented natural immunity in the liver and suggest that such organ-associated immune responses may play an important role in the antimetastatic effects of BRMs.This project has been funded at least in part with Federal funds from the Department of Health and Human Services, under contract number NO1-CO-23910 with Program Resources, Inc. the contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. GovernmentSupported by Grant SA 364/1-1 from the Deutsche Forschungsgemeinschaft, FRG     

Frequency-dependent variation in the two-dimensional beam pattern of an echolocating dolphin

Recent recordings of dolphin echolocation using a dense array of hydrophones suggest that the echolocation beam is dynamic and can at times consist of a single dominant peak, while at other times it consists of forward projected primary and secondary peaks with similar energy, partially overlapping in space and frequency bandwidth. The spatial separation of the peaks provides an area in front of the dolphin, where the spectral magnitude slopes drop off quickly for certain frequency bands. This region is potentially used to optimize prey localization by directing the maximum pressure slope of the echolocation beam at the target, rather than the maximum pressure peak. The dolphin was able to steer the beam horizontally to a greater extent than previously described. The complex and dynamic sound field generated by the echolocating dolphin may be due to the use of two sets of phonic lips as sound sources, or an unknown complexity in the sound propagation paths or acoustic properties of the forehead tissues of the dolphin.     

Augmentation of NK cell activity in tissue specific sites by liposomes incorporating MTP-PE

Liposomes incorporating a variety of immunomodulators have been shown to activate macrophages and monocytes for tumoricidal activity both in vivo and in vitro. We report that in addition to the activation of macrophages, the i.v. injection of liposomes (multilamellar vesicles) that have encapsulated muramyl tripeptide-phosphatidylethanolamine (MTP-PE) could also augment interstitial natural killer (NK) cell activity in the lung and the liver. In contrast, liposomes incorporating MTP-PE were unable to augment NK cell activity in the spleen, peripheral blood, or peritoneal cavity (after i.p. injection). In addition, liposomes did not augment splenic NK cell activity in vitro. This suggests that the augmentation of NK cell activity in the lungs and liver was not due to direct effects of the liposomes but may have been a secondary effect mediated by a monokine. The augmentation of pulmonary NK cell activity was paralleled by the nonspecific immunoprophylaxis of experimental pulmonary metastases. The augmented NK cell activity, as well as the enhanced nonspecific immunoprophylactic activity, was reduced by pretreatment of the mice with anti-asialo GM1 antiserum. Thus, the augmentation of organ-associated NK cell activity by liposomes incorporating MTP/PE plays a major role in the host's increased resistance to the formation of experimental metastases.     

Genetic deletion of trkB.T1 increases neuromuscular function

Neurotrophin-dependent activation of the tyrosine kinase receptor trkB.FL modulates neuromuscular synapse maintenance and function; however, it is unclear what role the alternative splice variant, truncated trkB (trkB.T1), may have in the peripheral neuromuscular axis. We examined this question in trkB.T1 null mice and demonstrate that in vivo neuromuscular performance and nerve-evoked muscle tension are significantly increased. In vitro assays indicated that the gain-in-function in trkB.T1(-/-) animals resulted specifically from an increased muscle contractility, and increased electrically evoked calcium release. In the trkB.T1 null muscle, we identified an increase in Akt activation in resting muscle as well as a significant increase in trkB.FL and Akt activation in response to contractile activity. On the basis of these findings, we conclude that the trkB signaling pathway might represent a novel target for intervention across diseases characterized by deficits in neuromuscular function.     

Hyporesponsiveness to augmentation of murine natural killer cell activity in different anatomical compartments by multiple injections of various immunomodulators including recombinant interferons and interleukin 2

Augmentation of natural killer (NK) cell activity has been observed after the single administration of a wide variety of biological response modifiers (BRM); however, multiple injections of BRM have resulted in hyporesponsiveness to NK augmentation in both preclinical and clinical studies. In these studies, hyporesponsiveness to augmentation of NK cell activity occurred after multiple injections of interferon (IFN recombinant human IFN-alpha A/D and recombinant IFN-gamma) and interleukin 2 and was found to be systemic (lungs, liver, blood, and spleen). In contrast, hyporesponsiveness to augmentation by multiple injections of maleicanhydride divinyl ether (MVE-2) or Propionibacterium acnes was limited to the spleen and peripheral blood lymphocytes, with continued augmentation of NK cell activity in the peritoneum, lungs, and liver. Despite the hyporesponsiveness to augmentation of NK activity by multiple IFN injections, NK activity could still be augmented by a single injection of another BRM. The NK cell hyporesponsiveness induced in the spleen by MVE-2 was also reversed by a single administration of IFN or polyinosinic-polycytidylic and poly-L-lysine solubilized by carboxymethyl cellulose but not by OK-432 or P. acnes. These results demonstrate that the nature of the hyporesponsiveness to NK augmentation, which is induced by multiple treatments with BRM, varies with the type of agent. The noncytokine BRM that were studied induced hyporesponsiveness only in specific lymphoid compartments but not in major nonlymphoid organs, whereas cytokine BRM induced a systemic hyporesponsiveness. The hyporesponsive state induced by the different types of BRM, also varied in regard to the pattern of susceptibility to augmentation of NK activity by unrelated BRM.     

The Structure of Plant Cell Walls: III. A Model of the Walls of Suspension-cultured Sycamore Cells Based on the Interconnections of the Macromolecular Components

Degradative enzymes have been used to obtain defined fragments of the isolated cell walls of suspension-cultured sycamore cells. These fragments have been purified and structurally characterized. Fragments released from endopolygalacturonase-pretreated cell walls by a purified endoglucanase and the fragments extracted from these walls by urea and alkali provide evidence for a covalent connection between the xyloglucan and pectic polysaccharides. Fragments released by a protease from endopolygalacturonase-endoglucanase-pretreated cell walls provide evidence for a covalent connection between the pectic polysaccharides and the structural protein of the cell wall. Based on these interconnections and the strong binding which occurs between the xyloglucan and cellulose, a tentative structure of the cell wall is proposed.     

Altered Diaphorase Activity in Optochin-resistant Pneumococci

Methylene blue (MB) reductase activities of washed cell suspensions and of cell-free extracts prepared from optochin-resistant mutant pneumococci were two times greater than and four to eight times more resistant to optochin inhibition than those of similar preparations from the optochin-sensitive parent strains. With whole cells, optochin hydrochloride was approximately four times more potent than quinine hydrochloride in inhibiting MB reductase activity. However, with cell-free extracts, both drugs had similar inhibitory activities. Glucose uptake was not affected by optochin hydrochloride, and both optochin-sensitive and optochin-resistant pneumococci had similar glucose uptake patterns. Diaphorase activities of cell-free extracts prepared from optochin-resistant pneumococci were two times greater than and four to eight times more resistant to optochin inhibition than those of cell-free extracts prepared from the optochin-sensitive parent strains. Flavin concentrations of cell-free extracts prepared from optochin-sensitive and optochin-resistant strains were similar.     

In vitro characteristics of metastatic variant subclones of restricted genetic origin

We have studied several metastatic variant cell lines derived from a common clonal origin and their transformed and untransformed parental cell lines. A number of in vitro characteristics were examined for each tumor line and these properties were correlated with the ability of the tumor cells to form pulmonary nodules in an experimental metastasis assay. Direct correlations with metastatic behavior in the lung colony assay were found to exist with the amount of cell-bound Concanavalin A and the procoagulant activities of cell lysates. In vitro parameters that did not correlate with the metastatic phenotype were: population doubling times in culture, saturation density achieved in culture, the number of colony-forming cells shed from confluent cultures, rates of cellular attachment to homotypic or heterotypic cell monolayers, plasminogen-activator production and procoagulant activity produced in serum-free conditioned medium.     

Identification and characterization of a novel gene disrupted by a pericentric inversion inv(4)(p13.1q21.1) in a family with cleft lip

Cleft lip with or without cleft palate is a common birth defect affecting 1 in every 700 live births. Several genetic loci are believed to be involved in the pathogenesis of syndromic and non-syndromic clefting. We identified a pericentric inversion of chromosome 4, inv(4)(p13q21) that segregates with cleft lip in a two-generation family. By using a combination of fluorescence in situ hybridization, yeast artificial chromosome, bacterial artificial chromosome contig mapping, and database searching we mapped and sequenced the inversion breakpoint region. The pericentric inversion disrupts a gene (ACOD4) on chromosome 4q21 that codes for a novel acyl-CoA desaturase enzyme. The 3.0 kb human ACOD4 cDNA spans approximately 170 kb and is composed of five exons of ACOD4. The inversion breakpoint is located in the second exon. The 3.0 kb mRNA is expressed at high level in fetal brain; a lower expression level was found in fetal kidney. No expression of ACOD4 was detected in fetal lung or liver or in adult tissues. The five exons code for a protein of 330 amino acids, with a predicted molecular weight of 37.5 kDa. The protein is highly similar to acyl-CoA desaturases from Drosophila melanogaster to Homo sapiens. The catalytically essential histidine clusters and the potential transmembrane domains are well conserved.