Augmented sympathetic tone alters muscle metabolism with exercise: lack of evidence for functional sympatholysis

Shoemaker, J. Kevin, Prasant Pandey, Michael D. Herr, DavidH. Silber, Qing X. Yang, Michael B. Smith, Kristen Gray, and LawrenceI. Sinoway. Augmented sympathetic tone alters muscle metabolismwith exercise: lack of evidence for functional sympatholysis. J. Appl. Physiol. 82(6):1932-1938, 1997.It is unclear whether sympathetic tone opposesdilator influences in exercising skeletal muscle. We examined highlevels of sympathetic tone, evoked by lower body negative pressure(LBNP, 60 mmHg) on intramuscular pH and phosphocreatine (PCr)levels (³¹P-nuclear magnetic resonance spectroscopy) duringgraded rhythmic handgrip (30 contractions/min; ~17, 34, 52 and 69%maximal voluntary contraction). Exercise was performedwith LBNP and without LBNP (Control). At the end of exercise, LBNPcaused lower levels of muscle pH (6.59 ± 0.09) comparedwith Control (6.78 ± 0.05; P < 0.05). PCr recovery, an index of mitochondrial respiration, was lessduring the recovery phase of the LBNP trial. Exercise mean arterialpressure was not altered by LBNP. The protocols were repeated withmeasurements of forearm blood flow velocity and deep venous samples(active forearm) of hemoglobin (Hb) saturation, pH, and lactate. WithLBNP, mean blood velocity was reduced at rest, during exercise, andduring recovery compared with Control (P < 0.05). Also, venous Hbsaturation and pH levels during exercise and recovery were lower withLBNP and lactate was higher compared with Control(P < 0.05). We concludethat LBNP enhanced sympathetic tone and reduced oxygen transport. Athigh workloads, there was a greater reliance on nonoxidativemetabolism. In other words, sympatholysis did not occur.

     

Differential manifestation of seed mortality induced by seed-specific expression of the gene for diphtheria toxin A chain in Arabidopsis and tobacco

Summary A pea vicilin promoter-diphtheria toxin A (DTx-A) chain gene fusion was introduced into Arabidopsis and tobacco. The chimeric Dtx-A gene behaves as a dominant, seed-lethal, Mendelian factor, and the segregation ratios are consistent with the numbers of integrated copies as revealed by Southern blotting. Germination deficiency results from distinct developmental abnormalities, thus allowing genetic dissection of seed development. The endosperm is affected first in both species. In Arabidopsis, full cellularization of the initially syncytial endosperm does not take place, which results in shrinkage and a shriveled appearance of the mature dry seed. The embryo, which appears structurally normal and lacks visible lesions, ceases to develop at the partially recurved cotyledon stage and does not use the remaining endosperm. In tobacco, peripheral degeneration and premature termination of cellular endosperm development occurs at the cotyledon initiation stage. Lesions appear in the cotyledons at the advanced cotyledon stage, but the embryo continues to grow and attains nearly the same size and level of differentiation as mature wild-type embryos before degeneration and intracellular disintegration take place throughout. Accumulation of protein bodies and other cytoplasmic inclusions is very limited and occurs only in few cells. The timing and distribution of lesions follow a pattern typical for accumulation of protein bodies in wild-type seeds. These observations are consistent with expression of the vicilin promoter in the enlargement phase of cell differentiation. A novel tissue interaction arises, when the embryo uses up all the arrested endosperm: the embryo proves to be capable of absorbing the parenchyma layers of the integument, which are normally obliterated by, and incorporated into, the endosperm. The mature seed thus consists of a seed coat of one rigid cell layer, and a degenerated embryo. The genetic ablation technique has thus contributed to the establishment of the sequence of events and elucidation of the role of different cell lineages and tissues in seed development.     

Structural characterization of the melano-macrophage centres (MMC) of goldfish Carassius auratus

In the present work we have studied the organization of melano-macrophage centres (MMCs) in the peripheral lymphoid organs, including spleen, pro- and mesonephros, of the goldfish, Carassius auratus, in an attempt to clarify their cellular composition, origins and functional relationships. Histological analysis demonstrated a similar organization in the three organs on the basis of closely packed phagocytic cells containing abundant pigment. The MMCs of Carassius auratus are found throughout the parenchyma of spleen and kidney and show a close association with the vascular system, i.e. splenic ellipsoids, sinusoids of red pulp and renal blood sinuses. They exhibit distinct degree of development from small groups of actively phagocytic macrophages to large, totally or partially encapsulated centres, where effete phagocytic cells are filled by cell debris. Ultrastructural and histochemical data suggest that the main inclusion observed in the MMCs of Carassius auratus is lipofuscin. Haemosiderin occurs in lesser amounts and melanin is almost restricted to kidney MMCs,--mainly mesonephros--. Our results suggest various non-specific physiological roles for the teleost MMCs, including tissue breakdown and erythrocyte catabolism.     

Biochemical and morphological characterization of the intra-acrosomal antigen SP-10 from human sperm

The human sperm protein SP-10 was previously defined as a "primary vaccine candidate" by a World Health Organization Taskforce on Contraceptive Vaccines. By one- and two-dimensional immunoblots, we show that SP-10, extracted from ejaculated human sperm, demonstrated a polymorphism of immunogenic peptides from 18 to 34 kDa, a pattern that was conserved from individual to individual and was not altered by reducing agents. The majority of the antigenic peptides possessed isoelectric points of approximately 4.9. Immunocytochemistry on testis sections indicated that SP-10 was localized to round spermatids and spermatozoa within the adluminal compartment of the seminiferous epithelium. Immunofluorescence showed that SP-10 was not associated with the surface of acrosome-intact, ejaculated sperm. Light and electron microscopic immunocytochemistry localized SP-10 throughout the acrosome, and electron microscopic evidence demonstrated a bilaminar array in association with the inner aspect of the outer acrosomal membrane and the outer aspect of the inner acrosomal membrane. After induction of the acrosome reaction with the ionophore A23187, SP-10 remained displayed on the sperm head in association with the inner acrosomal membrane and equatorial segment. The results indicate that the MHS-10 monoclonal antibody may be used as a marker of acrosome development in the human and as a probe to evaluate acrosome status. The results also support the hypothesis that inhibition of sperm-egg interaction by anti-SP-10 monoclonal antibody may occur as a result of antigen exposure following the acrosome reaction.     

Inflammatory changes in the epididymis after vasectomy in the Lewis rat

The epididymides of Lewis rats were studied at intervals up to 7 months after vasectomy, vasectomy followed 3 months later by vasovasostomy, or sham operations. Epididymal histology was related to testicular alterations and to serum antisperm antibodies as determined with an enzyme-linked immunosorbent assay. In vasectomy and vasovasostomy groups. 13 of 33 rats had testicular alterations, which consisted mainly of pronounced depletion of germ cells. Over half of the rats with testicular alterations also had severe epididymal lesions that included interstitial changes characteristic of an inflammatory response. These consisted of aggregates of mononuclear cells, including lymphocytes, plasma cells, and macrophages. The lumina of epididymides with interstitial changes contained polymorphonuclear leukocytes and/or macrophages. All animals with altered testes had greatly decreased numbers of epididymal sperm. In many instances, the lumen of the proximal cauda epididymidis was collapsed, and columnar cells of the epididymal epithelium contained many very large lysosomes. The distal cauda epididymidis was distended with sperm and debris. None of the rats that lacked testicular alterations showed epididymal changes. Mean serum antisperm antibody levels were significantly higher for rats with epididymal interstitial changes than for animals without such epididymal alterations. Infiltrations of inflammatory cells into the epididymal interstitium and lumen are part of the constellation of changes that occurs after immunization with testicular homogenates to produce experimental allergic orchitis. The observations reported here support the hypothesis that reproductive tract alterations after vasectomy in this model have an immune basis.     

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.     

Identification of Sphingolipid Metabolites That Induce Obesity via Misregulation of Appetite,Caloric Intake and Fat Storage in Drosophila

Obesity is defined by excessive lipid accumulation. However, the active mechanistic roles that lipids play in its progression are not understood. Accumulation of ceramide, the metabolic hub of sphingolipid metabolism, has been associated with metabolic syndrome and obesity in humans and model systems. Here, we use Drosophila genetic manipulations to cause accumulation or depletion of ceramide and sphingosine-1-phosphate (S1P) intermediates. Sphingolipidomic profiles were characterized across mutants for various sphingolipid metabolic genes using liquid chromatography electrospray ionization tandem mass spectroscopy. Biochemical assays and microscopy were used to assess classic hallmarks of obesity including elevated fat stores, increased body weight, resistance to starvation induced death, increased adiposity, and fat cell hypertrophy. Multiple behavioral assays were used to assess appetite, caloric intake, meal size and meal frequency. Additionally, we utilized DNA microarrays to profile differential gene expression between these flies, which mapped to changes in lipid metabolic pathways. Our results show that accumulation of ceramides is sufficient to induce obesity phenotypes by two distinct mechanisms: 1) Dihydroceramide (C_14:0) and ceramide diene (C_14:2) accumulation lowered fat store mobilization by reducing adipokinetic hormone- producing cell functionality and 2) Modulating the S1P: ceramide (C_14:1) ratio suppressed postprandial satiety via the hindgut-specific neuropeptide like receptor dNepYr, resulting in caloric intake-dependent obesity.     

Tetraspanin CD9 Promotes the Invasive Phenotype of Human Fibrosarcoma Cells via Upregulation of Matrix Metalloproteinase-9

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression.     

Rückkehr der Finnwale in die Antarktis

Return of the fin whales to Antarctica After their near-extirpation by commercial whaling, fin whales of the Southern Hemisphere have recently been observed to aggregate in large numbers along the islands of the Antarctic Peninsula. A dedicated research program currently employs a suite of non-lethal cetacean research methods to investigate population structure, abundance, habitat use and migratory origins in order to gain insights into population status and recovery.