E-1020, a water soluble imidazopyridine,has direct effects on Ca2+-dependent force and ATP hydrolysis of canine and bovine cardiac myofilaments

E-1020 is a cardiotonic agent that acts as a cyclic-AMP phosphodiesterase inhibitor but also may have actions which alter myofilament response to Ca²⁺. To identify direct actions of E-1020 on cardiac contractile proteins, effects of E-1020 on myofibrillar Ca²⁺ dependent MgATPase and force generation in chemically skinned fiber bundles were measured. In bovine cardiac myofibrils, E-1020 (100 M) significantly increased myofilament Ca²⁺ sensitivity and Ca²⁺-dependent ATPase activity at submaximal pCa values. At pCa 6.75, E-1020 significantly increased ATPase activity in bovine (10–100 pM) and canine (1–100 pM) cardiac myofibrils but had no effect on rat cardiac myofibrils. Moreover, in one population of canine ventricular fiber bundles, E-1020 (0.0–10 M) significantly increased isometric tension at pCa 6.5 and 6.0, whereas in another population of bundles E-1020 had no effect on tension. In no case was resting (pCa 8.0) or maximal tension (pCa 4.5) increased by E-1020. Measurements of Ca²⁺ binding to canine ventricular skinned fiber preparations demonstrated that E-1020 does not alter the affinity of myofilament troponin C for Ca²⁺. We conclude that part of the mechanism by which E-1020 acts as an inotropic agent may involve alterations in the responsiveness of contractile proteins to Ca²⁺. The lack of effect of E-1020 on some preparations may be dependent on isoform populations of myofilament proteins.     

Changes in PKB/Akt and calcineurin signaling during recovery in atrophied soleus muscle induced by unloading

Protein kinase B [PKB, also known as Akt (PKB/Akt)] and calcineurin (CaN) are postulated to play important roles in integrating intracellular signaling in skeletal muscle in response to disuse and increased muscle loading. These experiments investigated changes in signal transduction of the downstream pathways of PKB/Akt and CaN during recovery following disuse-induced muscle atrophy. A 10-day period of hindlimb unloading (HLU) via tail suspension (male rats) was used to produce soleus muscle atrophy. Muscle recovery was achieved by returning animals to normal ambulation for 3-10 days. HLU resulted in significant muscle atrophy and a slow-to-fast fiber transition as revealed by appearance of type IId/x and IIb myosin heavy chain (MHC) isoforms. Muscle mass in HLU animals recovered to control (Con) levels after 10 days of reloading, but the fast-to-slow shift in muscle MHC was incomplete, as indicated by the continued presence of type IId/x MHC. Ten days of HLU resulted in a significant decrease (-43%) in muscle levels of phosphorylated PKB/Akt. In contrast, muscle levels of phosphorylated PKB/Akt were greater (+56%) in HLU than in Con animals early after the onset of reloading (3 days). Soleus levels of phosphorylated p70S6K were significantly higher (+26%) in HLU animals after 3 days of muscle reloading. Muscle levels of phosphorylated PKB/Akt and phosphorylated p70S6K returned to Con levels by day 10 of recovery. Moreover, muscle CaN levels were significantly higher than Con levels after 10 days of muscle reloading. These findings are consistent with the hypothesis that PKB/Akt and its downstream mediators are active in the regrowth of muscle mass during the early periods of recovery from muscle atrophy. Our data support the concept that CaN is involved in muscle remodeling during the later phases of recovery from disuse muscle atrophy.     

Phylogenetic analysis using insertion sequence fingerprinting in Escherichia coli

Chromosomal DNA from 23 closely related, pathogenic strains of Escherichia coli was digested and probed for the insertion sequences IS1, IS2, IS4, IS5, and IS30. Under the assumption that elements residing in DNA restriction fragments of the same apparent length are identical by descent, parsimony analysis of these characters yielded a unique phylogenetic tree. This analysis not only distinguished among bacterial strains that were otherwise identical in their biochemical characteristics and enzyme electrophoretic mobilities, but certain aspects of the topology of the tree were consistent across several unrelated insertion elements. The distribution of IS elements was then reexamined in light of the inferred phylogenetic relationships to investigate the biological properties of the elements, such as rates of insertion and deletion, and to discover apparent recombinational events. The analysis shows that the pattern of distribution of insertion elements in the bacterial genome is sufficiently stable for epidemiological studies. Although the rate of recombination by conjugation has been postulated to be low, at least two such events appear to have taken place.      

Linkages between phosphorus transformations and carbon decomposition in a forest soil

Phosphorus mineralization is chemically coupled with organic matter (OM) decomposition in surface horizons of a mixed-conifer forest soil from the Sierra Nevada, California, and is also affected by the disturbance caused by forest harvesting. Solution¹³C nuclear magnetic resonance (NMR) spectroscopy of NaOH extracts revealed a decrease of O-alkyl and alkyl-C fractions with increasing degree of decomposition and depth in the soil profile, while carbonyl and aromatic C increased. Solid-state¹³C-NMR analysis of whole soil samples showed similar trends, except that alkyl C increased with depth. Solution³¹P-NMR indicated that inorganic P (P₁) increased with increasing depth, while organic-P (P_o) fractions decreased. Close relationships between P mineralization and litter decomposition were suggested by correlations between P₁ and C fractions (r = 0.82, 0.81, –0.87, and –0.76 for carbonyl, aromatic, alkyl and O-alkyl fractions, respectively). Correlations for diester-P and pyrophosphate with O-alkyl (r = 0.63 and 0.84) and inverse correlations with aromatics (r = –0.74 and –0.72) suggest that mineralization of these P fractions coincides with availability of C substrate. A correlation between monoester P and alkyl C (r = 0.63) suggests mineralization is linked to breakdown of structural components of the plant litter. NMR analyses, combined with Hedley-P fractionation, suggest that post-harvest buildup of labile P in decomposed litter increases the potential for leaching of P during the first post-harvest season, but also indicates reduced biological activity that transports P from litter to the mineral soil. Thus, P is temporarily stored in decomposed litter, preventing its fixation by mineral oxides. In the mineral horizons,³¹P-NMR provides evidence of decline in biologically-available P during the first post-harvest season.     

Improved method for bile collection in unrestrained conscious rats.

We describe an improved method for continuous collection of bile from unrestrained rats. Use of an externally accessible, continuous-loop cannula when cannulating the common bile duct allows for full recovery from anesthetic effects and maintenance of a normal bile salt pool until the cannula loop is cut. Bile resulting from the cut cannula is diverted into a surgically implanted glass collection vessel and removed periodically via an externalized sampling port. Bile flow over a 24-hour collection period averaged 0.98 +/- 0.04 ml/hr (Mean +/- SEM, n = 9) with no gross pathological changes noted upon necropsy. This technique offers the capability of reestablishing conditions as close to physiologic as possible postsurgery to minimize potential artifacts during bile collection.     

Lacrimal gland development and Fgf10-Fgfr2b signaling are controlled by 2-O- and 6-O-sulfated heparan sulfate

Heparan sulfate, an extensively sulfated glycosaminoglycan abundant on cell surface proteoglycans, regulates intercellular signaling through its binding to various growth factors and receptors. In the lacrimal gland, branching morphogenesis depends on the interaction of heparan sulfate with Fgf10-Fgfr2b. To address if lacrimal gland development and FGF signaling depends on 2-O-sulfation of uronic acids and 6-O-sulfation of glucosamine residues, we genetically ablated heparan sulfate 2-O and 6-O sulfotransferases (Hs2st, Hs6st1, and Hs6st2) in developing lacrimal gland. Using a panel of phage display antibodies, we confirmed that these mutations disrupted 2-O and/or 6-O but not N-sulfation of heparan sulfate. The Hs6st mutants exhibited significant lacrimal gland hypoplasia and a strong genetic interaction with Fgf10, demonstrating the importance of heparan sulfate 6-O sulfation in lacrimal gland FGF signaling. Altering Hs2st caused a much less severe phenotype, but the Hs2st;Hs6st double mutants completely abolished lacrimal gland development, suggesting that both 2-O and 6-O sulfation of heparan sulfate contribute to FGF signaling. Combined Hs2st;Hs6st deficiency synergistically disrupted the formation of Fgf10-Fgfr2b-heparan sulfate complex on the cell surface and prevented lacrimal gland induction by Fgf10 in explant cultures. Importantly, the Hs2st;Hs6st double mutants abrogated FGF downstream ERK signaling. Therefore, Fgf10-Fgfr2b signaling during lacrimal gland development is sensitive to the content or arrangement of O-sulfate groups in heparan sulfate. To our knowledge, this is the first study to show that simultaneous deletion of Hs2st and Hs6st exhibits profound FGF signaling defects in mammalian development.     

EDC3 phosphorylation regulates growth and invasion through controlling P‐body formation and dynamics

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P‐bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P‐body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P‐body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P‐bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin β1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer‐relevant functions and suggest that modulation of P‐body activity may represent a new paradigm for cancer treatment.     

Delta Opioid Receptors: The Link between Exercise and Cardioprotection

This study investigated the role of opioid receptor (OR) subtypes as a mechanism by which endurance exercise promotes cardioprotection against myocardial ischemia-reperfusion (IR) injury. Wistar rats were randomly divided into one of seven experimental groups: 1) control; 2) exercise-trained; 3) exercise-trained plus a non-selective OR antagonist; 4) control sham; 5) exercise-trained plus a kappa OR antagonist; 6) exercise-trained plus a delta OR antagonist; and 7) exercise-trained plus a mu OR antagonist. The exercised animals underwent 4 consecutive days of treadmill training (60 min/day at ∼70% of maximal oxygen consumption). All groups except the sham group were exposed to an in vivo myocardial IR insult, and the myocardial infarct size (IS) was determined histologically. Myocardial capillary density, OR subtype expression, heat shock protein 72 (HSP72) expression, and antioxidant enzyme activity were measured in the hearts of both the exercised and control groups. Exercise training significantly reduced the myocardial IS by approximately 34%. Pharmacological blockade of the kappa or mu OR subtypes did not blunt exercise-induced cardioprotection against IR-mediated infarction, whereas treatment of animals with a non-selective OR antagonist or a delta OR antagonist abolished exercise-induced cardioprotection. Exercise training enhanced the activities of myocardial superoxide dismutase (SOD) and catalase but did not increase the left ventricular capillary density or the mRNA levels of HSP72, SOD, and catalase. In addition, exercise significantly reduced the protein expression of kappa and delta ORs in the heart by 44% and 37%, respectively. Together, these results indicate that ORs contribute to the cardioprotection conferred by endurance exercise, with the delta OR subtype playing a key role in this response.