Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C₂C₁₂ myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C₂C₁₂ myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.     

Quantitative Live Imaging of Endogenous DNA Replication in Mammalian Cells

Historically, the analysis of DNA replication in mammalian tissue culture cells has been limited to static time points, and the use of nucleoside analogues to pulse-label replicating DNA. Here we characterize for the first time a novel Chromobody cell line that specifically labels endogenous PCNA. By combining this with high-resolution confocal time-lapse microscopy, and with a simplified analysis workflow, we were able to produce highly detailed, reproducible, quantitative 4D data on endogenous DNA replication. The increased resolution allowed accurate classification and segregation of S phase into early-, mid-, and late-stages based on the unique subcellular localization of endogenous PCNA. Surprisingly, this localization was slightly but significantly different from previous studies, which utilized over-expressed GFP tagged forms of PCNA. Finally, low dose exposure to Hydroxyurea caused the loss of mid- and late-S phase localization patterns of endogenous PCNA, despite cells eventually completing S phase. Taken together, these results indicate that this simplified method can be used to accurately identify and quantify DNA replication under multiple and various experimental conditions.     

Expression of Endogenous RNA C-Type Virus Group-Specific Antigens in Mammalian Cells

Highly sensitive and specific radioimmunoassays are described for quantitation of the intraspecies determinants of several mammalian C-type viral group-specific (gs) antigens. An interspecies (gs-3) immunoassay has been developed which has both the broad reactivity and great sensitivity necessary for detection of C-type viruses where intraspecies gs assays are not available. By using these immunoassays, the expression of endogenous virus-specified gs antigens in mammalian cells of different species has been studied. Whereas mouse gs antigen was clearly detectable in tissue culture cells of several mouse strains, the respective gs antigens of rat, cat, Chinese hamster, woolly monkey, and gibbon ape were not detectable in cells of those species, using assays of comparable sensitivity. Thus, differences exist in the level of endogenous virus expression in cells of different mammalian species.     

N-Acylethanolamines: Formation and Molecular Composition of a New Class of Plant Lipids

Recently, the biosynthesis of an unusual membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), was found to increase in elicitor-treated tobacco (Nicotiana tabacum L.) cells (K.D. Chapman, A. Conyers-Hackson, R.A. Moreau, S. Tripathy [1995] Physiol Plant 95: 120–126). Here we report that before induction of NAPE biosynthesis, N-acylethanolamine (NAE) is released from NAPE in cultured tobacco cells 10 min after treatment with the fungal elicitor xylanase. In radiolabeling experiments [¹⁴C]NAE (labeled on the ethanolamine carbons) increased approximately 6-fold in the culture medium, whereas [¹⁴C]NAPE associated with cells decreased approximately 5-fold. Two predominant NAE molecular species, N-lauroylethanolamine and N-myristoylethanolamine, were specifically identified by gas chromatography-mass spectrometry in lipids extracted from culture medium, and both increased in concentration after elicitor treatment. NAEs were found to accumulate extracellularly only. A microsomal phospholipase D activity was discovered that formed NAE from NAPE; its activity in vitro was stimulated about 20-fold by mastoparan, suggesting that NAPE hydrolysis is highly regulated, perhaps by G-proteins. Furthermore, an NAE amidohydrolase activity that catalyzed the hydrolysis of NAE in vitro was detected in homogenates of tobacco cells. Collectively, these results characterize structurally a new class of plant lipids and identify the enzymatic machinery involved in its formation and inactivation in elicitor-treated tobacco cells. Recent evidence indicating a signaling role for NAPE metabolism in mammalian cells (H.H.O. Schmid, P.C. Schmid, V. Natarajan [1996] Chem Phys Lipids 80: 133–142) raises the possibility that a similar mechanism may operate in plant cells.NAPE is a widespread, albeit minor, membrane phospholipid in animal and plant tissues (Schmid et al., 1990; Chapman and Moore, 1993). Its unusual structural features (a third fatty acid moiety linked to the amino head group of PE) impart stabilizing properties to membrane bilayers (Domingo et al., 1994; LaFrance et al., 1997). NAPE and its hydrolysis products, NAEs, are known to accumulate in vertebrate tissues under pathological conditions (for review, see Schmid et al., 1990). Recently, there has been renewed interest in NAEs because of the contention that anandamide (N-arachidonylethanolamine) is an endogenous ligand for the cannabinoid receptor in mammalian brain (Devane et al., 1992; Fontana et al., 1995; Schmid et al., 1996). The likely route for NAE formation in neural and nonneural tissues, although the matter of some debate, is via the signal-mediated hydrolysis of NAPE (DiMarzo et al., 1994; Schmid et al., 1996; Sugiura, et al., 1996).In plants little is known regarding the catabolism of NAPE. In cottonseed microsomes NAPE was metabolized to NAE or NAlysoPE by PLD- or PLA-type activities, respectively (Chapman et al., 1995b). However, the metabolic fate of NAPE in vivo and the factors that regulate NAPE hydrolysis remain largely unknown. We previously noted that the biosynthesis of NAPE was increased in elicitor-treated cell suspensions of tobacco (Nicotiana tabacum L.). Here we extend our investigations with this model system to examine NAPE catabolism by plant cells in vivo. NAE was released from NAPE, and it accumulated extracellularly. We identified by GC-MS these tobacco NAEs as N-lauroylethanolamine and N-myristoylethanolamine. These NAEs were increased in elicitor-treated cell suspensions. Furthermore, we detected the enzymatic machinery capable of the release and the degradation of NAEs in tobacco cells. To our knowledge this represents the first identification of the NAE molecular species in plant cells. It is tempting to speculate that NAPE hydrolysis in elicitor-treated plant cells may be involved in a signaling pathway analogous to that found in mammalian cells.     

Biological Effects of Leptospiral Lipids

Lipids were extracted from virulent Leptospira pomona and were purified. These lipids fixed complement in the presence of antiserum to L. pomona but did not stimulate the production of homologous agglutinins in rabbits, mice, or hamsters. When subsequently challenged, all of the mice and hamsters were fully susceptible to L. pomona. The lipid material was neither dermonecrotic nor lethal for mice or hamsters, but 382 mug of lipid from virulent or avirulent leptospires inhibited the growth of normal mice. Leptospiral lipids were toxic for peritoneal macrophages maintained in vitro, and when administered simultaneously with a million lethal doses of L. pomona, the lipids hastened death of the hamsters, presumably by inhibiting phagocytosis early in the course of the infection.     

Unusual Low Proton Permeability of Liposomes Prepared from the Endogenous Myelin Lipids

Abstract: In contrast with most other lipid substrates, in this article we show that liposomes prepared from the total myelin lipids exhibited a negligible proton permeability. Neither the generation of valinomycin-induced potassium diffusion potentials as high as -177 mV nor the imposition of large pH gradients (up to three units) was able to produce a substantial flux of protons through liposomal membranes, as determined by the distribution of [¹⁴C]-methylamine, or the changes in the fluorescence of the probes 9-aminoacridine, acridine orange, and pyranine. The presence of cations (Na⁺, K⁺, Ca²⁺) did not alter this behavior. Voltage clamping did not increase the trans-membrane ApH-driven proton permeability. However, II-posome diameter was found to be critical because small unilamellar vesicles displayed a much higher proton permeability than large unilamellar or multilamellar vesicles. This abnormally low proton permeability is interpreted by virtue of the characteristic biochemical composition of myelin lipid matrix, with a high content of cholesterol and sphingolipids and a very low level of free fatty acids. These results could be important for elucidating the role of myelin in the regulation of pH in the brain. In addition, the myelin lipid extract could be useful for reconstituting proteins that participate in the transport of H⁺ through the membrane.     

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

活络效灵丹抗炎镇痛作用的实验研究

目的：研究活络效灵丹的抗炎、镇痛、消肿等药理作用,为该药的临床研究提供基础。方法：用二甲苯致小鼠耳肿胀法和角又莱胶致大鼠足肿胀法研究抗炎作用,用热板法和扭体法研究镇痛作用。结果：活络效灵丹能较好地抑制二甲苯引起的小鼠耳廓炎性肿胀和大鼠甲醛致足跖肿胀,能显著提高热板试验小鼠的痛阈,有效抑制冰醋酸引起的小鼠扭体反应次数。结论：活络效灵丹具有良好的抗炎、镇痛、消肿等作用。     

On the Origins of the Universal Dynamics of Endogenous Granules in Mammalian Cells

Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics - both of which are crucial for the interpretation of the results from MPTM technique - are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms - molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.     

Multiple Effects of Introduced Mammalian Herbivores in a Temperate Forest

Introduced mammalian herbivores can significantly affect ecosystems. Here, I review evidence on effects of introduced mammalian herbivores in the temperate forest of the southern Andes. Available data suggest that introduced herbivores decrease the abundance of seedlings and saplings of dominant tree species in some forest types, which could impair forest regeneration. They also affect understory species composition. The mechanisms of the effects of introduced herbivores are complex, and include direct effects of browsing or trampling and more complex interactions such as indirect effects through other species. Some native mammalian and avian predators may benefit from increased food availability resulting from high densities of some introduced mammalian herbivores. In turn, enhanced populations of predators may have resulted in increased predation on native prey. Competition for resources and disease transmission have also been proposed as possible negative effects of introduced herbivores on native herbivores, but little evidence supports this claim. Little is known about effects on invertebrates.     

Anti-Diabetic Effects and Molecular Mechanisms of Amide Alkaloids from Piper longum Based on Network Pharmacology Integrated with Cellular Assays

Piper longum is a well-known spice and traditional medicine. It was revealed to possess anti-diabetic activity, but few information about its active component and underlying mechanism could be available. In this study, retrofractamides A ( 1 ) and C ( 2 ) isolated from P. longum showed potent inhibitory activity against PTP1B. Therefore, the potential mechanism was predicted by network pharmacology and molecular docking. PI3K/AKT was obtained as the most remarkable pathway against type 2 diabetes mellitus (T2DM), and AKT1 and GSK3β were yielded as the top two core targets of retrofractamides A ( 1 ) and C ( 2 ). Molecular docking of compounds with AKT1 and GSK3β showed strong binding affinity between them. Additionally, cellular experiments with a L6 cell model was conducted to further verify the above predictions. Results indicated that retrofractamides A ( 1 ) and C ( 2 ) exerted anti-diabetic effect via activating PI3K/AKT pathway, and they promoted glucose consumption, glucose uptake, glycogen synthesis and glycolysis.     

Reproductive Effects of the Mammalian Pineal

The reproductive systems of mammals are anatomically and physiologicallyaltered by the pineal gland. Characteristically, pineal removalaccelerates gonadal growth in immature animals and causes atransient modest enlargement of the reproductive organs if theoperations are done after adulthood is attained. Light deprivationrenders the pineal strongly antigonadotropic. In addition, thereare several conditions, e.g., androgen-sterilization and anosmia,which potentiate, probably indirectly, the gonad-inhibitingcapability of the rat's pineal. The chronic administration ofpineal extracts or pineal substances has effects opposite topinealectomy, i.e., they restrict gonadal development or growth.The pineal is possibly important in adjusting the level of reproductiveactivity to seasonal changes in environmental conditions.     

Calcium-dependent Modulation of the Agonist Affinity of the Mammalian Olfactory Cyclic Nucleotide-gated Channel by Calmodulin and a Novel Endogenous Factor

The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3′,5′-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mm Ca²⁺ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC₅₀) was reversibly increased from 3 μm to about 30 μm. This Ca²⁺-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg²⁺+ 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mm Ca²⁺ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 μm to about 200 μm. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca²⁺-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca²⁺-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM. Received: 26 December 1995/Revised: 14 March 1996     

Role of Guanine Nucleotides as Endogenous Ligands of a Kainic Acid Binding Site Population in the Mammalian Cerebellum

Abstract: An endogenous inhibitor of the membrane binding of kainic acid was extracted from pig brain tissue and purified. The substance was identified as GMP by structural analysis: Most likely it corresponds to an inhibitor previously extracted from the rat brain. The nucleotide is active as an inhibitor for kainate binding on goldfish brain synaptosomes, probably owing to direct displacement on receptor sites; it is also active on a low-affinity kainate site population in membranes from rat cerebellum. The interaction of GMP with the latter sites leads to a concentration-dependent kainate binding increase or inhibition, thus demonstrating that these sites can bind the nucleotide and cooperatively increase their affinity. Other guanine nucleotides show interaction with these sites, by either an increase (GTP) or inhibition (cyclic GMP or GDP) of kainate binding. These findings support the view that a guanine nucleotide is the endogenous ligand of a receptor in the mammalian cerebellum similar to the kainate binding protein present with high density in the cerebellum of lower vertebrates, whose function is probably connected to the role of the glial cells in this zone.     

Endocrine Effects of the Mammalian Pineal

The current status of knowledge concerning the mammalian pineal'sendocrine effects and the possible mechanisms for their mediationis reviewed and discussed. The diversity, physiological relations,and variability of the pineal's peripheral effects, and theconstancy, rapidity, and homeostatic nature of its central andbehavioral effects are consistent with the view that the pineal'sprimary action is on brain tissue. It remains probable thaton a seasonal basis, the pineal's vegetative and homeostaticeffects may contribute to the adaptive physiological changesin particular mammalian species under the influence of changesin photoperiod and possibly other environmental factors.     

Calcium Movements Mediated by Proteolipid Protein and Nucleotides in Liposomes Prepared with the Endogenous Lipids from Brain White Matter

A lipid extract with a composition similar to that of myelin was used to prepare liposomes and proteoliposomes containing the Folch-Lees proteolipid apoprotein. Freeze-fracture replicas of the proteoliposomes were prepared to demonstrate the presence of intramembrane protein particles in the fracture faces of the lipid bilayer. Experiments with 45CaCl2 showed that a steady calcium movement occurs across liposomal membranes, approaching equilibrium between intra- and extravesicular spaces. The most significant finding was that Mg-ATP, ATP analogues, and other nucleotides depressed significantly the calcium fluxes in proteoliposomes, having no effect on liposomes that lacked the proteolipid protein. It is suggested that this intrinsic protein, interacting with nucleotides and endogenous lipids, could be involved in the regulation of calcium levels in myelin by means of a conformational change mechanism. These observations could lead to implications concerning the pathophysiology of myelin.     

蜂胶的抗炎作用及其机制研究进展

本文通过查阅近年来蜂胶抗炎活性研究的相关文献,对近年来蜂胶改善炎症效果以及蜂胶和部分抗炎药物之间的相互作用进行了综述,并通过分析蜂胶中抗炎活性成分对蜂胶抗炎的作用途径及机制进行了探讨。指出蜂胶的抗炎活性研究对于探讨蜂胶对心血管疾病、消化系统疾病以及内分泌系统疾病的治疗作用机制有着重要的意义,蜂胶和部分抗炎药物之间呈现的协同作用对于开发复方抗炎药物提供了理论基础。     

The Shanghuang Mammalian Fauna, Middle Eocene of Jiangsu:History of Discovery and Significance

APaleogenemammalianfauna,herenamedtheShanghuangfauna,wasdiscoverednearthevillageofShanghuang,closetoLiyangCity,JiangsuProvince,China(Fig.1).Atpresent,rouglilysixtyspeciesoffossilmammalshavebeenrecogniedintheShanghuangfauna.Manyofthesemammaltaxaarenewtoscience,andothersrepresentnewoccurrencesfortheEoceneofChina.ParticularlywellrepresentedintheShanghuangfaunaaremicromammals(includingmarsupials,insectivores,rodents,lagomorphs,primates,andbats)thathaveseldomifeverbeenfoundinotherEoceneAsianl…