Kinetic studies of the Arf activator Arno on model membranes in the presence of Arf effectors suggest control by a positive feedback loop

Proteins of the cytohesin/Arno/Grp1 family of Arf activators are positive regulators of the insulin-signaling pathway and control various remodeling events at the plasma membrane. Arno has a catalytic Sec7 domain, which promotes GDP to GTP exchange on Arf, followed by a pleckstrin homology (PH) domain. Previous studies have revealed two functions of the PH domain: inhibition of the Sec7 domain and membrane targeting. Interestingly, the Arno PH domain interacts not only with a phosphoinositide (phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate) but also with an activating Arf family member, such as Arf6 or Arl4. Using the full-length membrane-bound forms of Arf1 and Arf6 instead of soluble forms, we show here that the membrane environment dramatically affects the mechanism of Arno activation. First, Arf6-GTP stimulates Arno at nanomolar concentrations on liposomes compared with micromolar concentrations in solution. Second, mutations in the PH domain that abolish interaction with Arf6-GTP render Arno completely inactive when exchange reactions are reconstituted on liposomes but have no effect on Arno activity in solution. Third, Arno is activated by its own product Arf1-GTP in addition to a distinct activating Arf isoform. Consequently, Arno activity is strongly modulated by competition with Arf effectors. These results show that Arno behaves as a bistable switch, having an absolute requirement for activation by an Arf protein but, once triggered, becoming highly active through the positive feedback effect of Arf1-GTP. This property of Arno might provide an explanation for its function in signaling pathways that, once triggered, must move forward decisively.     

EFFECTS OF DIURETIC HORMONE 31, DROSOKININ,AND ALLATOSTATIN A ON TRANSEPITHELIAL K+ TRANSPORT AND CONTRACTION FREQUENCY IN THE MIDGUT AND HINDGUT OF LARVAL Drosophila melanogaster

Recent studies have identified paracrine and endocrine cells in the midgut of larval Drosophila melanogaster as well as midgut and hindgut receptors for multiple neuropeptides implicated in the control of fluid and ion balance. Although the effects of diuretic factors on fluid secretion by isolated Malpighian tubules of D. melanogaster have been examined extensively, relatively little is known about the effects of such factors on gut peristalsis or ion transport across the gut. We have measured the effects of diuretic hormone 31 (DH31), drosokinin and allatostatin A (AST‐A) on both K⁺ transport and muscle contraction frequency in the isolated gut of larval D. melanogaster. K⁺ absorption across the gut was measured using K⁺‐selective microelectrodes and the scanning ion‐selective electrode technique. Allatostatin A (AST‐A; 1 μM) increased K⁺ absorption across the anterior midgut but reduced K⁺ absorption across the copper cells and large flat cells of the middle midgut. AST‐A strongly inhibited gut contractions in the anterior midgut but had no effect on contractions of the pyloric sphincter induced by proctolin. DH31 (1 μM) increased the contraction frequency in the anterior midgut, but had no effect on K⁺ flux across the anterior, middle, or posterior midgut or across the ileum. Drosokinin (1 μM) did not affect either contraction frequency or K⁺ flux across any of the gut regions examined. Possible functions of AST‐A, DH31, and drosokinin in regulating midgut physiology are discussed.     

Bacillus thuringiensis Cry4Aa insecticidal protein: Functional importance of the intrinsic stability of the unique α4–α5 loop comprising the Pro-rich sequence

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro¹⁹³Pro¹⁹⁴_Pro¹⁹⁶) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4–α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4–α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro¹⁹³. Further MD simulations revealed that the most critical residue−Pro¹⁹³ for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4–α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.     

乳酸菌阴道胶囊在宫颈糜烂患者宫颈环形电切术后的应用

目的探讨乳酸菌阴道胶囊在宫颈糜烂患者宫颈环形电切（LEEP）术后的应用。方法选取宫颈糜烂患者76例,随机分为观察组（n=38例）和对照组（n=38例）。两组患者均在月经干净后3-7 d行LEEP术,观察组患者加用乳酸菌阴道胶囊阴道放置,每晚1粒,连用14 d。观察两组患者术后阴道流血量、出血持续时间及创面愈合时间,术后8周复诊评估其临床疗效。结果观察组患者术后的阴道流血量、出血持续时间及创面愈合时间明显少于对照组（χ2=7.54、5.65和8.49,P〈0.05或P〈0.01）;术后8周复诊,观察组的临床总有效率明显优于对照组（94.74%vs.78.95%,χ2=4.15,P〈0.05）。结论乳酸菌阴道胶囊在宫颈糜烂患者宫颈环形电切术后的应用具有明确的效果,能明显减少阴道出血量和出血持续时间,有利于促进创面愈合。     

Effect of mental fatigue on induced tremor in human knee extensors

In this study, the effects of mental fatigue on mechanically induced tremor at both a low (3–6 Hz) and high (8–12 Hz) frequency were investigated. The two distinct tremor frequencies were evoked using two springs of different stiffness, during 20 s sustained contractions of the knee extensor muscles at 30% maximum voluntary contraction (MVC) before and after 100 min of a mental fatigue task, in 12 healthy (29 ± 3.7 years) participants. Mental fatigue resulted in a 6.9% decrease in MVC and in a 9.4% decrease in the amplitude of the agonist muscle EMG during sustained 30% MVC contractions in the induced high frequency only. Following the mental fatigue task, the coefficient of variation and standard deviation of the force signal decreased at 8–12 Hz induced tremor by 31.7% and 35.2% respectively, but not at 3–6 Hz induced tremor. Similarly, the maximum value and area underneath the peak in the power spectrum of the force signal decreased by 55.5% and 53.1% respectively in the 8–12 Hz range only. In conclusion, mental fatigue decreased mechanically induced 8–12 Hz tremor and had no effect on induced 3–6 Hz tremor. We suggest that the reduction could be attributed to the decreased activation of the agonist muscles.     

Histone Deacetylase-3 Mediates Positive Feedback Relationship between Anaphylaxis and Tumor Metastasis

Allergic inflammation has been known to enhance the metastatic potential of tumor cells. The role of histone deacetylase-3 (HDAC3) in allergic skin inflammation was reported. We investigated HDAC3 involvement in the allergic inflammation-promotion of metastatic potential of tumor cells. Passive systemic anaphylaxis (PSA) induced HDAC3 expression and FcϵRI signaling in BALB/c mice. PSA enhanced the tumorigenic and metastatic potential of mouse melanoma cells in HDAC3- and monocyte chemoattractant protein 1-(MCP1)-dependent manner. The PSA-mediated enhancement of metastatic potential involved the induction of HDAC3, MCP1, and CD11b (a macrophage marker) expression in the lung tumor tissues. We examined an interaction between anaphylaxis and tumor growth and metastasis at the molecular level. Conditioned medium from antigen-stimulated bone marrow-derived mouse mast cell cultures induced the expression of HDAC3, MCP1, and CCR2, a receptor for MCP1, in B16F1 mouse melanoma cells and enhanced migration and invasion potential of B16F1 cells. The conditioned medium from B16F10 cultures induced the activation of FcϵRI signaling in lung mast cells in an HDAC3-dependent manner. FcϵRI signaling was observed in lung tumors derived from B16F10 cells. Target scan analysis predicted HDAC3 to be as a target of miR-384, and miR-384 and HDAC3 were found to form a feedback regulatory loop. miR-384, which is decreased by PSA, negatively regulated HDAC3 expression, allergic inflammation, and the positive feedback regulatory loop between anaphylaxis and tumor metastasis. We show the miR-384/HDAC3 feedback loop to be a novel regulator of the positive feedback relationship between anaphylaxis and tumor metastasis.     

A Fundamental Trade-off in Covalent Switching and Its Circumvention by Enzyme Bifunctionality in Glucose Homeostasis

Covalent modification provides a mechanism for modulating molecular state and regulating physiology. A cycle of competing enzymes that add and remove a single modification can act as a molecular switch between “on” and “off” and has been widely studied as a core motif in systems biology. Here, we exploit the recently developed “linear framework” for time scale separation to determine the general principles of such switches. These methods are not limited to Michaelis-Menten assumptions, and our conclusions hold for enzymes whose mechanisms may be arbitrarily complicated. We show that switching efficiency improves with increasing irreversibility of the enzymes and that the on/off transition occurs when the ratio of enzyme levels reaches a value that depends only on the rate constants. Fluctuations in enzyme levels, which habitually occur due to cellular heterogeneity, can cause flipping back and forth between on and off, leading to incoherent mosaic behavior in tissues, that worsens as switching becomes sharper. This trade-off can be circumvented if enzyme levels are correlated. In particular, if the competing catalytic domains are on the same protein but do not influence each other, the resulting bifunctional enzyme can switch sharply while remaining coherent. In the mammalian liver, the switch between glycolysis and gluconeogenesis is regulated by the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). We suggest that bifunctionality of PFK-2/FBPase-2 complements the metabolic zonation of the liver by ensuring coherent switching in response to insulin and glucagon.     

Proteomic Analysis of Glycine Receptor β Subunit (GlyRβ)-interacting Proteins: EVIDENCE FOR SYNDAPIN I REGULATING SYNAPTIC GLYCINE RECEPTORS*

Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their β subunits (GlyRβ) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRβ, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378–455 of the large intracellular loop of GlyRβ as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRβ that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRβ in vitro and colocalized with GlyRβ upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRβ. Deletion and point mutation analysis disclosed that SdpI binding to GlyRβ is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.     

Abdominal stretch reception in Dipetalogaster maximus (Hemiptera: Reduviidae)

Each half abdominal segment in 5th-instar larvae of the giant bloodsucking reduviid, Dipetalogaster maximus, contains 3 stretch receptor neurones, one associated with the tergosternal muscles, one with the ventral intersegmental muscles and one with the dorsal intersegmental muscles. Each of the three receptors respond phasically to the onset of stretch in its respective muscle group, but none show persistent activity upon prolonged stretch. By contrast, stretch of the main abdominal nerves (which run between the thoracic ganglion and the ventral intersegmental muscles of each abdominal segment) is accompanied by a prolonged and sustained pattern of discharge by an as yet unidentified neurone, the rate of discharge being proportional to the degree of stretch. In life, the abdominal nerves become stretched to about 145% of their resting length when the larva takes a bloodmeal. Thus it appears that in Dipetalogaster stretch of the abdominal nerves themselves is the only mechanism for stretch reception after a blood meal.