Complexins regulate a late step in Ca2+-dependent neurotransmitter release

Synaptic vesicle fusion at synapses is triggered by increases in cytosolic Ca2+ levels. However, the identity of the Ca2+ sensor and the transduction mechanism of the Ca2+ trigger are unknown. We show that Complexins, stoichiometric components of the exocytotic core complex, are important regulators of transmitter release at a step immediately preceding vesicle fusion. Neurons lacking Complexins show a dramatically reduced transmitter release efficiency due to decreased Ca2+ sensitivity of the synaptic secretion process. Analyses of mutant neurons demonstrate that Complexins are acting at or following the Ca2+-triggering step of fast synchronous transmitter release by regulating the exocytotic Ca2+ sensor, its interaction with the core complex fusion machinery, or the efficiency of the fusion apparatus itself.     

Gamma-adaptin appendage domain: structure and binding site for Eps15 and gamma-synergin

The AP1 complex is one of a family of heterotetrameric clathrin-adaptor complexes involved in vesicular trafficking between the Golgi and endosomes. The complex has two large subunits, gamma and beta1, which can be divided into trunk, hinge, and appendage domains. The 1.8 A resolution structure of the gamma appendage is presented. The binding site for the known gamma appendage ligand gamma-synergin is mapped through creation of point mutations designed on the basis of the structure. We also show that Eps15, a protein believed to be involved in vesicle formation at the plasma membrane, is also a ligand of gamma appendage and binds to the same site as gamma-synergin. This observation explains the demonstrated brefeldinA (BFA)-sensitive colocalization of Eps15 and AP1 at the Golgi complex.     

Hydration,slaving and protein function

Protein dynamics is crucial for protein function. Proteins in living systems are not isolated, but operate in networks and in a carefully regulated environment. Understanding the external control of protein dynamics is consequently important. Hydration and solvent viscosity are among the salient properties of the environment. Dehydrated proteins and proteins in a rigid environment do not function properly. It is consequently important to understand the effect of hydration and solvent viscosity in detail. We discuss experiments that separate the two effects. These experiments have predominantly been performed with wild-type horse and sperm whale myoglobin, using the binding of carbon monoxide over a broad range of temperatures as a tool. The experiments demonstrate that data taken only in the physiological temperature range are not sufficient to understand the effect of hydration and solvent on protein relaxation and function. While the actual data come from myoglobin, it is expected that the results apply to most or all globular proteins.     

Balancing tissue perfusion demands: cardiovascular dynamics of Cancer magister during exposure to low salinity and hypoxia

Decapod crustaceans inhabit aquatic environments that are frequently subjected to changes in salinity and oxygen content. The physiological responses of decapod crustaceans to either salinity or hypoxia are well documented; however, there are many fewer reports on the physiological responses during exposure to these parameters in combination. We investigated the effects of simultaneous and sequential combinations of low salinity and hypoxia on the cardiovascular physiology of the Dungeness crab, Cancer magister. Heart rate, as well as haemolymph flow rates through the anterolateral, hepatic, sternal and posterior arteries were measured using a pulsed-Doppler flowmeter. Summation of flows allowed calculation of cardiac output and division of this by heart rate yielded stroke volume. When hypoxia and low salinity were encountered simultaneously, the observed changes in cardiac properties tended to be a mix of both factors. Hypoxia caused a bradycardia, whereas exposure to low salinity was associated with a tachycardia. However, the hypoxic conditions had the dominant effect on heart rate. Although hypoxia caused an increase in stroke volume of the heart, the low salinity had a more pronounced effect, causing an overall decrease in stroke volume. The patterns of haemolymph flow through the arterial system also varied when hypoxia and low salinity were offered together. The resulting responses were a mix of those resulting from exposure to either parameter alone. When low salinity and hypoxia were offered sequentially, the parameter experienced first tended to have the dominant effect on cardiac function and haemolymph flows. Low salinity exposure was associated with an increase in heart rate, a decrease in stroke volume and cardiac output, and a concomitant decrease in haemolymph flow rates. Subsequent exposure to hypoxic conditions caused a slight decrease in rate, but other cardiovascular variables were largely unaffected. In contrast, when low salinity followed acclimation to hypoxic conditions, apart from an increased heart rate, there were no other cardiovascular changes associated with the low salinity episode. The implications of these changes in cardiovascular dynamics are discussed in relation to physiological mechanisms and the ecology of decapod crustaceans, in hypoxic or low salinity environments.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension

Myostatin inhibits myogenesis. Therefore, we sought to determine if mice lacking the myostatin gene [Mstn(-/-)] would lose less muscle mass than wild-type mice during 7 days of hindlimb suspension (HS). Male Mstn(-/-) and wild-type (C57) mice were subjected to HS or served as ground-based controls (n = 6/group). Wild-type mice lost 8% of body mass and approximately 13% of wet mass from biceps femoris, quadriceps femoris, and soleus, whereas the mass of extensor digitorum longus (EDL) was unchanged after HS. Unexpectedly, Mstn(-/-) mice lost more body (13%, P < 0.05) and quadriceps femoris (17%, P < 0.05) mass than wild-type mice and lost 33% of EDL mass (P < 0.01) after HS. Protein expression of myostatin in biceps femoris and quadriceps femoris was not altered, whereas expression of MyoD, Myf-5, and myogenin increased in wild-type mice and tended to decrease in muscles of Mstn(-/-) mice. These data suggest that HS induced myogenesis in wild-type mice to counter atrophy, whereas myogenesis was not induced in Mstn(-/-) mice, thereby resulting in a greater loss of muscle mass.     

Synthesis of a highly substituted N(6)-linked immobilized NAD(+) derivative using a rapid solid-phase modular approach: suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N(6)-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N(6)-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 A) in an attempt to minimize nonbiospecific interactions. Analysis of the N(6)-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S(6)-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart l-lactate dehydrogenase (l-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. l-phenylalanine dehydrogenase (l-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and d-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S(6)-linked immobilized cofactor derivatives than for the new N(6)-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S(6)-linked immobilized NAD(+) derivative, but was locked-on strongly to the N(6)-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N(6)-linked immobilized NADP(+) derivative in the presence of d-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N(6)-linked and S(6)-linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N(6)-linkage as compared to thiol-linkage.     

Divergence and reticulation among montane populations of a jumping spider (Habronattus pugillis Griswold)

Populations of the jumping spider Habronattus pugillis Griswold isolated on nearby mountain ranges in southern Arizona are differentiated in many features of the males (color, shape, and orientation of setae on face; shape of carapace; markings of palpi and legs; motions during courtship behavior). These features are (mostly) consistent within a range and different between ranges. The concentration of differences in male courtship behavior and body parts exposed to the female during courtship and correlations between form and courtship behavior suggest sexual selection was involved in the differentiation. A phylogenetic analysis of the populations yields a tree that for the most part groups geographic neighbors, but the history of H. pugillis populations may not be adequately described by a tree. Geographic proximity of apparent convergences suggests that populations from at least some of the mountain ranges acquired characteristics through introgression. Lowering of the woodland habitat during the last glacial period probably brought some populations into contact, but it is not clear whether the interrange woodlands would have provided corridors for extensive mixing.