Putting the heat on sex determination

Sex determination and differentiation are inherently fascinating to both layperson and geneticist. Major advances have accelerated interest in the molecular genetic events mediating these processes in nematodes, flies, mice and humans. Far less attention has been paid to those organisms, particularly reptiles, where sex is determined by environmental cues. However, recent experimental evidence suggests that the two modes of sex determination may not only share common genetic elements, but may also be regulated by similar mechanisms. We argue that the ability to manipulate sex by temperature provides a particularly suitable model for exploring the molecular basis of this fundamental biological process.     

Persistence of Cell Division Synchrony in Spirogyra Insignis (Gamophyceae): Membrane Proteoglycans Transmitting Synchronizing Information Throughout Generations

Spirogyra insignis shows a long-term persistence of cell division synchrony in the absence of the synchronizing Zeitgeber, so that at least six generations are involved in the process. This tentatively suggests that a mechanism of transmission throughout generations of synchronizing information could maintain this synchrony. Apparently, a vital part of the molecular basis of this mechanism is a membrane proteoglycan complex. This complex could obtain temporal information from a synchronizing Zeitgeber and be transmitted to the progeny by distribution of plasma membrane between daughter cells.     

Benzylic alcohols as stereospecific substrates and inhibitors for aryl sulfotransferase

Aryl sulfotransferase IV catalyzes the 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent formation of sulfuric acid esters of benzylic alcohols. Since the benzylic carbon bearing the hydroxyl group can be asymmetric, the possibility of stereochemical control of substrate specificity of the sulfotransferase was investigated with benzylic alcohols. Benzylic alcohols of known stereochemistry were examined as potential substrates and inhibitors for the homogeneous enzyme purified from rat liver. For 1-phenylethanol, both the (+)-(R)- and (-)-(S)-enantiomers were substrates for the enzyme, and the kcat/Km value for the (-)-(S)-enantiomer was twice that of the (+)-(R)-enantiomer. The enzyme displayed an absolute stereospecificity with ephedrine and pseudoephedrine, and with 2-methyl-1-phenyl-1-propanol; that is, only (-)-(1R,2S)-ephedrine, (-)-(1R,2R)-pseudoephedrine, and (-)-(S)-2-methyl-1-phenyl-1-propanol were substrates for the sulfotransferase. In the case of 1,2,3,4-tetrahydro-1-naphthol, only the (-)-(R)-enantiomer was a substrate for the enzyme. Both (+)-(R)-2-methyl-1-phenyl-1-propanol and (+)-(S)-1,2,3,4-tetrahydro-1-naphthol were competitive inhibitors of the aryl sulfotransferase-catalyzed sulfation of 1-naphthalenemethanol. Thus, the configuration of the benzylic carbon bearing the hydroxyl group determined whether these benzylic alcohols were substrates or inhibitors of the rat hepatic aryl sulfotransferase IV. Furthermore, benzylic alcohols such as (+)-(S)-1,2,3,4-tetrahydro-1-naphthol represent a new class of inhibitors for the aryl sulfotransferase.     

Calmodulin structure and function: Implication of arginine in the compaction related to ligand binding

Calmodulin (CAM) is a modulatory protein that regulates cellular activity by binding to a large number of proteins. Key elements in the Ca²⁺-dependent mechanism of interaction between CAM and the proteins it activates are the selectivity for Ca²⁺ ions and the requirement for Ca²⁺-dependent conformational changes. We report on results from a series of molecular dynamics simulations that identified discrete steps in the mechanism of structural rearrangement of CAM. The findings implicate the side chains of arginine residues in the bending of the central alpha helix. Structural and energetic considerations point to a dynamic hydrogen bonding pattern around the arginine residues as a ratcheting-type mechanism, causing the kinking of the central helix in consecutive steps stabilized by each new pattern of hydrogen bonds. Initial model building studies to locate potential binding sites of ligands such as trifluoperazine (TFP) indicate that the compaction of CAM results in several structural changes, that explain the selective binding of molecules such as TFP in the N-terminal domain. The present studies identify specific residues involved in the process of compaction and point to specific CAM residues involved in the binding of the ligand. These insights lead directly to propositions for experimental engineering of the molecular structure of CAM in order to probe the hypotheses and their consequences for the function of this important protein.     

Differential effects of paraoxon on the M3 muscarinic receptor and its effector system in rat submaxillary gland cells

The effects of the organophosphorus anticholinesterase paraoxon on the binding of radioactive ligands to the M₃ subtype of the muscarinic receptor and receptor-coupled synthesis of second messengers in intact rat submaxillary gland (SMG) cells were investigated. The binding of [³H]quinuclidinyl benzilate ([³H]QNB) was most sensitive to atropine and the M₃-specific antagonist 4-DAMP followed by pirenzepine and least sensitive to the cardioselective M₂ antagonist AFDX116. This, and the binding characteristics of [³H]4-DAMP, confirmed that the muscarinic receptors in this preparation are of the M₃ subtype. Activation of these muscarinic receptors by carbamylcholine (CBC) produced both stimulation of phosphoinositide (PI) hydrolysis and inhibition of cAMP synthesis, suggesting that this receptor subtype couples to both effector systems. Paraoxon (100 μM) reduced B_max of [³H]4-DAMP binding from 27 ± 4 to 13 ± 3 fmol/mg protein with nonsignificant change in affinity, suggesting noncompetitive inhibition of binding by paraoxon. Like the agonist CBC, paraoxon inhibited the forskolininduced cAMP formation in SMG cells with an EC₅₀ of 200 nM, but paraoxon was > 500 fold more potent than CBC. However, while the inhibition by CBC was counteracted by 2 μM atropine, that by paraoxon was unaffected by up to 100 μM atropine. It suggested that this effect of paraoxon was not via binding to the muscarinic receptor. Paraoxon did not affect β-adrenoreceptor function in the preparation, since it did not affect the 10 μM isoproterenol-induced cAMP synthesis, which was inhibited totally by 10 μM propranolol and partially by CBC. Paraoxon had a small but significant effect on CBC-stimulated PI metabolism in the SMG cells. It is suggested that paraoxon binds to two different sites in these SMG cells. One is an allosteric site on the M₃ muscarinic receptor which affects ligand binding and may modulate receptor function. The other site may be on the G_i proteinadenylyl cyclase system, and produces CBC-like action, that is, inhibition of the forskolin-stimulated [³H]cAMP synthesis, and is unaffected by atropine inhibition of the muscarinic receptor. This adds to the complexity of paraoxon actions on muscarinic receptors and their effector systems.     

Eating, drinking and temperature responses to intracerebroventricular cholecystokinin in the chick

The central effect of cholecystokinin-octapeptide (CCK), SQ 19,844 or sincalide, on the intake of food and water and on colonic temperature (Tc) was investigated using the broiler cockerel. Four-week old chicks were maintained in a thermoneutral environment of 23-24 degrees C. After food was removed for a 24 hr interval, CCK was infused in a volume of 10.0 microliters into the lateral cerebral ventricle (ICV) in doses ranging from 10-150 ng. Although lower doses of CCK had no effect on food intake, 100 or 150 ng of CCK significantly reduced consumption of food in a dose-dependent manner; water drinking was significantly decreased by 100 ng of CCK. In addition, CCK at doses of 100 and 150 ng prevented the slow rise in Tc observed following infusions of control CSF. This latter effect appeared to be a result of feeding activity associated with caloric intake and the heat increment in the control birds rather than a specific thermoregulatory effect. Overall, our results suggest that CCK may comprise a part of the central mechanism underlying the neural control of short term satiety in an avian species similar to that proposed for the mammal.     

Evidence for a two-state mobile carrier mechanism in erythrocyte choline transport: Effects of substrate analogs on inactivation of the carrier by N-ethylmaleimide

Summary Choline transport in erythrocytes is irreversibly inhibited by N-ethylmaleimide. The hypothesis that the carrier alternates between outwardfacing and inward-facing forms and that only the latter reacts with the inhibitor (Martin, K. (1971)J. Physiol. (London) 213:647–667; Edwards, P.A. (1973)Biochim. Biophys. Acta 311:123–140) is here subjected to a quantitative test. In this test the effects of a series of substrate analogs upon rates of inactivation and rates of choline exit are compared. By hypothesis the effect of an analog in the external solution on the inactivation rate depends only on how it affects the proportion of the inward-facing carrier. Since¹⁴C-choline efflux is necessarily proportional to the concentration of free carrier in the inward-facing form, the analogs should have related effects on the two rates. In every case the observed effects were identical, whether the analogs accelerated transport or inhibited it. Analysis of the results demonstrates that (1) the transport mechanism depends on the operation of a mobile element; (2) distinguishable inward-facing and outward-facing conformations of the free carrier, carrier-substrate complex, and carrier-inhibitor complex exist, and only the inwardfacing forms react at a significant rate with N-ethylmaleimide; (3) carrier mechanisms involving a single form of free carrier or a single form of carriersubstrate complex are ruled out; and (4) dissociation of the carrier-substrate complex is a rapid step with all substrate analogs.     

Malate synthase from Gossypium hirsutum

Malate synthase was purified 2000-fold from cotyledons of dark-germinated cotton, Gossypium hirsutum. The purified enzyme had a pH optimum of 8.2, and an absolute requirement for a divalent cation. Only glyoxylate and acetyl-CoA served as condensation partners. Results obtained with functional-group directed inhibitors suggest the presence of lysine, tyrosine and histidine residues in the active site. Temperature optimum was 40°, and energy of activation was 3.3 kcal/mol. The MW of cotton malate synthase, determined by rate-zonal density gradient sedimentation, was 750 000. Initial-rate studies indicated Michaelis-Menten kinetics. Inhibition by substrate analogs, plus substrate-interaction kinetics gave results consistent with a sequential bireactant mechanism.     

Cytoplasmic streaming inParamecium

Summary Certain species ofParamecium demonstrate rotational cytoplasmic streaming, in which most cytoplasmic particles and organelles flow along permanent route, in a constant direction. By means of novel methods of immobilization, observation and recording, some dynamic properties of cytoplasmic streaming have been described. It was found that the velocity profiles of coaxial layers of cytoplasm have a (parabolic) paraboidal shape and the mean output of cytoplasm flow in different examined zones of streaming is constant. As the consequence of randomly distributed elementary propulsion units within the cytoplasm, particles, which serve as markers of movement, exhibit movements of a saltatory nature; this form of movement is seen inParamecium streaming only in cases of error due to polarization of the saltating particles. Interaction of actin filaments and myosin is likely to occur under specific conditions in microcompartments of cytoplasm where local solations are generated eventually leading to contractions which might propagate on gelated neighbouring areas. Places of elementary contractions are scattered. Therefore the motile effect appears as streaming. Rotational cytoplasmic streaming inParamecium may serve as a convenient model for the study of the dynamics and function of cytoplasmic motility.     

Mechanism of activation of cyclic GMP-dependent protein kinase from rat liver by multiple modulators

A number of polyanionic compounds, including DNA, RNA and polyglutamate, were shown to exhibit protein kinase stimulatory modulator activity as they were required for cyclic GMP to stimulate the phosphorylation of various cationic substrates by rat liver cyclic GMP-dependent protien kinase. Anionic proteins (casein, phosvitin) were phosphorylated poorly by the enzyme and their phosphorylation was not stimulated by the stimulatory modulators. Studies of the mechanism of action suggest that the modulators interact directly with the substrates to form a complex which is a better substrate than free histone. The observed effect of modulator is complex as it depends on the ratio of modulator to histone and the resultant state of the complex formed (better or poorer substrate than free histone). The observed effect is also dependent on the properties of the histone substrate as Michaelis-Menten kinetics are not observed in the phosphorylation of arginine-rich histone in the absence or presence of cyclic GMP.