SELECTION AND THE “SEX-RATIO” POLYMORPHISM IN NATURAL POPULATIONS OF DROSOPHILA PSEUDOOBSCURA

“Sex-ratio” (SR) is a naturally occurring X-linked meiotic drive system, where the SR-X chromosome is transmitted to nearly all progeny of SR males. It occurs at frequencies of up to 25% in some populations of Drosophila pseudoobscura. Because of the twofold drive advantage, SR should rapidly fix in populations, causing the extinction of the species, unless opposed by strong selection. I examine several of the adult components of fitness, including the frequencies of all genotypic mating combinations, fertilities, and fecundities of flies from two populations in southeastern Arizona. Significant reduction of fecundity of SR/SR females was observed in the Tucson population. No evidence was found for either lower fertility or reduced mating success of SR males, relative to standard males. Most selection opposing SR appears to be operating at the larval stages in nature.     

Na-Ca exchange and the trigger for sarcoplasmic reticulum Ca release: studies in adult rabbit ventricular myocytes.

The importance of Na-Ca exchange as a trigger for sarcoplasmic reticulum (SR) Ca release remains controversial. Therefore, we measured whole-cell Ca currents (ICa), Na-Ca exchange currents (INaCa), cellular contractions, and intracellular Ca transients in adult rabbit cardiac myocytes. We found that changing pipette Na concentration markedly affected the relationship between cell shortening (or Ca transients) and voltage, but did not affect the Ca current-voltage relationship. We then inhibited Na-Ca exchange and varied SR content (by changing the number of conditioning pulses before each test pulse). Regardless of SR Ca content, the relationship between contraction and voltage was bell-shaped in the absence of Na-Ca exchange. Next, we rapidly and completely blocked ICa by applying nifedipine to cells. Cellular shortening was variably reduced in the presence of nifedipine. The component of shortening blocked by nifedipine had a bell-shaped relationship with voltage, whereas the "nifedipine-insensitive" component of contraction increased with voltage. With the SR disabled (ryanodine and thapsigargin pretreatment), ICa could initiate late-peaking contractions that were approximately 70% of control amplitude. In contrast, nifedipine-insensitive contractions could not be elicited in the presence of ryanodine and thapsigargin. Finally, we recorded reverse Na-Ca exchange currents that were activated by membrane depolarization. The estimated sarcolemmal Ca flux occurring by Na-Ca exchange (during voltage clamp steps to +30 mV) was approximately 10-fold less than that occurring by ICa. Therefore, Na-Ca exchange alone is unlikely to raise cytosolic Ca concentration enough to directly activate the myofilaments. We conclude that reverse Na-Ca exchange can trigger SR Ca release. Because of the sigmoidal relationship between the open probability of the SR Ca release channel and pCa, the effects of ICa and INaCa may not sum in a linear fashion. Rather, the two triggers may act synergistically in the modulation of SR release.     

Ryanodine: a modifier of sarcoplasmic reticulum calcium release in striated muscle

We have proposed that the naturally occurring alkaloid ryanodine reduces the release of calcium from the sarcoplasmic reticulum (SR) in cardiac muscle cells. We summarize the data that support this hypothesis and discuss possible mechanisms for 1) the differences in sensitivity to ryanodine displayed by intact skeletal and cardiac muscle preparations vs. that of skinned cardiac cells and isolated SR membranes, 2) the ability of ryanodine to cause either an increase or a decrease in calcium accumulation by isolated skeletal muscle SR vesicles depending on experimental conditions, and 3) the positive inotropic effects produced by ryanodine in cardiac muscle preparations under certain experimental circumstances. In addition, we also show how ryanodine can be used to evaluate the contributions made by SR calcium release to cellular events in striated muscle.     

Stimulating the substrate folding activity of a single ring GroEL variant by modulating the cochaperonin GroES

In mediating protein folding, chaperonin GroEL and cochaperonin GroES form an enclosed chamber for substrate proteins in an ATP-dependent manner. The essential role of the double ring assembly of GroEL is demonstrated by the functional deficiency of the single ring GroEL(SR). The GroEL(SR)-GroES is highly stable with minimal ATPase activity. To restore the ATP cycle and the turnover of the folding chamber, we sought to weaken the GroEL(SR)-GroES interaction systematically by concatenating seven copies of groES to generate groES(7). GroES Ile-25, Val-26, and Leu-27, residues on the GroEL-GroES interface, were substituted with Asp on different groES modules of groES(7). GroES(7) variants activate ATP activity of GroEL(SR), but only some restore the substrate folding function of GroEL(SR), indicating a direct role of GroES in facilitating substrate folding through its dynamics with GroEL. Active GroEL(SR)-GroES(7) systems may resemble mammalian mitochondrial chaperonin systems.     

Modeling of Nonlinear Chemical Reaction Systems and Two-Parameter Stochastic Resonance

The idea of stochastic resonance (SR) is extended to two-parameter dynamical systems based on the Oregonator model of the Belousov-Zhabotinsky (BZ) reaction. The first case presents the photosensitivity of the reaction, and light flux and a flow rate are the two control parameters. The second case presents the effect of temperature on the oscillatory behaviors, and temperature and a flow rate are the control parameters. Stochastic resonance is demonstrated in the first case in which a signal and noise are applied to the different inputs, respectively. The scenario and novel aspects of SR in two-parameter systems are discussed, and the possibility of the analogous SR in biological systems is also pointed out.     

Muscle receptor organs do not mediate load compensation during body roll and defense response extensions in the crayfish Cherax destructor.

It has been proposed that the abdominal muscle receptor organ (MRO) of decapod crustaceans acts in a sensory feedback loop to compensate for external load. There is not yet unequivocal evidence of MRO activity during slow abdominal extension in intact animals, however. This raises the possibility that MRO involvement in load compensation is context-dependent. We recorded from MRO tonic stretch receptors (SRs) in freely behaving crayfish (Cherax destructor) during abdominal extension occurring during two different behaviors: body roll and the defense response. Abdominal extensions are similar in many respects in both behaviors, although defense response extensions are more rapid. In both situations, SR activity typically ceased when the abdominal extension commenced, even if the joint of the SR being monitored was mechanically prevented from extending by a block. Since extensor motor neuron activity increased when the abdomen was prevented from extending, we concluded that the load compensation occurring in these behaviors was not mediated by the MROs.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. III. Block of Ca2+-induced Ca2+ release by organic polyamines

Calcium ions that have been preloaded into isolated SR subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances in a manner that is effectively blocked by ruthenium red and other organic polyamines. Effective blocking substances include certain antibiotics (neomycin, gentamicin, streptomycin, clindamycin, kanamycin, and tobramycin), naturally occurring polyamines (spermine and spermidine), and a number of basic polypeptides and proteins (polylysine, polyarginine, certain histones, and protamine). These agents have only one feature in common: the presence of several amino groups. Ruthenium red, neomycin, spermine, and protamine all appear to act by blocking SR Ca2+ channels since unidirectional 45Ca2+ efflux from the vesicles is strongly inhibited by these agents. Functions ascribable to the SR Ca2+ pump are largely unaffected by these agents. Since inositol 1,4,5-trisphosphate is ineffective at inducing Ca2+ release under these conditions, we conclude that these polyamines may directly block SR Ca2+ channels at very low concentrations by a mechanism unrelated to effects on inositol 1,4,5-trisphosphate production.     

SR proteins are required for nematode trans-splicing in vitro.

SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, including modulation of alternative splicing, commitment of substrates to the splicing pathway, and splice site communication. The last of these, splice site communication, is particularly relevant to trans-splicing in which the 5' and 3' exons originate on separate molecules. The participation of SR proteins in naturally occurring, spliced leader RNA-dependent transsplicing has not been examined. Here, we have isolated SR proteins from an organism that performs both trans- and cis-splicing, the nematode Ascaris lumbricoides. To examine their activity in in vitro splicing reactions, we have also developed and characterized an SR protein-depleted whole-cell extract. When tested in this extract, the nematode SR proteins are required for both trans- and cis-splicing. In addition, the state of phosphorylation of the nematode SR proteins is critical to their activity in vitro. Interestingly, mammalian (HeLa) and A. lumbricoides SR proteins exhibit equivalent activities in cis-splicing, while the nematode SR proteins are much more active in trans-splicing. Thus, it appears that SR proteins purified from an organism that naturally trans-splices its pre-mRNAs promote this reaction to a greater extent than do their mammalian counterparts.     

Flow Cytometric Applications of Sulforhodamine 101 as a Fluorescent Stain for Total Cellular Protein

In this paper, the use of Sulforhodamine 101 (SR 101; C.I. 14318) as a fluorescent stain for flow cytometric determinations of total cellular protein (TCP) is described. Flow cytometric quantification of TCP fluorescence can provide a valuable analytical parameter for assessing both changes occurring in overall cellular protein content, such as in response to blast transformation, and heterogeneity in cellular size within a specimen, such as a tumor. Very little information is available in the literature pertaining to the use of SR 101 as a protein stain. Like fluorescein isothiocyanate (FITC), SR 101 can be excited at 488 nm; however, it binds ionically and has an emission maximum at 600 nm, which is advantageous in certain staining and filter combinations. In this report, the utility of SR 101 staining is demonstrated using pokeweed mitogen-stimu-lated lymphocytes and cycloheximide- and di-methylsufloxide-treated cells. Single, two- and three-color flow cytometric applications are possible, using SR 101 in combination with 4',6-diamidino-2-phenylindole (DAPI) and/or FITC.     

Dependence of the fluorescence of fluorescein labelled (Ca2+, Mg2+)-ATPase upon the lipid to protein ratio in sarcoplasmic reticulum reconstituted systems

Reconstituted sarcoplasmic reticulum (SR) vesicles have been prepared mixing fluorescein labelled SR, excess endogenous lipids and deoxycholate by a rapid dilution protocol and several freeze-thaw treatments. We have found that both the steady-state level and the polarization of fluorescein fluorescence of these reconstituted systems monotonically increase as a function of the lipid to protein ratio between 80 and 2000 (on a mole per mole basis). The magnitude of this increase is about 15%. Detergents, such as Triton X-100 and deoxycholate, when added to SR labelled vesicles below their critical micelle concentrations also induce similar changes in fluorescein fluorescence. We suggest that lipid dilution of protein in these reconstituted systems induce a decrease of the level of self-quenching by promoting dissociation of (Ca2+, Mg2+)-ATPase.     

Proton pump activity in bundle sheath tissues of broad-leaved trees in relation to leaf age

The fluorescent probe sulphorhodamine G (SR) has been previously used as an indicator of low extra-cellular pH and, by inference, of proton extrusion activity in living leaves. In legumes the SR uptake and proton extrusion was characteristic of the extended bundle sheath system (EBS) or paraveinal mesophyll, composed of bundle sheath cells and the related network of bridging cells between veins. This system has been identified as a site of temporary storage of amino carbon in soybean. A tree species. Populus deltoides Bartr. ex Marsh, was known both to have the EBS system in its leaves and to carry organic nitrogen in its xylem sap. It is now shown that P. deltoides also accumulates the SR probe in the EBS system. This association has been explored in 8 other broad-leaved tree species. Seven of the 8 species have EBS systems and accumulate SR in them in early summer. The 8th species, Tilia americana L. has no EBS system and shows weak SR accumulation. The capacity to accumulate SR (and by inference to scavenge solutes from the transpiration stream) disappeared in all species at various stages in late summer. In two species, in addition, SR accumulation is interrupted for several weeks during fruit growth. It is proposed that EBS systems will be found in many dicotyledonous leaves, and will be found to scavenge solutes, especially organic nitrogen, from the xylem sap.     

Hypophosphorylated ASF/SF2 binds TAP and is present in messenger ribonucleoproteins

Serine/arginine-rich proteins (SR proteins) function in precursor mRNA (pre-mRNA) splicing and may also act as adaptors for mRNA export. SR proteins are dynamically phosphorylated in their RS domain, and differential phosphorylation modulates their splicing activity and subcellular localization. In this study, we investigated the influence of phosphorylation on the function of SR proteins in events occurring during mRNA maturation. Immunoprecipitation experiments showed that the mRNA export receptor TAP associates preferentially with the hypophosphorylated form of shuttling SR proteins, including ASF/SF2. Overexpression of ASF induced subnuclear relocalization of TAP to SR protein-enriched nuclear speckles, suggesting their interaction in vivo. Moreover, the ASF found in a nucleoplasmic fraction rich in heterogeneous nuclear ribonucleoprotein (hnRNP) complexes is hyperphosphorylated, whereas mature messenger RNP (mRNP)-bound ASF is hypophosphorylated. Therefore, hypophosphorylation of ASF in mRNPs coincides with its higher affinity for TAP, suggesting that dephosphorylation of ASF promotes both its incorporation into mRNPs and recruitment of TAP for mRNA export. Thus, the phosphorylation state of RS domains may modulate the function of mammalian shuttling SR proteins during mRNA maturation or export.     

Influence of terrestrial runoff on phytoplankton species richness-biomass relationships: A double stress hypothesis

The relationship between biomass and species richness (SR) was studied along productivity gradients in coastal phytoplankton assemblages under the influence of terrestrial runoff. In particular, the investigation focused on (a) whether phytoplankton blooms affect the shape of the relationship, (b) whether the relationship is taxon specific (between dinoflagellates and diatoms), and (c) the potential mechanisms regulating the shape of the relationship. Analysis was based on phytoplankton and physicochemical data from six coastal areas in the Aegean Sea, Eastern Mediterranean, covering the whole range from oligotrophy to eutrophication. A SR-biomass relationship was not observed for the more oligotrophic areas, whereas hump-shaped curves were found in areas yielding higher maximum biomass values, corresponding to bloom events occurring mainly during winter. Furthermore, the observed hump-shaped relationships were taxon specific since they were more pronounced in the case of diatoms than dinoflagellates. It is hypothesized that SR at the leftmost part of the hump-shape is nutrient regulated, whereas at the rightmost part is limited by extreme abiotic stress occurring during the formation of blooms in coastal ecosystems. The results of this study suggest that a double stress mechanism related to abiotic factors may be responsible for the hump-shaped SR-biomass relationships occurring in coastal phytoplankton communities at various levels of productivity.     

Excitation signal processing times in Halobacterium halobium phototaxis.

Phototaxis responses of Halobacterium halobium were monitored with a computerized cell-tracking system coupled to an electronic shutter controlling delivery of photostimuli. Automated analysis of rates of change in direction and linear speeds provided detection of swimming reversals with 67 ms resolution, permitting measurement of distinct phases of the responses to attractant and repellent stimuli. After stimulation, there was a latency period in which the population reversal frequency was unchanged, followed by an excitation phase in which reversal frequency increased, and a slower adaptation phase in which reversal frequency returned to its prestimulus value. A step-decrease in illumination of the attractant receptor slow-cycling or sensory rhodopsin (SR) (lambda max, 587 nm) was interpreted by the cells as an unfavorable stimulus and, after a minimum latency of 0.70 +/- 0.14 s, induced swimming reversals with the peak response occurring 1.34 +/- 0.07 s after onset of the stimulus. Two distinct repellent responses in the near UV/blue were observed. One was a reversal response to 400 nm light, which was dependent on orange-red background illumination as expected for the photointermediate repellent form of SR (lambda max, 373 nm). The minimum latency of this response was approximately the same as that of the SR attractant system. The second was a reversal response with shorter minimum latency (0.40 +/- 0.07 s) to light of longer wavelength (450 nm) than absorbed by the known SR repellent form. This result confirms recent findings of an additional repellent photosystem in this spectral range. Further, the longer wavelength repellent response is independent of orange-red background illumination, indicating that the photoreceptor mediating this response is not a photointermediate of SR.     

Oxidative damage to Ca2+-ATPase sarcoplasmic reticulum by HOCl and protective effect of some antioxidants

Injury of rabbit skeletal sarcoplasmic reticulum (SR) induced by hypochlorous acid (HOCl) was studied. HOCl inhibited Ca2+-ATPase activity in a concentration-dependent manner (IC50=100 micromol/l). The concentration of 13.5 micromol/l HOCl reduced the level of sulfhydryl (SH) groups by 50%, yet it did not influence the enzyme activity. In comparison with SH group oxidation and enzyme activity inhibition, a significantly longer time was necessary for the generation of protein carbonyls in SR injured by HOCl. Protective effects of some antioxidants (stobadine, trolox, EGb 761, Pycnogenol) were studied in SR oxidatively injured by HOCl. Trolox and EGb 761 exerted a protective effect on ATPase activity and on SH groups of SR oxidatively modified by HOCl. Stobadine and Pycnogenol inhibited markedly protein carbonyl formation. Stobadine was the only antioxidant able to scavenge HOCl. In conclusion, the protective effects of antioxidants against decrease of Ca2+-ATPase activity induced by HOCl might be caused by protection of SH groups. The compounds with both antioxidant and Ca2+-ATPase protecting effect offer dual defense against tissue damage occurring, e.g. in aging process.     

Increased intracellular Ca2+ and SR Ca2+ load contribute to arrhythmias after acidosis in rat heart. Role of Ca2+/calmodulin-dependent protein kinase II

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.     

Effects of ryanodine in skinned cardiac cells

Ryanodine (1 X 10(-5) M) did not affect the Ca2+ sensitivity of the myofilaments of skinned (sarcolemma removed by microdissection) cardiac cells from the rat ventricle. Ryanodine (1 X 10(-5) M) inhibited three types of Ca2+ release from the sarcoplasmic reticulum (SR), which have different mechanisms: 1) Ca2+-induced release of Ca2+ triggered by a rapid and transient increase of [free Ca2+] at the outer surface of the SR; 2) caffeine-induced release of Ca2+; 3) spontaneous cyclic release of Ca2+ occurring in the continuous presence of a [free Ca2+] sufficient to overload the SR. These results suggest that the three types of Ca2+ release are through the same channel across the SR membrane, although the gating mechanisms are different for the three types. Ryanodine also diminished the rate of Ca2+ accumulation into the SR. Even in the presence of 1 X 10(-5) M ryanodine the SR accumulated Ca2+ that could be released when the SR was sufficiently overloaded with Ca2+. Thus, ryanodine pretreatment did not permit the direct activation of the myofilaments by externally applied Ca2+. The approximately 1000-fold difference in the effective concentrations of ryanodine in intact vs. skinned cardiac cells suggests that low concentrations of ryanodine act in the intact cardiac tissues through processes or on structures that are destroyed by the skinning procedure. No significant differences were observed in the effects of ryanodine in skinned cardiac cells from different adult mammalian species.     

Gingerol, a novel cardiotonic agent, activates the Ca2+-pumping ATPase in skeletal and cardiac sarcoplasmic reticulum

Gingerol, isolated as a potent cardiotonic agent from the rhizome of ginger, stimulated the Ca2+-pumping activity of fragmented sarcoplasmic reticulum (SR) prepared from rabbit skeletal and dog cardiac muscles. The extravesicular Ca2+ concentrations of the heavy fraction of the fragmented SR (HSR) were measured directly with a Ca2+ electrode to examine the effect of gingerol on the SR. Gingerol (3-30 microM) accelerated the Ca2+-pumping rate of skeletal and cardiac SR in a concentration-dependent manner. The rate of 45Ca2+ uptake of HSR was also increased markedly by 30 microM gingerol without affecting the 45Ca2+ efflux from HSR. Furthermore, gingerol activated Ca2+-ATPase activities of skeletal and cardiac SR (EC50, 4 microM). The activation of SR Ca2+-ATPase activity by gingerol (30 microM) was completely reversed by 100-fold dilution with the fresh saline solution. Kinetic analysis of activating effects of gingerol suggests that the activation of SR Ca2+-ATPase is uncompetitive and competitive with respect to Mg . ATP at concentrations of 0.2-0.5 mM and above 1 mM, respectively. Kinetic analysis also suggests that the activation by gingerol is mixed-type with respect to free Ca2+ and this enzyme is activated probably due to the acceleration of enzyme-substrate complex breakdown. Gingerol had no significant effect on sarcolemmal Ca2+-ATPase, myosin Ca2+-ATPase, actin-activated myosin ATPase and cAMP-phosphodiesterase activities, indicating that the effect of gingerol is rather specific to SR Ca2+-ATPase activity. Gingerol may provide a valuable chemical tool for studies aimed at clarifying the regulatory mechanisms of SR Ca2+-pumping systems and the causal relationship between the Ca2+-pumping activity of SR and muscle contractility.     

Activating and deactivating roles of lipid bilayers on the Ca(2+)-ATPase/phospholamban complex

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.