The effect of some organophosphorous and organochlorine compounds on calcium uptake by Sarcoplasmic reticulum isolated from insect and crustacean skeletal muscle

Summary Relatively pure mitochondrial-free Sarcoplasmic reticulum (SR) was isolated from crab and cockroach skeletal muscle, and the SR calcium uptake was studied in control and insecticide-treated conditions by incubation in Ca⁴⁵ media followed by millipore filtration and counting by liquid scintillation methods. All insecticides used (parathion, DDT, BHC and tri-cresyl phosphate) at 1 mM concentrations caused a slight fall in the intrinsic Ca²⁺ content of the SR, but this Ca²⁺ loss was insufficient to explain the contracture induction effect of these agents.All insecticides used (at 1 mM) caused a massive inhibition of SR Ca²⁺ uptake, and all were able to release Ca²⁺ previously bound to the SR during incubation in the presence of ATP. It is concluded that the contracture-induction effect of these insecticides is due to their inhibition of SR Ca²⁺ uptake, resulting in a massive rise in myoplasmic free Ca²⁺.     

Challenges of Improving the Evidence Base in Smaller Surgical Specialties,as Highlighted by a Systematic Review of Gastroschisis Management

Objective

To identify methods of improving the evidence base in smaller surgical specialties, using a systematic review of gastroschisis management as an example.

Background

Operative primary fascial closure (OPFC), and silo placement with staged reduction and delayed closure (SR) are the most commonly used methods of gastroschisis closure. Relative merits of each are unclear.

Methods

A systematic review and meta-analysis was performed comparing outcomes following OPFC and SR in infants with simple gastroschisis. Primary outcomes of interest were mortality, length of hospitalization and time to full enteral feeding.

Results

751 unique articles were identified. Eight met the inclusion criteria. None were randomized controlled trials. 488 infants underwent OPFC and 316 underwent SR. Multiple studies were excluded because they included heterogeneous populations and mixed intervention groups. Length of stay was significantly longer in the SR group (mean difference 8.97 days, 95% CI 2.14–15.80 days), as was number of post-operative days to complete enteral feeding (mean difference 7.19 days, 95%CI 2.01–12.36 days). Mortality was not statistically significantly different, although the odds of death were raised in the SR group (OR 1.96, 95%CI 0.71–5.35).

Conclusions

Despite showing some benefit of OPFC over SR, our results are tempered by the low quality of the available studies, which were small and variably reported. Coordinating research through a National Paediatric Surgical Trials Unit could alleviate many of these problems. A similar national approach could be used in other smaller surgical specialties.     

Theory of excitation-contraction coupling in cardiac muscle.

The consequences of cardiac excitation-contraction coupling by calcium-induced calcium release were studied theoretically, using a series of idealized models solved by analytic and numerical methods. "Common-pool" models, those in which the trigger calcium and released calcium pass through a common cytosolic pool, gave nearly all-or-none regenerative calcium releases (in disagreement with experiment), unless their loop gain was made sufficiently low that it provided little amplification of the calcium entering through the sarcolemma. In the linear (small trigger) limit, it was proven rigorously that no common-pool model can give graded high amplification unless it is operated on the verge of spontaneous oscillation. To circumvent this problem, we considered two types of "local-control" models. In the first type, the local calcium from a sarcolemmal L-type calcium channel directly stimulates a single, immediately opposed SR calcium release channel. This permits high amplification without regeneration, but requires high conductance of the SR channel. This problem is avoided in the second type of local control model, in which one L-type channel triggers a regenerative cluster of several SR channels. Statistical recruitment of clusters results in graded response with high amplification. In either type of local-control model, the voltage dependence of SR calcium release is not exactly the same as that of the macroscopic sarcolemmal calcium current, even though calcium is the only trigger for SR release. This results from the existence of correlations between the stochastic openings of individual sarcolemmal and SR channels. Propagation of regenerative calcium-release waves (under conditions of calcium overload) was analyzed using analytically soluble models in which SR calcium release was treated phenomenalogically. The range of wave velocities observed experimentally is easily explained; however, the observed degree of refractoriness to wave propagation requires either a strong dependence of SR calcium release on the rate of rise of cytosolic calcium or localization of SR release sites to one point in the sarcomere. We conclude that the macroscopic behavior of calcium-induced calcium release depends critically on the spatial relationships among sarcolemmal and SR calcium channels, as well as on their kinetics.     

Fluorescence and differential light absorption recordings with calcium probes and potential-sensitive dyes in skinned cardiac cells

This report describes an optical system for microspectrophotometry in a single cardiac cell from which the sarcolemma has been removed by microdissection (skinned cardiac cell). This system is attached to the high power inverted microscope used for the microdissection and includes (a) a single variable wavelength microspectrophotometer used to define the spectrum of a given dye or Ca2+ probe; and (b) a dual wavelength, differential microspectrophotometer used to record differentially between the optimum wavelength and a wavelength separated by 25--30 nm. Results are presented using the following optical methods: (a) fluorescence measurements with chlorotetracycline to monitor the amount of Ca2+ bound to the inner face of the sarcoplasmic reticulum (SR) membrane; (b) differential absorption measurements with arsenazo III to measure changes of myoplasmic [Ca2+]free resulting from Ca2+ release from the SR; (c)fluorescence and (or) differential absorption measurements with the potential-sensitive dyes merocyanine 540, NK 2367, and di-S-C3(5) to monitor changes of charge distribution on the SR membrane during Ca2+ accumulation in the SR, as well as before and during Ca2+-induced release of Ca2+ from the SR. A small and rapid signal is observed which precedes the Ca2+-induced release of Ca2+ from the SR. It is detected as an increase of CA2+ binding inside the SR with chlorotetracycline and as a "hyperpolarization" with potential-sensitive dyes, while no transient change of myoplasmic [Ca2+]free is detected with arsenazo III. This small and rapid signal preceding the Ca2+ release may be a first hint to an understanding of the mechanism whereby a small increase of [Ca2+]free outside the SR triggers Ca2+ release from the SR.     

Decavanadate is responsible for vanadate-induced two-dimensional crystals in sarcoplasmic reticulum

Two-dimensional protein crystals of the calcium pump protein of sarcoplasmic reticulum (SR) from fast skeletal muscle were induced using Na₃VO₃ as first described by Dux and Martonosi. These crystals exhibit repeat rows 11 nm apart which contain discrete units with 7 nm repeats. Four different methods of sample preparation for electron microscopy, i.e., negative staining, freezedrying, freeze-fracturing, and thin-sectioning electron microscopy, each give complimentary repeat units. The SR-membrane crystals exhibit surface structure by the freeze-drying technique and row-like structures on the normally smooth outer face of normal SR. The formation of the membrane crystals is dependent on the pH and concentration of the vanadate. Only conditions favoring the presence of decavanadate yield crystals. At low concentrations and neutral pH, decavanadate is unstable and with time converts to smaller oligomers and the monomer. The presence of membrane crystals was correlated with the life span of the decavanadate. Membrane crystals were obtained in the SR membrane from fast twitch muscle from light and heavy SR, referable to longitudinal and terminal cisternae as well as from reconstituted SR. Canine cardiac SR did not crystallize under these conditions.Abbreviations Tris (tris[hydroxymethyl])aminomethane - TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), 2-(2-hydroxy-1-bis[hydroxymethyl]ethyl)aminoethanesulfonic acid - SR sarcoplasmic reticulum - CPP calcium pump protein Dedicated to the memory of Prof. David E. Green, friend, mentor, and colleague.     

Sarcoplasmic reticulum Ca2+-pump density is higher in distal than in proximal segments of porcine left coronary artery

Densities of sarcoplasmic reticulum (SR) Ca²⁺-pump were compared in proximal and distal segments of pig left coronary artery using two biochemical methods: acylphosphate formation and immunoreactivity in Western blots, and a functional assay based on contraction to SR Ca²⁺-pump inhibitors. In the microsomes prepared from smooth muscle, the level of the 115 kDa SR Ca²⁺-pump acylphosphate was 7.1 ± 0.3 -fold greater in distal than in proximal segments. Similarly in Western blots using these microsomes, the reactivity of the 115 kDa band to an anti-SR Ca²⁺-pump antibody was 5.3 ± 0.8 -fold greater in distal than in proximal segments. Endothelium free coronary artery rings contracted to the SR Ca²⁺-pump inhibitors Cyclopiazonic acid (CPA, EC₅₀ = 0.19 ± 0.06 M) and thapsigargin (EC₅₀ = 0.0095 ± 0.0035 M). With 10 M CPA, the force of contraction per tissue wet weight was 4.2 ± 0.5-fold greater in distal than in proximal rings, and with 1 M thapsigargin it was 4.0 ± 1.0 -fold greater. The contractions produced by 60 mM KCl were used as a control. In contrast to the CPA and thapsigargin, the force per mg tissue weight produced by 60 mM KCl did not differ significantly between the proximal and distal segments. Thus, the results from the two biochemical methods and those from the contractility data were all consistent with the smooth muscle in the distal segments of the coronary artery containing a higher density of the SR Ca²⁺-pump than the proximal segments.Abbreviations CPA cyclopiazonic acid - DTT dithiothreitol - SR sarcoplasmic reticulum     

How source content determines intracellular Ca2+ release kinetics. Simultaneous measurement of [Ca2+] transients and [H+] displacement in skeletal muscle

In skeletal muscle, the waveform of Ca(2+) release under clamp depolarization exhibits an early peak. Its decay reflects an inactivation, which locally corresponds to the termination of Ca(2+) sparks, and is crucial for rapid control. In cardiac muscle, both the frequency of spontaneous sparks (i.e., their activation) and their termination appear to be strongly dependent on the Ca(2+) content in the sarcoplasmic reticulum (SR). In skeletal muscle, no such role is established. Seeking a robust measurement of Ca(2+) release and a way to reliably modify the SR content, we combined in the same cells the "EGTA/phenol red" method (Pape et al., 1995) to evaluate Ca(2+) release, with the "removal" method (Melzer et al., 1987) to evaluate release flux. The cytosol of voltage-clamped frog fibers was equilibrated with EGTA (36 mM), antipyrylazo III, and phenol red, and absorbance changes were monitored simultaneously at three wavelengths, affording largely independent evaluations of Delta[H(+)] and Delta[Ca(2+)] from which the amount of released Ca(2+) and the release flux were independently derived. Both methods yielded mutually consistent evaluations of flux. While the removal method gave a better kinetic picture of the release waveform, EGTA/phenol red provided continuous reproducible measures of calcium in the SR (Ca(SR)). Steady release permeability (P), reached at the end of a 120-ms pulse, increased as Ca(SR) was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting Ca(SR) (four cells). Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low Ca(SR). The increase in steady P upon depletion was associated with a slowing of the rate of decay of P after the peak (i.e., a slower inactivation of Ca(2+) release). These results are consistent with a major inhibitory effect of cytosolic (rather than intra-SR) Ca(2+) on the activity of Ca(2+) release channels.     

Dynamic Distribution and Interaction of the Arabidopsis SRSF1 Subfamily Splicing Factors

Ser/Arg-rich (SR) proteins are essential nucleus-localized splicing factors. Our prior studies showed that Arabidopsis (Arabidopsis thaliana) RSZ22, a homolog of the human SRSF7 SR factor, exits the nucleus through two pathways, either dependent or independent on the XPO1 receptor. Here, we examined the expression profiles and shuttling dynamics of the Arabidopsis SRSF1 subfamily (SR30, SR34, SR34a, and SR34b) under control of their endogenous promoter in Arabidopsis and in transient expression assay. Due to its rapid nucleocytoplasmic shuttling and high expression level in transient assay, we analyzed the multiple determinants that regulate the localization and shuttling dynamics of SR34. By site-directed mutagenesis of SR34 RNA-binding sequences and Arg/Ser-rich (RS) domain, we further show that functional RRM1 or RRM2 are dispensable for the exclusive protein nuclear localization and speckle-like distribution. However, mutations of both RRMs induced aggregation of the protein whereas mutation in the RS domain decreased the stability of the protein and suppressed its nuclear accumulation. Furthermore, the RNA-binding motif mutants are defective for their export through the XPO1 (CRM1/Exportin-1) receptor pathway, but retain nucleocytoplasmic mobility. We performed a yeast two hybrid screen with SR34 as bait and discovered SR45 as a new interactor. SR45 is an unusual SR splicing factor bearing two RS domains. These interactions were confirmed in planta by FLIM-FRET and BiFC and the roles of SR34 domains in protein-protein interactions were further studied. Altogether, our report extends our understanding of shuttling dynamics of Arabidopsis SR splicing factors.Ser/Arg-rich (SR) protein is the collective name given to a family of highly conserved splicing factors in Eukaryotes that regulate constitutive and alternative precursor mRNA splicing. SR proteins contain at least one RNA recognition motif (RRM) and an Arg/Ser-rich (RS) C-terminal domain (Manley and Krainer, 2010; Califice et al., 2012). The RRM appears to determine RNA-binding specificity, while the RS domain is involved in protein-protein and protein-RNA interactions (Shen et al., 2004). In human, twelve SR proteins have been described based on a set of formal criteria (Manley and Krainer, 2010). SR proteins have a modular organization: some SR proteins contain two RRMs while others contain a Zn-knuckle, which contributes to RNA binding. The activity of SR proteins is regulated by posttranslational modifications, such as Ser phosphorylation/dephosphorylation and Arg methylation. At steady-state, SR proteins accumulate in subnuclear speckles, which correspond to storage, assembly, and/or modification compartments for splicing factors. Several human SR proteins shuttle between the nucleus and the cytoplasm, and this dynamic shuttling is linked to their postsplicing activities in mRNA export, stability, and translation (Long and Caceres, 2009). The multiple roles and mechanisms of action of mammalian SR proteins have been extensively studied (for review, see Long and Caceres, 2009; Zhong et al., 2009; Kornblihtt et al., 2013; Änkö, 2014).The number of genes encoding SR proteins is higher in plants compared with metazoan. Plant genomes contain SR proteins homologous to the animal prototypes SRSF1/SRSF2/SRSF7, as well as plant-specific ones (Barta et al., 2010; Califice et al., 2012). Arabidopsis SR splicing factors localize into nuclear irregular dynamic domains similar to speckles, with no, only partial or complete colocalization (Tillemans et al., 2005; Lorković et al., 2008; Reddy et al., 2012). The functions of plant SR factors in postsplicing events remain unknown, though a nucleocytoplasmic shuttling activity has been described for RSZ22, a prototypic member of the SRSF7 subgroup (1 RRM, 1 Zn-knuckle) of Arabidopsis (Arabidopsis thaliana) SR protein family (Tillemans et al., 2006; Rausin et al., 2010).The nucleocytoplasmic transport of RNA and proteins occurs through nuclear pore complexes (NPCs), which require importin and exportin receptors (karyopherins or Kap) for trafficking of molecules larger than 40–90 kD. Kap often binds to cargo molecules that carry either nuclear localization signals (NLS) for nuclear import or nuclear export signals (NES) for nuclear export (Boruc et al., 2012). The best-known import pathway is mediated by the importin-α/β Kap that binds to NLS. Kap-β2 (or Transportin-SR, TRN-SR) was shown to function as the nuclear import receptor for human SRSF1 and SRSF2, and several Arabidopsis SR proteins (Yun et al., 2003; Xu et al., 2011). The human TRN-SR has recently been shown to embrace both the RRM and RS domains of SRSF1 for nuclear import (Maertens et al., 2014).XPO1 (Exportin-1, also named CRM1 in yeast [Saccharomyces cerevisiae]) is a well-characterized mammalian nuclear export receptor which recognizes Leu-rich NES (φ-X_2-3-φ-X_2-3-φ-X-φ, where φ is L, V, I, F, or M and X is any amino acid) on proteins implicated in snRNA and rRNA export (Natalizio and Wente, 2013). XPO1/CRM1 was also shown to mediate the export of unspliced (or partially spliced) viral mRNAs and of a small subset of mRNAs. XPO1 recruitment to mRNA is mediated by single adaptor proteins including Leu-rich pentatricopeptide repeat proteins (LRPPRC) and HuR (Natalizio and Wente, 2013). Apart from this, the bulk of mRNA is exported by the nonkaryopherin heterodimer Nxf1-Nxt1 (TAP-p15) in metazoans (Mex67-Mtr2 in yeast). The shuttling SR proteins are known to promote messenger ribonucleoprotein (mRNP) export through NPCs when dephosphorylated by interacting with export factor Nxf1 (Huang et al., 2003). Several human SR proteins are also part of the exon junction complex (EJC) deposited upstream of exon-exon junctions after splicing, consistent with a role of SR proteins in mRNP export and nonsense mediated RNA decay (Singh et al., 2012). The RS domain is necessary but not sufficient for the cytoplasmic export of shuttling SR proteins (Cáceres et al., 1997).We previously identified RSZ22 as a shuttling splicing factor whose nuclear export is at least partly controlled by the XPO1-dependent export pathway (Tillemans et al., 2006; Rausin et al., 2010). Mutating conserved residues within the RNA-binding motifs of this specific SR protein highlighted the in vivo dependence of RNA binding for proper subcellular dynamics (Rausin et al., 2010). However, the role of the different protein domains in directing the cellular dynamics may vary among SR proteins, and the role of the RS domain of RSZ22 had not been investigated. It is also unknown whether XPO1-dependent nuclear export also includes other Arabidopsis SR proteins. A more global understanding of the molecular mechanisms underlying the nucleocytoplasmic transport of plant SR factors therefore required further investigation.Here, we functionally characterized the four Arabidopsis SR proteins of the SRSF1 subfamily (orthologs of mammalian SRSF1) that contain two conserved RRM domains (Califice et al., 2012). We studied the expression profiles of SR30, SR34, SR34a, and SR34b, and attempted to investigate their shuttling activity. Among these SR proteins, SR30 showed a less active nuclear export rate, and SR34b protein was not detectable in any expression assay. Because of its stability and rapid shuttling, we further focused on the SR34 protein by generating a series of mutant versions of the RRMs and RS domains. We established the overall requirement of these protein domains to retain nucleocytoplasmic shuttling activity. Yeast two-hybrid (Y2H) assays also revealed strong interactions between SRSF1 subfamily members (SR30, SR34, and SR34a) and SR45, an atypical SR protein (two RS domains). We also investigated the importance of SR34 domains in protein-protein interactions. Collectively, our findings provide a more detailed mechanistic understanding of the role of the structural determinants regulating SR proteins dynamics, and insights into protein domain function in in vivo interactions.     

A quantitative study of synaptic ribbons in pinealocytes of adult Chinese hamsters (Cricetulus griseus) under different photoperiodic conditions

Summary Synaptic ribbons (SR) in pinealocytes of adult (120–130 day-old) male Chinese hamsters (Cricetulus griseus) were classified into types 1, 2 and 3; these have a central dense structure showing rod-like, various and ringlike profiles, respectively. The central structure of the type-2 SR usually appeared as round, oval or comma-like bodies, and occasionally as plates showing various profiles or clubshaped bodies. The quantity of each type of SR, expressed as the SR index, was determined over a 24-h period under a light/dark regime (LD) 1212 or LD 1410. On comparing the results obtained from adults with previously published data from young (60–70-days-old) animals under LD 1212, it was found that, in both young and adult animals, the type-1 and type-3 SR indices exhibited different 24-h variations, whereas the type-2 SR index remained constant over a 24-h period. In addition, the indices of the type-2 SR, but not those of the other SR types, were found to be significantly larger in adult than in young animals. In adult animals, the effects of the photoperiod were different between the three types of SR. A nocturnal increase in the type-1 SR index was observed under both LD 1212 and LD 1410, its time course being different for each of these photoperiods. Under LD 1410, the type-2 SR index showed a significant 24-h rhythm with larger values during the dark period; this was not observed under LD 1212. The type-3 SR index was almost the same under LD 1212 and LD 1410. The results suggest that pinealocyte SR of the Chinese hamster may be composed of three types of SR, each with a different functional role.     

Serine Racemase Regulated by Binding to Stargazin and PSD-95: POTENTIAL N-METHYL-d-ASPARTATE-α-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLEPROPIONIC ACID (NMDA-AMPA) GLUTAMATE NEUROTRANSMISSION CROSS-TALK*

d-Serine, an endogenous co-agonist for the glycine site of the synaptic NMDA glutamate receptor, regulates synaptic plasticity and is implicated in schizophrenia. Serine racemase (SR) is the enzyme that converts l-serine to d-serine. In this study, we demonstrate that SR interacts with the synaptic proteins, postsynaptic density protein 95 (PSD-95) and stargazin, forming a ternary complex. SR binds to the PDZ3 domain of PSD-95 through the PDZ domain ligand at its C terminus. SR also binds to the C terminus of stargazin, which facilitates the cell membrane localization of SR and inhibits its activity. AMPA receptor activation internalizes SR and disrupts its interaction with stargazin, therefore derepressing SR activity, leading to more d-serine production and potentially facilitating NMDA receptor activation. These interactions regulate the enzymatic activity as well as the intracellular localization of SR, potentially coupling the activities of NMDA and AMPA receptors. This shuttling of a neurotransmitter synthesizing enzyme between two receptors appears to be a novel mode of synaptic regulation.     

Three-dimensional arrangement of sarcoplasmic reticulum in crayfish as seen by high voltage electron microscope

Summary Sections several m thick of single fibres from the crayfish muscle were examined by means of a high voltage electron microscope to find out the geometry of the sarcoplasmic reticulum (SR). A sufficient contrast of the SR was achieved by lead acetate histochemical method for calcium. Longitudinally oriented files of SR vesicles at the level of A bands and interruptions in otherwise continuous SR net are the most conspicuous features.     

The effects of short pulses of light at night on numbers of pineal “synaptic” ribbons and serotonin N-acetyltransferase activity in male Sprague-Dawley rats

Summary To characterize further the functionally enigmatic synaptic ribbons (SR) of the mammalian pineal gland and to study possible relationships to melatonin synthesis, in the present investigation rats were exposed to short pulses of light at night when both SR numbers and serotonin N-acetyltransferase (NAT) activity are high in comparison to day-time values. Male Sprague-Dawley rats were killed at 13:00 and 01:00 h, respectively, and at 01:10 and 02:00 h after exposure to light for 10 and 60 min, respectively. The pineals were rapidly taken out and cut sagittally in half. One half was processed for electron-microscopic quantitation of SR numbers and the other half for NAT determinations. It was found that both SR numbers and NAT activity decreased significantly when the animals were exposed to light at night. Although both parameters showed corresponding changes, there was no clear-cut correlation between SR numbers and NAT activity in individual animals within a group, except after exposure to light for 60 min when a positive correlation (R = 0.939; p < 0.05) existed. After exposure to light the electron-lucent vesicles of the SR decreased in number, but the length of the SR was unchanged. These results show that numbers of pineal SR can be easily and quickly manipulated and that the presently used model may be ideal in studying the poorly understood mode in which degradation of SR occurs.Recipient of a DAAD stipend, on leave from Department of Zoology, University of Burdwan, Burdwan, India     

Cholesterol distribution and structural differentiation in the sarcoplasmic reticulum of rat cardiac muscle cells

Summary The polyene compound, filipin, was used as a probe to localize cholesterol in the membranes of the rat cardiac muscle cell, with particular reference to the sarcoplasmic reticulum (SR). Filipin binds specifically to cholesterol (and related 3--hydroxysterols) in membranes, producing distinct deformations which can be viewed by freeze-fracture and used as markers for the presence of cholesterol-rich regions in the membrane plane. In freeze-fracture replicas of filipin-treated rat myocardium, the muscle cells revealed abundant deformations in their plasma membranes, no deformations in mitochondrial membranes, and an intermediate response in the SR. These results are in agreement with the levels of cholesterol reported in isolated fractions of the different membrane types, and confirm the specificity of filipin action. Within the SR, the filipin-induced deformations were not randomly distributed but occurred more commonly in free SR at or near the Z-region of the sarcomere than in other parts of the free SR or the junctional SR. This finding is interpreted as evidence for a non-homogeneous distribution of cholesterol in cardiac muscle cell SR. The possible significance of cholesterol in relation to structural differentiation and function of the SR is discussed.     

Nuclear Compartmentalization of Serine Racemase Regulates d-Serine Production: IMPLICATIONS FOR N-METHYL-d-ASPARTATE (NMDA) RECEPTOR ACTIVATION*

d-Serine is a physiological co-agonist that activates N-methyl d-aspartate receptors (NMDARs) and is essential for neurotransmission, synaptic plasticity, and behavior. d-Serine may also trigger NMDAR-mediated neurotoxicity, and its dysregulation may play a role in neurodegeneration. d-Serine is synthesized by the enzyme serine racemase (SR), which directly converts l-serine to d-serine. However, many aspects concerning the regulation of d-serine production under physiological and pathological conditions remain to be elucidated. Here, we investigate possible mechanisms regulating the synthesis of d-serine by SR in paradigms relevant to neurotoxicity. We report that SR undergoes nucleocytoplasmic shuttling and that this process is dysregulated by several insults leading to neuronal death, typically by apoptotic stimuli. Cell death induction promotes nuclear accumulation of SR, in parallel with the nuclear translocation of GAPDH and Siah proteins at an early stage of the cell death process. Mutations in putative SR nuclear export signals (NESs) elicit SR nuclear accumulation and its depletion from the cytosol. Following apoptotic insult, SR associates with nuclear GAPDH along with other nuclear components, and this is accompanied by complete inactivation of the enzyme. As a result, extracellular d-serine concentration is reduced, even though extracellular glutamate concentration increases severalfold. Our observations imply that nuclear translocation of SR provides a fail-safe mechanism to prevent or limit secondary NMDAR-mediated toxicity in nearby synapses.     

Pharmacology of calcium release from sarcoplasmic reticulum

Calcium release from sarcoplasmic reticulum (SR) has been elicited in response to additions of many different agents. Activators of Ca²⁺ release are here tentatively classified as activators of a Ca²⁺-induced Ca²⁺ release channel preferentially localized in SR terminal or as likely activators of other Ca²⁺ efflux pathways. Some of these pathways may be associated with several different mechanisms for SR Ca²⁺ release that have been postulated previously. Studies of various inhibitors of excitation-contraction coupling and of certain forms of SR Ca²⁺ release are summarized. The sensitivity of isolated SR to certain agents is unusually affected by experimental conditions. These effects can seriously undermine attempts to anticipate effects of the same pharmacological agentsin situ. Finally, mention is made of a new preparation (sarcoballs) designed to make the pharmacological study of SR Ca²⁺ release more accessible to electrophysiologists, and some concluding speculations on the future of SR pharmacology are offered.     

FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3 complex, SR interacts preferentially with free FBXO22 species. In vivo ubiquitination and SR half-life determination indicate that FBXO22 does not target SR to the proteasome system. FBXO22 primarily affects SR subcellular localization and seems to increase d-serine synthesis by preventing the association of SR to intracellular membranes. Our data highlight an atypical role of FBXO22 in enhancing d-serine synthesis that is unrelated to its classical effects as a component of the ubiquitin-proteasome degradation pathway.     

Modification of an enzymic system of Ca2+ transport in sarcoplasmic reticulum during lipid peroxidation. In vivo damages in the development of pathological changes

The role of lipid peroxidation (LPO) in the damages of the enzymic system of Ca2+ transport in sarcoplasmic reticulum (SR) membranes of skeletal and cardiac muscles under conditions of vitamin E deficiency, ischemia and limb reoxygenation as well as in emotional-pain stress was investigated. It was shown that these processes are associated with activation of endogenous LPO in SR membranes "in vivo" and with simultaneous inhibition of Ca2+ transport, (i. e. decrease of the Ca2+/ATP ratio) and inactivation of Ca-ATPase. The degree of damage of the Ca2+ transport system was correlated with the concentration of LPO products accumulated in SR membranes "in vivo and during LPO induction by the Fe2+ + ascorbate system 'in vitro". Injection of natural and synthetic free radical scavengers (e. g. 4-methyl-2.6-ditretbutylphenol, alpha-tocopherol) to experimental animals resulted in practically complete suppression of LPO activation "in vivo" and in partial protection of the Ca2+-transporting capacity of SR membranes. A comparison of experimental results allowed to estimate the role of LPO in SR damage under pathological conditions. Model experiments with "contraction-relaxation" cycles including isolated components of muscle fibers (SR fragments and myofibrils) demonstrated that LPO induction in SR membranes by the Fe2+ + ascorbate system results in complete elimination of the relaxation step in myofibrils due to the loss of the SR affinity to decrease the concentration of Ca2+ in the incubation medium. This effect can be removed by free radical scavengers. The role of LPO in pathological changes of muscle contractility is discussed.     

The role of the sarcoplasmic reticulum in various types of cardiomyocytes

The relative importance of the sarcoplasmic reticulum (SR) as a source of Ca²⁺ in the excitation-contraction coupling of mammalian myocytes was tested. Shortening and intracellular Ca²⁺ transients of electrically paced, isolated,adult rat myocytes were found to be absolutely dependent on the presence of a functional SR and were completely abolished by the SR Ca²⁺-ATPase inhibitors cyclopiazonic acid and thapsigargin or by the Ca²⁺-release channel opener ryanodine.Neonatal rat cardiomyocytes, on the other hand, elicited consistent intracellular Ca²⁺-transients even after complete functional inhibition of the SR. The transients, however, were markedly prolonged. Also isolatedadult guinea pig myocytes maintained the ability to shorten after a complete inhibition of the SR Ca²⁺-ATPase by either thapsigargin or cyclopiazonic acid. The twitches and the intracellular Ca²⁺-transients, however, were considerably longer after inhibition of the SR Ca²⁺-ATPase. Different results were obtained after preincubation of the cells with 10 M ryanodine to induce emptying of the SR Ca²⁺ pool. In this case, Ca²⁺ spikes and twitches were also markedly reduced in size, in addition to being prolonged. When a SR Ca²⁺-pump inhibitor was added to ryanodine-treated cells, the size of the Ca²⁺-transients and the capacity of the cells to shorten increased. Ryanodine leaves the activity of the Ca²⁺-pump of the SR intact and thus leads to an underestimation of the amount of excitatory Ca²⁺-flowing into the cell.The results show that, while the significance of the SR in regulating the Ca²⁺-transients and shortening of cardiomyocytes varies depending on the species and the stage of development, SR function is of paramount importance for the occurrence of rapid twitches.Abbreviations EGTA ethylene glycol-bis-(beta amino ethyl ether)N,N,N,N tetraacetic acid - MOPS morpholinopropane sulfonic acid - SR sarcoplasmic reticulum - BSA bovine serum albumin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid