Identification of a Phosphoprotein Expressed During Somatic Embryogenesis in Wheat Leaf Base Cultures

2,4-D mediated induction of somatic embryogenesis in wheat is enhanced in the presence of Ca⁺⁺ and its removal by EGTA reduces the response significantly. Changes that occur at the polypeptide level following 2,4-D treatment were analysed. Intense cell division activity was discernable in the leaf base explants within an hour of treatment. Changes in protein profiles were prominent in the membrane fraction as compared to the soluble fraction. The protein profile of the leaf base culture with somatic embryos was distinct from the calli induced from mature embryos on a 2,4-D containing medium. The role of Ca²⁺ in the induction of somatic embryogenesis was demonstrated by the use of EGTA (a calcium chelator), verapamil, nifedipine (calcium channel blockers), W7 (calmodulin antagonist) and Li (PI inhibitor). ^{In vitro} protein phosphorylation studies showed that 2,4-D, calcium and related treatments inhibit phosphorylation of proteins. In the membrane fraction proteins, accumulation of polypeptides at the low molecular weight range was seen in samples treated with verapamil and W7, and a 30 kO polypeptide in the samples treated with calmodulin antagonist, W7. Autoradiography of membrane fraction proteins displayed the presence of a 16 kO protein phosphorylated in samples treated with verapamil, nifedipine and W7. It thus appears that 2,4-D and Ca⁺⁺ prevent the phosphorylation of this phosphoprotein. These results thus indicate the action of 2,4-D via the Ca²⁺-CaM signaling pathway in triggering the induction of somatic embryogenesis.     

Calcium-dependent activation of tryptophan hydroxylase by ATP and magnesium

Tryptophan hydroxylase [EC 1.14.16.4; L-tryptophan, tetrahydropteridine: oxygen oxidoreductase (5-hydroxylating)] in rat brainstem extracts is activated 2 to 2.5-fold by ATP and Mg⁺⁺ in the presence of subsaturating concentrations of the cofactor 6-methyltetrahydropterin (6MPH₄). The activation of tryptophan hydroxylase under these conditions results from a reduction in the apparent K_m for 6MPH₄ from 0.21 mM to 0.09 mM. The activation requires Mg⁺⁺ and ATP but is not dependent on either cAMP or cGMP. The effect of ATP and Mg⁺⁺ on enzyme activity was enhanced by μM concentrations of Ca⁺⁺ and totally blocked by EGTA. These data suggest that tryptophan hydroxylase can be activated by a cyclic nucleotide independent protein kinase which requires low calcium concentrations for the expression of its activity.     

The tumor promoter 12-0-tetradecanoylphorbol-13-acetate induces ornithine decarboxylase in proliferating basal cells but not in differentiating cells from mouse epidermis

The cultivation of mouse epidermal cells in medium of reduced calcium concentration (0.02–0.1 mM) selects for basal cell growth. Elevation of medium calcium levels above 0.1 mM results in rapid and well defined differentiative changes. This model was utilized to determine which cell type in mouse epidermis responds to the phorbol ester tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), by an induction of the enzyme ornithine decarboxylase (ODC). Previous data had shown that TPA induces ODC in primary mouse epidermal cells only during the first 36 hr after plating in medium containing 1.44 mM Ca²⁺. In contrast, the induction in cells grown in low calcium medium was 2–10-fold greater, and inducibility persisted for at least 4 weeks. The greater inducibility of ODC in low calcium cells is not paralleled by increased thymidine incorporation after TPA treatment, probably because these cells are already proliferating at a maximum rate. When low calcium cells grown in 0.07 mM Ca²⁺ medium were switched to 1.2 mM Ca²⁺, there was a rapid loss of ODC inducibility. These results strongly suggest that the basal cells of the epidermis constitute the major target cells for the induction of ODC by TPA. The induction of ODC by ultraviolet light was not enhanced by growth of cells in low calcium medium, indicating that extracellular calcium concentration per se does not determine ODC inducibility. When epidermal cells grown in 1.2 mM or 0.07 mM Ca²⁺ medium were exposed to both UV light and TPA, there was a significant synergistic effect of combined treatment over the sum of each individual response, suggesting that factors in addition to differentiation determine the extent of ODC induction.     

Involvement of cAMP and calcium in the induction of ornithine decarboxylase activity in an osteoblast cell line

The role of cAMP and calcium in the induction of ornithine decarboxylase (ODC, E.C.4.1.1.17) activity in the osteogenic sarcoma cell line, UMR 106-01, was studied, with particular interest for parathyroid hormone (PTH). PTH and forskolin dose-dependently induced the ODC activity and the cAMP production. Protein synthesis is involved in the effect of PTH and forskolin on ODC activity but not on cAMP production. Using quin2 we showed that 20 nM PTH and 10 microM forskolin increased the intracellular ionized calcium concentration ([Ca2+]i), thereby offering the possibility for calcium to play a role as cellular mediator in the action of PTH and forskolin in bone. Data obtained with A23187 showed that solely an increase of the [Ca2+]i is not sufficient to stimulate basal or potentiate PTH- and forskolin-induced ODC activity. However, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced ODC activity point to a specific role for calcium. Moreover, the effects of calcium channel blockers and EGTA on basal and PTH- and forskolin-induced cAMP production indicate that the involvement of calcium in the induction of ODC activity is primarily located at another site than the adenylate cyclase. These data indicate that calcium is involved in the control of basal ODC activity. Furthermore, these data suggest that both cAMP and calcium are involved in the induction of ODC activity by PTH and forskolin. More precisely, ODC activity in UMR 106-01 cells can be induced by PTH and forskolin via a calcium-dependent cAMP messenger system.     

Comparison of the Ca ++ requirement for the steroidogenic effect of ACTH acid dibutyryl cyclic AMP in rat adrenal cell suspension

Calcium requirement for ACTH and Dibutyryl cyclic AMP (DBCAMP) stimulation of steroidogenesis was compared in rat adrenal cell suspensions. In the absence of added calcium ACTH at low concentrations (< 1 mU/ml) was ineffective; however, the calcium requirement decreased when higher concentrations of ACTH were used. This was not the case with DBCAMP. At all levels of the nucleotide tested, the Ca⁺⁺ requirement was about the same. When the cells were preincubated with EGTA, the Ca⁺⁺ requirement became more pronounced for ACTH than for DBCAMP. The results indicate that the events before the formation of cyclic AMP show a greater dependence on Ca⁺⁺ than the events following its formation.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl₂, apparently because Ca⁺⁺ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg⁺⁺, Mn⁺⁺, Na⁺, K⁺, and NH₄⁺ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca⁺⁺ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca⁺⁺ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. $\text{in vivo}$ depends on the ability of that agent to chelate Ca⁺⁺.     

Abnormal lysosomal hydrolases excreted by cultured fibroblasts in I-cell disease (mucolipidosis II).

The characteristics of rat liver mitochondria swelling induced by diamide, an oxidizing agent for thiol groups, and by Ca ions are very similar. In both cases the swelling, which is initiated by addition of 0.5–1 mM phosphate or acetate, is prevented by FCCP, antimycin A, EGTA, Mg⁺⁺ and ruthenium red. Diamide potentiates the swelling action of Ca⁺⁺, while DTE potentiates that of Mg⁺⁺. The additive effects of calcium and diamide on rat liver mitochondria have been correlated with their synergic action in promoting the release of mitochondrial Mg⁺⁺. The results strongly indicate that some of the effects of diamide are mediated by a mobilization of endogenous divalent ions and that the antagonism between Ca⁺⁺ and Mg⁺⁺ is closely correlated with the redox state of membrane bound thiol groups.     

Levels of ornithine decarboxylase activation used as a simple marker of metal induced growth arrest in tissue culture.

Exposure of proliferating cells to specific water solube metal compounds at 0.1 or 1.0 mM concentrations inhibited cell growth and also depressed the induction of ornithine decarboxylase, an enzyme which is tightly coupled to the initiation of cell growth. Salts of Co⁺⁺, Ni⁺⁺, Cu⁺⁺, Cr⁺⁶ and Cd⁺⁺ significantly reduced incorporation of radiolabeled leucine, thymidine or uridine into trichloroacetic acid insoluble material, inhibited the doubling of Chinese hamster ovary cells, and blocked the the induction of ornithine decarboxylase. The addition of similar concentrations of other metals such as Fe⁺⁺, K⁺, Mg⁺⁺, Pb⁺⁺, Ca⁺⁺ or Sn⁺⁺ had no effect on ODC induction and also did not inhibit the other parameters associated with cell proliferation which were measured. These results suggest that ornithine decarboxylase induction can be used as a marker of metal induced growth arrest.     

Expression - Level Dependent Activation of Recombinant Human Parathyroid Hormone/Parathyroid Hormone-Related Peptide Receptor: Effect of Human Parathyroid Hormone (1-34), (1-31), and (28-48)

Abstract

A stable recombinant Chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10⁵ to 1.9xl0⁶ receptors per cell. The affinity of the receptors for hPTH-(l-34) was independent of the receptor number per cell (K<j = 8 nmol/1). The induction of cAMP by hPTH-(l-34) is maximal in clones expressing >2xl0⁵ receptors per cell and Ca⁺⁺ signals were maximal in cell lines expressing >1.4xl0⁶ receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca⁺⁺ are independent and Ca⁺⁺ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(l-31) showed also an increase in intracellular Ca⁺⁺. Even in cell lines expressing more than 10 receptors per cell the Ca⁺⁺/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.     

Ca++-dependent formation of brain adenylate cyclase-protein activator complex

In the presence of EGTA (ethyleneglycol-bis-(β-aminoethyl-ether) N,N′-tetraacetic acid), a Lubrol-PX solubilized rat brain adenylate cyclase (E.C. 4.4.1.1) and its protein activator were separated from each other in a Sephadex G-200 column. No activator was associated with the eluted enzyme, which required an exogenous activator for maximum activity. On the other hand, in the presence of Ca⁺⁺, some of the activator was eluted with the enzyme, which was independent of an exogenous activator for maximum activity. Because neither Ca⁺⁺ nor EGTA affected the elution profile of the activator in the filtration column, these results suggest that the formation of the enzyme-activator complex is dependent on Ca⁺⁺. Separate experiments indicated that the effect of Ca⁺⁺ on the formation of the enzyme-activator complex was immediate and reversible. Because the activator appears to be in excess of the enzyme, adenylate cyclase activityin vivo could be modulated by the cellular flux of Ca⁺⁺.     

Signaling Pathways for Ecdysteroid Hormone Synthesis in Crustacean Y-organs

The Y-organs of crustaceans secrete steroid hormones (ecdysteroids)which are responsible for molting and regeneration. The Y-organsin turn are controlled (negatively) by the eyestalk peptide,molt-inhibiting hormone (MIH). We are exploring the signalingpaths in Y-organ cells that lead to ecdysteroid generation whenactivated by the absence of MIH. The objective is to understandthe connections between MIH-receptor occupancy and the depressionof genes that express ecdysteroidogenic enzymes. MIH actionis mediated by a rise in cyclic 5' adenosine monophosphate (cAMP);cGMP also is involved in some species. That a cyclic nucleotideis a central regulatory component is indicated by the followingselection of results: dibutyryl cAMP, activators of adenylylcyclase or inhibitors of cyclic nucleotide phosphodiesteraseeach mimic the inhibitory action of MIH. Cyclic AMP inhibitsthe receptor-mediated uptake of cholesterol (the obligate ecdysteroidprecursor), by decreasing the number of receptor sites for thelipoprotein carrier of cholesterol. MIH via cAMP also depressesde novo protein synthesis upon which ecdysteroidogenesis dependsin part. A role for cellular free calcium (Ca⁺⁺) is indicatedby the ability of Ca⁺⁺ (or a Ca⁺⁺ionophore) to stimulate ecdysteroidproduction,thereby antagonizing MIH action. The mechanism involvesloweringcAMP levels by enhancing phosphodiesterase activity via calmodulin,not by affecting adenylate cyclase activity. Ca⁺⁺ counters thesuppressive action of MIH or cAMP on protein synthesis. Consistentwith the MIH-Ca⁺⁺ mutual antagonism, MIH increases Ca⁺⁺ effluxfrom ⁴⁵Ca-preloaded cells. Y-Organ cells contain protein kinaseC (PKC), the activation of which increases ecdysteroid production.PKC activity is not affected by MIH, but is stimulated by Ca⁺⁺.These and related experiments indicate that the PKC-activatedincrease in ecdysteroidogenesis involves events downstream fromthe production of cAMP and the degradation of cAMP by Ca⁺⁺.In relation to the latter, specific and non-specific inhibitorsof protein tyrosine kinases (PTK) inhibit ecdysteroid synthesisdose-dependently. The relationship of PTK with MIH-cAMP andCa⁺⁺-PKC systems is under study.     

Independent and interrelated regulation of ornithine decarboxylase by calcium and cAMP in fetal rat osteoblasts

The present study was undertaken to determine in fetal rat osteoblasts whether and how the intracellular messengers calcium and cAMP are involved in stimulation of ornithine decarboxylase (ODC) activity. For that purpose we used different drugs affecting [Ca2+]i and cAMP concentration. A23187 stimulates ODC activity in a biphasic way, with maximal stimulation at 100 nM A23187. At that concentration no stimulation of cAMP production was observed. Basal and A23187-stimulated (100 nM) ODC activity were inhibited by EGTA and trifluoperazine. Forskolin stimulated dose-dependently both ODC activity and cAMP production. Besides these effects forskolin (1 and 10 microM) increased the [Ca2+]i via an increased calcium influx. Addition of La3+, verapamil or EGTA, but not of trifluoperazine, significantly inhibited the forskolin-stimulated (10 microM) ODC activity. When forskolin (100 nM and 1 microM) was added together with 1 microM A23187, a synergistic stimulation of ODC activity was observed. These results implicate that calcium is involved in basal ODC activity, and that ODC activity can be stimulated via (1) a cAMP-independent calcium pathway, and (2) a calcium-dependent, cAMP pathway. It is proposed that ODC activity can be stimulated via interaction between calcium and cAMP.     

Intracellular calcium requirements for β1 integrin activation

Human polymorphonuclear leukocytes (PMNs) express β1 integrins that mediate adhesion to extracellular matrix proteins following stimulation with agonists that induce an increase in intracellular calcium. The purpose of these studies was to determine the contribution made by alterations in intracellular calcium ([Ca⁺⁺]_i) to inside-out activation of β1 integrins using dimethyl sulfoxide (DMSO)-differentiated granulocytic HL60 cells as a model of human PMNs. Activation of β1 integrins was determined by measuring the expression of an activation-dependent epitope on the β1 subunit that is recognized by monoclonal antibody (mAb) 15/7. Exposure of granulocytic HL60 cells to calcium ionophore ionomycin (800 nM) alone did not increase the binding of mAb 15/7 to the cell surface, nor did it increase β1 integrin-mediated adhesion of the cells to fibronectin. Similarly, exposure of the cells to the direct protein kinase C (PKC) activator, dioctanoylglycerol (di-C₈) at 100 μM, neither increased binding of mAb 15/7 to these cells nor adhesion to fibronectin. Simultaneous addition of di-C₈ and ionomycin, however, caused a significant increase in the expression of the 15/7 epitope and cell adhesion, suggesting synergy between elevating [Ca⁺⁺]_i and stimulating PKC in β1 integrin activation. Chelation of [Ca⁺⁺]_i with Quin-2 and EGTA reduced both basal (unstimulated) expression of the 15/7 epitope and basal adhesion of granulocytic HL60 cells to fibronectin. In addition, chelation of [Ca⁺⁺]_i caused a significant decrease in 15/7 binding and adhesion stimulated by low (1 ng/ml) concentrations of phorbol myristate acetate (PMA). The inhibitory effect of [Ca⁺⁺]_i chelation on β1 integrin activation was reversed by repleting [Ca⁺⁺]_i with ionomycin in a Ca⁺⁺-containing buffer, or by the addition of higher concentrations of PMA (10 ng/ml). These data suggest a role for [Ca⁺⁺]_i in inside-out activation of β1 integrins, probably through a synergistic effect with PKC activation. J. Cell. Physiol. 175:193–202, 1998. © 1998 Wiley-Liss, Inc.     

Sugar reception in the blowfly: a possible Ca(++) involvement

The present study investigates the effects of W-7 (a calmodulin antagonist involved in the Ca⁺⁺ cascade) on the response of the ‘sugar’ and ‘water’ cells of labellar chemosensilla in the blowfly Protophormia terraenovae to stimulation with sucrose or fructose. In order to ascertain whether Ca⁺⁺ conductance is involved, the effects of EGTA, one of the most used Ca⁺⁺ chelating agent, and of SK&F-96365, an inhibitor of receptor mediated calcium influx, were also studied. Our electrophysiological data indicate that W-7 addition strongly depresses the ‘sugar’ chemoreceptor response to both sugars and in the case of sucrose stimulation also influences adaptation rate. The Ca⁺⁺ chelator has no significant effects on the response of the ‘sugar’ cell following stimulation with sucrose, but lowers fructose stimulating effectiveness. In the presence of SK&F-96365 both sucrose and fructose responses are inhibited. A possible transduction mechanism for sugar reception is discussed.     

Does cyclic AMP mobilize Ca2+ for amylase secretion from rat parotid cells?

Both dibutyryl cAMP and carbachol stimulated amylase are released from rat parotid cells incubated in Ca²⁺-free medium containing 1 mM EGTA. Cells preincubated with 10 μM carbachol in Ca²⁺-free, 1 mM EGTA medium for 15 min lost responsiveness to carbachol, but maintained responsiveness to dibutyryl cAMP. Dibutyryl cAMP still evoked amylase release from cells preincubated with 1 μM ionophore A23187 and 1 mM EGTA for 20 min. Although carbachol stimulated net efflux of ⁴⁵Ca from cells preequilibrated with ⁴⁵Ca for 30 min, dibutyryl cAMP did not elicit any apparent changes in the cellular ⁴⁵Ca level. Inositol trisphosphate, but not cAMP, evoked ⁴⁵Ca release from saponin-permeabilized cells. These results suggest that cAMP does not mobilize calcium for amylase release from rat parotid cells.     

ROLE OF CALCIUM IN THE CALLOSE RESPONSE OF SELF-POLLINATED BRASSICA STIGMAS

In Brassica oleracea, sporophytic self-incompatibility prevents germination of self pollen, or normal growth of self pollen tubes. After self-pollination, the papillae of stigmas synthesize callose. The role of Ca⁺⁺ in the formation of stigmatic callose was tested by adding compounds that interact with Ca⁺⁺ to suspensions of pollen that were known to induce callose formation in self stigmas. The calcium channel antagonist, lanthanum, and the calcium chelating agent, EGTA, reduced or abolished the callose response to self-pollen suspensions. In the presence of Ca⁺⁺, the calcium ionophore, A23187, induced callose in stigmatic papillae when added to pollen suspensions, or alone. Therefore, callose deposition in response to incompatible pollinations appears to be a calcium-dependent process. Pretreatment of pistils with 100 μm 2-deoxy-D-glucose abolished the callose response to self-pollination, while self pollen remained inhibited and cross pollen grew normally in treated pistils. Thus, callose formation in the stigma is not an essential part of the self-incompatibility mechanism preventing the growth of self pollen in Brassica.     

Mitochondrial and calcium perturbations in rat CNS neurons induce calpain-cleavage of Parkin: Phosphatase inhibition stabilizes pSer65Parkin reducing its calpain-cleavage

Mitochondrial impairment and calcium (Ca⁺⁺) dyshomeostasis are associated with Parkinson's disease (PD). When intracellular ATP levels are lowered, Ca⁺⁺-ATPase pumps are impaired causing cytoplasmic Ca⁺⁺ to be elevated and calpain activation. Little is known about the effect of calpain activation on Parkin integrity. To address this gap, we examined the effects of mitochondrial inhibitors [oligomycin (Oligo), antimycin and rotenone] on endogenous Parkin integrity in rat midbrain and cerebral cortical cultures. All drugs induced calpain-cleavage of Parkin to ~36.9/43.6 kDa fragments. In contrast, treatment with the proinflammatory prostaglandin J2 (PGJ2) and the proteasome inhibitor epoxomicin induced caspase-cleavage of Parkin to fragments of a different size, previously shown by others to be triggered by apoptosis. Calpain-cleaved Parkin was enriched in neuronal mitochondrial fractions. Pre-treatment with the phosphatase inhibitor okadaic acid prior to Oligo-treatment, stabilized full-length Parkin phosphorylated at Ser⁶⁵, and reduced calpain-cleavage of Parkin. Treatment with the Ca⁺⁺ ionophore A23187, which facilitates Ca⁺⁺ transport across the plasma membrane, mimicked the effect of Oligo by inducing calpain-cleavage of Parkin. Removing extracellular Ca⁺⁺ from the media prevented oligomycin- and ionophore-induced calpain-cleavage of Parkin. Computational analysis predicted that calpain-cleavage of Parkin liberates its UbL domain. The phosphagen cyclocreatine moderately mitigated Parkin cleavage by calpain. Moreover, the pituitary adenylate cyclase activating peptide (PACAP27), which stimulates cAMP production, prevented caspase but not calpain-cleavage of Parkin. Overall, our data support a link between Parkin phosphorylation and its cleavage by calpain. This mechanism reflects the impact of mitochondrial impairment and Ca⁺⁺-dyshomeostasis on Parkin integrity and could influence PD pathogenesis.     

Chronic heat-shock treatment driven differentiation induces apoptosis in Leishmania donovani

The present study investigates the role of apoptosis in the regulation of cell numbers of Leishmania donovani during the in vitro differentiation of promastigote stage to amastigote stage in axenic conditions. We report that apoptosis is induced in Leishmania donovani due to chronic heat-shock treatment of 37 ^°C that also mediates the differentiation of promastigotes to amastigotes. This is characterized by the fragmentation of DNA, blebbing in the parasite cell membrane, nuclear condensation, formation of preapoptotic bodies and involvement of Ca⁺⁺ in the apoptotic process. The flowcytometric analysis shows an early and steep rise in percentage apoptotic nuclei till 48-hour stage of differentiation and then a gradual decline, suggesting synergistic action of Ca⁺⁺ ATPase and probably Hsp70. Hsp70 might be rescuing cells from apoptosis in the death signaling pathway. Incubation of the culture with Ca⁺⁺ chelator EGTA (1 mM) brings down the percentage of apoptotic nuclei considerably showing thereby that calcium is needed for the process of cell death here that occurs by apoptosis. The survival of the infective individuals appears to be decided by the parasite in the early stages of its differentiation. Our studies show the potential of the physiological temperature of 37 ^°C in inducing apoptosis in Leishmania donovani and the therapeutic use it can be put to.     

Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation

Summary Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the⁴⁵Ca-exchange method to determine free cellular Ca⁺⁺, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca⁺⁺). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca⁺⁺] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca⁺⁺ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca⁺⁺] results in a release of the fusion-block and an increase in cellular Ca⁺⁺ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca⁺⁺] must exist. The latter suggests further that cell surface Ca⁺⁺ was also critical.