Changes in cell surface proteins and glycoproteins during the encystation of Entamoeba invadens

Changes in cell surface components of axenically grown trophozoites of Entamoeba invadens which occur during encystation were followed. Protein patterns of trophozoites, immature and mature cyst forms, were analyzed by sodium dodecyl sulphate gel electrophoresis. Total protein profiles of trophozoites and cyst forms stained by Coomassie blue gave similar patterns. In contrast, a number of different bands were observed in gels stained with the carbohydrate-specific Schiff's reagent as well as when nitrocellulose blottings were treated with 125I-radiolabelled wheat germ or soybean agglutinins. The most notable differences were bands at 250 and 95-105 kDa present in the cyst forms and absent in the trophozoites, and two bands at 70 and 75 kDa present in the latter and missing in the cysts. Labelling of trophozoites and cyst cell surfaces by iodination with lactoperoxidase revealed a number of protein bands which were exposed on the trophozoite surface and missing in the cysts. Moreover, gel electrophoresis patterns of non-reduced or reduced samples also differed considerably, indicating that a number of proteins are linked by disulphide bonds. This study shows that specific glycoproteins are produced during cyst formation.     

Rat vas deferens SERCA2 is modulated by Ca2+/calmodulin protein kinase II-mediated phosphorylation

Ca²⁺ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca²⁺-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca²⁺-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca²⁺ (Ca_0.5 = 780 nM) and a low sensitivity to vanadate (IC₅₀ = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca²⁺ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca²⁺ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca²⁺ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca²⁺ pumping activity.     

Effect of calcium antagonists, calcium channel blockers and calmodulin inhibitors on the growth and encystation of Entamoeba histolytica and E. invadens

The effects of calcium antagonists, calcium channel blockers, and calmodulin inhibitors on the growth of Entamoeba histolytica and the growth and encystation of Entamoeba invadens were examined. Calcium chelators ethyleneglycol bis (beta-aminoethyl ether)-N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA) inhibited the growth of both Entamoeba and also the encystation of E. invadens in a dose-dependent manner, with EDTA being more effective than EGTA. A putative antagonist of intracellular calcium flux, 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited both growth and encystation, with the E. histolytica being more sensitive than E. invadens, and with the growth of E. invadens being more sensitive than encystation. The slow Na+-Ca2+ channel blockers bepridil and verapamil inhibited both growth and encystation. Bepridil was more effective than verapamil. The calmodulin (CaM) inhibitors, W-7 (N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide) and trifluoperazine (TFP), were also inhibitory for both the growth and encystation; TFP was more effective than W-7, and encystation was more sensitive than growth in E. invadens. These results indicate that extracellular calcium ions, amebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the growth and encystation of Entamoeba.     

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Thapsigargin has been reported to inhibit ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca²⁺ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca²⁺ content of the SR. The SR was first depleted of Ca²⁺ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 M was required to inhibit Ca²⁺ loading by 50%. In contrast, another SR Ca²⁺ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 M and total inhibition at 50 M. When cyclopiazonic acid (100 M) was applied after, rather than during, Ca²⁺ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 M), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca²⁺ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.     

Nitrotyrosine-modified SERCA2: a cellular sensor of reactive nitrogen species

The endo-/sarcoplasmic reticulum Ca(2+)-Mg(2+)-adenosine triphosphatase (SERCA2) isoform of the sarco/endoplasmic reticulum Ca(2+)-ATPase is sensitive to cellular conditions of inflammation and oxidative stress as evidenced by the common appearance of 3-nitrotyrosine-modified forms of SERCA2 in aging and disease in both striated and smooth muscle of humans and rodent models. Structure-function studies of nitrated SERCA2 in aging heart and skeletal muscle demonstrate stoichiometric nitration of vicinal tyrosines, Tyr(294) and Tyr(295), on the lumenal side of the membrane-spanning helix, M4, which correlates with partial inhibition of Ca(2+)-ATPase activity suggesting a possible regulatory function in down-regulating mitochondrial energy production and the associated generation of reactive oxygen/nitrogen species. This review discusses recent work regarding the nitrative and oxidative sensitivity of SERCA2 in muscle with respect to general cellular mechanisms of turnover and repair of modified proteins.     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

Effect of proteasome inhibitors on the growth,encystation, and excystation of <Emphasis Type="Italic">Entamoeba histolytica and Entamoeba invadens</Emphasis>

The effect of three proteasome inhibitors, lactacystin, clasto-lactacystin beta-lactone, and MG-132, on the growth, encystation, and excystation of Entamoeba histolytica and Entamoeba invadens was examined. All of these drugs blocked E. histolytica growth in a concentration-dependent manner; lactacystin was most potent for the inhibition and MG-132 showed the inhibitory effect only at higher concentrations. E. invadens was more resistant to these drugs than E. histolytica. Encystation of E. invadens was also inhibited and was more sensitive to the drugs than was growth. Beta-lactone was the most potent encystation inhibitor. The inhibitory effect of lactacystin and the beta-lactone on encystation was slightly and little abrogated by the removal of the drug, respectively. Multinucleation occurred in E. histolytica trophozoites treated with these drugs, being most marked with lactacystin. In contrast, no multinucleation was observed in E. invadens treated with the drugs. Electron microscopy revealed that the treatment of E. histolytica trophozoites with lactacystin led to an increase in the number of cells with many glycogen granules in the cytoplasm. Lactacystin, beta-lactone and MG-132 had no or little effect on the excystation and metacystic development of E. invadens. These results suggest that proteasome function plays an important role for Entamoeba growth and encystation, but has no obvious effect on excystation or metacystic development.     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca²⁺-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca²⁺-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca²⁺. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca²⁺-activated K⁺ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca²⁺ influx was triggered through the Na⁺/Ca²⁺ exchanger and the other Ca²⁺ entry pathways were blocked. Thapsigargin, another specific SR Ca²⁺-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca²⁺-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca²⁺ influx presumably via nifedipine-sensitive Ca²⁺ channels. This study suggests the important role of SR Ca²⁺-ATPase in the modulation of the muscle tone and the function of SR as a “buffer barrier” to Ca²⁺ entry in the cat gastric fundus smooth muscle. Received: 10 August 1995/Received after revision: 9 November 1995/Accepted: 10 November 1995     

Fast- and slow-twitch isoforms (SERCA1 and SERCA2a) of sarcoplasmic reticulum Ca-ATPase are expressed simultaneously in chronically stimulated muscle fibers

 Using an immunohistochemical double-labeling technique, we observed that different isoforms of sarcoplasmic reticulum Ca-ATPase are co-expressed in single fibers of canine fast-twitch skeletal muscles stimulated chronically at low frequency. By 7 days of neuromuscular stimulation, the population of hybrid fibers expressing both SERCA1 and SERCA2a [fast- and slow-twitch isoforms of sarco(endo)plasmic reticulum Ca²⁺-ATPase] had increased from 1.5% to 9.2% of fibers. By 14 days of stimulation 90% of the pure fast-twitch fibers (expressing only SERCA1) were replaced by hybrid fibers. An additional 28 days of stimulation caused all fast-twitch fibers to express SERCA2a at the same level as found in nonstimulated slow-twitch fibers (expressing only SERCA2a). At this time, one-half of the previously hybrid fibers had become pure slow-twitch fibers. The remaining one-half of the hybrid fibers expressed SERCA1 at a very low level. Extending stimulation to 70 days did not further change the percentage of fibers that were slow-twitch or hybrid. Immunoblot studies at the whole-muscle level confirmed that changes in SERCA expression at 42 days of neuromuscular stimulation were complete. Immunohistochemical analysis of longitudinal sections of muscle showed that the changes in SERCA protein were uniform along the length of the muscle fiber, indicating that nuclei along its length responded equally to chronic stimulation. Received: 12 November 1996 / Received after revision and accepted: 16 December 1996     

The effect of phosphatase inhibitors and agents increasing cyclic-AMP-dependent phosphorylation on calcium channel currents in cultured rat dorsal root ganglion neurones: interaction with the effect of G protein activation

Ca²⁺ channel currents have been recorded in cultured rat dorsal root ganglion neurones. The amplitude of I _Ba(GTPS), recorded in the presence of GTP[S] (200 M) in the patch pipette solution, is enhanced by external application of forskolin (10 M), and there is an increase in the proportion of the rapidly activating component of the current. When forskolin (1 M) is present in the bathing solution at the start of recording, or when 8-bromocyclic AMP (100 M) is present in the patch pipette solution, the amplitude and rate of activation of I _Ba(GTPS) are also increased compared to control I _Ba(GTPS). The effect is mimicked by internal application of a 5 M solution of a phosphopeptide fragment of inhibitor 1 (I1PP), which inhibits phosphatase 1. The enhancement of I _Ba(GTPS) caused by I1PP is not additive with that due to forskolin. Furthermore, the enhancement due to I1PP is reversibly lost when the holding potential is shifted from –80 mV to –30 mV, as was the enhancement due to forskolin and 8-bromocyclic AMP. I1PP also produced a less marked stimulation of the control Ca²⁺ channel current in the absence of G protein activation. The results suggest that phosphorylation regulates the interaction between calcium channels and G proteins in these neurones, and that phosphatase 1 is tonically active to dephosphorylate the relevant protein(s).     

Action of 1,25-Dihydroxycholecalciferol on cartilage mineralization and on endosteal lining cells of bone

Summary The aim of our study was to answer the following questions: 1)Does 1,25-dihydroxycholecalciferol influence the mineralization process directly? 2)How does 1,25-D₃ act on the endosteal cell envelope of bone, which is thought to be engaged in the moment to moment regulation of calcium homeostasis? We studied the endosteal surface of calvaria and the epiphyseal cartilage of rachitic rats in a tissue culture system in order to avoid the secondary effects of endogenous hormones or mineral sources. Investigations were carried out using transmission and scanning electron microscopy. Compared with untreated rachitic control specimens, 1,25-D₃ (4×10^–8 M) did not enhance the mineralization process. We therefore conclude that the established in vivo effect of 1,25-D₃ on the mineralization process must be an indirect one. Within 2 h the flat-shaped surface lining cells of calvaria showed a rapid transformation into globiform cells and a loss of cell adhesion. Within two days a second slower transformation into spindle-shaped fibroblast-like proliferating cells occured. All changes of cell shape were reversible within several days in the absence of 1,25-D₃. Obviously, bone matrix fiber texture influences the spatial movement of proliferating cells. The endosteal cell envelope is considered to act as a functional membrane of bone. Starting from this consideration we conclude that the changes of cellular shape described are the morphological basis for the action of 1,25-D₃ in the regulation of calcium homeostasis in bone at the cellular level.The paper was presented in part at the 4th Workshop on Vitamin D, Berlin 1979With the support of VolkswagenstiftungTo Prof. Doerr on the occasion of his 65th birthday     

The relationship of calbindin-containing neurons with substance P, Leu-enkephalin and cholecystokinin fibres: An immunohistochemical study in the rat thalamus

In the rat thalamus, immunoreactivity for the calcium binding protein calbindin (Cb) is mostly confined to neuronal cell bodies, sometimes revealing proximal dendrites, of the midline, intralaminar and posterior regions. Substance P (SP)-, cholecystokinin (CCK)- and Leu-enkephalin (L-ENK)-immunoreactive (ir) elements in the thalamus are fibre-like structures, intermingled with punctate elements probably representing axonal arborizations and their synaptic boutons. These peptidergic fibres are unevenly distributed in several thalamic domains, including the areas that contain Cb-ir neurons. The relationship between Cb-ir cell bodies and these three different peptidergic systems of thalamic innervation was studied with immunohistochemistry. Single-labelling experiments on adjacent sections and double immunostaining on the same section were performed. A considerable overlap between Cb-ir perikarya and SP-ir fibres was found in most thalamic nuclei. In particular, in the intralaminar nuclei and posterior complex. SP-ir punctate elements were frequently observed in close proximity to Cb-ir cell bodies and dendrites. On the other hand, no consistent topographical correspondence between Cb-ir perikarya and CCK- or L-ENK-ir fibres was evident. Altogether, the present data suggest a selective anatomical and, possibly, functional relationship between SP and Cb in at least a subpopulation of rat thalamic neurons.     

异丙嗪对家兔EGTA性发热的解热作用及其机制研究

目的：观察异丙嗪（ｐｒｏｍｅｔｈａｚｉｎｅ,ＰＭＺ）对家兔ＥＧＴＡ性发热的影响并探讨其作用机制。方法：侧脑室和静脉给药。用Ｆｕｒａ－２荧光分光光度法测定细胞内游离钙浓度（［Ｃａ２＋］ｉ）。结果：（１）侧脑室灌注０６μｍｏｌＥＧＴＡ引起家兔明显的发热反应,侧脑室灌注０６μｍｏｌＥＧＴＡ２０ｍｉｎ后,静脉注射ＰＭＺ（５ｍｇ／ｋｇ）明显抑制ＥＧＴＡ引起的结肠温度上升,其３ｈ发热反应指数明显低于侧脑室灌注０６μｍｏｌＥＧＴＡ２０ｍｉｎ后静脉注射生理盐水（ＮＳ）组。而侧脑室灌注人工脑脊液（ＡＣＳＦ）２０ｍｉｎ后静脉注射ＰＭＺ（５ｍｇ／ｋｇ）组家兔结肠温度明显低于ＡＣＳＦ＋ＮＳ对照组。（２）体外实验发现,向下丘脑细胞悬液中加入终浓度为０７４ｍｍｏｌ／Ｌ的ＰＭＺ,下丘脑细胞［Ｃａ２＋］ｉ从（１５９２±１８８）ｎｍｏｌ／Ｌ升高到（５３３７±９０１）ｎｍｏｌ／Ｌ（Ｐ＜００５）。结论：ＰＭＺ诱导体温调节中枢神经细胞［Ｃａ２＋］ｉ升高可能是ＰＭＺ抑制家兔ＥＧＴＡ性发热的中枢机制之一。     

Differential modulation of pharmacologically distinct components of Ca2+ currents by protein kinase C activators and phosphatase inhibitors in nerve-growth-factor-differentiated rat pheochromocytoma (PC12) cells

We have studied the effects of protein kinase C (PKC) activators 4-phorbol 12-myristate 13-acetate (4-PMA) and 1-oleoyl-2-acetylglycerol (OAG) and of phosphatase inhibitors (okadaic acid and calyculin A) on voltage-activated Ca²⁺ and K⁺ channels in nerve growth factor-(NGF)-differentiated pheochromocytoma (PC12) cells. Whole-cell Ba²⁺ and K⁺ currents were recorded at room temperature with the patch-clamp technique. By using -conotoxin (CgTX) and isradipine, two specific Ca²⁺ channel blockers, we found three types of Ba²⁺ currents (I _Ba): (1) a -CgTX-sensitive I _Ba; (2) an isradipine-sensitive I _Ba; and (3) a -CgTX plus isradipine-resistant I _Ba. The external application of 4 -PMA or OAG down-modulated the isradipine-sensitive I _Ba whereas the two other I _Ba were not affected. 4-PMA-induced inhibition of I _Ba was prevented by staurosporine (a protein kinase inhibitor) and PKC (19–31) (a specific PKC inhibitor). The delayed rectifier K⁺ current (I _K) was unaffected by PKC activators. Both okadaic acid and calyculin A affected the components of the I _Ba in different manners. The presence of okadaic acid decreased the isradipine-sensitive I _Ba more than the -CgTX-sensitive I _Ba. The -CgTX plus isradipine-resistant I _Ba was not affected. Calyculin A down-modulated all three components of I _Ba to a similar degree. Our results suggest a differential modulation of voltage-activated Ca²⁺ and K⁺ channels by the PKC signalling pathway in NGF-differentiated PC12 cells.     

Developmental changes in the actions of phosphatase inhibitors on calcium current of rabbit heart cells

We used whole-cell voltage clamp to compare the modulation of calcium current density (I _Ca, picoampere per picofarad) of freshly isolated, adult and newborn rabbit heart in response to intracellular application of microcystin and okadaic acid, both of which block phosphatase activity of phosphatase types 1 and 2A. Newborn cells showed a much larger response to the intracellular application of either microcystin or okadaic acid than did adult cells. In newborn cells, the application of microcystin produced an increase in I _Ca which appeared to maximize I _Ca, as shown by the rise in I _Ca to levels which could be reached by application of 10 M forskolin or by the intracellular application of 200 M 3,5-cyclic adenosine monophosphate (cAMP). In adult cells, the maximal response to microcystin was considerably less than that obtainable with forskolin or cAMP. After achieving a maximal response with microcystin, the addition of forskolin increased I _Ca further in adult cells but elicited no additional response in newborn cells. The treatment of cells with 0.1 M isoproterenol, a concentration approximately equal to that required for a half-maximal response, strongly potentiated the effect of microcystin in newborn cells, but not in adult cells. We propose that newborn rabbit heart cells compared with adult rabbit heart cells have a greater level of protein phosphatase activity (perhaps combined with a somewhat greater kinase activity), a greater proportion of the protein phosphatase activity in the form of protein phosphatase type 1 (which is inhibited by isoproterenol) and a greater dependence on the inhibition of protein phosphatase as a mechanism of action of isoproterenol, compared with the increase in kinase activity on calcium channels.     

Interaction of the epithelial Ca2+ channels TRPV5 and TRPV6 with the intestine- and kidney-enriched PDZ protein NHERF4

The epithelial Ca²⁺ channels TRPV5 and TRPV6 constitute the apical Ca²⁺ influx pathway in epithelial Ca²⁺ transport. PDZ proteins have been demonstrated to play a crucial role in the targeting or anchoring of ion channels and transporters in the apical domain of the cell. In this study, we describe the identification of NHERF4 (Na-P_i Cap2/IKEPP/PDZK2) as a novel TRPV5- and TRPV6-associated PDZ protein. NHERF4 was identified using two separate yeast two-hybrid screens with the carboxyl termini of TRPV5 and TRPV6 as bait. Binding of the carboxyl termini of TRPV5 and TRPV6 with NHERF4 was confirmed by GST pull-down assays using in-vitro-translated NHERF4 or lysates of Xenopus laevis oocytes expressing NHERF4. Furthermore, the interaction was confirmed by GST pull-down and co-immunoprecipitation assays using in-vitro-translated full-length TRPV5 and Xenopus oocytes or HEK293 cells co-expressing NHERF4 and TRPV5/TRPV6, respectively. The fourth PDZ domain of NHERF4 was sufficient for the interaction, although PDZ domain 1 also contributed to the binding. The binding site for NHERF4 localized in a conserved region in the carboxyl terminus of TRPV5 and was distinct from the binding site of the PDZ protein NHERF2. NHERF4 predominantly localized at the plasma membrane of X. laevis oocytes and HeLa cells. This localization was independent of the presence of TRPV5. Therefore, we hypothesize a role for this novel PDZ protein as a putative plasma membrane scaffold for the epithelial Ca²⁺ channels.     

The effect of inhibitors of protein biosynthesis on the infectivity of alfalfa mosaic virus: role of coat protein

Cycloheximide, when present in the inoculum at a concentration of 50 μg/ml decreases the infectivity of nucleoproteins of strain 425 of alfalfa mosaic virus (AMV) by more than 90%. Infectivity of the nucleoproteins of the AMV strain yellow spot mosaic virus (YSMV) and the Strasbourg strain were much less sensitive to cycloheximide; at 50 μg/ml of the antibiotic 60–80% of the normal infectivity was found. However, when chloramphenicol and cycloheximide were given simultaneously, the infectivity of these strains was as much reduced as that of AMV 425 in the presence of cycloheximide alone. As was shown earlier, infectious RNA preparations consist of 4 RNA species, 3 large RNAs constituting the complete genome, and a small monocistronic RNA, the top component a RNA. When the latter is removed, the RNA preparation is no longer infectious. A mixture of bottom, middle, and top component b RNAs can be activated by the coat protein. The infectivity of the 4 RNAs from YSMV and AMV 425 was equally sensitive to cycloheximide. A combination of YSMV RNA activated by AMV 425 coat protein was as sensitive to cycloheximide as AMV 425 nucleoprotein. This suggests that the coat protein plays a role in the localization of translation, which is in accordance with the previous finding that sensitivity to cycloheximide is determined by the top component b RNA, which contains the genetic information for the coat protein.     

Identification and biochemical characterisation of a protein phosphatase 5 homologue from Plasmodium falciparum

We report the identification of a new serine/threonine phosphatase from Plasmodium falciparum at the DNA and protein levels. A 1.8 kb cDNA fragment encoding the protein phosphatase was identified via PCR amplification. The sequence has a coding capacity of 594 amino acids. Immunoblot analysis of P. falciparum extracts showed that antibodies generated against the His₆-fusion protein recognise a protein of approximately 80 kDa. The deduced amino acid sequence shares 55% identity with a mouse protein, identified as Protein Phosphatase 5 (PP5). We show that the P. falciparum PP5 homologue (PfPP5) has all structural and functional characteristics of this class of enzymes. It contains three tetratricopeptide repeats (TPR) and a nuclear targeting sequence at its N-terminus and a highly conserved C-terminal catalytic domain. Southern blot results are compatible with the existence of PfPP5 as a single copy gene. Purified recombinant protein, like the native protein enriched from P. falciparum extracts exhibited phosphatase activity that can be enhanced by both arachidonic and oleic acids, but not by myristic or stearic acid. In addition, the activity is inhibited by okadaic acid (OA) with an IC₅₀ of 4 nM. Immunofluorescence microscopy has localised PfPP5 preferentially to the nucleus. The function of PfPP5 is presently unclear, but like other PP5s of many eukaryotic organisms, it may have important regulatory functions in the parasite cell cycle.