Cross Talk Between Substance P and Melittin-Activated Cellular Signaling Pathways in Rat Lactotroph-Enriched Cell Cultures

Abstract: We have investigated the possible interaction (cross talk) between the phospholipase A₂ (PLA₂) and inositol 1,4,5-trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph-enriched cell cultures. Melittin, a bee venom peptide, stimulated release of [³H]-arachidonic acid ([³H]AA) from [³H]AA-labeled enriched lactotrophs in a dose-dependent manner. Moreover, melittin and exogenous AA induced a redistribution of PKC catalytic activity and PKCα and β immunoreactivity from the soluble to the particulate fraction in resting and substance P (SP)-stimulated cells. Mellitin had no effect on phospholipase C (PLC) activity. Pretreatment of cell cultures with the PLA₂ inhibitors quinacrine and aristolochic acid resulted in a dose-dependent inhibition of melittin-stimulated PKC isozyme translocation as did the inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cyclooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) dose-dependently increased levels of [³H]AA released from cells. Pretreatment of cell cultures with quinacrine reduced the effect of SP on [³H]AA formation. After long-term treatment (24 h) of cells with TPA, the effect of TPA on [³H]AA production was not different from control, whereas SP still displayed [³H]AA-releasing abilities although not at full scale. Pretreatment of cells with thapsigargin, U 73122, methoxyverapamil, and RHC 80267, an inhibitor of diacylglycerol lipase, all resulted in reduced SP-stimulated [³H]AA liberation. Treatment of cell cultures with pertussis toxin (PTX) reduced the release of [³H]AA induced by SP, whereas PTX had no effect on SP-stimulated generation of ³H-inositol phosphates. On the basis of these results, it is concluded that (1) the PLA₂ pathways interfere with the phosphoinositide-PLC signaling system at the level of PKC isozymes α and β, the product responsible for this interaction being either AA or a metabolite produced by the action of lipoxygenase; (2) SP and TPA are able to activate the PLA₂ pathway at a level at or beyond PLA₂, and this effect is mediated, in part, through PKCα and β species and (for SP) intracellular Ca²⁺ recruited from internal stores as well as from external sources; and (3) SP also activates PLA₂ through a PTX-sensitive pathway distinct from the one coupled to phosphoinositide-PLC, which is PTX insensitive.     

Coexistence of three major isoactins in a single sarcoma 180 cell

Actin in transformed sarcoma 180 cells is composed of the nonmuscle β and γ species and of a third, more acidic stable variant termed ζ. Two-dimensional peptide analysis shows that ζ is similar to β actin, differing in the mobility of only one tryptic peptide. Several lines of evidence indicate that ζ is not a modified β-actin species. This third actin species comprises 20% of the total labeled actin, has the same molecular weight as the β and γ actins and has a different mobility in isoelectric focusing gels from that of the known a actins from skeletal, cardiac and vascular smooth muscle. Like β and γ actin, ζ can be extracted with the actin depolymerizing factor from slime mold. Two-dimensional gel electrophoresis (isoelectric focusing) of the ³⁵S-methionine-labeled polypeptides synthesized by a single sarcoma 180 cell showed that all three major actin species coexist within the same cell. This analysis also showed for the first time the coexistence of α and β tubulin, vimentin, α actinin and three other polypeptides present in intermediate-filament-enriched cytoplast cytoskeletons (spots 12, 24 and 31). Determination of the ratio of γ plus β to ζ actin in different cytoskeletal preparations of intact and enucleated sarcoma 180 cells indicated that this actin species is not localized specifically to any of the major actin-containing structures preserved in the cytoskeletons.     

Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P₁) phosphorylation was studied. Activation of S1P₁ receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P₁ phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P₁ phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P₁ phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P₁ immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P₁ phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P₁.     

Antioxidant Effect of Tocopherols on Autoxidation of Milk Fat

Antioxidant activity of d-α-, dl-β-, d-γ- and d-δ-tocopherol was investigated with fatty acid methylester of milk fat from which unsaponifiable matter had been removed. Autoxidation was carried out at 50°C and its degree was indicated by peroxide value, α- or β-Tocopherol was more effective at lower concentrations (0.003 and 0.01%) than at higher concentrations (0.05, 0.1 and 0.5%). The antioxidant activity of γ- and δ-tocopherol was increased with the increase of tocopherol concentration within the range of 0.001 to 0.5%. The order of antioxidant activity of these tocopherols, which was compared in terms of the time to reach 30 meq of peroxide value, varied with the concentration; γ > β > δ > α at 0.001%, α > γ > β > δ at 0.003%, γ > δ > β > α at 0.01%, and δ > γ > β > α at the concentrations more than 0.05%. α-Tocopherol at the concentration of 0.003%, which corresponded to the concentration in original milk fat, was more effective than other tocopherols at the same concentration and α-tocopherol at other concentrations. Synergism due to the combination of β-, γ-, or δ-tocopherol with 0.003% of α-tocopherol was not observed.     

Modulation of Adenylylcyclase by Protein Kinase C in Human Neurotumor SK-N-MC Cells: Evidence that the α Isozyme Mediates Both Potentiation and Desensitization

Abstract: Exposure of human SK-N-MC neurotumor cells to 4β-phorbol 12-myristate 13-acetate (PMA) increased isoproterenol stimulation of cyclic AMP levels by severalfold. This potentiation was blocked by inhibitors of protein kinase C (PKC) and did not occur in cells in which PKC had been down-regulated. PMA treatment also enhanced the stimulation by dopamine, cholera toxin, and forskolin. Thus, the effect of PMA on the adenylylcyclase system was postreceptor and involved either the guanine nucleotide binding regulatory (G) proteins or the cyclase itself. As PMA treatment did not impair the inhibition of isoproterenol stimulation by neuropeptide Y, an involvement of the inhibitory G protein G_i was unlikely. Cholate extracts of membranes from control and PMA-treated cells were equally effective in the reconstitution of adenylylcyclase activity in S49 cyc^? membranes, which lack the stimulatory G protein subunit G_sα; thus, G_s did not appear to be the target of PMA action. Membranes from PMA-treated cells exhibited increased adenylylcyclase activity to all stimulators including Mn²⁺ and Mn²⁺ plus forskolin. In addition, activity was increased when control membranes were incubated with ATP and purified PKC from rat brain. This is consistent with a direct effect of PKC on the adenylylcyclase catalyst in SK-N-MC cells. PMA treatment also resulted in a shift to less sensitivity in the K_act for isoproterenol but not for dopamine or CGP-12177 (a β₃-adrenergic agonist) stimulation. Thus, the β₁ but not the D₁ or β₃ receptors were being desensitized by PKC activation. Analysis of SK-N-MC cells by western blotting with antibodies against different PKC isozymes revealed that both the α and ζ isozymes were present in these cells. Whereas PKC-α was activated and translocated from cytosol to membrane by phorbol esters, the ζ isozyme was not. Thus, PKC-α, which has been implicated in desensitization in other cell lines, also appears to potentiate adenylylcyclase activity.     

Cyclic strain stimulates isoform-specific PKC activation and translocation in cultured human keratinocytes

Previous studies have demonstrated that cyclic strain induces keratinocyte proliferative and morphological changes. Since protein kinase C (PKC) is known to play an important role in the regulation of keratinocyte growth and differentiation, the objective of this study was to determine the role of the PKC signaling pathway as a mediator of strain modulation of the keratinocyte phenotype. In particular, we tested the following specific hypotheses: (1) cyclic strain stimulates PKC activity and translocation, (2) cyclic strain activates PKC in an isoform-specific manner, and (3) PKC mediates the strain activated proliferative and morphological response in cultured human keratinocytes. To test these hypotheses, keratinocytes were subjected to vacuum-generated cyclic strain (10% average strain), followed by measurement of PKC activity, PKC isoform distribution by Western blot analysis and confocal microscopy, and examination of the effect of PKC inhibitors (calphostin C and staurosporine) on strain induced proliferative and morphological changes. We observed stimulation of PKC activity (62.3 ± 5.1% increase) coupled with translocation of PKC from the cytosolic to the membrane fraction in keratinocytes subjected to acute cyclic strain. Cyclic strain also caused translocation of PKC α and δ, but not ζ isoforms, from the cytosolic to the membrane fraction as demonstrated by both Western blot analysis and confocal microscopy. PKC β was not detected in these cells. PKC inhibitors, calphostin C (10 nM), and staurosporine (5 nM), inhibited strain-induced PKC activation and keratinocyte proliferation, but did not block the effects of strain on cellular morphology or alignment. We conclude that these data support our hypothesis that cyclic strain stimulates PKC activity and translocation in an isoform-specific manner in cultured human keratinocytes. Moreover, our studies with PKC inhibitors support the hypothesis that strain-induced changes in the keratinocyte phenotype may be selectively modulated by PKC. J. Cell. Biochem. 67:327–337, 1997. © 1997 Wiley-Liss, Inc.     

佛波酯引起蛋白激酶C下降调节的专一性

探讨了佛波酯（ＰＭＡ）对蛋白激酶的下降调节是否有激酶专一性及亚型专一性．用组蛋白Ｈ１作为蛋白激酶Ｃ（ＰＫＣ）和蛋白激酶Ａ（ＰＫＡ）的受体底物,加入ＰＫＣ和ＰＫＡ的特异性激活剂区分ＰＫＣ和ＰＫＡ,用聚谷酪（４１）为酪氨酸蛋白激酶（ＴＰＫ）的专一性受体底物,以３２Ｐ－ＡＴＰ为３２Ｐ共同供体底物测定三种蛋白激酶的活力,并用免疫组化法测定ＰＫＣ亚型．结果发现ＰＭＡ对人７７２１肝癌细胞只引起ＰＫＣ而不引起ＰＫＡ和ＴＰＫ的下降调节,ＰＫＣ的非特异性抑制剂槲皮素和特异性抑制剂Ｄ－鞘氨醇能大部分取消ＰＭＡ对ＰＫＣ的下降调节,但ＴＰＫ抑制剂ｇｅｎｅｓｔｅｉｎ则没有阻断下降调节的作用．用ＨＬ－６０细胞还证明ＰＭＡ只对含量丰富的ＰＫＣα和ＰＫＣβⅡ亚型而不对含量很少的ＰＫＣβⅠ亚型发生下降调节．上述结果说明ＰＭＡ对蛋白激酶的下降调节有激酶和亚型专一性．     

TWO MOTILE STAGES OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE): MORPHOLOGY,GROWTH, AND TOXICITY1

Eight months after the 1988 bloom of Chrysochromulina polylepis Manton et Parke in Skagerrak and Kattegat, off the coasts of Norway, Sweden, and Denmark, an alternate cell type carrying a scale complement different from that of authentic C. polylepis appeared in some clones isolated from the bloom. The cultures were recloned, and the development of the new clones was monitored. In clones with 100% cells of the alternate type, authentic cells reappeared, suggesting that the alternate cell type is a stage in the life cycle of C. polylepis and that transition between cell types occurs in both directions. Growth rates of clone cultures (termed a cultures) producing exclusively authentic cells, and of clone cultures (termed β cultures), capable of producing the alternate cell type, were compared at various combinations of temperature and photon fluence rate. The β cultures were less tolerant of high temperatures and photon fluence rates (≤20° C, 570 μmol photons·m^?2·s^?1) than were the α cultures. At lower temperatures and photon fluence rates (≤16° C, ≤90 μmol photons·m^?2·s^?1ss), β cultures grew better than α cultures. Relative abundance of the two cell types in β cultures changed in an apparently random manner during these experiments. Preliminary results from flow cytometric analyses indicated that cells of the alternate type were about twice the size and contained an equal or smaller amount of DNA per cell compared to the authentic cells. The β cultures were less toxic to Artemia nauplii than were the a cultures. Three other Chrysochromulina species tested were apparently nontoxic.     

Effects of Depolarization and NMDA Antagonists on the Survival of Cerebellar Granule Cells: A Pivotal Role for Protein Kinase C Isoforms

Abstract: Primary cultures of cerebellar granule cells (CGCs) grown in high-K⁺ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3–4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K⁺ concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC α, γ, μ, λ, and ι from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC ε and βII, respectively, whereas no significant change was observed for βI. The isoforms of δ, θ, η, and ζ were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC ι and ε were decreased, those of PKC α, βI, βII, γ, and λ were unchanged, whereas that of PKC μ was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2–4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K⁺ cultures, levels of PKC α, μ, λ, and ι, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC γ, λ, ι, and μ at 10 and 14 DIV. In addition, the PKC ε level was increased at 14 DIV but decreased at early stages, whereas PKC α, βI, and βII levels were unchanged, as compared with K25 culture alone. Taken together, induction and up-regulation of PKC isoforms may play an important role in the maintenance of CGC survival by depolarization and MK-801.     

Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (α, β₁, β₂, γ, δ and ɛ) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10 mM NaN₃, combined with 2 mM 2-deoxyglucose (chemical ischemia); (iii) 1 h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC β₁, δ and ɛ isoforms total levels (cytosol + particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while α isoform was slightly reduced and γ isoform was no longer detectable. After reperfusion, the changes displayed by α, β₁, γ, δ and ɛ were maintained and even potentiated, moreover, an increase in β₂ (by 41 ± 12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC α isoform, which following reperfusion was found only in the cytosol. PKC β₁ and δ isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC β₂ and ɛ isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, 1 mM, but not the N-methyl-d-asparate receptor antagonist, MK-801, 1 μM, prevented the translocation of β₁ isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in β₂ and ɛ isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.     

Protein kinase C {delta}-specific activity reporter reveals agonist-evoked nuclear activity controlled by Src family of kinases

Conventional and novel protein kinase C (PKC) isozymes transduce the abundance of signals mediated by phospholipid hydrolysis; however redundancy in regulatory mechanisms confounds dissecting the unique signaling properties of each of the eight isozymes constituting these two subgroups. Previously, we created a genetically encoded reporter (C kinase activity reporter (CKAR)) to visualize the rate, amplitude, and duration of agonist-evoked PKC signaling at specific locations within the cell. Here we designed a reporter, δCKAR, that specifically measures the activation signature of one PKC isozyme, PKC δ, in cells, revealing unique spatial and regulatory properties of this isozyme. Specifically, we show two mechanisms of activation: 1) agonist-stimulated activation at the plasma membrane (the site of most robust PKC δ signaling), Golgi, and mitochondria that is independent of Src and can be triggered by phorbol esters and 2) agonist-stimulated activation in the nucleus that requires Src kinase activation and cannot be triggered by phorbol esters. Translocation studies reveal that the G-protein-coupled receptor agonist UTP induces the translocation of PKC δ into the nucleus by a mechanism that depends on the C2 domain and requires Src kinase activity. However, translocation from the cytosol into the nucleus is not required for the Src-dependent regulation of nuclear activity; a construct of PKC δ prelocalized to the nucleus continues to be activated by UTP by a mechanism dependent on Src kinase activity. These data identify the nucleus as a signaling hub for PKC δ that is driven by receptor-mediated signaling pathways (but not phorbol esters) and differs from signaling at plasma membrane and Golgi in that it is controlled by Src family kinases.     

Identification of protein kinase C isozymes involved in the anti-proliferative and pro-apoptotic activities of 10-Methyl-aplog-1, a simplified analog of debromoaplysiatoxin,in several cancer cell lines

10-Me-aplog-1 is a simplified analog of the tumor-promoting compound debromoaplysiatoxin (DAT) and a unique protein kinase C (PKC) activator with limited tumor-promoting and pro-inflammatory activities. 10-Me-aplog-1 inhibits the growth of several cancer cell lines, but the inhibitory mechanism involving PKC isozymes remains unclear. We quantified the amount of PKC isozymes in nine human cancer cell lines that differ in 10-Me-aplog-1 sensitivity. PKCα and δ were the predominant isozymes expressed in all cell lines, but there was no significant correlation between expression levels and anti-proliferative activity. Knocking down PKCα, and/or PKCδ in the three aplog-sensitive cell lines indicated their involvement in the anti-proliferative and pro-apoptotic activities of 10-Me-aplog-1. This finding suggests that PKCα and/or PKCδ activation could be effective for treating certain cancers. Since the mechanism underlying 10-Me-aplog-1's anti-proliferative activities resembles that of DAT, 10-Me-aplog-1 may be regarded as a special key derived from pleiotropic DAT as a bunch of keys.     

Protein electrophoresis in Andean condors (Vultur gryphus): Reference values and differences between wild and rehabilitating individuals

The study of wildlife health greatly contributes to understanding population dynamics and detecting conservation threats. The determination of the different fractions of plasma proteins (proteinogram) is an important laboratory tool to study wildlife health. The aim of this study was to characterize protein electrophoresis in wild Andean condors (Vultur gryphus) from north‐western Patagonia and to evaluate differences according to age and sex classes. Once reference values of wild, apparently healthy individuals, were established, we compared these values to those of individuals received at the Buenos Aires Zoo in Argentina for rehabilitation due to various health problems. Reference proteinograms from wild Andean condors differed only in the α 1 and β 2‐fractions between sex categories. Males showed higher concentrations of these protein fractions than females. We found clear differences between wild birds and rehabilitating individuals. Total proteins, globulins, α 1‐globulins, total α‐globulins, β 2‐globulins, total β‐globulins, and γ‐globulins were significantly higher in rehabilitating than in wild individuals, whereas albumin, α 2, and β1‐globulins were similar between these groups. The albumin/globulin ratio, as a general indicator of health, was significantly lower in rehabilitating than in wild individuals. The results indicate the effects on different protein fractions of pathologic processes occurring in individuals undergoing rehabilitation. Our results provide useful insights, contributing to improving diagnoses and prognoses in this species. This information may also be useful to assess the health status of Andean condors in studies of wild populations and for comparisons with other bird species.     

Rottlerin,an inhibitor of protein kinase Cdelta (PKCdelta), inhibits astrocytic glutamate transport activity and reduces GLAST immunoreactivity by a mechanism that appears to be PKCdelta-independent

Protein kinase C (PKC) regulates the activity and/or cell surface expression of several different neurotransmitter transporters, including subtypes of glutamate transporters. In the present study, the effects of pharmacological inhibitors of PKC were studied in primary astrocyte cultures that express the glutamate aspartate transporter (GLAST) subtype of glutamate transporter. We found that general inhibitors of PKC, bisindolylmaleimide I (Bis I), bisindolylmaleimide II (Bis II), staurosporine and an inhibitor of classical PKCs, Gö6976, had no effect on Na⁺‐dependent glutamate transport activity. However, rottlerin, a putative specific inhibitor of PKCδ, decreased transport activity with an IC₅₀ value (less than 10 µm ) that is comparable to that reported for inhibition of PKCδ. The effect of rottlerin was very rapid (maximal effect within 5 min) and was due to a decrease in the capacity (V_max) for transport. Rottlerin also caused a drastic loss of GLAST immunoreactivity within 5 min, suggesting that rottlerin accelerates GLAST degradation/proteolysis. Rottlerin had no effect on cell surface or total expression of the transferrin receptor, providing evidence that the effect on GLAST cannot be attributed to a non‐specific internalization/degradation of plasma membrane proteins. Down‐regulation of PKCδ with chronic phorbol ester treatment did not block rottlerin‐mediated inhibition of transport activity. These results suggest a novel mechanism for regulation of the GLAST subtype of glutamate transporter and indicate that there is a rottlerin target that is capable of controlling the levels of GLAST by controlling the rate of degradation or limited proteolysis. It appears that the target for rottlerin may not be PKCδ.     

Crystal structure of DNA polymerase III β sliding clamp from Mycobacterium tuberculosis

The sliding clamp is a key component of DNA polymerase III (Pol III) required for genome replication. It is known to function with diverse DNA repair proteins and cell cycle-control proteins, making it a potential drug target. To extend our understanding of the structure/function relationship of the sliding clamp, we solved the crystal structure of the sliding clamp from Mycobacterium tuberculosis (M. tuberculosis), a human pathogen that causes most cases of tuberculosis (TB). The sliding clamp from M. tuberculosis forms a ring-shaped head-to-tail dimer with three domains per subunit. Each domain contains two α helices in the inner ring that lie against two β sheets in the outer ring. Previous studies have indicated that many Escherichia coli clamp-binding proteins have a conserved LF sequence, which is critical for binding to the hydrophobic region of the sliding clamp. Here, we analyzed the binding affinities of the M. tuberculosis sliding clamp and peptides derived from the α and δ subunits of Pol III, which indicated that the LF motif also plays an important role in the binding of the α and δ subunits to the sliding clamp of M. tuberculosis.     

Indolepyruvate ferredoxin oxidoreductase from Pyrococcus sp. KOD1 possesses a mosaic structure showing features of various oxidoreductases

Indolepyruvate ferredoxin oxidoreductase (IOR) catalyzes the oxidative decarboxylation of arylpyruvates. Gene cloning and sequencing analysis of the IOR gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was performed. Two genes, iorA and iorB, encoding α and β subunits of IOR were found to be tandemly arranged, which suggests that gene expression is translationaly coupled. Sequence analysis showed the C-terminal region of the α subunit to have a typical ferredoxin-type [4Fe-4S] cluster motif (CXXCXXCXXCXXXCP), which is similar to that present in the δ subunits of other oxidoreductases such as pyruvate ferredoxin oxidoreductase (POR) and 2-ketoisovalerate ferredoxin oxidoreductase (VOR). We suggest that the α subunit of KOD1-IOR has a mosaic structure composed of features characteristic of the α, β and δ subunits from POR and VOR. KOD1-IOR was overproduced in anaerobically incubated Escherichia coli cells and the crude enzyme was extracted under anaerobic conditions. The optimal temperature for activity of recombinant IOR was 70°?C and the half-life of this enzyme in the presence of air was 15 min at 25°?C.     

Tetrahymena Tubulins and In Vitro Translation of Tetrahymena RNA*

SYNOPSIS. Tetrahymena outer doublet tubulin was compared with neurotubulin and Chlamydomonas flagellar tubulin on SDS-polyacrylamide gels. Tetrahymenaα tubulin did not comigrate with either brain or flagellar α tubulins, although brain, flagellar, and ciliary β tubulins all comigrated. Axonemal tubulin from Tetrahymena strain ST was compared with this tubulin from strains W. S. HSM, and E, and all were found to have the same mobilities. Poly-A containing RNA was separated from whole cell Tetrahymena RNA by oligo-dT cellulose chromatography. Poly-A+ RNA from 24-h cultures (early exponential growth) stimulated greater incorporation of amino acids into polypeptides in the wheat germ cell-free translation system than did poly-A+ RNA from 36-h and 49-h cultures. When separated on SDS-polyacrylamide gels, the translation products of the 24-h poly-A + RNA had 2 prominent protein bands which comigrated with α and β tubulin isolated from Tetrahymena cilia. These bands were not found in the translation products of poly-A+ RNA isolated from 49-h cultures or in the translation products ofpoly-A- RNA.     

Antibiotic Resistant Group A Streptococci

A series of Streptococcus pyogenes strains, including strains isolated from patients, mutants which had acquired in vitro resistance to penicillin (Pc), mitomycin C (MC), tetracycline (TC) and chloramphenicol (CM), ultraviolet light induced α hemolytic mutants, as well as β hemolytic mutants (β mutants) derived from α hemolytic mutants (α mutants) were compared as to their antibiotic sensitivity, and physiological, biochemical and serological properties. To obtain β mutants from α mutants the following procedures were employed: (1) serial mouse passage, (2) serial serum-broth transfers, (3) cultivation in heat-killed cultures of parent strains, and (4) cultivation in broth containing bacterial DNA extracted from parent streptococcus cells. From the results obtained these strains could be divided into two major groups, each with two subgroups. Group 1 strains produce soluble hemolysins and are sensitive to Pc. Subgroup 1–1 strains are sensitive to other antibiotics too; subgroup 1–2 are resistant to certain antibiotics other than Pc, bacitracin and MC. Group 2 strains do not produce soluble hemolysins and resistant to Pc. Subgroup 2-1 strains are α hemolytic on horse blood agar and subgroup 2–2 are β hemolytic on the same medium. Pc resistance in group 2 strains was more than 100-fold higher than that of sensitive strains, and was accompanied by MC resistance, but to a lesser degree. Pc resistance in group 2 mutants could be induced by antibiotics other than Pc and also by ultraviolet irradiation. Although group 1 cells retained the characteristics of typical S. pyogenes, group 2 cells, both α and β hemolytic, lost most of the physiological, biochemical and serological properties of this species. The similarity of group 2 strains to group D or group N streptococcal strains in their general properties is discussed.     

Expression of α and β parvalbumin is differentially regulated in the rat organ of corti during development

The expression of two calcium‐binding proteins of the parvalbumin (PV) family, the α isoform (αPV) and the β isoform known as oncomodulin (OM), was investigated in the rat cochlea during postnatal development and related to cholinergic efferent innervation. Using RT‐PCR analysis, we found that OM expression begins between postnatal day 2 (P2) and P4, and peaks as early as P10, while αPV mRNA begins expression before birth and remains highly expressed into the adult period. Both in situ hybridization and immunoreactivity confirm that OM is uniquely expressed by the outer hair cells (OHCs) in the rat cochlea and occurs after efferent innervation along the cochlear spiral between P2 and P4. In contrast to OM expression, αPV immunoreactivity is expressed in both inner hair cells (IHCs) and OHCs at birth. Following olivocochlear efferent innervation, OHCs demonstrate weak OM immunoreactivity beginning at P5 and diminished αPV immunoreactivity after P10. In organ cultures isolated prior to the efferent innervation of OHCs, OM immunoreactivity failed to develop in OHCs, but αPV immunoreactivity remained present in both IHCs and OHCs. In contrast, organ cultures isolated after efferent innervation of OHCs show OHCs with low levels of OM immunoreactivity and high levels of αPV immunoreactivity. This study suggests that OM and αPV are differentially regulated in OHCs during cochlear development. Our findings further raise the possibility that the expression of PV proteins in OHCs may be influenced by efferent innervation. © 2003 Wiley Periodicals, Inc. J Neurobiol 58: 479–492, 2004     

Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1)

Background: Phosphorylation critically regulates the catalytic function of most members of the protein kinase superfamily. One such member, protein kinase C (PKC), contains two phosphorylation switches: a site on the activation loop that is phosphorylated by another kinase, and two autophosphorylation sites in the carboxyl terminus. For conventional PKC isozymes, the mature enzyme, which is present in the detergent-soluble fraction of cells, is quantitatively phosphorylated at the carboxy-terminal sites but only partially phosphorylated on the activation loop.Results: This study identifies the recently discovered phosphoinositide-dependent kinase 1, PDK-1, as a regulator of the activation loop of conventional PKC isozymes. First, studies in vivo revealed that PDK-1 controls the amount of mature (carboxy-terminally phosphorylated) conventional PKC. More specifically, co-expression of the conventional PKC isoform PKC βII with a catalytically inactive form of PDK-1 in COS-7 cells resulted in both the accumulation of non-phosphorylated PKC and a corresponding decrease in PKC activity. Second, studies in vitro using purified proteins established that PDK-1 specifically phosphorylates the activation loop of PKC α and βII. The phosphorylation of the mature PKC enzyme did not modulate its basal activity or its maximal cofactor-dependent activity. Rather, the phosphorylation of non-phosphorylated enzyme by PDK-1 triggered carboxy-terminal phosphorylation of PKC, thus providing the first step in the generation of catalytically competent (mature) enzyme.Conclusions: We have shown that PDK-1 controls the phosphorylation of conventional PKC isozymes in vivo. Studies performed in vitro establish that PDK-1 directly phosphorylates PKC on the activation loop, thereby allowing carboxy-terminal phosphorylation of PKC. These data suggest that phosphorylation of the activation loop by PDK-1 provides the first step in the processing of conventional PKC isozymes by phosphorylation.