Saccharomyces cerevisiae DNA repair processes: an update

The effect of incubating T3-1 cells with phorbol 12,13-dibutyrate (PDBu) on the protein kinase C (PKC) isoform content (predominantly , and isoforms) was assessed by immunoblotting, enzyme activity assay and [³H]PDBu binding. After exposure to PDBu for 17 h the immunoreactivity detected for both PKC and PKC had disappeared from cytosol and had increased slightly in membranes. Immunoreactivity for PKC was present as two bands in cytosol; after PDBu treatment both bands decreased in intensity, the higher molecular weight band more than the lower. The lower molecular weight band corresponded with a component of constitutive PKC activity eluting from DEAE cellulose that was defined by inhibition of basal activity with GF 109203X or H7. Investigation of very short treatment times with PDBu using binding, immunoblot and activity measurements (in the presence/absence of Ca²⁺) indicated that translocation of PKC and was very rapid — detectable by 10 sec, maximal within minutes. Reduction of these isoforms in membranes took much longer, and was not apparent up to 150 min. The immunoblot data for PKC in cytosol showed no detectable effect of PDBu treatment on the low molecular weight band up to 150 min although it was reduced at 17 h. Translocation of the upper band was detectable at 10 sec but this band may have resulted from cross-reaction with other PKC isoforms. The constitutive activity and low molecular weight (authentic) PKC immunoreactivity were partially affected after long exposure only, suggesting an action of PDBu on PKC secondary to activation of the other PKC isoforms. An endogenous receptor agonist, luteinising hormone-releasing hormone (LHRH), was also used to assess by immunoblotting, translocation of the PKC isoforms. Although all the isoforms did translocate from cytosol to membrane fractions, they did so with distinctly different time courses: PKC moved more rapidly than PKC which appeared to translocate more quickly than PKC . After downregulation of the responsive PKC isoforms with PDBu, the remaining PKC was not translocated by LHRH. (Mol Cell Biochem 165: 65–75, 1996)     

Phorbol ester stimulation of phosphatidylcholine synthesis in four cultured neural cell lines: Correlations with expression of protein kinase C isoforms

Phosphatidylcholine (PtdCho) can provide lipid second messengers involved in signal transduction pathways. As a measure of phospholipid turnover in response to extracellular stimulation, we investigated differential enhancement of [³H]choline incorporation into PtdCho by phorbol esters. In C6 rat glioma and SK-N-SH human neuroblastoma cells, [³H]PtdCho synthesis was 2–4 fold stimulated by -12-O-tetradecanoylphorbol-13-acetate (-TPA) when [³H]choline was incubated simultaneously with, or 15 min prior to, -TPA treatment. By contrast, in N1E-115 mouse and SK-N-MC human neuroblastoma cells, phorbol esters had no appreciable effect on [³H]choline incorporation; however, in all cells, 200 M oleic acid enhanced PtdCho synthesis, indicating a stimulable process. Alterations by thymeleatoxin (TMT), an activator of conventional PKC isoforms (, and ), were similar to -TPA. We investigated whether expression of specific PKC isoforms might correlate with these effects of phorbol esters on PtdCho synthesis. All cell lines bound phorbol esters, had PKC activity that was translocated by phorbol esters and differentially expressed isoforms of PKC. Northern and western blot analyses, using specific cDNA and antibodies for PKC-,-,-,-,-, and-, revealed that expression of -isoform predominated in C6 and SK-N-SH cells. In contrast, TPA-responsive -isoform predominated in SK-N-MC cells. -PKC was not detected in any cells and only in C6 cells was PKC- present and translocated by -TPA treatment. PKC- was not detected in SK-N-MC cell lines but translocated with TPA treatment in the other three cell lines. PKC- was present in all cells but was unaltered by TPA treatment. Accordingly, stimulation of PtdCho turnover by phorbol esters correlated only with expression of PKC-; presence of PKC- alone was insufficient for a TPA response.Abbreviations DAG diacylglycerol - DMEM Dulbecco's modified Eagle's medium - dPPA 12-deoxyphorbol-13-phenylacetate-20-acetate - PKC protein kinase C - cPKC conventional PKC - PtdCho phosphatidylcholine - TPA 12-O-tetradecanoylphorbol-13-acetate - TMT thymeleatoxin     

\4 integrin expression onin vitro andin vivo metastatic variants of lewis lung carcinoma

The expression of 4, 6, and 1 integrin subunits has been investigated on somein vitro andin vivo murine metastatic variants derived from Lewis lung carcinoma (3LL). By the use of monoclonal antibodies which recognizes different epitopes of 6, 1, and 4 subunits we demonstrate that 6 and 1 subunits are expressed in all metastatic variants of 3LL irrespective of their metastatic potential, whereas 4 subunit is expressed only in highly metastasizing cells of 3LL. Northern blots of different metastatic variants probed with 1 and 4 subunits demonstrate thata) significant amounts of 1 mRNA were detected in all metastatic variants of 3LL;b) mRNA corresponding to the described entire coding sequence of 4 subunit is expressed only on highly metastasizing cells of 3LL. We conclude that 4 subunit is specifically expressed in highly metastasizig cells of 3LL while is undetectable in lower metastasizing ones.     

Identification of ‘Amigo’ and ‘Kavkaz’ translocations in Ohio soft red winter wheats (Triticum aestivum L.)

One cultivar (GR876) and two advanced Ohio soft red winter wheat lines (OH413 and OH414), with Kavkaz in their pedigrees, were examined for the presence of the Kavkaz, 1RS/1BL rye/wheat chromosome translocation. Another advanced line (OH416), with Amigo in its pedigree, was examined for the presence of the Amigo, 1RS/1AL translocation. Only two satellited chromosomes were observed in most mitotic root-tip cells from GR876, OH413, and OH414, compared to four in most cells from OH416. Heteromorphic bivalents were observed in most PMCs from hybrids produced by crossing GR876, OH413, and OH414 as females to Chinese Spring. No heteromorphic bivalents were observed in PMCs from OH416 x Chinese Spring hybrids. When GR876 and the Ohio lines were hybridized with Chinese Spring dimonotelosomic-1B, telosomic trivalents, consisting of the short- and longarm telosomes paired with chromosome 1B, were only observed in PMCs from 43-chromosome hybrids involving OH416. The long-arm telosome paired with the translocation chromosome, while the short-arm telosome remained unpaired in all other 43-chromosome hybrids. Separation of gliadin proteins from GR876 and the Ohio lines by PAGE revealed that secalin bands for GR876, OH413, and OH414, migrated similarly to the secalins for Kavkaz. Bands for OH416, identified as possible secalins, migrated similarly to those for Amigo. Cultivar GR876 and advanced Ohio soft red winter wheat lines OH413 and OH414 carry the Kavkaz translocation, while OH416 carries the Amigo translocation.Communicated by K. Tsunewaki     

Protein kinase C isoforms in pituitary cells displaying differential sensitivity to phorbol ester

Investigations with protein kinase C (PKC) isoform-specific antisera, revealed distinct profiles of PKC isoform content amongst pituitary tissues. Western analysis revealed the and isoforms of PKC are present in rat anterior and posterior pituitary tissue as well as in the GH₃ somatomammotrophic cell line. AtT-20/D16-V corticotrophic and T3-1 gonadotrophic murine cell lines contained no PKC-. The or isoforms were undetected in any pituitary tissue. PKC activity measurements revealed Ca²⁺-independent PKCs in T3-1 and GH₃ cells which were more sensitive to activation by phorbol-dibutyrate (PDBu) than the corresponding PKC activity found in COS cells. However, Ca²⁺-dependent PKC activities were of similar sensitivity to PDBu in GH₃, T3-1 and COS cells, indicating that functional differences observed in PDBu-sensitivity in these cells may be due to differential activation of Ca²⁺-independent PKC isoforms. Moreover, substrate-specificity of these PKCs were also compared indicating that the amount of Ca²⁺-dependency of the observed PKC activity from the same pituitary tissue is dependent upon the substrate utilized by the PKC isotypes present. These findings explain differential sensitivities of PKC-mediated actions that have previously been observed in a range of pituitary cells. (Mol Cell Biochem 000-000, 1999)     

Structure and Sites of Phosphorylation of 14-3-3 Protein: Role in Coordinating Signal Transduction Pathways

The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of adaptor protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition. It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions. 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX_1,2SpXP. The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases. The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins. We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms.     

Signalling by protein kinase C isoforms in the heart

Understanding transmembrane signalling process is one of the major challenge of the decade. In most tissues, since Fisher and Krebs's discovery in the 1950's, protein phosphorylation has been widely recognized as a key event of this cellular function. Indeed, binding of hormones or neurotransmitters to specific membrane receptors leads to the generation of cytosoluble second messengers which in turn activate a specific protein kinase. Numerous protein kinases have been so far identified and roughly classified into two groups, namely serine/threonine and tyrosine kinases on the basis of the target amino acid although some more recently discovered kinases like MEK (or MAP kinase kinase) phosphorylate both serine and tyrosine residues.Protein kinase C is a serine/threonine kinase that was first described by Takai et al. [1] as a Ca- and phospholipid-dependent protein kinase. Later on, Kuo et al. [2] found that PKC was expressed in most tissues including the heart. The field of investigation became more complicated when it was found that the kinase is not a single molecular entity and that several isoforms exist. At present, 12 PKC isoforms and other PKC-related kinases [3] were identified in mammalian tissues. These are classified into three groups. (1) the Ca-activated -, -,and -PKCs which display a Ca-binding site (C2); (2) the Ca-insensitive -, -, -, -, and -PKCs. The kinases that belong to both of these groups display two cystein-rich domains (C1) which bind phorbol esters (for recent review on PKC structure, see [4]). (3) The third group was named atypical PKCs and include , , and -PKCs that lack both the C2 and one cystein-rich domain. Consequently, these isoforms are Ca-insensitive and cannot be activated by phorbol esters [5]. In the heart. evidence that multiple PKC isoforms exist was first provided by Kosaka et al. [6] who identified by chromatography at least two PKC-related isoenzymes. Numerous studies were thus devoted to the biochemical characterization of these isoenzymes (see [7] for review on cardiac PKCs) as well as to the identification of their substrates.This overview aims at updating the present knowledge on the expression, activation and functions of PKC isoforms in cardiac cells. (Mol Cell Biochem 157: 65–72, 1996)     

Phorbol ester and diacylglycerol activation of native protein kinase C species from various tissues

The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca²⁺ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC were included for comparison.The presence of specific PKC isoforms in different tissues was determined using Western blot analysis. Protein kinase C , ₁, , , and / were all present in rat midbrain cytosolic extract, PKC , ₁, , and / were present in spleen cytosol, and PKC and / were present in COS 7 cell cytosol. The predominance of and activities in COS 7 and spleen extracts respectively was confirmed by enzymic assay.The PKC activity assay was configured such that the Ca²⁺ dependence of the PKC activity induced by different PKC activators could be determined. Phorbol 12,13-dibutyrate (PDBu) was virtually equipotent on the Ca²⁺-dependent PKC activity from midbrain and spleen and slightly less potent on that from COS 7 cells. In the absence of Ca²⁺, PDBu was considerably less potent overall (as, indeed, were the other PKC activators) and was less potent on COS 7 cell PKC than on those from midbrain or spleen. Mezerein was more potent than PDBu at inducing PKC activity in COS 7 cell extracts in either the absence or presence of Ca²⁺ whereas in the presence of Ca²⁺, mezerein was slightly less potent on midbrain and spleen than PDBu and equipotent in the absence of Ca²⁺. Maximum values for Ca²⁺-independent activation by mezerein indicated that this activator was particularly effective in recruiting Ca²⁺-dependent PKC isoform activity in a Ca²⁺ free environment. The greater potency of mezerein on PKC was confirmed using PKC and further purified from rat spleen by hydroxylapatite (HAP) chromatography. The effects of both PDBu and mezerein were investigated using anterior pituitary tissue where a particularly high potency of mezerein in the absence of Ca²⁺ was noted. The diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DOG), appeared to cause little or no activation of native Ca²⁺-dependent isoforms in Ca²⁺ free conditions unlike another longer chain diacylglycerol, 1,2-dioleoyl-sn-glycerol. Also DOG activated midbrain PKCs more potently than PKCs from spleen or COS 7 cells (or lung and pituitary tissue) in the presence of Ca²⁺. The concentration-dependence of DOG was examined on PKC and PKC further purified from brain by HAP chromatography, revealing that DOG was equally potent on both of these isoforms derived from brain and on recombinant PKC . However, [³H]PDBu binding data using PKC purified from several sources gave very different IC₅₀ values when DOG was used as a displacer, and in general these values correlated with the EC₅₀ values recorded from the activity assay.The data presented here indicate that there are distinct differences in the activator pharmacology of different native PKC isoforms and between the same isoform expressed in different tissues, either because of post-translational modifications or some other tissue specific factor.     

Post-translational modifications of α-tubulin in<Emphasis Type="Italic"> Zea mays</Emphasis> L. are highly tissue specific

To further understand post-translational modifications (PTMs) of plant -tubulin, post-translationally modified -tubulin isoforms from selected tissues of Zea mays L. were examined using two-dimensional electrophoresis and immunoblotting. Except for polyglycylated tubulin, tyrosinated, detyrosinated, acetylated and polyglutamylated -tubulin isoforms were all present in maize tissues. Tyrosinated -tubulin was the predominant variant in all cases, with isoforms 1–4 (5) being the most common components. Leaves exhibited a striking difference in PTM patterns of -tubulin isoforms compared to other tissues examined. In leaves, several major specific isoforms were highly modified by detyrosination, acetylation and polyglutamylation. In pollen and anthers, only the most abundant isoform 3 was acetylated to an appreciable extent, and no acetylated isoform was found in roots. Similarly, in pollen, anthers and roots, only 3 was appreciably polyglutamylated. Additionally, a detyrosinated isoform 6 was present in anthers and in leaves, while the tyrosinated isoform 6 seemed to be pollen specific. These results indicate that certain types of PTM of plant -tubulin preferentially occur in a tissue-specific way.Abbreviations 1-, 2-D one-, two-dimensional - MT microtubule - PTM post-translational modification     

Preliminary studies on the genetic variability of six Hungarian common carp strains using microsatellite DNA markers

Genetic variation in six Hungarian common carp (Cyprinus carpio L.) strains was evaluated using 12 microsatellite loci. The domesticated (Tatai, Biharugrai and Szarvasi) strains were derived from fish farms. Two of wild strains (Tiszai and Dunai) were sampled from brood stocks maintained at fish farms for breeding, and Kis-Balatoni wild carp were sampled from the Small Balaton Lake. Pairwise Fst-values (0.013–0.161) were highly significant (p0.0001), demonstrating differentiation among strains. The mean number of alleles ranged between 3.9 and 8.2. Overall mean observed heterozygosity was lower (0.557) than the mean expected heterozygosity (0.700). By strain, the only exception to this trend was the Dunai (Danubian), which showed higher mean observed heterozygosity (0.764) than expected (0.602). For five loci the Dunai strain showed extremely high levels of heterozygosity (1.00). Two wild strains exhibited a number of loci (Tiszai, 4; Dunai, 6) that were not in Hardy–Weinberg equilibrium. A relatively high number of private alleles overall (n=26), as well as differences in allele frequencies supported our ability to assign most individual fish (over 90%) to each strain.     

Lipopolysaccharide Induction of MARCKS-Related Protein and Cytokine Secretion are Differentially Impaired in Microglia from LPS-nonresponsive (C3H/HeJ) Mice

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (100 ng/ml) than in normal (C3H/OuJ) microglia (10 ng/ml). By contrast, TNF production was not significantly increased in C3H/HeJ microglia even at 1 g LPS/ml. Microglia expressed PKC isoforms , , , and (but not and ); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lps^d mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.     

The effects of mitochondrial energetics inhibitors on the fluorescence of potential-sensitive dyes rhodamine 123 and DiS-C3-(5) in lymphocyte suspensions

The effects of uncouplers (FCCP, DNF), oligomycin, and rotenone on the fluorescence of potential-sensitive dyes, rhodamine 123 and diS-C₃-(5), in lymphocyte suspensions were compared. The fluorescence of these optical probes gradually increased at higher FCCP concentrations. The dependences of fluorescence intensities and FCCP concentrations were similar for both dyes, and only diS-C₃-(5) fluorescence started increasing at lower FCCP concentrations. Rotenone (1 µM) significantly increased rhodamine 123 fluorescence. TMPD-induced and uncoupler-induced diS-C₃-(5) fluorescence changes increased 1.5- to 2-fold if the incubation mixture was supplemented with oligomycin (0.1–0.2 µg/ml). The fluorescence responses of the dyes in the lymphocyte suspension correlate with the effects of mitochondrial energetics inhibitors on _m in isolated mitochondria. The results suggest the possibility of using these dyes for estimating the direction of the _m changes in the lymphocyte suspension.Abbreviations _m difference in electrical potential across the mitochondrial inner membrane - _p difference in electrical potentials across the plasma membrane - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - DNP 2,4-dinitrophenol - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - diS-C₃-(5) 3,3-dipropylthiodicarbocyanine - MOPS morpholinopropane sulfonic acid     

Regulation of cellular Gsα levels and basal adenylyl cyclase activity by expression of the β2-adrenoceptor in neuroblastoma cell lines

Mouse neuroblastoma x rat glioma hybrid NG108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human 2 adrenoceptor cDNA under the control of the actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (N22) but not one expressing only low levels of the receptor (N17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg²⁺ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn²⁺. The elevated basal activity was partially reversed by addition of the -adrenoceptor antagonist propranolol. Agonist activation of N22 but not N17 cells led to a large selective down-regulation of cellular G_s levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [³H] forskolin was achieved with substantially greater potency (some 30 fold) in N22 cell membranes than in N17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [³H] forskolin binding which was equipotent in each of N22, N17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of G_s down-regulation in each cell type. Expression of an epitope tagged variant of G_s in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type G_s. Isolation of clones of NCB20 cells expressing high levels of the 2 adrenoceptor also resulted in a specific agonist-induced down-regulation of G_s.     

Regulation ofN-acetylglucosaminyltransferase V by protein kinases

When 7721 human hepatocarcinoma cells were treated with 100nm phorbol-12-myristate-13-acetate (PMA), the activity ofN-acetylglucosaminyltransferase V(GnT-V) in the cells varied in accordance with the activity of membranous protein kinase C (PKC), but not with that of cytosolic PKC. Quercetin, a non-specific inhibitor of Ser/Thr protein kinase, andd-sphingosine and staurosporine, two specific inhibitors of PKC, blocked the activation of membranous PKC and GnT-V by PMA. Among the three inhibitors, quercetin was least effective. The inhibitory rates of quercetin and staurosporine toward membranous PKC and GnT V were proportional to the concentrations of the two inhibitors. The activities of GnT V and membranous protein kinase A (PKA) were also induced in parallel by dibutyryl cAMP (db-cAMP) and this induction was blocked by a specific PKA inhibitor. When cell free preparations of 7721 cells and human kidney were treated with alkaline phosphatase (ALP) to remove the phosphate groups, the GnT V activities were decreased. These results suggest that GnT V may be activated by membranous PKC or PKA, indirectly or directly, via phosphorylation of Ser/Thr residues.Abbreviations UDP uridine diphospho- - GnT N-acetylglucosaminyltransferase - GlcNAc Gn N-acetylglucosamine - M mannose - PMA phorbol-12-myristate-13-acetate - PKC protein kinase C - PKA protein kinase A - cAMP adenosine 3, 5-cyclic monophosphate - db-cAMP dibutyryl cAMP - TPK tyrosine protein kinase - MES 2-[N-morpholino]ethanesulfonic acid - DTT dithiothreitol - PMSF phenylmethylsulfonyl fluoride - EDTA ethylene diamine tetraacetic acid - EGTA glycol-bis-(-aminoethyl) etherN,N,N,N-tetraacetic acid - PA 2-aminopyridine - ALP alkaline phosphatase - C₂C₂ GlcNAc1-2 Man1-6(GlcNAc1-2Man1-3)ManR - C_2,4C₂ GlcNAc1-2Man1-6(GlcNAc1-4[GlcNAc1-2]Man1-3)ManR - C₂C_2,6 GlcNAc1-6[GlcNAc1-2]Man1-6(GlcNAc1-2Man1-3)ManR - C_2,4C_2,6 GlcNAc1-6[GlcNAc1-2]Man1-6(GlcNAc1-4[GlcNAc1-2]Man1-3)ManR where R=1-4GlcNAc1-4GlcNAcAsnX - Gn₂M₃Gn₂-PA C₂C₂ where R=1-4GlcNAc1-4GlcNAc-PA - Gn₃M₃Gn₂-PA C₂C_2,6 where R=1-4GlcNAc1-4GlcNAc-PA     

Nest und Nestbau von Winter- und Sommergoldh?hnchen(Regulus regulus undR. ignicapillus)

Beobachtungen über Nest und Nestbautechnik von Winter- und Sommergoldhähnchen(Regulus regulus, R. ignicapillus) im Freiland und in Volieren zeigen:

Further evidence for abnormal protein kinase C regulation of macromolecule secretion in fibroblasts from cystic fibrosis patients

In comparison to skin fibroblasts from normal subjects, those from patients with cystic fibrosis (CF): (1) bound [20-³H] phorbol 12,13-dibutyrate (PDBu) with a higher affinity (K_d=25.8 vs 12.8 nM respectively) but expressed a similar number of total phorbol ester binding sites (about 2.5 pmol PDBu bound/mg of protein); (2) exhibited a faster and higher response to 4-phorbol 12-myristate 13-acetate (PMA) for the stimulation of [³⁵S]-labelled glycoconjutate release, but were equally sensitive to the synergistic effect of A23187 on this process; and (3) secreted glycoconjugates with similar [³⁵S]-sulfate and [¹⁴C]-leucine to [¹⁴C]-glucosamine labelling ratios. Taken together, these results provide further evidence for abnormal protein kinase C (PKC) regulation of macromolecule secretion in CF disease.Abbreviations BSA Bovine serum albumin - DBcAMP Dibutyryl cyclic AMP - DMEM Dulbecco's modified Eagle's medium - DMSO Dimethylsulfoxide - LDH Lactate dehydrogenase - PBS Phosphate-buffered saline - PDBu 4-phorbol 12,13-dibutyrate - 4-PDD 4-phorbol 12,13-didecanoate - PMA 4-phorbol 12-myristate 13-acetate - TCA Trichloroacetic acid     

Protein kinase C isoenzymes in mouse Harderian gland. Differential expression of the α- and ε-isoforms during pregnancy

Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell lines and tissues. Experiments were designed to determine whether the Harderian gland of CD-1 mouse produces PKC isoenzymes and whether the expression of the isoforms changes during pregnancy. The presence of the isoenzymes was assessed by immunoblotting experiments using extract of total Harderian gland and polyclonal antisera specific for nine different PKC isoforms. Antisera giving a positive staining on Western blots were subsequently used for immunohistochemical investigation using a secondary antibody conjugated to alkaline phosphatase. Immunoblotting experiments revealed that the Harderian gland from female mouse expresses PKC isoforms-,-,- and-. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the - and -isoforms. The 80-kDa native form of PKC- almost doubled in the pregnant mouse in comparison with normal female mouse whereas the amount of 50-kDa catalytic domain did not change. Protein kinase C- appeared as a 92- to 93-kDa form and a 67-kDa form. While the 92- to 93-kDa protein was expressed to a similar extent in both types of mouse, the 67-kDa form was more abundant inthe Harderian gland from normal female mouse. These data were corroborated by immunohistochemical experiments and showing a diffuse and granular staining of the adenomeres. These observations demonstrate for the first time (to our knowledge) that the mouse Harderian gland produces several PKC isoenzymes that could be involved in the regulation of exocytosis and/or other functions. Moreover, the expression of the - and -isoforms could be regulated by sexual hormones, as suggested by the differential abundance of these two proteins in the gland of pregnant mouse compared with normal female mouse.