Interaction of casein kinase 1 delta (CK1delta) with post-Golgi structures, microtubules and the spindle apparatus

Members of the casein kinase 1 family of serine/threonine kinases are highly conserved from yeast to mammals and seem to play an important role in vesicular trafficking, DNA repair, cell cycle progression and cytokinesis. We here report that in interphase cells of various mammalian species casein kinase 1 delta (CK1delta) specifically interacts with the trans Golgi network and cytoplasmic, granular particles that associate with microtubules. Furthermore, at mitosis CK1delta is recruited to the spindle apparatus and the centrosomes in cells, which have been exposed to DNA-damaging agents like etoposide or gammairradiation. In addition, determination of the affinity of CK1delta to different tubulin isoforms in immunoprecipitation-Western analysis revealed a dramatically enhanced complex formation between CK1delta and tubulins from mitotic extracts after introducing DNA damage. The high affinity of CK1delta to the spindle apparatus in DNA-damaged cells and its ability to phosphorylate several microtubule-associated proteins points to a regulatory role of CK1delta at mitosis.     

The alpha1 subunit EGL-19, the alpha2/delta subunit UNC-36, and the beta subunit CCB-1 underlie voltage-dependent calcium currents in Caenorhabditis elegans striated muscle

Voltage-gated calcium channels, which play key roles in many physiological processes, are composed of a pore-forming α1 subunit associated with up to three auxiliary subunits. In vertebrates, the role of auxiliary subunits has mostly been studied in heterologous systems, mainly because of the severe phenotypes of knock-out animals. The genetic model Caenorhabditis elegans has all main types of voltage-gated calcium channels and strong loss-of-function mutations in all pore-forming and auxiliary subunits; it is therefore a useful model to investigate the roles of auxiliary subunits in their native context. By recording calcium currents from channel and auxiliary subunit mutants, we molecularly dissected the voltage-dependent calcium currents in striated muscle of C. elegans. We show that EGL-19 is the only α1 subunit that carries calcium currents in muscle cells. We then demonstrate that the α2/δ subunit UNC-36 modulates the voltage dependence, the activation kinetics, and the conductance of calcium currents, whereas another α2/δ subunit TAG-180 has no effect. Finally, we characterize mutants of the two β subunits, CCB-1 and CCB-2. CCB-1 is necessary for viability, and voltage-dependent calcium currents are abolished in the absence of CCB-1 whereas CCB-2 does not affect currents. Altogether these results show that EGL-19, UNC-36, and CCB-1 underlie voltage-dependent calcium currents in C. elegans striated muscle.     

Expression and regulation of delta5-desaturase,delta6-desaturase,stearoyl-coenzyme A (CoA) desaturase 1, and stearoyl-CoA desaturase 2 in rat testis

In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and Delta5- and Delta6-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of Delta5- and Delta6-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, Delta5- and Delta6-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. Delta5- and Delta6-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)2cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.     

Conversion of 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one to delta 16-C19 steroids by the reconstituted delta 16-C19-steroid synthetase system

The substrate specificity of the reconstituted delta 16-C19-steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3 beta-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and NADH-cytochrome b5 reductase all from pig testicular microsomes. It was found that 5 alpha-reduced C21 steroids such as 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 alpha-ol and 5 alpha-androst-16-en-3 beta-ol, respectively. The results suggest that 5 alpha-reduced delta 16-C19 steroids might be synthesized from pregnenolone and progesterone via 5 alpha-reduced C21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5 alpha-reduced delta 16-C19 steroids from pregnenolone and progesterone.     

Casein kinase 1 delta (CK1delta) interacts with the SNARE associated protein snapin

In this study we identified snapin as an interaction partner of the CK1 isoform delta (CK1delta) in the yeast two-hybrid system and localized the interacting domains of both proteins. The interaction of CK1delta with snapin was confirmed by co-immunoprecipitation. Snapin was phosphorylated by CK1delta in vitro. Both proteins localized in close proximity in the perinuclear region, wherein snapin was found to associate with membranes of the Golgi apparatus. The identification of snapin as a new substrate of CK1delta points towards a possible function for CK1delta in modulating snapin specific functions.     

Sexually dimorphic and ontogenetic expression of dmrt1, cyp19a1a and cyp19a1b in Gobiocypris rarus

Fish have diverse sex determination and differentiation. DMRT1 and aromatase are conserved in the phyla and play pivotal roles in sex development. Gobiocypris rarus is a small fish used as a model in aquatic toxicology in China and has been used to study the effects of environmental endocrine disruptors on gene expression, but its sexual development remains elusive. Here, we report the full-length cDNA of G. rarus dmrt1 and its expression along with the expression of cyp19a1a and cyp19a1b, two genes encoding gonad and brain type aromatases, in adults and during ontogenesis. Both cyp19a1a and dmrt1 are expressed in the ovary and testis but show sexual dimorphism. Expression of cyp19a1a in the ovary is higher than in testes and dmrt1 follows the opposite pattern. Juvenile gonad histology changes at 15 days after hatching. The dimorphic expression of dmrt1 and cyp19a1a appears from 5 days after hatching, which is earlier than histological change. cyp19a1b is expressed coordinately with cyp19a1a until 15 days after hatching. These results show that dmrt1 and cyp19a1a play important roles in sex determination and sex differentiation in G. rarus.     

The TIPP opioid peptide family: development of delta antagonists, delta agonists, and mixed mu agonist/delta antagonists

The discovery of the prototype delta opioid antagonists TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) in 1992 was followed by extensive structure-activity relationship studies, leading to the development of analogues that are of interest as pharmacological tools or as potential therapeutic agents. Stable TIPP-derived delta opioid antagonists with subnanomolar delta receptor binding affinity and extraordinary delta receptor selectivity include TIPP[Psi] (H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH] and TICP[Psi] (H-Tyr-TicPsi[CH(2)NH]Cha-Phe-OH); Cha: cyclohexylalanine), which are widely used in opioid research. Theoretical conformational analyses in conjunction with the pharmacological characterization of conformationally constrained TIPP analogues led to a definitive model of the receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related delta opioid antagonists, which is characterized by all-trans peptide bonds. Further structure-activity studies revealed that the delta antagonist vs delta agonist behavior of TIP(P)-derived compounds depended on very subtle structural differences in diverse locations of the molecule and suggested a delta receptor model involving a number of different inactive receptor conformations. A further outcome of these studies was the identification of a new class of potent and very selective dipeptide delta agonists of the general formula H-Tyr-Tic-NH-X (X = arylalkyl), which are of interest for drug development because of their low molecular weight and lipophilic character. Most interestingly, TIPP analogues containing a C-terminal carboxamide group displayed a mixed mu agonist/delta antagonist profile, and thus were expected to be analgesics with a low propensity to produce tolerance and physical dependence. This turned out to be the case with the TIPP-derived mu agonist/delta antagonist DIPP-NH(2)[Psi] (H-Dmt-TicPsi[CH(2)NH]Phe-Phe-NH(2)); Dmt: 2',6'- dimethyltyrosine).     

delta 1-Tetrahydrocannabinol and 1 alpha, 2 alpha-epoxyhexahydrocannabinol: mutagenicity investigation in the Ames test.

1,2-Epoxyhexahydrocannabinol is a metabolite of delta 1-tetrahydrocannabinol. Because many epoxides are mutagens, we investigated 1,2-epoxyhexahydrocannabinol as well as delta 1-tetrahydrocannabinol for mutagenicity with Salmonella typhimurium TA1535, TA1537, TA98 and TA100 in the presence and in the absence of S9 mix from liver homogenate of rats treated with Aroclor 1254. Additionally, an epoxide hydratase inhibitor was used in some experiments. Whereas several other epoxides and further positive controls, not requiring activation or activated under the same conditions, respectively, showed strong mutagenicity, no indications of a mutagenic hazard by 1,2-epoxyhexahydrocannabinol or by delta 1-tetrahydrocannabinol were found.     

Analysis of the interacting partners of the neuronal calcium-binding proteins L-CaBP1, hippocalcin, NCS-1 and neurocalcin delta

Intracellular Ca2+ signals are transduced by the binding of Ca2+ to sensor proteins, which subsequently modify the activity of their target proteins. Identification of these target proteins is, therefore, important for an understanding of cellular signalling processes. We have investigated the binding partners of four EF-hand Ca2+-binding proteins. Three proteins of the neuronal calcium sensor (NCS) family, hippocalcin, NCS-1 and neurocalcin delta were prepared as N-terminally tagged GST fusion proteins, and the less closely related protein L-CaBP1 was prepared in both N- and C-terminally tagged forms, the latter requiring generation of a new vector. Immobilised fusion proteins were used to purify binding partners from bovine brain cytosol and membrane extracts in the presence of 1 microM free Ca2+. Bound proteins were eluted with Ca2+-free and high-salt buffers and eluted proteins were identified by MALDI-MS and Western blotting. New protein targets detected included ARF1, Ca2+-dependent activator protein for secretion 1, cyclic nucleotide 3',5'-phosphodiesterase, the vacuolar ATPase, AP1 and AP2 complexes and the type I TGF-beta receptor. While certain of these interactions occurred with more than one of the Ca2+-binding proteins, others were found to be specific targets for particular Ca2+ sensors, and many of these did not overlap with known calmodulin-binding proteins. These findings provide new clues to the functional roles of the neuronal calcium sensor proteins.     

Structural studies of duck delta 1 and delta 2 crystallin suggest conformational changes occur during catalysis

Duck delta1 and delta2 crystallin are 94% identical in amino acid sequence, and while delta2 crystallin is the duck orthologue of argininosuccinate lyase (ASL) and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, the delta1 isoform is enzymatically inactive. The crystal structures of wild type duck delta1 and delta2 crystallin have been solved at 2.2 and 2.3 A resolution, respectively, and the refinement of the turkey delta1 crystallin has been completed. These structures have been compared with two mutant duck delta2 crystallin structures. Conformational changes were observed in two regions of the N-terminal domain with intraspecies differences between the active and inactive isoforms localized to residues 23-32 and both intra- and interspecies differences localized to the loop of residues 74-89. As the residues implicated in the catalytic mechanism of delta2/ASL are all conserved in delta1, the amino acid substitutions in these two regions are hypothesized to be critical for substrate binding. A sulfate anion was found in the active site of duck delta1 crystallin. This anion, which appears to mimic the fumarate moiety of the argininosuccinate substrate, induces a rigid body movement in domain 3 and a conformational change in the loop of residues 280-290, which together would sequester the substrate from the solvent. The duck delta1 crystallin structure suggests that Ser 281, a residue strictly conserved in all members of the superfamily, could be the catalytic acid in the delta2 crystallin/ASL enzymatic mechanism.     

Identification of novel protein kinase CK1 delta (CK1delta) inhibitors through structure-based virtual screening

In eukaryotes, protein phosphorylation of serine, threonine or tyrosine residues by protein kinases plays an important role in many cellular processes. Members of the protein kinase CK1 family usually phosphorylate residues of serine that are close to other phosphoserine in a consensus motif of pS-X-X-S, and they are implicated in the regulation of a variety of physiological processes as well as in pathologies like cancer and Alzheimer's disease. Using a structure-based virtual screening (SBVS) approach we have identified two anthraquinones as novel CK1delta inhibitors. These amino-anthraquinone analogs (derivatives 1 and 2) are among the most potent and selective CK1delta inhibitors known today (IC(50)=0.3 and 0.6 microM, respectively).     

Expression of KCNQ1OT1, CDKN1C,H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine

Background

Beckwith-Wiedemann syndrome (BWS) is a loss-of-imprinting pediatric overgrowth syndrome. The primary features of BWS include macrosomia, macroglossia, and abdominal wall defects. Secondary features that are frequently observed in BWS patients are hypoglycemia, nevus flammeus, polyhydramnios, visceromegaly, hemihyperplasia, cardiac malformations, and difficulty breathing. BWS is speculated to occur primarily as the result of the misregulation of imprinted genes associated with two clusters on chromosome 11p15.5, namely the KvDMR1 and H19/IGF2. A similar overgrowth phenotype is observed in bovine and ovine as a result of embryo culture. In ruminants this syndrome is known as large offspring syndrome (LOS). The phenotypes associated with LOS are increased birth weight, visceromegaly, skeletal defects, hypoglycemia, polyhydramnios, and breathing difficulties. Even though phenotypic similarities exist between the two syndromes, whether the two syndromes are epigenetically similar is unknown. In this study we use control Bos taurus indicus X Bos taurus taurus F1 hybrid bovine concepti to characterize baseline imprinted gene expression and DNA methylation status of imprinted domains known to be misregulated in BWS. This work is intended to be the first step in a series of experiments aimed at determining if LOS will serve as an appropriate animal model to study BWS.

Results

The use of F1 B. t. indicus x B. t. taurus tissues provided us with a tool to unequivocally determine imprinted status of the regions of interest in our study. We found that imprinting is conserved between the bovine and human in imprinted genes known to be associated with BWS. KCNQ1OT1 and PLAGL1 were paternally-expressed while CDKN1C and H19 were maternally-expressed in B. t. indicus x B. t. taurus F1 concepti. We also show that in bovids, differential methylation exists at the KvDMR1 and H19/IGF2 ICRs.

Conclusions

Based on these findings we conclude that the imprinted gene expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 ICRs are conserved between human and bovine. Future work will determine if LOS is associated with misregulation at these imprinted loci, similarly to what has been observed for BWS.     

Blocking of the effect of delta1 tetrahydrocannabinol (delta1THC) on spontaneous motor activity in the rat by immunization with a protein conjugate of delta1THC

Rats actively immunized with porcine gamma globulin- hemisuccinate-Δ¹-tetrahydrocannabinol (PγG-HS-Δ¹THC) showed higher spontaneous motor activity after intraperitoneal administration of Δ¹THC at a dose of 10 mg. per kg. than did rats immunized with a control antigen, porcine gamma globulin-hemisuccinate-phenol (PγG-HS-Phenol). The capacity to neutralize the effect of Δ¹THC was found to depend on the degree of immunization; thus, the difference in mean spontaneous motor activity after injection of Δ¹THC was significant in rats which had received five injections of the immunogen over a period of 86 days, and not in those which had received only two injections over a period of 34 days.In view of the observations that Δ¹-tetrahydrocannabinol induces a decrease in spontaneous motor activity in rats, the observed neutralization of the effect of δ¹THC in animals receiving multiple injections of protein conjugates of Δ¹THC may be due to the binding of the drug by anti-THC antibodies (which are expected to be produced on active immunization with these conjugates), thus preventing Δ¹THC from reaching drug-receptor sites.     

Region-dependent G-protein activation by mu-, delta 1- and delta 2-opioid receptor agonists in the brain: comparison between the midbrain and forebrain

The ability of selective mu- ([D-Ala2, NHPhe4, Gly-ol]enkephalin: DAMGO), delta1- ([D-Pen2, Pen5]enkephalin: DPDPE) and delta2- ([D-Ala2]deltorphin II: DELT II) opioid receptor agonists to activate G-proteins in the midbrain and forebrain of mice and rats was examined by monitoring the binding of guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The levels of [35S]GTPgammaS binding stimulated by DAMGO in the mouse and rat midbrain were significantly greater than those by DPDPE or DELT II. However, relatively lower levels of stimulation of [35S]GTPgammaS binding by all of the agonists than would have been predicted from the receptor densities were observed in either the limbic forebrain or striatum of mice and rats. The effects of DAMGO, DPDPE and DELT II in all three regions were completely reversed by selective mu-, delta1- and delta2-antagonists, respectively. The results indicate that the levels of mu-, delta1- and delta2-opioid receptor agonist-induced G-protein activation in the midbrain are in good agreement with the previously determined distribution densities of each receptor type. Furthermore, the discrepancies observed in the forebrain might reflect differential catalytic efficiencies of receptor-G-protein coupling.