BACKGROUND Cyclosporine (CsA) use has been associated to the development of cholelithiasis in transplant recipients. We herein explored the role of time under CsA on this association in asymptomatic adult kidney transplant recipients (KTR). METHODS A cross-sectional study was conducted in 140 KTR with variable post-transplant follow-up (PTFU), and without history of symptomatic biliary disease. Upper abdominal ultrasound was performed in all patients. According to the immunosuppressive schedule, patients were classified in three groups: Azathioprine + prednisone (group 1, n = 37), azathioprine + prednisone < 24 months CsA (group 2, n = 58), or azathioprine + prednisone > or = 24 months CsA (group 3, n = 45). Age at time of ultrasound performance, gender, PTFU, chronic viral liver disease, parity, oral contraceptives, serum lipids, diabetes and body mass index were analyzed concomitantly. RESULTS Median age was 38, 31, and 36 years in groups 1, 2, and 3, respectively. The male:female ratio in the same groups was 1.5:1, 1:1, and 2:1. Mean PTFU was 130, 48, and 53 months, respectively (p = 0.0001). Gallstones were found in three (8%) group 1 KTR, in nine (16%) group 2 KTR, and in 10 (22%) group 3 KTR (p = 0.214). Adjusting for PTFU, the association between length of CsA and prevalence of lithiasis was significantly stronger among those with longer use of CsA (odds ratio = 6.1, p = 0.046). No significant differences were found among groups in the other variables. CONCLUSIONS KTR receiving CsA for more than two years show increased prevalence of gallstones.

The following amino acids of the Xenopus laevis beta subunit of protein kinase CK2 (casein kinase 2) were changed to alanine: Pro-58 (beta P-->A); Asp-59 and Glu-60 and Glu-61 (beta DEE-->AAA); His-151-153 (beta HHH-->AAA). The last 37 amino acids of the carboxyl end were deleted (beta delta 179-215). Stimulation of CK2 alpha catalytic subunit activity was measured with casein as substrate and the following relative activities were observed: beta P-->A > beta DEE-->AAA >>> beta WT > beta HHH-->AAA >>> beta delta 179-215. The beta DEE-->AAA and beta P-->A were similar to beta WT in reducing CD2 alpha binding to DNA but beta delta 179-215 was less active. The results indicate that both Pro-58 and the surrounding acidic cluster play roles in dampening the activation of CK2 alpha and that the carboxyl end of beta is involved in the interaction with CK2 alpha.

Northern-blot analysis of RNAs from different tissues demonstrated that the mRNA for the protein kinase CK2 alpha subunit is very abundant in the ovary of Xenopus laevis. The competitive reverse-PCR technique has been used to quantitate the mRNA for both CK2 alpha and CK2 beta subunits during oogenesis. The results obtained using eight different animals consistently show an increment of 2-3-fold in the mRNA for both subunits in vitellogenic oocytes (stages II-VI). Each stage-VI oocyte contains approximately 5 x 10(-7) molecules CK2 alpha mRNA and 1 x 10(-7) molecules CK2 beta mRNA. These amounts are considerably higher than many other mRNAs analyzed in these cells. Activity measurements of CK2 using casein or a specific model peptide revealed increments of about 10-12-fold during oogenesis, and also indicated that the amount of enzyme in the nucleus accounted for 15-30% of the total enzyme in the oocyte at all stages. Western-blot analysis of CK2 alpha indicated that the amount of this protein subunit also increased during oogenesis in a parallel fashion with the increment of enzymic activity.

The recombinant alpha subunit of protein kinase CK2 (casein kinase 2) from Xenopus laevis is inhibited by the addition of single stranded or double stranded DNA. This inhibition is competitive with the casein substrate, having an apparent Ki of 160 nM for an 86 bp DNA fragment. Assays with a fragment containing the putative promoter of the human CK2 beta gene indicated that the affinity of CK2 for this fragment was not greater than that of other unrelated DNA. The inhibitory capacity of DNA toward the protein phosphorylating activity of CK2 alpha is greatly reduced by the presence of the beta subunit which can completely reverse the inhibition, The interaction of CK2 alpha with DNA can also be assayed by the nitrocellulose filter binding assay. This assay demonstrates that the interaction of CK2 alpha with the tested DNAs is not sequence specific and that the beta subunit can also greatly diminish the binding of CK2 alpha to DNA. Casein at substrate concentrations also is inhibitory to CK2 alpha DNA binding, Likewise, polyanionic inhibitors of the CK2 catalytic activity, such as heparin, poly(U), and copoly(alu:Tyr) polypeptides, can compete for and inhibit the binding of DNA to CK2 alpha. However, quercetin, which also inhibits CK2 phosphorylation activity, and ATP do not affect DNA binding. A mutant CK2 alpha in which glutamic acids replace two lysine residues in positions 75 and 76 of the alpha peptide chain is less susceptible to DNA inhibition, indicating that this basic region of the molecule is involved in its interaction with DNA.

The cDNA gene coding for the α subunit of Xenopus laevis casein kinase II was mutated using the overlap extension PCR method. The mutation substituted glutamic acids for Lys 75 and Lys 76 , changing the charge distribution of a very basic sequence found in the α subunit. Expression of the mutated cDNA in a pT7‐7 vector in E. coli yielded an active mutant recombinant protein that was extensively purified. This mutant was not significantly affected in its app. K m for casein or a model peptide substrate, nor in its interaction with the activating β subunit. Inhibition by quercetin and by 5,6‐dichloro‐1‐β‐ d ‐ribofuranosyl benzimidazole was also the same for mutant and wild type subunits. However, the CKII αE 75 E 76 mutant was at least one order of magnitude less sensitive to inhibition by polyanionic inhibitors such as heparin, poly U, copolyglutamic acid:tyrosine (4:1) and 2,3 diphosphoglycerate.

Casein kinase II purified from the nuclei of Xenopus laevis oocytes as well as the recombinant alpha and beta subunits of the X. laevis CKII, produced in E. coli from the cloned cDNA genes, were tested with different divalent metal ions. The enzyme from both sources was active with either Mg2+, Mn2+, or Co2+. Optimal concentrations were 7-10 mM for Mg2+, 0.5-0.7 mM for Mn2+ and 1-2 mM for Co2+. In the presence of Mn2+ or Co2+ the enzyme used GTP more efficiently than ATP as a phosphate donor while the reverse was true in the presence of Mg2+. The apparent Km values for both nucleotide triphosphates were greatly decreased in the presence of Mn2+ as compared with Mg2+. Addition of Zn2+ (above 150 microM) to an assay containing the optimal Mg2+ ion concentration caused strong inhibition of both holoenzyme and alpha subunit. Inhibition of the holoenzyme by 400 microM Ni2+ could be reversed by high concentrations of Mg2+ but no reversal of this inhibition was observed with the alpha subunit.

Casein kinase II (CKII) is a ubiquitous protein kinase, found predominantly in cell nuclei, which has two subunits in a tetrameric alpha 2 beta 2 or alpha alpha' beta 2 conformation. The catalytic center is present in the alpha subunit which is active by itself while beta is a regulatory subunit that can greatly enhance the activity of alpha. The cDNA genes of Xenopus laevis coding for the alpha and beta subunits of CKII have been expressed in Escherichia coli and extensively purified. The recombinant subunits reconstitute a fully active holoenzyme when incubated in stoichiometric amounts. Mutations that change serines in positions 2 and 3 of the beta subunit for glycines completely eliminate the autophosphorylation site present in this subunit but do not significantly affect the capacity of beta to activate alpha. A fusion protein composed of glutathione transferase linked to the X. laevis CKII beta subunit can also activate alpha. This fusion protein binds to glutathione-agarose beads and can mediate the binding of the alpha subunit to this matrix. Conversely, the alpha subunit was found to bind to glass fiber filters in an active form that can still be activated by beta to an extent similar to that seen in solution. Using peptides containing tyrosine and glutamic acid as inhibitors of the activity of the isolated alpha subunit and of the holoenzyme, the effect of beta on the specificity of inhibition was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

The level of blood lipids in children with Down's syndrome was determined with the purpose of establishing possible differences in total cholesterol, triglyceride and HDL-cholesterol levels with those of healthy children. LDL-cholesterol fraction was calculated. Blood samples were obtained from 66 healthy children (controls) and 72 patients who suffered from clinically diagnosed Down's syndrome. All the children were grouped according to age. The variables of body weight, height, and blood lipids gave a distribution of values that allows one to distinguish the group of children with Down's syndrome from the normal group. The values obtained for triglycerides, total cholesterol, and LDL-cholesterol ranged higher, with a constant deficit of HDL-cholesterol in all age groups. The lipid pattern encountered in the Down's syndrome patient suggests the existence of unknown, possibly genetically determined mechanisms, that provoke a disorder in lipid metabolism.

Casein kinase II purified from nuclei of Xenopus laevis oocytes is inhibited by several specific nucleic acids. This kinase, the main phosphorylating activity of the oocyte nucleus, is markedly inhibited by poly U at 10 μg/ml, and this polymer is a competitive inhibitor of the phosporylation of the substrate casein (Kiapp 80 nM). M 13 phage ssDNA and unfractionated yeast tRNA also inhibit between 50 and 200 μg/ml. Poly C, poly A, poly AG, dsDNA and Escherichia coli rRNA do not alter activity significantly at similar concentrations. Inhibitions are reversed by RNase (poly U, tRNA) or S1 nuclease (ssDNA). Oocyte casein kinase I or rabbit cAMP‐dependent protein kinase are not inhibited by poly U at 200 μg/ml. The sensitivity of the casein kinase II to these inhibitors suggests a regulatory role for nucleic acids in nuclear phosphorylation reactions.

Polylysine-containing peptides are found to affect membrane protein kinases, phosphatidylinositol kinases, and adenylate cyclase. Poly(L-lysine), poly(D-lysine), random copolymers of lysine and serine or lysine and alanine, and poly(L-ornithine) produced large increases in the in vitro phosphorylation of some membrane proteins present in Xenopus laevis oocyte membranes. Poly(L-arginine) did not cause a similar stimulation. In these membranes the phosphorylation of polydisperse protein of approximately 25 kDa was also greatly increased by 1 mM spermine and spermidine, by 10 microM histone H1, or by 200 microM peptide containing the 14-residue sequence at the carboxyl terminus of the human c-Ki-ras 2 gene product, which has eight lysines. Similar specific stimulation of protein phosphorylation was observed with membranes of NG-108-15 nerve cells in culture. Polylysine peptides, including the c-Ki-ras 2 segment, also stimulate the in vitro phosphorylation of membrane inositolphospholipids, to produce mainly phosphatidylinositol 4-phosphate and less phosphatidylinositol 4,5-bisphosphate. Polylysine also alters the activity of oocyte adenylate cyclase, assayed in the presence of either F- or 5'-guanylyl imidodiphosphate.