Regulation of casein kinase-2 (CK2) activity by inositol phosphates

Casein kinase 2 (CK2) was one of the first protein kinases to be discovered and has been suggested to be responsible for as much as one-fifth of the eukaryotic phosphoproteome. Despite being responsible for the phosphorylation of a vast array of proteins central to numerous dynamic cellular processes, the activity of CK2 appears to be unregulated. In the current study, we identified a protein kinase activity in rat liver supernatant that is up-regulated by inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6). The substrate for the inositol phosphate-regulated protein kinase was identified as a phosphatidylcholine transfer protein-like protein. Using the phosphorylation of this substrate in an assay, we purified the inositol phosphate-regulated protein kinase and determined it to be CK2. Bacterially expressed recombinant CK2, however, showed very high basal activity and was only modestly activated by IP6 and not regulated by IP. We found that an endogenous component present in rat liver supernatant was able to inhibit both recombinant and liver-purified CK2 basal activity. Under these conditions, recombinant CK2 catalytic activity could be increased substantially by IP4, inositol 1,3,4,5,6-pentakisphosphate (IP5), and IP6. We concluded that, contrary to the previously held view, CK2 can exist in a state of low constitutive activity allowing for its regulation by inositol phosphates. The ability of the higher inositol phosphates to directly stimulate CK2 catalytic activity provides the first evidence that these signaling molecules can operate via a direct control of protein phosphorylation.     

Signal transduction and transforming properties of the TEL-TRKC fusions associated with t(12;15)(p13;q25) in congenital fibrosarcoma and acute myelogenous leukemia

The TEL-TRKC fusion is expressed as a consequence of t(12;15)(p13;q25), and is associated with two human cancers: congenital fibrosarcoma and acute myelogenous leukemia (AML). We report that the T/T(F) and T/T(L) fusion variants associated with congenital fibrosarcoma and AML, respectively, are constitutively tyrosine phosphorylated, and confer factor-independent growth to the murine hematopoietic cell line Ba/F3. Retroviral transduction of T/T(L) causes a rapidly fatal myeloproliferative disease in a murine bone marrow transplant (BMT) model, whereas T/T(F) causes a long-latency, pre-B-cell lymphoblastic lymphoma. TEL-TRKC variants are potent activators of the MAP kinase pathway, but neither variant activates Stat5 or other Stat family members. T/T(L), but not T/T(F), induces tyrosine phosphorylation of phospholipase Cgamma (PLCgamma), phosphoinositol-3 kinase and SHC. However, mutation analysis demonstrates that PLCgamma tyrosine phos phorylation by T/T(L) is dispensable for induction of the myeloproliferative phenotype by T/T(L). Collectively, these data demonstrate that the TEL-TRKC fusion variants are oncoproteins that activate the MAP kinase pathway, and do not require activation of either PLCgamma or Stat5 for efficient induction of a myeloproliferative phenotype in the murine BMT model.     

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.     

Synthesis and properties of uniquely modified oligoribonucleotides: yeast tRNA(Phe) fragments with 6-methyluridine and 5,6-dimethyluridine at site-specific positions

The phosphoramidites of 6-methyluridine and 5,6-dimethyluridine were synthesized and the modified uridines site-selectively incorporated into heptadecamers corresponding in sequence to the yeast tRNA(Phe) anticodon and TpsiC domains. The oligoribonucleotides were characterized by NMR, MALDI-TOF MS and UV-monitored thermal denaturations. The 6-methylated uridines retained the syn conformation at the polymer level and in each sequence location destabilized the RNAs compared to that of the unmodified RNA. The decrease in RNA duplex stability is predictable. However, loss of stability when the modified uridine is in a loop is sequence context dependent, and can not, at this time, be predicted from the location in the loop.     

The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74).

The substantial variations in the responses of cells to the anaphylatoxin C5a and its desarginated form, C5adR(74), suggest that more than one type of cell surface receptor for these ligands might exist. However, only a single receptor for C5a and C5adR(74), CD88, has been characterized to date. Here we report that the orphan receptor C5L2/gpr77, which shares 35% amino acid identity with CD88, binds C5a with high affinity but has a 10-fold higher affinity for C5adR(74) than CD88. C5L2 also has a moderate affinity for anaphylatoxin C3a, but cross-competition studies suggest that C3a binds to a distinct site from C5a. C4a was able to displace C3a, suggesting that C5L2, like the C3a receptor, may have a low binding affinity for this anaphylatoxin. Unlike CD88 and C3a receptor, C5L2 transfected into RBL-2H3 cells does not support degranulation or increases in intracellular [Ca(2+)] and is not rapidly internalized in response to ligand binding. However, ligation of C5L2 by anaphylatoxin did potentiate the degranulation response to cross-linkage of the high affinity IgE receptor by a pertussis toxin-sensitive mechanism. These results suggest that C5L2 is an anaphylatoxin-binding protein with unique ligand binding and signaling properties.     

Imidazo[4,5-b]pyridines as corticotropin releasing factor receptor ligands

A series of high affinity CRF receptor ligands with an imidazo[4,5-b]pyridine core is described. Individual analogues were synthesized and tested in a rat CRF receptor binding assay. The best compounds were further tested in the dog N-in-1 pharmacokinetic model to assess plasma levels at 1mg/kg (po) and in the rat situational anxiety model to assess anxiolytic efficacy at 3mg/kg (po). The structure-activity relationships for good receptor binding affinity are described herein.     

Comparison of the expression-linked extra copy (ELC) and basic copy (BC) genes of a trypanosome surface antigen

A recombinant clone of an expression-linked extra copy (ELC) gene of a trypanosome-variable surface glycoprotein was sequenced. In addition the sequences of the corresponding cDNA and portions of the two basic copy genes were determined. Comparison of these sequences reveals that the 5' boundary of the ELC-transposed segment (2.2 kb) occurs within a repetitive sequence about 700 bp upstream from the start codon of the coding sequence. This sequence does not contain internal symmetries and is not homologous with the repetitive sequence at the 3' boundary. The first 35 nucleotides of the cDNA are different than the corresponding ELC sequence and presumably were transcribed from another genomic location. A restriction fragment containing predominantly sequences outside of the 5' boundary hybridizes to a Pst I fragment whose length is variable in different trypanosome clones. This hybridization pattern is similar to that observed using probes for surface glycoprotein genes that are expressed via the nonduplication-associated (NDA) mechanism rather than the ELC mechanism. This indicates that there is a sequence correlation between these two DNA rearrangement mechanism.     

Structural and functional roles of the N1- and N3-protons of psi at tRNA's position 39.

Pseudouridine at position 39 (Psi(39)) of tRNA's anticodon stem and loop domain (ASL) is highly conserved. To determine the physicochemical contributions of Psi(39)to the ASL and to relate these properties to tRNA function in translation, we synthesized the unmodified yeast tRNA(Phe)ASL and ASLs with various derivatives of U(39)and Psi(39). Psi(39)increased the thermal stability of the ASL (Delta T (m)= 1.3 +/- 0.5 degrees C), but did not significantly affect ribosomal binding ( K (d)= 229 +/- 29 nM) compared to that of the unmodified ASL (K (d)= 197 +/- 58 nM). The ASL-Psi(39)P-site fingerprint on the 30S ribosomal subunit was similar to that of the unmodified ASL. The stability, ribosome binding and fingerprint of the ASL with m(1)Psi(39)were comparable to that of the ASL with Psi(39). Thus, the contribution of Psi(39)to ASL stability is not related to N1-H hydrogen bonding, but probably is due to the nucleoside's ability to improve base stacking compared to U. In contrast, substitutions of m(3)Psi(39), the isosteric m(3)U(39)and m(1)m(3)Psi(39)destabilized the ASL by disrupting the A(31)-U(39)base pair in the stem, as confirmed by NMR. N3-methylations of both U and Psi dramatically decreased ribosomal binding ( K (d)= 1060 +/- 189 to 1283 +/- 258 nM). Thus, canonical base pairing of Psi(39)to A(31)through N3-H is important to structure, stability and ribosome binding, whereas the increased stability and the N1-proton afforded by modification of U(39)to Psi(39)may have biological roles other than tRNA's binding to the ribosomal P-site.     

Clinical features of symptomatic patellofemoral joint osteoarthritis

Introduction

Patellofemoral joint osteoarthritis (OA) is common and leads to pain and disability. However, current classification criteria do not distinguish between patellofemoral and tibiofemoral joint OA. The objective of this study was to provide empirical evidence of the clinical features of patellofemoral joint OA (PFJOA) and to explore the potential for making a confident clinical diagnosis in the community setting.

Methods

This was a population-based cross-sectional study of 745 adults aged ≥50 years with knee pain. Information on risk factors and clinical signs and symptoms was gathered by a self-complete questionnaire, and standardised clinical interview and examination. Three radiographic views of the knee were obtained (weight-bearing semi-flexed posteroanterior, supine skyline and lateral) and individuals were classified into four subsets (no radiographic OA, isolated PFJOA, isolated tibiofemoral joint OA, combined patellofemoral/tibiofemoral joint OA) according to two different cut-offs: ''any OA'' and ''moderate to severe OA''. A series of binary logistic and multinomial regression functions were performed to compare the clinical features of each subset and their ability in combination to discriminate PFJOA from other subsets.

Results

Distinctive clinical features of moderate to severe isolated PFJOA included a history of dramatic swelling, valgus deformity, markedly reduced quadriceps strength, and pain on patellofemoral joint compression. Mild isolated PFJOA was barely distinguished from no radiographic OA (AUC 0.71, 95% CI 0.66, 0.76) with only difficulty descending stairs and coarse crepitus marginally informative over age, sex and body mass index. Other cardinal signs of knee OA - the presence of effusion, bony enlargement, reduced flexion range of movement, mediolateral instability and varus deformity - were indicators of tibiofemoral joint OA.

Conclusions

Early isolated PFJOA is clinically manifest in symptoms and self-reported functional limitation but has fewer clear clinical signs. More advanced disease is indicated by a small number of simple-to-assess signs and the relative absence of classic signs of knee OA, which are predominantly manifestations of tibiofemoral joint OA. Confident diagnosis of even more advanced PFJOA may be limited in the community setting.