Metabolic labeling experiments followed by immunoprecipitation were performed to investigate the kinetics, location and inhibitor sensitivity of degradation of both wild‐type (wt) and mutant (delta F508) cystic fibrosis conductance transmembrane regulator (CFTR). At the earliest stages of the biosynthetic process, both wt and delta F508 CFTR were found to be susceptible to degradation by endogenous proteases. Virtually all delta F508 CFTR and 45‐80% of wt CFTR were rapidly degraded with a similar half‐life (t1/2 approximately 0.5 h). The remaining wt CFTR attained a protease‐resistant configuration regardless of whether traffic between the endoplasmic reticulum (ER) and Golgi was operational. Metabolic energy is required for the conformational transition, but not to maintain the stability of the protease‐resistant wt CFTR. Intracellular degradation of delta F508 CFTR and of incompletely folded wt CFTR occurs in a non‐lysosomal, pre‐Golgi compartment, as indicated by the sensitivity of proteolysis to different inhibitors and temperature. Accordingly, products of the degradation of delta F508 CFTR could be detected by immunoblotting in isolated ER, but not in the Golgi. Together, these results suggest a dynamic equilibrium between two forms of wt CFTR in the ER: an incompletely folded, protease‐sensitive form which is partially converted by an ATP‐dependent process to a more mature form that is protease‐resistant and capable of leaving the ER. The inability delta F508 CFTR to undergo such a transition renders it susceptible to complete and rapid degradation in a pre‐Golgi compartment.

Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important in B-lymphocyte development, differentiation, and signaling. Btk is a member of the Tec family of kinases. Mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signaling events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor-kappaB (NF-kappaB) and nuclear factor of activated T cells (NFAT). In B cells, NF-kappaB was shown to bind to the Btk promoter and induce transcription, whereas the B-cell receptor-dependent NF-kappaB signaling pathway requires functional Btk. Moreover, Btk activation is tightly regulated by a plethora of other signaling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin-1. For example, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and steady state levels of Btk. It is intriguing that PKC and Pin1, both of which are negative regulators, bind to the pleckstrin homology domain of Btk. To this end, we describe here novel mutations in the pleckstrin homology domain investigated for their transforming capacity. In particular, we show that the mutant D43R behaves similar to E41K, already known to possess such activity.

Transcript accumulation for the psbA, psbD, psbD-C, rbcL-S and rrn genes in Synechocystis 6803 was followed under different light conditions. psbA, psbD, psbD-C and rbcL-S transcripts required light to accumulate and the relative abundance of these transcripts differed between high and low light conditions. Under high light conditions, steady-state levels of psbA, psbD and psbD-C transcripts were higher while levels of rbcL-S transcripts were lower than under low light conditions. rrn transcripts accumulated in the dark and the transcript levels were the same under illuminated conditions. Analyses of constructed Synechocystis 6803 mutants showed that both psbA-2 and psbA-3 could produce high levels of transcripts under illuminated conditions. No psbA-1 transcripts were detected.

Cytoplasmic protein-tyrosine kinases (PTKs) are enzymes involved in transducing a vast number of signals in metazoans. The importance of the Tec family of kinases was immediately recognized when, in 1993, mutations in the gene encoding Bruton's tyrosine kinase (Btk) were reported to cause the human disease X-linked agammaglobulinemia (XLA). Since then, additional kinases belonging to this family have been isolated, and the availability of full genome sequences allows identification of all members in selected species enabling phylogenetic considerations. Tec kinases are endowed with Pleckstrin homology (PH) and Tec homology (TH) domains and are involved in diverse biological processes related to the control of survival and differentiation fate. Membrane translocation resulting in the activation of Tec kinases with subsequent Ca2+ release seems to be a general feature. However, nuclear translocation may also be of importance. The purpose of this essay is to characterize members of the Tec family and discuss their involvement in signaling. The three-dimensional structure, expression pattern and evolutionary aspects will also be considered.

We have combined a peptide nucleic acid (PNA) with the SV40 core nuclear localization signal (NLS), to create a bifunctional PNA-NLS peptide. The PNA-NLS peptide increased the nuclear uptake of oligonucleotides and enhanced the transfection efficacy of plasmids. Gene expression from an enhanced green fluorescent protein plasmid and a lacZ plasmid was preserved when hybridized to PNA-NLS. In combination with the transfection agent polyethyleneimine, we have improved both the nuclear translocation of fluorescence-marked oligonucleotides, and the efficacy of plasmid transfection, up to eightfold. The technique obviates the use of cumbersome coupling procedures of the vector due to DNA-PNA duplex formation or displacement of the antisense plasmid DNA strand by a PNA molecule.

Bruton's tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B-lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full-length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype-phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering-RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA-, Xid-, and Btk-knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid- and Btk-KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C-missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.

We demonstrate that fusion proteins consisting of the her-COS cells transfected with tk alone. Tumours established pes simplex virus (HSV) transport protein VP22 linked in in mice with neuroblastoma cell lines expressing VP22-tk frame to HSV thymidine kinase (tk) retain the ability to be regressed upon administration of ganciclovir. Furthermore transported between cells. In vivo radiolabelling experitumours established from 50:50 mixtures of VP22-tk transments and in vitro assays show that the fusion proteins duced and nontransduced cells also regressed while no also retain tk activity. When transfected COS cells, acting significant effect was observed in similar experiments with as a source of the VP22-tk chimera, were co-plated on to cells transduced with tk alone. VP22 mediated transport gap junction-negative neuroblastoma cells, ganciclovir may thus have application in a clinical setting to amplify treatment induced efficient cell death in the recipient neurodelivery of the target protein in enzyme-prodrug protocols. blastoma cell monolayer. No such effect was observed with

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