Sharad Kumar

Nedd4-2 Functionally Interacts with ClC-5 INVOLVEMENT IN CONSTITUTIVE ALBUMIN ENDOCYTOSIS IN PROXIMAL TUBULE CELLS

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl– channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 μg/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 μg/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

NEDD4-2 as a potential candidate susceptibility gene for epileptic photosensitivity.

Photosensitive seizures occur most commonly in childhood and adolescence, usually as a manifestation of complex idiopathic generalized epilepsies (IGEs). Molecular mechanisms underlying this condition are yet to be determined because no susceptibility genes have been identified. The NEDD4‐2 (Neuronally Expressed Developmentally Downregulated 4) gene encodes a ubiquitin protein ligase proposed to regulate cell surface levels of several ion channels, receptors and transporters involved in regulating neuronal excitability, including voltage‐gated sodium channels (VGSCs), the most clinically relevant of the epilepsy genes. The regulation of NEDD4‐2 in vivo involves complex interactions with accessory proteins in a cell type specific manner. We screened NEDD4‐2 for mutations in a cohort of 253 families with IGEs. We identified three NEDD4‐2 missense changes in highly conserved residues; S233L, E271A and H515P in families with photosensitive generalized epilepsy. The NEDD4‐2 variants were as effective as wild‐type NEDD4‐2 in downregulating the VGSC subtype Nav1.2 when assessed in the Xenopus oocyte heterologous expression system showing that the direct interaction with the ion channel was not altered by these variants. These data raise the possibility that photosensitive epilepsy may arise from defective interaction of NEDD4‐2 with as yet unidentified accessory or target proteins.

A POXVIRUS BIDIRECTIONAL PROMOTER ELEMENT WITH EARLY/LATE AND LATE FUNCTIONS

A novel bidirectional promoter element of fowlpox virus (FPV) was characterized by transcription analysis, transient expression assays, and recombinant virus construction. This promoter element contained an early/late and a late function in opposite orientation, all within 42 bp of the DNA sequence. The 42-bp sequence was sufficient to express two reporter genes simultaneously in a temporally regulated manner. Both early and late mRNA from the early/late promoter originated at the same TAAAT motif and lacked a long 5poly(A) leader sequence. Late mRNA, initiated from a TAAAT motif of the oppositely oriented late promoter strand, had a leader sequence of approximately 26 bases. Sequence alignment of two strands of the bidirectional element showed that 28 of 42 bases matched. Because of its small and defined size as well as unique structure, this bidirectional promoter should prove to be an important tool in defining the sequences required for the temporal regulation of poxvirus genes.

A defect in DNA topoisomerase II activity in ataxia-telangiectasia cells

DNA topoisomerase type I and II activities were determined by serial dilution in nuclear extracts from control and ataxia-telangiectasia lymphoblastoid cells. Topoisomerase I activity, assayed by relaxation of supercoiled plasmid DNA, was found to be approximately the same in both cell types. In order to remove interference from topoisomerase I, the activity of topoisomerase II was measured by the unknotting of knotted P4 phage DNA in the presence of ATP. The activity of topoisomerase II was markedly reduced in two ataxia-telangiectasia cell lines, AT2ABR and AT8ABR, compared to controls. This reduction in activity was detected with increasing concentration of protein and in time course experiments at a single protein concentration. A third cell line, AT3ABR, did not have a detectably lower activity of topoisomerase II when assayed under these conditions. The difference in topoisomerase II activity in the ataxia-telangiectasia cell lines examined may reflect to some extent the heterogeneity observed in this syndrome.

Activity of a fowlpox virus late gene promoter in vaccinia and fowlpox virus recombinants

SummaryCharacterization of a late promoter of fowlpox virus (FPV) and a study of its activity in FPV and vaccinia virus (VV) was carried out. The 5′-mRNA start site of the FPV late gene mapped to a TAAAT sequence near the translation start site (ATG). A cloned DNA fragment of FPV genome (PFL1) comprising of the 5′-end of the late gene was used to express the LacZ gene ofE. coli in FPV and VV recombinants. A comparative analysis of β-galactosidase (BG) expression from the LacZ gene under the control of the FPV promoter and a VV late promoter (PL11) was performed. Like FPV-PL11-LacZ and VV-PL11-LacZ constructs, FPV-PFL1-LacZ and VV-PFL1-LacZ virus recombinants expressed BG indicating that essential features of transcription were conserved in the two viruses. Furthermore, the LacZ transcripts originating from PFL1 in FPV and VV recombinants mapped to the expected TAAAT sequence. Time course analysis of BG expressed by VV and FPV recombinants suggested that although the transcription machinery in the two viruses was essentially conserved, subtle differences in the efficiency of transcription or translation may exist.

Isolation of DNA and RNA from ascidians

A procedure is described for the extraction of DNA and RNA from solitary and colonial ascidians obtained on the east coast of Australia. Modifications to more established procedures have been made to avoid nuclease digestion, binding to polyphenolic compounds and other less well characterized pigments that interfere with the use of nucleic acids for restriction-enzyme analysis and gene-cloning. Tissue is homogenized in liquid nitrogen to produce a powder which is washed three times in 0.25 M ammonium acetate, pH 5.0, to remove a variety of contaminants. This is followed by lysis and immediate extraction in a number of different phenol/CHCl3/isoamyl alcohol mixtures. Additional steps are similar to those described in previous methods. Extraction of RNA by this procedure precludes the necessity for addition of ribonuclease inhibitors.

Coupling of histone mRNA levels to radioresistant DNA synthesis in ataxia-telangiectasia cells

Cloned genomic DNA for human histone H1, H3 and H4 genes has been used to determine the effects of γ-radiation on histone mRNA levels and synthesis in ataxia-telangiectasia cells. Synthesis of histone mRNA was determined in cells synchronized with aphidicolin. Effects of irradiation on DNA synthesis and passage through S phase were also monitored. Irradiation was found to slow the passage of control cells through the cell cycle but had no effect on progression of ataxia-telangiectasia cells. H1 and core histone mRNA synthesis was inhibited by radiation in two control cell lines after release from aphidicolin block. No inhibition was observed in one ataxia-telangiectasia cell line and a small degree of inhibition in a second. An increased level of mRNA was observed in both irradiated control and ataxia-telangiectasia cells at 5–7 h post-irradiation compared to unirradiated cells. Similar results were obtained in log phase cells. These results demonstrate that histone mRNA synthesis is radioresistant in ataxia-telangiectasia cells and is coupled to radioresistant DNA synthesis in these cells.

Rearrangements of t-cell receptor β-chain genes in human leukaemias

Abstract Rearrangements of the T-cell receptor (TCR), β-chain genes and immunoglobulin (Ig) heavy chain genes in several T-cell leukaemias (T-ALL and ATL), and some B-cell and myelogenous leukaemias were investigated. Two out of 15 cases of T-cell leukaemia tested failed to show a rearrangement pattern of TCR β genes although both expressed mRNA for this gene. The remaining 13 cases showed diverse patterns of rearrangements involving either C β 1 , C β 2 or both. C β 1 , but not C β 2 was deleted in s the T-cell leukaemias. Polyclonal T cells from four normal individuals showed the germ line pattern and an additional two bands in Hind III digested DNA. Except for one, all cases of C-ALL (B-cell leukaemia) showed a rearranged J H locus which was not evident in any of T-cell leukaemias studied. One case of B-cell leukaemia showed a rearrangement of both TCR β genes and J H genes. The results of these studies suggest that rearrangement of TCR and Ig genes occurs at a very early stage of differentiation of stem cells and does not appear to play a direct role in leukaemogenesis per se .

Study of chromatin structure in ataxia-telangiectasia cells

Micrococcal nuclease was used as a probe to study chromatin structure in control and ataxia-telangiectasia cells. The rate and extent of release of acid-soluble nucleotide was similar in both cell types. Production of mono- and oligonucleosomes by micrococcal nuclease as determined by gel electrophoresis also failed to reveal differences in chromatin structure between control and ataxia-telangiectasia cells. Radiation exposure did not significantly alter the kinetics of digestion. These results indicate that there are no gross alterations in chromatin structure in ataxia-telangiectasia cells.