Acute renal allograft rejection following pegylated IFN-alpha treatment for chronic HCV in a repeat allograft recipient on hemodialysis: a case report.

Interferon alpha (IFN-alpha) can be effective therapy for patients with chronic kidney disease who have chronic hepatitis C (HCV). However, acute allograft rejection has been reported in association with IFN-alpha following kidney transplantation, and therefore IFN therapy is recommended prior to, rather than after, kidney transplantation whenever feasible. The special case of repeat allograft recipients who contract HCV after the first transplantation presents special difficulties. This report features the case of a repeat allograft recipient who presented with neutropenic fevers after 5 months of pegylated IFN-alpha therapy, initiated 6 months following the functional loss of his third graft and the reinitiation of hemodialysis (HD). Physical exam, radiographic and laboratory findings led to allograft nephrectomy. The pathologic findings supported a diagnosis of acute-on-chronic rejection. This represents a rare case of IFN-alpha induced rejection following allograft failure and return to HD in a repeat allograft recipient. It also calls attention to the need for a high index of suspicion for the development of allograft rejection, which may require allograft nephrectomy even after allograft 'failure'.

Array comparative genomic hybridisation analysis of boys with X linked hypopituitarism identifies a 3.9 Mb duplicated critical region at Xq27 containing SOX3.

INTRODUCTION: Array comparative genomic hybridisation (array CGH) is a powerful method that detects alteration of gene copy number with greater resolution and efficiency than traditional methods. However, its ability to detect disease causing duplications in constitutional genomic DNA has not been shown. We developed an array CGH assay for X linked hypopituitarism, which is associated with duplication of Xq26-q27. METHODS: We generated custom BAC/PAC arrays that spanned the 7.3 Mb critical region at Xq26.1-q27.3, and used them to search for duplications in three previously uncharacterised families with X linked hypopituitarism. RESULTS: Validation experiments clearly identified Xq26-q27 duplications that we had previously mapped by fluorescence in situ hybridisation. Array CGH analysis of novel XH families identified three different Xq26-q27 duplications, which together refine the critical region to a 3.9 Mb interval at Xq27.2-q27.3. Expression analysis of six orthologous mouse genes from this region revealed that the transcription factor Sox3 is expressed at 11.5 and 12.5 days after conception in the infundibulum of the developing pituitary and the presumptive hypothalamus. DISCUSSION: Array CGH is a robust and sensitive method for identifying X chromosome duplications. The existence of different, overlapping Xq duplications in five kindreds indicates that X linked hypopituitarism is caused by increased gene dosage. Interestingly, all X linked hypopituitarism duplications contain SOX3. As mutation of this gene in human beings and mice results in hypopituitarism, we hypothesise that increased dosage of Sox3 causes perturbation of pituitary and hypothalamic development and may be the causative mechanism for X linked hypopituitarism.

GRKO mice express an aberrant dexamethasone-binding glucocorticoid receptor, but are profoundly glucocorticoid resistant.

The introduction of a targeted insertion mutation into exon 2 of the gene coding for the glucocorticoid receptor (GR) enabled production of glucocorticoid receptor knock-out (GRKO) mice. GRKO mice on a C57BL/6/129sv mixed genetic background show a variable phenotype, with 90% of -/- mice dying at birth with respiratory insufficiency but 10% of mutant mice surviving to maturity. To investigate the possibility of residual GR expression in surviving GRKO mice we have measured binding of the synthetic glucocorticoid dexamethasone in tissue extracts from adrenalectomized mice. High affinity binding of dexamethasone in protein extracts of liver, kidney, lung and brain from adult GRKO mice is found at levels 30-60% those in wild-type mice, with heterozygotes (+/-) having intermediate levels. PCR and ribonuclease protection analysis showed comparable levels of GR mRNA on the 3' side of the gene-targeted insertional mutation in exon 2 of the GR gene, with almost no GR mRNA detected from exons 1 and 2 on the 5' side of the gene-targeted insertional mutation. Western blot analysis using a C-terminal specific GR antibody detects a 39 kDa GR fragment in extracts from adult GRKO mice. Despite the evidence for expression of a ligand-binding domain fragment of the glucocorticoid receptor these mice are profoundly glucocorticoid resistant, with elevated levels of plasma ACTH and corticosterone. Thymocytes from adult and fetal GRKO mice are resistant to dexamethasone-induced apoptosis and cultured fetal hepatocytes from GRKO mice are completely refractory to glucocorticoid induction of the gluconeogenic enzyme glucose-6-phosphatase. Thus although the surviving adult homozygous GRKO mice express a dexamethasone-binding GR fragment, their classic target tissues remain profoundly glucocorticoid insensitive.

Pk92B: a Drosophila melanogaster protein kinase that belongs to the MEKK family.

A Drosophila melanogaster cDNA, encoding a protein with sequence similarity to the MEKK family of Ser/Thr kinases, was isolated from an eye-antennal imaginal disc cDNA library using a PCR-based approach. The deduced protein, Pk92B, has a kinase domain that is 40-48% identical to MEKK family members. The Pk92B gene was mapped to 92B8-10 on the third chromosome by in situ hybridization to polytene chromosomes.

Protein phosphatase 2A positively and negatively regulates Ras1-mediated photoreceptor development in Drosophila.

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase present in most tissues and cell types, has been implicated in the regulation of cell cycle progression, DNA replication, transcription, and translation. Here we present genetic evidence suggesting that PP2A functions downstream of Ras1 in the Sevenless receptor tyrosine kinase (RTK) signal transduction pathway that specifies R7 photoreceptor cell fate in the developing Drosophila eye. Ras1 and downstream cytoplasmic kinases, Raf, MEK, and MAPK, comprise an evolutionarily conserved cascade that mediates the transmission of signals from RTKs at the plasma membrane to specific factors in the nucleus. Using transgenic flies expressing constitutively activated Ras1 or Raf proteins that function independently of upstream signaling events, we show that a reduction in the dose of the gene encoding the catalytic subunit of PP2A stimulates signaling from Ras1 but impairs signaling from Raf. This suggests that PP2A both negatively and positively regulates the Ras1 cascade by dephosphorylating factors that function at different steps in the cascade.

KSR, a novel protein kinase required for RAS signal transduction.

We have identified and characterized two genes in Drosophila whose products are required for activated RAS to signal with normal efficiency, but do not appear to effect signaling by activated RAF. One encodes the beta subunit of type I geranylgeranyl transferase, a prenylation enzyme essential for targeting RAS to the plasma membrane. The other encodes a protein kinase that we have named kinase suppressor of ras (ksr). By genetic criteria, we show that KSR functions in multiple receptor tyrosine kinase pathways. We have isolated mammalian homologs of KSR that, together with the Drosophila gene, define a novel class of kinases. Our results suggest that KSR is a general and evolutionarily conserved component of the RAS signaling pathway that acts between RAS and RAF.

phyllopod functions in the fate determination of a subset of photoreceptors in Drosophila.

phyllopod (phyl) encodes a novel protein required for fate determination of photoreceptors R1, R6, and R7, the last three photoreceptors to be recruited into the ommatidia of the developing Drosophila eye. Genetic data suggests that phyl acts downstream of Ras1, raf, and yan to promote neuronal differentiation in this subset of photoreceptors. Ectopic expression of phyl in the cone cell precursors mimics the effect of ectopic activation of Ras1, suggesting that phyl expression is regulated by Ras1. phyl is also required for embryonic nervous system and sensory bristle development.

dachshund encodes a nuclear protein required for normal eye and leg development in Drosophila.

Neural specification and differentiation in the Drosophila eye sweep across the unpatterned epithelial monolayer of the eye imaginal disc following a developmental wave termed the morphogenetic furrow. The furrow begins at the posterior margin of the eye imaginal disc and moves anteriorly as a linear front. Progression of the furrow requires the function of hedgehog, which encodes a secreted signaling protein. We characterize mutations in dachshund, a gene that encodes a novel nuclear protein required for normal cell-fate determination of imaginal disc cells. In the absence of dachshund function, cells at the posterior margin of the eye disc fail to follow a retinal differentiation pathway and appear to adopt a cuticle fate instead. These cells are therefore unable to respond to pattern propagation signals such as hedgehog and furrow initiation does not occur. In contrast, cells in more anterior portions of the eye disc are able to differentiate as retinal cells in the absence of dachshund activity and respond normally to patterning signals. These results suggest that posterior margin cells are distinct from other cells of the eye imaginal disc by early stages of development. dachshund is also necessary for proper differentiation of a subset of segments in the developing leg. Null mutations in dachshund result in flies with no eyes and shortened legs.

The Drosophila melanogaster ribosomal S6 kinase II-encoding sequence.

A cDNA encoding the Drosophila melanogaster p90 ribosomal S6 kinase II (RSK) was isolated from an eye-antennal imaginal disc library and sequenced. The conceptually translated protein is 60-63% identical to vertebrate RSK homologs and contains a perfectly conserved mitogen-activated protein kinase phosphorylation site. The gene was mapped to the base of the X chromosome in division 20 by in situ hybridization to polytene chromosomes.

Enhanced catalysis by active-site mutagenesis at aspartic acid 153 in Escherichia coli alkaline phosphatase.

Bacterial alkaline phosphatase catalyzes the hydrolysis and transphosphorylation of phosphate monoesters. Site-directed mutagenesis was used to change the active-site residue Asp-153 to Ala and Asn. In the wild-type enzyme Asp-153 forms a second-sphere complex with Mg2+. The activity of mutant enzymes D153N and D153A is dependent on the inclusion of Mg2+ in the assay buffer. The steady-state kinetic parameters of the D153N mutant display small enhancements, relative to wild type, in buffers containing 10 mM Mg2+. In contrast, the D153A mutation gives rise to a 6.3-fold increase in kcat, a 13.7-fold increase in kcat/Km (50 mM Tris, pH 8), and a 159-fold increase in Ki for Pi (1 M Tris, pH 8). In addition, the activity of D153A increases 25-fold as the pH is increased from 7 to 9. D153A hydrolyzes substrates with widely differing pKa's of their phenolic leaving groups (PNPP and DNPP), at similar rates. As with wild type, the rate-determining step takes place after the initial nucleophilic displacement (k2). The increase in kcat for the D153A mutant indicates that the rate of release of phosphate from the enzyme product complex (k4) has been enhanced.