Anti-PRKCB (aa 261-341) polyclonal antibody

Aims/hypothesis Activation of protein kinase C (PKC) isoforms has been implicated as a central mediator in the pathogenesis of diabetic nephropathy. Although high glucose levels stimulate catalytic activity of PKC, the effects of high glucose levels on the expression of genes encoding PKC isoforms are unknown. We sought to determine whether in addition to activation, diabetes may lead to increased transcription of two PKC isoforms that have been implicated in the pathogenesis of diabetic nephropathy, PKC-alpha and PKC-beta. Methods Recent advances in molecular biological techniques now permit quantitative analysis of mRNA from archival, formalin-fixed, paraffin-embedded tissue sections. RNA was extracted from scraped 6 mu m sections of biopsy tissue, and PRKC-alpha and PRKC-beta (also known as PRKCA and PRKCB) mRNA measured using real-time PCR. Expression of genes encoding PKC isoforms was examined in renal biopsies (n = 25) with classical histological features of diabetic nephropathy and compared with that in normal control tissue (n = 6). Peptide localisation of PKC-alpha, PKC-beta and the activated forms phosphorylated PKC-alpha and -beta was also performed on matched paraffin-embedded sections of renal biopsies using immunohistochemistry. The effects of high glucose on PRKC-beta expression and peptide production in cultured human proximal tubular epithelial cells were assessed. Results Quantitative real-time PCR demonstrated a 9.9-fold increase in PRKC-beta mRNA in kidney biopsies of diabetic patients relative to control (p < 0.001). No increase in PRKC-alpha expression was seen. In addition, a correlation between renal PRKC-beta mRNA and HbA(1c) was observed in diabetic patients (r = 0.63, p < 0.05). There was co-localisation of PKC-beta and phospho-PKC-beta predominantly to proximal tubules. A 60% increase in PRKC-beta mRNA and peptide in cultured human proximal tubular epithelial cells exposed to high glucose (p < 0.05) was seen in vitro. Conclusions/interpretation PKC-beta is upregulated at the gene expression level in human diabetic nephropathy. PRKC-beta mRNA correlates closely with serum HbA(1c), possibly partially explaining the relationship between glycaemic control and progression of diabetic nephropathy. Archival human tissue provides a valuable resource for molecular analyses.

P>Background Although it has been shown that T-cell dysfunction in psoriasis correlates with various endogenous and exogenous stimuli, there are also intensive and complicated genetic background effects indicating that intrinsic abnormalities may play a more predominant role such as in haematopoietic cells, the haematopoietic microenvironment and the development of haematopoietic cells into T cells. Objective To reveal the activity of haematopoietic stem cells from patients with psoriasis. Methods Bone marrow CD34+ cells were isolated and expression levels were tested by reverse transcriptase-polymerase chain reaction and Western blotting. The binding site for RUNX1 located between SLC9A3R1 and NAT9 and the apoptotic index of CD34+ cells were also investigated. Results We found that the expression of RUNX1, SLC9A3R1, HLA-C and PRKCB mRNA and RUNX1 protein was significantly higher in patients with psoriasis than in the controls, and SLC9A3R1 expression showed a significant positive correlation with the Psoriasis Area and Severity Index score. There was no statistical significance for PDCD1 mRNA and PKC-beta I protein expression levels between the patients with psoriasis and controls. Mutation of RUNX1 binding sites between SLC9A3R1 and NAT9 was not detected, and the apoptotic index in patients with psoriasis showed no statistical significance compared with the controls. Conclusion Expression of the critical transcription factor RUNX1, which regulates CD34+ cell development and differentiation, was abnormal, and augmentation of these expression levels might induce dysfunction of marrow haematopoietic stem cells and the haematopoietic microenvironment and be involved in the progression of psoriasis.