Kurt Kofler

Protein Kinase C θ Affects Ca2+ Mobilization and NFAT Activation in Primary Mouse T Cells

Protein kinase C (PKC)θ is an established component of the immunological synapse and has been implicated in the control of AP-1 and NF-κB. To study the physiological function of PKCθ, we used gene targeting to generate a PKCθ null allele in mice. Consistently, interleukin 2 production and T cell proliferative responses were strongly reduced in PKCθ-deficient T cells. Surprisingly, however, we demonstrate that after CD3/CD28 engagement, deficiency of PKCθ primarily abrogates NFAT transactivation. In contrast, NF-κB activation was only partially reduced. This NFAT transactivation defect appears to be secondary to reduced inositol 1,4,5-trisphosphate generation and intracellular Ca2+ mobilization. Our finding suggests that PKCθ plays a critical and nonredundant role in T cell receptor–induced NFAT activation.

Identification of genes involved in estrogenic action in the human prostate using microarray analysis.

Estrogens have profound effects on the developing prostate and are suspected to contribute to the development of benign prostatic hyperplasia, but the mechanism by which this hormone elicits its regulatory function still remains largely unknown. Using complementary RNA microarrays comprising approximately 10,000 oligonucleotide gene targets we compared differences in mRNA expression of estradiol-treated and untreated prostatic stromal cells in vitro. Based on a threshold of greater than twofold change, 228, 241, and 464 of the expressed genes were found to be regulated by estradiol after 10, 24, and 48 h of treatment, respectively. The secondary analysis of one estradiol-activated transcript, namely lipopolysaccharide-binding protein, and four estradiol-repressed genes, namely ras homolog gene family member E (RhoE/Rnd3), ubiquitin thiolesterase, interleukin 6, and interleukin 8 (IL-8), by real-time quantitative PCR confirmed the results of the microarray analysis. Moreover, IL-8 and RhoE were found to be down-regulated by estradiol at the protein level as well. We identified a set of genes involved in a wide range of cellular functions that are potentially important for understanding the molecular basis of estradiol action in the prostate.

Molecular genetics and structural genomics of the human protein kinase C gene module

BackgroundProtein kinase C (PKC) has become a major focus among cell biologists interested in second-messenger signal transduction and much has been learned about differences in the cellular localization and function of its different isotypes. In this study we systematically address the genomic locations and gene structures of the human PKC gene module.ResultsWe first carried out fine chromosomal mapping of all nine PKC genes by fluorescence in situ hybridization (FISH), using cosmid and BAC probes. The PKC genes are found to be dispersed throughout the genome, and in some positions distinct from those previously reported: PKCα is at 17q24, PKCβ at 16p12, PKCγ at 19q13.4, PKCδ at 3p21.2, PKCε at 2p21, PKCζ at 1p36.3, PKCη at 14q22-23, PKCθ at 10p15 and PKCι at 3q26. For PKCι, an additional FISH signal mapped on Xq21.3 revealed a pseudogene (derived by retrotransposition). PKCγ, ζ, and θ are found to map to the most distal positions on the chromosomes, potentially implicating telomere position effects in their expression. Using the complete human genome draft sequence and bioinformatics tools, we then carried out a systematic analysis of PKC gene structure, including determination of the occurrence of single-nucleotide polymorphisms corresponding to the PKC loci.ConclusionThis resource of genomic information now facilitates investigation of the PKC gene module in structural chromosomal abnormalities and human disease locus mapping studies.

Counting the repetitive kringle-IV repeats in the gene encoding human apolipoprotein(a) by fibre-FISH

Counting the repetitive kringle-IV repeats in the gene encoding human apolipoprotein(a) by fibre-FISH

Molecular characterization of the human protein kinase C theta gene locus (PRKCQ).

Members of the protein kinase C (PKC) family of serine/threonine kinases, in particular PKCθ, play critical roles in the regulation of differentiation and proliferation of T lymphocytes. In this study the genomic structure of the human PRKCQ gene that encodes PKCθ was determined. Two genomic P1 clones were isolated from human P1 libraries using the PKCθ cDNA as a probe and have been used to confirm the assignment of the single PRKCQ locus to chromosome 10p15 by FISH analysis. The PRKCQ locus, the first mammalian PKC gene locus characterized so far, spans approximately 62 kb and is composed of 15 coding exons and 14 introns, varying in size between 98 and 16 000 bp. All exon-intron boundaries have been determined by long-range PCR and subsequent DNA sequence analysis. Comparison with other known genomic PKC genes reveals a high degree of homology to the genomic organization of the Drosophila melanogaster dPRKC gene. Alignment of the intron positions in the PRKCQ gene with the intron locations in the dPRKC gene indicates that the sites of seven of the 14 PRKCQ introns are exactly conserved. Exons 5 (32 bp), 11 (174 bp) and 12 (92 bp) share highest similarity in size, organization and primary structure with their counterparts in the Drosophila gene. On the basis of this knowledge of the genomic PRKCQ locus, a directed search for potential genetic polymorphisms and/or genetic abnormalities involved in human genetic disease(s) can now be initiated.

PERSISTENT DETRUSOR INSTABILITY AFTER TRANSURETHRAL RESECTION OF THE PROSTATE IS ASSOCIATED WITH REDUCED PERFUSION OF THE URINARY BLADDER

OBJECTIVES To elucidate, in patients with benign prostatic hyperplasia (BPH), how often detrusor overactivity (DOA) is persistent after transurethral resection of the prostate (TURP) and if perfusion of the lower urinary tract influences postoperative outcomes. PATIENTS AND METHODS Fifty men with urodynamically confirmed DOA and bladder outlet obstruction due to BPH had a TURP. Before and 1 year after TURP the International Prostate Symptom Score (IPSS), quality of life (QoL) score, prostate-specific antigen (PSA) level and total prostatic volume (TPV) were evaluated. Also, the lower urinary tract was evaluated using pressure-flow studies and transrectal colour Doppler ultrasonography to assess the vascular resistive index (RI) as a variable of the perfusion of the lower urinary tract. RESULTS After TURP the IPSS, QoL score, PSA level and TPV decreased. Cystometric measurements showed that in 15 (30%) patients DOA was persistent after TURP. The mean (sd) maximum urinary flow rate increased from 9.20 (4.03) to 15.98 (4.62) mL/s and postvoiding residual urine volumes decreased from 109.38 (73.71) to 29.24 (45.00) mL. When men with persistent DOA (15 patients; group 1) were compared with those with no DOA after TURP (35; group 2) there was a statistically significantly higher RI of the bladder vessels in group 1, at 0.86 (0.068) than in group 2, at 0.68 ( 0.055) (P < 0.001). CONCLUSIONS Persistent DOA in men after TURP seems to be associated with increased vascular resistance of the bladder vessels with subsequent reduced perfusion and hypoxia.