A duplication in chromosome 4q35 is associated with hereditary benign intraepithelial dyskeratosis.

Hereditary benign intraepithelial dyskeratosis (HBID) is an autosomal dominant disorder characterized by elevated epithelial plaques on the ocular and oral mucous membranes. It has been reported primarily, but not exclusively, in individuals of American Indian heritage in North Carolina. We have examined and obtained DNA on two large families affected by HBID. Using genetic linkage analysis we have localized the HBID gene to chromosome 4 (4q35) with a peak LOD score of 8.97. Molecular analysis of these data reveals that all individuals affected with HBID in both families demonstrate the presence of three alleles for two tightly linked markers, D4S1652 and D4S2390, which map to the telomeric region of 4q35. This suggests the presence of a duplication segregating with the disease phenotype that is most likely involved in its causation.

Programmed cell death induced by ceramide.

Sphingomyelin hydrolysis and ceramide generation have been implicated in a signal transduction pathway that mediates the effects of tumor necrosis factor-alpha (TNF-alpha) and other agents on cell growth and differentiation. In many leukemic cells, TNF-alpha causes DNA fragmentation, which leads to programmed cell death (apoptosis). C2-ceramide (0.6 to 5 microM), a synthetic cell-permeable ceramide analog, induced internucleosomal DNA fragmentation, which was inhibited by zinc ion. Other amphiphilic lipids failed to induce apoptosis. The closely related C2-dihydroceramide was also ineffective, which suggests a critical role for the sphingolipid double bond. The effects of C2-ceramide on DNA fragmentation were prevented by the protein kinase C activator phorbol 12-myristate 13-acetate, which suggests the existence of two opposing intracellular pathways in the regulation of apoptosis.

High-dose chemotherapy-induced platelet defect: inhibition of platelet signal transduction pathways.

Patients receiving high-dose chemotherapy and autologous bone marrow transplantation acquire a platelet secretion defect. The role of chemotherapeutic agents and their metabolites in mediating this platelet defect was investigated. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU), but not cyclophosphamide or cis-platinum, was found to inhibit platelet aggregation in vitro in response to activation by either ADP, thrombin, or collagen. Inhibition by BCNU was dose dependent and required preincubation of platelets with BCNU. After a 60-min preincubation, 30 microM BCNU produced 50% inhibition of platelets in platelet-rich plasma. The cyclophosphamide metabolites acrolein and 4-hydroperoxycyclophosphamide also inhibited platelet aggregation in a dose-dependent manner, with a requirement for preincubation. Platelet inhibition occurred at clinically relevant concentrations of BCNU and metabolites of cyclophosphamide. The effects of acrolein were totally prevented by coincubation with the sulfhydryl-protecting agents N-acetylcysteine and 2-mercaptoethanesulfonic acid, whereas the effects of BCNU were incompletely prevented. The mechanism of platelet inhibition was investigated next by examining protein phosphorylation in response to platelet agonists. Acrolein inhibited thrombin- and phorbol ester-induced phosphorylation of a 40-kDa polypeptide and other substrates, indicating a cellular defect in protein kinase C signaling. BCNU did not interfere with protein phosphorylation, indicating preservation of initial signaling pathways. Thus, chemotherapeutic agents and their metabolites inhibit platelet function by inhibiting distinct components of the intracellular activation pathways.

Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.

The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.

Plasma assay of salbutamol by means of high-performance liquid chromatography with amperometric determination using a loop column for injection of plasma extracts. Application to the evaluation of subcutaneous administration of salbutamol.

An isocratic high-performance liquid chromatography method with amperometric detection for the assay of plasma salbutamol is described. The plasma extract is injected into the chromatographic system via a loop column. This insures the purification of the injected extracts and allows a simple and rapid liquid-solid extraction procedure. The good reliability, as shown by the low limit of detection (0.5 ng/ml) and a precision ranging between 5 and 10%, has permitted the investigation of a new mode of administration of salbutamol using a portable subcutaneous infusion pump. Our results show that subcutaneous administration yields plasma levels comparable with those obtained after usual intravenous doses.

In vitro kinetics of 8-methoxypsoralen penetration into human lymphoid cells.

Extracorporeal photochemotherapy (ECPC) requires ex vivo UVA irradiation of blood lymphocytes during the time of the theoretical peak 8-methoxsalen (8-MOP) concentration. The aims of this study were to determine the mechanism of cellular uptake of 8-MOP, its possible saturation and the time needed to reach maximal concentration (Tmax) in lymphoid cells. 8-MOP was measured by liquid chromatography in the supernatant of lymphoid cell suspensions incubated with a known amount of 8-MOP. The kinetics of cellular uptake were determined and showed that equilibrium had already been reached after 2 min and remained constant for at least 60 min. The uptake was independent of temperature (4, 25 and 37 degrees C) and was proportional to the 8-MOP concentration in the supernatant. This indicated that 8-MOP penetrated into lymphoid cells by passive diffusion, rather than by active transport or facilitated diffusion, and was thus a non-saturable process. In addition, intracellular metabolism was negligible. These findings demonstrated that the plasma and lymphocytic Tmax were reached simultaneously and statistical analysis showed them to be significantly correlated, thereby validating the standard ECPC protocol for drug ingestion and lymphocyte irradiation.

Transcriptional regulation of protein kinase C by 1,25-dihydroxyvitamin D3 in HL-60 cells.

Vitamin D3 treatment of the human promyelocytic cell line, HL-60, is accompanied by an increase in phorbol ester receptor number (Martell, R. E., Simpson, R. U., and Taylor, J. M. (1987) J. Biol. Chem. 262, 5570-5575). In this study, the mechanism and significance of vitamin D3-induced changes in protein kinase C levels were investigated. Treatment of HL-60 cells with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) resulted in a 2-3-fold increase in phorbol dibutyrate binding at 24 h. This was accompanied by a 4.2-fold increase in steady state mRNA levels for the beta isoenzyme of protein kinase C and by a 3.8-fold increase in its transcriptional rate as determined from nuclear run-off studies. Protein kinase C alpha mRNA, which was approximately 15% of the beta isoenzyme levels, showed similar increases in mRNA and transcriptional rates in response to 1,25-(OH)2D3. Protein kinase C gamma mRNA was not detected. The increases in protein kinase C levels were accompanied by increases in activation of the enzyme by phorbol esters. More importantly, 1,25-(OH)2D3 caused a 1.5-2.0-fold increase in the endogenous phosphorylation of protein kinase C substrates independent of exogenous activators or endogenous formation of diacylglycerol. This is the first report of transcriptional activation of protein kinase C. This mechanism of up-regulation of protein kinase C may explain the increased activity of protein kinase C in vitamin D3-treated HL-60 cells, and may constitute an important mechanism for the long term modulation of the protein kinase C pathway of cell regulation independent of diacylglycerol generation.