The mixed lineage kinase leucine-zipper protein kinase exhibits a differentiation-associated localization in normal human skin and induces keratinocyte differentiation upon overexpression.

Leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase is a recently described protein serine/threonine kinase which belongs to the mixed lineage kinase family. The overall pattern of expression of the leucine-zipper protein kinase/dual leucine zipper bearing kinase/mitogen-activated protein kinase-upstream kinase gene in embryonic and adult mouse tissues suggested that this kinase could be involved in the regulation of epithelial cell proliferation and differentiation. In order to get more insights into the potential role of leucine-zipper protein kinase in these cellular processes, we characterized its expression in normal human skin, both at the mRNA and protein levels. In situ hybridization, western blotting, and indirect immunofluorescence studies were conducted to localize leucine-zipper protein kinase on various human skin tissues. This is one of the first reports that leucine-zipper protein kinase has a very precise pattern of expression in human skin epithelia, as both mRNA and protein are restricted to the granular layer of the epidermis and inner root sheath of hair follicles. Detection of leucine-zipper protein kinase protein on skin from various body sites, donors of different ages as well as on reconstructed human skin always reveals that leucine-zipper protein kinase is present only in the very differentiated keratinocytes of epidermis and hair follicles. To determine directly whether leucine-zipper protein kinase exhibits any effect on cell growth and differentiation, keratinocytes were transfected with an expression vector harboring the leucine-zipper protein kinase cDNA. The presence of this construct in keratinocytes results in growth arrest together with a concomitant increase in filaggrin expression. Collectively, our results indicate that leucine-zipper protein kinase plays an active part in cellular processes related to terminal differentiation of epidermal keratinocytes.

Protein Changes during the Stratification of Malus domestica Borkh. Seed.

Apple seeds (Malus domestica Borkh. cv Golden Delicious) were stratified at 5 and 15 degrees C for various lengths, weighed, and soluble protein of axis and cotyledon tissue was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only seeds treated at 5 degrees C germinated; seeds treated at 15 degrees C did not germinate. Optimal germination required 63 days of stratification. Excised embryos required less stratification time for germination than intact seeds. When stratification was less than 35 days, the resulting seedlings from 5 degrees C stratified embryos were dwarfed and epinastic. After 63 days of stratification, axes from 5 and 15 degrees C treated intact seeds had increased in fresh weight by 72 and 28% (w/w), respectively. The dry weights of the axes did not change significantly and both fresh and dry weights of cotyledons remained unchanged during stratification. Total soluble protein in axes and cotyledons changed very little during stratification. However, axis polypeptide profiles changed. Most obvious was the occurrence of a new polypeptide and the increase of four other clearly identifiable polypeptides during 5 degrees C treatment. The levels of the five most predominant axis proteins decreased at the same time. We observed no changes in the profiles of soluble cotyledon proteins. Control seeds kept at -10 degrees C showed none of the reported changes.

Reevaluation of the cyanide resistance of seed germination.

Although high levels of KCN (53 micromoles per gram fresh weight of seed, corresponding to 3.2 millimolar) failed to block germination of lettuce seeds incubated in covered Petri dishes, the same levels totally blocked germination in sealed dishes. Inhibition was reversed by removing the seal. Placement of KCN remote from seeds also blocked germination in closed systems. Cyanide effectiveness was enhanced by acidifying the KCN solution but negated by the presence of a trap containing strong alkali. Low levels of aqueous HCN (2.6 micromoles HCN per gram, corresponding to 0.16 millimolar) injected into sealed dishes gave maximal inhibition of germination, suggesting that the effectiveness of KCN was due to formation of HCN in KCN solutions. Studies with nine additional crop species generally supported the interpretation that cyanide inhibition of germination has been underestimated in the past due to escape of volatile HCN from open systems.

Cyanide-insensitive, Salicylhydroxamic Acid-sensitive Processes in Potentiation of Light-requiring Lettuce Seeds.

Phytochrome-mediated germination of Lactuca sativa L. cv. Waldmann's Green seeds was inhibited strongly by 10 millimolar salicylhydroxamic acid (SHAM), but only slightly delayed by the same level of KCN. SHAM was most effective if applied within the 8-hour potentiation period (release from dormancy) following red light treatment, but much less effective with completely potentiated seeds. SHAM at 3 millimolar actually hastened completion of potentiation, whereas concentrations of 6.6 millimolar or higher retarded the process. A temporary upsurge of O(2) consumption was particularly evident during the period of most rapid potentiation (3 hours after red light), especially in the seed sections containing the embryonic axis. The embryonic axis obtained from dormant seeds also contained most of the SHAM-sensitive O(2) uptake. However, 8 hours of potentiation caused loss of SHAM sensitivity from axes and a simultaneous gain of SHAM sensitivity by cotyledons. Concomitant with this increased sensitivity to SHAM, O(2) uptake by cotyledonary tissues lost some sensitivity to KCN. Red light-stimulated metabolic processes leading to germination were blocked more effectively by SHAM than by KCN, but O(2) consumption by both dormant and nondormant seeds was much less sensitive to 10 millimolar SHAM than to the same concentration of KCN. This apparent contradiction between effects of SHAM on potentiation and O(2) uptake may be a result of: (a) compensatory electron flow through the cytochrome pathway at the expense of the alternate pathway; (b) a functional site of action of SHAM that differs from the organized, energy-coupled respiratory system; or (c) a combination of these possibilities.