Lectin binding as a probe of proliferative and differentiative phases in primary monolayer cultures of cutaneous keratinocytes

The surface of cells in the cutaneous epidermis of the newborn rat exhibits a discrete change in lectin-binding specificity from Griffonia simplicifolia I-B4 (GS I-B4), specific for alpha-D-galactosyl residues, to Ulex europeus agglutinin I (UEA), specific for alpha-L-fucose, as the cell leaves the basal layer and differentiates. Primary monolayer cultures of rat keratinocytes maintained in low Ca2+ medium (0.08 mM) exhibited a characteristic unimodal pattern in the ratio of bound UEA to bound GS I-B4 (UEA/B4 ratio) over a 7-day culture period as determined by a quantitative fluorometric assay. The UEA/B4 ratio was initially low between Days 1 and 2 (0.56 +/- 0.05), steadily increased to a maximum of 0.84 +/- 0.09 between Days 2 and 4, and then gradually decreased to 0.41 +/- 0.07 between Days 6 and 7. Estimation of DNA synthesis showed (a) a higher [3H]thymidine incorporation when the UEA/B4 ratio was low and (b) a steady but lower incorporation between Days 3 and 4, coincident with the higher UEA/B4 ratio. Autoradiographic results further showed that cells stained intensely with UEA failed to incorporate [3H]thymidine into their nuclei. Electrophoresis of [3H]fucose-labeled material isolated on UEA-Sepharose 4B revealed that the changes in labeling by [3H]fucose, bound UEA, and the UEA/B4 ratio in the monolayer were related in part to variable expression of "96K-associated UEA-bound" radioactivity corresponding to a major class of lectin-specific cell-surface glycoproteins (GP96 fraction) identified in situ. Overall, the results suggest that (a) the increase in the UEA/B4 ratio between Days 2 and 4 reflects the progression of a proportion of the cells in the monolayer to an early spinous cell stage, the ultimate fate of which is desquamation into the medium and (b) the decrease in the UEA/B4 ratio between Days 5 and 7 reflects a consequent proliferative response to this loss of cells. This system should be useful for studying environmental influences on the homeostasis of cell proliferation and differentiation in the cutaneous epidermis.     

Activity of maize leaf phosphoenolpyruvate carboxylase in relation to tautomerization and nonenzymatic decarboxylation of oxaloacetate

The keto form of oxaloacetate (OAA), a product of phosphoenolpyruvate carboxylase (PEPC) activity, can undergo various nonenzymatic conversions which make conventional methods of assaying the enzyme difficult, because the products may either act as inhibitors or go undetected. In studies with PEPC isolated from leaves of maize, an assay coupled with reduction of OAA to malate was compared with product analysis using high-performance liquid chromatography and an assay based on Pi release. The results show that activity of the enzyme in the assay coupled to malate dehydrogenase is underestimated, to varying extents, depending on magnesium concentration, buffer, and pH. In the assay coupled to malate dehydrogenase, inaccuracies occur due to conversion of the keto form of OAA to the enol form, which is not utilized as a substrate, and due to loss of OAA by decarboxylation to pyruvate. The assay based on Pi formation is considered to give the true rate of catalysis. With this assay the pH optimum is 7.8, compared to 8.3-8.5 for the assay coupled to malate dehydrogenase. The metal enol complex of oxaloacetate (M-OAAenol) is an inhibitor of PEPC and conditions which are favorable for forming this tautomer, high pH with divalent metal ions or high concentrations of Tris buffer at a pH below its pKa value, limit catalysis. Glycine stimulates enzyme activity, and it may have its effect by preventing the formation of the hydrated M-OAAenol complex and maintaining more of the OAA in the keto form. This interpretation is consistent with glycine stimulation of malate synthesis in the assay of PEPC coupled to malate dehydrogenase, with glycine stimulation of the decarboxylation of OAA, and with a reduction in the level of the M-OAAenol complex in the presence of glycine.     

用三维图象量测卵裂前蛙卵膜分子的三维运动

两栖类卵第一次卵裂前,缩时电影显示出卵表面有收缩波。由于卵的体积未变,在收缩时卵的高度必然增加。Sawai(1978)利用棱镜侧面摄取了蝾螈卵轮廓的高度变化,证实了卵裂前卵最高处的高度确有增加。但这一方法不能测知整个卵表面各处的高度变化,而且仅是二维的。在林蛙卵上,我们用荧光漂白恢复技术发现第一次卵裂前卵表面分子有规律性的流动,推测这是卵球的张弛。为了进一步     

Canopy transpiration and water fluxes in the xylem of the trunk of Larix and Picea trees — a comparison of xylem flow,porometer and cuvette measurements

Summary Leaf gas exchange, transpiration, water potential and xylem water flow measurements were used in order to investigate the daily water balance of intact, naturally growing, adult Larix and Picea trees without major injury. The total daily water use of the tree was very similar when measured as xylem water flow at breast height or at the trunk top below the shade branches, or as canopy transpiration by a porometer or gas exchange chamber at different crown positions. The average canopy transpiration is about 12% lower than the transpiration of a single twig in the sun crown of Larix and Picea. Despite the similarity in daily total water flows there are larger differences in the actual daily course. Transpiration started 2 to 3 h earlier than the xylem water flow and decreased at noon before the maximum xylem water flow was reached, and stopped in the evening 2 to 3 h earlier than the water flow though the stem. The daily course of the xylem water flow was very similar at the trunk base and top below the lowest branches with shade needles. The difference in water efflux from the crown via transpiration and the water influx from the trunk is caused by the use of stored water. The specific capacitance of the crown wood was estimated to be 4.7 x 10^-8 and 6.3 x 10^-8 kg kg^-1 Pa^-1 and the total amount of available water storage was 17.8 and 8.7 kg, which is 24% and 14% of the total daily transpiration in Larix and Picea respectively. Very little water was used from the main tree trunk. With increasing transpiration and use of stored water from wood in the crown, the water potential in the foliage decreases. Plant water status recovers with the decrease of transpiration and the refilling of the water storage sites. The liquid flow conductance in the trunk was 0.45 x 10^-9 and 0.36 x 10^-9 mol m^-2s^-1 Pa^-1 in Larix and Picea respectively. The role of stomata and their control by environmental and internal plant factors is discussed.     

Synthesis and LTD4-antagonist activity of desamino-2-nor-leukotriene analogs

A series of desamino-2-nor-leukotriene analogs has been prepared by the reaction of various thiols with several methyl trans-4,5-epoxy-6Z-alkenoates, followed by deprotection. The products were assessed for their ability to antagonize the LTD4-induced contraction of the isolated guinea pig trachea. Several compounds displayed potent leukotriene antagonist activity, i.e., KB values in the sub-micromolar range, while only minimally affecting basal airway tone. The most potent analog, 4-hydroxy-5-(2-carboxyethylthio)-6Z-nonadecenoic acid, antagonized both LTD4- and LTE4-induced contractions of the trachea in an apparently competitive fashion. These agents possess increased potency relative to SK&F 101132, the first leukotriene analog identified as having LT-antagonist activity. Thus, these results demonstrate that deletion of the peptide amino group can produce leukotriene analogs which have minimal intrinsic contractile activity on the isolated guinea pig trachea, yet possess potent leukotriene-antagonistic effects.     

Partial purification and properties of Galleria mellonella larvae proteolytic inhibitors acting on Metarhizium anisopliae toxic protease

Gel permeation, preparative isoelectric focusing, and affinity chromatography were used to purify three inhibitors of proteolytic activity from perchloric acid extracts of last instar Galleria mellonella larvae. Electrofocusing experiments revealed three isoinhibitors with different isoelectric points: inhibitor I-1 with p1 of pH 5.6, inhibitor I-2, pH 7.7, and inhibitor I-3 (of small inhibitory activity), pH 8.6. By affinity chromatography on trypsin-Sepharose 4B the I-1 was purified 9.7 ×, but 71.1% of inhibitory activity was lost. Molecular mass of the inhibitory complex was 12,600 Da. I-1 and I-2 are relatively stable to heat at several pHs with minor stability at pH 10. I-1 and I-2 inhibit serine proteases about 2.5 times as much as sulfhydryl proteases. In the same ratio protease P-1 and protease P-2 from Metarhizium anisopliae are inhibited.     

Purification and characterization of maize ribulose-1,5-bisphosphate carboxylase

The ribulose-1,5-bisphosphate carboxylase/oxygenase purified from maize (a C₄ monocot) to homogeneity has a MW of532 000 and sedimentation coeffici