Sequence, purification, and cloning of an intracellular serine protease, quiescent cell proline dipeptidase

We recently observed that specific inhibitors of post-proline cleaving aminodipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell proline dipeptidase (QPP). QPP activity was purified from CD26(-) Jurkat T cells. The protein was identified by labeling with [(3)H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.     

Glucocorticoids regulate transendothelial fluid flow resistance and formation of intercellular junctions

The regulation of transendothelial fluid flow by glucocorticoidswas studied in vitro with use of human endothelial cells cultured fromSchlemm's canal (SCE) and the trabecular meshwork (TM) in conjunctionwith computer-linked flowmeters. After 2-7 wk of 500 nMdexamethasone (Dex) treatment, the following physiological, morphometric, and biochemical alterations were observed: a 3- to 5-foldincrease in fluid flow resistance, a 2-fold increase in therepresentation of tight junctions, a 10- to 30-fold reduction in themean area occupied by interendothelial "gaps" or preferential flow channels, and a 3- to 5-fold increase in the expression of thejunction-associated protein ZO-1. The more resistive SCE cells expressed two isoforms of ZO-1; TM cells expressed only one. To investigate the role of ZO-1 in the aforementioned Dex effects, itsexpression was inhibited using antisense phosphorothioate oligonucleotides, and the response was compared with that observed withthe use of sense and nonsense phosphorothioate oligonucleotides. Inhibition of ZO-1 expression abolished the Dex-induced increase inresistance and the accompanying alterations in cell junctions and gaps.These results support the hypothesis that intercellular junctions arenecessary for the development and maintenance of transendothelial flowresistance in cultured SCE and TM cells and are likely involved in themechanism of increased resistance associated with glucocorticoid exposure.

     

The central role of PhEIN2 in ethylene responses throughout plant development in petunia

The plant hormone ethylene regulates many aspects of growth and development. Loss-of-function mutations in ETHYLENE INSENSITIVE2 (EIN2) result in ethylene insensitivity in Arabidopsis, indicating an essential role of EIN2 in ethylene signaling. However, little is known about the role of EIN2 in species other than Arabidopsis. To gain a better understanding of EIN2, a petunia (Petunia x hybrida cv Mitchell Diploid [MD]) homolog of the Arabidopsis EIN2 gene (PhEIN2) was isolated, and the role of PhEIN2 was analyzed in a wide range of plant responses to ethylene, many that do not occur in Arabidopsis. PhEIN2 mRNA was present at varying levels in tissues examined, and the PhEIN2 expression decreased after ethylene treatment in petals. These results indicate that expression of PhEIN2 mRNA is spatially and temporally regulated in petunia during plant development. Transgenic petunia plants with reduced PhEIN2 expression were compared to wild-type MD and ethylene-insensitive petunia plants expressing the Arabidopsis etr1-1 gene for several physiological processes. Both PhEIN2 and etr1-1 transgenic plants exhibited significant delays in flower senescence and fruit ripening, inhibited adventitious root and seedling root hair formation, premature death, and increased hypocotyl length in seedling ethylene response assays compared to MD. Moderate or strong levels of reduction in ethylene sensitivity were achieved with expression of both etr1-1 and PhEIN2 transgenes, as measured by downstream expression of PhEIL1. These results demonstrate that PhEIN2 mediates ethylene signals in a wide range of physiological processes and also indicate the central role of EIN2 in ethylene signal transduction.     

Monitoring Migration and Measuring Biomass in Benthic Biofilms: The Effects of Dark/far-red Adaptation and Vertical Migration on Fluorescence Measurements

Pulse modulated fluorescence has increasingly been used as an ecological tool to examine changes in the vertical distribution of microphytobenthic cells within the upper layers of estuarine sediments (most often using the minimum fluorescence yield F(o)) as well as to indicate the health of the community (using the maximum PS II quantum efficiency F(v)/F(m)). However, the practicalities of in situ measurements, often dictates that short dark adaptation periods must be used ( approximately 15 min). The use of far-red light as an alternative to dark adaptation was investigated in natural migratory microphytobenthic biofilms and artificial non-migratory biofilms. Prolonged periods of darkness ( approximately 24 h) were not adequate to achieve 'true' measurements of F(o) and F(v)/F(m), which require complete oxidation of Q(A) and full reversal of non-photochemical quenching (NPQ). In some instances, stable values were only achieved using far-red light. Prolonged exposure to dark/far-red light led to a downwards migration of cells in natural assemblages, as seen by a reduction in both F(o) and the maximum fluorescence yield (F(m)). In non-migratory biofilms, F(m) increased in the dark and far-red treatments, indicating a reversal of NPQ, whereas F(o) decreased in far-red light but increased in the dark. It is suggested that far-red light and darkness differentially affected the balance between NPQ reversal and Q(A) oxidation that lead to the measured F(o) yield. The use of far-red light as an alternative to dark adaptation is discussed and the implications of short (e.g., 15 min) dark adaptation times used in situ are discussed with reference to the vertical migration of cells within sediment biofilms.     

Comparing the consequences of induced and constitutive plant resistance for herbivore population dynamics

Although it has been suggested that induced and constitutive plant resistance should have different effects on insect herbivore population dynamics, there is little experimental evidence that plant resistance can influence herbivore populations longer than one season. We used a density-manipulation experiment and model fitting to examine the effects of constitutive and induced resistance on herbivore dynamics over both the short and long term. We used likelihood methods to fit population dynamic models to recruitment data for populations of Mexican bean beetles on soybean varieties with no resistance, constitutive resistance, or induced resistance. We compared model configurations that fit parameters for resistance types separately to models that did not account for resistance type. Models representing the hypothesis that the three resistance types differed in their effects on beetle dynamics received the most support. Induced resistance resulted in lower population growth rates and stronger density dependence than no resistance. Constitutive resistance resulted in lower population growth rates and stronger density dependence than induced resistance. Constitutive resistance had a stronger effect on both short-term beetle recruitment and predicted beetle population dynamics than induced resistance. The results of this study suggest that induced and constitutive resistance can differ in their effects on herbivore populations even in a relatively complex system.     

Vertebrate phylogeny of antigen D10: identification of a conserved foregut cell lineage

The monoclonal antibody 5HL-5D11-D10 to antigen D10 identifies a cell lineage that is restricted to certain tissues of the human foregut. We investigated the tissue distribution of antigen D10 in mammals, birds, reptiles, amphibians and fish by immunohistochemical staining. Tissue from human and each of ten other mammalian species showed staining of gastric mucous neck cells and glands of the cardia and antrum, Brunner's glands, peribiliary glands and periductal glands of the pancreas. Six of the mammalian species also expressed antigen D10 in mucosa of the larger bronchi, and five expressed it to varying degree in small bowel distal to the duodenum and in colon (three of these five species). Antigen was not detected in any of the three species of bird studied. Both reptiles and amphibians showed strong staining for antigen D10 in the gastric mucous neck cells and pyloric glands, and in a subpopulation of secretory cells in the oesophagus, with the amphibian also expressing antigen in some epithelial cells of the mouth and lung. Although absent from two species of bony fish, antigen D10 was expressed by small groups of epithelial cells of the intestine of a shark, and generally by the epithelial and connective tissue cells of the gut and gills, and hepatocytes of one species of ray. The presence of antigen D10 in different tissues and species was confirmed by both an indirect ELISA and immunoblot analysis of tissue extracts. Our observations suggest that the D10 epitope characterises a subpopulation of mucus-secreting cells, predominantly of the foregut and associated organs, which has been conserved throughout terrestrial vertebrate evolution.     

Substrate specificity in glycoside hydrolase family 10. Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of Pseudomonas cellulosa xylanase 10A

The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes beta1, 4-linked xylans but exhibits very low activity against aryl-beta-cellobiosides. The family 10 enzyme, Cex, from Cellulomonas fimi, hydrolyzes aryl-beta-cellobiosides more efficiently than does Xyl10A, and the movements of two residues in the -1 and -2 subsites are implicated in this relaxed substrate specificity (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The three-dimensional structure of Xyl10A suggests that Tyr-87 reduces the affinity of the enzyme for glucose-derived substrates by steric hindrance with the C6-OH in the -2 subsite of the enzyme. Furthermore, Leu-314 impedes the movement of Trp-313 that is necessary to accommodate glucose-derived substrates in the -1 subsite. We have evaluated the catalytic activities of the mutants Y87A, Y87F, L314A, L314A/Y87F, and W313A of Xyl10A. Mutations to Tyr-87 increased and decreased the catalytic efficiency against 4-nitrophenyl-beta-cellobioside and 4-nitrophenyl-beta-xylobioside, respectively. The L314A mutation caused a 200-fold decrease in 4-nitrophenyl-beta-xylobioside activity but did not significantly reduce 4-nitrophenyl-beta-cellobioside hydrolysis. The mutation L314A/Y87A gave a 6500-fold improvement in the hydrolysis of glucose-derived substrates compared with xylose-derived equivalents. These data show that substantial improvements in the ability of Xyl10A to accommodate the C6-OH of glucose-derived substrates are achieved when steric hindrance is removed.     

Identification of a new gene (rat TM6P1) encoding a fasting-inducible, integral membrane protein with six transmembrane domains

We describe the isolation of a new gene that encodes a membrane-integrated protein with six transmembrane domains, termed TM6P1. A 403-bp expressed sequence tag was isolated from fasted rat liver subtracted cDNA library, and its full-length cDNA is 1482 bp long. It contains an open reading frame of 816 bp and is predicted to encode a 271-amino acid protein with a deduced mass of 29520 Da. A sequence homology search failed to show significant correspondence to any known protein in the databank. TM6P1 has six highly hydrophobic domains that are predicted to be transmembrane helices. Consistent with this prediction, the TM6P1-EGFP fusion protein was shown to localize to the plasma membrane. TM6P1 mRNA is widely expressed in rat tissues, with placenta and liver being the most abundant sites. Fasting increased TM6P1 mRNA nearly two-fold in liver. Taken together, our data suggest that TM6P1 is a unique new membrane integral protein that might have a function important during fasting-induced catabolism.