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.

     

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.     

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.     

Advice to use topical or oral ibuprofen for chronic knee pain in older people: randomised controlled trial and patient preference study

Objective To determine whether older patients with chronic knee pain should be advised to use topical or oral non-steroidal anti-inflammatory drugs (NSAIDs).Design Randomised controlled trial and patient preference study.Setting 26 general practices.Participants People aged ≥50 with knee pain: 282 in randomised trial and 303 in preference study. Interventions Advice to use topical or oral ibuprofen.Primary outcome measures WOMAC (Western Ontario and McMaster Universities) osteoarthritis index, major and minor adverse effects.Results Changes in global WOMAC scores at 12 months were equivalent. In the randomised trial the difference (topical minus oral) was two points (95% confidence interval −2 to 6); in the preference study, it was one point (−4 to 6). There were no differences in major adverse effects in the trial or study. The only significant differences in secondary outcomes were in the randomised trial. The oral group had more respiratory adverse effects (17% v 7%,95% confidence interval for difference −17% to −2%), the change in serum creatinine was 3.7 mmol/l less favourable (0.9 µmol/l to 6.5 µmol/l); and more participants changed treatments because of adverse effects (16% v 1%, −16% to −5%). In the topical group more participants had chronic pain grade III or IV at three months, and more participants changed treatment because of ineffectiveness.Conclusions Advice to use oral or topical preparations has an equivalent effect on knee pain over one year, and there are more minor side effects with oral NSAIDs. Topical NSAIDs may be a useful alternative to oral NSAIDs.Trial registration ISRCTN 79353052.     

Stochastic context-free grammars for tRNA modeling.

Stochastic context-free grammars (SCFGs) are applied to the problems of folding, aligning and modeling families of tRNA sequences. SCFGs capture the sequences' common primary and secondary structure and generalize the hidden Markov models (HMMs) used in related work on protein and DNA. Results show that after having been trained on as few as 20 tRNA sequences from only two tRNA subfamilies (mitochondrial and cytoplasmic), the model can discern general tRNA from similar-length RNA sequences of other kinds, can find secondary structure of new tRNA sequences, and can produce multiple alignments of large sets of tRNA sequences. Our results suggest potential improvements in the alignments of the D- and T-domains in some mitochondrial tRNAs that cannot be fit into the canonical secondary structure.     

Biannual Spawning and Temporal Reproductive Isolation in Acropora Corals

Coral spawning on the oceanic reef systems of north-western Australia was recently discovered during autumn and spring, but the degree to which species and particularly colonies participated in one or both of these spawnings was unknown. At the largest of the oceanic reef systems, the participation by colonies in the two discrete spawning events was investigated over three years in 13 species of Acropora corals (n = 1,855 colonies). Seven species spawned during both seasons; five only in autumn and one only in spring. The majority of tagged colonies (n = 218) spawned once a year in the same season, but five colonies from three species spawned during spring and autumn during a single year. Reproductive seasonality was not influenced by spatial variation in habitat conditions, or by Symbiodinium partners in the biannual spawner Acropora tenuis. Colonies of A. tenuis spawning during different seasons separated into two distinct yet cryptic groups, in a bayesian clustering analysis based on multiple microsatellite markers. These groups were associated with a major genetic divergence (G”_ST = 0.469), despite evidence of mixed ancestry in a small proportion of individuals. Our results confirm that temporal reproductive isolation is a common feature of Acropora populations at Scott Reef and indicate that spawning season is a genetically determined trait in at least A. tenuis. This reproductive isolation may be punctuated occasionally by interbreeding between genetic groups following favourable environmental conditions, when autumn spawners undergo a second annual gametogenic cycle and spawn during spring.