Cowpea bruchid midgut transcriptome response to a soybean cystatin--costs and benefits of counter-defence.

The insect digestive system is the first line of defence protecting cells and tissues of the body from a broad spectrum of toxins and antinutritional factors in its food. To gain insight into the nature and breadth of genes involved in adaptation to dietary challenge, a collection of 20 352 cDNAs was prepared from the midgut tissue of cowpea bruchid larvae (Callosobruchus maculatus) fed on regular diet and diets containing antinutritional compounds. Transcript responses of the larvae to dietary soybean cystatin (scN) were analysed using cDNA microarrays, followed by quantitative real-time PCR (RT-PCR) confirmation with selected genes. The midgut transcript profile of insects fed a sustained sublethal scN dose over the larval life was compared with that of insects treated with an acute high dose of scN for 24 h. A total of 1756 scN-responsive cDNAs was sequenced; these clustered into 967 contigs, of which 653 were singletons. Many contigs (451) did not show homology with known genes, or had homology only with genes of unknown function in a Blast search. The identified differentially regulated sequences encoded proteins presumptively involved in metabolism, structure, development, signalling, defence and stress response. Expression patterns of some scN-responsive genes were consistent in each larval stage, whereas others exhibited developmental stage-specificity. Acute (24 h), high level exposure to dietary scN caused altered expression of a set of genes partially overlapping with the transcript profile seen under chronic lower level exposure. Protein and carbohydrate hydrolases were generally up-regulated by scN whereas structural, defence and stress-related genes were largely down-regulated. These results show that insects actively mobilize genomic resources in the alimentary tract to mitigate the impact of a digestive protease inhibitor. The enhanced or restored digestibility that may result is possibly crucial for insect survival, yet may be bought at the cost of weakened response to other stresses.

Phenytoin half-life and clearance during maintenance therapy in adults and elderly patients with epilepsy.

BACKGROUND: Phenytoin (PHT) is widely used to treat epilepsy in elderly patients, but information on its pharmacokinetics in this population is limited. OBJECTIVE: The purpose of this study was to investigate the effects of age and sex on PHT pharmacokinetics using stable-labeled (SL) isotopes of PHT or fosphenytoin (FOS) administered IV or IM while patients remained on their oral maintenance regimen. METHODS: Subjects were patients 18 years or older with epilepsy, but otherwise healthy, on a maintenance regimen of PHT who were not taking interacting medications. Subjects were given a single injection of a 100 mg dose of SL-PHT or SL-FOS followed by their usual morning PHT dose less 100 mg. Serial blood samples were collected up to 196 hours after the SL dose. Plasma PHT and SL-PHT concentrations were measured by a gas chromatographic-mass spectrometric assay. PHT pharmacokinetics were characterized using a population-based, nonlinear, mixed-effects model. RESULTS: Sixty-three subjects completed the study, 45 of whom were 65 years or older. There was no difference between adult and elderly or men and women in PHT clearance, distribution volume, and elimination half-life. The mean elimination half-life was 40 hours. CONCLUSIONS: Healthy elderly adults appear to have the same phenytoin (PHT) pharmacokinetics as younger adults. Reduced PHT dosage requirements may be due to age-related changes in patients' sensitivity to the therapeutic and toxic effects of the drug. The prolonged elimination half-life suggests that most patients can take PHT once daily and the time to reach steady-state may extend to 2-3 weeks.

Functional expression of an insect cathepsin B-like counter-defence protein.

Insects are capable of readjusting their digestive regimes in response to dietary challenge. Cowpea bruchids (Callosobruchus maculatus) strongly induce C. maculatus cathepsin B-like cysteine protease 1 (CmCatB1) transcripts when fed diet containing a soybean cysteine protease inhibitor soyacystatin N (scN). CmCatB1 shares significant sequence similarity with cathepsin B-like cysteine proteases. In this study, we isolated another cDNA, namely CmCatB2 that encodes a protein sequence otherwise identical to CmCatB1, but lacking a 70-amino-acid internal section. CmCatB1 and CmCatB2 probably resulted from alternate splicing events. Only the CmCatB1 transcript, however, exhibited differential expression in response to dietary scN. Further, this expression was only detectable in larvae, which is the developmental stage associated with food ingestion. The scN-activated and developmentally regulated CmCatB1 expression pattern suggests it may have a unique function in insect counter-defence against antinutritional factors. Heterologously expressed recombinant CmCatB1 protein exhibited enzymatic activity in a pH-dependent manner. Activity of the protein was inhibited by both the cysteine protease inhibitor E-64 and the cathepsin B-specific inhibitor CA-074, verifying its cathepsin B-like cysteine protease nature. Interestingly, the enzymatic activity was unaffected by the presence of scN. Together, we have provided functional evidence suggesting that CmCatB1 confers inhibitor-insensitive enzymatic activity to cowpea bruchids, which is crucial for insect survival when challenged by dietary protease inhibitors.

Cowpea bruchid Callosobruchus maculatus counteracts dietary protease inhibitors by modulating propeptides of major digestive enzymes.

Cowpea bruchids, when challenged by consumption of the soybean cysteine protease inhibitor scN, reconfigure expression of their major CmCP digestive proteases and resume normal feeding and development. Previous evidence indicated that insects selectively induced CmCPs from subfamily B, that were more efficient in autoprocessing and possessed not only higher proteolytic, but also scN-degrading activities. In contrast, dietary scN only marginally up-regulated genes from the more predominant CmCP subfamily A that were inferior to subfamily B. To gain further molecular insight into this adaptive adjustment, we performed domain swapping between the two respective subfamily members B1 and A16, the latter unable to autoprocess or degrade scN even after intermolecular processing. Swapping the propeptides did not qualitatively alter autoprocessing in either protease isoform. Incorporation of either the N- (pAmBA) or C-terminal (pAmAB) mature B1 segment into A16, however, was sufficient to prime autoprocessing of A16 to its mature form. Further, the swap at the N-terminal mature A16 protein region (pAmBA) resulted in four amino acid changes. Replacement of these amino acid residues by the corresponding B1 residues, singly and pair-wise, revealed that autoprocessing activation in pAmBA resulted from cumulative and/or coordinated individual effects. Bacterially expressed isolated propeptides (pA16 and pB1) differed in their ability to inhibit mature B1 enzyme. Lower inhibitory activity in pB1 is likely attributable to its lack of protein stability. This instability in the cleaved propeptide is necessary, although insufficient by itself, for scN-degradation by the mature B1 enzyme. Taken together, cowpea bruchids modulate proteolysis of their digestive enzymes by controlling proCmCP cleavage and propeptide stability, which explains at least in part the plasticity cowpea bruchids demonstrate in response to protease inhibitors.

Functional roles of specific bruchid protease isoforms in adaptation to a soybean protease inhibitor.

Upon challenge by the soybean cysteine protease inhibitor soyacystatin N (scN), cowpea bruchids reconfigure their major digestive cysteine proteases (CmCPs) in adaptation to the inhibitor and resume normal feeding and development. We have previously shown that CmCPB transcripts were 116.3-fold more abundant in scN-adapted bruchid guts than in unadapted guts, while CmCPA transcripts were only 2.5-fold higher. In order to further elucidate the functional significance of this differential regulation, we expressed three CmCPA and one CmCPB isoforms (A9, A13, A16 and B1) using a bacterial expression system, and characterized their activities. In contrast to the precursors of CmCPAs (proCmCPAs), proCmCPB1 exhibited more efficient autocatalytic conversion from the latent proenzyme to its active mature protease form, and demonstrated higher intrinsic proteolytic activity. Among proCmCPAs, dependence on exogenous enzymatic processing varies: while maturation of proCmCPA13 and proCmCPA16 was impaired in the absence of external proteolytic activity, proCmCPA9 appeared to utilize a two-step autoprocessing mechanism. Although all CmCPs are scN-sensitive, scN was degraded by CmCPB1 when outnumbered by the protease, but scN remained intact in the presence of excessive CmCPA9. These results provide further evidence that differential expression of CmCPs under scN challenge brings about adaptation to the inhibitor. High induction of unique cysteine protease isoforms with superior autoprocessing and proteolytic efficacy represents a strategy cowpea bruchids use to cope with dietary scN.

Transcriptional regulation in cowpea bruchid guts during adaptation to a plant defence protease inhibitor.

Cowpea bruchid, when fed on a diet containing the soybean cysteine protease inhibitor soyacystatin N (scN), activates an array of counter-defence genes to adapt to the negative effects of the inhibitor and regain its normal rate of feeding and development. A collection of 1920 cDNAs was obtained by differential subtraction with cDNAs prepared from guts of the 4th instar larvae of scN-adapted (reared on scN-containing diet) and scN-unadapted (reared on regular scN-free diet) cowpea bruchids. Subsequent expression profiling using DNA microarray and Northern blot analyses identified ninety-four transcript species from this collection that are responsive to dietary scN. scN-adapted insects induced genes encoding protein and carbohydrate digestive enzymes, probably to help meet their carbon and nitrogen requirements. Up-regulation of antimicrobial and detoxification protein genes may represent a generalized defence response. Genes down-regulated by scN reflected physiological adjustments of the cowpea bruchids to scN challenge. A large portion of the responsive genes, presumably involved in carrying out the counter-defence response, were of unknown function. The full-length cDNA of an scN-inducible cathepsin B-like cysteine protease was obtained. Its transcriptional response to scN during larval development contrasts with the pattern of the cathepsin L family, the major digestive enzymes. These results suggest cathepsin B-like cysteine proteases may play a crucial role in cowpea bruchid adaptation to dietary scN.

Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor.

The soybean cysteine protease inhibitor, soyacystatin N (scN), negatively impacts growth and development of the cowpea bruchid, Callosobruchus maculatus[Koiwa et al. (1998) Plant J 14: 371-379]. However, the developmental delay and feeding inhibition caused by dietary scN occurred only during the early developmental stages (the 1st, 2nd and 3rd instars) of the cowpea bruchid. The 4th instar larvae reared on scN diet (adapted) exhibited rates of feeding and development which were comparable to those feeding on an scN-free diet (unadapted) prior to pupation. Total gut proteolytic capacity at this larval stage significantly increased in the scN-adapted insects. The elevated enzymatic activity was attributed to a differential expression of insect gut cysteine proteases (representing the major digestive enzymes), and of aspartic proteases. scN degradation by the gut extract was observed only in adapted bruchids, and this activity appeared to be a combined effect of scN-induced cysteine and aspartic proteases. Thirty cDNAs encoding cathepsin L-like cysteine proteases were isolated from insect guts, and they were differentially regulated by dietary scN. Our results suggest that the cowpea bruchid adapts to the challenge of scN by qualitative and quantitative remodelling of its digestive protease complement, and by activating scN-degrading protease activity.

Epitheliomesenchymal transdifferentiation of cultured RPE cells.

Retinal pigment epithelium (RPE) cells of the proliferative vitreoretinopathy (PVR) membrane take on the shape of fibroblasts and participate in fibrosis, thus deviating from the character of epithelial cells. This study was undertaken to evaluate RPE cell transdifferentiation in vitro. During the culture of porcine RPE cells, primary and 10th-passaged RPE cells were investigated for cell growth in response to transforming growth factor (TGF) beta(2), change of phenotype and amount in collagen synthesis as well as expression of alpha-smooth-muscle actin (alpha-SMA). TGF-beta(2) inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, while the spindle-shaped 10th-passaged RPE cells were not inhibited by TGF-beta(2). The 10th-subcultured cells did not show much difference in the quality of collagen synthesis, other than type VIII collagen which was not produced. Collagen synthesis was dose-dependently stimulated by TGF-beta(2). The stimulation by TGF-beta(2) in the 10th-passaged RPE cells was much greater than in primary RPE cells. The 10th-subcultured RPE cells produced substantial alpha-SMA compared to alpha-SMA production by primary RPE cells. These results were also observed by confocal laser microscopy. These findings indicated that RPE metaplasia resulting in a change of biological cell behavior might be a necessary predisposing step in the development of PVR.

Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3.

Using homology-based PCR, we have isolated cDNA encoding a novel member (491 amino acids) of the angiopoietin (Ang) family from human adult heart cDNA and have designated it angiopoietin-3 (Ang3). The NH2-terminal and COOH-terminal portions of Ang-3 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in other known Angs. Ang3 has a highly hydrophobic region at the N-terminus (approximately 21 amino acids) that is typical of a signal sequence for protein secretion. Ang3 mRNA is most abundant in adrenal gland, placenta, thyroid gland, heart and small intestine in human adult tissues. Additionally, Ang3 is a secretory protein, but is not a mitogen in endothelial cells.

The effect of photorefractive keratectomy in Korea.

To assess the predictability and accuracy of photorefractive keratectomy (PRK) in myopia, we reviewed the charts of 182 patients who underwent PRK and were followed up for more than 12 months. The mean visual acuity was improved from 0.11 to 0.80 at 12 months postoperatively, and the mean refraction change shows from -7.94D to -0.89D. 47% of the treated eyes achieved final visual acuity of 1.0, and 97% achieved 0.5 or better. In the case of postoperative refraction, 96% of cases who were less than -6 D myopia achieved final refraction within +/- 1D, while 66% of cases who were more than -6 D myopia did. These results suggest that PRK is a safe and predictable procedure in treating mild to moderate myopia.