A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests

Angiotensin I converting enzyme (ACE) inhibitory peptides can induce antihypertensive effects after oral administration. By means of an ACE inhibitory peptide database, containing about 500 reported sequences and their IC(50) values, the different proteins in pea and whey were quantitatively evaluated as precursors for ACE inhibitory peptides. This analysis was combined with experimental data from the evolution in ACE inhibitory activity and protein degradation during in vitro gastrointestinal digestion. Pea proteins produced similar in silico scores and were degraded early in the in vitro digestion. High ACE inhibitory activity was observed after the simulated stomach phase and augmented slightly in the simulated small intestine phase. The major whey protein beta-lactoglobulin obtained the highest in silico scores, which corresponded with the fact that degradation of this protein in vitro only occurred from the simulated small intestine phase on and resulted in a 10-fold increase in the ACE inhibitory activity. Whey protein obtained total in silico scores of about 124 ml/mg, compared to 46 ml/mg for pea protein, indicating that whey protein would be a richer source of ACE inhibitory peptides than pea protein. Although beta-lactoglobulin is only partially digested, a higher ACE inhibitory activity was indeed found in the whey (IC(50) = 0.048 mg/ml) compared to the pea digest (IC(50) = 0.076 mg/ml). In silico gastrointestinal digestion of the highest scoring proteins in pea and whey, vicilin and albumin PA2, and beta-lactoglobulin, respectively, directly released a number of potent ACE inhibitory peptides. Several other ACE inhibitory sequences resisted in silico digestion by gastrointestinal proteases. Briefly, the quantitative in silico analysis will facilitate the study of precursor proteins on a large scale and the specific release of bioactive peptides.     

The angiotensin converting enzyme inhibitor captopril reduces oviposition and ecdysteroid levels in Lepidoptera

The role of angiotensin converting enzyme (ACE, peptidyl dipeptidase A) in metamorphic- and reproductive-related events in the Egyptian cotton leafworm, Spodoptera littoralis (Lepidoptera, Noctuidae) was studied by using the selective ACE inhibitor captopril. Although oral administration of captopril had no effect on larval growth, topical administration to new pupae resulted in a large decrease of successful adult formation. Oviposition and overall appearance of adults emerging from treated larvae did not differ significantly from those emerging from non-treated larvae. In contrast, topical or oral administration of captopril to newly emerged adults caused a reduction in oviposition. By evaluating the effect of captopril on ecdysteroid titers and trypsin activity, we revealed an additional physiological role for ACE. Captopril exerted an inhibitory effect on ecdysteroid levels in female but not in male adults. Larvae fed a diet containing captopril exhibited increased trypsin activity. A similar captopril-induced increase in trypsin activity was observed in female adults. In male adults, however, captopril elicited reduced levels of trypsin activity. Our results suggest that captopril downregulates oviposition by two independent pathways, one through ecdysteroid biosynthesis regulation, and the other through regulation of trypsin activity. Apparently, fecundity is influenced by a complex interaction of ACE, trypsin activity, and ecdysteroid levels.     

Nocardia asteroides culture filtrates cause dopamine depletion and cytotoxicity in PC12 cells

Experimental infection of BALB/c mice with the gram-positive bacterium Nocardia asteroides produces marked loss of nigrostriatal dopamine neurons, resulting in striatal dopamine depletion. To investigate the mechanism(s) responsible for this neuronal loss, we evaluated the influence of N. asteroides cell-free culture filtrates on rat pheochromocytoma PC12 cells, an in vitro model for dopamine neurons. Changes in cell viability and cell numbers were minimal after 24 h, but increased with longer incubation. In contrast, dopamine depletion occurred after 30 min incubation, and was greater with GUH-2 filtrate than with filtrate from the less virulent strain 10905. Incubation with the culture filtrate decreased viability in neuroblastoma and glioma cell lines, indicating that cytotoxic effects were not limited to dopaminergic cells. These findings suggest that the loss of nigrostriatal dopamine neurons and concomitant striatal dopamine depletion in Nocardia-infected mice may be due, at least in part, to the neurotoxicity of nocardial secretory products.     

Microbial populations involved in cycling of dimethyl sulfide and methanethiol in freshwater sediments

Although several microorganisms that produce and degrade methanethiol (MT) and dimethyl sulfide (DMS) have been isolated from various habitats, little is known about the numbers of these microorganisms in situ. This study reports on the identification and quantification of microorganisms involved in the cycling of MT and DMS in freshwater sediments. Sediment incubation studies revealed that the formation of MT and DMS is well balanced with their degradation. MT formation depends on the concentrations of both sulfide and methyl group-donating compounds. A most-probable number (MPN) dilution series with syringate as the growth substrate showed that methylation of sulfide with methyl groups derived from syringate is a commonly occurring process in situ. MT appeared to be primarily degraded by obligately methylotrophic methanogens, which were found in the highest positive dilutions on DMS and mixed substrates (methanol, trimethylamine [TMA], and DMS). Amplified ribosomal DNA restriction analysis (ARDRA) and 16S rRNA gene sequence analysis of the total DNA isolated from the sediments and of the DNA isolated from the highest positive dilutions of the MPN series (mixed substrates) revealed that the methanogens that are responsible for the degradation of MT, DMS, methanol, and TMA in situ are all phylogenetically closely related to Methanomethylovorans hollandica. This was confirmed by sequence analysis of the product obtained from a nested PCR developed for the selective amplification of the 16S rRNA gene from M. hollandica. The data from sediment incubation experiments, MPN series, and molecular-genetics detection correlated well and provide convincing evidence for the suggested mechanisms for MT and DMS cycling and the common presence of the DMS-degrading methanogen M. hollandica in freshwater sediments.     

Automated subcellular localization and quantification of protein expression in tissue microarrays

The recent development of tissue microarrays-composed of hundreds of tissue sections from different tumors arrayed on a single glass slide-facilitates rapid evaluation of large-scale outcome studies. Realization of this potential depends on the ability to rapidly and precisely quantify the protein expression within each tissue spot. We have developed a set of algorithms that allow the rapid, automated, continuous and quantitative analysis of tissue microarrays, including the separation of tumor from stromal elements and the sub-cellular localization of signals. Validation studies using estrogen receptor in breast carcinoma show that automated analysis matches or exceeds the results of conventional pathologist-based scoring. Automated analysis and sub-cellular localization of beta-catenin in colon cancer identifies two novel, prognostically significant tumor subsets, not detected by traditional pathologist-based scoring. Development of automated analysis technology empowers tissue microarrays for use in discovery-type experiments (more typical of cDNA microarrays), with the added advantage of inclusion of long-term demographic and patient outcome information.     

Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells

Characterization of the surface exposed membrane subproteome of human mammary epithelial cells (strain 184 A1L5) implemented lysine specific in situ labeling of the proteins using sulfosuccinimidyl-6-(biotinamido)hexanoate, followed by enrichment of the biotinylated, tryptically digested peptides, and then liquid chromatography-tandem mass spectrometry analysis of the labeled peptides. Probing the membrane subproteome in this manner yielded unambiguous identification of proteins situated on the cell surface. The method reported can be adapted to include stable isotope labeling of proteins for quantitation of changes occurring on the cell surface in response to specific perturbations.     

Different responses of tobacco antioxidant enzymes to light and chilling stress

The effect of elevated light treatment (25 degrees C, PPFD 360 mumol m-2 sec-1) or chilling temperatures combined with elevated light (5 degrees C, PPFD 360 mumol m-2 sec-1) on the activity of six antioxidant enzymes, guaiacol peroxidases, and glutathione peroxidase (GPx, EC 1.11.1.9) protein accumulation were studied in tobacco Nicotiana tabacum cv. Petit Havana SR1. Both treatments caused no photooxidative damage, but chilling caused a transient wilting. The light treatment increased the activities of ascorbate peroxidase (APx, EC 1.11.1.11) and guaiacol peroxidases while catalase (EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were unchanged. In contrast, chilling treatment did not increase any of the antioxidant enzyme activities, but decreased catalase and to a lesser extent DHAR activities. Glutathione peroxidase protein levels increased sporadically under light treatment and constantly under chilling. Both chilling and light stress caused induction of glutathione synthesis and accumulation of oxidised glutathione, although the predominant part of the glutathione pool remained in the reduced form. Antioxidant enzymes from the chilling treated plants were measured at both 25 degrees C and 5 degrees C. Measurements at 5 degrees C revealed a 3-fold reduction in catalase activity, compared with that measured at 25 degrees C, indicating that the overall reduction in catalase after four days of chilling was approximately 10-fold. The overall reduction in activity for the other antioxidant enzymes after four days of chilling was 2-fold for GR and APx, 1.5-fold for MDHAR, 3.5-fold for DHAR. The activity of SOD was the same at 25 and 5 degrees C. These results indicate that catalase and DHAR are most strongly affected by the chilling treatment and may be the rate-limiting factor of the antioxidant system at low temperatures.     

Helicoverpa armigera granulovirus interference with progression of H. zea nucleopolyhedrovirus disease in H. zea larvae

Capsular proteins from Helicoverpa armigera granulovirus (HaGV) have previously been shown to enhance H. armigera nucleopolyhedrovirus (HaSNPV) infection in H. armigera larvae. Yet, HaGV and HaS-NPV, as viable viruses, interfered with one another. In our study, we have examined the effects of co-infection of the slow-killing virus HaGV with the fast-killing virus Helicoverpa zea NPV (HzSNPV) on H. zea larvae. The mortality parameter measured was survival time. Virus stocks had 50% lethal concentrations of 3.2x10(-9) g HaGV-infected cadavers (GVC) (HaGV) and 32 occlusion bodies (HzSNPV) per cup. Average survival times were 16.8 and 5.5 days for larvae treated with HaGV and HzSNPV, respectively; death of HzSNPV-treated larvae was as early as 72 h posttreatment. In co-infection experiments in which larvae were treated concurrently with both viruses, the viruses competed in typical fashion for host resources. However, interference with disease progression in HzSNPV-fed larvae occurred even when HaGV was fed to larvae up to 36 h after the NPV, a time at which NPV infection should have been well established in host larvae. At death, co-infected larvae were observed microscopically to be filled with HaGV granules rather than HzSNPV polyhedra. The time study results imply that HaGV might be outcompeting HzSNPV by inhibiting its replication. We also observed that H. zea larvae treated with high dosages of HaGV (> or =3x10(-5) g GVC) were initially stunted but had survival times similar to those of larvae treated with lower dosages.