Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Quantifying the relationship between disease severity and the amount of Aphanomyces euteiches detected in roots of alfalfa and pea with a real-time PCR assay

A real-time PCR assay was used to quantify the relationship in alfalfa and pea between disease severity and the amount of Aphanomyces euteiches detected in roots. The study included isolates of race 1 and race 2 of the alfalfa pathovar of A. euteiches and an isolate obtained from diseased pea. Spearman rank correlations between pathogen DNA content and disease severity index (DSI) ratings were positive ( ? 0.57) and significant (P  0.0007) for individual alfalfa plants, bulked alfalfa plant samples, and individual pea plants. In all experiments, significantly more pathogen was detected in susceptible populations than in resistant populations. The results clearly demonstrate that resistance to A. euteiches in both alfalfa and pea is characterized by a reduction in pathogen colonization relative to levels observed for susceptible reactions. The assay was very specific for A. euteiches, producing very linear assays with DNA extracted from pathogen isolates obtained from alfalfa, pea, and bean. Possible applications of the assay in conjunction with other real-time PCR assays specific to other legume pathogens are discussed in relation to simultaneous disease screening for multiple plant pathogens and the study of microbial population dynamics in mixed plant infections.     

Geographical and temporal variation in the diet of Bank Cormorants Phalacrocorax neglectus in South Africa

The Bank Cormorant Phalacrocorax neglectus is endemic to the Benguela upwelling ecosystem off southwest Africa and is classified as Endangered owing to a recent large reduction in its number. It is thought that food scarcity, including a decreased abundance of West Coast rock lobster Jasus lalandii, has been a major driver of the decrease, yet its diet in South Africa is poorly known. We collected 941 pellets regurgitated by Bank Cormorants, at 18 South African breeding colonies during 1975–1985, and 1 523 pellets at 17 colonies during 1995–2002. The species composition of the diet (% numbers) was significantly different between the two periods, with widespread decreases in proportions of rock lobster in the west and of octopus and cuttlefish Sepia spp. at most localities. These taxa were replaced in the diet by fish, including Gobiidae and Clinidae. The pelagic goby Sufflogobius bibarbatus, an important prey of Bank Cormorants in Namibia, was absent from pellets collected in 1975–1985 but common at northern localities from 1995–2002. Composition of the diet by frequency of occurrence was only determined for 1995–2002, when rock lobster was present in 67% of all samples collected, cuttlefish in 39%, and Clinidae in 32%. Data for 1975–1985 and 1995–2002 showed that carapace lengths of rock lobsters eaten by Bank Cormorants averaged 56 mm (range 22–82 mm) and 50 mm (range 22–75 mm), respectively, which compares to the minimum legal size of 75 mm for fisheries in South Africa. This energy- rich prey item was an important constituent of the diet in the winter breeding period.     

In vitro biosynthesis, core glycosylation and membrane integration of opsin

A membrane-integrated , core-glycosylated form of bovine opsin was synthesized in vitro when bovine retina mRNA was translated in a wheat germ cell-free system supplemented with dog pancreas microsomal vesicles; glycosylation and integration of opsin into membranes were coupled to translation. Proteolysis with themolysin was used to probe the orientation of opsin within the dog pancreas microsomal membrane, and to compare it with that of opsin in rod cell disk membranes isolated from bovine retina. Intact microsomal or disk vesicles were required for production of discrete, membrane-associated thermolysin fragments of opsin; no discrete opsin fragments were detected when membranes were incubated with thermolysin in the presence of the nonionic detergent, Triton X-100. The major opsin fragments produced by themosylin treatment of intact microsomal vesicles resembled those from disk vesicles in their size, oligosaccharide content, and order of appearance. In each case, the first cleavage of opsin took place at the COOH-terminus, generating a glycosylated fragment, O’, which was only slightly smaller than intact opsin. Both the microsomal and disk membrane forms of O’ were next cleaved internally; glycosylated fragments of similar sizes in both cases were detected which were derived from the NH(2)-terminal portion of O’. Several smaller NH(2)-terminal fragments of opsin were detected only in thermolysin-treated microsomal membranes, and not in disk membranes. The data suggest that the topology of opsin integrated into dog pancreas microsomal vesicles is similar to that in rod cell disk vesicles, although not identical. In each case, the glycosylated NH(2)-terminal region of opsin is located within the lumen of the vesicle, while discrete COOH-terminal and internal segments of opsin apparently emerge at the outer, cytoplasmic face of the membrane. Thus, opsin in the heterologous microsomal membrane, like its counterpart in the native disk membrane, may cross the bilayer at least three times. The internal domain of the polypeptide that emerges at the outer membrane surface is apparently more highly exposed in the case of opsin in microsomal membranes, evidenced by the additional internal thermolysin cleavage sites detected.     

Attentional modulation of central odor processing

Two studies were conducted to investigate the influence of attention on the components of the chemosensory event-related potential (CSERP). In the first study the odors linalool and eugenol were delivered to six male subjects, in the second study three male and two female subjects were presented with their own body odor (axillary hair) and the body odor of a same sex donor. In both studies the odors were presented in an oddball paradigm under ignore and attend conditions via a constant- flow olfactometer. In the ignore condition attention was diverted from the odors with a distractor task, while in the attend condition the subjects were asked to respond to the infrequently occurring odor. In both studies the allocation of attention led to a decrease in the latency of the early components (N1, P2, N2) and to an increase in the amplitude of the late positivities. The modulation of the early components suggests that attentional gating in olfaction might already be effective at an early processing level.      

Effect of fruit maturity,seed weight and storage time on the viability and germination of the seed of candelilla (<i>Euphorbia antisiphylitica</i> Zucc.)

Candelilla (Euphorbia antisiphylitica Zucc.) is a very important plant resource in the arid lands of Northern Mexico. This is because the wax content coating the stem has unique properties which have been useful for multiple applications in the food industry, electronics, cosmetics, etc. However, the intensive exploitation of this resource has caused a great decrease in the populations of this species making necessary to consider strategies for their conservation and sustainable use. One of the primary needs with regeneration purposes is to know their reproductive processes, particularly the biotic and/or abiotic factors that determine the viability and germination of seeds. The present study evaluated the (1) germination and seed viability in relation to the ripeness degree of the fruit at the time of collection, (2) weight of the seed (low, medium and high), and (3) storage time (1, 3, and 5 months). Fruits from four locations, two in the State of Coahuila (Las Coloradas and Candela) and two in the State of Nuevo Leon (Icamole 1 and Icamole 2), were collected. Three germination assays were carried out corresponding to each month of storage. Seed viability was determined by the tetrazolium test. The average weight of the candelilla seeds was 0.0029 ± 0.0010 g, with extreme average values of 0.0018 ± 0.0006 g at Las Coloradas and 0.0036 ± 0.0010 g in Icamole 2. Those seeds with heavier weight obtained from red fruits and with 1 month of storage showed the highest average percentage of viability (66.87 ± 24.19%). At the same time, seeds with around average weight, obtained from red fruits and five months of storage, showed the highest average germination percentage (50.00 ± 9.42%).     

Evaluating eukaryotic secreted protein prediction

Background  

Improvements in protein sequence annotation and an increase in the number of annotated protein databases has fueled development of an increasing number of software tools to predict secreted proteins. Six software programs capable of high throughput and employing a wide range of prediction methods, SignalP 3.0, SignalP 2.0, TargetP 1.01, PrediSi, Phobius, and ProtComp 6.0, are evaluated.     

Experience in the application of Java Technologies in telemedicine

Java language has been demonstrated to be an effective tool in supporting medical image viewing in Russia. This evaluation was completed by obtaining a maximum of 20 images, depending on the client's computer workstation from one patient using a commercially available computer tomography (CT) scanner. The images were compared against standard CT images that were viewed at the site of capture. There was no appreciable difference. The client side is a lightweight component that provides an intuitive interface for end users. Each image is loaded in its own thread and the user can begin work after the first image has been loaded. This feature is especially useful on slow connection speed, 9.6 Kbps for example. The server side, which is implemented by the Java Servlet Engine works more effective than common gateway interface (CGI) programs do. Advantages of the Java Technology place this program on the next level of application development. This paper presents a unique application of Java in telemedicine.     

Comparison of computational methods for identifying translation initiation sites in EST data

Background

Expressed Sequence Tag (EST) sequences are generally single-strand, single-pass sequences, only 200–600 nucleotides long, contain errors resulting in frame shifts, and represent different parts of their parent cDNA. If the cDNAs contain translation initiation sites, they may be suitable for functional genomics studies. We have compared five methods to predict translation initiation sites in EST data: first-ATG, ESTScan, Diogenes, Netstart, and ATGpr.

Results

A dataset of 100 EST sequences, 50 with and 50 without, translation initiation sites, was created. Based on analysis of this dataset, ATGpr is found to be the most accurate for predicting the presence versus absence of translation initiation sites. With a maximum accuracy of 76%, ATGpr more accurately predicts the position or absence of translation initiation sites than NetStart (57%) or Diogenes (50%). ATGpr similarly excels when start sites are known to be present (90%), whereas NetStart achieves only 60% overall accuracy. As a baseline for comparison, choosing the first ATG correctly identifies the translation initiation site in 74% of the sequences. ESTScan and Diogenes, consistent with their intended use, are able to identify open reading frames, but are unable to determine the precise position of translation initiation sites.

Conclusions

ATGpr demonstrates high sensitivity, specificity, and overall accuracy in identifying start sites while also rejecting incomplete sequences. A database of EST sequences suitable for validating programs for translation initiation site prediction is now available. These tools and materials may open an avenue for future improvements in start site prediction and EST analysis.