Genomic mapping of cAMP receptor protein (CRPMt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor

Chromatin immunoprecipitation identified 191 binding sites of Mycobacterium tuberculosis cAMP receptor protein (CRPMt) at endogenous expression levels using a specific alpha-CRPMt antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRPMt binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRPMt during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRPMt-binding site represented only a minor portion of this transcriptional reprogramming with similar to 19% of those representing transcriptional regulators potentially controlled by CRPMt. The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRPMt can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRPMt-binding sites.

New insight into the architecture of oxy-anion pocket in unliganded conformation of GAT domains: A MD-simulation study

Human Guanine Monophosphate Synthetase (hGMPS) converts XMP to GMP, and acts as a bifunctional enzyme with N-terminal ``glutaminase'' (GAT) and C-terminal ``synthetase'' domain. The enzyme is identified as a potential target for anticancer and immunosuppressive therapies. GAT domain of enzyme plays central role in metabolism, and contains conserved catalytic residues Cys104, His190, and Glu192. MD simulation studies on GAT domain suggest that position of oxyanion in unliganded conformation is occupied by one conserved water molecule (W1), which also stabilizes that pocket. This position is occupied by a negatively charged atom of the substrate or ligand in ligand bound crystal structures. In fact, MD simulation study of Ser75 to Val indicates that W1 conserved water molecule is stabilized by Ser75, while Thr152, and His190 also act as anchor residues to maintain appropriate architecture of oxyanion pocket through water mediated H-bond interactions. Possibly, four conserved water molecules stabilize oxyanion hole in unliganded state, but they vacate these positions when the enzyme (hGMPS)-substrate complex is formed. Thus this study not only reveals functionally important role of conserved water molecules in GAT domain, but also highlights essential role of other non-catalytic residues such as Ser75 and Thr152 in this enzymatic domain. The results from this computational study could be of interest to experimental community and provide a testable hypothesis for experimental validation. Conserved sites of water molecules near and at oxyanion hole highlight structural importance of water molecules and suggest a rethink of the conventional definition of chemical geometry of inhibitor binding site.

Resonant Raman characterisation of ultra-thin nano-protective carbon layers for magnetic storage devices

Carbon thin films are very important as protective coatings for a wide range of applications such as magnetic storage devices. The key parameter of interest is the sp3 fraction, since it controls the mechanical properties of the film. Visible Raman spectroscopy is a very popular technique to determine the carbon bonding. However, the visible Raman spectra mainly depend on the configuration and clustering of the sp2 sites. This can result in the Raman spectra of different samples looking similar albeit having a different structure. Thus, visible Raman alone cannot be used to derive the sp3 content. Here we monitor the carbon bonding by using a combined study of Raman spectra taken at two wavelengths (514 and 244 nm). We show how the G peak dispersion is a very useful parameter to investigate the carbon samples and we endorse it as a production-line characterisation tool. The dispersion is proportional to the degree of disorder, thus making it possible to distinguish between graphitic and diamond-like carbon. © 2003 Elsevier B.V. All rights reserved.

Report on Marine Mammal Unusual Mortality Event UMESE0501Sp: Multispecies Mass Stranding of Pilot Whales (Globicephala macrorhynchus), Minke Whale (Balaenoptera acutorostrata), and Dwarf Sperm Whales (Kogia sima) in North Carolina on 15-16 January 2005

On 15-16 January 2005, three offshore species of cetaceans (33 short-finned pilot whales, Globicephala macrorhynchus, one minke whale, Balaenoptera acutorostrata, and two dwarf sperm whales, Kogia sima) stranded alive on the beaches of North Carolina. The pilot whales stranded near Oregon Inlet, the minke whale in northern North Carolina, and the dwarf sperm whales near Cape Hatteras. Live strandings of three species in one weekend was unique in North Carolina and qualified as an Unusual Mortality Event. Gross necropsies were conducted on 16-17 January 2005 on 27 pilot whales, two dwarf sperm whales, and the minke whale. Samples were collected for clinical pathology, parasitology, gross pathology, histopathology, microbiology and serology. There was variation in the number of animals sampled for each collection type, however, due to carcasses washing off the beach or degradation in carcass condition during the course of the response. Comprehensive histologic examination was conducted on 16 pilot whales, both dwarf sperm whales, and the minke whale. Limited organ or only head tissue suites were obtained from nine pilot whales. Histologic examination of tissues began in February 2005 and concluded in December 2005 when final sampling was concluded. Neither the pilot whales nor dwarf sperm whales were emaciated although none had recently ingested prey in their stomachs. The minke whale was emaciated; it was likely a dependent calf that became separated from the female. Most serum biochemistry abnormalities appear to have resulted from the stranding and indicated deteriorating condition from being on land for an extended period. Three pilot whales had clinical evidence of pre-existing systemic inflammation, which was supported by histopathologic findings. Although gross and histologic lesions involving all organ systems were noted, consistent lesions were not observed across species. Verminous pterygoid sinusitis and healed fishery interactions were seen in pilot whales but neither of these changes were causes of debilitation or death. In three pilot whales and one dwarf sperm whale there was evidence of clinically significant disease in postcranial tissues which led to chronic debilitation. Cardiovascular disease was present in one pilot whale and one dwarf sperm whale; musculoskeletal disease and intra-abdominal granulomas were present in two pilot whales. These lesions were possible, but not definitive, causal factors in the stranding. Remaining lesions were incidental or post-stranding. The minke whale and three of five tested pilot whales had positive morbillivirus titers (≥1:8 with one at >1:256), but there was no histologic evidence of active viral infection. Parasites (nematodes, cestodes, and trematodes) were collected from 26 pilot whales and two dwarf sperm whales. Sites of collection included stomach, nasal/pterygoid, peribullar sinuses, blubber, and abdominal cavity. Parasite species, locations and loads were within normal limits for free-ranging cetaceans and were not considered causative for the stranding event. Gas emboli lesions which were considered consistent with or diagnostic of sonarassociated strandings of beaked whales or small cetaceans were not found in the whales stranded as part of UMESE0501Sp. Twenty-five heads were examined with nine specific anatomic locations of interest: extramandibular fat, intramandibular fat, auditory meatus, peribullar acoustic fat, peribullar soft tissue, peribullar sinus, pterygoid sinus, melon, and brain. The common finding in all examined heads was verminous pterygoid sinusitis. Intramandibular adipose tissue reddening, typically adjacent to the vascular plexus, was observed in some individuals and could represent localized hemorrhage resulting from vascular rete rupture, hypostatic congestion, or erythrocyte rupture during the freeze/thaw cycle. One cetacean had peracute to acute subdural hemorrhage that likely occurred from thrashing on the beach post-stranding, although its occurrence prior to stranding cannot be excluded. Information provided to NMFS by the U.S. Navy indicated routine tactical mid-frequency sonar operations from individual surface vessels over relatively short durations and small spatial scales within the area and time period investigated. No marine mammals were detected by marine mammal observers on operational vessels; standard operating procedure for surface naval vessels operating mid-frequency sonar is the use of trained visual lookouts using high-powered binoculars. Sound propagation modeling using information provided to NMFS indicated that acoustic conditions in the vicinity likely depended heavily on position of the receivers (e.g., range, bearing, depth) relative to that of the sources. Absent explicit information on the location of animals meant that it was not possible to estimate received acoustic exposures from active sonar transmissions. Nonetheless, the event was associated in time and space with naval activity using mid-frequency active sonar. It also had a number of features in common (e.g., the “atypical” distribution of strandings involving multiple offshore species, all stranding alive, and without evidence of common infectious or other disease process) with other sonar-related cetacean mass stranding events. Given that this event was the only stranding of offshore species to occur within a 2-3 day period in the region on record (i.e., a very rare event), and given the occurrence of the event simultaneously in time and space with a naval exercise using active sonar, the association between the naval sonar activity and the location and timing of the event could be a causal rather than a coincidental relationship. However, evidence supporting a definitive association is lacking, and, in particular, there are differences in operational/environmental characteristics between this event and previous events where sonar has apparently played a role in marine mammal strandings. This does not preclude behavorial avoidance of noise exposure. No harmful algal blooms were present along the Atlantic coast south of the Chesapeake Bay during the months prior to the event. Environmental conditions, including strong winds, changes in upwelling- to downwelling-favorable conditions, and gently sloping bathymetry, were consistent with conditions which have been correlated with other mass strandings. In summary, we did not find commonality in gross and histologic lesions that would indicate a single cause for this stranding event. Three pilot whales and one dwarf sperm whale had debilitating conditions identified that could have contributed to stranding, one pilot whale had a debilitating condition (subdural hemorrhage) that could have been present prior to or resulting from stranding. While the pilot and dwarf sperm whale strandings may have had a common cause, the minke whale stranding was probably just coincidental. On the basis of examination of physical evidence in the affected whales, however, we cannot definitively conclude that there was or was not a causal link between anthropogenic sonar activity or environmental conditions (or a combination of these factors) and the strandings. Overall, the cause of UMESE0501Sp in North Carolina is not and likely will not be definitively known. (PDF contains 240 pages)

Characterization of the benthos, marine debris and bottom fish at Gray’s Reef National Marine Sanctuary

Executive Summary: Baseline characterization of resources is an essential part of marine protected area (MPA) management and is critical to inform adaptive management. Gray’s Reef National Marine Sanctuary (GRNMS) currently lacks adequate characterization of several key resources as identified in the 2006 Final Management Plan. The objectives of this characterization were to fulfill this need by characterizing the bottom fish, benthic features, marine debris, and the relationships among them for the different bottom types within the sanctuary: ledges, sparse live bottom, rippled sand, and flat sand. Particular attention was given to characterizing the different ledge types, their fish communities, and the marine debris associated with them given the importance of this bottom type to the sanctuary. The characterization has been divided into four sections. Section 1 provides a brief overview of the project, its relevance to sanctuary needs, methods of site selection, and general field procedures. Section 2 provides the survey methods, results, discussion, and recommendations for monitoring specific to the benthic characterization. Section 3 describes the characterization of marine debris. Section 4 is specific to the characterization of bottom fish. Field surveys were conducted during August 2004, May 2005, and August 2005. A total of 179 surveys were completed over ledge bottom (n=92), sparse live bottom (n=51), flat sand (n=20), and rippled sand (n=16). There were three components to each field survey: fish counting, benthic assessment, and quantification of marine debris. All components occurred within a 25 x 4 m belt transect. Two divers performed the transect at each survey site. One diver was responsible for identification of fish species, size, and abundance using a visual survey. The second diver was responsible for characterization of benthic features using five randomly placed 1 m2 quadrats, measuring ledge height and other benthic structures, and quantifying marine debris within the entire transect. GRNMS is composed of four main bottom types: flat sand, rippled sand, sparsely colonized live bottom, and densely colonized live bottom (ledges). Independent evaluation of the thematic accuracy of the GRNMS benthic map produced by Kendall et al. (2005) revealed high overall accuracy (93%). Most discrepancies between map and diver classification occurred during August 2004 and likely can be attributed to several factors, including actual map or diver errors, and changes in the bottom type due to physical forces. The four bottom types have distinct physical and biological characteristics. Flat and rippled sand bottom types were composed primarily of sand substrate and secondarily shell rubble. Flat sand and rippled sand bottom types were characterized by low percent cover (0-2%) of benthic organisms at all sites. Although the sand bottom types were largely devoid of epifauna, numerous burrows indicate the presence of infaunal organisms. Sparse live bottom and ledges were colonized by macroalgae and numerous invertebrates, including coral, gorgonians, sponges, and “other” benthic species (such as tunicates, anemones, and bryozoans). Ledges and sparse live bottom were similar in terms of diversity (H’) given the level of classification used here. However, percent cover of benthic species, with the exception of gorgonians, was significantly greater on ledge than on sparse live bottom. Percent biotic cover at sparse live bottom ranged from 0.7-26.3%, but was greater than 10% at only 7 out of 51 sites. Colonization on sparse live bottom is likely inhibited by shifting sands, as most sites were covered in a layer of sediment up to several centimeters thick. On ledge bottom type, percent cover ranged from 0.42-100%, with the highest percent cover at ledges in the central and south-central region of GRNMS. Biotic cover on ledges is influenced by local ledge characteristics. Cluster analysis of ledge dimensions (total height, undercut height, undercut width) resulted in three main categories of ledges, which were classified as short, medium, and tall. Median total percent cover was 97.6%, 75.1%, and 17.7% on tall, medium, and short ledges, respectively. Total percent cover and cover of macroalgae, sponges, and other organisms was significantly lower on short ledges compared to medium and tall ledges, but did not vary significantly between medium and tall ledges. Like sparse live bottom, short ledges may be susceptible to burial by sand, however the results indicate that ledge height may only be important to a certain threshold. There are likely other factors not considered here that also influence spatial distribution and community structure (e.g., small scale complexity, ocean currents, differential settlement patterns, and biological interactions). GRNMS is a popular site for recreational fishing and boating, and there has been increased concern about the accumulation of debris in the sanctuary and potential effects on sanctuary resources. Understanding the types, abundance, and distribution of debris is essential to improving debris removal and education efforts. Approximately two-thirds of all observed debris items found during the field surveys were fishing gear, and about half of the fishing related debris was monofilament fishing line. Other fishing related debris included leaders and spear gun parts, and non-gear debris included cans, bottles, and rope. The spatial distribution of debris was concentrated in the center of the sanctuary and was most frequently associated with ledges rather than at other bottom types. Several factors may contribute to this observation. Ledges are often targeted by fishermen due to the association of recreationally important fish species with this bottom type. In addition, ledges are structurally complex and are often densely colonized by biota, providing numerous places for debris to become stuck or entangled. Analysis of observed boat locations indicated that higher boat activity, which is an indication of fishing, occurs in the center of the sanctuary. On ledges, the presence and abundance of debris was significantly related to observed boat density and physiographic features including ledge height, ledge area, and percent cover. While it is likely that most fishing related debris originates from boats inside the sanctuary, preliminary investigation of ocean current data indicate that currents may influence the distribution and local retention of more mobile items. Fish communities at GRNMS are closely linked to benthic habitats. A list of species encountered, probability of occurrence, abundance, and biomass by habitat is provided. Species richness, diversity, composition, abundance, and biomass of fish all showed striking differences depending on bottom type with ledges showing the highest values of nearly all metrics. Species membership was distinctly separated by bottom type as well, although very short, sparsely colonized ledges often had a similar community composition to that of sparse live bottom. Analysis of fish communities at ledges alone indicated that species richness and total abundance of fish were positively related to total percent cover of sessile invertebrates and ledge height. Either ledge attribute was sufficient to result in high abundance or species richness of fish. Fish diversity (H`) was negatively correlated with undercut height due to schools of fish species that utilize ledge undercuts such as Pareques species. Concurrent analysis of ledge types and fish communities indicated that there are five distinct combinations of ledge type and species assemblage. These include, 1) short ledges with little or no undercut that lacked many of the undercut associated species except Urophycis earlii ; 2) tall, heavily colonized, deeply undercut ledges typically with Archosargus probatocephalus, Mycteroperca sp., and Pareques sp.; 3) tall, heavily colonized but less undercut with high occurrence of Lagodon rhomboides and Balistes capriscus; 4) short, heavily colonized ledges typically with Centropristis ocyurus, Halichoeres caudalis, and Stenotomus sp.; and 5) tall, heavily colonized, less undercut typically with Archosargus probatocephalus, Caranx crysos and Seriola sp.. Higher levels of boating activity and presumably fishing pressure did not appear to influence species composition or abundance at the community level although individual species appeared affected. These results indicate that merely knowing the basic characteristics of a ledge such as total height, undercut width, and percent cover of sessile invertebrates would allow good prediction of not only species richness and abundance of fish but also which particular fish species assemblages are likely to occur there. Comparisons with prior studies indicate some major changes in the fish community at GRNMS over the last two decades although the causes of the changes are unknown. Species of interest to recreational fishermen including Centropristis striata, Mycteroperca microlepis, and Mycteroperca phenax were examined in relation to bottom features, areas of assumed high versus low fishing pressure, and spatial dispersion. Both Mycteroperca species were found more frequently when undercut height of ledges was taller. They often were found together in small mixed species groups at ledges in the north central and southwest central regions of the sanctuary. Both had lower mode size and proportion of fish above the fishery size limit in heavily fished areas of the sanctuary (i.e. high boat density) despite the presence of better habitat in that region. Black sea bass, C. striata, occurred at 98% of the ledges surveyed and appeared to be evenly distributed throughout the sanctuary. Abundance was best explained by a positive relationship with percent cover of sessile biota but was also negatively related to presence of either Mycteroperca species. This may be due to predation by the Mycteroperca species or avoidance of sites where they are present by C. striata. Suggestions for monitoring bottom features, marine debris, and bottom fish at GRNMS are provided at the end of each chapter. The present assessment has established quantitative baseline characteristics of many of the key resources and use issues at GRNMS. The methods can be used as a model for future assessments to track the trajectory of GRNMS resources. Belt transects are ideally suited to providing efficient and quantitative assessment of bottom features, debris, and fish at GRNMS. The limited visibility, sensitivity of sessile biota, and linear nature of ledge habitats greatly diminish the utility of other sampling techniques. Ledges should receive the bulk of future characterization effort due to their importance to the sanctuary and high variability in physical structure, benthic composition, and fish assemblages. (PDF contains 107 pages.)

Deep reef fish surveys by submersible on Alderdice, McGrail, and Sonnier Banks in the Northwestern Gulf of Mexico

Submersible surveys at numerous reefs and banks in the northwestern Gulf of Mexico (NWGOM) were conducted as part of the Sustainable Seas Expedition (SSE) during July/August 2002 to identify reef fish communities, characterize benthic habitats, and identify deep coral reef ecosystems. To identify the spatial extent of hard bottom reef communities, the Flower Garden Banks National Marine Sanctuary (FGBNMS) and the U.S. Geological Survey (USGS) mapped approximately 2000 km2 of the Northwestern Gulf of Mexico (NWGOM) continental shelf during June 2002 with high-resolution multibeam bathymetry. Previous investigations conducted on the features of interest (with the exceptions of East and West Flower Garden and Sonnier Banks, accessible by SCUBA) had not been conducted since the 1970s and 1980s, and did not have the use of high-resolution maps to target survey sites. The base maps were instrumental in navigating submersibles to specific features at each study site during the Sustainable Seas Expedition (SSE)—a submersible effort culminating from a partnership between the National Atmospheric and Oceanic Administration (NOAA) and the National Geographic Society (NGS). We report the initial findings of our submersible surveys, including habitat and reef fish diversity at McGrail, Alderdice, and Sonnier Banks. A total of 120 species and 40,724 individuals were identified from video surveys at the three banks. Planktivorous fishes constituted over 87% by number for the three banks, ranging from 81.4% at Sonnier Banks to 94.3% at Alderdice Bank, indicating a direct link to pelagic prey communities, particularly in the deep reef zones. High numbers of groupers, snappers, jacks, and other fishery species were observed on all three features. These sites were nominated as Habitat Areas of Particular Concern (HAPC) by the Gulf of Mexico Fishery Council in March 2004. Data obtained during this project will contribute to benthic habitat characterization and assessment of the associated fish communities through future SCUBA, ROV, and submersible missions, and allow comparisons to other deep reef ecosystems found throughout the Gulf of Mexico and western Atlantic Ocean.

Transport of pollutants through the water and subsurface soil using simulated acid rain

The mobility of heavy metals (Zn, Cd, Pb and Ni) was studied in the laboratory acidic leaching two different soils around Ibadan with simulated acid rain. The sampling was carried out from two different sites viz: Orogun and Ilupeju respectively. For Orogun site a depth of 128cm was reached (consisting of four horizons). Different length of polyvinyl chloride (PVC) pipes were cut for different soil horizon depth as observed on the field. The PVC pipes were packed with requires masses of soil. This is then leached using simulated acid rain of different pH of 2.0, 4.0, 6.0 and 8.0 after spiking with known volume of standard solution of metals of interest. It was found that simulated acid rain enhanced the mobility of metals in solution. The pH, Cation Exchange capacity, % clay and organic matter were found to contributed majority to the mobility of metals. Generally as observed, the mobility of metal was to follow the order Zn>Ni>Pb>Cd as the soil is becoming more acidic

Natural habitat and population control mechanism of Crassula helmsii (Australian Swamp Stonecrop) in Australia

The plant Crassula helmsii (Kirk) Cochayne, was likely to become widely distributed and to dominate many damp and wet areas of nature reserves, recreational waters and agricultural drainage of Britain. The aim of this report was to study Australian Swamp Stonecrop in its natural habitat where it is in balance with its environment. This contrasts with its rapid and widespread distribution in the U.K. where its growth interferes with the use of fisheries and amenity lakes but also reduces the value of nature reserves and sites of special scientific interest by suppressing native flora. It was proposed to observe its growth at a variety of sites over its natural distribution and to include some environmental factors, e.g. water-level, water-chemistry (nutrients, acidity and alkalinity), frost-tolerance, salinity, with the help of portable sensors, locally-available services or data. 8 weeks of travel in Australia allowed time to study the plant in its natural habitat including the coastal areas of the southern half of the continent i.e . Western Australia, South Australia, New South Wales, Victoria, Tasmania and southern Queensland. The overall objective was to determine the environmental range by visits to selected sites of Crassula helmsii over its geographic range.

Report on the 2001 stock assessment of the River Darwen catchment

The River Darwen is a highly impacted Lancashire river with very little known about its fishery interest above the impassable weir at Salmesbury Bottoms. Below the weir there are populations of coarse fish around the confluence with the River Ribble. To the knowledge of local bailiff staff, prior to 1996 the fish population in the middle and upper River Darwen had never been surveyed by electric fishing. In order to address this lack of knowledge, a survey was undertaken during the summer of 1996 with the aim of evaluating the salmonid and cyprinid fish population in the river. Twenty two sites were surveyed by electric fishing between June 11th and July 11th 1996. Information was gathered on the presence and density of fish populations in the river catchment, and analysed according to the National Fisheries Classification Scheme in order to determine how these populations compare nationally with sites of similar habitat features. From this report, recommendations were made to improve and develop the fishery potential in relation to water quality and habitat prioritising areas classed as being Ashless. It was recommended that juvenile coarse fish should be stocked in the Houghton Bottoms area. This area has excellent fishery habitat and was found to contain a minor coarse fish population. Water quality in this stretch of river was thought to be good enough to establish a major coarse fish population. Fish were introduced for the first time in 1998 at Houghton Bottoms from the Agency's Leyland Fish Farm. 3000 each of Roach, Chub and Dace were introduced. Further fish introductions occurred in 2000 with the stocking of 1000 Chub, again from the Agency's Leyland Fish Farm in the Lower Darwen and Witton areas of the main river on a trial basis.

An evaluation of volumetric interest points

This paper presents the first performance evaluation of interest points on scalar volumetric data. Such data encodes 3D shape, a fundamental property of objects. The use of another such property, texture (i.e. 2D surface colouration), or appearance, for object detection, recognition and registration has been well studied; 3D shape less so. However, the increasing prevalence of depth sensors and the diminishing returns to be had from appearance alone have seen a surge in shape-based methods. In this work we investigate the performance of several detectors of interest points in volumetric data, in terms of repeatability, number and nature of interest points. Such methods form the first step in many shape-based applications. Our detailed comparison, with both quantitative and qualitative measures on synthetic and real 3D data, both point-based and volumetric, aids readers in selecting a method suitable for their application. © 2011 IEEE.

A performance evaluation of volumetric 3D interest point detectors

This paper presents the first performance evaluation of interest points on scalar volumetric data. Such data encodes 3D shape, a fundamental property of objects. The use of another such property, texture (i.e. 2D surface colouration), or appearance, for object detection, recognition and registration has been well studied; 3D shape less so. However, the increasing prevalence of 3D shape acquisition techniques and the diminishing returns to be had from appearance alone have seen a surge in 3D shape-based methods. In this work, we investigate the performance of several state of the art interest points detectors in volumetric data, in terms of repeatability, number and nature of interest points. Such methods form the first step in many shape-based applications. Our detailed comparison, with both quantitative and qualitative measures on synthetic and real 3D data, both point-based and volumetric, aids readers in selecting a method suitable for their application. © 2012 Springer Science+Business Media, LLC.

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples. Copyright (C) 2004 John Wiley Sons, Ltd.

Characterization of the acid sites in dealuminated nanosized HZSM-5 zeolite with the probe molecule trimethylphosphine

The acid sites in dealuminated HZSM-5 zeolite with crystal sizes down to the nanoscale were firstly characterized by the probe molecule trimethylphosphine (TMP). As evidenced by the combination of P-31 CP/MAS NMR, Al-27 MAS and H-1 --> Al-27 CP/MAS NMR measurements, the Bronsted acid sites of both microsized and nanosized HZSM-5 could be decreased upon the dealumination of zeolitic framework after hydrothermal treatment. At the same time, the appearance of Lewis acid sites was observed. The dealuminated nanosized HZSM-5 is easier to form Lewis acid sites than microsized HZSM-5, and the type of Lewis acid sites in nanosized HSM-5 is more than one. In addition, the origin of Lewis acid sites is mainly associated with the aluminum at ca. 30 ppm, in the Al-27 MAS NMR spectra, and only a part of which in the dealuminated HZSM-5 zeolite acts as Lewis acid sites. (C) 2002 Elsevier Science B.V. All rights reserved.

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site. proved to be the preferential one, being 6.50 x 10(5) 1.94 x 10(6) and 8.94 x 10(5). respectively. In addition. it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions. (C) 2002 Elsevier Science B.V. All rights reserved.

Do diabetic veterans use the Internet? Self-reported usage, skills, and interest in using My HealtheVet Web portal.

OBJECTIVE: The Veterans Health Administration has developed My HealtheVet (MHV), a Web-based portal that links veterans to their care in the veteran affairs (VA) system. The objective of this study was to measure diabetic veterans' access to and use of the Internet, and their interest in using MHV to help manage their diabetes. MATERIALS AND METHODS: Cross-sectional mailed survey of 201 patients with type 2 diabetes and hemoglobin A(1c) > 8.0% receiving primary care at any of five primary care clinic sites affiliated with a VA tertiary care facility. Main measures included Internet usage, access, and attitudes; computer skills; interest in using the Internet; awareness of and attitudes toward MHV; demographics; and socioeconomic status. RESULTS: A majority of respondents reported having access to the Internet at home. Nearly half of all respondents had searched online for information about diabetes, including some who did not have home Internet access. More than a third obtained "some" or "a lot" of their health-related information online. Forty-one percent reported being "very interested" in using MHV to help track their home blood glucose readings, a third of whom did not have home Internet access. Factors associated with being "very interested" were as follows: having access to the Internet at home (p < 0.001), "a lot/some" trust in the Internet as a source of health information (p = 0.002), lower age (p = 0.03), and some college (p = 0.04). Neither race (p = 0.44) nor income (p = 0.25) was significantly associated with interest in MHV. CONCLUSIONS: This study found that a diverse sample of older VA patients with sub-optimally controlled diabetes had a level of familiarity with and access to the Internet comparable to an age-matched national sample. In addition, there was a high degree of interest in using the Internet to help manage their diabetes.