Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

A finite-rate-of-innovation signal sampling perspective of the source-filter model of speech production

We consider the speech production mechanism and the asso- ciated linear source-filter model. For voiced speech sounds in particular, the source/glottal excitation is modeled as a stream of impulses and the filter as a cascade of second-order resonators. We show that the process of sampling speech signals can be modeled as filtering a stream of Dirac impulses (a model for the excitation) with a kernel function (the vocal tract response),and then sampling uniformly. We show that the problem of esti- mating the excitation is equivalent to the problem of recovering a stream of Dirac impulses from samples of a filtered version. We present associated algorithms based on the annihilating filter and also make a comparison with the classical linear prediction technique, which is well known in speech analysis. Results on synthesized as well as natural speech data are presented.

Effect of a natural mutation in the 5 ` untranslated region on the translational control of p53 mRNA

Tumor-suppressor protein p53, the `guardian of the genome', is critical in maintaining cellular homeostasis and genomic stability. Earlier, we have reported the discovery of internal ribosome entry sites (IRESs) within the p53 mRNA that regulate the translation of the full length and its N-terminal-truncated isoform, Delta N-p53. Polypyrimidine tract-binding protein (PTB) is an IRES trans-acting factor that positively regulates the IRES activities of both p53 isoforms by relocating from nucleus to the cytoplasm during stress conditions. Here we have demonstrated the putative contact points of PTB on the p53 IRES RNA. Studies on mutations that occur naturally in the 5' untranslated region (5' UTR) in p53 mRNA were lacking. We have investigated a naturally occurring C-to-T single-nucleotide polymorphism (SNP) first reported in human melanoma tumors. This SNP is at position 119 in the 5' UTR of p53 mRNA and we demonstrate that it has consequences on the translational control of p53. Introduction of this SNP has led to decrease in cap-independent translation from p53 5' UTR in bicistronic reporter assay. Further, the effects of this SNP on cap-independent translation have been studied in the context of p53 cDNA as well. Interestingly, the 5' UTR with this SNP has shown reduced binding to PTB that can be corroborated to its weaker IRES activity. Previously, it has been shown that G2-M checkpoint, DNA-damaging stress and oncogenic insult favor IRES-mediated translation. Under similar conditions, we demonstrate that this SNP interferes with the enhancement of the IRES activity of the 5' UTR. Taken together, the results demonstrate for the first time that SNP in the 5' UTR of the p53 mRNA might have a role in translational control of this critical tumor-suppressor gene.

Reversible induction of translational isoforms of p53 in glucose deprivation

Tumor suppressor protein p53 is a master transcription regulator, indispensable for controlling several cellular pathways. Earlier work in our laboratory led to the identification of dual internal ribosome entry site (IRES) structure of p53 mRNA that regulates translation of full-length p53 and Delta 40p53. IRES-mediated translation of both isoforms is enhanced under different stress conditions that induce DNA damage, ionizing radiation and endoplasmic reticulum stress, oncogene-induced senescence and cancer. In this study, we addressed nutrient-mediated translational regulation of p53 mRNA using glucose depletion. In cell lines, this nutrient-depletion stress relatively induced p53 IRES activities from bicistronic reporter constructs with concomitant increase in levels of p53 isoforms. Surprisingly, we found scaffold/matrix attachment region-binding protein 1 (SMAR1), a predominantly nuclear protein is abundant in the cytoplasm under glucose deprivation. Importantly under these conditions polypyrimidine-tract-binding protein, an established p53 ITAF did not show nuclear-cytoplasmic relocalization highlighting the novelty of SMAR1-mediated control in stress. In vivo studies in mice revealed starvation-induced increase in SMAR1, p53 and Delta 40p53 levels that was reversible on dietary replenishment. SMAR1 associated with p53 IRES sequences ex vivo, with an increase in interaction on glucose starvation. RNAi-mediated-transient SMAR1 knockdown decreased p53 IRES activities in normal conditions and under glucose deprivation, this being reflected in changes in mRNAs in the p53 and Delta 40p53 target genes involved in cell-cycle arrest, metabolism and apoptosis such as p21, TIGAR and Bax. This study provides a new physiological insight into the regulation of this critical tumor suppressor in nutrient starvation, also suggesting important functions of the p53 isoforms in these conditions as evident from the downstream transcriptional target activation.

Demodulation of Narrowband Speech Spectrograms Using the Riesz Transform

We propose a two-dimensional (2-D) multicomponent amplitude-modulation, frequency-modulation (AM-FM) model for a spectrogram patch corresponding to voiced speech, and develop a new demodulation algorithm to effectively separate the AM, which is related to the vocal tract response, and the carrier, which is related to the excitation. The demodulation algorithm is based on the Riesz transform and is developed along the lines of Hilbert-transform-based demodulation for 1-D AM-FM signals. We compare the performance of the Riesz transform technique with that of the sinusoidal demodulation technique on real speech data. Experimental results show that the Riesz-transform-based demodulation technique represents spectrogram patches accurately. The spectrograms reconstructed from the demodulated AM and carrier are inverted and the corresponding speech signal is synthesized. The signal-to-noise ratio (SNR) of the reconstructed speech signal, with respect to clean speech, was found to be 2 to 4 dB higher in case of the Riesz transform technique than the sinusoidal demodulation technique.

An Alternating l(p) - l(2) Projections Algorithm (ALPA) for Speech Modeling using Sparsity Constraints

We address the problem of separating a speech signal into its excitation and vocal-tract filter components, which falls within the framework of blind deconvolution. Typically, the excitation in case of voiced speech is assumed to be sparse and the vocal-tract filter stable. We develop an alternating l(p) - l(2) projections algorithm (ALPA) to perform deconvolution taking into account these constraints. The algorithm is iterative, and alternates between two solution spaces. The initialization is based on the standard linear prediction decomposition of a speech signal into an autoregressive filter and prediction residue. In every iteration, a sparse excitation is estimated by optimizing an l(p)-norm-based cost and the vocal-tract filter is derived as a solution to a standard least-squares minimization problem. We validate the algorithm on voiced segments of natural speech signals and show applications to epoch estimation. We also present comparisons with state-of-the-art techniques and show that ALPA gives a sparser impulse-like excitation, where the impulses directly denote the epochs or instants of significant excitation.

Automatic transcription of conversational telephone speech

This paper discusses the Cambridge University HTK (CU-HTK) system for the automatic transcription of conversational telephone speech. A detailed discussion of the most important techniques in front-end processing, acoustic modeling and model training, language and pronunciation modeling are presented. These include the use of conversation side based cepstral normalization, vocal tract length normalization, heteroscedastic linear discriminant analysis for feature projection, minimum phone error training and speaker adaptive training, lattice-based model adaptation, confusion network based decoding and confidence score estimation, pronunciation selection, language model interpolation, and class based language models. The transcription system developed for participation in the 2002 NIST Rich Transcription evaluations of English conversational telephone speech data is presented in detail. In this evaluation the CU-HTK system gave an overall word error rate of 23.9%, which was the best performance by a statistically significant margin. Further details on the derivation of faster systems with moderate performance degradation are discussed in the context of the 2002 CU-HTK 10 × RT conversational speech transcription system. © 2005 IEEE.

An environmental assessment of the John Pennekamp Coral Reef State Park and the Key Largo Coral Reef Marine Sanctuary (Unpublished 1983 Report)

The Pennekamp Coral Reef State Park was established in 1960 and the Key Largo National Marine Sanctuary in 1975. Field studies, funded by NOAA, were conducted in 1980 - 1981 to determine the state of the coral reefs and surrounding areas in relation to changing environmental conditions and resource management that had occurred over the intervening years. Ten reef sites within the Sanctuary and seven shallow grass and hardbottom sites within the Park were chosen for qualitative and quantitative studies. At each site, three parallel transects not less than 400 m long were run perpendicular to the reef or shore, each 300 m apart. Observations, data collecting and sampling were done by two teams of divers. Approximately 75 percent of the bottom within the 18-m isobath was covered by marine grasses, predominantly turtle grass. The general health of the seagrasses appeared good but a few areas showed signs of stress. The inner hardbottom of the Park was studied at the two entrances to Largo Sound. Though at the time of the study the North Channel hardbottom was subjected to only moderate boat traffic, marked changes had taken place over the past years, the most obvious of which was the loss of the extensive beds of Sargassum weed, one of the most extensive beds of this alga in the Keys. Only at this site was the green alga Enteromorpha encountered. This alga, often considered a pollution indicator, may denote the effects of shore run off. The hardbottom at South Channel and the surrounding grass beds showed signs of stress. This area bears the heaviest boat traffic within the Park waters causing continuous turbidity from boat wakes with resulting siltation. The offshore hardbottom and rubble areas in the Sanctuary appeared to be in good health and showed no visible indications of deterioration. Damage by boat groundings and anchors was negligible in the areas surveyed. The outer reefs in general appear to be healthy. Corals have a surprising resiliency to detrimental factors and, when conditions again become favorable, recover quickly from even severe damage. It is, therefore, a cause for concern that Grecian Rocks, which sits somewhat inshore of the outer reef line, has yet to recover from die-off in 1978. The slow recovery, if occurring, may be due to the lower quality of the inshore waters. The patch reefs, more adapted to inshore waters, do not show obvious stress signs, at least those surveyed in this study. It is apparent that water quality was changing in the keys. Water clarity over much of the reef tract was observed to be much reduced from former years and undoubtedly plays an important part in the stresses seen today over the Sanctuary and Park. (PDF contains 119 pages)

Coral Remote Sensing Workshop: Proceedings and Recommendations, 17-18 September, Sheraton, Brickell Ave, Miami FL

Coral reefs exist in warm, clear, and relatively shallow marine waters worldwide. These complex assemblages of marine organisms are unique, in that they support highly diverse, luxuriant, and essentially self-sustaining ecosystems in otherwise nutrient-poor and unproductive waters. Coral reefs are highly valued for their great beauty and for their contribution to marine productivity. Coral reefs are favorite destinations for recreational diving and snorkeling, as well as commercial and recreational fishing activities. The Florida Keys reef tract draws an estimated 2 million tourists each year, contributing nearly $800 million to the economy. However, these reef systems represent a very delicate ecological balance, and can be easily damaged and degraded by direct or indirect human contact. Indirect impacts from human activity occurs in a number of different forms, including runoff of sediments, nutrients, and other pollutants associated with forest harvesting, agricultural practices, urbanization, coastal construction, and industrial activities. Direct impacts occur through overfishing and other destructive fishing practices, mining of corals, and overuse of many reef areas, including damage from souvenir collection, boat anchoring, and diver contact. In order to protect and manage coral reefs within U.S. territorial waters, the National Oceanic and Atmospheric Administration (NOAA) of the U.S. Department of Commerce has been directed to establish and maintain a system of national marine sanctuaries and reserves, and to monitor the condition of corals and other marine organisms within these areas. To help carry out this mandate the NOAA Coastal Services Center convened a workshop in September, 1996, to identify current and emerging sensor technologies, including satellite, airborne, and underwater systems with potential application for detecting and monitoring corals. For reef systems occurring within depths of 10 meters or less (Figure 1), mapping location and monitoring the condition of corals can be accomplished through use of aerial photography combined with diver surveys. However, corals can exist in depths greater than 90 meters (Figure 2), well below the limits of traditional optical imaging systems such as aerial or surface photography or videography. Although specialized scuba systems can allow diving to these depths, the thousands of square kilometers included within these management areas make diver surveys for deeper coral monitoring impractical. For these reasons, NOAA is investigating satellite and airborne sensor systems, as well as technologies which can facilitate the location, mapping, and monitoring of corals in deeper waters. The following systems were discussed as having potential application for detecting, mapping, and assessing the condition of corals. However, no single system is capable of accomplishing all three of these objectives under all depths and conditions within which corals exist. Systems were evaluated for their capabilities, including advantages and disadvantages, relative to their ability to detect and discriminate corals under a variety of conditions. (PDF contains 55 pages)

Connectivity: Science, people and policy in the Florida Keys National Marine Sanctuary

Executive Summary: The Connectivity Colloquium evolved from an exhortation by Dan Basta, Director of the National Marine Sanctuary Program, to come together and assess what we know about the condition of our natural resources, identify information gaps and how to fill them, and transform science and management from an emphasis on documentation to a nexus for action. This purpose in some ways reflects the initiation of the Florida Keys National Marine Sanctuary itself, which was designated by an act of the U.S. Congress in 1990 in the aftermath of the 1989 Exxon Valdez oil spill in Alaska and three major ship groundings of the Florida Reef Tract in late 1989. Over the next seven years NOAA worked with federal, state, and local partners to develop a comprehensive management plan for the Sanctuary implemented under a co-trustee partnership between NOAA and the State of Florida. (PDF contains 270 pages; 14Mb)

Collision tumour involving a rectal gastrointestinal stromal tumour with invasion of the prostate and a prostatic adenocarcinoma

Background: Gastrointestinal stromal tumours (GISTs) are the most common primary mesenchymal neoplasia in the gastrointestinal tract, although they represent only a small fraction of total gastrointestinal malignancies in adults (<2%). GISTs can be located at any level of the gastrointestinal tract; the stomach is the most common location (60-70%), in contrast to the rectum, which is most rare (4%). When a GIST invades into the adjacent prostate tissue, it can simulate prostate cancer. In this study, we report on a case comprising the unexpected collision between a rectal GIST tumour and a prostatic adenocarcinoma. Findings: We describe the complexity of the clinical, endoscopic and radiological diagnosis, of the differential diagnosis based on tumour biopsy, and of the role of neoadjuvant therapy using imatinib prior to surgical treatment. Conclusions: Although isolated cases of coexisting GISTs and prostatic adenocarcinomas have reviously been described, this is the first reported case in the medical literature of a collision tumour involving a rectal GIST and prostatic adenocarcinoma components.

2002-03 Florida Keys National Marine Sanctuary science report: An ecosystem report card after five years of marine zoning

Executive Summary: Circulation and Exchange of Florida Bay and South Florida Coastal Waters The coastal ecosystem of South Florida is comprised of distinct marine environments. Circulation of surface waters and exchange processes, which respond to both local and regional forcings, interconnect different coastal environments. In addition, re-circulating current systems within the South Florida coastal ecosystem such as the Tortugas Gyre contribute to retention of locally spawned larvae. Variability in salinity, chlorophyll, and light transmittance occurs on a wide range of temporal and spatial scales, in response to both natural forcing, such as seasonal precipitation and evaporation and interannual “El Niño” climate signals, and anthropogenic forcing, such as water management practices in south Florida. The full time series of surface property maps are posted at www.aoml.noaa.gov/sfp. Regional surface circulation patterns, shown by satellite-tracked surface drifters, respond to large-scale forcing such as wind variability and sea level slopes. Recent patterns include slow flow from near the mouth of the Shark River to the Lower Keys, rapid flow from the Tortugas to the shelf of the Carolinas, and flow from the Tortugas around the Tortugas Gyre and out of the Florida Straits. The Southwest Florida Shelf and the Atlantic side of the Florida Keys coastal zone are directly connected by passages between the islands of the Middle and Lower Keys. Movement of water between these regions depends on a combination of local wind-forced currents and gravitydriven transports through the passages, produced by cross-Key sea level differences on time scales of several days to weeks, which arise because of differences in physical characteristics (shape, orientation, and depth) of the shelf on either side of the Keys. A southeastward mean flow transports water from western Florida Bay, which undergoes large variations in water quality, to the reef tract. Adequate sampling of oceanographic events requires both the capability of near real-time recognition of these events, and the flexibility to rapidly stage targeted field sampling. Capacity to respond to events is increasing, as demonstrated by investigations of the 2002 “blackwater” event and a 2003 entrainment of Mississippi River water to the Tortugas. (PDF contains 364 pages.)

Movement of yellowtail snapper (Ocyurus chrysurus Block 1790) and black grouper (Mycteroperca bonaci Poey 1860) in the northern Florida Keys National Marine Sanctuary as determined by acoustic telemetry

We tagged a total of 14 yellowtail snapper (Ocyurus chrysurus Bloch 1790) and black grouper (Mycteroperca bonaci Poey 1860) inside the Conch Reef Research Only Area (a no-take marine reserve) in the northern Florida Keys National Marine Sanctuary in November 2001. Both species are heavily exploited in the region. Our objective was to characterize site fidelity and movement behavior along the reef tract to the north and south of the release point. Fishes were collected by baited hook and line from the surface, surgically-tagged with coded-acoustic transmitters, and returned to the reef by snorkelers. Tracking of fish movement behavior was conducted by five acoustic receivers deployed on the seafloor from Davis Reef in the south to Pickles Reef in the north. Fishes were tracked for up to eight months. Results indicated that the majority of signal detections for individual fish from both species were recorded at the two Conch Reef receivers. Limited movement from Conch Reef to Davis Reef was recorded, but no signal detections were recorded at the two sites to the north of Conch Reef. These results suggest that both species show site fidelity to Conch Reef. Future studies will seek to characterize this site fidelity with increased temporal and spatial resolution at Conch Reef. (PDF contains 25 pages.)

Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC''. The calpain-mediated ACT processing allows trafficking of the "soluble AC'' domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools'', which would play different roles in the cell pathophysiology.

Structural Integrity of the Contralesional Hemisphere Predicts Cognitive Impairment in Ischemic Stroke at Three Months

After stroke, white matter integrity can be affected both locally and distally to the primary lesion location. It has been shown that tract disruption in mirror's regions of the contralateral hemisphere is associated with degree of functional impairment. Fourteen patients suffering right hemispheric focal stroke (S) and eighteen healthy controls (HC) underwent Diffusion Weighted Imaging (DWI) and neuropsychological assessment. The stroke patient group was divided into poor (SP; n = 8) and good (SG; n = 6) cognitive recovery groups according to their cognitive improvement from the acute phase (72 hours after stroke) to the subacute phase (3 months post-stroke). Whole-brain DWI data analysis was performed by computing Diffusion Tensor Imaging (DTI) followed by Tract Based Spatial Statistics (TBSS). Assessment of effects was obtained computing the correlation of the projections on TBSS skeleton of Fractional Anisotropy (FA) and Radial Diffusivity (RD) with cognitive test results. Significant decrease of FA was found only in right brain anatomical areas for the S group when compared to the HC group. Analyzed separately, stroke patients with poor cognitive recovery showed additional significant FA decrease in several left hemisphere regions; whereas SG patients showed significant decrease only in the left genu of corpus callosum when compared to the HC. For the SG group, whole brain analysis revealed significant correlation between the performance in the Semantic Fluency test and the FA in the right hemisphere as well as between the performance in the Grooved Pegboard Test (GPT) and theTrail Making Test-part A and the FA in the left hemisphere. For the SP group, correlation analysis revealed significant correlation between the performance in the GPT and the FA in the right hemisphere. Palabras clave