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At least 847 records · Page 47Linked to original sources
PubMed2002

Characterization of a gene which encodes a mannosyltransferase homolog of Paracoccidioides brasiliensis.

We screened an expression library of the yeast form of Paracoccidioides brasiliensis with a pool of human sera that was pre-adsorbed with mycelium, from patients with paracoccidioidomycosis (PCM). A sequence (PbYmnt) was obtained and characterized. A genomic clone was obtained by PCR of P. brasiliensis total DNA. The sequence contained a single open reading frame (ORF) encoding a protein of 357 amino acid residues, with a molecular mass of 39.78 kDa. The deduced amino acid sequence exhibited identity to mannosyl- and glycosyltransferases from several sources. A DXD motif was present in the translated gene and this sequence is characteristic of the glycosyltransferases. Hydropathy analysis revealed a single transmembrane region near the amino terminus of the molecule that suggested a type II membrane protein. The PbYmnt was expressed preferentially in the yeast parasitic phase. The accession number of the nucleotide sequence of PbYmnt and its flanking regions is AF374353. A recombinant protein was generated in Escherichia coli. Our data suggest that PbYmnt encodes one member of a glycosyltransferase family of proteins and that our strategy was useful in the isolation of differentially expressed genes.

Amino Acid Motifs↗
PubMed2006

Functional imaging of sequence learning in Parkinson's disease.

Sequence learning, a cognitive task linked to cortico-striatal function, is impaired in Parkinson's disease (PD). We chose this task as a behavioral paradigm to study the functional architecture of PD in treated and untreated conditions. In our studies, participants were scanned with H(2)(15)O while performing a kinematically controlled motor sequence learning task and a matching motor baseline task. Experiments revealed that a specific sequence learning network predicts learning in normal subjects, and in independent cohorts of early and advanced PD patients. The analysis of the relationship of network activity to learning performance revealed diverging influences of dopaminergic therapy and deep brain stimulation (DBS). DBS of the internal GP and of STN increased network activity and task performance, while levodopa decreased both measures. In separate studies, we investigated the role of dopaminergic modulation on brain activation during sequence learning. In healthy subjects dopamine transporter (DAT) binding correlated with learning-related brain activation in prefrontal, premotor and cingulate cortices, and in the thalamus. By contrast, in PD most of these regional relationships were lost. Only ventral and dorsolateral prefrontal cortex activation correlated with caudate dopaminergic input. In a final set of studies, we found a significant decline in learning performance in early stage PD patients followed over the course of 2 years. Longitudinal declines in learning-related activation were found in parietal areas, while concomitant increases were localized to the left hippocampus. These observations support hypotheses on disease-stage and task-specific effects within the different cortico-striato-pallido-thalamocortical loops and the mesocortical system in PD.

Aged↗
PubMed2006

MMP-20 is predominately a tooth-specific enzyme with a deep catalytic pocket that hydrolyzes type V collagen.

Matrix metalloproteinase-20 (MMP-20, enamelysin) has a highly restricted pattern of expression. In healthy tissues, MMP-20 is observed in the enamel organ and pulp organ of developing teeth and is present only as an activated enzyme. To identify other tissues that may express MMP-20, we performed a systematic mouse tissue expression screen. Among the non-tooth tissues assayed, MMP-20 transcripts were identified only in minute quantities within the large intestine. The murine Mmp20 promoter was cloned, sequenced, and assessed for potential tooth-specific regulatory elements. In silico analysis identified four promoter modules that were common to Mmp20 and at least two of three coregulated predominantly tooth-specific genes that encode ameloblastin, amelogenin, and enamelin. We asked if the highly restricted MMP-20 expression pattern was associated with a broad substrate specificity that might preclude its expression in other tissues. An iterative mixture-based random doedecamer peptide library screen with Edman sequencing of MMP-20 cleavage products revealed that, among MMPs previously screened, MMP-20 had unique substrate preferences. These preferences indicate that MMP-20 has a deep and wide catalytic pocket that can accommodate substrates with large aromatic residues in the P1' position. On the basis of matrices derived from the peptide library data, we identified and then confirmed that type V collagen is an MMP-20 substrate. Since type V collagen is not present in dental enamel but is an otherwise widely distributed collagen, and since only active MMP-20 has been observed in teeth, our data suggest that control of MMP-20 activity is primarily regulated by transcriptional means.

Animals↗
PubMed1999

Nanoliter-scale sample preparation methods directly coupled to polymethylmethacrylate-based microchips and gel-filled capillaries for the analysis of oligonucleotides.

We are currently developing miniaturized, chip-based electrophoresis devices fabricated in plastics for the high-speed separation of oligonucleotides. One of the principal advantages associated with these devices is their small sample requirements, typically in the nanoliter to sub-nanoliter range. Unfortunately, most standard sample preparation protocols, especially for oligonucleotides, are done off-chip on a microliter-scale. Our work has focused on the development of capillary nanoreactors coupled to micro-separation platforms, such as micro-electrophoresis chips, for the preparation of sequencing ladders and also polymerase chain reactions (PCRs). These nanoreactors consist of fused-silica capillary tubes (10-20 cm x 20-50 microns I.D.) with fluid pumping accomplished using the electroosmotic flow generated by the tubes. These reactors were situated in fast thermal cyclers to perform cycle sequencing or PCR amplification of the DNAs. The reactors could be interfaced to either a micro-electrophoresis chips via capillary connectors micromachined in polymethylmethacrylate (PMMA) using deep X-ray etching (width 50 microns; depth 50 microns) or conventional capillary gel tubes using zero-dead volume glass unions. For our chips, they also contained an injector, separation channel (length 6 cm; width 30 microns; depth 50 microns) and a dual fiber optic, near-infrared fluorescence detector. The sequencing nanoreactor used surface immobilized templates attached to the wall via a biotin-streptavidin-biotin linkage. Sequencing tracks could be directly injected into gel-filled capillary tubes with minimal degradation in the efficiency of the separation process. The nanoreactor could also be configured to perform PCR reactions by filling the capillary tube with the PCR reagents and template. After thermal cycling, the PCR cocktail could be pooled from multiple reactors and loaded onto a slab gel or injected into a capillary tube or microchip device for fractionation.

Electrophoresis, Capillary↗
PubMed2003

Cloning of a muscle-specific calpain from the American lobster Homarus americanus: expression associated with muscle atrophy and restoration during moulting.

A cDNA (1977 bp) encoding a crustacean calpain (Ha-CalpM; GenBank accession no. AY124009) was isolated from a lobster fast muscle cDNA library. The open reading frame specified a 575-amino acid (aa) polypeptide with an estimated mass of 66.3 kDa. Ha-CalpM shared high identity with other calpains in the cysteine proteinase domain (domain II; aa 111-396) and domain III (aa 397-575), but most of the N-terminal domain (domain I; aa 1-110) was highly divergent. Domain II contained the cysteine, histidine and asparagine triad essential for catalysis, as well as two conserved aspartate residues that bind Ca(2+). In domain III an acidic loop in the C2-like region, which mediates Ca(2+)-dependent phospholipid binding, had an expanded stretch of 17 aspartate residues. Ha-CalpM was classified as a non-EF-hand calpain, as it lacked domain IV, a calmodulin-like region containing five EF-hand motifs. Northern blot analysis, relative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR showed that Ha-CalpM was highly expressed in skeletal muscles, but at much lower levels in heart, digestive gland, intestine, integument, gill, nerve cord/thoracic ganglion and antennal gland. An antibody raised against a unique N-terminal sequence recognized a 62 kDa isoform in cutter claw and crusher claw closer muscles and a 68 kDa isoform in deep abdominal muscle. Ha-CalpM was distributed throughout the cytoplasm, as well as in some nuclei, of muscle fibers. Purification of Ha-CalpM showed that the 62 kDa and 68 kDa isoforms co-eluted from gel filtration and ion exchange columns at positions consistent with those of previously described Ca(2+)-dependent proteinase III (CDP III; 59 kDa). Ha-CalpM mRNA and protein did not change during the moulting cycle. The muscle-specific expression of Ha-CalpM and the ability of Ha-CalpM/CDP III to degrade myofibrillar proteins suggest that it is involved in restructuring and/or maintaining contractile structures in crustacean skeletal muscle.

Amino Acid Sequence↗
PubMed1975

The variable fine structure of elastin visualized with Verhoeff's iron hematoxylin.

Verhoeff's iron hematoxylin (VIH) followed by lead citrate (LC) applied to epoxy thin sections stained the dense component of elastic fibers heavily and the peripheral microfibrillar component lightly in guinea pig trachea and mouse testis fixed with a glutaraldehyde-osmium tetroxide sequence. This method stained large fimbriated fibers beneath tracheal epithelium, small fibers and stacked aggregates thereof in the deep lamina propria, cartilage and adventitia of the trachea and large stacked fibers in the fibroelastic band of the trachea. Fibers of the fetus differed from those of the adult, especially in the subepithelial elastic lamina of the trachea. Elastic fibers were intimately associated with fibroblasts and particularly slender fibroblast processes in tracheal stroma and with chondrocytes in tracheal cartilage. Fibroblasts associated with elastic fibers in the tracheal subepithelial lamina propria were often closely bordered by eosinophils, mast cells, or monocytes. Occasional mast cells extended slender processes around elastic fibers in the subepithelial lamina propria. In mouse testis and in many regions of the trachea, small elastic fibers were identified which were below the limits of resolution for the light microscope and were not apparent at the ultrastructural level in routinely stained thin sections.

Animals↗
PubMedDate not supplied

Non-natural CBP2 binding peptides and peptomers modulate carcinoma cell adhesion and invasion.

A combinatorial approach that utilized a repertoire of bacteriophage-peptides has identified a number of non-natural CBP2 binding peptides. Moreover, co-localization of some of these peptides with CBP2 in a number of tumor cell lines demonstrated that the peptides were directed to an intracellular location spatially coincident with the normal distribution of CBP2 [Sauk et al., 2000]. From among these sequences WHYPWFQNWAMA and LDSRYSLQAAMY were the most effective CBP2 binding peptides and best fulfilled the combinatorial motif containing deep hydrophobic pockets. When the hydropathic profiles of collagen alpha1(IV) and alpha2 (IV) were compared with these dodecapeptides, the hydropathic profiles of WHYPWFQNWAMA and LDSRYSLQAAMY closely matched those of alpha1(IV) 414-452 and alpha1(IV)531-543. These peptides were shown to be functional peptidomimics and possessed the ability to alter cell adhesion and invasion of human squamous cell carcinoma cell lines. Peptomers were formed of these non-natural peptides to explore the role that a repetitive peptide may have on cell adhesion. The enhanced cell adhesion observed with the peptomers required both CBP2 antibodies and integrin antibodies for inhibition. The enhanced adhesion observed even in the face of combined antibody inhibition was consistent with such complexes possessing correspondingly slower dissociation rates. Thus, suggesting that peptomers may function in a like manner to multimeric peptide MHC complexes (tetramers) binding more than one cell receptor on a specific cell. These findings evoke both peptidomimics of native ligands and their peptomers as potential reagents by which to target tumor cells for chemotherapy, imaging, or retargeting viral vectors for gene therapy.

Bacteriophages↗
PubMed2000

Molecular adaptation to an extreme environment: origin of the thermal stability of the pompeii worm collagen.

The annelid Alvinella pompejana is probably the most heat-tolerant metazoan organism known. Previous results have shown that the level of thermal stability of its interstitial collagen is significantly greater than that of coastal annelids and of vent organisms, such as the vestimentiferan Riftia pachyptila, living in colder parts of the deep-sea hydrothermal environment. In order to investigate the molecular basis of this thermal behavior, we cloned and sequenced a large cDNA molecule coding the fibrillar collagen of Alvinella, including one half of the helical domain and the entire C-propeptide domain. For comparison, we also cloned the 3' part of the homologous cDNA from Riftia. Comparison of the corresponding helical domains of these two species, together with that of the previously sequenced domain of the coastal lugworm Arenicola marina, showed that the increase in proline content and in the number of stabilizing triplets correlate with the outstanding thermostability of the interstitial collagen of A. pompejana. Phylogenetic analysis showed that triple helical and the C-propeptide parts of the same collagen molecule evolve at different rates, in favor of an adaptive mechanism at the molecular level.

Adaptation, Physiological↗
PubMed1988

Histochemical heterogeneity of dermal mast cells in athymic and normal rats.

Mucosal mast cells (MMC) and connective tissue mast cells (CTMC) of the rat contain different proteoglycans, which can be distinguished using histochemical methods. The chondroitin sulphate proteoglycan of the MMC, unlike the heparin of the CTMC, does not show fluorescent berberine binding, is susceptible to aldehyde fixatives and stains preferentially with Alcian Blue in a staining sequence with Safranin. The majority of the dermal mast cells are typical CTMC and are located in the deep part of the dermis. Subepidermal mast cells are comparatively few in normal rats but numerous in athymic rats and mice. These cells differ from other dermal mast cells in that they stain preferentially with Alcian Blue and they appear to contain little histamine. We examined some of the histochemical properties of the skin mast cells of female PVG-rnu/rnu rats and their heterozygous littermates aged from 5 to 29 weeks. The thiazine dye-binding of the subepidermal mast cells was partially blocked by formaldehyde fixation and only about half of them showed a weakly fluorescent berberine binding. The critical electrolyte concentration of the Alcian Blue staining of the subepidermal mast cells was between that of CTMC and MMC. Deaminative cleavage with nitrous acid abolished the staining of all skin mast cells, while that of the MMC was unaffected. There were no statistically significant differences in the staining patterns of the dermal mast cells between different ages or groups of rat. These results indicate that the subepidermal mast cells contain a heparin proteoglycan which is, however, different from that of the typical CTMC of other sites. They thus appear to represent a second example of a mast cell within a defined anatomical location exhibiting a distinct proteoglycan expression.

Animals↗
PubMed1989

Collagen fibril surface structures: freeze-etching data and computer modelling.

In order to investigate the correlation between primary structure and surface profile of collagen, freeze-fractured and deep-etched (dehydrated) collagen fibrils have been compared with a fibril model built directly from the amino acid sequence. The model appeared to be almost identical to the freeze-etched collagen fibril with respect to the most relevant features, such as the gap-overlap ratio and the location of the two main intraperiod ridges (X3 and X2). However, it did not show the minor ridges observed in the gap zone (X1 and Y1), thus suggesting that these structures, although consistently present in collagen fibrils, may in fact be due to post-translational modifications or to non-collagenic material adhering to the fibril.

Amino Acid Sequence↗
PubMedDate not supplied

Lateral approach to the sciatic nerve block in the popliteal fossa: correlation between evoked motor response and sensory block.

BACKGROUND AND OBJECTIVES: The purpose of this study was to identify which of two motor responses of the foot (plantar flexion versus dorsiflexion) best predicts complete sensory blockade of the sciatic nerve when is used for lateral popliteal sciatic nerve block. METHODS: Thirty American Society of Anesthesiologist physical status I or II patients scheduled for foot and ankle surgery under lateral popliteal sciatic nerve block were enrolled in the study. During each block, the needle was placed to evoke one of the following motor responses of the foot: plantar flexion or dorsiflexion. Thirty milliliters of 0.75% ropivacaine was injected after the motor response was elicited at <0.5 mA. The sequence of elicited motor response was randomized. Sensory blockade of the areas of the foot innervated by the deep peroneal, superficial peroneal, posterior tibial, and sural nerves was checked in a blinded manner. Time required for onset of sensory and motor block of the foot was recorded. RESULTS: The 2 groups were similar with regard to demographic variables and type of surgery. The total of nerves blocked (deep and superficial peroneal, posterior tibial, and sural nerves) after elicited plantar flexion was greater (complete sensory block in 58 of 60 nerve distributions) than after elicited dorsiflexion (34 of 60 nerve distributions) (P <.05). Onset of complete sensory and motor blockade of the foot was faster after elicited plantar flexion (16.6 +/- 5.1 minutes, 20.1 +/- 5.1 minutes, respectively) than after elicited dorsiflexion (24.3 +/- 5.1 minutes, 28.1 +/- 5.0 min, P <.05). CONCLUSIONS: After stimulation of the sciatic nerve, plantar flexion better predicts complete sensory blockade of the foot than dorsiflexion when using the lateral approach to the popliteal fossa. The findings of the present study apply to a single injection of 30 mL of ropivacaine 0.75%.

Adolescent↗
PubMed1998

Determining whether a parotid tumor is in the superficial or deep lobe using magnetic resonance imaging.

PURPOSE: This study examined the usefulness of magnetic resonance imaging (MRI) in assessing whether parotid tumors were located in the superficial or deep lobe. PATIENTS AND METHODS: Eight patients with parotid gland tumors underwent MRI using a spin echo pulse sequence. T1- and T2-weighted images were obtained. To assess tumor localization in the parotid gland, two anatomic landmarks were used: 1) a line connecting the lateral surface of posterior belly of digastric muscle and lateral surface of the cortical bone of ascending ramus (facial nerve [FN] line) and 2) the relationship to the retromandibular vein (RV). Results of all MRI examinations were compared with the surgical findings. RESULTS: Seven of eight cases were correctly diagnosed using the FN line criterion. Five of eight cases were correctly diagnosed using the RV criterion. CONCLUSION: MRI is an excellent modality to show tumor localization in the parotid gland.

Adenocarcinoma↗
PubMed1998

The structure of SAICAR synthase: an enzyme in the de novo pathway of purine nucleotide biosynthesis.

BACKGROUND: The biosynthesis of key metabolic components is of major interest to biologists. Studies of de novo purine synthesis are aimed at obtaining a deeper understanding of this central pathway and the development of effective chemotherapeutic agents. Phosphoribosylaminoimidazolesuccinocarboxamide (SAICAR) synthase catalyses the seventh step out of ten in the biosynthesis of purine nucleotides. To date, only one structure of an enzyme involved in purine biosynthesis has been reported: adenylosuccinate synthetase, which catalyses the first committed step in the synthesis of AMP from IMP. RESULTS: We report the first three-dimensional structure of a SAICAR synthase, from Saccharomyces cerevisiae. It is a monomer with three domains. The first two domains consist of antiparallel beta sheets and the third is composed of two alpha helices. There is a long deep cleft made up of residues from all three domains. Comparison of SAICAR synthases by alignment of their sequences reveals a number of conserved residues, mostly located in the cleft. The presence of two sulphate ions bound in the cleft, the structure of SAICAR synthase in complex with ATP and a comparison of this structure with that of other ATP-dependent proteins point to the interdomain cleft as the location of the active site. CONCLUSIONS: The topology of the first domain of SAICAR synthase resembles that of the N-terminal domain of proteins belonging to the cyclic AMP-dependent protein kinase family. The fold of the second domain is similar to that of members of the D-alanine:D-alanine ligase family. Together these enzymes form a new superfamily of mononucleotide-binding domains. There appears to be no other enzyme, however, which is composed of the same combination of three domains, with the individual topologies found in SAICAR synthase.

Adenosine Triphosphate↗
PubMed2004

Aftershocks driven by a high-pressure CO2 source at depth.

In northern Italy in 1997, two earthquakes of magnitudes 5.7 and 6 (separated by nine hours) marked the beginning of a sequence that lasted more than 30 days, with thousands of aftershocks including four additional events with magnitudes between 5 and 6. This normal-faulting sequence is not well explained with models of elastic stress transfer, particularly the persistence of hanging-wall seismicity that included two events with magnitudes greater than 5. Here we show that this sequence may have been driven by a fluid pressure pulse generated from the coseismic release of a known deep source of trapped high-pressure carbon dioxide (CO2). We find a strong correlation between the high-pressure front and the aftershock hypocentres over a two-week period, using precise hypocentre locations and a simple model of nonlinear diffusion. The triggering amplitude (10-20 MPa) of the pressure pulse overwhelms the typical (0.1-0.2 MPa) range from stress changes in the usual stress triggering models. We propose that aftershocks of large earthquakes in such geologic environments may be driven by the coseismic release of trapped, high-pressure fluids propagating through damaged zones created by the mainshock. This may provide a link between earthquakes, aftershocks, crust/mantle degassing and earthquake-triggered large-scale fluid flow.

Journal Article↗
PubMed2005

Environments shape the nucleotide composition of genomes.

To test the impact of environments on genome evolution, we analysed the relative abundance of the nucleotides guanine and cytosine ('GC content') of large numbers of sequences from four distinct environmental samples (ocean surface water, farm soil, an acidophilic mine drainage biofilm and deep-sea whale carcasses). We show that the GC content of complex microbial communities seems to be globally and actively influenced by the environment. The observed nucleotide compositions cannot be easily explained by distinct phylogenetic origins of the species in the environments; the genomic GC content may change faster than was previously thought, and is also reflected in the amino-acid composition of the proteins in these habitats.

Amino Acids↗
PubMed1995

Molecular cloning and differential expression patterns of avian glutamate receptor mRNAs.

We have identified and studied potential ionotropic glutamate receptor genes in pigeon brain. Three cDNA clones exhibit significant amino acid sequence identity to members of a rodent ligand-gated ion channel family. One of them, GluP-II, encodes a full-length AMPA-sensitive glutamate receptor GluR2 (GluR-B) homologue, whereas the other two partial clones, designated as GluP-III and -IV, are nearly identical to rodent GluR3 (GluR-C) and GluR4 (GluR-D) receptor subunits. Northern analysis demonstrated that the avian genes are widely expressed in the brain. Within the brain regions analyzed by in situ hybridization histochemistry, the three avian GluR subunits showed distinct and regionally specific mRNA expression patterns in the adult. Most of the differences in their expression were observed in cell types of the telencephalon, certain thalamic nuclei, the optic tectum, and the cerebellar cortex. A particularly striking finding was the expression of GluP-II in Golgi epithelial/Bergmann glial cells. In contrast, Bergmann glial cells in rat cerebellum do not express GluR2 (GluR-B) subunit genes. Immunoreactivity for a monoclonal sequence-specific antipeptide antibody was widespread and most prominent in Purkinje cell perikarya and their dendrites, neuronal cell bodies of the ectostriatum, and the deep optic tectum. These results demonstrate the existence of multiple subunits of the ionotropic glutamate receptor channel family in avians. Excitatory amino acid receptor genes appear to be highly conserved during evolution.

Amino Acid Sequence↗
PubMed2000

Turbidite Megabeds in an Oceanic Rift Valley Recording Jökulhlaups of Late Pleistocene Glacial Lakes of the Western United States.

Escanaba Trough is the southernmost segment of the Gorda Ridge and is filled by sandy turbidites locally exceeding 500 m in thickness. New results from Ocean Drilling Program (ODP) Sites 1037 and 1038 that include accelerator mass spectrometry (AMS) 14C dates and revised petrographic evaluation of the sediment provenance, combined with high-resolution seismic-reflection profiles, provide a lithostratigraphic framework for the turbidite deposits. Three fining-upward units of sandy turbidites from the upper 365 m at ODP Site 1037 can be correlated with sediment recovered at ODP Site 1038 and Deep Sea Drilling Program (DSDP) Site 35. Six AMS 14C ages in the upper 317 m of the sequence at Site 1037 indicate that average deposition rates exceeded 10 m/k.yr. between 32 and 11 ka, with nearly instantaneous deposition of one approximately 60-m interval of sand. Petrography of the sand beds is consistent with a Columbia River source for the entire sedimentary sequence in Escanaba Trough. High-resolution acoustic stratigraphy shows that the turbidites in the upper 60 m at Site 1037 provide a characteristic sequence of key reflectors that occurs across the floor of the entire Escanaba Trough. Recent mapping of turbidite systems in the northeast Pacific Ocean suggests that the turbidity currents reached the Escanaba Trough along an 1100-km-long pathway from the Columbia River to the west flank of the Gorda Ridge. The age of the upper fining-upward unit of sandy turbidites appears to correspond to the latest Wisconsinan outburst of glacial Lake Missoula. Many of the outbursts, or jökulhlaups, from the glacial lakes probably continued flowing as hyperpycnally generated turbidity currents on entering the sea at the mouth of the Columbia River.

Journal Article↗