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PubMedDate not supplied

Magnetization transfer MR of the normal adult brain.

PURPOSE: To establish a normal baseline of the percent magnetization transfer of gray (cortical and deep) and white matter structures in the brain in healthy adults and to determine whether there are adult age-related differences in these measurements. METHODS: Axial T1-weighted scans (800/20 [repetition time/echo time]) with and without magnetization transfer were prospectively performed on a 1.5-T MR imaging unit on 68 healthy patients (aged 20 to 76 years). Presaturation and postsaturation magnetization transfer images were obtained using an on-resonance binomial pulse. All patients had normal MR scans on all pulse sequences. A calculated "difference" image was used to calculate the percent magnetization transfer in multiple specific regions of the brain. In each hemisphere, 9 discrete areas of cortical and deep gray matter and 29 areas of white matter were measured in 68 patients to generate age-related changes in percent magnetization transfer in these anatomic regions. Ranges of normal percent magnetization transfer in each of the 38 measures were established. RESULTS: The percent magnetization transfer of the gray matter (28% +/- 2%) was lower than that of the white matter (36% +/- 2%). There was no statistically significant difference in the percent magnetization transfer in different areas of gray matter. Deep white matter in the different lobes (percent magnetization transfer, 31% to 38%) also showed no differences by age. Percent magnetization transfer was the highest in the genu of the corpus callosum (42%), and this was statistically significant compared with other white matter measurements. CONCLUSION: There were no statistically significant age-related variations in the percent magnetization transfer in healthy adults in gray or white matter. These percent magnetization transfer measurements provide baseline normative data, which can be used to measure the extent and severity of white matter changes in disease states.

Adult↗
PubMed1996

[Recent progress in molecular diagnostic technology in clinical mycology].

Diagnosing deep-seated mycoses continues to be a major challenge for the clinician. The non-culture-dependent laboratory assay with high sensitivity and specificity are needed for rapid diagnosis of deep-seated mycoses. The most promising methodology that has this potential is the detection of fungus-specific DNA. Over the last five years, increasing numbers of papers were published which document the molecular biological assays feasible to detect fungus-specific DNA sequences in clinical specimens. In almost all of the assays thus far reported, DNA was detected following in vitro amplification using the RCR technology. This review will deal with those PCR assays which were developed, with the aim of diagnosing or screening deep-seated mycoses, for the detection of DNA from Candida species, Aspergillus species, Penicillium marneffei and Cryptococcus neoformans. Some of the PCR assays described here have been successfully used with clinical specimens to detect fungi, more rapidly and with a greater degree of sensitivity and/or specificity than by conventional means. It suggests us that PCR-based amplification strategies may help pave the way for modern clinical laboratory to better assist the clinician in diagnosing deep-seated mycoses.

Aspergillosis↗
PubMed2002

Integrated imaging in peripheral nerve lesions in type 1 neurofibromatosis.

PURPOSE: Our aim was to evaluate the role of Ultrasonography (US) and Magnetic Resonance (MR) in the staging and follow-up of peripheral nerve lesions in type 1 Neurofibromatosis (NF1). MATERIAL AND METHODS: We evaluated forty-six NF1 patients (16 males and 30 females, age range 12-65 years, mean age 34 years) affected by 51 soft tissue lesions, clinically diagnosed as subcutaneous (18 cases) and plexiform (33 cases) neurofibromas. The lesions were studied to identify site, size, extension, relationship with surrounding structures, and any features indicating malignant transformation. All patients underwent at least one US examination performed with a 7.5 - 10 MHz linear multi-frequency probe. All subcutaneous neurofibromas larger than 2 cm, all superficial or deep plexiform neurofibromas not clearly defined at US, and all large plexiform neurofibromas with massive involvement of surrounding tissues were studied by MR using 0.2 T magnet (Artoscan) or 1.5 T magnet (Magnetom Vision Plus Siemens). Fifteen patients underwent surgery; the remaining cases (31) were followed up according to the National Institutes of Health (NIH) protocol. RESULTS: The subcutaneous neurofibromas smaller than 2 cm showed homogeneous hypoechoic echotexture (10 cases), whereas lesions equal to or larger than 2 cm showed a characteristic target pattern (peripheral hypoechoic rim with hyperechoic core). All lesions larger than 2 cm studied by MRI displayed the target pattern in T2-weighted sequences, with peripheral signal hyperintensity and central signal hypointensity. At US, the 33 plexiform neurofibromas had an irregular shape with undefined borders; the patterns were classified as follows: 1) superficial plate-like lesions (14 cases); 2) superficial lesions with deep digitations (8 cases); 3) deep multi-lobulated lesions (5 cases); 4) extensive lesions massively involving soft tissues (6 cases). MRI was performed in 11 cases, showing an isointense or moderately hyperintense signal on T1-weighted sequences, high signal intensity on T2-weighted sequences, and, after intravenous gadolinium administration, variable enhancement on T1-weighted sequences. CONCLUSIONS: The results obtained in subcutaneous neurofibromas indicate a clear relationship between lesion size and US-MR imaging, with similar local staging. Since MRI failed to provide additional information, US may be considered the first-choice, and sometimes decisive, examination in these cases. As regards plexiform neurofibromas, US only proved useful in staging superficial lesions and partly deep multi-lobulated lesions as it accurately depicted lesion size, but not local extension; MRI is therefore useful in the preoperative staging of lesions. In extensive lesions, US proved unable to provide accurate and complete local staging of the lesions, so that MRI should be systematically used in these cases.

Adolescent↗
PubMed2001

Isolation of the rpoD gene encoding the principal sigma factor of the deep-sea piezophilic bacterium Shewanella violacea strain DSS12 and its overexpression in Escherichia coli.

The gene encoding the principal a factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced. The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3% identity to those of Escherichia coli and Pseudomonas putida, respectively. Comparison with E. coli sigma70 and P. putida sigma70 showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding. Using an expression plasmid harboring the rpoD gene, the S. violacea sigma70 factor was overexpressed in E. coli and successfully purified to near homogeneity.

Amino Acid Sequence↗
PubMed1996

X-ray structure of an anti-fungal chitosanase from streptomyces N174.

We report the 2.4 A X-ray crystal structure of a protein with chitosan endo-hydrolase activity isolated from Streptomyces N174. The structure was solved using phases acquired by SIRAS from a two-site methyl mercury derivative combined with solvent flattening and non-crystallographic two-fold symmetry averaging, and refined to an R-factor of 18.5%. The mostly alpha-helical fold reveals a structural core shared with several classes of lysozyme and barley endochitinase, in spite of a lack of shared sequence. Based on this structural similarity we postulate a putative active site, mechanism of action and mode of substrate recognition. It appears that Glu 22 acts as an acid and Asp 40 serves as a general base to activate a water molecule for an SN2 attack on the glycosidic bond. A series of amino-acid side chains and backbone carbonyl groups may bind the polycationic chitosan substrate in a deep electronegative binding cleft.

Binding Sites↗
PubMed1999

The phylogenetic relationships of Caulobacter, Asticcacaulis and Brevundimonas species and their taxonomic implications.

The phylogenetic relationships among the species of Caulobacter, Asticcacaulis and Brevundimonas were studied by comparison of their 16S rDNA sequences. The analysis of almost complete sequences confirmed the early evolutionary divergence of the freshwater and marine species of Caulobacter reported previously [Stahl, D. A., Key, R., Flesher, B. & Smit, J. (1992). J Bacteriol 174, 2193-2198]. The freshwater species formed two distinct clusters. One cluster contained the species Caulobacter bacteroides, Caulobacter crescentus, Caulobacter fusiformis and Caulobacter henricii. C. bacteroides and C. fusiformis are very closely related (sequence identity 99.8%). The second cluster was not exclusive and contained the specis Caulobacter intermedius, Caulobacter subvibrioides and Caulobacter variabilis, as well as Brevundimonas diminuta and Brevundimonas vesicularis. The marine species Caulobacter halobacteroides and Caulobacter maris were very closely related, with a sequence identity of 99.7%. These two species were most closely but distantly related to the marine hyphal/budding bacteria Hyphomonas jannaschiana and Hirschia baltica, which formed a deep phylogenetic line with Rhodobacter sphaeroides and Rhodobacter capsulatus. Caulobacter leidyia is unrelated to the other species of Caulobacter and belongs to the alpha-4 subclass of the Proteobacteria, forming a distinct cluster with Asticcacaulis excentricus and Asticcacaulis biprosthecium. The taxonomic implications of the polyphyletic nature of the genus Caulobacter and the absence of a type culture for the type species of the genus Caulobacter vibrioides, are discussed.

Caulobacter↗
PubMed1991

Mitochondrial resolution of a deep branch in the genealogical tree for perching birds.

Animal mitochondrial DNA (mtDNA) is known to contain information about the genealogical relations among closely related species and is shown here to yield information about distant relations as well. Our results also draw attention to the need for caution in using third positions of codons for tree construction. This is evident from comparative studies of the cytochrome b gene in 13 species representing major groups within the order of perching birds (Passeriformes). Sequences of a 924 base-pair segment of this gene were obtained from each of these species via the polymerase chain reaction and a novel set of versatile primers. With a woodpecker sequence as an outgroup, trees that separate songbirds from other perching birds and resolve the ancient branch leading to songbirds were obtained utilizing the conservative first and second positions of codons. Analysis of positions within codons suggests that, for deep branches, the skewed base composition at the fast-changing third positions can result in phylogenetic disinformation, which conflicts with the information retained in the first and second positions. The mitochondrial tree shows broad concordance with that based on hybridization of nuclear DNA; however, parsimony and maximum likelihood methods suggest a close kinship between thrushes and Australian babblers, in agreement with the traditional morphological classification.

Amino Acid Sequence↗
PubMed1993

Cloning of a crustacean myosin heavy chain isoform: exclusive expression in fast muscle.

A clone of a fast isoform of myosin heavy chain (HC) gene was isolated from a cDNA1 expression library made from mRNA purified from the deep abdominal flexor muscle of the lobster, Homarus americanus. The cDNA (1.5 kb) contained the 3' untranslated region (UTR) and the coding sequence for the last 413 amino acid residues of the carboxyl terminus of the polypeptide. The deduced amino acid sequence showed high homology with that of myosin HCs from Drosophila (73% identity), nematode (57% identity), and vertebrates (49% identities). Hydropathy plots showed a 28-amino acid periodicity that is consistent with the alpha-helical coiled coil structure of the rod region of native myosin. Northern blot analysis and in situ hybridization showed that the fast myosin HC isoform was expressed only in fast fibers; the probes did not hybridize to mRNA from slow fibers. The message consisted of a single transcript of 6.6 kb. The intracellular localization of the fast myosin mRNA was not uniform. The mRNA was largely confined to the intermyofibrillar spaces and to the subsarcolemmal cytoplasm of the fiber periphery and large infoldings of the cell membrane. Within these regions the mRNA was concentrated in the cytoplasm immediately surrounding the nuclei. This constitutes the first report of the cloning and expression of a myosin HC gene from a crustacean species.

Amino Acid Sequence↗
PubMed2001

Cloning, expression, and structure analysis of carbamate kinase-like carbamoyl phosphate synthetase from Pyrococcus abyssi.

Pyrococcus abyssi, a hyperthermophilic archaeon found in the vicinity of deep-sea hydrothermal vents, grows optimally at temperatures around 100 degrees C. Carbamoyl phosphate synthetase (CPSase) from this organism was cloned and sequenced. The active 34-kDa recombinant protein was overexpressed in Escherichia coli when the host cells were cotransformed with a plasmid encoding tRNA synthetases for low-frequency Escherichia coli codons. Sequence homology suggests that the tertiary structure of P. abyssi CPSase, resembling its counterpart in Pyrococcus furiosus, is closely related to the catabolic carbamate kinases and is very different from the larger mesophilic CPSases. P. furiosus CPSase and carbamate kinase form carbamoyl phosphate by phosphorylating carbamate produced spontaneously in solution from ammonia and bicarbonate. In contrast, P. abyssi CPSase has intrinsic bicarbonate-dependent ATPase activity, suggesting that the enzyme can catalyze the phosphorylation of the isosteric substrates carbamate and bicarbonate.

Amino Acid Sequence↗
PubMedDate not supplied

Thrombus organization plays no major role in late neointimal formation after angioplasty in porcine coronary arteries.

Thrombus organization has been suggested to play a major role in late neointimal formation after coronary angioplasty. We sought to describe the time sequence of lesion formation after angioplasty in porcine coronary arteries and to quantify the relation between early thrombosis and late neointimal formation. Deep vessel wall injury was induced by conventional balloon angioplasty in the circumflex (CX) and right coronary (RCA) arteries and by retraction of a chain-encircled balloon in the left anterior descendent artery (LAD). Lesions were assessed by histomorphometry at days 0, 1, 4, 7, 14, 28, and 56 after angioplasty. A response-to-injury index (lesion area/injury length) was determined for each artery. Angioplasty led to rupture/removal of media. Thrombus was present at the exposed adventitia at days 0, 1, and 4. From day 7, neointima was observed on the luminal side of the arterial wall. All thrombus had disappeared at day 28, at which only neointima was observed. Histomorphometry revealed that lesion formation after angioplasty was a gradually increasing process from day 0 to day 28 with no further growth from day 28 to day 56. Maximal thrombus size (day 4, RCA: 0.07+/-0.04 mm, CX: 0.23+/-0.16 mm, LAD: 0.15+/-0.11 mm) was significantly smaller than late neointimal formation (day 28, RCA: 0.68+/-0.18 mm, CX: 0.63+/-0.23 mm, LAD: 0.71+/-0.18 mm) in all three arteries (p < .03). Lesion formation after angioplasty is a gradually increasing process for 4 weeks. Maximal thrombus size is about four times smaller than late neointimal formation. Thus, thrombus organization plays no major role in late neointimal formation.

Angioplasty, Balloon, Coronary↗
PubMed2002

Crystal structure of a complex between the aminoglycoside tobramycin and an oligonucleotide containing the ribosomal decoding a site.

Aminoglycoside antibiotics target the decoding aminoacyl site (A site) on the 16S ribosomal RNA and induce miscoding during translation. Here, we present the crystal structure, at 2.54 A resolution, of an RNA oligonucleotide containing the A site sequence complexed to the 4,6-disubstituted 2-deoxystreptamine aminoglycoside tobramycin. The three aminosugar rings making up tobramycin interact with the deep-groove atoms directly or via water molecules and stabilize a fully bulged-out conformation of adenines A(1492) and A(1493). The comparison between this structure and the one previously solved in the presence of paromomycin confirms the importance of the functional groups on the common neamine part of these two antibiotics for binding to RNA. Furthermore, the analysis of the present structure provides a molecular explanation to some of the resistance mechanisms that have spread among bacteria and rendered aminoglycoside antibiotics inefficient.

Anti-Bacterial Agents↗
PubMed2003

Solution structure and backbone dynamics of the TGFbeta type II receptor extracellular domain.

Isoforms of transforming growth factor beta (TGFbeta) are 25 kDa homodimeric polypeptides that signal by binding and bringing together two related, functionally distinct cell surface receptors designated as TbetaR1 and TbetaR2. Here, we report the solution structure of the 13.8 kDa extracellular domain of human TbetaR2 (ecTbetaR2) as calculated from N(N)-H(N), C(alpha)-H(alpha), and C(alpha)-C(O) residual dipolar coupling restraints in conjunction with NOE distance, dihedral angle, and scalar coupling restraints. Comparison of the free ecTbetaR2 solution structure with the TGFbeta3-bound ecTbetaR2 crystal structure reveals backbone conformations that superimpose with RMSDs of 1.0 A over the regions of regular secondary structure and 1.4 A overall. The differences in structure fall mainly in loop regions that are either poorly defined by the available NMR data or are involved in crystal contacts. The noted similarities between the NMR structure of the free form and the crystal structure of the TGFbeta-bound form are also consistent with the close correspondence, 0.16 A RMSD for regions of secondary structure and 0.51 A RMSD overall, for the crystal structure of free ecTbetaR2 as compared to the crystal structure of TGFbeta3-bound ecTbetaR2. Despite the apparent similarities between the free and the bound forms, there appears to be small but significant differences in structure involving the interfacial contact region of the receptor. Measurements of backbone (15)N relaxation times and interpretation of these by the model-free formalism with axial diffusional anisotropy further reveal significant ms to micros time scale motions centered about two of the conserved disulfide bonds and in several residues that comprise the TGFbeta binding surface. Together, these observations indicate that binding likely occurs through a mechanism with a small component of induced fit character, whereby flexibility within the receptor facilitates the transition to the TGFbeta-bound state.

Amino Acid Sequence↗
PubMed1993

A conserved helix motif complements the protein kinase core.

Residues 40-300 of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase define a conserved bilobal catalytic core shared by all eukaryotic protein kinases. Contiguous to the core is an extended amphipathic alpha-helix (A helix). Trp30, a prominent feature of this helix, fills a deep hydrophobic pocket between the two lobes on the surface opposite to the active site. The C subunit in Dictyostelium discoideum shows sequence conservation of residues 40-350 with the mouse enzyme but contains an N-terminal extension of 332 residues. A sequence corresponding to the A helix contiguous to the core is absent. However, we have now identified a remote A-helix motif (residues 77-98). When the core of the Dictyostelium C subunit was modeled, based on the mouse C subunit, complementarity between this putative A helix and the surface of the core was found to be conserved. Analysis of other protein kinases reveals that the A-helix motif is not restricted to cAMP-dependent protein kinase. In the Src-related family of protein kinases, for example, an A helix is very likely contiguous to the core, thus serving as a linker between the conserved catalytic core and the Src homology 2 domain. We predict that an A-helix motif complementary to the core will be a conserved feature of most eukaryotic protein kinases.

Amino Acid Sequence↗
PubMed1994

Crystal structures at 2.5 angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate.

Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 A resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions in a deep hydrophilic cleft formed by the antiparallel beta sheet and surrounding loops of the synthetase catalytic domain. Four regions in the primary sequence are involved in the interactions, including the motif 2 and 3 regions of class 2 synthetases. Apart from the specific recognition of the serine side chain, the interactions are likely to be similar in all class 2 synthetases.

Adenosine↗
PubMed1996

The telomeric and centromeric ends of HLA class I: MCD maps of YAC derived cosmids and sequence analysis of 740 kb of genomic DNA.

We previously isolated and characterized a set of overlapping yeast artificial chromosome (YAC) clones spanning 2.4 Mb, including the entire MHC class I region, as a first step towards a detailed genetic analysis. We report here the genomic sequence of the two ends of HLA class I. The centromeric portion of HLA class I, extending from TNF to HLA-C was determined using two different sources of genomic DNA. As a first source, we sequenced cosmids (provided by T. Spies) derived from a total human DNA library which were mapped with conventional restriction digestion and fingerprinting. A second more generalizable approach was used to obtain cosmids for the remainder of the region. The new technology of Multiple-Complete-Digest (MCD) mapping, developed in Maynard Olson's laboratory, was used to map cosmids derived from YACs. This technique involves screening deep cosmid libraries derived from selected YACs and subjecting the cosmids to complete digestion with restriction enzymes, followed by computational assembly into completed maps. This method was also used to obtain material for sequence from three overlapping YACs covering a contiguous region from HLA-G to a point 330 kb telomeric. Among the plethora of new genetic information is a detailed picture of the organization of new multigene families contained within the telomeric end and of members of families spread throughout HLA class I. A clear relationship between the telomeric and centromeric ends of HLA class I has been defined, suggesting that large portions of these regions derived from a common ancestor. Our results demonstrate genomic sequencing to be one of the most effective and efficient means of identifying new genes, yielding information about genomic structure, regulation, and offering new insights into the meaning of physical relationships among functionally interacting genes.

Centromere↗
PubMed1998

Mitochondrial DNA phylogeography of European hedgehogs.

European hedgehog populations belonging to Erinaceus europaeus and E. concolor have been investigated by mitochondrial DNA analysis. A 383 bp fragment of the cytochrome b gene has been sequenced and maximum parsimony and neighbour-joining trees of Tamura-Nei genetic distance values have been constructed. Similar topologies have been produced by both methods, showing a deep divergence between E. europaeus and E. concolor and a further subdivision of each species into a western and an eastern clade. A comparison with previously published allozyme data is made, and concordant and discordant patterns are discussed. The influence of Pleistocene glaciations on the observed pattern of divergence is inferred.

Animals↗
PubMed1996

Molecular cloning of the first metazoan beta-1,3 glucanase from eggs of the sea urchin Strongylocentrotus purpuratus.

We report the molecular cloning of the first beta-1,3 glucanase from animal tissue. Three peptide sequences were obtained from beta-1,3 glucanase that had been purified from eggs of the sea urchin Strongylocentrotus purpuratus and the gene was cloned by PCR using oligonucleotides deduced from the peptide sequences. The full-length cDNA shows a predicted enzyme structure of 499 aa with a hydrophobic signal sequence. A 3.2-kb message is present in eggs, during early embryogenesis, and in adult gut tissue. A polyclonal antibody to the native 68-kDa enzyme recognizes a single band during early embryogenesis that reappears in the adult gut, and recognizes a 57-kDa fusion protein made from a full-length cDNA clone for beta-1,3 glucanase. The identity of this molecule as beta-1,3 glucanase is confirmed by sequence homology, by the presence of all three peptide sequences in the deduced amino acid sequence, and by the recognition of the bacterial fusion protein by the antibody directed against the native enzyme. Data base searches show significant homology at the amino acid level to beta-1,3 glucanases from two species of bacteria and a clotting factor from the horseshoe crab. The homology with the bacteria is centered in a 304-aa region in which there are seven scattered regions of high homology between the four divergent species. These four species were also found to have two homologous regions in common with more distantly related plant, fungal, and bacterial proteins. A global phylogeny based on these regions strongly suggests that the glucanases are a very ancient family of genes. In particular, there is an especially deep split within genes taken from the bacterial genus Bacillus.

Amino Acid Sequence↗