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PubMed1999

Kluyveromyces nonfermentans sp. nov., a new yeast species isolated from the deep sea.

Eleven strains of a new species of the genus Kluyveromyces, characterized as having evanescent asci and Q-6 as the major ubiquinone, were isolated from sediments, a clam and a crab collected at depths of 1000-2000 m in Suruga Bay and Sagami Bay, Japan. A phylogenetic tree based on small-subunit (18S) rRNA gene sequences placed these isolates into a cluster of Kluyveromyces. DNA complementarity and phylogenetic trees of internal transcribed spacer (ITS) regions and 5.8S rRNA genes showed that the isolates are closely related to Kluyveromyces aestuarii, but that these two species are genetically distinct. The isolates are described as Kluyveromyces nonfermentans sp. nov. Because this species lacks the fermentative ability considered to be an important criterion for the genus Kluyveromyces, the definition of the genus has been emended. The type strain of K. nonfermentans is strain SY-33T (= JCM 10232T).

Animals↗
PubMed1999

The recognition of distorted DNA structures by HMG-D: a footprinting and molecular modelling study.

The high mobility group (HMG) domain is a DNA binding motif found in some eukaryotic chromosomal proteins and transcription factors. This domain binds in the minor groove of DNA inducing a sharp bend and also preferentially binds to certain distorted DNA structures. Although structures of sequence-specific HMG domains with their cognate double-helical DNA binding sites have been solved, the nature of the interaction of the domain with distorted DNA remains to be established. In this study we have investigated the interaction of HMG-D, a Drosophila counterpart of the vertebrate HMG1, with a DNA oligomer containing a bulge of two adenine residues. We show by footprinting that HMG-D binds preferentially on one side of the bulged DNA. Based on these data and on the published NMR structures of the HMG domain of HMG-D and the LEF-1-DNA complex, we modelled the HMG-D - bulged DNA complex. This model predicts that two residues, Val32 and Thr33, in the loop between alpha-helices I and II are inserted deep into the "hole" in the DNA formed by the two missing bases on one strand of the DNA bulge. Mutation of these residues confirmed that both are required for the efficient binding and bending of DNA by HMG-D. We discuss both the role of this loop in the recognition of distorted DNA structures by non-sequence specific HMG domain proteins and that of the basic tail in stabilising the induced DNA bend.

Amino Acid Sequence↗
PubMed1990

In vivo T1 measurements of canine brain for deep tissue temperature measurements with MRI.

The possibility of deep tissue temperature imaging with a magnetic resonance (MR) imager was studied in 30 mongrel dogs. The coronary view of the brain of a dog anesthetized with muscle relaxant and pentobarbital and ventilated automatically with air was taken with a special head coil by a 0.1 Tesla MR imager, Asahi Mark-J, driven by a spin warp pulse sequence. T1s in the imaging of the right and left white matter, grey matter, thalamus and caudate nucleus were measured with rectal temperature in group I (10 dogs) for a control study every 30 min until 120 min without intervention, in group II (10 dogs) for hyperthermia with extracorporeal bypass method, before heating, at 41 and 42 degrees C, in group III (10 dogs) for hypothermia, before cooling, at 35 and 30 degrees C and after rewarming, respectively. In groups I, II and III, the T1s at all sites except the white matter at 42 degrees C did not change significantly at any rectal temperature. T1 prolongation in the white matter at 42 degrees C may be due to edema, probably because of biological reaction to hyperthermia. The present study suggests that deep tissue temperature imaging in vivo by T1s measured with the MR imager may be impossible in spite of obvious evidence of T1 dependance on temperature in theoretical, in vitro and ex vivo studies already achieved.

Animals↗
PubMed2005

Phylogenetic resolution and habitat specificity of members of the Photobacterium phosphoreum species group.

Substantial ambiguity exists regarding the phylogenetic status of facultatively psychrophilic luminous bacteria identified as Photobacterium phosphoreum, a species thought to be widely distributed in the world's oceans and believed to be the specific bioluminescent light-organ symbiont of several deep-sea fishes. Members of the P. phosphoreum species group include luminous and non-luminous strains identified phenotypically from a variety of different habitats as well as phylogenetically defined lineages that appear to be evolutionarily distinct. To resolve this ambiguity and to begin developing a meaningful knowledge of the geographic distributions, habitats and symbiotic relationships of bacteria in the P. phosphoreum species group, we carried out a multilocus, fine-scale phylogenetic analysis based on sequences of the 16S rRNA, gyrB and luxABFE genes of many newly isolated luminous strains from symbiotic and saprophytic habitats, together with previously isolated luminous and non-luminous strains identified as P. phosphoreum from these and other habitats. Parsimony analysis unambiguously resolved three evolutionarily distinct clades, phosphoreum, iliopiscarium and kishitanii. The tight phylogenetic clustering within these clades and the distinct separation between them indicates they are different species, P. phosphoreum, Photobacterium iliopiscarium and the newly recognized 'Photobacterium kishitanii'. Previously reported non-luminous strains, which had been identified phenotypically as P. phosphoreum, resolved unambiguously as P. iliopiscarium, and all examined deep-sea fishes (specimens of families Chlorophthalmidae, Macrouridae, Moridae, Trachichthyidae and Acropomatidae) were found to harbour 'P. kishitanii', not P. phosphoreum, in their light organs. This resolution revealed also that 'P. kishitanii' is cosmopolitan in its geographic distribution. Furthermore, the lack of phylogenetic variation within 'P. kishitanii' indicates that this facultatively symbiotic bacterium is not cospeciating with its phylogenetically divergent host fishes. The results of this fine-scale phylogenetic analysis support the emerging view that bacterial species names should designate singular historical entities, i.e. discrete lineages diagnosed by a significant divergence of shared derived nucleotide characters.

Animals↗
PubMed2000

Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH.

Long-depth imaging of specific gene expression in the midgestation whole-mount mouse embryo (WME) is demonstrated with single-photon excitation (1PE) confocal fluorescence microscopy and fluorescence in situ hybridization. Expression domains of Pax-6 mRNA transcripts were labeled with an in situ hybridization probe that is a RNA sequence complementary to the cloned gene fragment and were rendered visible using two fluorochrome-conjugated antibodies that fluoresce at peak wavelengths of lambda(F) = 0.525 microm and lambda(F) = 0. 580 microm, respectively. Distributions of Pax-6 mRNA domains as deep as 1000 microm in the day 9.5 WME were imaged with a long-working-distance (13.6 mm) objective lens (magnification 5x). The scattering problem posed by the optically thick WME sample is alleviated by careful control of the detector pinhole size and the application of simple but fast postdetection image enhancement techniques, such as space and wavelength averaging to produce high-quality fluorescence images. A three-dimensional reconstruction that clearly shows the Pax-6 mRNA expression domains in the forebrain, diencephalon, optic cup, and spinal cord of the day 9.5 WME is obtained. The advantages of 1PE confocal fluorescence imaging over two-photon excitation fluorescence imaging are discussed for the case of long-depth imaging in highly scattering media. Imaging in midgestation WMEs at optical depths of more than 350 microm has not yet been realized with two-photon fluorescence excitation.

Animals↗
PubMed1998

Orientation-dependent changes in MR signal intensity of articular cartilage: a manifestation of the "magic angle" effect.

OBJECTIVE: To study magnetic resonance (MR) imaging pattern of normal hyaline articular cartilage in the knee joint with regard to the contribution of the "magic angle" effect to the MR signal. DESIGN: Thirty-two healthy volunteers were imaged in a standard supine position in a 1.5-T unit using spin echo and gradient echo sequences. Nine volunteers were reimaged with the knee flexed. The signal behavior of the hyaline cartilage of the femoral condyles was evaluated qualitatively and quantitatively. The extended and flexed positions of the nine volunteers were compared. RESULTS: A superficial and a deep hyperintense layer and a hypointense middle cartilage layer were observed. Segments of increased signal intensity were visible along the condyles; a magic angle effect on signal intensity was evident in the hypointense middle layer with both gradient echo and spin echo images. CONCLUSION: The MR signal behavior of hyaline cartilage is influenced by the alignment of the collagen fibers within the cartilage in relation to the magnetic field. Failure to recognize this effect may lead to inaccurate diagnosis.

Cartilage, Articular↗
PubMed1997

Pituitary dwarfism in the R271W Pit-1 gene mutation.

UNLABELLED: The Pit-1 gene encodes the POU-domain transcription factor Pit-1 which is important for the differentiation of the anterior pituitary and regulation of the PRL, GH and TSH genes. As a member of the POU domain transcription factors, Pit-1 contains a DNA-binding region, consisting of a POU-specific domain and a POU homeodomain. Mutation of the Pit-1 gene causes hypoplasia of the pituitary gland and deficiencies of GH, PRL and TSH. In a DNA sample from a 3-month-old girl with severe growth deficiency from birth, single stranded conformational polymorphism analysis of the Pit-1 gene identified a gel shift in exon 6. DNA-sequencing disclosed a single base mutation in codon 271 (CGG to TGG) that changes arginine to tryptophan (R271W) in the POU homeodomain. The patient presented distinct facial features with prominent forehead, marked mid-facial hypoplasia with depressed nasal bridge, deep-set eyes and a short nose with anteverted nostrils. MRI examination showed a hypoplastic pituitary gland. Low serum GH did not respond to insulin-arginine provocation or GHRH tests. PRL levels below the detection limit did not increase in response to a TRH test. T4 and free T4 was below detection limit (< 20 nmol/l and < 4 pmol/l). TSH was 2.0 mU/l and showed a blunt response to 6.0 mU/l following TRH test. TBG was normal. In spite of inappropriately low TSH and very low T4, T3 was in the low normal range (1.4-1.6 nmol/l) and she was clinically euthyroid. The thyroid function tests are consistent with increased monodeiodination activity and increased conversion of T4 to T3, possibly related to the Pit-1 gene mutation. GH and T4 treatment resulted in catch-up growth continued during 5 years of therapy. CONCLUSION: Reports of nine other cases of R271W mutations of different populations as well as the present Norwegian patient suggest codon 271 of exon 6 to be a "hot spot" for Pit-1 mutations. To enable rapid and simple detection of this type of de novo mutation we have designed a specific amplification-created-restriction-site assay to check for the R271W mutation in patients suspected to have this rare form of genetic defect in growth hormone production.

Arginine↗
PubMed2002

Unusual deep intronic mutations in the COL4A5 gene cause X linked Alport syndrome.

The X-linked form of Alport syndrome is caused by mutations in the COL4A5 gene in Xq22. This large multiexonic gene has, in the past, been difficult to screen, with several studies detecting only about 50% of mutations. We report three novel intronic mutations that may, in part, explain this poor success rate and demonstrate that single base changes deep within introns can, and do, cause disease: one mutation creates a new donor splice site within an intron resulting in the inclusion of a novel in-frame cryptic exon; a second mutation results in a new exon splice enhancer sequence (ESE) that promotes splicing of a cryptic exon containing a stop codon; a third patient exhibits exon skipping as a result of a base substitution within the polypyrimidine tract that precedes the acceptor splice site. All three cases would have been missed using an exon-by-exon DNA screening approach.

Amino Acid Sequence↗
PubMed1985

A sequential representation of the occiput, arm, forearm and hand across the rostrocaudal dimension of areas 1, 2 and 5 in macaque monkeys.

Receptive fields were determined for recording sites in cortical areas 1, 2 and 5 in macaque monkeys. While a mixture of cutaneous and deep receptors were represented in most regions of area 2, a specific mediolateral level of area 2 was highly responsive to cutaneous input. Anteroposterior rows of recording sites across areas 1, 2 and 5 at this mediolateral location revealed a continuous sequence of cutaneous receptive fields proceeding from the occiput and face in area 1 to the forearm and hand in area 5.

Animals↗
PubMed2005

Muscle growth patterns and regulation during fish ontogeny.

In fish, the skeletal muscle of the trunk and the tail derives from the somites which form in the paraxial mesoderm in a rostro-caudal sequence. The development of the fish myotome begins with the onset of myogenic regulatory factors expression and continues with the formation of a distinct superficial layer of slow muscle fibres that covers a bulk of fast muscle fibres located in the deep portion of the myotome. Muscle fibres of the slow-twitch lineage originate in fish embryos from adaxial cells, a distinct subpopulation of the paraxial mesoderm that flanks the notochord. During the early maturation of the somite these adaxial cells migrate away from the notochord towards the lateral part of the somite where they form the superficial slow fibres. Lateral presomitic cells that remain deep in the myotome differentiate into fast muscle fibres. Morphogens of the hedgehog family secreted by the notochord have a pivotal role in inducing the slow-twitch lineage. In late embryos, additional fibres are added from discrete germinal zones situated at the ventral and dorsal extremes of the developing myotome. This regionalised process has been termed "stratified hyperplasia." In fish which grow to a large final size this is followed by a mosaic hyperplastic process that leads to the formation of new fibres throughout the whole myotome. Current knowledge about the endocrine and autocrine factors that potentially regulate the proliferation and the differentiation of muscle cells within the embryonic and larval fish myotome is reviewed.

Animals↗
PubMed2004

Membrane insertion of the N-terminal alpha-helix of equinatoxin II, a sea anemone cytolytic toxin.

Equinatoxin II (Eqt-II) is a member of the actinoporins, a unique family of cytotoxins comprising 20 kDa pore-forming proteins isolated from sea anemones. Actinoporins bind preferentially to lipid membranes containing sphingomyelin, and create cation-selective pores by oligomerization of three to four monomers. Previous studies have shown that regions of Eqt-II crucial for its cytolytic mechanism are an exposed aromatic cluster and the N-terminal region containing an amphipathic alpha-helix. In the present study, we have investigated the transfer of the N-terminal alpha-helix into the lipid membrane by the use of three mutants containing an additional tryptophan residue in different positions within the amphipathic alpha-helix (Ile18-->Trp, Val22-->Trp and Ala25-->Trp). The interaction of the mutants with different model systems, such as lipid monolayers, erythrocytes and ghost membranes, was extensively characterized. Intrinsic fluorescence measurements and the use of vesicles containing brominated phospholipids indicated a deep localization of the N-terminal amphipathic helix in the lipid bilayer, except for the case of Val22-->Trp. This mutant is stabilized in a state immediately prior to final pore formation. The introduction of additional tryptophan residues in the sequence of Eqt-II has proved to be a suitable approach to monitor the new environments that surround defined regions of the molecule upon membrane interaction.

Animals↗
PubMed2006

A novel enzyme, lambda-carrageenase, isolated from a deep-sea bacterium.

A lambda-carrageenan-degrading Pseudoalteromonas bacterium, strain CL19, was isolated from a deep-sea sediment sample. A lambda-carrageenase from the isolate was purified to homogeneity from cultures containing lambda-carrageenan as a carbon source. This is the first report of the isolation of lambda-carrageenase together with the gene sequence for the enzyme. The molecular mass of the purified enzyme was approximately 100 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that the enzyme is a monomer. The optimal pH and temperature for activity were about 7 and 35 degrees C, respectively. The enzyme had specific activity of 253 U/mg protein. The enzyme required monovalent salts for the activity. Carbohydrates, such as sorbitol, sucrose, trehalose, improved the enzyme stability. The pattern of lambda-carrageenan hydrolysis showed that the enzyme is an endo-type lambda-carrageenase, and the final main product was a tetrasaccharide of the lambda-carrageenan ideal structure with galactose 2,6-disulfate at the reducing end, indicating the enzyme cleaves the beta-1,4 linkages of its backbone structure. Furthermore, the gene (cglA) encoding the enzyme was sequenced. It encoded a mature protein of 103 kDa (917 amino acids). Remarkably, the deduced amino acid sequence showed no similarity to any reported proteins.

Amino Acid Sequence↗
PubMed1996

Localization of ATP-gated ion channels in cerebellum using P2x2R subunit-specific antisera.

The distribution of the P2x2 purinoceptor subunit protein, which forms ATP-gated ion channels by homo- and hetero-multimeric assembly, was examined in the adult rat and guinea-pig cerebellum using two novel antisera generated against separate 18 amino acid sequences located in the predicted extracellular domain of this subunit. These antisera, the first available for labelling the P2x2R subunit protein, were validated by selective labelling of a fusion protein containing the target amino acid sequences, and in cerebellum, by peptide specific block of immunoreactivity and by comparison with the distribution of P2x2R mRNA. P2x2R-like immunoreactivity was seen in Purkinje cells, specifically the soma and dendrites, neurons in the granular and molecular layers and deep cerebellar nuclei. The identification of P2x2R-like immunoreactivity within the cerebellar neural circuitry is consistent with a role for extracellular ATP acting as a fast neurotransmitter in motor learning and coordination of movement. Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis.

Adenosine Triphosphate↗
PubMed1988

Epithelial restitution in the gastrointestinal tract.

Injury to the gastrointestinal mucosa can be divided into two types: (a) deep injury involving extensive hemorrhage and large areas of tissue necrosis, and (b) superficial injury confined to the upper regions of the mucosa and not involving hemorrhagic lesions. Mucosal repair differs according to the severity of the damage. Repair of deep injury takes weeks because large regions of the mucosa must be replaced with new tissue, a process involving mitosis. Superficial injury is initially repaired rapidly over hours by epithelial restitution that does not involve mitosis but proceeds in the following sequence of events: First, the damaged surface epithelial cells are shed and form a layer that protects the restituting mucosa. Then the viable epithelial cells that remain attached to the mucosa at the margin of the wound become flattened and rapidly migrate over the denuded basal lamina. The superficial epithelium is re-established when migrating cells touch, form new tight junctions, and repolarize their organelles. This rapid protective mechanism, called restitution, has recently been documented in the stomach, duodenum, colon, and rectum. The cellular mechanisms, speed of migration, recovery of transmucosal electrical potential difference, and specific peculiarities of rapid epithelial restitution in the various regions of the gastrointestinal tract are reviewed here.

Animals↗
PubMed2001

Genomic anatomy of a premier major histocompatibility complex paralogous region on chromosome 1q21-q22.

Human chromosomes 1q21-q25, 6p21.3-22.2, 9q33-q34, and 19p13.1-p13.4 carry clusters of paralogous loci, to date best defined by the flagship 6p MHC region. They have presumably been created by two rounds of large-scale genomic duplications around the time of vertebrate emergence. Phylogenetically, the 1q21-25 region seems most closely related to the 6p21.3 MHC region, as it is only the MHC paralogous region that includes bona fide MHC class I genes, the CD1 and MR1 loci. Here, to clarify the genomic structure of this model MHC paralogous region as well as to gain insight into the evolutionary dynamics of the entire quadriplication process, a detailed analysis of a critical 1.7 megabase (Mb) region was performed. To this end, a composite, deep, YAC, BAC, and PAC contig encompassing all five CD1 genes and linking the centromeric +P5 locus to the telomeric KRTC7 locus was constructed. Within this contig a 1.1-Mb BAC and PAC core segment joining CD1D to FCER1A was fully sequenced and thoroughly analyzed. This led to the mapping of a total of 41 genes (12 expressed genes, 12 possibly expressed genes, and 17 pseudogenes), among which 31 were novel. The latter include 20 olfactory receptor (OR) genes, 9 of which are potentially expressed. Importantly, CD1, SPTA1, OR, and FCERIA belong to multigene families, which have paralogues in the other three regions. Furthermore, it is noteworthy that 12 of the 13 expressed genes in the 1q21-q22 region around the CD1 loci are immunologically relevant. In addition to CD1A-E, these include SPTA1, MNDA, IFI-16, AIM2, BL1A, FY and FCERIA. This functional convergence of structurally unrelated genes is reminiscent of the 6p MHC region, and perhaps represents the emergence of yet another antigen presentation gene cluster, in this case dedicated to lipid/glycolipid antigens rather than antigen-derived peptides.

Antigens, CD1↗
PubMed1995

An rpoE-like locus controls outer membrane protein synthesis and growth at cold temperatures and high pressures in the deep-sea bacterium Photobacterium sp. strain SS9.

Many deep-sea bacteria have evolved specialized adaptations for life at cold temperatures and high pressures. A locus required for both psychro- and baro-adaptation in the psychrophilic, moderate barophile, Photobacterium species strain SS9 was identified among SS9 transposon mutants. DNA sequence analysis of this locus identified four complete open reading frames (ORFs), which appear to comprise an operon, and a fifth incomplete ORF. All transposon insertions isolated are in ORF3. Extensive sequence similarity exists between the translation products of ORFs 1-3 and a collection of gene products proposed to include alternative RNA polymerase sigma factors and modifiers of sigma-factor activity involved in extracytoplasmic sensing and regulation. Based on the similarity between ORF1 and Escherichia coli rpoE, we have tentatively designated this locus the rpoE locus. SS9 rpoE locus ORF3 insertion mutants showed altered abundances of numerous outer membrane proteins and were both baro- and psychro-sensitive. ORF3 mutant revertants that displayed enhanced high-pressure growth also displayed concomitant enhanced low-temperature growth. Most of these revertants possessed DNA rearrangements at the site of the transposon insertion, further demonstrating the importance of the rpoE locus to high-pressure and cold-temperature growth. Complementation analyses indicated that ORF3 functions in OMP synthesis regulation while ORF4 is required for baro- and psychro-adaptation.

Bacterial Outer Membrane Proteins↗
PubMed1997

The Cretaceous-Tertiary impact crater and the cosmic projectile that produced it.

Evidence gathered to date from topographic data, geophysical data, well logs, and drill-core samples indicates that the buried Chicxulub basin, the source crater for the approximately 65 Ma Cretaceous-Tertiary (K/T) boundary deposits, is approximately 300 km in diameter. A prominent topographic ridge and a ring of gravity anomalies mark the position of the basin rim at approximately 150 km from the center. Wells in this region recovered thick sequences of impact-generated breccias at 200-300 m below present sea level. Inside the rim, which has been severely modified by erosion following impact, the subsurface basin continues to deepen until near the center it is approximately 1 km deep. The best planetary analog for this crater appears to be the 270 km-diameter Mead basin on Venus. Seismic reflection data indicate that the central zone of downward displacement and excavation (the transient crater is approximately 130 km in diameter, consistent with previous studies of gravity anomaly data). Our analysis of projectile characteristics utilizes this information, coupled with conventional scaling relationships, and geochemical constraints on the mass of extraterrestrial material deposited within the K/T boundary layer. Results indicate that the Chicxulub crater would most likely be formed by a long-period comet composed primarily of nonsilicate materials (ice, hydrocarbons, etc.) and subordinate amounts (< or = 50%) primitive chondritic material. This collision would have released the energy equivalent to between 4 x 10(8) and 4 x 10(9) megatons of TNT. Studies of terrestrial impact rates suggest that such an event would have a mean production rate of approximately 1.25 x 10(-9) y-1. This rate is considerably lower than that of the major mass extinctions over the last 250 million years (approximately 5 x 10(-7) y-1). Consequently, while there is substantial circumstantial evidence establishing the cause-effect link between the Chicxulub basin forming event and the K/T biological extinctions, the results of our analysis do not support models of impact as a common or singular causative agent of mass extinctions on Earth.

Biological Evolution↗
PubMed2002

Comparative genomic analysis of archaeal genotypic variants in a single population and in two different oceanic provinces.

Planktonic crenarchaeotes are present in high abundance in Antarctic winter surface waters, and they also make up a large proportion of total cell numbers throughout deep ocean waters. To better characterize these uncultivated marine crenarchaeotes, we analyzed large genome fragments from individuals recovered from a single Antarctic picoplankton population and compared them to those from a representative obtained from deeper waters of the temperate North Pacific. Sequencing and analysis of the entire DNA insert from one Antarctic marine archaeon (fosmid 74A4) revealed differences in genome structure and content between Antarctic surface water and temperate deepwater archaea. Analysis of the predicted gene products encoded by the 74A4 sequence and those derived from a temperate, deepwater planktonic crenarchaeote (fosmid 4B7) revealed many typical archaeal proteins but also several proteins that so far have not been detected in archaea. The unique fraction of marine archaeal genes included, among others, those for a predicted RNA-binding protein of the bacterial cold shock family and a eukaryote-type Zn finger protein. Comparison of closely related archaea originating from a single population revealed significant genomic divergence that was not evident from 16S rRNA sequence variation. The data suggest that considerable functional diversity may exist within single populations of coexisting microbial strains, even those with identical 16S rRNA sequences. Our results also demonstrate that genomic approaches can provide high-resolution information relevant to microbial population genetics, ecology, and evolution, even for microbes that have not yet been cultivated.

Amino Acid Sequence↗