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PubMed1995

Crystallographic structure of a PLP-dependent ornithine decarboxylase from Lactobacillus 30a to 3.0 A resolution.

Ornithine decarboxylase from Lactobacillus 30a (L30a OrnDC) is representative of the large, pyridoxal-5'-phosphate-dependent decarboxylases that act on lysine, arginine or ornithine. The crystal structure of the L30a OrnDC has been solved to 3.0 A resolution using MIR phases in combination with density modification (space group P6; a = 195.6 A, c = 97.6 A; dimer of 1460 amino acid residues/asymmetric unit; VM = 3.26 A3/Da). The refined crystallographic R-value was 0.219 (Rfree = 0.268) using 2-fold restraints with a 4 sigma cutoff and 8.0 to 3.0 A resolution data. Six dimers related by C6 symmetry compose the enzymatically active dodecamer (approximately 10(6) Da). Each monomer of L30a OrnDC can be described in terms of five sequential folding domains. The amino-terminal domain, residues 1 to 107, consists of a five-stranded beta-sheet termed the "wing" domain. Two wing domains of each dimer project inward towards the center of the dodecamer and contribute to dodecamer stabilization. The "linker" domain, residues 108 to 160, consists of short alpha-helices separated by a loop that fills in the PLP pocket. The third domain, residues 161 to 413, is an alpha/beta domain containing a seven stranded beta-sheet that resembles the PLP-binding domain of the aspartate aminotransferases. The fourth domain, residues 414 to 569, resembles the "small" domain of the aspartate aminotransferases, but is significantly larger due to insertions. The remaining carboxy-terminal domain, residues 570 to 730, is organized into multiple antiparallel loops and seven alpha-helices that help form a deep channel leading to the PLP-binding site.

Amino Acid Sequence↗
PubMed1994

Detection of Candida albicans DNA in serum by polymerase chain reaction.

Polymerase chain reaction (PCR) was applied in order to improve the diagnosis of disseminated candidosis. A nested PCR technique with 2 primer pairs was used to increase the sensitivity. We were able to detect Candida DNA in serum and tissue samples from experimentally infected mice as well as in serum samples from candidemia patients and patients with deep-seated Candida infection. Our PCR could detect as little as 1 pg Candida albicans DNA. The PCR method was more sensitive than culture in both the mouse experiments and the patients with deep candidosis (5/7 were PCR positive and 0/7 blood culture positive), and of similar sensitivity in candidemia patients (11/17 were 15/17 blood culture positive). The relatively short processing time of PCR, when compared with culturing, its sensitivity, as well as the possibility of using serum samples for analysis, all helped improve the diagnostics in deep-seated candidosis and candidemia.

Adult↗
PubMed2006

Stratified communities of active Archaea in deep marine subsurface sediments.

Archaeal 16S rRNA was extracted from samples of deep marine subsurface sediments from Peru Margin site 1227, Ocean Drilling Program leg 201. The amounts of archaeal 16S rRNA in each extract were quantified by serial dilution and reverse transcription (RT)-PCR. The results indicated a 1,000-fold variation in rRNA content with depth in the sediment, with the highest concentrations found near the sediment surface and in the sulfate-methane transition zone (SMTZ). The phylogenetic composition of the active archaeal population revealed by cloning and sequencing of RT-PCR products changed with depth. Several phylotypes affiliated with marine benthic group B (MBGB) dominated clone libraries from the upper part of the SMTZ and were detected only in this layer. Members of the miscellaneous crenarchaeotal group (MCG) dominated clone libraries from the other layers. These results demonstrate that archaeal communities change in activity and community composition over short distances in geochemically distinct zones of deep subseafloor sediments and that these changes are traceable in the rRNA pool. It was shown for the first time that members of both the MCG and MBGB Archaea are more active in the SMTZ than in layers above and below. This indicates that they benefit either directly or indirectly from the anaerobic oxidation of methane. They also appear to be ecophysiologically flexible, as they have been retrieved from a wide range of marine sediments of various geochemical properties.

Archaea↗
PubMed2002

Phylogenetic diversity of marine cyanophage isolates and natural virus communities as revealed by sequences of viral capsid assembly protein gene g20.

In order to characterize the genetic diversity and phylogenetic affiliations of marine cyanophage isolates and natural cyanophage assemblages, oligonucleotide primers CPS1 and CPS8 were designed to specifically amplify ca. 592-bp fragments of the gene for viral capsid assembly protein g20. Phylogenetic analysis of isolated cyanophages revealed that the marine cyanophages were highly diverse yet more closely related to each other than to enteric coliphage T4. Genetically related marine cyanophage isolates were widely distributed without significant geographic segregation (i.e., no correlation between genetic variation and geographic distance). Cloning and sequencing analysis of six natural virus concentrates from estuarine and oligotrophic offshore environments revealed nine phylogenetic groups in a total of 114 different g20 homologs, with up to six clusters and 29 genotypes encountered in a single sample. The composition and structure of natural cyanophage communities in the estuary and open-ocean samples were different from each other, with unique phylogenetic clusters found for each environment. Changes in clonal diversity were also observed from the surface waters to the deep chlorophyll maximum layer in the open ocean. Only three clusters contained known cyanophage isolates, while the identities of the other six clusters remain unknown. Whether or not these unidentified groups are composed of bacteriophages that infect different Synechococcus groups or other closely related cyanobacteria remains to be determined. The high genetic diversity of marine cyanophage assemblages revealed by the g20 sequences suggests that marine viruses can potentially play important roles in regulating microbial genetic diversity.

Bacteriophages↗
PubMedDate not supplied

Preliminary technical, experimental and clinical results of the use of the HPLR 27 system for the treatment of deep-seated tumours by hyperthermia.

Based on the theoretical deep penetration of radio frequencies, we have developed several 27.12 MHz ridged wave guides, with large apertures, and filled with circulating deionized water. Different low impedance matching networks have been tested and a 0-800 W power generator has been designed for this particular clinical use. This can be driven easily by a microcomputer which is used to regulate the temperature at depth by varying the sequencing time and/or by adjusting the RF power. The complete system, known as HPRL 27, is manufactured by Sairem Ets. Studies of the spatial power density distributions show a relatively homogeneous RF field for an aperture greater than 30 X 14 cm. Preliminary heating patterns studied in phantoms indicate the possibility of treating volumes greater than 2000 cm3 within the 50 per cent isotherm. Maximum heating is observed between 2 and 5 cm deep with passive cooling, and between 3 and 6 cm deep with active cooling, in which case the 50 per cent isotherm is situated between 2 and 10 cm deep. Preliminary phase I clinical results on 20 patients with pelvic or thoracic carcinomas show the possibility of heating deep-seated tumours to above 42 degrees C with a good patient tolerance. Temperature is controlled by implantable catheters or in the case of pelvic heating, by inserting catheters into the vagina, the rectum and the bladder. This new device is well adapted for the treatment of pelvic tumours.

Aged↗
PubMed2000

Idiomarina gen. nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp. nov. and Idiomarina zobellii sp. nov.

Two bacterial strains, KMM 227T and 231T, were isolated from seawater samples collected from the north-western Pacific Ocean at a depth of 4000-5000 m and were characterized using polyphasic taxonomy. Both were Gram-negative, psychrotolerant, heterotrophic, aerobic and required NaCl for growth (0.6-15.0%). The temperature for growth was 4-30 degrees C. Both strains were rod-shaped, with a single flagellum. However, strain KMM 231T revealed a single long fimbrium. Cellular fatty acids detected in the isolates were predominantly odd-numbered and iso-branched, with 15 and 17 carbons (ca. 70%). Also present were saturated and monounsaturated straight-chain fatty acids. Results of phylogenetic analyses, employing three tree-making methods, strongly indicated that the two strains formed a distinct lineage within a clade containing the genera Alteromonas, Colwellia and Pseudoalteromonas, in the gamma-Proteobacteria. The two strains shared 16S rDNA sequence similarity of 96.9% and genomic DNA relatedness of 27%; the latter was determined by dot-blot hybridization. The strains were differentiated by the presence of fimbria, production of chitinase, ability to grow on 15% NaCl and BIOLOG profiles. Given the polyphasic evidence accumulated in this study, it is proposed that the two deep-sea isolates be classified in the genus Idiomarina gen. nov., as Idiomarina abyssalis sp. nov. (type strain is KMM 227T) and Idiomarina zobellii sp. nov. (type strain is KMM 231T).

Base Composition↗
PubMed1989

Ultrastructural development of Leishmania chagasi in its vector, Lutzomyia longipalpis (Diptera: Psychodidae).

The development of Leishmania chagasi, etiologic agent of American visceral leishmaniasis, was studied by light and electron microscopy in the gut of the sand fly, Lutzomyia longipalpis, a natural vector. New aspects of suprapylarian Leishmania behavior were elucidated. In the sand fly midgut, amastigotes transformed into promastigotes (division promastigote I) during a first division sequence within the bloodmeal. Secondary division of these promastigotes resulted in a second form (division promastigote II), and these subsequently elongated into nectomonad promastigotes. Nectomonads existed in long and short populations which divided in the bloodmeal and throughout the midgut lumen after escape from the peritrophic membrane. Nectomonads adhered to the midgut cells in a highly organized manner, with their flagella embedded deep into microvilli and cytoplasm. Migration of parasites from the posterior midgut into the cardia/stomodeal valve region at 36 hr was associated with breakdown of the peritrophic membrane anteriorly. Posterior breakdown at 48 hr resulted in a peritrophic tube open at both ends containing some parasites within the digesting bloodmeal for up to 6 days postinfection. At the stomodeal valve, a myriad of slender and rounded promastigotes attached to the intima by flagellar hemidesmosomes; these may represent a transformation sequence from slender nectomonads to pear-shaped haptomonads. Pear-shaped forms appear to be precursors of paramastigotes, which also attached to the valve intima. Both rounded haptomonads and paramastigotes were found in the esophagus, dividing in a complex sequence initiated by posterior cleavage of the cytoplasm producing unique heart-shaped forms. Dividing paramastigotes also colonized the pharynx up to the cibarial valve. The ultrastructure of paramastigotes suggested that they may be infective forms, capable of some motility in the foregut. Free-swimming "infective" promastigotes were observed throughout the midgut and foregut, were attached in the pharynx (armature region), and were associated with the labrum-epipharynx of the proboscis in 3.6% of flies (16 days). The fine structure of hemidesmosomes in the foregut showed regional specializations, including the presence of plasmalemmar bridges in the gap space.

Animals↗
PubMed1993

Identification of Legionella pneumophila in various specimens by the polymerase chain reaction.

Amplification of the mip sequence with polymerase chain reaction (PCR) proved to be specific for Legionella pneumophila. With nested PCR, the sensitivity of the test was markedly increased. The lower limit of detection for nested PCR in the aqueous medium for live and heat-inactivated dead L. pneumophilia was approximately 1-10 bacteria/ml. The sensitivity of the method, however, was reduced by a factor of 10 to 100 when the bacteria were added to homogenised pulmonary tissue. Fixing the bacteria in aqueous suspension or in tissue homogenate with buffered 4% formalin (pH 7.3) reduced the sensitivity of the PCR by a factor of about 100. After intravenous injection of heat-inactivated bacteria in mice L. pneumophila was detected in deep-frozen samples of plasma and various tissues. The molecular biological technique of nested PCR is proposed as an additional method for the diagnosis of legionella.

Base Sequence↗
PubMed1992

Use of an instrument-holding clip for deeply recessed anastomoses of the rectum and esophagus.

Posterior layer closure of deeply located anastomoses including those performed in the course of low anterior rectal resection, total gastrectomy, and esophagogastrectomy, are often difficult. Usually five to six seromuscular or through-and-through posterior wall sutures are inserted sequentially and the loop or cut end of bowel is advanced over these parallel sutures. The sutures are then tied down. Entanglement of these sutures in a deep surgical field with limited access can lead to tears in the bowel wall. In addition, uneven and nonsequential tying of these sutures may compromise the anastomosis itself. We have described here how manual anastomosis in deep, hard to reach surgical fields can be better accomplished with the use of an instrument-holding clip. Use of the described instrument-holding clip, to clearly delineate and anchor each suture in sequence, eliminates these problems. An easily available device, it can save operative time, facilitate a more even anastomosis, and decrease the chance of bowel wall tearing, thus enhancing the security of the anastomosis.

Anastomosis, Surgical↗
PubMed2006

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases.

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7-8 and at temperature close to 35 degrees C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40-45 degrees C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.

Alaska↗
PubMed2005

Dermatan carriers for neovascular transport targeting, deep tumor penetration and improved therapy.

A new approach to functional tumor imaging and deep interstitial penetration of therapeutic agents is to target the upregulated transport activities of neovascular endothelium. Agents are formulated with the anionic glycosaminoglycan, 435-type dermatan sulfate (DS 435, 22.2 kDa), chemically enriched for oligosaccharide sequences that confer high heparin cofactor II binding and correlate with high tumor uptake. A magnetic resonance (MR) imaging agent is prepared as self-assembling, 5-nm nanoparticles of Fe(+3):deferoxamine (Fe:Df) bound by strong ion pairing to DS, which forms the outer molecular surface (Zeta potential -39 mV). On intravenous (i.v.) injection, Fe:Df-DS rapidly (<7 min) and selectively targets and transports at high capacity across the neovascular endothelium of large (2-cm) Dunning prostate R3327 AT1 rat tumors; releases from the abluminal surface, due to reversible binding of its multivalent, low-affinity (K(d) 10(-4) to 10(-5)) oligosaccharide ligands; and progressively penetrates the interstitium from its initial site of high uptake in the well-perfused outer tumor rim, into the poorly perfused central subregion. By gamma camera imaging of (67)Ga:Df-DS, the agent avoids normal site uptake and clears through the kidneys with a t(1/2) of 18 min. A therapeutic formulation of DS-doxorubicin (DS-dox) is prepared by aqueous high-pressure homogenization of the drug and DS 435, which produces 11-nm nanoparticles of doxorubicin cores coated with DS (Zeta potential -39 mV) that are stable to lyophilization. Microscopic analysis of tumor sections 3 h after i.v. injection shows much higher overall tumor fluorescence and deeper matrix penetration for DS-dox than conventional doxorubicin (dox): >75 vs. <25 microm between the nearest microvessels. DS-dox also results in enhanced tumor-cell internalization and nuclear localization of the drug. Therapeutic efficacies in established (250 +/- 15 mg) MX-1 human breast tumor xenografts at maximum tolerated doses (MTDs) are (control vehicle, dox, dox-DS) (a) median days to 7-fold tumor growth: 8.3, 25.6 (p = 0.0007), 43.2 (p = 0.0001); (b) complete 90-day tumor regressions: 0/10, 0/10, 4/10. These results demonstrate the potential to develop a novel class of carbohydrate-targeted neovascular transport agents for sensitive, high-resolution (100-microm) MR imaging and improved treatment of larger sized human tumor metastases.

Angiogenesis Inhibitors↗
PubMed2004

Structural insights into the EB1-APC interaction.

EB1 proteins bind to microtubule ends where they act in concert with other components, including the adenomatous polyposis coli (APC) tumor suppressor, to regulate the microtubule filament system. We find that EB1 is a stable dimer with a parallel coiled coil and show that dimerization is essential for the formation of its C-terminal domain (EB1-C). The crystal structure of EB1-C reveals a highly conserved surface patch with a deep hydrophobic cavity at its center. EB1-C binds two copies of an APC-derived C-terminal peptide (C-APCp1) with equal 5 microM affinity. The conserved APC Ile2805-Pro2806 sequence motif serves as an anchor for the interaction of C-APCp1 with the hydrophobic cavity of EB1-C. Phosphorylation of the conserved Cdc2 site Ser2789-Lys2792 in C-APCp1 reduces binding four-fold, indicating that the interaction APC-EB1 is post-translationally regulated in cells. Our findings provide a basis for understanding the dynamic crosstalk of EB1 proteins with their molecular targets in eukaryotic organisms.

Amino Acid Sequence↗
PubMed2002

Petrotoga olearia sp. nov. and Petrotoga sibirica sp. nov., two thermophilic bacteria isolated from a continental petroleum reservoir in Western Siberia.

Strictly anaerobic, thermophilic bacteria (strains SL24T, SL25T, SL27, SL29 and SL32) were isolated from a deep, continental oil reservoir in Western Siberia (Russia). These motile, rod-shaped organisms were surrounded by a sheath-like structure, a feature characteristic of the Thermotogales. On the basis of partial 16S rDNA sequences (500 nucleotides), strains SL25T, SL27, SL29 and SL32 were identical. Therefore, only strains SL24T and SL25T were studied in detail. The optimum temperature for growth of both strains was 55 degrees C. Their optimum pH for growth was 7.5 and their optimum NaCl concentration was between 20 and 30 g l(-1). The novel isolates reduced elemental sulfur and cystine, but not thiosulfate or sulfate, to hydrogen sulfide. The G+C contents of the genomic DNA of strains SL24T and SL25T were respectively 35 and 33 mol%. Phylogenetically, both strains are most closely related to Petrotoga miotherma, there being 98.9-99.4% similarity between their 16S rDNA sequences. Phenotypic properties and DNA-DNA hybridization experiments indicate that the strains belong to two novel species, for which the names Petrotoga olearia (type strain SL24T = DSM 13574T = JCM 11234T) and Petrotoga sibirica (type strain SL25T= DSM 13575T = JCM 11235T) are proposed.

Base Composition↗
PubMed1999

Molecular phylogenetic analysis of archaeal intron-containing genes coding for rRNA obtained from a deep-subsurface geothermal water pool.

Molecular phylogenetic analysis of a naturally occurring microbial community in a deep-subsurface geothermal environment indicated that the phylogenetic diversity of the microbial population in the environment was extremely limited and that only hyperthermophilic archaeal members closely related to Pyrobaculum were present. All archaeal ribosomal DNA sequences contained intron-like sequences, some of which had open reading frames with repeated homing-endonuclease motifs. The sequence similarity analysis and the phylogenetic analysis of these homing endonucleases suggested the possible phylogenetic relationship among archaeal rRNA-encoded homing endonucleases.

Archaea↗
PubMed1989

[Deep interscapular temperature and thermogenesis of brown fat during sleep].

Deep interscapular temperature measured just below the brown fat lobes was studied in rats during sleep at two ambient temperatures (24 degrees C and 4 degrees C) before and after adaptation (9 days) to cold (4 degrees C). The results show that in the cold ambient deep interscapular temperature decreases during desynchronized sleep independently of adaptation. Such change in temperature is probably the result of the depression in sympathetic vasoconstrictor influences on heat exchangers producing blood and brown fat cooling in sequence during this stage of sleep.

Adipose Tissue, Brown↗
PubMed1989

Some basic features of the pathological and normal motor system studied by chronic deep electrodes.

Our stereotactic experiences in agreement with the literature showed, that different target points could influence the same motor disturbance. To choose the best target point or target point combinations we generally implant the electrodes into VL, Vim, CM, P, dentate nucleus and motor cortex. To ensure the correct sequence of therapeutic lesions we developed an investigation system, taking into consideration the resting and the working state of the motor system. We elicited events centrally (stimulation of the different target points) and peripherally (reflexes) and recorded the evoked potentials at the non-stimulated sites along with the motor and motor modulation effects in the appropriate muscles. The elicited events depend on the site of stimulation and registration and on the state of muscle activity. The centrally and peripherally elicited events influence each other. With our technique the elicited events and their functional dependency is most explicit within the motor system. The results help to explain some basic motor functions and help to answer some of our therapeutic questions.

Athetosis↗
PubMed2001

Crystal structure of paromomycin docked into the eubacterial ribosomal decoding A site.

BACKGROUND: Aminoglycoside antibiotics interfere with translation in both gram-positive and gram-negative bacteria by binding to the tRNA decoding A site of the 16S ribosomal RNA. RESULTS: Crystals of complexes between oligoribonucleotides incorporating the sequence of the ribosomal A site of Escherichia coli and the aminoglycoside paromomycin have been solved at 2.5 A resolution. Each RNA fragment contains two A sites inserted between Watson-Crick pairs. The paromomycin molecules interact in an enlarged deep groove created by two bulging and one unpaired adenines. In both sites, hydroxyl and ammonium side chains of the antibiotic form 13 direct hydrogen bonds to bases and backbone atoms of the A site. In the best-defined site, 8 water molecules mediate 12 other hydrogen bonds between the RNA and the antibiotics. Ring I of paromomycin stacks over base G1491 and forms pseudo-Watson-Crick contacts with A1408. Both the hydroxyl group and one ammonium group of ring II form direct and water-mediated hydrogen bonds to the U1495oU1406 pair. The bulging conformation of the two adenines A1492 and A1493 is stabilized by hydrogen bonds between phosphate oxygens and atoms of rings I and II. The hydrophilic sites of the bulging A1492 and A1493 contact the shallow groove of G=C pairs in a symmetrical complex. CONCLUSIONS: Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.

Amino Acid Motifs↗
PubMed1999

Microsporidia are related to Fungi: evidence from the largest subunit of RNA polymerase II and other proteins.

We have determined complete gene sequences encoding the largest subunit of the RNA polymerase II (RBP1) from two Microsporidia, Vairimorpha necatrix and Nosema locustae. Phylogenetic analyses of these and other RPB1 sequences strongly support the notion that Microsporidia are not early-diverging eukaryotes but instead are specifically related to Fungi. Our reexamination of elongation factors EF-1alpha and EF-2 sequence data that had previously been taken as support for an early (Archezoan) divergence of these amitochondriate protists show such support to be weak and likely caused by artifacts in phylogenetic analyses. These EF data sets are, in fact, not inconsistent with a Microsporidia + Fungi relationship. In addition, we show that none of these proteins strongly support a deep divergence of Parabasalia and Metamonada, the other amitochondriate protist groups currently thought to compose early branches. Thus, the phylogenetic placement among eukaryotes for these protist taxa is in need of further critical examination.

Animals↗