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At least 1,117 records · Page 62Linked to original sources
PubMed2002

PCR detection of psychrotolerant clostridia associated with deep tissue spoilage of vacuum-packed chilled meats.

AIMS: To develop a practical molecular procedure that directly (without isolation) and specifically detects the presence of clostridia, which cause the deep tissue spoilage condition. METHODS AND RESULTS: A primer set was designed and a PCR amplification procedure developed to detect the presence of Clostridium algidicarnis and Cl. putrefaciens 16S rDNA gene fragments in meat. The procedure yielded amplicons of the expected size with homologous DNA templates, but failed to give PCR products with DNAs from 47 food clostridia and common meat spoilage micro-organisms. The minimum level of detection was 10(4) cfu g-1 for nonenriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, the presence of Cl. algidicarnis and/or Cl. putrefaciens was confirmed in a swab sample taken from the cartilage of an ovine stifle joint, which on opening exhibited strong offensive odours. CONCLUSIONS: The developed method can be used for rapid detection of clostridia causing deep tissue spoilage in commercial vacuum packs. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of deep tissue clostridial spoilage in commercial vacuum-packed chilled meats.

Animals↗
PubMed1999

Changes in motor network expression related to moulting behaviour in lobster: role of moult-induced deep hypoxia

The well known rhythmically active pyloric neural network in intact and freely behaving lobsters Homarus gammarus was monitored prior to and following ecdysis. Despite long-lasting hormonal and metabolic alterations associated with this process, spontaneous pyloric network activity remained largely unaltered until the last 12-48 h before exuviation. At this time, the most notable change was a progressive lengthening of pyloric cycle period, which eventually attained 500-600 % of control values. It was only in the very last minutes before ecdysis that burst patterning became irregular and the otherwise strictly alternating motor sequence broke down. After the moult, coordinated rhythmicity was re-established within 10 min. Concomitant with these final changes in motor network expression at ecdysis was a drastic reduction in blood oxygen levels which led to a temporary near-anoxia. By imposing similarly deep hypoxic conditions both on intermoult animals and on the pyloric network in vitro, we mimicked to a large extent the moult-induced changes in pyloric network performance. Our data suggest that, despite major surrounding physiological perturbations, the pyloric network in vivo retains stable pattern-generating properties throughout much of the moulting process. Moreover, some of the most significant modifications in motor expression just prior to ecdysis can be related to a substantial reduction in oxygen levels in the blood.

Journal Article↗
PubMed1986

Successive waves of differentiation of cutaneous afferents in rat mystacial skin.

The present study has traced the sequence of maturation of sensory receptors in the mystacial pad of postnatal rats. At birth the follicle-sinus complexes (F-SC) are well innervated by deep vibrissal nerves although the number of axons entering the sinus is less than that in the adult. The innervation of the F-SC by the conus or superficial vibrissal nerves derived from skin nerves that form the superficial dermal nerve plexus is limited to the Merkel rete ridge collar at birth, and the innervation to the inner conical body is conspicuously absent. The inner conical body innervation begins to appear 3-4 days after birth and rapidly matures over the week. By 3 weeks of age the F-SCs have a mature sensory innervation. At birth small guard hairs are present in the intervibrissal pelage and are associated with scant axons of the superficial dermal nerve plexus, but no mature sensory terminals are present. The sensory innervation of the intervening pelage begins to differentiate during the second week and mature piloneural complexes can be recognized by 3 weeks of age. Innervation to vellus hairs is still developing at 3-4 weeks of age. These maturational changes in peripheral sensory innervation correlate with gradual changes in the structure of barrels in the first somatosensory cortex (SI). Sequential waves of differentiation of sensory receptors appear to be a general feature of neural development.

Animals↗
PubMed2001

Genetic transformation system for a psychrotrophic deep-sea bacterium: isolation and characterization of a psychrotrophic plasmid.

A versatile system that permits genetic manipulation of a psychrotrophic deep-sea bacterium, Pseudoalteromonas sp. PS1M3, has been developed. A cryptic indigenous plasmid, pPS1M3, of 3.1 kb from the above strain was isolated and characterized. The nucleotide sequence analysis of plasmid pPS1M3 revealed the presence of one open reading frame, and its deduced amino acid sequence was identified as the essential protein for plasmid maintenance. Transformation with the pPS1M3 harboring antibiotic resistance genes by electroporation was fully successful using the pPS1M3-cured strain as a host. This plasmid was quite stable under nonselective culture conditions for about 100 generations at 4 degrees C. The copy number of this plasmid in the cell was about 5 copies per chromosome.

Journal Article↗
PubMed1981

Some principles of flexor reflex generation in human leg muscles.

Flexor reflexes, evoked by stimulation of the tibial and deep peroneal nerves at the ankle level, were investigated in several ipsi and contralateral human leg muscles. On both sides, the reflex consist of a stereotyped oscillating sequence of inhibition and facilitation, exhibiting specifity to the stimulated site only in the initial ipsilateral phases. The responsible afferents were identified as belonging to a group of non-nociceptice skin afferents with a maximum conduction velocity of about 40-54 m/sec. Interdependence was observed between both latencies and amplitudes of the individual phases of the reflex sequences in synergistic and antagonistic pairs of muscles. It is concluded that the function of such a conjoint reflex generation in different muscles is to ensure an organized and adequate withdrawal.

Electric Stimulation↗
PubMed2004

Characterisation of proteoglycans and their catabolic products in tendon and explant cultures of tendon.

Tendons are collagenous tissues made of mainly Type I collagen and it has been shown that the major proteoglycans of tendons are decorin and versican. Little is still known about the catabolism of these proteoglycans in tendon. Therefore, the aim of the study was to characterise the proteoglycans including their catabolic products present in uncultured bovine tendon and in the explant cultures of tendon. In this study, the proteoglycans were extracted from the tensile region of deep flexor tendon and isolated by ion-exchange chromatography and after deglycosylation analysed by SDS-polyacrylamide electrophoresis, Western blotting and amino-terminal amino acid sequence analysis. Based on amino acid sequence analysis, approximately 80% of the total proteoglycan core proteins in fresh tendon was decorin. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V(0) and/or V(1)) and aggrecan. Approximately 35% of decorin present in the matrix showed carboxyl-terminal proteolytic processing at a number of specific sites. The analysis of small proteoglycans lost to the medium of tendon explants showed the presence of biglycan and decorin with the intact core protein as well as decorin fragments that contained the amino terminus of the core protein. In addition, two core protein peptides of decorin starting at residues K(171) and D(180) were observed in the matrix and one core protein with an amino-terminal sequence commencing at G(189) was isolated from the culture medium. The majority of the large proteoglycans present in the matrix of tendon were degraded and did not contain the G1 globular domain. Furthermore the aggrecan catabolites present in fresh tendon and lost to the medium of explants were derived from aggrecanase cleavage of the core protein at residues E(373)-A(374), E(1480)-G(1481) and E(1771)-A(1772). The analysis of versican catabolites (splice variants V(0) and/or V(1)) also showed evidence of degradation of the core protein by aggrecanase within the GAG-beta subdomain, as well as cleavage by other proteinase(s) within the GAG-alpha and GAG-beta subdomains of versican (variants V(0) and/or V(2)). Degradation products from the amino terminal region of type XII collagen were also detected in the matrix and medium of tendon explants. This work suggests a prominent role for aggrecanase enzymes in the degradation of aggrecan and to a lesser extent versican. Other unidentified proteinases are also involved in the degradation of versican and small leucine-rich proteoglycans.

Amino Acid Sequence↗
PubMed2006

Dissecting the ancient rapid radiation of microgastrine wasp genera using additional nuclear genes.

Previous estimates of a generic level phylogeny for the ubiquitous parasitoid wasp subfamily Microgastrinae (Hymenoptera) have been problematic due to short internal branches deep in the phylogeny. These short branches might be attributed to a rapid radiation among the taxa, the use of genes that are unsuitable for the levels of divergence being examined, or insufficient quantity of data. We added over 1200 nucleotides from four nuclear genes to a dataset derived from three genes to produce a dataset of over 3000 nucleotides per taxon. While the number of well-supported short branches in the phylogeny increased, we still did not obtain strong bootstrap support for every node. Parametric and nonparametric bootstrap simulations projected that an enormous, and likely unobtainable, amount of data would be required to get bootstrap support greater than 50% for every node. However, a marked increase in the number of well-supported nodes was seen when we conducted a Bayesian analysis of a combined dataset generated from morphological characters added to the seven gene dataset. Our results suggest that, in some cases, combining morphological and genetic characters may be the most practical way to increase support for short branches deep in a phylogeny.

Animals↗
PubMed2002

Green sulfur bacteria from hypersaline Chiprana Lake (Monegros, Spain): habitat description and phylogenetic relationship of isolated strains.

The 'Salada de Chiprana' (Chiprana Lake) is a hypersaline (30-73 per thousand), permanent and shallow lake of endorheic origin in a semi-arid region of the Ebro depression (Aragon, Spain). Magnesium sulfate and sodium chloride represent the main salts of this athalassohaline environment. Anoxic conditions occurred periodically in the bottom layers of the lake during the study period. When stratified, high sulfide concentrations (up to 7 mM) were measured in the hypolimnion. Physical and chemical conditions gave rise to the development of very dense green sulfur bacteria blooms (10.7 mg l(-1) of BChl c and 16.7 mg l(-1) of BChl d) at 0.5-1 m from the bottom. Microscopic observations revealed that cells morphologically similar to Chlorobium vibrioforme were dominant in the phototrophic bacterial community, but Prosthecochloris aestuarii was also found sometimes at lower concentrations, as revealed by both microscopic observation and flow cytometric analyses. Deep agar dilution series allowed to obtain several axenic cultures of phototrophic bacteria. They were identified according to their morphology, pigment composition and phylogenetic relationships (16S rDNA sequence analysis). Two of the sequenced strains (CHP3401 and CHP3402) belonged to the green sulfur bacteria and were related to Prosthecochloris aestuarii SK413(T) and Chlorobium vibrioforme DSM260(T), respectively. HPLC analyses of both natural samples and Chlorobium vibrioforme isolates indicated that these strains contained both BChl c and BChl d. Phylogenetic results suggested that Chlorobium vibrioforme strains DSM260(T) and CHP3402, all sequenced strains of Prosthecochloris aestuarii and strain CIB2401 constitute a separate cluster of green sulfur bacteria, all of them isolated from marine to hypersaline habitats.

Journal Article↗
PubMed1995

Intracellular coexistence of methano- and thioautotrophic bacteria in a hydrothermal vent mussel.

The coexistence of two phylogenetically distinct symbiont species within a single cell, a condition not previously known in any metazoan, is demonstrated in the gills of a Mid-Atlantic Ridge hydrothermal vent mussel (family Mytilidae). Large and small symbiont morphotypes within the gill bacteriocytes are shown to be separate bacterial species by molecular phylogenetic analysis and fluorescent in situ hybridization. The two symbiont species are affiliated with thioautotrophic and methanotrophic symbionts previously found in monospecific associations with closely related mytilids from deep-sea hydrothermal vents and hydrocarbon seeps.

Animals↗
PubMed1998

Involvement of waaY, waaQ, and waaP in the modification of Escherichia coli lipopolysaccharide and their role in the formation of a stable outer membrane.

The waaY, waaQ, and waaP genes are located in the central operon of the waa (formerly rfa) locus on the chromosome of Escherichia coli. This locus contains genes whose products are involved in the assembly of the core region of the lipopolysaccharide molecule. In the R1 core prototype strain, E. coli F470, there are nine genes in this operon, and all but waaY, waaQ, and waaP have been assigned function. In this study, the waaY, waaQ, and waaP genes were independently mutated by insertion of a non-polar antibiotic resistance cassette, and the structures of the resulting mutant core oligosaccharides were determined by chemical analyses and phosphorus-nuclear magnetic resonance spectroscopy. All three of these mutations were shown to affect the modification of the heptose region of the core, a region whose structure is critical to outer membrane stability. Mutation of waaY resulted in a core oligosaccharide devoid of phosphate on HepII. Mutation of waaQ resulted in loss of the branch HepIII residue on HepII and impeded the activity of WaaY. Mutation of waaP resulted in loss of phosphoryl substituents on HepI and obviated WaaQ and WaaY activity. Only mutation of waaP resulted in hypersensitivity to novobiocin and sodium dodecyl sulfate, a characteristic of deep-rough mutations.

Base Sequence↗
PubMed1994

Patterns of intracellular compartmentalization, trafficking and acidification of 5'-fluorescein labeled phosphodiester and phosphorothioate oligodeoxynucleotides in HL60 cells.

We have examined the intracellular compartmentalization and trafficking of fluorescein labeled (F) phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos) in HL60 cells. A series of F-oligos (PO and PS) were incubated for 6 hrs. with HL60 cells and the mean intracellular fluorescence determined by flow cytometry. The F signal was normalized by the addition of the ionophore monensin. An increase in signal intensity following addition of monensin indicated that the oligo was resident in an acidic intracellular environment. F-PS, but not F-PO oligos were found to reside in an acidic environment. An exception was a PO homopolymer of 15 cytidine bases (FOdC15) which was acidified. Using two different methods, the average resident intracellular pH of F-PS oligos and F-OdC15 was shown to be approximately 1 pH unit lower than that of F-PO oligos. Acidification of F-PS oligos could be blocked by the antibiotic bafilomycin, indicating that acidification was occurring in endosomes or vacuoles. F-PO and F-PS oligos were effluxed from HL60 cells from two intracellular compartments. However, approximately 60% of internalized F-PO oligo resided in a 'shallow' compartment that was turned over rapidly (t1/2 = 5-10 min.) whereas only 20% of F-PS oligo resided in this compartment. Conversely, approximately 80% of the internalized F-PS oligo but only 40% of F-PO oligo resided in a 'deep' compartment that turned over with t1/2 = 2-5 hrs. This report is the first quantitative demonstration that PO and PS oligos, and PO oligos of different sequences are trafficked differently by HL60 cells.

Cell Line↗
PubMed1991

Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase.

The crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase complexed with a 20-amino acid substrate analog inhibitor has been solved and partially refined at 2.7 A resolution to an R factor of 0.212. The magnesium adenosine triphosphate (MgATP) binding site was located by difference Fourier synthesis. The enzyme structure is bilobal with a deep cleft between the lobes. The cleft is filled by MgATP and a portion of the inhibitor peptide. The smaller lobe, consisting mostly of amino-terminal sequence, is associated with nucleotide binding, and its largely antiparallel beta sheet architecture constitutes an unusual nucleotide binding motif. The larger lobe is dominated by helical structure with a single beta sheet at the domain interface. This lobe is primarily involved in peptide binding and catalysis. Residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases. Most of the invariant amino acids in this conserved catalytic core are clustered at the sites of nucleotide binding and catalysis.

Adenosine Triphosphate↗
PubMed2003

Novel eukaryotes from the permanently anoxic Cariaco Basin (Caribbean Sea).

Present knowledge of microbial diversity is decidedly incomplete (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000). Protistan phylogenies are particularly deficient and undoubtedly exclude clades of principal ecological and evolutionary importance (S. L. Baldauf, Science 300:1703-1706, 2003). The rRNA approach has been extraordinarily successful in expanding the global prokaryotic record (S. J. Giovannoni and M. S. Rappé, p. 47-84, in D. Kirchman, ed., Microbial Ecology of the Oceans, 2000; E. Stackebrandt and T. M. Embley, p. 57-75, in R. R. Colwell and D. J. Grimes, ed., Nonculturable Microorganisms in the Environment, 2000) but has rarely been used in protistan discovery. Here we report the first application of the 18S rRNA approach to a permanently anoxic environment, the Cariaco Basin off the Venezuelan coast. On the basis of rRNA sequences, we uncovered a substantial number of novel protistan lineages. These included new clades of the highest taxonomic level unrelated to any known eukaryote as well as deep branches within established protistan groups. Three novel lineages branch at the base of the eukaryotic evolutionary tree preceding, contemporary with, or immediately following the earliest eukaryotic branches. These newly discovered protists may retain traits reminiscent of an early eukaryotic ancestor(s).

Animals↗
PubMed2025

AlphaGenome Enhances Personal Gene Expression Prediction but Retains Key Limitations.

In recent years, numerous genome AI models have been developed to elucidate the relationship between DNA sequence and gene expression. However, these models have faced criticism for their limited accuracy in predicting individual-specific gene expression. AlphaGenome, the current state-of-the-art in genome AI, achieves exceptional performance across a range of sequence-based predictive tasks, but its utility for personal expression prediction has not yet been assessed. In this study, we evaluate AlphaGenome's ability to predict personal gene expression and find that it significantly outperforms its predecessor. Using GTEx data, AlphaGenome improves the prediction of expression direction over Enformer, achieving an odds ratio of 3.0. In some cases, it even reverses previously observed negative correlations into positive ones. Moreover, AlphaGenome demonstrates improved performance for genes with known nonlinear sequence-expression relationships, though it uncovers mechanisms distinct from those identified by tree-based models.

deep learning↗
PubMed1998

[Cognitive impairment and cardiovascular disease risk factors. Project CASCADE Kraków. VI. Magnetic resonance imaging of the aging brain in elderly persons (65-78 years old)].

Magnetic resonance imaging (MRI) is the most sensitive non-invasive method for the detection of brain lesions. The aim of the study was to examine the prevalence of brain abnormalities (white matter lesions, atrophy, and infarction) on MRI in an elderly population. Sample of 174 men and women, aged from 65 to 78 years, was examined. MRI scans were made using a mobile MRI 1.0 Tesla machine (Siemens). Imaging was performed with standard spin-echo sequences (slice thickness 5-6 mm thick and 20% gap): proton density, T1 and T2 images. All MRI scans were read using standardized methodology of assessment of deep white matter lesions, periventricular lesions, cortical atrophy and infarction. Medium subcortical white matter lesions (4-10 mm) were present in 55% participants. Large subcortical white matter lesions (> or = 10 mm) were observed in 27% people, 24% had periventricular white matter lesions (score > or = 3), 83% had cortical atrophy (score > or = 4) and 18% had at least 1 infarct. Frequency of cortical atrophy increased gradually by age group (p < 0.01). The cortical atrophy, deep and periventricular white matter lesions were more frequent only in the oldest group (75-78 years).

Aged↗
PubMed1999

Diagnostic strategies in venous thromboembolism.

BACKGROUND AND OBJECTIVE: Diagnosis of acute deep vein thrombosis (DVT) and of pulmonary embolism (PE) is often difficult: symptomatic patients are usually investigated employing several diagnostic tests, which should be appropriately selected and sequenced, taking into account their sensitivity, specificity, safety and cost. The objective of this paper is to evaluate the performance of the new diagnostic tests and their combination in rational diagnostic strategies. DESIGN AND METHODS: A literature review was made using a Medline(R) database search for the period 1988-1998 on the following key words in various combinations: diagnosis, diagnostic strategy, venous thrombosis, pulmonary embolism, venous thromboembolism. Results of a new study by our group on diagnosis of DVT in hospitalized patients are also discussed. RESULTS: In patients with symptoms or signs suggestive of DVT, compression ultrasound (CUS) appears to be the diagnostic test of first choice, since it is a noninvasive test with high specificity and sensitivity for proximal DVT (about 97%). When CUS gives a negative result it is usually recommended that the test is repeated after one week, since its sensitivity for calf DVT is poor. The positive and negative predictive values (PPV and NPV) of CUS in symptomatic outpatients can be improved if adequate consideration is given to clinical diagnosis, using a standardized model (ref. #9), which allows symptomatic outpatients to be categorized as having a high, moderate or low probability of DVT. In case of agreement between clinical diagnosis and CUS results, no further testing is needed: patients with high or intermediate clinical probability and positive CUS results are treated, while in patients with low clinical probability and negative CUS results the diagnosis of DVT is excluded. In the case of discrepancy between clinical diagnosis and CUS results, D-dimer test and/or venography are requested. However in patients who develop signs or symptoms of DVT in the hospital the clinical model does not work, and diagnosis should be based on an appropriate mix of CUS, D-dimer (DD) test and venography. In patients presenting with signs or symptoms of pulmonary embolism, the ventilation/perfusion (V/P) lung scan remains a pivotal diagnostic test, and pulmonary angiography the reference standard, but both methods have limitations and in recent years other diagnostic tests such as echocardiography, helical (or spiral) computerized tomography, and magnetic resonance imaging have been introduced into clinical practice. Moreover, all four diagnostic tools mentioned for DVT diagnosis can be considered. Several diagnostic strategies have been proposed and evaluated in comparative studies but there is still debate over the most efficient test combination or sequence. INTERPRETATION AND CONCLUSIONS: Diagnostic strategies which include adequate consideration of clinical diagnosis using standardized models have the potential of being more efficient for outpatients (but not for inpatients) with symptoms or signs suggesting DVT of lower limbs. For patients with suspected PE, several diagnostic strategies have been assessed: V/P lung scan remains a pivotal diagnostic test, but its limitations have been increasingly recognized and newer non-invasive techniques are gaining credit. A consensus is still to be reached over the most appropriate combination of diagnostic tests.

Angiography↗
PubMed1998

[Type I protein C deficiency caused by a novel protein C gene mutation].

OBJECTIVE: To study the phenotype and genotype of a thrombophilia family. METHODS: Antigens and activities of protein C, antithrombin III, protein S, plasminogen and activated protein C resistance were assayed in 13 members from four generations of the family. RESULTS: Type I protein C deficiency was revealed in 5 members including the 3 members with deep vein thrombosis. All the exons and intron/exon junctions of the protein C gene were amplified by PCR. No abnormal band was found in SSCP assay. DNA sequencing identified a novel mutation 3444C-->A in exon VI of protein C gene leading to His134Asn. This mutation erased a Hph I site. PCR/Hph I analysis demonstrated that 6 members including 5 protein C deficiency members had the same mutations. CONCLUSION: His134Asn is a novel mutation causing type I protein C deficiency.

Adolescent↗
PubMed2005

Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer-Villiger monooxygenase catalysed lactone synthesis.

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L(-1) glycerol). Following induction of CHMO activity biomass concentrations of up to 6 gDCW L(-1) were obtained. Cells from each fermentation were subsequently used for the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L(-1)) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 microL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.

Acinetobacter calcoaceticus↗