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At least 631 records · Page 35Linked to original sources
PubMed2006

Differential protein expression of murine macrophages upon interaction with Candida albicans.

Numerous studies highlight the importance of macrophages for optimal host protection against systemic Candida albicans infections. We chose the murine macrophage cell line RAW 264.7 and the wild-type strain C. albicans SC5314 to study of the induced expression/repression of proteins in macrophages when they are in contact with C. albicans, based on 2-DE, comparison between different gels and protein identification. RAW 264.7 cells were allowed to interact with C. albicans cells for 45 min, and a significant differential protein expression was observed in these macrophages compared to controls. Gels were stained with SYPRO Ruby, allowing a better quantification of the intensity of the protein spots. Fifteen spots were up-regulated, whereas 32 were down-regulated; 60 spots appeared and 49 disappeared. Among them, we identified 11 proteins: annexin I, LyGDI (GDID4), Hspa5 (Grp78, Bip), tropomyosin 5 and L-plastin, that augment; and Eif3s5, Hsp60, Hspa9a, Grp58 (ER75), and Hspa8a (Hsc70), that decrease. The translation elongation factor (Eef2p) is modified in some of its different protein species. Many processes seem to be affected: cytoskeletal organisation, oxidative responses (superoxide and nitric oxide production) and protein biosynthesis and refolding.

Animals↗
PubMed2006

Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum.

Using proteomic analyses, a study was carried out aimed at understanding the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection. The DNA-damage inducible protein was found to be induced and glycosylated in F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.

Chloroplasts↗
PubMed2006

Proteomic analysis of the ventromedial nucleus of the hypothalamus (pars lateralis) in the female rat.

The use of proteomics to study changes in the expression of CNS proteins, which may underlie the regulation of physiological and/or behavioral responses, represents an emerging application of this technology. In the current study, the Palkovits' microdissection method was evaluated as a means of obtaining proteomic data from discrete brain nuclei. The pars lateralis of the ventromedial nucleus of the hypothalamus (VMN) was chosen for the initial studies because of its established role in the expression of gonadal hormone dependent female sexual behavior. The VMN from ovariectomized rats was microdissected from 300 microm frozen brain sections using a 500 microm punch. Total proteins were separated using 2-DE. A group consensus of 432 protein spots, visualized by SYPRO Ruby stain, was obtained from gels from four independent VMN samples. A low mean CV and high gel-to-gel correlation coefficients indicate that reproducible 2-DE gels can be generated from microdissected tissue samples. Proteins from the mediobasal hypothalamus (MBH) were also separated on 2-DE gels. Evaluation of the 2-DE maps from the VMN and the MBH revealed different protein profiles, and indicates that microdissection improves the detection of low-abundance proteins, and reduces the relative occurrence of abundant proteins on 2-DE maps.

Animals↗
PubMed2007

A comparative proteome and phosphoproteome analysis of differentially regulated proteins during fertilization in the self-incompatible species Solanum chacoense Bitt.

We have used 2-DE for a time-course study of the changes in protein and phosphoprotein expression that occur immediately after fertilization in Solanum chacoense. The phosphorylation status of the detected proteins was determined with three methods: in vivo labeling, immunodetection, and phosphoprotein-specific staining. Using a pI range of 4-7, 262 phosphorylated proteins could be mapped to the 619 proteins detected by Sypro Ruby staining, representing 42% of the total proteins. Among these phosphoproteins, antibodies detected 184 proteins from which 78 were also detected with either of the other two methods (42%). Pro-Q Diamond phosphoprotein stain detected 111 proteins, of which 76 were also detected with either of the other two methods (68%). The 32P in vivo labeling method detected 90 spots from which 78 were also detected with either of other two methods (87%). On comparing before and after fertilization profiles, 38 proteins and phosphoproteins presented a reproducible change in their accumulation profiles. Among these, 24 spots were selected and analyzed by LC-MS/MS using a hybrid quadrupole-TOF (Q-TOF) instrument. Peptide data were searched against publicly available protein and EST databases, and the putative roles of the identified proteins in early fertilization events are discussed.

Fertilization↗
PubMed2004

Attenuating effect of a traditional korean formulation, Paeng-Jo-Yeon-Nyeon-Baek-Ja-In-Hwan (PJBH), on hydrogen peroxide-induced injury in PC12 cells.

The Paeng-Jo-Yeon-Nyeon-Baek-Ja-In-Hwan (PJBH) prescription is a dried decoctum consisting of a mixture of 18 medicinal herbs that include Semen Biotae, Fructus Torilis seu cnidii, Fructus Rubi, Herba Dendrobii, Radix Morindae officinalis, Cortex Eucommiae, Radix Aspragi, Radix Polygalae, Radix Dipsaci, Ramulus Cinnamomi, Rhizoma Acori graminei, Rhizoma Alismatis, Rhizoma Dioscoreae, Radix Ginseng, Radix Rehmanniae preparata, Fructus corni, Fructus Schisandrae and Herba Cistanches. The effect of PJBH extracts on H2O2-induced toxicity in the rat pheochromocytoma line PC12 was examined by measurements of cell lesion, level of lipid peroxidation and antioxidant enzyme activities, since free radicals are involved in neurodegeneration in Alzheimer's disease (AD). After a 30 min exposure of the cells to H2O2 (150 microM), a marked decrease in cell survival, activities of glutathione peroxidase and catalase as well as an increased production of malondialdehyde (MDA) were found. Pretreatment of the cells with PJBH (0.5-10 microg/ml) prior to H2O2 exposure significantly elevated cell survival, antioxidant enzyme activities and resulted in a decrease in the level of MDA. The effects of the PJBH on hydrogen peroxide-induced injury in PC12 cells were also examined. PJBH had a remarkable elevating effect on catalase and GSH-Px activities as well as cell survival, suggesting that cytoprotective effects of the PJBH are involved in stimulation against intermediate concentrations of H2O2-induced PC12 cell injury. The above-mentioned neuroprotective effects were also compared with the effect of tacrine. The results suggest that PJBH has potential for use as a novel neuronal therapeutic agent.

Alzheimer Disease↗
PubMed2002

Identification of proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing as a prefractionation step followed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation mass spectrometry.

Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain-specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two-dimensional gel electrophoresis (2-DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid-phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO-Ruby-stained 2-D gels, with final protein identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). To optimise the focusing of the protein spots on the 2-D gel, the ampholyte concentration in liquid-phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2-D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2-D gels from prefractionated CSF compared with direct 2-DE separations of CSF. Several proteins, including cystatin C, IgM-kappa, hemopexin, acetyl-coenzyme A carboxylase-alpha, and alpha-1-acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. alpha-1-acid glycoprotein and alpha-2-HS glycoprotein, can be enriched, thus better resolved and detected on the 2-D gel. Liquid-phase IEF, as a prefractionation step prior to 2-DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI-MS analysis.

Cerebrospinal Fluid Proteins↗
PubMed2005

Rapid enrichment and analysis of yeast phosphoproteins using affinity chromatography, 2D-PAGE and peptide mass fingerprinting.

A combination of affinity purification, 2D-PAGE and peptide mass fingerprinting was employed to study the phosphoprotein complement of Saccharomyces cerevisiae. Protein extracts were first passed through a phosphoprotein affinity column, and the phosphoprotein-enriched eluate fractions were then separated on 2D gels and visualized by staining with SYPRO Ruby. Proteins were excised from the gels and identified by peptide mass fingerprinting; 11/13 protein spots identified from a gel of the phosphoprotein-enriched fraction had prior published evidence indicating that they were phosphoproteins. Additional experiments using a specific stain for phosphoproteins, prior incubation of the protein extract with alkaline phosphatase and blotting with monoclonal antibodies to phosphothreonine, phosphoserine and phosphotyrosine demonstrated that the phosphoprotein affinity column was an effective method for enriching phosphoproteins. Further validating the method, growth of yeast in the presence of sorbic acid resulted in altered phosphorylation of 17 proteins, 13 of which had prior published evidence that they were phosphoproteins or had ATP binding activity.

Alkaline Phosphatase↗
PubMed1994

Sequence analysis of a 10 kb fragment of yeast chromosome XI identifies the SMY1 locus and reveals sequences related to a pre-mRNA splicing factor and vacuolar ATPase subunit C plus a number of unidentified open reading frames.

We report the DNA sequence analysis of a region on the left arm of chromosome XI of Saccharomyces cerevisiae extending over 10 kb. The region contains five open reading frames (ORFs) of greater than 100 amino acids which do not show significant overlap with other ORFs. YKL408 contains a sequence with strong similarity to the RNA helicase pre-mRNA splicing factors PRP2, PRP16 and PRP22 (Burgess et al., 1990; Company et al., 1991; Ruby et al., 1991). YKL409 corresponds to the gene SMY1, the sequence of which was previously reported by Lillie and Brown (1992). YKL410 is identical to ATPase subunit C (Beltran et al., 1992) except for an N-terminal extension. YKL406 and YKL407 show no significant identity with any sequences in the databases searched.

Adenosine Triphosphatases↗
PubMed1999

Environmental light enhances survival and axonal regeneration of axotomized retinal ganglion cells in adult cats.

A segment of peripheral nerve was transplanted to the cut stump of the optic nerve to facilitate axonal regeneration of retinal ganglion cells (RGCs) in adult cats. The cats were reared under different light environment: 12 h light-12 h darkness, additional flash light under conventional light cycle, or 24 h darkness. After 60 days, the density and morphology of RGCs with regenerated axons were examined with retrograde labeling by fluoro-ruby and intracellular injections of Lucifer Yellow. In the retina of cats reared in darkness, densities of RGCs with regenerated axons were 11-42% of those in the retina of cats reared under conventional light and dark cycle. More than half of the labeled RGCs were degenerative in the retina of cats reared in darkness, while most RGCs were normal under conventional environment or flash light. We conclude that environmental light is essential for the survival and axonal regeneration of axotomized RGCs.

Animals↗
PubMed2001

Sorting of regenerating rat sciatic nerve fibers with target-derived molecules.

The functional outcome of microsurgical repair of divided nerves is disappointing since many regenerating axons fail to reach appropriate targets. Sorting of regenerating axons according to target tissue might be used to improve functional regeneration. The aim of the present study is to see if regenerating axons can be sorted into functionally different bundles with target-derived molecules. The proximal stump of the adult rat sciatic nerve was sutured into the inlet of a silicon Y-tube. The two branches of the Y-tube were filled with agarose primed with filtrates prepared from skin and muscle homogenates from the operated rat. The tibial and sural nerves were inserted in the two branches of the Y-tube. Six weeks later the sciatic nerve axons showed vigorous regeneration into both branches. Electron microscopic examination of regenerated nerve segments showed numerous myelinated and unmyelinated axons. The proportion of myelinated axons was significantly larger in the muscle-gel branch than in the skin-gel branch. Retrograde tracing from the nerve regenerates with Fast Blue and Fluoro-Ruby showed that ventral horn neurons at L4-L5 segmental levels were preferentially labeled from the muscle-gel branch. Neurons in corresponding dorsal root ganglia were labeled from both Y-tube branches (no significant numerical difference). A few neurons of both types contained both tracers. Measurements revealed that sensory neurons labeled from the muscle-gel branch were significantly larger (mean perikaryal area 870 microm(2)) than neurons labeled from the skin-gel branch (mean area 580 microm(2)). We conclude that regenerating motor and sensory axons can be sorted with target-derived molecules.

Animals↗
PubMed1998

Chromosomal localization and genomic characterization of the mouse melastatin gene (Mlsn1).

We recently described a novel gene, melastatin, whose expression is inversely correlated with melanoma aggressiveness. Chromosomal localization of this gene places it on mouse chromosome 7 and in the 15q13-q14 region of the human genome. Although expression patterns and chromosomal localization in the mouse are consistent with involvement of melastatin mutations in the mouse ruby-eye-2 defect, congenic analysis showed genetic segregation of the two loci. Cloning of the full-length human cDNA revealed a much larger transcript than we had previously identified, corresponding to a 1533-amino-acid protein product with homology to members of the transient receptor potential (Trp) family of calcium channels. The mouse melastatin gene contains 27 exons and spans at least 58 kb of genomic DNA. The promoter region of Mlsn1 contains four potential microphthalmia binding sites including an M box, a transcriptional regulatory element unique to genes with a restricted melanocytic expression pattern. A 1-kb PvuII fragment from this region was capable of driving high levels of luciferase expression in B16 melanoma cells.

Amino Acid Sequence↗
PubMed1998

Simulation methods for looping transitions.

Looping transitions occur in field-swept electron magnetic resonance spectra near avoided crossings and involve a single pair of energy levels that are in resonance at two magnetic field strengths, before and after the avoided crossing. When the distance between the two resonances approaches a linewidth, the usual simulation of the spectra, which results from a linear approximation of the dependence of the transition frequency on magnetic field, breaks down. A cubic approximation to the transition frequency, which can be obtained from the two resonance fields and the field-derivatives of the transition frequencies, along with linear (or better) interpolation of the transition-probability factor, restores accurate simulation. The difference is crucial for accurate line shapes at fixed angles, as in an oriented single crystal, but the difference turns out to be a smaller change in relative intensity for a powder spectrum. Spin-3/2 Cr3+ in ruby and spin-5/2 Fe3+ in transferrin oxalate are treated as examples.

Computer Simulation↗
PubMed1991

Arctiid moth clicks can degrade the accuracy of range difference discrimination in echolocating big brown bats, Eptesicus fuscus.

Four big brown bats (Eptesicus fuscus) born and raised in captivity were trained using the Yes/No psychophysical method to report whether a virtual sonar target was at a standard distance or not. At threshold bats were able to detect a minimum range difference of 6 mm (a delta t of 36 microseconds). Following threshold determinations, a click burst 1.8 ms long containing 5 pulses from the ruby tiger moth, Phragmatobia fuliginosa (Arctiidae), was presented randomly after each phantom echo. The sound energy of the click burst was -4 dB relative to that of the phantom echo. Clicks presented for the very first time could startle naive bats to different degrees depending on the individual. The bats' performance deteriorated by as much as 4000% when the click burst started within a window of about 1.5 ms before the phantom echo. Even when one of ten phantom echoes was preceded by a click burst, the range difference discrimination worsened by 200%. Hence, clicks falling within the 1.5 ms time window seem to interfere with the bat's neural timing mechanism. The clicks of arctiid moths appear to serve 3 functions: they can startle naive bats, interfere with range difference determinations, or they can signal the moth's distastefulness, as shown in earlier studies.

Animals↗
PubMed1995

Overdrainage and shunt technology. A critical comparison of programmable, hydrostatic and variable-resistance valves and flow-reducing devices.

When vertical body position is simulated, conventional differential pressure valves show an absolutely unphysiological flow, which is 2-170 times the normal liquor production rate. Although this is compensated in part by the resistance of the silicon tubes, which may produce up to 94% of the resistance of the complete shunt system, a negative intracranial pressure (ICP) of up to 30-44 cmH2O is an unavoidable consequence, which can be followed by subdural hematomas, slit ventricles, and other well-known complications. Modern shunt technology offers programmable, hydrostatic, and "flow-controlled" valves and anti-siphon devices; we have tested 13 different designs from 7 manufacturers (56 specimens), using the "Heidelberg Valve Test Inventory" with 16 subtests. "Programmable" valves reduce, but cannot exclude, unphysiological flow rates: even in the highest position and in combination with a standard catheter typical programmable Medos-Hakim valves allow a flow of 93-232 ml/h, Sophy SU-8-valves 86-168 ml/h with 30 cmH2O. The effect of hydrostatic valves (Hakim-Lumbar, Chhabra) can be inactivated by movements of daily life. The weight of the metal balls in most valves was too low for adequate flow reduction. Antisiphon devices are highly dependent on external, i.e. subcutaneous, pressure which has unpredictable influences on shunt function, and clinically is sometimes followed by shunt insufficiency. Two new Orbis-Sigma valves showed relatively physiological flow rates even when the vertical position (30 cmH2O) was simulated. One showed an insufficient flow (5.7 ml/h), and one was primarily obstructed. These have by far the smallest outlet of all valves. Additionally, the ruby pin tends to stick. Therefore, a high susceptibility to obliterations and blockade is unavoidable. Encouraging results obtained in pediatric patients contrast with disappointing experiences in some German and Swedish hospitals, which suggests that our laboratory findings are confirmed by clinical results. The concept of strict flow limitation seems to be inadaequate for adult patients, who need a relatively high flow during (nocturnal) ICP crises. The problem of shunt overdrainage remains unsolved.

Adult↗
PubMed1994

Identification of Agrobacterium strains by PCR-RFLP analysis of pTi and chromosomal regions.

Chromosomes and Ti plasmids of 41 Agrobacterium strains, belonging to biovars 1, 2, 3, and Agrobacterium rubi species were characterized by the restriction fragment length polymorphism of PCR-amplified DNAs. Profiles that were obtained by the analysis of the amplified 16S rDNA confirmed the grouping of the strains according to their species. Higher polymorphism was detected in the intergenic spacer between the 16S rDNA and 23S rDNA genes, allowing efficient discrimination of strains. Identification of most strains was possible, and the genetic relatednesses of Agrobacterium strains could be estimated. The analysis of the plasmid Ti encoded regions between the tmr and nos genes, and the virA and virB2 genes, allowed fingerprinting of Ti plasmids. Genomic typing by the rapid PCR-RFLP method is thus shown to be useful for an independent identification of strains and of the conjugative Ti plasmids.

Arginine↗
PubMed1994

Molecular map of chromosome 19 including three genes affecting bleeding time: ep, ru, and bm.

The mouse ruby eye (ru) and pale ear (ep) pigment dilution genes cause platelet storage pool deficiency (SPD) and prolonged bleeding times. The brachymorphic (bm) gene, in addition to causing skeletal abnormalities, is also associated with prolonged bleeding times. All three hemorrhagic genes are found within 10 cM on Chromosome (Chr) 19. In this study, 15 microsatellite markers and five cDNAs, spanning 21 cM of Chr 19, were mapped in relation to the bm, ep, and ru genes in 457 progeny of an interspecific backcross utilizing the highly inbred strain PWK derived from the Mus musculus musculus species. Several markers were found to be closely linked to the three genes and should be useful as entry points in their eventual molecular identification.

Animals↗
PubMed1984

Studies on Tn951 (lac+) expression in Agrobacterium.

None of the Agrobacterium tumefaciens and A. rubi strains tested produces detectable amounts of beta-galactosidase although they are capable of utilizing lactose as sole source of carbon. This opportunity was taken to investigate the expression of lac transposon Tn951 (Cornelis et al. 1978) in Agrobacterium with the ultimate goal of using this system to investigate alien gene expression. When the transposon was introduced with the help of a broad-host range plasmid, RP1, the transconjugants produced significant quantities of beta-galactosidase which was inducible by isopropyl-beta-D-thiogalactopyranoside. Tn951 was capable of restoring the Lac+ phenotype to an A. tumefaciens mutant not capable of using lactose. Cellobiose, a known inducer of aldohexopyranoside: cytochrome c oxidoreductase (which regulates the characteristic 3-ketolactose production in Agrobacterium; van Beeumen and De Ley (1968), had no effect on beta-galactosidase activity.

Cellobiose↗
PubMed1981

A model approach to laser coagulation of dermal vascular lesions.

The quantitative absorption of light by blood and by human skin is discussed in relation to laser coagulation therapy of dermal vascular lesions. The skin is represented by epidermis and dermis. Both layers are assumed to be planparallel. Absorption and scattering of light by turbid materials was accounted for by the Kubelka theory. Reflection and transmission data of blood and of skin were taken from the literature. This model shows that, firstly, argon-laser coagulation at 488/514.5 nm of dermal vascular lesions is a selective method because vascular absorption exceeds non-vascular dermal absorption by a factor of 10. Secondly, a coagulation depth of about 1 mm is predicted in agreement with the literature. Thirdly, the epidermis absorbs the 488/514.5 nm wavelengths about half as strong as blood and is therefore non-transparent. The consequences of this have been discussed in relations to the final cosmetic result. Fourthly, there is theoretical evidence that better cosmetic results may be expected when coagulation is performed at either 420 nm, 540 nm, or 575 nm. This suggests application of the krypton-laser with lines at 407, 413, and 415 nm (violet), at 531 nm, or at 568 nm. The krypton 647 nm line and the ruby 694 nm line are of little value for this purpose.

Absorption↗