Centrifugation Pellet Research Articles

Fluorescence Detection of a Lipid-induced Tetrameric Intermediate in Amyloid Fibril Formation by Apolipoprotein C-II

The misfolding and self-assembly of proteins into amyloid fibrils that occurs in several debilitating and age-related diseases is affected by common components of amyloid deposits, notably lipids and lipid complexes. We have examined the effect of the short-chain phospholipids, dihexanoylphosphatidylcholine (DHPC) and dihexanoylphosphatidylserine (DHPS), on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Micellar DHPC and DHPS strongly inhibited apoC-II fibril formation, whereas submicellar levels of these lipids accelerated apoC-II fibril formation to a similar degree. These results indicate that the net negative charge on DHPS, compared with the neutrally charged DHPC, is not critical for either the inhibition or activation process. We also investigated the mechanism for the submicellar, lipid-induced activation of fibril formation. Emission data for fluorescently labeled apoC-II indicated that DHPC and DHPS stimulate the early formation and accumulation of oligomeric species. Sedimentation velocity and equilibrium experiments using a new fluorescence detection system identified a discrete lipid-induced tetramer formed at low apoC-II concentrations in the absence of significant fibril formation. Seeding experiments showed that this tetramer was on the fibril-forming pathway. Fluorescence resonance energy transfer experiments established that this tetramer forms rapidly and is stabilized by submicellar, but not micellar, concentrations of DHPC and DHPS. Several recent studies show that oligomeric intermediates in amyloid fibril formation are toxic. Our results indicate that lipids promote on-pathway intermediates of apoC-II fibril assembly and that the accumulation of a discrete tetrameric intermediate depends on the molecular state of the lipid.

Morphological evidence for phages in Xylella fastidiosa

Presumptive phage particles associated with Xylella fastidiosa strain Temecula-1 grown in PW broth were observed by transmission electron microscopy (TEM) in ultrathin sections of bacterial cell-containing low speed centrifugation pellets and in partially purified preparations from CsCl equilibrium centrifugation density gradients. Ultrathin-sectioned cell pellets contained icosahedral particles of about 45 nm in diameter. Samples collected from CsCl density gradients revealed mostly non-tailed icosahedral but also tailed particles. The icosahedral particles could be divided into two types: a large type (about 45 nm) and a small type (about 30 nm). Filamentous phage-like particles (17 × 120 to 6,300 nm) were also observed. The presence of different types of phage-like particles resembling to those in several bacteriophage families provides new physical evidence, in addition to X. fastidiosa genomic information, that X. fastidiosa possesses active phages. This is the first report of phage particles released in X. fastidiosa cultures.

Propagating the missing bacteriophages: a large bacteriophage in a new class

The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly). As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter), tail (486 × 26 nm), corkscrew-like tail fibers (187 × 10 nm) and genome (221 Kb) that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305φ8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage), has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305φ8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305φ8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305φ8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion.

Mesure de la sensibilité aux antibiotiques selon les conditions standards directement à partir de liquides biologiques infectés et d'hémocultures positives

Determining the conditions, which would allow us to apply bacterial antibiotic susceptibility tests directly to infected biological fluids and positive blood cultures and in accordance with the standard conditions of these tests. For infected fluids (N=23), a correlation was determined between the bacterial count observed by microscopic field on a cystopin centrifugation pellet and the dilution required to obtain bacterial inocula corresponding to those used in the standard antibiogram and E-test methods. For blood cultures detected positive with the BactALERT system (26 Enterobacteriaceae, 29 Staphylococcus and 11 Pneumococcus), the broth dilution required to obtain such inocula was determined for each bacterial type. For infected fluids, the dilution required for the antibiogram of enterobacterial and staphylococcal isolates was respectively of 10(-1) and 10(0) when there were 50 to 100 bacteria/field, 10(-2) and 10(-1) for 100 to 500, 10(-3) and 10(-2) for 500 to 1000, and 10(-4) and 10(-3) for>1000. A minimum of 50 to 100 bacteria/field was required to determine beta-lactam MICs towards a pneumococcal isolate present in cerebrospinal fluid. For blood cultures, a broth dilution of 10(-4) for Enterobacteriaceae and of 10(-2) for Staphylococcus and Pneumococcus allowed to reproduce the standard antibiogram. To reproduce the standard conditions of the E-test method for beta-lactam MICs towards a pneumococcal isolate, the broth dilution was 10(-1). Following the procedure described, antibiotic susceptibility can be available 24 h earlier.

P-842: Motility characteristics and DNA integrity of spermatozoa processed with different concentrations of reduced glutathione (GSH)

Sperm processing methods with repeated centrifugal pelleting of unselected spermatozoa for assisted conception have been reported to result in sperm damage due to enhanced generation of reactive oxygen species (ROS) resulting in subsequent failure of pregnancy in some cases. The main objective of the present study was to assess the effect of in vitro supplementation of GSH during pellet swim-up method. Prospective study Liquefied semen samples of normozoospermic (n=20) patients attending the andrology laboratory at University of Calcutta were mixed and centrifuged with minimal essential medium (MEM with 4mg/ml HSA) containing 2.5 mM or 5 mM GSH along with proper controls. Care was taken each time to dissolve the GSH powder to the processing media just prior to the preparation to get the maximum antioxidant capacity. The resultant pellet was layered with Quinn’s sperm preparation media for swim-up. The resultant swim-up fraction was analyzed for motility parameters according to WHO standards and DNA integrity was analyzed by acridine orange method. There was no significant improvement in % motility and DNA integrity between the control and GSH groups. However an improvement was observed in the % rapid forward motility in both the doses of GSH employed (Table1) with 5mM group showing a statistically significant value (P value - 0.036).Tabled 1 The protective effect of GSH can be attributed to its strong antioxidant activity, which might be helpful in minimizing the ROS generation during the pellet swim-up procedure. However care should be taken to the use of GSH as it may act also as a pro-oxidant. Instant addition of GSH to the processing media may be helpful rather than storage of GSH supplemented media in frozen conditions and repeated usage of frozen thawed GSH containing media.

Pellet injectors for steady state plasma fuelling

Successful steady state operation of a fusion reactor should be supported by repetitive pellet injection of solidified hydrogen isotopes in order to produce high performance plasmas. This paper presents pneumatic pellet injectors and its implementation for long discharge on the LHD and TORE SUPRA, and a new centrifuge pellet injector test results. All injectors are fitted with screw extruders well suited for steady state operation.

Renal ultrasonography and detection of pseudomycelium in urine as means of diagnosis of renal fungus balls in neonates

To present a series of neonates with renal fungus balls diagnosed by ultrasonography, urine culture and/or by the detection of Candida pseudomycelium in urine. We revised the clinical records of neonates for whom the diagnosis of renal fungus ball was established by ultrasound and laboratory studies; these patients had been hospitalized at the National Institute of Pediatrics in Mexico between January 1st, 1999 and December 31st, 2002. During the study period, 9 neonates were diagnosed with renal fungus ball. In 7 cases, the ethiologic agent was Candida albicans; whereas it was C. tropicalis in one case and C. parapsilosisin the other. Urine culture was positive (> or =10,000 UFC/ml) in 8 cases, whereas the fungal density was only 2400 UFC/ml in the last sample. Pseudohyphae were present in all cases and ultrasonography showed fungus ball in every case. All patients received a single antifungal drug, either amphotericin B or fluconazole. All the patients recovered and none of them required surgical treatment. Control postreatment by ultrasound studies showed that the fungus balls had disappeared in every case. The diagnosis of Candida renal fungus balls based on the ultrasound study and urine culture is also substantiated by the detection of pseudomycelium in the centrifugation pellet of urine samples, which is a fast diagnostic method. This approach permitted an early diagnosis and treatment of Candida renal fungus balls.

Development of Pellet Technologies for Plasma Fueling

This contribution presents recent results of pellet technologies development for plasma fuelling in magnetic confinement machines with open or closed magnetic configuration. The current status of ITV7 pellet injector for GOL3 multimirror linear machine, PGS2.2 pellet guide system of ITV4 in-situ pellet injector for TUMAN-3M tokamak and ITV5 centrifuge pellet injector for Globus-M spherical tokamak is reported. New results on modeling of tangential pellet injection into TUMAN-3M tokamak are discussed as well.

Technological development and progress of plasma performance on the JT-60U

The plasma performance exploration of JT-60U in advanced tokamak regimes based on high βp and reversed shear mode operation has been conducted intensively, by using 500 keV negative-ion based neutral beam injection (N-NBI) and 110 GHz electron cyclotron (EC) systems for plasma heating and current drive, and a repetitive centrifugal pellet injector for efficient core particle fueling. High power injection of 6.2 MW at 381 keV for 1.7 s and a long pulse injection of 10 s at 2.6 MW and 355 keV were achieved. In the EC system capable of generating 4 MW of rf power for 5 s, the maximum injected EC power reached 3.0 MW for 2.7 s so far. Pellet injection at a velocity of 100 m/s from the mid-plane in the HFS has also performed well. With application of N-NBI power of 5.7 MW at 402 keV, a high fusion triple product was increased to n i (0) τ E T i(0), of 3×10 20 keV s/m 3 under a full non-inductive current drive condition. By using steerable antennas of the EC system and a tracking technique of the MHD island, suppression of the neoclassical tearing mode in real time has been demonstrated successfully. High-magnetic-field-side pellet injection extended the plasma density up to 0.7 n GW with H 89p kept at ∼2.

Partial purification and characterization of a soybean β-glucosidase with high specific activity towards isoflavone conjugates

A β-glucosidase with high specific activity towards isoflavone conjugates was purified from soybean [ Glycine max] roots by high salt extraction from a low speed centrifugal pellet and subsequent anion and cation exchange chromatography. Purification required stabilization throughout fractionation in 10% glycerol. The enzyme is most likely a dimer (approximate M r 165 kDa) with potential subunits of M r 80 and/or 75 kDa. The pH and temperature optima are pH 6 and 30 °C, respectively. The enzyme was highly heat-stable. Of the various potential effectors examined, silver and mercury ions were the most inhibitory. The IC 50 of silver ions was increased from 140 μM to 14 mM in the presence of 250 μM β-mercaptoethanol. Glucono-δ-lactone was not strongly inhibitory (IC 50 24 mM). The activity was highly active against isoflavone conjugates, with a specificity constant 160–1000 fold higher for isoflavone conjugates over the generic chromogenic substrate, p-nitrophenyl β-glucoside. The enzyme was inactive against the flavonol glycosides tested. The partially purified enzyme had similar K m and k cat towards 7- O-glucosyl- and 7- O-glucosyl-6″-malonyl-isoflavones, suggesting that it may be able to cleave the esterified glucosyl conjugate. We hypothesize that the enzyme is involved in the release of daidzein and genistein, both of which play central roles in soybean defense.

A repetitive pellet injection system for JT-60U

A centrifugal pellet injector and a guide tube for magnetic high-field side (HFS) injection were developed in JT-60U. The injector can eject trains of up to 40 cubic (1.9 mm) 3 deuterium pellets at frequencies of 1–10 Hz and velocities of 0.2–1.0 km/s. Pellet injection experiments into ohmic heating (OH) plasma were performed. From plasma density measurement, central fueling and enhanced fueling rate compared to gas puffing were observed for magnetic low-field side (LFS) injections. The results of HFS injection through the guide tube connected to the upper port of the vessel showed that the pellet velocity exceeding 220 m/s caused pellet destruction inside the tube.

Test of an extruder feed system on the Tore Supra centrifugal pellet injector

A deuterium ice extruder feed system has been developed by ORNL for adaptation to the existing ORNL Centrifugal Pellet Injector installed on Tore Supra. A detailed assessment of this feed system coupled to the centrifugal launcher has been carried out in a dedicated test bed at Cadarache, to complete and understand the first results obtained at ORNL and to improve them. The results presented in this paper are related to the study of the pellet trajectory from the deuterium ice cutting to the collection of the accelerated pellets in a guide tube. The overall reliability of the injector has been shown to be very sensitive to deviations in the free flight part of the trajectory, before capture by the rotating launcher.

Identification of soybean elicitation competency factor, CF-1, as the soybean Kunitz trypsin inhibitor

The soybean defence responses that occur in cells immediately adjacent (proximal) to the hypersensitive response (HR) can be induced by the cell wall glucan elicitor (WGE) from Phytophthora sojae, but in some cultivars only in cells that have been pre-conditioned by endogenous competency factors associated with wounded or HR-dying cells. Competency factor, CF-1, causes a general amplification of phenylpropanoid responses to elicitor and greatly enhances the phenolic polymer response to elicitor. CF-1 activity, which is heat-labile and associated with a low speed centrifugal pellet of soybean tissues, can be partially mimicked by reduced glutathione (GSH). Here it is reported that GSH releases CF-1 activity into intercellular washing fluids, suggesting it is associated with the apoplast. Likewise, GSH can be used to enrich CF-1 activity greatly in extracts from low speed pellets. This enrichment allowed the purification of CF-1 to near homogeneity and its subjection to mass spectral and sequence analysis. CF-1 was identified as the soybean Kunitz trypsin inhibitor (SKTI). Although plant protease inhibitors have been implicated in insect and pathogen resistance, to the authors' knowledge this is the first suggestion of a regulatory role in activation of cellular defence responses. Protease inhibitors play a role in many signaling processes in eukaryotic systems, and SKTI has intriguing three-dimensional homologies to interleukin 1-α and 1-β and fibroblast growth factor.

Mutations in the X-linked RP2 gene cause intracellular misrouting and loss of the protein.

Mutations in RP2 cause the second most frequent form of X-linked retinitis pigmentosa, a severe retinal degeneration that leads to loss of visual acuity and blindness. The RP2 gene encodes a protein with homology to cofactor C, a tubulin-folding chaperone. By searching protein sequence databases, we identified a whole set of similar molecules from diverse organisms. Protein sequence alignments show that RP2 and cofactor C represent members of two distinct orthologous groups. All previously identified missense mutations affect amino acid residues which are conserved in all RP2 orthologues or both orthologous groups. Intracellular localization of the wild-type protein and mutated variants was determined by fluorescence microscopy of cells expressing RP2 with a green fluorescent protein tag. A mutation in the N-terminus of RP2 abolishes localization to the plasma membrane in HeLa cells. C-terminal protein truncation mutations, which account for 2/3 of the pathogenic RP2 variants, lead to scattered fluorescent foci in the cytoplasm of COS-7 and HeLa cells. Analysis of protein extracts from the respective cells with anti-RP2 antibodies identified truncated proteins of expected size in a low-speed centrifugation pellet while the wild-type protein appeared in the supernatant. Moreover, no protein was detected in immortalized cell lines from patients with protein truncation mutations while mRNA was still present. Thus, loss of the protein and/or aberrant intracellular distribution might be the basis for the photoreceptor cell degeneration in most RP2 cases.

Status of the Russian pellet injector R&D program for plasma fuelling

Basic trends of research within the Russian pellet injector R&D program for 1999–2001 are discussed. The attention is focused on injectors with a screw extruder and those with a porous generator capable of injecting pellets in a repetitive continuous steady state mode. Over 12 000 deuterium pellets of 2 mm diameter were produced and accelerated to 200 m/s at a rate of 10 Hz with a reliability over 99% by a pneumatic injector equipped with a screw extruder. Over 2000 hydrogen pellets of 2.4 mm diameter and 4 mm length were accelerated to 1 km/s at a rate of 0.3–0.8 Hz by a pneumatic injector equipped with a porous generator with no moving parts. The injector designs and experimental results are presented and a concept of a new centrifuge pellet injector is discussed.

Lack of toxicity to adults of the Mexican fruit fly (Diptera: Tephritidae) of beta-exotoxin in Bacillus thuringiensis endotoxin preparations.

beta-Exotoxin (thuringiensin) was found in high titers in centrifugation supernatants and acetone/lactose powders produced from centrifugation pellets of strains Guat 1 and HD 2 of Bacillus thuringiensis (Berliner). Diets containing powders of either strain were toxic, diets containing Guat 1 supernatant were not toxic, diets containing HD 2 supernatant were slightly toxic, and diets containing powders or supernatants from uninoculated culturing medium spiked with beta-exotoxin were not toxic. Most mortality occurred within 3 d when flies fed on powders but not until 6-7 d when flies fed on HD 2 supernatant. These results indicated that the primary toxic principals of the powders were endotoxins/spores and that beta-exotoxin alone was not toxic to adult flies at the concentrations found in the supernatants or powders.

Transmission electron microscope study of bacterial morphotypes on the anterior dorsal surface of human tongues

The human tongue has been the subject of many cytological and histological studies. When a literature search disclosed no reports of the ultrastructure of the morphotypes of bacteria residing on the tongue's surface, a transmission electron microscope study of ultrathin sections of bacteria obtained by scraping eight human tongues was undertaken. The scrapings from the anterior dorsal tongue surfaces, processed conventionally for electron microscope study, revealed 33-35 different bacterial morphotypes. Several of the morphotypes were unique to a tongue. Morphotype differences were also related to donor characteristics such as smoking, tongue site, location in centrifuge pellet, diet, and medications. The predominant morphotypes were Gram-positive cocci. These preliminary findings suggest that the microbiota of the human tongue and variations in that microbiota, related to physical condition, lifestyle, medications, and dietary preferences, merit more attention from anatomists.

Improved stop cylinder loading scheme for a centrifuge pellet accelerator

In order to perform plasma refueling experiments with cryogenic hydrogen pellets at the midsize tokamak ASDEX Upgrade, a centrifuge injector was developed based on the novel stop cylinder principle. Following the demonstration of excellent performance with this instrument, similar injectors were constructed for two of the world’s largest tokamaks, JET and JT-60U. The extended fuel consumption in these tokamaks enforced the employment of continuous extruders instead of a storage cryostat, requiring an increase in pellet temperature from 8 to 14 K. Degradation of the injector performance was found at these higher pellet temperatures due to increased gas evaporation rates but could be eliminated by a revised acceleration scheme. Applying the adapted setup, reliable and stable operation has been realized with JET and JT-60U injectors.

Laccase activity of forest litter

Laccases are blue-copper enzymatic proteins involved in the transformation of polyphenolic compounds, such as lignin, into forest litter. A method to measure their activity was developed using an evergreen oak litter as a model system. We considered factors that affect the extraction of these enzymes and the measurement of their activity. The factorsstudied included powdering of the leaves, adding polyvinylpolypyrrolidone or Tween 80, adsorption of enzymes on centrifugation pellets, concentration of the extracts, nature and molarity of the extraction solvent, amount of powdered leaves used for enzymatic extraction, extraction time, pH, and temperature during the measurement of the activity.

A thermostable esterase activity from newly isolated moderate thermophilic bacterial strains

Thirty-nine bacterial moderate thermophilic strains growing on triolein as sole carbon and energy source were isolated from soil samples by an enrichment culture on a mineral medium. When grown on a Tween 80 medium, most of the strains showed very high specific growth rates in the range of 1–2 h −1 which corresponded to doubling times of 20–40 min. A high esterase activity was detected in the cell culture of all strains when assayed on soluble p-nitrophenyl caprylate (pNPC8). Strain MAS2 showed the highest esterase activity (around 40 U l −1). This activity was equally recovered in the culture supernatant and cell fraction. The majority of the cell esterase activity was recovered in the supernatant after high speed centrifugation, suggesting a soluble localization. Activity on pNPC16 was only recovered in the high speed centrifugation pellet, suggesting a membrane localization for the lipase activity. The best medium for pNPC8 production was a synthetic medium containing both glucose and yeast extract. The culture medium supernatant showed the highest activity on p-nitrophenyl acetate (pNPC2) than on pNPC8 and no activity on pNPC16, indicating the presence of esterase rather than lipase activity. This pNPC8 activity was stable (98%) after a 1 h treatment at 70°C which renders this new esterase very attractive for biotechnological applications.