Quench correction in Cerenkov counting: channels ration and external source channels ratio methods

Channels ratio and external source ratio methods which may be used for accurate quench correction in ?erenkov counting systems have been developed. In general, the channels ratio method will be more accurate for quench correction in high count rate samples where good ratio statistics are available. For low level samples, the external source channels ratio method will provide more accurate quench correction if efficiencies are greater that 10%.     

Charring does not affect wood infestation by subterranean termites

Fire is an important part of forest ecosystems, as is the insect fauna. Changes in wood brought about by fire may alter the ability of termites to use the wood, interrupting the decay cycle of woody debris. The ability of termites to find, infest, and feed upon wood after it had been charred was evaluated in the laboratory and field. Eastern subterranean termites, Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae), fed on char from burned wood had significantly reduced numbers of protozoa compared to termites fed on pine shavings, but significantly more than starved termites. The ability of termites to find and infest wood was not affected by surface charring. In a laboratory choice test, there were no significant differences in the onset of feeding by termites between charred and non‐charred wood boards. Likewise in the field, no differences were observed in the time to initial attack by termites on charred and non‐charred wood boards or bolts. Because termites will likely survive fires of low to moderate intensity, in most cases, there should be no disruption of the termite contribution to forest nutrient and carbon cycles.     

Low-pH-sensitive PEG-stabilized plasmid-lipid nanoparticles: preparation and characterization

The acid-labile poly(ethyleneglycol)-diorthoester-distearoylglycerol lipid (POD), was used with a cationic lipid-phosphatidylethanolamine mixture to prepare stabilized plasmid-lipid nanoparticles (POD SPLP) that could mediate gene transfer in vitro by a pH triggered escape from the endosome. Nanoparticles of 60 nm diameter were prepared at pH 8.5 using a detergent dialysis method. The DNA encapsulation efficiency in the nanoparticles was optimal between 10 and 13 mol % ratio of cationic lipid and at a POD content of 20 mol %. The apparent zeta potential of the nanoparticles at 1 mM salt and pH 7.5 was positive, indicating cationic lipid on the external surface. However, the external layer of the nanoparticles was depleted in the cationic component compared to the starting mole ratio. Low pH sensitivity of the POD SPLP was characterized by a lag phase followed by a rapid collapse; at pH 5.3 the nanoparticles collapsed in 100 min. Nanoparticles prepared from a pH-insensitive PEG-lipid, PEG-distearoylglycerol had similar physicochemical characteristics as the POD SPLP but did not collapse at low pH. The POD SPLP had up to 3 orders of magnitude greater gene transfer activity than did the pH-insensitive nanoparticles. Both the pH-sensitive and pH-insensitive nanoparticles were internalized to a qualitatively similar extent in a punctate pattern into cultured cells within 2 h of incubation with the cells; thus, increased gene transfer of the POD SPLP was due to a more rapid escape from the endosome rather than to greater cell association of these nanoparticles. These results suggest that the pH-sensitive stabilized plasmid-lipid nanoparticles may be a useful component of a synthetic vector for parenterally administered gene therapy.     

HPLC法测定低pH静脉注射用人血丙种球蛋白麦芽糖含量

本文采用HPLC法测定低pH静脉注射用血丙种球蛋白中麦芽糖含量,所用色谱柱为氨基柱。首先用磺基水杨酸沉淀蛋白并离心分离,上清液上样分析。通过外标法建立三级校正曲线来测定样品中麦芽糖含量,该法准确快速较化学方法好。     

Domestic burnt offerings and sacrifices at Roman and pre-Roman Pompeii, Italy

Excavations below the A.D. 79 destruction levels in two houses at the Roman town of Pompeii, Italy included programmes of sieving and flotation for the recovery of biological remains. In addition to the usual finds of charred plant material and bones representing crop processing or food waste, and mineralised remains from sewage, were numerous burnt offerings. Cones and seeds of stone pine, fruit such as fig and grape and nuts such as walnut and hazel were present in many of them. Some of the offerings contained burnt bone, with the heads and feet of cocks (male domestic fowl) being particularly well represented. There was some change in the plants and animals used for the burnt offerings with time. These offerings could be related to domestic worship described in literary sources and depicted as offerings on altars in Pompeiian wall paintings at shrines to the Lares (household gods). The burnt offerings resulted in the preservation by charring of fruit that are not usually burnt during their processing for consumption. They also presented a taphonomic problem because re-worked charred material from burnt offerings was likely to have been a major component of other charred assemblages from the sites. Received November 22, 2001 / Accepted February 26, 2002     

Experiments on the effects of charring on <Emphasis Type="Italic">Setaria italica</Emphasis> (foxtail millet)

This study aims to better understand taphonomical effects relevant to Setaria italica (foxtail millet), particularly the deformation caused by charring in fully-formed grains, and the potential preservation of underdeveloped seeds. Foxtail millet is a staple grain commonly found in Neolithic and later sites across Eurasia after initial domestication in northern China. Precise control of atmospheric conditions enabled determination of ideal parameters for charring without seed destruction. These experiments were able to produce charred seeds that strongly resemble archaeological specimens, making three key findings: (1) lateral expansion found in many ancient foxtail millet seeds indicates that charring occurred with the seeds in the husks. (2) Oxidizing conditions produced far better results in terms of seed preservation and retention of identifiable characteristics. (3) Smaller and less developed or ‘filled’ seeds survived in the same conditions as larger, plump seeds. These results allow for better interpretation of depositional context of millet seeds, and point to heat treatment during the de-husking process as a common way for seeds to enter the archaeological record.     

Reconstitution of expressed KCa channels from Xenopus oocytes to lipid bilayers.

Reconstitution of large conductance calcium-activated potassium (KCa) channels from native cell membranes into planar lipid bilayers provides a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, KCa channels derived from the Drosophila Slowpoke (Slo) gene have been cloned and heterologously expressed in Xenopus oocytes. In this report, we describe the reconstitution of cloned and expressed Slo KCa channels from Xenopus oocyte membranes into lipid bilayers. The reconstituted channels demonstrate functional properties characteristic of native KCa channels. They possess a mean unitary conductance of approximately 260 pS in symmetrical potassium (250 mM), and they are voltage- and calcium-sensitive. At 50 microM Ca2+, their half-activation potential was near -20 mV; and their affinity for calcium is in the micromolar range. Reconstituted Slo KCa channels were insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethylammonium (KD = 158 microM, at 0 mV) and internal Ba2+ (KD = 76 microM, at 40 mV). In addition, they were blocked by internally applied "ball" inactivating peptide (KD = 480 microM, at 40 mV). These results demonstrate that cloned KCa channels expressed in Xenopus oocytes can be readily incorporated into lipid bilayers where detailed mechanistic studies can be performed under controlled internal and external experimental conditions.     

A new assay method for lipid peroxides using a methylene blue derivative

To determine the absolute amount of lipid hydroperoxides in biological materials, a simple and sensitive colorimetric method was newly developed, based on the reaction of lipid hydroperoxides with a leucomethylene blue derivative in the presence of hemoglobin. The amount of methylene blue formed was measured by its absorbance at 666 nm to calculate the amount of lipid hydroperoxides using cumene hydroperoxide as external standard. By this method, lipid hydroperoxide concentrations of less than 7.5 nmol/tube were accurately determined.     

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.     

Mass spectrometric analysis demonstrates that BODIPY 581/591 C11 overestimates and inhibits oxidative lipid damage

BODIPY C11 is being used with increasing frequency to quantify lipid oxidation; however, it is not known whether signals from the dye yield accurate information. To determine the quantitative accuracy of signals from this dye we utilized a triple-quadrupole mass spectroscopy method to measure concentrations of 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine (SAPC) as it underwent oxidative damage, and compared these results to fluorescence signals from the dye. Results indicate that BODIPY C11 was significantly more sensitive to oxidative damage than SAPC lipid. As a consequence, BODIPY C11 overreported the extent of oxidative lipid damage, underreported the antioxidant effect of alpha-tocopherol, and exhibited an antioxidant effect of its own. We conclude that BODIPY C11 fluorescence does not yield quantitative information about lipid oxidation, although the dye remains a sensitive indicator of free radical processes that have the potential to oxidize lipids in membranes.     

Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.     

Effects of charring on the carbon isotopic composition of grass (Poaceae) epidermis

Charred modern grass epidermis preserves the carbon isotopic composition of the parent plant photosynthetic pathway. Fifty-nine modern grasses and sedges were collected in lowland western Uganda. All charred epidermal samples from C₄ grasses or sedges preserve a carbon isotopic value within the range typical for C₄ plants (−17 to −10‰), and charred epidermal fragments from C₃ plants have carbon isotopic values between −30 and −26‰. The process of charring results in a slightly enriched carbon isotopic signature (−11.9‰ mean charred value as compared to −12.8‰ mean unaltered grass tissue value). δ¹³C measurements of replicate samples from the same plant vary within 1–2‰, yet all values for the same plant stay within the expected values for the photosynthetic pathway of the plant. δ¹³C measurements on >180-μm charred grass epidermal fragments extracted from surface sediment samples from three lakes on the lowland western Ugandan landscape confirm the predominant lowland C₄ grass input (δ¹³C=−16 to −19‰). These results demonstrate the utility of using carbon isotopic analysis of charred grass epidermis to reconstruct C₃ vs. C₄ grassland assemblages on the landscape. Furthermore, such downcore δ¹³C profiles can be used to highlight key zones of C₃ vs. C₄ grass change for which taxonomic analysis of fossil grass epidermis could provide more detailed information regarding grassland community composition.     

Sources of error in the channels ratio method for efficiency determination in liquid scintillation counting

The reliability of the channels ratio method for determining counting efficiency in liquid scintillation counting was investigated. It was found that the efficiency of counting gels, cloudy samples, two-phase samples, samples in which the radioactive material was precipitating, and samples on solid supports could not be reliably determined from a normal quench correction curve. A curve constructed from external standard channels ratios was unreliable when mixing different vial sizes and sample volumes, but one constructed from sample channels ratios was not. It was also found that variation in instrument performance can result in large errors unless samples and standards are counted together. Statistical error changed relatively little within the range of ratios 0.3 to 0.8.     

l-Acylimidazoles with Broad-spectrum Fungicidal Activity

The membrane lipids of six higher plants that differ in salt tolerance were analyzed and compared. The root lipids increased in a ratio of glycolipid/phospholipid with increasing salt- tolerance. A similar increase in the ratio was observed with increasing external salinity when halophytic orach and salt-sensitive cucumber were exposed to varying salinity, although the latter plant was limited to only a little increase. Measurements of ion-transport rates with artificial lipid membranes revealed that the root lipids from a salt-resistant plant formed a more permeable membrane than those from a salt-sensitive species. It was found that the membrane permeability was related to the glycolipid/phospholipid ratio in the membrane lipids, where the glycolipids were stimulative and the phospholipids were repressive for ion-flow. These different effects of the two lipid classes may be attributed to their molecular species and head groups.     

Translocation of histone proteins across lipid bilayers and Mycoplasma membranes

We show that the three core histones H2A, H3 and H4 can transverse lipid bilayers of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). In contrast, the histone H2B, although able to bind to the liposomes, fails to penetrate the unilamellar and the multilamellar vesicles. Translocation across the lipid bilayer was determined using biotin-labeled histones and an ELISA-based system. Following incubation with the liposomes, external membrane-bound biotin molecules were neutralized by the addition of avidin. Penetrating biotin-histone conjugates were exposed by Triton treatment of the neutralized liposomes. The intraliposomal biotin-histone conjugates, in contrast to those attached only to the external surface, were attached to the detergent lysed lipid molecules. Thus, biotinylated histone molecules that were exposed only following detergent treatment of the liposomes were considered to be located at the inner leaflet of the lipid bilayers. The penetrating histone molecules failed to mediate translocation of BSA molecules covalently attached to them. Translocation of the core histones, including H2B, was also observed across mycoplasma cell membranes. The extent of this translocation was inversely related to the degree of membrane cholesterol. The addition of cholesterol also reduced the extent of histone penetration into the MLVs. Although able to bind biotinylated histones, human erythrocytes, erythrocyte ghosts and Escherichia coli cells were impermeable to them. Based on the present and previous data histones appear to be characterized by the same features that characterize cell penetrating peptides and proteins (CPPs).     

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.     

Microbial lipid production by Rhodosporidium toruloides under sulfate-limited conditions

Novel biochemical approaches remain to be developed to improve microbial lipid technology. This study demonstrated that sulfate limitation was effective to promote accumulating substantial amounts of intracellular lipid by the oleaginous yeast Rhodosporidium toruloides Y4. When it was cultivated using a medium with an initial carbon-to-sulfur (C/S) molar ratio of 46,750, cellular lipid content reached up to 58.3%. The time courses of cell growth, lipid accumulation and nutrient depletion were analyzed and discussed in terms of lipid biosynthesis. Moreover, lipid accumulation under sulfate-limited conditions was effective regardless of the presence of a high concentration of nitrogen sources. Thus, lipid contents almost held constant at near 57% in the media with an initial C/S molar ratio of 11,380 although the carbon-to-nitrogen molar ratio ranged from 28.3 to 5.7. Taken together, our results established the sulfate-limitation approach to control lipid biosynthesis, which should be valuable to explore nitrogen-rich raw materials as the feedstock for lipid production.     

Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma

In a previous study we demonstrated that highly purified lipid-transfer protein facilitated the transfer of triglyceride, cholesteryl ester, and phosphatidylcholine between plasma lipoproteins. It remained unclear, however, whether these lipids were transferred by independent sites on the lipid-transfer protein. To address this point, we have studied the protein-mediated transfer of triglyceride, cholesteryl ester, and phosphatidylcholine as a function of the concentration and lipid composition of donor and acceptor lipoproteins. Lipoproteins labeled in vitro, reconstituted lipoproteins of defined lipid composition, and phosphatidylcholine liposomes with or without triglyceride and/or cholesteryl ester have been used to investigate the inter-relationships of lipids transferred by the lipid-transfer protein. In studies of initial (less than or equal to 10-13%) transfer, we found that, although absolute transfer rates were affected, the ratio of cholesteryl ester to triglyceride transferred was independent of donor and acceptor lipoprotein concentrations and acceptor lipoprotein lipid composition. With reconstituted lipoproteins as donor, we demonstrated that this ratio was linearly related to the ratio of cholesteryl ester to triglyceride in the donor particle; the sum of triglyceride and cholesteryl ester transferred remained constant and independent of the lipid composition of the donor. Experiments with intact lipoproteins labeled in vitro and with small unilamellar vesicles in the presence and absence of p-chloromercuriphenylsulfonate, confirmed the interdependence of triglyceride and cholesteryl ester transfer. In contrast, under all assay conditions, no correlation was found between the amount of phosphatidylcholine transferred and the transfer of triglyceride and/or cholesteryl ester. We conclude that triglyceride and cholesteryl ester compete for transfer and that the extent of transfer for each lipid is determined by its relative concentration in the donor particle, whereas phosphatidylcholine transfer is independent of triglyceride and cholesteryl ester transfer. The data also strongly support the conclusion that lipid transfer protein promotes both the exchange and net transfer of triglyceride and cholesteryl ester and that the net transfer process proceeds by a reciprocal exchange of triglyceride and cholesteryl ester without net transfer of core lipid between lipoproteins.     

How does fire affect the nature and stability of soil organic nitrogen and carbon? A review

After vegetation fires considerable amounts of severely or partly charred necromass (referred to here as char) are incorporated into the soil, with long-term consequences for soil C and N dynamics and thus N availability for primary production and C and N transport within the soil column. Considering results reported in the pyrolysis literature in combination with those obtained from controlled charring of plant material and soil organic matter (SOM), it has become clear that common models claiming char as a graphite-like material composed mainly of highly condensed polyaromatic clusters may be oversimplified. Instead, I suggest a concept in which char is a heterogeneous mixture of heat-altered biopolymers with domains of relatively small polyaromatic clusters, but considerable substitution with N, O and S functional groups. Such a concept allows fast oxidation facilitating both microbial attack and dissolution. Although, char is commonly believed to degrade more slowly than litter, over the long term and under oxic conditions, char may degrade to an extent that it becomes indistinguishable from naturally formed SOM. Oxygen depletion or environments with low microbial activity may be necessary for char to survive without major chemical alteration and in considerable amounts for millennia or longer.     

Conditions controlling tumor cytotoxicity of rat liver macrophages mediated by liposomal muramyl dipeptide

Activation of rat liver macrophages with free and liposome-encapsulated muramyl dipeptide (MDP) to a tumorcytotoxic state was characterized by employing various experimental conditions. Macrophage-mediated tumor cytotoxicity was determined using two standard assay systems: a [methyl-3H]thymidine release assay to measure the extent of tumor cell lysis and a [methyl-3H]thymidine incorporation assay to measure the combined effects of tumor cell lysis and stasis. The extent of cell lysis was not affected by the ratio of macrophages to tumor cells within the ratio range of 30:1 to 5:1, provided that the macrophages form a confluent monolayer. Tumor cell lysis, however, was significantly influenced by macrophage density; a low macrophage density for example resulted in a low percentage of tumor cell lysis. Tumor target cells used in this study, i.e., C26 adenocarcinoma, B16 melanoma and P815 mastocytoma, differed in their susceptibility towards macrophage-mediated cell lysis, whereas no differences were observed with respect to tumor cell stasis. Non-tumorigenic cell lines such as human fibroblastic cells and LLC monkey kidney cells were not lysed by activated macrophages, although proliferation of these cells was markedly inhibited. Additionally, the effects of liposomal lipid composition on macrophage activation were studied. With a basic composition of phospholipid/cholesterol/dicetylphosphate, we used either egg-yolk, dipalmitoyl-, distearoyl- or dihexadecylphosphatidylcholine as the bulk phospholipid constituent. Although these liposomes display a widely different susceptibility to lysosomal phospholipase activities, we could not detect any significant difference in either the extent or the duration of the tumoricidal activity induced by MDP encapsulated in these different types of liposomes.