Reduced sensitivity of Niemann-Pick C1-deficient cells to θ-toxin (perfringolysin O): sequestration of toxin to raft-enriched membrane vesicles

-Toxin (perfringolysin O) binds to cell surface cholesterol and forms oligomeric pores that cause membrane damage. Both in cytotoxicity and cell survival assays, a mutant Chinese hamster ovary cell line NPC1(–) that lacked Niemann-Pick C1 showed reduced sensitivity to -toxin, compared with wild-type (wt) cells. BC is a derivative of -toxin that retains cholesterol-binding activity but lacks cytotoxicity. Confocal and electron microscopy revealed the presence of multiple vesicles which bound BC, both on the cell surface and in the extracellular space of these cells. BC binding to raft microdomains was verified by its resistance to 1% Triton X-100 at 4°C and recovery of bound BC in floating low-density fractions on sucrose density gradient fractionation. BC-labeled vesicles were abolished when NPC1(–) cells were depleted of lipoproteins and also when treated with a Rho-associated kinase inhibitor Y-27632. In addition, similar vesicles were observed in wt cells treated with progesterone. In parallel with these results, -toxin sensitivity of NPC1(–) cells was increased when cells were depleted of lipoproteins or treated with Y-27632, whereas that of wt cells was decreased by progesterone. Our findings suggest that sequestration of toxin to raft-enriched cell surface vesicles may underlie reduced sensitivity of NPC1-deficient cells to -toxin.     

Receptive field of the stemmata in the swallowtail butterflyPapilio

Summary Receptive fields of individual retinular cells in the stemmata ofPapilio xuthus L. were examined electrophysiologically, and the receptive field of the complete stemmatal system was reconstructed (Fig. 8).In stemmata I-IV, proximal retinular cells have narrow receptive fields (acceptance angles of = 1.7–5 °, Fig. 5) and small inclinations of the visual axes (inclinations of = 0.7–1.5 °, Fig. 2) with respect to the axis of the stemma, while distal ones have wide fields ( =7–13 °, Fig. 5) and large inclinations of the visual axes ( = 5–10 °, Fig. 3). In stemmata V and VI, both proximal and distal retinular cells have wide receptive fields ( = 7–26 °, Fig. 6) and have large inclinations of their visual axes ( = 9–19 °) with respect to the axis of the stemma except for one proximal cell ( = 0 °) (Fig. 4).The spatial properties of distal and proximal retinular cells, combined with the finding that distal cells are homogeneous in the spectral sensitivity while proximal ones are heterogeneous (Ichikawa and Tateda 1980), suggest that the distal cells may be concerned largely with the detection of objects and proximal cells are involved with the discrimination of the color and shape of the detected objects.     

Changes of fluorescence lifetime and rotational correlation time of bovine serum albumin labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride in guanidine and thermal denaturations

The nanosecond fluorescence depolarization method was applied to measure the fluorescence lifetime () and the rotational correlation time () of bovine serum albumin (BSA) labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-Cl). Changes of and of dansyl BSA in the guanidine denaturation and in the thermal denaturation were examined. In parallel, the secondary structural change of dansyl BSA was followed by circular dichroism measurements. The magnitude of was almost unchanged between 1 and 2 M guanidine, where the secondary structure of the protein was predominantly disrupted; whereas that of began to increase before the disruption of secondary structure in the guanidine denaturation. In the thermal denaturation, in contrast, changes of both and occurred in a temperature range where the secondary structure was predominantly disrupted. The volume of equivalent sphere (V _e ) and the axial ratio () for the BSA were 3.6–3.8×10^–19 cm³ and 3.6 at 2M guanidine as against 2.1×10^–19 cm³ and 2.2 in the absence of guanidine (25°C), respectively. The magnitudes ofV _e and were 4.9×10^–19 cm³ and 4.5 at 65°C, respectively. Although the secondary structural change of dansyl BSA was irreversible in the thermal denaturation,V _e and were reversible.     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, chlorine dioxide (ClO₂), and a commercial raw fruit and vegetable sanitizer (Fit powder) were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5–11.0) ClO₂ (200 mg/mL) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log cfu/mL, respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log cfu/mL resulting from treatment with 200 mg/mL chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5-log and 0.4-log cfu/mL reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO₂ (85 mg/mL), acidified (pH 3.4) ClO₂ (85 mg/mL), and a mixture of ClO₂ (85 mg/mL) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log cfu/mL, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/mL organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO₂ (400 mg/mL) for 30 min reduced populations by 4.6 and 5.2 log cfu/mL, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO₂.Published by permission of the International Association for Food Protection: Journal of Food Protection (2004) 60:1702–1708This revised version was published online in April 2005 with corrections to the text and the section heading.In section Preparation of treatment solutions the phrase 22-28°C was replaced by 22±2°C.     

Assortative mating and the genetic correlation

Summary The effect of assortative mating on the genetic correlation between traits X and Y is considered. Assortation on trait X changes the magnitude of the genetic correlation but not its sign. There are two situations depending on the signs of the correlation between mates () and of the random mating genetic correlation (): 1) if sign () = sign (), then >, where is the genetic correlation at equilibrium after continued assortation, and 2) if sign () = sign (), then < . However, negative assortative mating is virtually powerless to alter the magnitude of the genetic correlation. The consequences of a mixed assortation model, e.g., high milk production females mated to fast growing males and lesser productive females mated to slower growing sires, were also studied. Mixed positive assortation always increases the genetic correlation, but negative assortation decreases it. The implications of assortative mating on correlated responses to selection and on the equilibrium covariances between relatives for pairs of traits are discussed.     

The measurement of mean temperatures in plant and soil studies by the sucrose inversion method

Summary A polarimetric procedure is described for determining exponential mean temperatures (_e) from the rate of inversion of sucrose solutions buffered at pH values between 1 and 3. An equation is given for calculating the pH values appropriate for various temperature ranges and time scales. The procedure used for monitoring air and soil temperatures over a 5 year period at a network of sites in Aberdeenshire was to expose 5 ml volumes of sucrose solution at pH 2.06 in sealed polythene tubes. Each month the changes in optical rotation were measured with a sensitive automatic polarimeter and fresh tubes exposed. The values of _e were 2°C higher than arithmetic means obtained from standard thermometric devices. Observations at elevations between 275 and 686 m a.s.l. demonstrate the lapse of temperature with altitude and illustrate the usefulness of _e measurements.     

Beta-1,3-glucanase from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. Comparison with beta-1,3-glucanases of marine and terrestrial mollusks

-1,3-Glucanase (Lu) was isolated from unfertilized eggs of the sea urchin Strongylocentrotus intermedius. A comparative study of some properties of -1,3-glucanase Lu and -1,3-glucanases with different action types—endo--1,3-glucanase from crystalline style of the marine mollusk Spisula sachalinensis (LIV) and exo--1,3-glucanase from the terrestrial snail Eulota maakii (LII)—was performed. It was found that -1,3-glucanase Lu hydrolyzes laminaran with a high yield of glucose in the reaction products. The enzyme hydrolyzes substrates with retention of the glycosidic bond configuration, is able to cleave modified substrates, and exhibits transglycosylating activity. All properties of -1,3-glucanase from S. intermedius were more similar to those of the endo--1,3-glucanase from the marine mollusk (LIV) than exo--1,3-glucanase LII from the terrestrial snail. The differences in the effect of LIV and Lu on laminaran are probably related to the functions of -1,3-glucanase Lu from sea urchin eggs (which, in contrast to LIV, is not a digestive enzyme).     

Mapping dominant markers using F2 matings

The development of efficient methods for amplifying random DNA sequences by the polymerase chain reaction has created the basis for mapping virtually unlimited numbers of mixed-phase dominant DNA markers in one population. Although dominant markers can be efficiently mapped using many different kinds of matings, recombination frequencies and locus orders are often mis-estimated from repulsion F₂ matings. The major problem with these matings, apart from excessive sampling errors of recombination frequency () estimates, is the bias of the maximum-likelihood estimator (MLE) of ( _ML). when the observed frequency of double-recessive phenotypes is 0 and the observed frequency of double-dominant phenotypes is less than 2/3 — the bias for those samples is — . We used simulation to estimate the mean bias of _ML. Mean bias is a function of n and and decreases as n increases. Valid maps of dominant markers can be built by using sub-sets of markers linked in coupling, thereby creating male and feamle coupling maps, as long as the maps are fairly dense (about 5 cM) — the sampling errors of increase as increases for coupling linkages and are equal to those for backcross matings when =0. The use of F₂ matings for mapping dominant markers is not necessarily proscribed because they yield twice as many useful markers as a backcross population, albeit in two maps, for the same number of DNA extractions and PCR assays; however, dominant markers can be more effeciently exploited by using doubled-haploid, recombinant-inbred, or other inbred populations.     

Changes in Cortical Activity in Altered States of Consciousness: The Study of Meditation by High-Resolution EEG

The specific features of the topology of spectral powers and coherent interregional interrelationships in the narrow, individually determined -, -, ₁-, ₂-, and ₃-frequency bands were studied by means of high-resolution EEG (62 channels) in novice and experienced meditators (NMs and EMs) at rest and under the conditions of generation of an altered state of consciousness characterized by inactivation of cognitive activity and the occurrence of a positive emotional experience of happiness. EMs in the meditation-free state were found to be characterized by a shift in the values of the individual frequency to a lower-frequency region of the spectrum, along with higher, compared to NMs, -, ₁-, ₂-, and ₃-band power values, which probably reflects the cumulative character of the influence of long-term meditative practice. The effective achievement of altered states of consciousness in EMs was associated with an increase in the local - and ₁ powers in the anterior cortical areas, as well as long-distance coherence between the prefrontal and posterior associative cortex with the formation of a center of gravity in the left prefrontal region (lead AF ₃). According to the data of the correlation analysis of the EEG power values and the data of subjective scaling of the meditation state, the -power values were positively associated with positive emotional experiences and negatively associated with the level of mental activity. The results of this study are consistent with current concepts that the and activities in narrow frequency bands reflect the activity of multifunctional neuronal networks selectively associated with processes of cognitive and affective activity.     

Phosphorus depletion in the rhizosphere as influenced by soil moisture

To study the influence of soil moisture on phosphorus (P) depletion in the rhizosphere, maize (Zea mays cv. Trak) was pre-grown in vermiculite filled-PVC tubes for 9 days and then the plants with the tubes were transplanted into soil columns maintained at two soil moisture levels () of 0.14 and 0.20 cm³ cm^–3 for 10 days. The soil columns were separated at 1 cm depth by a nylon screen of 53 m inner mesh size, into 1 cm soil layer above and 3 cm soil column below screen. A root mat developed over the screen, but root hairs only could penetrate it. Regardless of the soil moisture level in the columns, and adequate and equal water and nutrients supply was maintained via wicks from an external nutrient solution to the plant roots in vermiculite. After 10 days, the soil columns were separated from the root mats, quickly frozen in liquid nitrogen and sliced into thin layers (0.2mm) using a refrigerated microtome to give soil samples at defined distances from the root mats for analyses. Lower soil moisture (=0.14) resulted in narrower and steeper depletion profile of 0.5 M NaHCO₃ extractable P (NaHCO₃-P_i) as compared to higher soil moisture (=0.20). Depletion of P in soil solution in the immediate vicinity of root mats did not differ much but the extension of the depletion zones was 0.10 cm at =0.14 and 0.20 cm at =0.20. The depletion up to 0.05cm with =0.14 and up to 0.07 cm with =0.20 was uniform, and may be attributed to the depletion in the root hair zone. Beyond the root hair zones, the theory of diffusion and mass flow was able to explain the observed differences in shape and extent of the P depletion profiles at the two soil moisture levels.     

Nucleotide sequence of the humanθ 1-globin gene

We have cloned and sequenced the human ₁-globin gene. The nucleotide sequence and organization of the human ₁ gene (exons, introns, promoter, and polyadenylation signals) are similar to those reported for the orangutan ₁-globin gene. If these genes are functional, the sequences of their ₁-globin chains would differ by only one amino acid residue (at position 137).This research was supported by USPHS Research Grants HLB-05168 and HLB-15158. This is contribution No. 1085 from the Department of Cell and Molecular Biology at the Medical College of Georgia in Augusta.     

Spectral Characteristics of the EEG in Students with Different Levels of Result Achievement under the Conditions of Examination Stress

The EEG correlates of the performance of examination tests and special cognitive tests under the conditions of everyday study (common studying conditions, CSCs) and immediately before examination (examination conditions, ECs) were analyzed in 39 male students aged 18–20 years. The results of the examinations strongly correlated with the relative spectral power (SP) of the EEG rhythm before the examination. Therefore, the students were divided into two groups with different directions of changes in the -range SP under the ECs compared to the CSCs. Students from group 1 were characterized by increased relative - and -rhythm SPs under the ECs and a good examination performance; students of group 2, by decreased - and -rhythm SPs, an increased index of the EEG of the central and frontal cortical regions under the ECs, and a poor examination performance.     

Ultrastructure of Bacillus pulvifaciens

The ultrastructure of vegetative cells and spores of Bacillus pulvifaciens was studied by CTEM and SEM methods. The vegetative cells are rods, 1.6–4.5 m long and 0.4–0.6 m wide, exhibiting typical ultrastructural features of Gram-positive bacteria. The spores are of ellipsoidal shape, 0.6×1.2 m in size, with six longitudinal ribs reaching up to 130 nm in height. There are satelite ribs on both sides of the longitudinal ribs, reaching up to 20 nm in height. Between the longitudinal ribs, additional transversal ribs were observed in SEM. A special tubular layer, separating the outer and inner coat of the spores, was revealed in ultrathin sections. This layer seems to be a typical ultrastructural feature of Bacillus pulvifaciens spores.     

Microbial degradation of dehydrodiconiferyl alcohol,a lignin substructure model

Bacteria, yeasts, and molds which grew in a medium containing a synthetic lignin — a dehydrogenation polymer (DHP) of coniferyl alcohol — as a sole carbon source, were isolated from soil. One fungus, Fusarium solani M-13-1, was found to degrade the DHP most vigorously among the isolated organisms. It was shake-cultured in a medium containing dehydrodiconiferyl alcohol (DHCA) (I), an important lignin model compound, and the following six metabolic products were isolated and identified: 1) Phenylcoumaran--aldehydic (II) and -carboxylic compounds, 2) phenylcoumaran--aldehydic compound (IV), formed by release of a 2-carbon fragment from the phenylcoumaran--carboxylic compound, 3) 5-acetylvanillyl alcohol (V), formed by cleavage of the coumaran ring and reduction of the -aldehyde group, 4) 5-carboxyvanillyl alcohol (VI), formed by subsequent oxidation of the acetyl group, and 5) the -ether of DHCA (VII), considered to be a by-product. A degradation pathway for DHCA was proposed on the basis of these metabolic products.Non-Standard Abbreviations DHP dehydrogenation polymer - DHCA dehydrodiconiferyl alcohol - DDQ dichlorodicyano-p-benzoquinone - DDHQ dichlorodicyano-p-hydroquinone - Ar aromatic - TLC thin layer chromatography - GC-MS gas chromatography-mass spectrometry     

Comparison of laboratory- and field-derived soil water retention curves for a fine sand soil using tensiometric,resistance and capacitance methods

The approximate range from 100 to 50% of plant-available water in Apopka fine sand (loamy, siliceous, hyperthermic Grossarenic Paleudult) is 0.08–0.04 cm³ cm^–3 soil water content () or –5 to –15 kPa of soil water matric potential (). This narrow range of plant-available soil water is extremely dry for most soil water sensors. Knowledge of the soil water retention curves for these soils is important for effective irrigation of crops in fine sand soils of subtropical and tropical regions of the world. The primary objective of this study was to compare sandy soil water retention curves in the field as measured by tensiometer and resistance block values and capacitance sensor . The second objective was to compare these curves to one developed on a Florida fine sand soil using a pressure plate apparatus. Tensiometer and resistance block values were compared to values from capacitance sensors calibrated gravimetrically. The effective range of both tensiometers and resistance sensors in fine sand soils is between –5 and –20 kPa . Soil water potential values for both sensors were within 2 kPa of the mean for each sensor. Change in was similar over the range of 0.04–0.08 cm³ cm^–3 . Curves for the two sensors were different by 4 kPa at 0.04 cm³ cm^–3. The relationship between and were similar at 10–20, 20–30 and 40–50 cm depths. This was not true for a laboratory determined soil water retention curve for the same soil type. These differences are significant in soils with very low water holding capacities. Differences between laboratory- and field-determined retention curves could be due to a combination of entrapped air in the field soil and/or alteration in bulk density in the laboratory samples.     

Soil phosphorus levels needed for equal P uptake from four soils with different water contents at the same water potential

Soil volumetric water contents, , at –33 kPa potential may vary with soil from 0.06 to 0.70. Because P diffusion depends on , most economic P fertilizer rates required for different soils may require adjusting according to their soil-water relationships. The objective of this study was, after experimentally verifying a mechanistic nutrient uptake model on a series of soils varying in at –33 kPa potential, to use the model to predict labile P levels needed for each of these soils to achieve equal P uptake by maize (Zae mays L.) and verify these predictions. Maize was grown in a pot experiment using four soils having of 0.13, 0.20, 0.26, and 0.40 at –33 kPa each at 0, 200, and 400 mg kg^-1 of added P. When root parameters obtained experimentally were used, predicted P uptake with the uptake model agreed with observed P uptake, y=0.99x+9.08 (r²=0.98). When P uptake was plotted vs. soil solution P, C_li, the relation varied with soil. The higher the the lower the C_li needed for equal P uptake. A similar relation was found between P uptake and diffusible soil P, C_si. Differences between the two plots occurred because of differences among soils in buffer power, C_si/C_li. The C_si vs. P added relation was used to calculate differences among soils in the C_si needed to obtain equal P uptake. The C_si values ranged from 1.3 to 4.0 mmol kg^–1. The calculated values were used in a second pot experiments to verify the predictions. No significant difference (=0.05) in P uptake occurred. The results of this research indicate that the mechanistic nutrient uptake model can be used to predict the degree of adjustments in C_si needed to obtain the most economic P fertilizer rates among soils varying in .Journal Paper No. 13072. Purdue Univ. Agric. Exp. Stn., West Lafayette, IN 47907.     

Macrophage-activating factor extracted from mycoplasmas

Summary Mycoplasmas (M. gallisepticum, chicken mycoplasmas), in concert with interferon (IFN), were effective in activating macrophages (M) to be tumoricidal. The M-activating capacity of mycoplasmas was maintained after treatment with heat, 0.1 M NaOH, 1 M HC1, or trypsin. M-activating factor was extracted from mycoplasmas with chloroform/methanol and water (Mf-B). Mf-B was also effective in activating M in the presence of IFN. The threshold dose of Mf-B for M of ordinary C3H/He mice and that for those of C3H/HeJ mice, the latter being known to be low responders to bacterial lipopolysaccharide, were actually the same. This seems to indicate that the effectiveness of Mf-B was not attributable to possibly contaminating lipopolysaccharides, and that the pathway of activity of Mf-B is different from that of lipopolysaccharides. Since the M-activating principle was only a very small part of Mf-B, we have not yet succeeded in identifying it, but there was no evidence that it was protein, nucleic acid, sugar, or lipid. The cytotoxicity of M activated by Mf-B plus IFN was dependent onl-arginine in the culture, suggesting that arginine metabolites are involved in M cytotoxicity. Mf-B induced a small amount of tumor necrosis factor in M, and this induction was markedly enhanced by IFN.     

Effect of lectin from the ascidian on the growth and the adhesion of HeLa cells

Cell adhesion molecules, some of which are lectins, play a key role in the control of normal and pathological processes of various living organisms. We found herein that NacetylDglucosaminespecific lectin, isolated from the ascidian Didemnum ternatanum (DTL), alters the growth properties of HeLa tumor cells depending on the anchorage. DTL was shown to increase the proliferation of HeLa cells grown in soft agar greatly (in anchorageindependent fashion). In contrast, DTL inhibits the proliferative activity of HeLa cells grown on solid substrate and acts as inductor of differentiation, slowing cell growth, increasing the cell attachment and spreading. Scanning electron microscopic data have demonstrated that DTL treatment resulted in pronounced changes of the shape and surface of HeLa cells. Changes of cellular morphology correlated with essential redistribution of actin microfilaments.     

The quantum efficiency of photosystem II and its relation to non-photochemical quenching of chlorophyll fluorescence; the effect of measuring-and growth temperature

The relation between the quantum yield of oxygen evolution of open photosystem II reactions centers (_p), calculated according to Weis and Berry (1987), and non-photochemical quenching of chlorophyll fluorescence of plants grown at 19°C and 7°C was measured at 19°C and 7°C. The relation was linear when measured at 19°C, but when measured at 7°C a deviation from linearity was observed at high values of non-photochemical quenching. In plants grown at 7°C this deviation occurred at higher values of non-photochemical quenching than in plants grown at 19°C. The deviations at high light intensity and low temperature are ascribed to an increase in an inhibition-related, non-photochemical quenching component (q_I).The relation between the quantum yield of excitation capture of open photosystem II reaction centers (_exe), calculated according to Genty et al. (1989), and non-photochemical quenching of chlorophyll fluorescence was found to be non-linear and was neither influenced by growth temperature nor by measuring temperature.At high PFD the efficiency of overall steady state electron transport measured by oxygen-evolution, correlated well with the product of q _N and the efficiency of excitation capture (_exe) but it deviated at low PFD. The deviations at low light intensity are attributed to the different populations of chloroplasts measured by gas exchange and chlorophyll fluorescence and to the light gradient within the leaf.Abbreviations F₀ basic fluorescence - F₀ basic fluorescence, thylakoid in energized state - F_m maximal fluorescence - F_m maximum fluorescence in energized state - F_s steady state fluorescence - F_v maximal variable fluorescence - PFD photon flux density - PS II_rc Photosystem II reaction center - q_F0 quenching of basic fluorescence - q_E energy related quenching - q_N non-photochemical quenching:-q_f-total quenching - q_I inhibition-related quenching - q_p photochemical quenching - q_r quenching due to state transition - R_d dark respiration - _p PS II efficiency of excitation capture of open PS II_rc - _pe extrapolated minimal value of _p - _p0 extrapolated maximal value of _p - _si quantum efficiency of linear electron transport, calculated from gas exchange measurements based on incident light - _sf quantum efficiency of linear electron transport, calculated from fluorescence measurements, based on incident measuring light