Identification of P-57, a serine proteinase, from human erythrocyte membranes, which cleaves both chains of human third component (C3) of complement

A proteinase, which cleaves human third component of complement, was solubilized from erythrocyte membranes then purified by gel filtration chromatography, fluid phase electrophoresis, and hydroxylapatite chromatography. Labeling of the purified material by 125I or 3H-DFP and measurement of proteolytic activity subsequently isolated by SDS-polyacrylamide gel electrophoresis allowed to identify a 57 kDa single band, in non reducing conditions. Inhibition of this activity by PMSF supports covalent modification of an active serine residue. This membrane serine proteinase cleaved alpha and beta chains of human third component of complement, suggesting that p-57 is distinct from plasma serine proteinases.     

Effect of the essential oil fromAchillea fragrantissima onEscherichia coli cells

Escherichia coli cells treated with the essential oil from the plantAchillea fragrantissima released five polypeptides as well as K⁺ ions into the incubation medium. The oil also inhibited the respiration ofE. coli cells and reduced their ATP content. Electron micrographs showed that oil-treated cells were permeable to uranyl acetate. The effect of the essential oil on the cell membrane is discussed.     

Effect of neutralization and addition of urea,sucrose, and various glycols on phosphorus absorption and leaf damage from foliar-applied phosphate

Summary Inclusion of sucrose in the solution applied to soybean (Glycine max L. merr.) leaves much reduced the severity of the damage to the leaves from application of urea and, to a lesser extent, from application of phosphorus (P) as orthophosphoric acid. Sucrose had no evident effect on P absorption. Damage to the leaves from joint application of orthophosphoric acid and urea exceeded the sum of the damage caused by the substances individually. Urea did not seem to influence P absorption, but the effect, if any, was not readily determined because nearly all values for P absorption exceeded 90%.Neutralization of orthophosphoric acid with nitrogen-containing organic bases, including choline, guanidine, and guanyl urea, did not prove useful as a technique for increasing the quantity of orthophosphate that could be applied without damage to the leaves.Absorption and translocation of orthophosphate by corn (Zea mays L.) and soybean leaves were not influenced by the pH of the solution within the range from 2 to 10. Absorption of tripolyphosphate by corn leaves decreased with an increase in pH of the solution applied, but translocation of the absorbed P was not influenced by pH. With soybeans, absorption of tripolyphosphate decreased with an increase in pH of the solution. Translocation of P applied to soybean leaves as tripolyphosphate was less than 5% of the amount absorbed within the first 24 hr and decreased with an increase in pH after 10 days.     

Galactosyltransferase activities in subcellular fractions of two YC8 lymphoma cell variants: does a relationship exist between antigenic determinants and acceptor sites for galactose?

Experiments have been undertaken to correlate physiological changes, observed in two YC8 cells variants (P and L) and some of their immunological and enzymatic properties. These cell lines show different responses towards antilymphocyte and anti-Moloney sera. Subcellular fractionations have been made. The A fractions (d: 1.14/1.16) have the highest ouabain-inhibited Mg²⁺-stimulated (Na⁺-K⁺)-dependent ATPase and galactosyltransferase activities. Some properties of the latter enzyme have been studied: whereas optima pH and requirements for Mn²⁺ ions have been found to be the same for both cell line enzymes, on the contrary, different kinetic parameters have been shown with respect to sugar donor (UDP-galactose) on endogeneous or exogeneous (ovomucoid) acceptors. Apparent Km for UDP-galactose is 1.7 × 10⁻⁶ M (P-cells) and 3.3 × 10⁻⁶ M (L-cells), on endogeneous acceptors, and P-cell V max < L-cell V max; on ovomucoid it is 0.61 × 10⁻⁶ M, for both cell lines. These results suggest the presence on L-cells of more endogeneous acceptor sites, the higher affinity of P-cells for UDP-galactose being balanced by less endogeneous acceptor sites for galactose. When ovomucoid is added, galactose transfer on endogeneous acceptor sites of both cells is negligible. Apparent Km for ovomucoid is 8.6 × 10⁻⁵ M (P-cells) and 4.3 × 10⁻⁵ M (L-cells). These data support the above-mentioned hypothesis: L-cell enzymes would be more rapidly saturated than P-cell enzymes because of the higher number of endogeneous sites on L-cells.This supposed acquired character of L-cells as well as their immunological behaviour could explain the modified properties of L-cells as compared to P-cells.     

Analysis of phenotypic diversity among host-independent mutants of Bdellovibrio bacteriovorus 109J

Host-independent (H-I) mutants of the obligate bacterial parasite Bdellovibrio bacteriovorus were isolated from wild-type strain 109J. Seven H-I mutants differed in morphological features such as cell length (2-30 microm) and shape (short or long spirals or rod-like), plaque size, and pigmentation (from almost colorless to bright orange). The mutants exhibited widely different growth capabilities in rich medium, with biomass doubling times and final biomass varying by a factor of two or more. Growth was always enhanced by the addition of host cell extract or divalent cations to the growth medium, but the effect varied widely between the mutants. Analysis of the hit region, mutations in which were previously proposed to be associated with the H-I phenotype, revealed that changes in the nucleotide sequence in this region occurred only in three of the seven mutants.     

Novel Technique for Quantifying Adhesion of Metarhizium anisopliae Conidia to the Tick Cuticle

The present study describes an accurate quantitative method for quantifying the adherence of conidia to the arthropod cuticle and the dynamics of conidial germination on the host. The method was developed using conidia of Metarhizium anisopliae var. anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae) and engorged Rhipicephalus annulatus (Say) (Arachnida: Ixodidae) females and was also verified for M. anisopliae var. acridum Driver et Milner (Hypocreales: Clavicipitaceae) and Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae. This novel method is based on using an organic solvent (dichloromethane [DCM]) to remove the adhered conidia from the tick cuticle, suspending the conidia in a detergent solution, and then counting them using a hemocytometer. To confirm the efficacy of the method, scanning electron microscopy (SEM) was used to observe the conidial adherence to and removal from the tick cuticle. As the concentration of conidia in the suspension increased, there were correlating increases in both the number of conidia adhering to engorged female R. annulatus and tick mortality. However, no correlation was observed between a tick''s susceptibility to fungal infection and the amount of adhered conidia. These findings support the commonly accepted understanding of the nature of the adhesion process. The mechanism enabling the removal of the adhered conidia from the host cuticle is discussed.The entomopathogenic fungus Metarhizium anisopliae var. anisopliae (Metschn.) Sorokîn (1883) infects a broad range of arthropod hosts and can be used as a biopesticide against different insect and tick species (8, 22, 35, 36). The adhesion of the conidia of entomopathogenic fungi to the host cuticle is the initial stage of the pathogenic process and includes both passive and active events (5, 10). The hydrophobic epicuticular lipid layer plays an important role during both the attachment process and the germination of the conidia on the surface of the host (15, 19). According to Boucias et al. (7), the attachment of conidia to the host cuticle is based on nonspecific hydrophobic and electrostatic forces. The conidia of most entomopathogenic fungi, including M. anisopliae, have an outer cell layer made up of rodlets (6). The hydrophobins, specific proteins present in the rodlet layer, mediate the passive adhesion of conidia to hydrophobic surfaces, such as the cuticles of arthropods (16, 45, 46). However, as germination commences, the hydrophobins are replaced by an adhesion-like protein, Mad1, which promotes tighter and more-specific adhesion between the conidia and the host (44). Many factors may affect the adhesion and persistence of conidia on the host cuticle (i.e., characteristics of the pathogen, including its virulence [2, 18, 48], conditions under which the pathogen is cultured [17], type of spores [7, 16], topographical and chemical properties of the host cuticle [9, 38, 42], host surface hydrophobicity [15, 23], host behavior [31, 33], and environmental conditions [33]). Conidia of M. anisopliae have shown an affinity to cuticular regions containing setae or spines (7, 38) and to highly hydrophobic cuticle regions, such as mosquitoes'' siphon tubes (23) and intersegmental folds (43). Sites with higher numbers of adhered conidia varied among host species. However, in general, the membranous intersegmental regions were often particularly attractive sites for conidial attachment (26). Variation in the distribution of conidia across different anatomical regions has also been noted in studies of several tick species inoculated with entomopathogenic fungi (3, 21, 22). An evaluation of the attachment of Beauveria bassiana conidia to three tick species, Dermacentor variabilis, Rhipicephalus sanguineus, and Ixodes scapularis, demonstrated that the distribution patterns of the different conidia on the ticks'' bodies were not uniform (22). The density of the conidia and their germination varied dramatically across different anatomical regions of Amblyomma maculatum and A. americanum that had been inoculated with B. bassiana (21). Arruda et al. (3) demonstrated that mass adhesion of M. anisopliae conidia to engorged Boophilus microplus females occurs predominantly on ticks'' legs, suggesting its association with the presence of setae.There are a few approaches for assessing the adhesion of conidia to the host cuticle that are based on direct observation of the conidia on the arthropod cuticle. They involve examining a few areas on the surface of an arthropod by means of scanning electron microscopy (SEM) (11, 15, 30), transmission electron microscopy (TEM) (4), or fluorescence microscopy following vital staining of the conidia (2, 28, 29, 37). These methods are expensive, time-consuming, and relatively inaccurate due to the uneven distribution of conidia on the host surface.In this work, we describe a quantitative method for determining the total amount of conidia that have adhered to a whole host cuticle. This method is based on removing adhered conidia from the tick cuticle using an organic solvent, separating the conidia from the extract by centrifugation, resuspending the conidia in a detergent solution, and then counting the conidia in a hemocytometer. The efficacy of the method was evaluated by comparing the results of this procedure with those of a supplementary examination of conidial removal using SEM.The term “adhered” is often used to define conidia in different states: washed or unwashed after inoculation, present on the host cuticle immediately after inoculation, or kept for several hours (1, 2, 38). In this paper, the term “adhered conidia” refers to conidia that remained on the cuticle after washing by vortexing the inoculated and dried host in an aqueous solution of Triton X-100 and rinsing of the material under tap water.     

Availability of water affects renewal of tissues in migratory blackcaps during stopover

Migrating blackcaps (Sylvia atricapilla) were used to test the predictions that (1) the rebuilding of the digestive tract, as reflected by mass-specific consumption of food on the first 2-3 days of a stopover, is faster in birds with access to drinking water than in birds without, and (2) that adipose tissue and pectoral muscles grow faster and to a greater extent in birds with unlimited access to water. We simulated migratory stopover in two experiments. In Experiment I, each of 31 birds was randomly assigned to one of three experimental groups for 6 days. Along with mealworms (～64% water) ad libitum, Group 1 received drinking water ad libitum; Group 2 had 0.5 h/day access to water; and Group 3 had no access to water. In Experiment II, 30 birds were offered a mixed diet for insectivorous birds (～33% water) ad libitum for 6 days, while randomly assigned to two groups: (1) Water ad libitum-control; and (2) 30 min access to water twice a day. We measured lean mass and fat mass using dual energy X-ray absorptiometry, as well as body mass (m(b)), pectoral muscle index (PMI), and daily intake of food and water. Mean daily water intake was significantly different among the groups in both experiments. However, the availability of drinking water positively affected the rates of gain of lean and fat mass only in birds fed with the mixed, relatively dry diet. Furthermore, mass-specific daily food intake was affected by the availability of drinking water only in the mixed diet experiment, in which birds with unlimited access to drinking water reached an asymptote, 1 day earlier than birds in the water-restricted group. We suggest that in birds consuming diets with low water content, the lack of sufficient drinking water may result in slower rebuilding of the digestive tract, or may influence biochemical processes in the gut that result in slower growth of tissue. Although blackcaps obtained sufficient water from preformed and metabolic water to renew lost tissues when eating mealworms, given access to water, the birds drank prodigiously. Our results also suggest that if drinking water is unavailable to migrating blackcaps, their choices are restricted to water-rich foods, which may constrain their rate of feeding and thus the rate at which they deposit fat. Consequently, drinking water may have an important influence on birds' migratory strategies with respect to habitat selection, use of energy, and the saving of time.     

Somple flow-cell for the fluorimetric study of conformational changes of proteins on agarose matrix

The construction and utilization of a new cylindrical cell for fluorescence measurements on protein-Sepharose 4B conjugates is described. This experimental device proved very convenient for fluorimetry of proteins covalently bound to agarose gels, for measurements on proteins in solution, and finally for monitoring the adsorption of proteins in the course of affinity chromatography. With the aid of this cell, the fluorescence spectra of human α-lactalbumin in solution and in an insoluble state were compared. The α-lactalbumin-Sepharose 4B complex gives a spectrum which closely resembles that of the native protein. Fluorescence spectra were recorded with as little as 50 μliters gel in the cell, which corresponds to approximately 0.015–23 nmoles of chemically bound protein. The fluorescence intensity was within experimental error proportional to protein concentration from 0.03 to 0.20 nmole bound protein/mg dry resin. The application of this fluorimetric method to conformational studies on membrane bound enzymes such as the proteins of the lactose synthetase function is discussed.