Biochemical approaches to the study of plant-fungal interactions in arbuscular mycorrhiza

This communication compares some biochemical methods for quantifying colonization by arbuscular mycorrhizal (AM) fungi. The degree of mycorrhizal colonization can conveniently be measured by determining fungal specific sterols. AM-colonized plants show a specific synthesis of 24-methylene cholesterol and an enhanced level of campesterol (=24-methyl cholesterol). A gene probe for nitrate reductase, the key enzyme for nitrogen assimilation, has been developed, which allows the monitoring of the distribution of this enzyme in fungi. Among the phytohormones tested, only abscisic acid (ABA) is found at a considerably higher level in AM-colonized plants than in controls. The concentration of ABA is about twenty times higher in spores and hyphae of the AM fungusGlomus than in maize roots. Other phytohormones (auxins, cytokinins) do not show such alterations after mycorrhizal colonization. The roots of gramineous plants become yellow as a result of mycorrhizal colonization. The yellow pigment(s) formed is (are) deposited in larger quantities in the vacuole(s) of the root parenchyma and endodermis cells during the development of the gramineous plants. A substance isolated from such roots has now been identified as a C-14 carotenoid with two carboxylic groups, and named mycorradicin.     

A simple method for the preparation of antibodies to the mitochondrial biotin-dependent carboxylases

Summary Heterologous antiserum to the 3 biotin-dependent carboxylases was prepared by selective removal of these enzymes from human liver on an avidin-sepharose column. A carboxylase-avidin-sepharose matrix was used as an antigen to produce anti-carboxylase antibodies. The resultant antisera can be used to purifiy the specific carboxylases, to prepare monoclonal antibodies to these enzymes or to study inherited carboxylase deficiencies and biotin-dependent intermediary metabolism.The authors thank Dr Emmanuel Shapira for his helpful suggestions and Patricia Tuck for her technical assistance. This work was supported by NIH research grants AM25675 and AM26127. Barry Wolf is a recipient of an NIH Research Career Development Award (AM00677) and is aided by a Basil O'Connor Starter Research Grant from the National Foundation—March of Dimes (5–263). This is paper No. 137 from the Department of Human Genetics — Medical College of Virginia.     

Conformational dynamics of the beta2-microglobulin C terminal in the cell-membrane-anchored major histocompatibility complex type I

We have recently described an anti-beta2-microglobulin (beta2-m) monoclonal antibody (mAb 14H3) capable of recognizing the epitope 92-99 of the protein in the monomeric native state as well as in the fibrillar polymeric state, but not in the major histocompatibility complex type I (MHCI) anchored to the cell membrane. In the present study, we investigated the molecular basis for the inaccessibility of the C-terminal end of beta2-m in the MHCI complex, and demonstrated that mAb 14H3 binds the soluble fraction of the MHCI complex with a Kd of 0.3 microM. An interaction between the complex and the membrane protects beta2-m from immunological recognition at the MHCI level. This protection from antibody recognition can be weakened by procedures such as heat shock or gamma irradiation that perturb the membrane structure and commit the cell to the apoptotic pathway. mAb 14H3 can recognize MHCI in a transient state that most likely precedes beta2-m shedding and may be proposed as a useful tool for dynamic analysis of MHCI conformational modifications.     

Quaternary structure-dependent idiotope and antigen binding of a monoclonal antibody specific for conformational epitope on type II collagen

We previously generated a monoclonal antibody (mAb) against a putative pathogenic epitope on native type II collagen (CII) for the induction of collagen-induced arthritis in mice (mAb1), and an anti-idiotypic mAb which appears to possess the internal image of the CII epitope (mAb2). In the present study, the structural basis of the antigen/mAb1 and mAb1/mAb2 interactions was examined. When partially SH-reduced mAb1 was analysed on Western blots, only fragments containing both heavy (H) and light (L) chains were recognized by mAb2. When mAb2 was partially SH-reduced, only fragments containing both H and L chains were recognized by mAb1. H and L chains were separated from mAb1 in a reduced, denatured condition, and each chain and a mixture of the two were refolded. mAb2 reacted specifically to the renatured whole IgG molecule of mAb1, but not to the refolded L or to H chains. Recombinant single chain Fv (scFv) generated from mAb1 and mAb2 had properties of the original mAbs, whereas genetical ly constructed chimeric scFvs, consisting of V_H from mAb1 and an irrelevant V_L , or V_L of mAb1 and an irrelevant V_H , did not react either to CII or to mAb2. Thus, interactions among CII, mAb1 and mAb2 appear to depend on quaternary structures containing different protein subunits. These observations support the internal image property of the mAb2. In addition, this dependency on quaternary structure for recognition of proteins may also be relevant to other protein-protein interactions. Received 29 July 1996; received after revision 13 September 1996; accepted 18 October 1996     

Monoclonal antibodies against antigens on breast cancer cells

Summary Of 360 mAb obtained in a cell fusion experiment with the spleen cells of a mouse immunized with a mixture of different human breast carcinoma cells lines, 30 mAb were selected which reacted more strongly with tumor cells than with (noncancerous) fibroblasts. Theses mAb were tested for reactivity with additional types of cancerous and noncancerous tissues. Two mAb showed high tumor selectivity, but the corresponding epitopes on individual tumor cells were heterogeneously expressed. The mAb will be evaluated for in vivo applications.     

Monoclonal antibodies against antigens on breast cancer cells

Of 360 mAb obtained in a cell fusion experiment with the spleen cells of a mouse immunized with a mixture of different human breast carcinoma cell lines, 30 mAb were selected which reacted more strongly with tumor cells than with (noncancerous) fibroblasts. These mAb were tested for reactivity with additional types of cancerous and noncancerous tissues. Two mAb showed high tumor selectivity, but the corresponding epitopes on individual tumor cells were heterogeneously expressed. The mAb will be evaluated for in vivo applications.     

Initiation of germination of Clostridium difficile spores by lysozyme]

The germination rate of spores of C. difficile which is usually lower than 10(-5) is raised to about 5.10(-3) in presence of lysozyme. All spores are initiated by lysozyme when previously treated by sodium thioglycolate. These spores are indeed lysozyme-dependent for germination.     

Different effects of D-glucose anomers for respiration of bacterial germinated spores.

Effects of alpha- or beta-D-glucose on the respiration of germinated spores (only germinated spores not including swollen spores and elongated spores) of Bacillus subtilis and B. megaterium were studied. In our conditions, net amount of oxygen consumed by 10(10) germinated spores of B. subtilis per min after addition of alpha- or beta-D-glucose was 1.6 microgram or 6.6 microgram (beta/alpha = 4.13), while that by B.megaterium was 4.5 microgram of 6.8 microgram (beta/alpha = 1.51), respectively. However, the net amounts of oxygen consumed by 10(10) vegetative cells per min after addition of alpha- or beta-D-glucose were identical, for B.subtillis in both cases 443.0 microgram and for B.megaterium in both cases 604.4 microgram.     

Effects of the light-dark cycle and scheduled feeding on behavioral and reproductive rhythms of the cyprinodont fish,Medaka,Oryzias latipes

Summary Medaka were maintained on a 168 light-dark cycle and fed once daily on one of 5 different feeding schedules. The daily rhythm of agonistic behavior rapidly entrained to the scheduled feeding time and maintained this entrainment during a 3-day starvation period. In contrast the daily rhythms of egg laying and courtship stayed entrained to the L:D cycle regardless of the feeding schedule. Thus, temporal integration of this fish with its daily environment can involve multistimuli which concurrently and differentially entrain externally expressed circadian systems.We thank Mark Goodrich and Steve Huber for fish maintenance and technical assistance; and Don Dovala for aid in developing the automatic feeder. The research was funded in part by NIH, AM 25191 and NIEHS, ES No. 01985.     

Polyreactive antibodies in adaptive immune responses to viruses

B cells express immunoglobulins on their surface where they serve as antigen receptors. When secreted as antibodies, the same molecules are key elements of the humoral immune response against pathogens such as viruses. Although most antibodies are restricted to binding a specific antigen, some are polyreactive and have the ability to bind to several different ligands, usually with low affinity. Highly polyreactive antibodies are removed from the repertoire during B-cell development by physiologic tolerance mechanisms including deletion and receptor editing. However, a low level of antibody polyreactivity is tolerated and can confer additional binding properties to pathogen-specific antibodies. For example, high-affinity human antibodies to HIV are frequently polyreactive. Here we review the evidence suggesting that in the case of some pathogens like HIV, polyreactivity may confer a selective advantage to pathogen-specific antibodies.     

Expression and immunolocalisation of odorant-binding and chemosensory proteins in locusts

We have identified, cloned and expressed a new chemosensory protein (CSP) in the desert locust Schistocerca gregaria belonging to a third sub-class of these polypeptides. Polyclonal antibodies stained a band of 14 kDa, as expected, in the extracts of antennae and palps of the adults, but not in the 4th and 5th instars. In the related species Locusta migratoria, instead, the same antibodies cross-reacted only with a band of apparent molecular mass of 35 kDa in the extract of 1st–5th instars, but not in the adults. The recombinant protein binds the fluorescent probe N-phenyl-1-naphthylamine, but none of the compounds so far reported as pheromones for S. gregaria. The expression of the odorant-binding protein (OBP) and of CSPs of sub-classes I and II was also monitored in antennae, tarsi, palpi, wings and other organs of solitary and gregarious locusts in their nymphal and adult stages. OBP was found to be antenna specific, where it is expressed at least from the 3rd instar in both solitary and gregarious locusts. CSPs, instead, appear to be more ubiquitous, with different expression patterns, according to the sub-class. Immunocytochemistry experiments revealed that OBP is present in the sensillum lymph of sensilla trichodea and basiconica, while CSP-I and CSP-III were found in the outer sensillum lymph of sensilla chaetica and in the sub-cuticular space between epidermis and cuticle of the antenna. Sensilla chaetica on other parts of the body showed the same expression of CSP-I as those on the antenna.Received 11 Janury 2005; received after revision 21 February 2005; accepted 18 March 2005X. Jin and A. Brandazza contributed equally to this work.     

Inverse regulation of spore germination and growth by cyclic AMP inStreptomyces hygroscopicus

Summary The cyclic AMP level in germinating spores ofStreptomyces hygroscopicus rises to a maximum at outgrowth of germ tubes. Exogenous cyclic AMP results in an inverse effect on germination speed and growth.     

Display of proteins on Bacillus subtilis endospores

The targeting and anchoring of heterologous proteins and peptides to the outer surface of bacteriophages and cells is becoming increasingly important, and has been employed as a tool for fundamental and applied research in microbiology, molecular biology, vaccinology, and biotechnology. Less known are endospores or spores produced by some Gram-positive species. Spores of Bacillus subtilis are surrounded by a spore coat on their outside, and a few proteins have been identified being located on the outside layer and have been successfully used to immobilize antigens and some other proteins and enzymes. The major advantage of spores over the other published systems is their synthesis within the cytoplasm of the bacterial cell. Therefore, any heterologous protein to be anchored on the outside does not have to cross any membrane. Furthermore, spores are extremely resistant against high temperature, irradiation and many chemicals, and can be stored for many years at room temperature.     

Ecdysteroids in vitro promote differentiation in the accessory glands of male mealworm beetles

Summary Physiological peak doses of 20-hydroxyecdysone were added to organ cultures of young pupal accessory glands of maleTenebrio molitor. During subsequent culture in vitro or in vivo, the glands accumulated adult-specific antigens. Control organ cultures showed no such antigen accumulation. In this system, ecdysteroid controls not only cell cycles but also differentiation.21 October 1986This research was supported in part by a National Institutes of Health Research Service Award F32 AM7515 (KAG) and NIH grants GM26140 and AI15662 (GMH). We thank Toshinobu Yaginuma for help in developing the transplantation technique.     

Lethal effects of heat on bacterial physiology and structure

High temperatures have profound effects on the structural and physiological properties of sporulating and non-sporulating bacteria, with membranes, RNA, DNA, ribosomes, protein and enzymes all affected. Nevertheless, it is apparent that no one single event is responsible for cell death. The induction of intracellular heat-shock proteins and the activation of extracellular alarmones in vegetative cells exposed to mildly lethal temperatures are important cell responses. In bacterial spores, several factors contribute to the overall resistance to moist (wet) and dry heat; the latter, but not the former, induces mutations. Heat resistance develops during sporulation, when spore-specific heat-shock proteins are also produced. Heat sensitivity is regained during germination of spores.     

Immunological comparison between albumins of three species of mice (genusMus)

Summary Three closely related species of short-tailed mice (Mus musculus musculus, M. spretoides andM. spicilegus) were tentatively discriminated using immunological techniques based on albumin cross-reactivity. Different fractionations of crude albumin antisera allowed the recovery of antibody populations specific to theM. m. musculus albumin, whereas antibody population differences do not seem to exist betweenM. spicilegus andM. spretoides. Moreover, immunoreactivities tested with native and S-carboxymethylated albumins revealed that species-specific antibodies correspond to antigenic determinants depending on the amino acid sequence (sequential determinants). The observed immunological differences are related to species divergence and albumin sequences.     

极性分子对甲基丙烯酸丁酯/丙烯酰胺共聚合的影响

以自由基本体聚合方法制备了甲基丙烯酸丁酯/丙烯酰胺(BMA/AM)共聚物.将极性分子环己醇引入到BMA和AM的共聚体系中,研究了环己醇对单体转化率和BMA/AM共聚物特性黏度的影响.随着环己醇加入量的增加,AM在反应体系中溶解的量明显增大,单体转化率增加,BMA/AM共聚物的特性黏度存在峰值.借助傅里叶变换红外(FTIR)、核磁共振(13C NMR)、动态力学分析法(DMA)、差示扫描量热法(DSC)和热重分析法(TGA)研究了环己醇对BMA/AM共聚物的结构和性能的影响,结果表明:AM只能与BMA进行共聚反应,随着环己醇的加入量的增加,BMA/AM共聚物的含量将增加,由此发泡阶段的酰亚胺化反应程度就越高,BMA/AM共聚物分子链中的酰亚胺环结构就越多,从而显著提高BMA/AM共聚物的性能,有望制备高性能的聚甲基丙烯酰亚胺(PMI)泡沫塑料.     

Changes in beta-2 adrenergic receptor sensitivity with maturation of erythroid progenitor cells

Summary Erythroid burst forming units (BFU-E) were much more sensitive to the beta-2 selective adrenergic drug, salbutamol, than erythroid colony forming units (CFU-E) in an in vitro study of erythroid progenitor cells.This work was supported by USPHS Grant AM13211. B. Beckman is a USPHS Postdoctoral Fellow AM05960.     

Inhibition of virus multiplication and alteration of cyclic AMP level in cell cultures by flavonoids

Summary The inhibitory effect of four flavonoid compounds on virus multiplication and their influence on the intracellular cyclic AMP (cAMP) level were studied in cell cultures. Quercetin and quercitrin reduced the yields ofHuman (alpha) herpesvirus 1 (HSV-1) andSuid (alpha) herpesvirus 1 (pseudorabies virus), but hesperidin and rutin had no effect. Further, quercetin and quercitrin elevated the intracellular level of cAMP, whereas hesperidin and rutin did not alter the cAMP level. Both antiviral activity and cAMP-enhancing effect were dependent on the concentrations of the flavonoids, and these effects turned out to be parallel.This study suggests that a relation exists between the antiviral effect and the cAMP-enhancing activity of flavonoids.     

Effect of plant lectins on Ustilago maydis in vitro

Ustilago maydis is an edible parasitic basidiomycete, which specifically infects corn (Zea mays) and teocintle (Z. diploperennis). To characterise the interaction between the basidiomycete and its host organism, we tested the effect of plant lectins with well-known sugar specificity on the growth and germination of U. maydis spores. Lectins specific for N-acetyl-D-galactosamine, such as those from Dolichos biflorus and Phaseolus lunatus, and the wheatgerm agglutinin specific for N-acetyl-D-glucosamine inhibited spore germination, but were ineffective in modifying U. maydis cell growth. The galactose-specific lectin from the corn coleoptyle inhibited both germination and cell growth, while the lectin concanavalin A (mannose/glucose specific) activated spore germination and growth. Our results suggest that specific saccharide-containing receptors participate in regulating the growth and maturation of U. maydis spores.