Difference in Topology of Normal and Tumour Cell Membranes shown by Different Surface Distributions of Ferritin-conjugated Concanavalin A

THE observations of Burger and Goldberg¹ on the differential agglutinability of malignant cells by the saccharide-binding protein, wheat germ agglutinin (WGA), have prompted other investigators to study the nature of tumour cell surfaces using similar reagents. A variety of other plant agglutinins specifically agglutinate certain oncogenic virus-transformed cells at much lower concentrations than those required to agglutinate their normal parental lines. In addition to WGA^1,2 (specific for binding N-acetyl-D-glucosamine-like residues¹), these include concanavalin A (Con A)^3,4 (specific for binding α-D-glucose or α-D-mannose-like residues⁵), soy bean agglutinin (SBA)^6,7 (specific for binding N-acetyl-D-galactosamine and D-galactose-like residues⁶) and Ricinus communis agglutinin (specific for binding D-galactose and L-arabinose-like residues) (G. L. N. and J. Blaustein, in preparation). To explain this differential agglutinability phenomenon, Burger² proposed that the agglutinin-binding sites on normal cells could have undergone three possible types of change after transformation: (a) the normal parent cell may have no agglutinin sites and after transformation a complete de novo synthesis of agglutinin sites occurs; (b) the normal parent cell may have agglutinin sites, but not enough for agglutination and transformation results in an increase in the number of agglutinin sites; or (c) the normal parent cell agglutinin sites remain constant in number after transformation; but this process results in an exposure of “cryptic” agglutinin sites and the transformed cell is rendered agglutinable. Several workers^8,10,19 postulate a fourth type of change: (d) the normal parent cell agglutinin sites remain constant in number after transformation but the topological distribution of agglutinin sites changes to a distribution more favourable for agglutination. The first and second mechanisms have proved to be unlikely as originally suggested², because agglutinin surface receptors are present on normal cells² and they are present in the same amounts on normal or transformed cells. This latter finding has been demonstrated in saturation binding experiments using purified ¹²⁵I-labelled WGA⁸, Con A^8,9 and SBA¹⁰, with results contrary to earlier reports using ⁶³Ni-labelled Con A³.     

Enzyme-linked lectinosorbent assay of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 10⁶ and 5 × 10⁵ cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.     

Altered glycosylation,expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C,hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients

Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients’ sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients’ group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients’ group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients’ group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients’ groups, to the contrary high glycan branching was observed in HCC patients’ group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients’ group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC without the need of liver biopsy.     

Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from <Emphasis Type="Italic">Arachis hypogaea</Emphasis>

Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic engineering. In this study, a full-length AhLPAT4 gene was isolated via cDNA library screening and rapid amplification of cDNA ends (RACE); our data demonstrated that AhLPAT4 had 1631 nucleotides, encoding a putative 43.8 kDa protein with 383 amino acid residues. The deduced protein included a conserved acyltransferase domain and four motifs (I–IV) with putative LPA and acyl-CoA catalytic and binding sites. Bioinformatic analysis indicated that AhLPAT4 contained four transmembrane domains (TMDs), localized to the endoplasmic reticulum (ER) membrane; detailed analysis indicated that motif I and motifs II–III in AhLPAT4 were separated by the third TMD, which located on cytosolic and ER luminal side respectively, and hydrophobic residues on the surface of AhLPAT4 protein fold to form a hydrophobic tunnel to accommodate the acyl chain. Subcellular localization analysis confirmed that AhLPAT4 was a cytoplasm protein. Phylogenetic analysis revealed that AhLPAT4 had a high homology (63.7–78.3%) with putative LPAT4 proteins from Glycine max, Arabidopsis thaliana and Ricinus communis. AhLPAT4 was ubiquitously expressed in diverse tissues except in flower, which is almost undetectable. The expression analysis in different developmental stages in peanut seeds indicated that AhLPAT4 did not coincide with oil accumulation.     

Interactions of human serum albumin with bioactive 3<Emphasis Type="Italic">H</Emphasis>-imidazo[4,5-<Emphasis Type="Italic">a</Emphasis>]acridines: Insights from fluorescence spectroscopic studies

Several 3H-imidazo[4,5-a]acridine derivatives were conveniently synthesized by the reaction of imidazo[4,5-a]acridones in boiling POCl3. The imidazoacridones were obtained by rearrangement of 3H-imidazo[4',5':3,4]benzo[c]isoxazoles in concentrated sulfuric acid containing nitrous acid at room temperature. The structures of all newly synthesized compounds were confirmed by IR, ¹H NMR, and mass spectral data. The interactions of 3H-imidazo[4,5-a]acridines with human serum albumin (HSA) were studied by fluorescence spectroscopy. The binding of 3H-imidazo[4,5-a]acridines quenches the HSA fluorescence, revealing a 1: 1 interaction with a binding constant of about 2.34 × 10⁵–3.16 × 10⁶ M^–1. A decrease in fluorescence intensity at 339 nm, when excited at 280 nm, is attributed to changes in the environment of the protein fluorophores caused by the presence of the ligand. The differences in interactions of 3H-imidazo[4,5-a]acridines with HSA were observed using spectrofluorimetry technique.     

Correlations between the ages of<Emphasis Type="Italic">Alnus</Emphasis> host species and the genetic diversity of associated endosymbiotic<Emphasis Type="Italic">Frankia</Emphasis> strains from nodules

Nodule samples were collected from four alder species:Alnus nepalensis, A. sibirica, A. tinctoria andA. mandshurica growing in different environments on Gaoligong Mountains, Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS betweennifD andnifK genes was directly applied to unculturedFrankia strains in the nodules. A total of 21 restriction patterns were obtained. TheFrankia population in the nodules ofA. nepalensis had the highest genetic diversity among all fourFrankia populations; by contrast, the population in the nodules ofA. mandshurica had the lowest degree of divergence; the ones in the nodules ofA. sibirica andA. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated thatFrankia strains fromA. sibirica andA. tinctoria had a close genetic relationship.Frankia strains fromA. nepalensis might be the ancestor ofFrankia strains infecting otherAlnus species. From these results and the inference of the ages ofAlnus host species, it is deduced that there was a co-evolution betweenAlnus and its microsymbiontFrankia in China.     

Regulation of the photosynthetic electron transport and specific photoprotective mechanisms in <Emphasis Type="Italic">Ricinus communis</Emphasis> under drought and recovery

Ricinus communis is one of the major commercial non-edible oilseed crops grown in semiarid and arid environments worldwide and is reported as a drought tolerant species. Surprisingly, little is known about the mechanisms achieving this tolerance, especially in relation to photoprotection. The aim of this study was to analyze the association of the regulation of the photosynthetic electron transport and photoprotective mechanisms with drought tolerance in R. communis. Drought induced decreases in the relative water content, water potential and growth in R. communis exposed to 9 days of drought. After 6 days of rehydration, these parameters were completely recovered, demonstrating a potential of drought tolerance in this species. In addition, drought inhibited photosynthesis by stomatal and metabolic limitations (V _cmax, J _max, and Rubisco activity), with partial recovery after rehydration. Leaves displayed transient photoinhibition after 6 days of drought, which was completely recovered after 6 days of dehydration. The effective quantum yields and the electron transport rates of PSII and PSI were modulated to face drought avoiding the excess energy produced by decreases in CO₂ assimilation. NPQ was increased during drought, and it was maintained higher than control after the recovery treatment. In addition, the estimated cyclic electron flow was induced under drought and decreased after recovery. Photorespiration was also increased under drought and maintained at higher levels after the recovery treatment. Furthermore, antioxidative enzymes activities (SOD, APX, and CAT) were increased under drought to avoid ROS harmful effects. Altogether, we clearly showed that the modulation of photoprotective mechanisms and antioxidant enzymes are crucial to this species under drought. The implication of these strikingly strategies to drought tolerance is discussed in relation to agricultural and natural systems.     

Hybridization between Arctostaphylos viscida and A. canescens in Oregon

Evidence of natural hybridization betweenArctostaphylos viscida andA. canescens in southwestern Oregon was obtained from morphological studies of field populations. Hybridization occurs where populations ofA. viscida andA. canescens meet in areas where serpentine and nonserpentine soils abut. At these contacts,A. viscida forms large populations only on serpentine andA. canescens only on nonserpentine. Most of the indentifiable hybrids survive on either transitional or nonserpentine soils. The small proportion that are found on serpentine are considered to be backcrosses toA. viscida. The hybrid plants are similar to several putative species maintained in the standard floras, and it is suggested that these no longer be given taxonomic recognition.     

Photochemical studies on the binding of an organic fluoride to bovine serum albumin

The interaction of fipronil (FPN), a pesticide containing fluorine, to bovine serum albumin (BSA) was studied by spectroscopy including fluorescence spectra, UV–Visible absorption, scattering spectra, circular dichroism (CD) spectra, synchronous and three-dimensional fluorescence spectra. The number of binding sites n and observed binding constant Kb was measured by fluorescence quenching method. The thermodynamic parameters ΔH, ΔG, ΔS at different temperatures were calculated and the results indicate that hydrophobic forces played major role in the reaction. The distance r between donor (BSA) and acceptor (FPN) was obtained according to the Förster theory of non-radiation energy transfer. The structural change of BSA molecules with addition of FPN was analyzed and the results may be helpful to biologists, chemists and therapeutists.     

Extraction,purification and characterization of a novel cysteine protease from the latex of plant <Emphasis Type="Italic">Vallaris solanacea</Emphasis>

Plant proteases with excellent catalytical properties perform many functions in biological systems. A novel plant protease Vallaris solanacea, was identified. Its proteolytic activity was screened using the substrate casein. This protein activity was specifically inhibited by p-chloromercuribenzoate, which showed that it is a cysteine protease. Preliminary investigations such as pH effect and temperature dependence on the caseinolytic activity of crude protease were done. Stability towards temperature and pH were also evaluated. The activity curves drawn in relation to pH, temperature and stability suggested the presence of one protease in the latex of Vallaris solanacea. In the present study, separation and purification of the latex cysteine protease solanain from Vallaris solanacea to a state of near homogeneity was also done using ion exchange and size exclusion chromatography. SDS PAGE was used to determine molecular weight of the solanain (28–29 kDa). The molecular weight was confirmed as 28.9 kDa using MALDI-TOF. Purified protease was named solanain and it was further characterized. An internal tryptic fragment was identified by MALDI-TOF, and this peptide showed a homology (66% sequence similarity) with target sequence of cysteine endopeptidase from Ricinus communis.     

Localization of acid phosphatase in the differentiating root meristem

The localization of acid phosphatase was studied by Gomori’s newer technique and by azo-coupling methods (α-naphthyl phosphate + fast red ITR or fast garnet GBC; AS or AS D phosphate + fast blue B or fast red violet LB) in the root tips ofVicia faba L. on paraffin sections (fixation with Wolman’s acidified ethanol) and on frozen sections (fixation with Baker’s calcium formol). Analogous results were obtained on the material treated in various ways and using different methods. In the broad bean, the reaction is most intense in the cap. In the meristematic zone, the primary core is more intensely stained than the ground parenchyma of the central cylinder. The phloem and xylem poles are usually strongly positive. Using both types of methods on Wolman fixed paraffin embedded material, essentially the same localization of acid phosphatase was found in the root tips ofRicinus communis L.,Lupinus luteus L.,Sinapis alba L.,Allium cepa L. as in the broad bean. InZea mays L. the rhizodermis and the hypodermic layers of the primary core were found to be most active. On sections of Wolman fixed paraffin embedded broad bean the most intense reaction was observed at pH 4.2–4.8. In the same material, both the azo-coupling and the Gomori reaction is inhibited by 10⁼² M NaF, but 10^?2 M tartaric acid only inhibits the Gomori reaction.     

Genomic modulation of diabetes (<Emphasis Type="Italic">db/db)</Emphasis> and obese (<Emphasis Type="Italic">ob/ob</Emphasis>) mutation-induced hypercytolipidemia: cytochemical basis of female reproductive tract involution

Infertility and hypercytolipidemic utero-ovarian involution are recognized consequences of the diabetes-obesity syndrome (DOS) in C57BL mice with either obese (ob/ob) or diabetes (db/db) single gene mutations. We have evaluated the interdependent deleterious influences of both mutation types and differences in the genomic background on utero-ovarian dysfunction in C57BL mice. Control (+/?) C57BL mice were matched with littermate ob/ob and db/db mutants expressed on either the /KsJ or /6 background. Both ob/ob and db/db mutations increased body weights of /KsJ and /6 background strains relative to +/? groups. In contrast, uterine and ovarian weights were depressed by ob/ob and db/dbmutations relative to +/?, regardless of the background strain, but especially when expressed on the /KsJ background. Functionally, both ob/ob and db/db mutations induced hyperglycemic-hyperinsulinemic states coupled with depressed serum estradiol-17-β and progesterone concentrations when expressed on a /KsJ background. Microscopic analysis of utero-ovarian tissue samples revealed marked hypercytolipidemia in the follicular granulosa and endometrial epithelial tissue layers of both ob/oband db/db mutant groups relative to normal +/? cytoarchitecture. The db/db mutation consistently promoted more severe hypercytolipidemic profiles than the ob/obmutation, regardless of background strain. Thus, the severity of utero-ovarian hypercytolipidemia following the expression ofob/ob and db/db mutations in C57BL mice is influenced, or moderated, by the genomic background on which the mutation is expressed.     

Chromosome number variability in central european members of the<Emphasis Type="Italic">Festuca ovina</Emphasis> and<Emphasis Type="Italic">F. pallens</Emphasis> groups (sect.<Emphasis Type="Italic">Festuca</Emphasis>)

Chromosome numbers for 98 plants ofF. pallens, 19 ofF. psammophila, F. belensis andF. vaginata, and 44 ofF. ovina (originating from Austria, the Czech Republic, Germany, Slovakia and Latvia) are given. In addition to theF. ovina andF. pallens groups, chromosome counts for the following taxa are also reported:F. alpestris (2n=14) reported for the first time in this work,F. amethystina subsp.amethystina (2n=28),F. brevipila (2n=42),F. cinerea (2n=28),F. rupicola subsp.rupicola (2n=42) andF. versicolor subsp.versicolor (2n=14).InF. pallens, two ploidy levels (2n=2x=14+0-1B, 2n=4x=28+0-1B) as well as two natural triploid plants (2n=21+0-1B), were found. In addition to the fourF. pallens types that have been distinguished in Austria, one new tetraploid type (F. pallens “scabrifolia”) from the Czech Republic and Germany is reported and its taxonomy is discussed. The distributions of the Oberösterreich-Niederösterreich and Pannonisches-HügellandF. pallens types outside of Austria are documented.Only the diploid chromosome number (2n=14) was found inF. psammophila andF. vaginata. Chromosome numbers forF. psammophila subsp.muellerstollii andF. belensis (both 2n=14) were determined here for the first time. Two ploidy levels, 2n=14+0-5B corresponding toF. ovina subsp.ovina and 2n=28 corresponding toF. ovina subsp.guestphalica andF. cf.duernsteinensis were confirmed inF. ovina. Differences in chromosome structure (simple and multiple secondary constrictions) betweenF. pallens as opposed toF. psammophila andF. vaginata are discussed. A complete survey of published chromosome counts for Central European species from theF. ovina andF. pallens groups is included.     

The variability of critical species ofLotus L. in Iran and in the neighbouring countries I. the circle ofL. corniculatus L. andL. gebelia Vent.

The region of Iran, Iraq, Afghanistan and the neighbouring countries is important for some groups of the speciesLotus L., especially those of the circle ofL. corniculatus L. andL. gebelia Vent. The first group is represented by the speciesL. corniculatus L. with 4 subspecies (3 of which are important for this region), andL. tenuis Waldst. etKit. which here attains the eastern boundary of the continuous area of distribution, and by the eastern speciesL. krylovii Schischk. etSerg. andL. rechingeri Chrtková-?ertová. The second group is represented by the speciesL. gebelia Vent.,L. michauxianus Ser. in DC. andL. libanoticus Boiss. their areas of distribution covering mostly those regions. Most of the species show considerable variability within the species.     

Effects of Lectins on initial attachment of cariogenic <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.     

Effect of light and temperature on the daily rhythm of adult eclosion in the blood-sucking mosquitoes <Emphasis Type="Italic">Aedes communis</Emphasis> De Geer and <Emphasis Type="Italic">Culex pipiens</Emphasis> L. (Diptera,Culicidae)

A comparative study on the effect of light and temperature on the daily rhythm of adult eclosion was carried out on the blood-sucking mosquitoes Aedes (Ochlerotatus) communis De Geer and Culex pipiens pipiens L. Two forms of C. p. pipiens were investigated: the autogenous form molestus occurring in the underground habitats (the urban mosquito), and the anautogenous form pipiens inhabiting mostly the above-ground biotopes. The study included both field observations and laboratory experiments. Under natural conditions (southern Karelia, 62°N) at optimal temperatures A. communis and C. p. p. f. pipiens demonstrated a bimodal eclosion rhythm with morning and evening peaks, whereas in C. p. p. f. molestus only an evening maximum was observed. The fraction of adults eclosing in the middle of the day increased at lower temperatures. In all the mosquitoes studied, the daily eclosion dynamics in the nature did not differ significantly between the variants with different levels of illumination, suggesting that temperature was the main factor regulating the eclosion rhythm. However, experiments with constant temperature showed that light also could influence the timing of eclosion. The responsiveness to light was different in two studied forms of C. p. pipiens. Under a constant temperature and light-dark cycles the rhythm became weak or disappeared completely in the mosquitoes from above-ground biotopes (A. communis and C. p. p. f. pipiens) but persisted in the urban mosquito. The effect of gradual twilight transitions was more prominent than that of switching the light on or off abruptly. The observed differences in the light and temperature dependence of the eclosion rhythm are discussed in relation to the ecological conditions in different habitats.     

The genera Amblostoma,Lanium, and Stenoglossum (Orchidaceae)

The generaAmblostoma, Lanium, andStenoglossum are discussed, and it is concluded that all three should be included inEpidendrum.Lanium is treated as a section, and new names are proposed for two species whose epithets are preoccupied inEpidendrum: E. macrum (forAmblostoma gracile) andE. stiliferum (forLanium subulatum).     

Reactions and resistance ofChenopodium species to tobacco mosaic virus

Chenopodium species react on infection with tobacco mosaic virus by the formation of chlorotic or necrotic lesions and later by the abscission of infected leaves. A transition of local infection into the stem has been observed exceptionally inChenopodium quinoa, C. hybridum, andC. rubrum, but no systemic infection of the leaves followed. Systemic infection was demonstrated only inC. polyspermum andC. murale. The recovery of new sprouts was demonstrated in C.murale in the late chronic phase of infection.