The potential energy surface of methyl 2-O-(α-D-mannopyranosyl)-α-D-mannopyranoside in aqueous solution: Conclusions derived from optical rotation

The optical rotation of methyl 2-O -(α-D -mannopyranosyl)-α-D -mannopyranoside is calculated semiempirically as a function of the linkage dihedral angles ? (H1-C1-O1-C2′) and ψ (C1-O1-C2′-H2′). Although the rotation calculated for the global energy minimum conformation found in several rigid-residue modeling calculations (?,ψ = ?40°,?20°) is in good agreement with the observed solution rotation, the observed rotation is also compatible with the limited flexibility inferred from more recent relaxed residue modeling calculations on a structurally related rhamnose disaccharide © 1994 John Wiley & Sons, Inc.     

Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 -induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.     

Effects of ethanol,octanoic and decanoic acids on fermentation and the passive influx of protons through the plasma membrane of Saccharomyces cerevisiae

Ethanol, octanoic and decanoic acids are known toxic products of alcoholic fermentation and inhibit yeast functions such as growth and fermentation. pH-stat measurements showed that, in a concentration range up to 20 mg/l, octanoic and decanoic acids increase the rate of passive H⁺ influx across the plasma membrane of Saccharomyces cerevisiae IGC 3507. Decanoic acid was more active than octanoic acid, which agrees with its higher liposolubility. The fatty acids probably act as H⁺ carriers, since the magnitude of the effect depended on pH and correlated with the concentration of protonated fatty acids. Esterification of the fatty acids partially abolished the enhancing effect on passive H⁺ influx. Passive H⁺ influx showed saturation kinetics with half-maximal activity at 6.6 M H⁺ (pH 5.2). Contrary to previous findings, ethanol inhibited H⁺ influx exponentially up to a concentration of 8% (v/v). At higher concentrations, ethanol reactivated H⁺ influx; the original rate of H⁺ uptake was reached at 14% (v/v) ethanol. In the same concentration ranges that affected passive H⁺ influx, ethanol, octanoic and decanoic acids inhibited the fermentation rate. This inhibitory effect of the fatty acids on fermentation rate depended on liposolubility, pH, and esterification in the same way as that found for their effect on passive H⁺ influx. Inhibition of fermentation by octanoic and decanoic acids could therefore result from their effect on the rate of passive H⁺ influx. Correspondence to: S. Stevens     

Autoproteolysis of herpes simplex virus type 1 protease releases an active catalytic domain found in intermediate capsid particles.

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.     

Glycoprotein B is a specific determinant of herpes simplex virus type 1 neuroinvasiveness.

Herpes simplex virus type 1 strains ANG and KOS lack neuroinvasiveness when inoculated on the footpads of mice, and because the strains are able to complement each other, the genes associated with this phenotype differ. In this study, we used marker rescue techniques to show that at least two genes cloned from ANG are required to restore neuroinvasiveness to KOS. One of the two fragments required is the 6.3-kb BamHI-A/EcoRI-D fragment (0.15 to 0.19 map units). The second has been identified as the sequence encoding glycoprotein B (gB) (UL27). Analysis of ANG and KOS DNA sequences in the relevant region of the gB gene revealed two nucleotide differences which result in amino acid differences in the gB protein. One appears to be unique to the strain of KOS used in our laboratory. The second, at codon 523 of the mature gB protein, encodes a valine in KOS and an alanine in ANG. Recombinant KOS viruses which contained ANG sequences in this region were constructed, and two independently selected recombinants demonstrated increased neuroinvasiveness in mice. From these results, we conclude that gB significantly influences neuroinvasiveness. Mechanisms by which this might occur are discussed.     

Growth inhibition of selected food-borne bacteria by tannic acid, propyl gallate and related compounds

Tannic acid, propyl gallate, gallic acid and ellagic acid were tested for their inhibitory effects on selected food-borne bacteria by the well assay technique. Tannic acid and propyl gallate were inhibitory whereas gallic acid and ellagic acid were not.     

The effect of wiping and spray-wash temperature on bacterial retention on abraded domestic sink surfaces

The relative cleanability of artificially abraded stainless steel, enamelled steel, mineral resin and polycarbonate domestic sinks was assessed by examining bacterial retention after cleaning. Two cleaning regimes were used: the mechanical action of wiping combined with a spray-rinse, and spray-washing at a range of temperatures. After wiping, stainless steel retained 0.5–1 log order fewer bacteria than the enamel sinks which in turn were 0.5 log order cleaner than the mineral resin and polycarbonate sinks. After spray-washing, stainless steel retained 0.5 log order fewer bacteria than enamel which in turn was 0.5 log order cleaner than the polycarbonate and mineral resin. Extending the number of wipes or increasing spray-wash temperature enhanced bacterial removal but, in general, did not change the relative cleanability of the sink materials. As a cleaning technique, wiping was shown to be more effective than spray-washing in reducing bacterial numbers. SEM studies showed that bacteria were typically retained in surface imperfections, particularly pits and crevices such that surfaces which sustained the most extensive damage due to abrasion retained higher numbers of bacteria.     

Am improved procedure for the isolation of chloroplast DNA fromChlamydomonas reinhardtii

The isolation of chloroplast DNA fromChlamydomonas reinhardtii requires the efficient separation of this AT-rich genome from the GC-rich nuclear genome by density-gradient centrifugation. We describe a simple and efficient method for separating these DNA fractions by using a sodium iodide gradient in combination with the DNA-binding dye, bisbenzimide. The yield of chloroplast DNA is close to the theoretical maximum and the DNA is suitable for restriction enzyme analysis and cloning. This method is applicable to the isolation of AT-rich plastid genomes from other organisms and may be appropriate as a general method for separating species of DNA that differ in their AT/GC ratios. An erratum to this article is available at .     

Photo-induction of electrical current with the cyanobacteriumAgmenellum quadruplicatum PR-6

We investigated the possibility of eliciting a measurable photoinduced electrical current from the cyanobacteriumAgmenellum quadruplicatum PR-6 (Synechococcus PCC 7002). This proved virtually impossible for intact cells. However, treated PR-6 cells fixed in an alginate matrix on tin oxide as the active electrode in a three electrode electrochemical cell gave rise to a significant light response. Cell treatments involving toluene, chloroform or detergents were effective and gave current responses up to 250 nA. Drying the cyanobacterial matrix increased the current yield at least fifty-fold. These effects were observed for light wavelengths > 650 nm and were not influenced by inhibitors or enhancers of photosynthesis nor by sustained argon bubbling of the electrolyte.French pressure cell lysates facilitated distinction between two light induced current components. Lysates prepared without CaCl₂ gave current induction kinetics that were indistinguishable from those on chemically treated cells i.e. slowly rising to a stable maximum in 10–15 min. When CaCl₂ was present during lysis, a rapidly induced (<1 s) unstable component was observed. Toluenization of PR-6 either prior to or post lysing abolished the CaCl₂ related effect. CaCl₂ had no effect on current induction in strain PR-6008, which lacked the and subunits of phycocyanin and exhibited slow current induction kinetics.The observed effects are interpreted as responses of components of the photosystems of PR-6 rather than in terms of an integrated photosynthetic process.     

Carbohydrate distribution and cellular injuries in acid rain and cold-treated spruce needles

Summary The cellular structures of acid rain-irrigated needles of several provenances of Norway spruce (Picea abies L. Karst) seedlings were studied after winter experimental freezing. Frost injuries and recovery were characterized by visual damage scoring and classification of mesophyll cell alterations, also using histochemical methods for carbohydrate fluorescent staining. The treatment with-30° C during the late dormancy period was sufficient to cause significant injuries and intracellular degradation in the tissues of the green needles. The most affected seedlings in terms of visual injury scoring were found among those treated with clean water or at pH 3, while freezing injury, defined as an occlusion of phenolic substances in the central vacuole of the mesophyll cells, was most abundant in the needles from spruces irrigated either with clean water or at pH 4 or pH 3. Electron microscopy revealed the details of the injury, e. g. thinning out of the cytoplasm and chloroplast stroma, darkening of the chloroplasts and eventually swelling of the chloroplasts and protoplast. PAS and ConA reactions in the needle tissue revealed intense starch accumulation in the mesophyll and transfusion tissues as early as in March, with a tendency to increase, especially in the untreated needles during the recovery period. Plasma membrane disturbances were indicated by histochemical identification of callose deposits in the mesophyll cell walls, these being most abundant in the acid rain-treated needles. All these findings suggest that freezing at –30° C was more deleterious to the seedlings pretreated with acid or clean water than to those not given additional irrigation.