Identification and purification of a family of dimeric major cold shock protein homologs from the psychrotrophic Bacillus cereus WSBC 10201.

Whole-cell protein patterns of a psychrotrophic Bacillus cereus strain from cultures grown at 7 and 30 degrees C were compared. This analysis revealed that at least three major proteins are expressed at a significantly higher rate at 7 degrees C than at 30 degrees C. The most abundant of these cold-induced proteins was a small polypeptide of 7.5 kDa, designated CspA, of B. cereus. In addition, four small proteins very similar in size to CspA were seen on both 7 degrees C and 30 degrees C two-dimensional protein gels. Immunoblot analysis using B. cereus anti-CspA antibodies indicated that the five proteins described above plus an additional sixth protein not visible on silver-stained two-dimensional gels are members of a B. cereus cold shock protein family. This hypothesis was corroborated by cloning and sequencing of the genes encoding five proteins of this family. The protein sequences deduced are highly similar and show homology to small procaryotic cold shock proteins and to the cold shock domain of eucaryotic Y-box proteins. Besides CspA, only one of the additional five CspA homologs was slightly cold inducible. In the presence of 100 mM NaCl, the two purified members of the protein family (CspA and CspE) elute as dimers at an apparent molecular mass of 15 kDa from a gel filtration column. At higher salt concentrations, they dissociate into their monomers. Their ability to bind to the ATTGG motif of single-stranded oligonucleotides was demonstrated by band shift analysis.     

Induction of accessory cell function of human alveolar macrophages by inhalation of human natural interleukin-2

 Accessory function allows antigen-presenting cells to produce sufficient secondary signals for optimum T cell proliferation and interleukin-2 (IL-2) production. Alveolar macrophages are inferior accessory cells compared to monocytes (PBM). We report here that the accessory index (AI) of alveolar macrophages and PBM of patients with lung metastases of solid tumors treated with inhalations of human natural IL-2 (hnIL-2) increased following its administration (P<0.005). The accessory index was significantly elevated from baseline values after 2 weeks of inhalation of 300 000 IU hnIL-2/day (8.2±10.2 compared to 1.1±1; P<0.001). The inhalation of 150 000 IU also induced increases in the index (AI = 2.3±1.9), however, without reaching statistical significance. In addition at 300 000 IU IL-2/day a significant increase in the accessory index was observed for PBM (4±2.5; P<0.05). The indices of PBM and alveolar macrophages prior to inhalation showed a significant negative correlation with the age of the patients (r _s =  – 0.5; r _s =  – 0.8, respectively; P<0.03 for all comparisons). Our data demonstrate that the inhalational application of hnIL-2 enhances the accessory function of alveolar macrophages and, to lesser extent, the accessory index of PBM, indicating the occurrence of pharmacological immunostimulation. Received: 16 August 1995 / Accepted: 4 January 1996     

Simulation of the effect of mixing,scale-up and pH-value regulation during glutamic acid fermentation

A mixing model is coupled with fermentation kinetics in order to simulate a fermentation as a function of mixing conditions and scale-up. The mixing model for a batch stirred tank with three stirrers consists of three regions, each of them characterized by an ideally mixed compartment around the stirrer and two macromixers, i.e. cascades of tank-in-series, describing the recirculation flow. The model contains four parameters — radial and axial circulation time, volume of the ideally mixed stirrer compartment and the number of tanks in each cascade. These values, determined by Mayr et al. in function of the operational conditions and scale-up, were choosen to simulate the fermentation of glutamic acid to show the pH-fluctuation at different control and scale conditions. By choosing optimal regulation properties, such as input flow rate and/or concentration of the base, regulation span, position of the pH-electrode and base input location, etc., fluctuations of the pH-value in the bio-reactor can be minimized. However, the negative effect of insufficient mixing conditions can be reduced only by an increasing number of the base input places. In large scale fermentors, the axial circulation time is rather high, about 5–10 times larger than the radial one. This might result in a large amplitude of the pH-fluctuation. As it is shown, using an input place for base in each stirrer region, the negative impact of the insufficient axial mixing on the fermentation can be diminished perfectly. In this case ammonia should be fed into the reactor as an aqueous solution.     

Strange limits of stability in host-parasitoid systems

The classical Nicholson-Bailey model for a two species host-parasitoid system with discrete generations assumes random distributions of both hosts and parasitoids, randomly searching parasitoids, and random encounters between the individuals of the two species. Although unstable, this model induced many investigations into more complex host-parasitoid systems. Local linearized stability analysis shows that equilibria of host parasitoid systems within the framework of a generalized Nicholson-Bailey model are generally unstable. Stability is only possible if host fertility does not exceede ⁴=54.5982 and if superparasitism is unsuccessful. This special situation has already been discovered by Hassell et al. (1983) in their study of the effects of variable sex ratios on host parasitoid dynamics. We discuss global behaviour of the Hassell-Waage-May model using KAM-theory and illustrate its sensitivity to small perturbations, which can give rise to radically different patterns of the population dynamics of interacting hosts and parasitoids.     

Investigation of the dithiocarbamate-induced coupling of allylidene and carbonyl ligands on a tungsten center

Reaction of the allylidene tungsten complex [W(CPhCHCHMe)Br₂(CO)₂(4-picoline)] (1) with the dithiocarbamates MS₂CNR₂ (a: M=Na, R=Et; b: M=Na, R=Me; c: M=Li, R=Ph) in THF at 50 °C affords the vinylketene tungsten complexes [W(S₂CNR₂)₂(OCCPhCHCHMe)(CO)] (2a–c). At lower temperatures, four reaction intermediates (3–6) may be discerned. Spectroscopic studies indicate that these compounds contain η⁴-allyldithiocarbamate ligands which are generated by addition of dithiocarbamate across the metal-carbon double bond of the allylidene-tungsten unit in 1. The structures of [W(S₂CNEt₂)₂(OCCPhCHCHMe)(CO)] (2a) and of one intermediate, [W(η⁴-Et₂NCS₂CPhCHCHMe)(S₂CNEt₂)(CO)₂] (5a) were elucidated by X-ray crystallography.     

Glycosylation in cardenolide biosynthesis

The glycosylation and deglycosylation of cardiac glycosides was investigated using cell suspension cultures and shoot cultures, both established from Digitalis lanata EHRH. plants, as well as isolated enzymes. Shoots were capable of glucosylating digitoxigenin, evatromonoside, digiproside, glucodigitoxigenin and digitoxin. Suspension cultured Digitalis cells glucosylated all the substrates mentioned but digiproside, whereas the UDP-glucosedependent cardinolide glucosyltransferase isolated from that source did not accept digitoxigenin and digiproside as substrates. It is concluded that at least three different glucosyltransferases are involved in cardiac glycoside formation in Digitalis. Similar experiments carried out with glucosylated cardenolides which were administered to cultured cells, shoots and a cardenolide -glucosidase isolated from young leaves revealed that at least two different glucosidases occur in Digitalis lanata, albeit in different tissues or during different phases of development. The biotransformation of glucoevatromonoside was investigated using unlabelled compound and [¹⁴C-glucose]-glucoevatromonoside synthesized enzymatically. After 7 d of incubation almost no radioactivity could be recovered from the cardenolide fraction, indicating that the terminal glucose of glucoevatromonoside was now incorporated into volatile, hydrophilic and insoluble compounds. Since, on the other hand, large amounts of cardenolides were found in the experiments with unlabelled glucoevatromonoside it is assumed that steady state or pool size regulation is achieved by the coordinated action of a cardenolide glucosidase and a glucosyltransferase.Abbreviations Acdox D-acetyldigitoxose - dgen digoxigenin - dox D-digitoxose - dten digitoxigenin - dtl D-digitalose - fuc D-fucose - gten gitoxigenin - qun D-quinovose - CGH cardenolide 16-O-glucohydrolase - DFT UDP-fucose:digitoxigenin 3-O-fucosyltransferase - DGT UDP-glucose:Digitoxin 16-O-glucosyltransferase - DQT UDP-quinovose:digitoxigenin 3-O-quinovosyltransferase     

Gibberellic acid induces cytoplasmic acidification in maize coleoptiles

Indole-3-acetic acid (IAA), fusicoccin and weak acids all lower the cytoplasmic pH (pH_i) and induce elongation growth of maize (Zea mays L.) coleoptiles. Gibberellic acid (GA₃) also induces elongation growth and we have used confocal laser scanning microscopy to study the effects of GA₃ on pH_i employing the pH-indicator dyes, 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein and carboxy-semi-naphthorhodafluor-1. We confirm that GA₃ induces growth significantly in light-grown but only slightly or not at all in dark-grown coleoptiles. The growth induced by IAA treatment was similar in light- and dark-grown coleoptiles. The pH_i decreased by up to 0.6 units during the first 7 min of GA₃ or IAA treatment of both light- and dark-grown coleoptiles. Gibberellic acid inhibited IAA-induced growth of dark-grown coleoptiles. Hence, in dark-grown coleoptiles GA₃ may activate either directly or indirectly reactions that interfere with the signalling pathway leading to elongation growth. The possible role of pH_i in growth is discussed.Abbreviations ABA abscisic acid - AM acetoxymethyl ester - BCECF 2,7-bis(2-carboxyethyl)-5-(and-6) carboxyfluorescein - [Ca²⁺]_i cytoplasmic free calcium - GA_(n) gibberellin A_(n) - GA₃ gibberellic acid - IAA indole-3-acetic acid - PGR plant growth regulator - pH_i cytoplasmic pH - Pipes piperazine-N,N-bis[2-ethanesulfonic acid] - Snarf-1 carboxy-semi-naphthorhodafluor-1 We thank Dr R. King (CSIRO, Canberra) for providing the GA₁ and T. Phillips for processing the photographic material. H.R. Irving was supported by an Australian Research Council Research Fellowship and the work was supported by an Australian Research Council grant.     

Ozone-induced microscopical changes and quantitative carbohydrate contents of hybrid poplar (Populus × euramericana)

Summary Cuttings of hybrid poplar (Populus × euramericana var. Dorskamp) were exposed to ozone (80 g/m³ from 2100 hours to 0700 hours, 180 g/m³ from 0700 hours to 2100 hours) for 3 months. Ozone reduced the starch content in leaves and stem bark, whereas starch granules accumulated in bundle sheath cells along small leaf veins. At the same time, sucrose and inositol content increased in the leaves. Mesophyll cells in the vicinity of the stomata were injured first, and droplet-like material appeared on their walls. In the sieve plates of fumigated trees, the pores showed a higher degree of narrowing than those of the control treatment. Cell collapse in the leaves was accompanied by water loss and an increase in air space. In the stems, the ozone treatment led to a reduced radial width, particularly in the xylem tissue. These results are discussed in relation to reduced or inhibited phloem loading and ozone-induced drought stress. The plants injured by ozone showed quite distinct patterns of metabolite responses as well as enzyme activities (PEP- and RubP-carboxylase) in the leaves from the top to the bottom. There were also remarkable differences in the reaction of sucrose and inositol between leaves and stem bark. Future research should therefore increasingly follow a whole-plant approach for a better understanding of complex plant reactions.     

Role of the Cys 2-Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1.

The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.