Synthesis of ribonucleic acids during the germination of Rhizopus stolonifer sporangiospores

A number of ribonucleic acids initiated synthesis during the first 15 min of germination of Rhizopus stolonifer sporangiospores. They included ribosomal, 5S, and transfer RNA, and a heterogeneously-sedimenting fraction that composed about 5% of the total cellular RNA; this fraction was unmethylated, did not compete with ribosomal RNA for hybridization to DNA, and was bound to poly dT-cellulose in a manner characteristic of RNAs containing polyadenylate segments. The nearly simultaneous onset of synthesis of the various RNAs with germination contrasts with the sequential onset of RNA synthesis reported for other fungal spores.     

Extracellular Ribonuclease Production by Rhizopus stolonifer: Influence of Metal Ions

A strain of Rhizopus stolonifer produced high levels of extracellular ribonuclease (RNase) when grown on YPG (yeast extract, peptone, glucose) medium. Influence of various medium components on the production of extracellular RNase activity showed that divalent metal ions had a marked effect on growth and enzyme production. Maximum enzyme activity (3000 U/ml) was obtained in 5 days when the culture was grown in YPG medium containing Mg²⁺ (12 mM), Mn²⁺, and Fe²⁺ (2 ppm each). Inorganic phosphate, however, repressed enzyme production. Antibodies raised against the purified extracellular RNase were then used to establish the relationship between intra- and extracellular enzymes.     

Nuclease Rsn from Rhizopus stolonifer: specificity and mode of action

Nuclease Rsn from Rhizopus stolonifer catalyzes the hydrolysis of ss- and dsDNA in a ratio of approximately 2:1. Time course of 3' and 5' terminal analysis of the hydrolytic products of ss- and dsDNA showed that nuclease Rsn does not show any strict base preference and cleaves DNA in a non-specific manner. Moreover, separation of the hydrolytic products of ss- and dsDNA in the presence of Mg2+, Mn2+ or Co2+ showed the predominance of tetra-, tri-, and dinucleotides followed by mononucleotides, suggesting an endo mode of action.     

Mycelial Mattress from a Sporangia Formation-Delayed Mutant of Rhizopus stolonifer as Wound Healing-Enhancing Biomaterial

A mycelial mattress of Rhizopus stolonifer obtained from a liquid static culture was utilized for wound dressing and biomedical use. Following screening of mutants induced by UV radiation, F6, exhibiting delayed sporangium formation was selected because its sporangium maturation exhibited a 5-day delay without significant loss of mycelial weight compared to the wild type. The sporangium-free mycelial mattress from the sporangiospore culture of F6 was treated with 1N sodium hydroxide NaOH at 85°C for 2 h to produce a sponge-like membrane named Rhizochitin. The trifluoroacetic acid hydrolysate of Rhizochitin contained 36% N-acetylglucosamine and 53% hexose respectively detected by the Elson-Morgen and phenol-sulfuric acid methods. Results indicated the wound area in rats covered with Rhizochitin was 40% less than that of the uncovered group. Rhizochitin decreased the expression of PDGF in the proliferation stage, increased the expression of TGF-β in the inflammation and proliferation stages, and increased the expression of VEGF in the inflammation and proliferation stages. Rhizochitin inhibited secretion of matrix metalloproteinase-9 on days 1, 7, 9, and 12 and matrix metalloproteinase-2 on days 3, 7, 9, and 12. It was concluded that Rhizochitin has beneficial properties of biocompatible, biodegradable, and wound healing.     

Expression and functional analysis of a glycoside hydrolase family 45 endoglucanase from Rhizopus stolonifer

A novel endoglucanase gene was cloned from Rhizopus stolonifer and expressed in Escherichia coli, the gene product EG II (45 kDa) was assigned to Glycoside Hydrolase Family 45 (GH45), and its specific activity on phosphoric acid-swollen cellulose (PASC) was 48 IU/mg. To solve the problem of substrate accumulation in the cellulose hydrolysis and enhance the catalytic efficiency of endoglucanase, the eg2 gene was modified by site directed mutagenesis. Mutations generated by overlapping PCR have been proven to increase its catalytic activity on carboxymenthyl cellulose, microcrystalline cellulose (Avicel) and PASC, among which the mutant EG II-E containing all 6 mutations (N39S, V136D, T251G, D255G, P256S and E260D) peaked 121 IU/mg on PASC. The bioinformatic analysis showed that 2 key catalytic residues (D136 and D260) moved closer with the opening of a loop after mutagenesis, and a tunnel was formed by structural transformation. This structure was conducive for the substrate to access the active centre, and D136 played an indispensable role in the substrate recognition.     

The mitochondrial respiratory chain of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill

Rhizopus stolonifer (Ehrenb.:Fr.) Vuill mitochondria contain the complete system for oxidative phosphorylation, formed by the classical components of the electron transport chain (complexes I, II, III, and IV) and the F₁F₀-ATP synthase (complex V). Using the native gel electrophoresis, we have shown the existence of supramolecular associations of the respiratory complexes. The composition and stoichiometry of the oxidative phosphorylation complexes were similar to those found in other organisms. Additionally, two alternative routes for the oxidation of cytosolic NADH were identified: the alternative NADH dehydrogenase and the glycerol-3-phosphate shuttles. Residual respiratory activity after inhibition of complex IV by cyanide was inhibited by low concentrations of n-octyl gallate, indicating the presence of an alternative oxidase. The K_0.5 for the respiratory substrates NADH, succinate, and glycerol-3-phosphate in permeabilized cells was higher than in isolated mitochondria, suggesting that interactions of mitochondria with other cellular elements might be important for the function of this organelle.     

Ribonuclease Rs from Rhizopus stolonifer: lowering of optimum temperature in the presence of urea

RNase Rs showed an approx. 2-fold increase in its activity when incubated in the presence of 2 M urea at 37 degrees C. The increase in its activity, in the presence of urea, was comparable to the activity at its optimum temperature, i.e. 45 degrees C. Compared to the native enzyme at 37 degrees C, the K(m) and V(max) of RNase Rs at 45 degrees C and in the presence of 2 M urea at 37 degrees C showed an increase while k(cat)/K(m) decreased. Arrhenius plots in the presence and absence of urea showed a decrease in the activation energy in the presence of urea. Though there was no change in the secondary structure of the protein in the presence of urea, minor changes were observed in the tertiary structure. Hence, the increase in the activity of RNase Rs, in the presence of 2 M urea at 37 degrees C, is due to the lowering of the activation energy as a result of changes in the microenvironment of the active site.     

Equilibrium unfolding of RNase Rs from Rhizopus stolonifer: pH dependence of chemical and thermal denaturation

The conformational stability of RNase Rs was determined with chemical and thermal denaturants over the pH range of 1-10. Equilibrium unfolding with urea showed that values of D(1/2) (5.7 M) and DeltaG(H(2)O) (12.8 kcal/mol) were highest at pH 5.0, its pI and the maximum conformational stability of RNase Rs was observed near pH 5.0. Denaturation with guanidine hydrochloride (GdnHCl), at pH 5.0, gave similar values of DeltaG(H(2)O) although GdnHCl was 2-fold more potent denaturant with D(1/2) value of 3.1 M. The curves of fraction unfolded (f(U)) obtained with fluorescence and CD measurements overlapped at pH 5.0. Denaturation of RNase Rs with urea in the pH range studied was reversible but the enzyme denatured irreversibly >pH 11.0. Thermal denaturation of RNase Rs was reversible in the pH range of 2.0-3.0 and 6.0-9.0. Thermal denaturation in the pH range 4.0-5.5 resulted in aggregation and precipitation of the protein above 55 degrees C. The aggregate was amorphous or disordered precipitate as observed in TE micrographs. Blue shift in emission lambda(max) and enhancement of fluorescence intensity of ANS at 70 degrees C indicated the presence of solvent exposed hydrophobic surfaces as a result of heat treatment. Aggregation could be prevented partially with alpha-cyclodextrin (0.15 M) and completely with urea at concentrations >3 M. Aggregation was probably due to intermolecular hydrophobic interaction favored by minimum charge-charge repulsion at the pI of the enzyme. Both urea and temperature-induced denaturation studies showed that RNase Rs unfolds through a two-state F right arrow over left arrow U mechanism. The pH dependence of stability described by DeltaG(H(2)O) (urea) and DeltaG (25 degrees C) suggested that electrostatic interactions among the charged groups make a significant contribution to the conformational stability of RNase Rs. Since RNase Rs is a disulfide-containing protein, the major element for structural stability are the covalent disulfide bonds.     

Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of - single strand preferential - deoxyribonuclease activity

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M(r) of the purified enzyme determined by native PAGE was 67? omitted?000 and it is a tetramer and each protomer consists of two unidentical subunits of M(r) 21? omitted?000 and 13? omitted?000. It is an acidic protein with a pI of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg(2+), Mn(2+) and Co(2+) for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg(2+) was 40 degrees C, it showed higher optimum temperature (45 degrees C) for dsDNA hydrolysis in presence of Mn(2+) and Co(2+). Nuclease Rsn was inhibited by divalent cations like Zn(2+), Cu(2+) and Hg(2+), inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3'- or 5'-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3'-hydroxyl and 5'-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.     

Surface layer protein EA1 is not a component of Bacillus anthracis spores but is a persistent contaminant in spore preparations

EA1 is an abundant, highly antigenic, surface layer protein of Bacillus anthracis vegetative cells. Recent studies indicate that EA1 is also a component of B. anthracis spores and a potential marker for spore detection. We show here that EA1 is not a spore component but a persistent contaminant in spore preparations.     

A cilium is not a cilium is not a cilium: signaling contributes to ciliary morphological diversity

Mukhopadhyay and colleagues reveal in this issue of Developmental Cell that signaling mediated by a specialized neuronal cilium in C. elegans affects its structure. The finding that this cilium is modified in response to the cues it transduces suggests that cilia may not be static antennae, but organelles whose functions are shaped by their signaling activities.     

Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.     

Concanavalin A is not a ferritin

Contrary to recent claims, in vitro evidence has been obtained to establish that Concanavalin A (Con A) is not a ferritin. Four techniques including immunoprecipitation, gel filtration, sucrose density gradient ultracentrifugation and CsCl centrifugation were employed. None of them showed that Con A is a ferritin.     

Oligo(dT) is not a correct native PAGE marker for single-stranded DNA

Polyacrylamide gel electrophoresis is a widely used method to study short DNA fragments in solution. It is, however, a relative method requiring length markers to assess mobility, shape, flexibility, and molecularity of the DNA structures of interest. In recent literature we have encountered the use of oligo(dT) fragments as the native PAGE length markers. We show here that this practice is inadequate because oligo(dT) migration is strongly retarded in native polyacrylamide gels. This conclusion is qualitatively true irrespective of the conditions of electrophoresis, oligo(dT) length, and gel concentration. Depending on their length, oligo(dT) fragments migrate 2--4 times slower than that would correspond to their nucleotide number. This leads to erroneous conclusions, e.g., determination of the number of associated molecules in guanine quadruplexes or other DNA complexes.     

Naiveté is not forever: responses of a vulnerable native rodent to its long term alien predators

Alien predators have wreaked havoc on isolated endemic and island fauna worldwide, a phenomenon generally attributed to prey naiveté, or a failure to display effective antipredator behaviour due to a lack of experience. While the failure to recognise and/or respond to a novel predator has devastating impacts in the short term after predators are introduced, few studies have asked whether medium to long term experience with alien predators enables native species to overcome their naiveté. In Australia, introduced dogs Canis lupus familiaris, foxes Vulpes vulpes and cats Felis catus have caused rapid extinctions and declines in small–medium sized native mammals since they were introduced ～150 years ago. However, native wildlife have had ～4000 years experience with another dog – the dingo Canis lupus dingo. Native bush rats Rattus fuscipes remain common despite predation from these predators. We predicted that prior experience with dingoes would mean that bush rats recognise and respond to dogs, but suspect that hundreds of years experience may not be enough for effective responses to cats and foxes. To test these predictions, we combined the giving‐up density (GUD) with analysis of remote camera footage to measure bush rat foraging and behavioural responses to body odour from dogs, foxes, cats and native spotted‐tail quolls Dasyurus maculatus. Bush rats responded strongly to dogs with increased GUDs, increased vigilance and decreased foraging. However, mixed responses to foxes and cats suggest that at least some individuals remain naïve towards these predators. Naiveté is not necessarily forever: alien predators devastate many native prey species, but others may learn or adapt to the new threat.