Purification and properties of an endopolygalacturonase produced byRhizopus stolonifer

Summary A polygalacturonase from culture filtrates of a strain ofRhizopus stolonifer was purified about 80 fold by ethanol precipitation, followed by ion exchange chromatography (CM-Sepharose 6B) and gel filtration (Sephadex G-100). The purified preparation was homogeneous when examined by PAGE. The enzyme is an endopolygalacturonase with an optimum catalytic activity at pH 5.0 and 45°C, and a molecular weight of 57,000±500 daltons. The activity was stimulated by Fe⁺⁺⁺, Mg⁺⁺, Co⁺⁺, and inhibited by Mn⁺⁺ and Zn⁺⁺. The enzyme was stable in the pH range of 3.0 to 5.0. The purified enzyme was specific for nonmethoxylate polygalacturonic acid, with K_m and V_max values respectively of 0.19 mg/ml and 1.3 mol/g/min. In addition, this enzymatic preparation degraded pectic substances in organge peel.     

A new filter for cell separation

Summary A novel filter housing has been designed that permits the use of a coarse sintered stainless steel cylindrical filter element as a cross-flow filtration unit. Cultures of the fungal micro-organism Trichoderma reesei have been used to study the performance characteristics of the unit and filtration rates of 1.0m³/m²/h have been achieved in a sterile manner.     

Release of periplasmic enzymes fromEscherichia coli using tangential flow filtration

Summary Escherichia coli cells producing the periplasmic enzymes nuclease, -lactamase and alkaline phosphatase were exposed to osmotic shock treatment in a closed system consisting of a tank, a pump and a filtration unit. The enzymes were released by circulating the cell suspensions in the filtration system and separated from cells and spheroplasts by filtration. This novel releasing method was shown to be equally effective as osmotic shock treatments performed by agitation and centrifugation. Since the extraction is performed in a closed system, aerosol formation is avoided. In addition the method may easily be scaled up.     

Agrobacterium — and microprojectile — mediated viral DNA delivery into barley microspore-derived cultures

Summary Anther cultures of barley (Hordeum vulgare L. var. Igri) were used as targets for Agrobacterium-mediated DNA transfer and direct DNA uptake by particle bombardment. A wheat dwarf virus construct which can replicate to a high copy number in cereal cells provided a sensitive marker for successful DNA delivery. Although DNA delivery was achieved using both procedures, particle bombardment gave more reproducible and higher levels of infection. The ability to deliver DNA into cereal cells which have a high regeneration capacity may provide a route for stable transformation.Abbreviations WDV Wheat dwarf virus - Gus -glucuronidase - ^mA N⁶-methyladenine     

Modeling the influence of slurry concentration on Saccharomyces cerevisiae cake porosity and resistance during microfiltration

Filtration of an isotonic suspension of baker's yeast through a 0.45‐μm membrane was studied at two different pressures, 40 and 80 kPa, for yeast concentrations ranging from 0.14 to 51 kg/m³ (dry weight). For a yeast volume fraction above 0.06 (～21.8 kg/m³), the porosity of the yeast cake is less dependent on the suspension concentration. For highly diluted suspensions, the specific cake resistance approaches a minimum that depends on the filtration pressure. Correlation functions of cake porosity and specific cake resistance were obtained for the concentration range investigated showing that the Kozeny–Carman coefficient increases when the applied pressure increases. Both filtration pressure and slurry concentration can be process controlled. In the range of moderate yeast concentration, the filtrate flux may be increased by manipulating the filtration pressure and the slurry concentration, thereby improving the overall process efficiency. The complex behavior of yeast cakes at high slurry concentration can be described by a conventional model as long as part of yeast cells are assumed to form aggregates, which behave as single bigger particles. The aggregation effect may be accounted for using a binary mixture model. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

The Effect of Universal Fluorinated Nucleobases on the Catalytic Activity of Ribozymes

Abstract

Four fluoro modified universal nucleobases have been synthesized. The universal nucleobases 1 and 2 , containing a 2,4-difluorobenzene as nucleobase and a 4,6-difluorobenzimidazole, respectively, were chemically incorporated into a selected hammerhead ribozyme sequence which has already been retrovirally expressed as an anti-HIV ribozyme to investigate their effect on the catalytic activity of the ribozymes. The substitution of the natural nucleosides with either 1 or 2 results only in a small decrease of the catalytic activity. The K_m value for the monosubstituted ribozyme with a 2,4-difluorobenzene is 309 nM^?1, the corresponding k_cat is 2.91 · 10^?3 min^?1. A disubstituted hammerhead ribozyme carrying one of each modification has also been synthesized. For a further stabilization of the ribozyme/substrate complex 2′-(β-aminoethoxy) modified fluorinated nucleosides 15 and 16 have been developed.     

Plasmid stability during continuous culture in aSaccharomyces cerevisiae double mutant transformed by a plasmid carrying a eukaryotic gene

Summary An investigation of plasmid stability in aSaccharomyces cerevisiae double mutant has been performed. The host was a double recombinantura3 furl mutant containing a plasmid bearing the yeast URA3⁺ allele and an expression cassette for human ₁-antitrypsin. The mutant was grown in continuous culture employing a semi-defined medium containing added uracil to provide non selective growth conditions. After 150 generations of continuous growth, no cured cells had been detected: the specific expression level of ₁-antitrypsin remained constant throughout the experiment.     

New cell recycle ethanol fermentation with periodic cleaning of filter with gas

High ethanol productivities were obtained by cell recycle cultures of yeast and bacterial strains at a dry cell concentration of 200 kg cells m^–3 using a new membrane bioreactor system. The filtration rates of the cultures were stabilized by removing the microbial cake on the filter with periodic back flows of the fermentation gas through the filter. For instance, the filtration flux of 0.023 m³m^–2h^–1 was maintained for 30 h with the periodic cleaning of the filter, whereas it decreased at a half time of 2 h without the cleaning. Ethanol productivity, ethanol concentration and filtration flux attained were: 68.7 kg/(m³ · h), 62.7 kg/m³ and 0.029 m³m^–2h^–1 for Saccharomyces carlsbergensis, LAM1068, the respective values for Zymomonas mobilis, ZM4, were: 93.7, 33 5 and 0.074.     

Purification and characterisation of the extracellular maltase ofPaecilomyces varioti CMI 16196

Summary The extracellular maltase of the fungusPaecilomyces varioti CMI 16196 was purified by acetone precipitation and Sephadex G-150 gel filtration. The enzyme functioned optimally at pH 3.5–4.0 and 60°C. The maltase hydrolysed maltose and maltotriose preferentially but was inactive on both the aryl-and alkyl--D-glucosides. The Km for maltose was 1.31 mM. End product configuration was maintained and transferase activity was observed.     

Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH,NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8–9 U mg^?1 protein (µmoles mg^?1 min^?1) for NADPH and 3–4 U mg^?1 protein for NADH formation with reduced methylviologen (MV⁺⁺) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg^?1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.

The K_m-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.

Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg^?1 protein of an NADP⁺-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV⁺⁺ regeneration. The regeneration of MV⁺⁺ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg^?1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)⁺ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.

Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (D_s-(+)-isocitrate), L_s-(-)-isocitrate and 6-phosphogluconate.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

Organogenesis in cell cultures of leafy spurge (Euphorbiaceae) accessions from Europe and North America

Plants were regenerated from leafy spurge (Euphorbia esula L.) cell suspensions obtained from stem callus. A North Dakota accession was highly regenerable, but two accessions from Oregon and Austria formed only a few plantlets. Organogenesis occurred in media without growth regulators, under fluorescent lights (30 to 90 E m^–2 s^–1, 14 h photoperiod). Organogenesis was greatest in larger size clumps subcultured during maximum cell growth into media containing a reduced:oxidized nitrogen ratio of 33:67. Roots formed first and some clumps produced shoots. Organogenic suspension cultures also were initiated from hypocotyl and root segments of germinated seedlings, directly in liquid medium. Plantlets of the North Dakota accession formed in vitro adapted to greenhouse conditions. They were phenotypically similar to the parent plants.     

Production of terpenes by differentiated shoot cultures of Mentha citrata transformed with Agrobacterium tumefaciens T37

Crown gall initiation on Mentha × piperita var. citrata (Ehrh.) Briq. (mint) was investigated using a range of wild type and mutant strains of Agrobacterium tumefaciens. Axenic transformed shoot cultures of Mentha citrata were established on plant stems inoculated with the nopaline strain T37 of Agrobacterium tumefaciens. The presence of T-DNA in the transformed tissues and the absence of bacterial contamination was established by Southern Blot hybridisation, using ³²P labelled fragments of the T-DNA and virulence region of the Ti plasmid as probes. The shoot cultures synthesised a mint oil fraction which contained the major terpenes characteristic of the parent plant in quantities similar to those found in intact tissue. Oil glands were observed to be present on the leaves of the transformed culture using scanning electron microscopy.Abbreviations aux 1 Tryptophan monoxygenase - aux 2 Indoleacetamide hydrolase - ipt Isopentenyl transferase - tzs Trans-Zeatin secretion locus - 2,4-D 2,4-Dichloro phenoxyacetic acid - oct Octopine - nop Nopaline - suc L,L-succinamopine - T-DNA transferred DNA     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

Cytldlne Cyclic (3′, 5′) Monophosphate: Cyclic Nucleotide With a Non-Characteristic Ribose Ring Conformation

Abstract

Cytidine 3′,-5′-cyclic phosphate (cCMP) occurs in nature and has growth stimulatory activity on L-1210 cells. The initiation of cell growth by cCMP, under conditions where CAMP, cGMP and cUMP delay the onset of proliferation suggests that cCMP may play a regulatory role in the cell metabolism. It has been reported that in 3′,5′-cyclic nucleotides, the phosphate ring fused to the furanose ring resuicts the conformation of the furanose ring to the twist form C(3′) endo C(4′) exo (³T₄), in contrast to the C(2′) endo C(3′) endo (²T₃) and C(3′) endo C(2′) exo (³T₂) twist forms normally found in nucleotides and nucleosides. We have carried out an accurate crystal structure of cCMP and found that the furanose ring in cCMP has the C(3′) endo C(2′) exo conformation (³T₂), with a pseudo rotation amplitude (P) of 44° and phase angle τ_m of 12°. cCMP is in low anti conformation (X_CN = 15.4°) and O(5′) has the fixed g conformation. The phosphate ring is constrained to the chair conformation, as in other cyclic nucleotides. The two exocyclic P-O bond distances are short (1.489, 1.476Å) and the ring angle at N(3) is large (125.2°) suggesting that the molecule in the solid state is a zwitterion with a plus charge on N(3). The crystals are hydrated and highly unstable. The three water molecules are highly disordered in ten locations. The crystals of cCMP 3H₂O are hexagonal, a = 16.294(3), b = c = 11.099(4)Å, space group P6₁, final R value is 0.067 for 1620 reflections 230.     

A negative selection scheme for tobacco protoplast-derived cells expressing the T-DNA gene 2

The amido hydrolase encoded by the T-DNA gene 2 catalyzes the conversion of indole-acetamide, -naphthalene acetamide, and other substrate analogues into the corresponding auxins. As a result, only gene 2-expressing protoplast-derived tobacco cells can grow in medium containing low concentrations (0.2–1 M) of -naphthalene acetamide as auxin precursor. However, in a mixture of SR1 and SR1, gene 2 ⁺ protoplast-derived cells, cross-feeding occurs and consequently no positive selection for gene 2 is obtained. A 100-times higher concentration of -naphthalene acetamide (between 30 and 300 M) provides a negative selection scheme. Only the tobacco cells expressing gene 2 are sensitive to the high naphthalene acetamide concentration and cannot grow to colonies, while cells lacking the gene 2 product regenerate calli even in mixed gene 2 ⁺ and gene 2 ^– cell populations. Thus, gene 2 might provide a unique biochemically defined marker to investigate mutations and gene inactivation.     

P1-(Thymidine 5′-)P1-amino-triphosphate: Synthesis,Hydrolysis and Reaction with Cytidine in Alkali1

Abstract

The title compound 1 is prepared from thymidine 5′-phos-phorodiamidate (2) and inorganic pyrophosphate (3) in anhydrous DMF, at 30–32°C. The products of alkaline hydrolysis of 1, at room temperature, are: thymidine 5′-phosphoramidate (4), thymidine 3′-phosphoramidate (8) and thymidine (9) as well as 3 and inorganic trimetaphosphate (10). In 1 N NH₄OH, 1 reacts with cytidine (15) to form cytidylyl-/2^T(3′)-5′/-thymidine (16) and a mixture of cytidine 2′,3′-cyclic phosphate (17) and 9.     

Expression of interferon genes in murine macrophages: Possible role of endogenous interferon in the modulation of cell differentiation

The expression of interferon (IFN)- gene was studied in mouse peritoneal macrophages (PM) harvested from normal mice (lps ⁿ ) or LPS-hyporesponsive mice (lps ^d ). A strong direct correlation between the LPS response of PM and their capacity to expressing basal levels of IFN was found. The results suggest that the constitutive expression of IFN- gene can play an important role not only in the resistance to viral infection but also in the modulation of cell differentiation.