A cold acclimation protein with refolding activity on frozen denatured enzymes

We found that a cold acclimation protein from an ice-nucleating bacterium, Patoea ananas KUIN-3, has refolding activity on frozen denatured protein. Based on a SDS-PAGE analysis, we confirmed that the cold shock-treated cells of strain KUIN-3 could produce some cold acclimation proteins that inhibit their syntheses by the addition of chloramphenicol during the cold acclimation. Among such proteins, Hsc25 had refolding activity similar to GroELS. Hsc25 was purified to apparent homogeneity by (NH4)2SO4 precipitation and some chromatographies. The purified Hsc25 was composed of 8 subunits of 25,000 each with a molecular mass of 200,000 and had refolding activity against denatured enzymes, which were denatured by heat-treatment at 100 degrees C, cryopreservation at -20 degrees C, or guanidine hydrochloride, in a manner similar to GroELS. The N-terminal sequence of Hsc25 was Met-Arg-Ala-Ser-Thr-Tyr-His-Ala-Ala-Arg-. Furthermore, Hsc25 had a high level of activity at low temperature (12 degrees C). Also, the dissociation constants, KD (M) as the binding specificity for enolase, mutarotase, isocitrate dehydrogenase, and lactate dehydrogenase were 1.82x10(-10), 4.35x10(-9), 8.98x10(-12), and 3.05x10(-11), respectively. The affinity of Hsc25 for frozen danatured enzymes was higher than the affinity for heat denatured enzymes when compared with the affinity of GroEL. These results are the first report on the characterization of a purified chaperon that was induced by cold acclimation.     

Cinnamtannin A2, a Tetrameric Procyanidin,Increases GLP-1 and Insulin Secretion in Mice

The ice-nucleating bacterium, Pantoea agglomerans IFO12686, induces the cryoptotective protein (CRP) by cold acclimation at 12°C. The CRP was purified to apparent homogeneity by various chromatographies. We found that the purified CRP was a monomer of approximately 29,000 according to gel filtration chromatography and SDS-PAGE, and was a heat-stable protein. The CRP could protect freeze-labile enzymes, lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH) and isocitrate dehydrogenase (iCDH), against freezing-thawing denaturation. The activity of the CRP was about 3.5×10⁴ times more effective than bovine serum albumin (BSA) and 2×10⁶ times than COR26 from the ice-nucleating bacterium Pseudomonas fluorescens KUIN-1. We confirmed that the CRP was a novel protein, as judged by the a different molecule mass from the already-known cryoprotectants, and has an extremely high cryoprotective activity.     

Characterization of Odorous Compounds in Rotten Blue-green Algae

An ice-nucleating bacterium, strain KUIN-1, was isolated from the leaves of field beans (Phaseolus vulgaris L.). Strain KUIN-1 was identified as Pseudomonas fluorescens from its taxonomical characteristics. Ice-nucleating activity was obtained when strain KUIN-1 was cultured aerobically in a medium containing Koser citrate broth (pH 7.0) for 24 hr at 18°C. The ice- nucleating activity did not appear until the bacterial cell concentration reached 10⁷ to 10⁸/ml. Nucleation at — 3.0°C was detected in suspensions (1.8 × 10⁹ cells/ml) of cells that had been grown on the medium containing Koser citrate broth. Strain KUIN-1 produced a lower nucleation frequency (i.e. the number of ice nuclei/cell) than did ice-nucleating Pseudomonas syringae No. 31 suspensions, particularly at temperatures above — 5°C. The nucleation frequency of strain KUIN- 1-suspensions was similar to that obtained for an ice-nucleating Erwinia herbicola No. 26 at — 5°C.     

Isolation and Characterization of a Novel Ice-nucleating Bacterium,Pseudomonas sp. KUIN-4, Which Has Stable Activity in Acidic Solution

A novel ice-nucleating bacterium, KUIN-4 was isolated from a cherry leaf, which was unsusceptible to frost injury. Strain KUIN-4 was identified as Pseudomonas sp. from its characteristics and taxonomies; the optimum temperature and pH for its growth were 18°C and 4.5, respectively. When strain KUIN-4 was cultured aerobically in CYE medium (pH 4.5) for 48 h at 18°C, the ice-nucleating activities of strain KUIN-4 cells and culture broth (extracellular ice-nucleating matter) after removal of the cell were obtained. The ice-nucleating temperature, T₅₀ (°C), was indicated to be –2.8°C in cell suspensions (4.25 x 10⁷ cell/ml) of strain KUIN-4. Also, it had become apparent that the ice-nucleating activity involved class A, B, and C structures as judged by its freezing difference spectrum in D₂O versus H₂O. The ice-nucleating activity of this strain as well as other ice-nucleating bacteria was significantly decreased by heat treatment (40°C, 30 min). The ice-nucleating activity from this strain had unique features, which was stable under acidic conditions (pH 3.5–5.0) and was weakly inhibited by denaturants and protein-modifying reagents.     

Identification of a Compound in Chamaecyparis taiwanensis Inhibiting the Ice-nucleating Activity of Pseudomonas fluorescens KUIN-1

Inactivation of the ice-nucleating activity of Pseudomonas fluorescens KUIN-1 by compounds in the leaves from coniferous trees were investigated, and the inactivated material was identified. Intact cells of the strain KUIN-1 and the acetone or methanol extracts of leaves of various coniferous trees were allowed to react for 30 min at 18°C. Antinucleation compounds were obtained from Chamaecyparis taiwanensis. When the acetone extract from the leaves of coniferous trees was added to the cell suspension (about 10⁶ cells/ml) in 50 mM potassium phosphate buffer (pH 7.0), the ice nucleating temperature, T₅₀, was significantly decreased (T₅₀<-5°C). This inhibitor was isolated by using TLC, then identified as hinokitiol based on UV-VIS, IR, and mass spectral data. When intact cells of the strain KUIN-1 were incubated with hinokitiol, limonene, and α-pinene of the principal constituent of the leaves of coniferous trees in 50 mM potassium phosphate buffer (pH 7.0), the ice-nucleating activity decreased, but not in α-terpinene. Furthermore, the ice-nucleating activities from other ice-nucleating bacteria also decreased in the presence of hinokitiol. This inhibition was proportional to the concentration of hinokitinol. The pH and thermal stabilities of the ice-nucleating activity of the cells were changed by the addition of hinokitiol (10 mM).     

Preparation of recombinant human thymic stromal lymphopoietin protein

Human thymic stromal lymphopoietin (hTSLP) protein plays a central role in inflammation. Characterizing properties of hTSLP requires a recombinant overexpression system that produces correctly folded, active hTSLP. In this report, an efficient overexpression system for the production of hTSLP was developed. We constructed expression plasmids of the full-length hTslp gene with or without the signal peptide and transformed the plasmids into Escherichia coli. The design of the recombinant proteins included an N-terminal His-tag, which facilitated purification. An affinity gradient elution method was used to improve recovery and concentration levels of denatured hTSLP, with 90% purity observed following affinity chromatography. Refolding of the denatured hTSLP was tested using four different protein refolding approaches. The optimal refolding conditions involved stepwise buffer exchanges to reduce the urea concentration from 4 to 0?M in 50?mM Tris (pH 8.0), 1?mM EDTA, 50?mM NaCl, 10% glycerol, 400?mM L-Arg, 0.2?mM oxidized glutathione, and 2?mM reduced glutathione. The activity of the refolded recombinant hTSLP protein was measured by an ELISA assay. Interestingly, the presence of N-terminal signal peptide inhibited the overexpression of hTSLP in E. coli. The amount of recombinant hTSLP protein purified reached a level of 2.52?×?10^?3?mg/L.     

Determination of the Chemical Form of Fluorine in Tea Infusions by 19F-NMR

A novel marine ice-nucleating bacterium, KUIN-5, was isolated from a marine algae, Monostroma latissum. Strain KUIN-5 was identified as a Pseudomonas sp. from its characteristics and taxonomies; the optimum temperature and pH for its growth were 25°C and 6.0, respectively. When strain KUIN-5 was aerobically cultured in Carlucci-Pramer medium (pH 6.0) for 50 h at 25°C, the highest ice-nucleating activity of the cells among the media for marine bacteria was obtained, and the ice-nucleating temperature, T₅₀, was indicated to be ? 3.2°C. Also, the optimum concentration of NaCl for the growth in this medium, which was prepared with distilled water instead of seawater, was 2.0% (w/v) and then the ice-nucleating activity was inversely proportional to the NaCl concentration. Moreover, when strain KUIN-5 was cultured in Davis medium under optimum conditions, it produced insoluble polysaccharide (IPS) in the culture. The maximum amount of IPS production by strain KUIN-5 was 84.5 mg/ml of medium under optimum conditions. Therefore, this IPS was isolated and could be identified as cellulose, based on TLC or HPLC of the acid hydrolysate, and GC-MS of the acetylated polyalcohol prepared by periodate oxidation and Smith degradation of this polysaccharide. This is the first report of cellulose production by a marine ice-nucleating bacterium.     

Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry

A mechanically transmissible virus obtained from symptomless plants of a red raspberry selection imported into Scotland from Quebec, Canada was indistinguishable serologically from a cherry isolate of cherry rasp leaf virus (CRLV). The raspberry isolate, CRLV-R, was graft transmitted to several virus indicator species and cultivars of Rubus without inducing noticeable symptoms. In Chenopodium quinoa sap, CRLV-R lost infectivity after dilution to 10^-5 or heating for 10 min at 60°C but was infective after 16 days (the longest period tested) at 18°, 4° or - 15°C. The virus particles are isometric, c. 28 nm in diameter, and were purified with difficulty from infected C. murale and C. quinoa plants. The particles comprise two nucleoprotein components with sedimentation coefficients of 89 and 115 S and are prone to aggregate during purification. When centrifuged to equilibrium in CS₂SO₄ solution, purified virus preparations formed two major components with p= 1·28 and 1·36 g/cm³. Virus particles contained two RNA species which, when denatured in glyoxal and electrophoresed in agarose gels, had estimated mol. wt of 2·56 × 10⁶ (RNA-1) and 1·26 × 10⁶ (RNA–2). Infectivity of CRLV-R RNA was abolished by treatment with proteinase K, suggesting that the RNA is linked to protein necessary for infectivity; RNA molecules contained polyadenylate. In reticulocyte lysates, CRLV-R RNA stimulated the incorporation of ³H-leucine, mainly into two polypeptides of estimated mol. wt 200 000 and 102 000. When electrophoresed in polyacrylamide gels, protein obtained from CRLV-R particles purified by centrifugation to equilibrium in Cs₂SO₄ separated into three bands with estimated mol. wt 26 000 , 23 000 and 21 000.     

Konjac-mannanase from the Tubers of Amorphophallus konjac C. Koch

A crude enzyme preparation hydrolyzing konjac mannan was extracted from germinating konjac tubers, and purified by chromatography with DEAE-cellulose and alkali-swollen cellulose, and by gel-filtration on Sephadex G-100. The purified enzyme preparation showed optimal activity at pH 4.7, optimum temperature at 40°C. It was considerably stable at pH’s between 4.0 and 8.0, but inactivated rapidly by temperaters above 50°C. Hydrolysis of the mannan by this enzyme proceeded by typical random mechanism, and the rate was in agreement with an empirical equation, p=0.43 E^0.77 to^0.5. As the Km and V_max values for mannan, 7.14×10^-2(%)and 23.8×10^-3 (ΔOD_500nm) were obtained, respectively.     

The role of the photosynthetic apparatus in cold acclimation of <Emphasis Type="Italic">Lolium multiflorum</Emphasis>. Characteristics of novel genotypes low-sensitive to PSII over-reduction

During cold acclimation by higher plants, temperature perception via changes in redox state of Photosystem II (PSII) and subsequent acclimation of the photosynthetic apparatus to cold is very important for achieving freezing tolerance. These properties were studied in two groups (A and B) of the same backcross 3 (BC₃) progeny derived from a triploid hybrid of Festuca pratensis (2×) × Lolium multiflorum (4×) backcrossed three times onto diploid L. multiflorum cultivars. Leaves of Group A plants formed at 20°C at medium-low light were unable to acclimate their photosynthetic apparatus to cold. Compared to Group B, the Group A plants were also more frost sensitive. This acclimation ability correlated with the freezing tolerance of the plants. However, leaves of the same Group A plants developed at 20°C, but under higher-light conditions had increased ability to acclimate their photosynthetic apparatus to cold. It was concluded that Group A plants may have impaired PSII temperature perception, and this then resulted in their poor capability to cold acclimate.     

Equilibrium studies on neutral red-DNA binding.

Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat-denatured, sonicated, calf thymus DNA in a 0.2M ionic strength buffer containing Tris–sodium acetate–potassium chloride at 25°C. The pK_A of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5–8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 10³ M^?1 and 0.136 × 10³ M^?1, respectively, for native DNA and 7.48 × 10³ M^?1 and 0.938 × 10³ M^?1, respectively, for denatured DNA. The pK_A value of the neutral red–DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms.     

Differential in vitro inhibition of polyphenoloxidase from a wild edible mushroom Lactarius salmonicolor

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K_M and V_Max values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 × 10^{? 3} M & 0.723 EU/mL and 9.465 × 10^{? 3} M & 0.722 EU/mL, respectively.

Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5–11.0 and 5–50°C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC₅₀ values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, β-mercaptoethanol and syringic acid were determined as 9.1 × 10^{? 4}, 2.3 × 10^{? 4} M, 1.5 × 10^{? 4} M, 3.8 × 10^{? 7} M, 1.2 × 10^{? 4} M, 4.9 × 10^{? 4} M, and 4 × 10^{? 4} M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.     

Identification and properties of steroid-binding proteins in nesting Chelonia mydas plasma

We report for the first time the presence of a sex steroid-binding protein in the plasma of green sea turtles Chelonia mydas, which provides an insight into reproductive status. A high affinity, low capacity sex hormone steroid-binding protein was identified in nesting C. mydas and its thermal profile was established. In nesting C. mydas testosterone and oestradiol bind at 4°C with high affinity (K _a = 1.49 ± 0.09 × 10⁹ M⁻¹; 0.17 ± 0.02 × 10⁷ M⁻¹) and low binding capacity (B _max = 3.24 ± 0.84 × 10⁻⁵ M; 0.33 ± 0.06 × 10⁻⁴ M). The binding affinity and capacity of testosterone at 23 and 36°C, respectively were similar to those determined at 4°C. However, oestradiol showed no binding activity at 36°C. With competition studies we showed that oestradiol and oestrone do not compete for binding sites. Furthermore, in nesting C. mydas plasma no high-affinity binding was observed for adrenocortical steroids (cortisol and corticosterone) and progesterone. Our results indicate that in nesting C. mydas plasma temperature has a minimal effect on the high-affinity binding of testosterone to sex steroid-binding protein, however, the high affinity binding of oestradiol to sex steroid-binding protein is abolished at a hypothetically high (36°C) sea/ambient/body temperature. This suggests that at high core body temperatures most of the oestradiol becomes biologically available to the tissues rather than remaining bound to a high-affinity carrier.     

牛小脑肌醇磷脂激酶PI(4)K高产率纯化与特征

对牛小脑膜区肌醇磷脂激酶进行了11 500倍纯化,过程包括：TritonX-100抽提,硫酸铵沉淀,阳离子交换层析(phosphocellulose),亲和层析(Heparin Sepharose CL-6B)和阴离子交换层析(DEAE10,FPLC)等.纯化程度可达95％以上,对SDS-PAGE电泳结果进行扫描分析测其分子质量为56 ku.纯化的肌醇磷脂激酶的特异活性为450 nmol/mg·min, 动力学性质表现为ATP的表观K_m值为7.9×10^-7 mol/L,PI的表观K_m值为6.6×10^-7 mol/L. 腺嘌呤核苷是该酶的有效抑制剂,3.5×10^-7 mol/L腺嘌呤核苷可使该酶活力降低约50％,而TritonX-100对该酶活力具有刺激作用,0.5% TritonX-100可使该酶表现为最高活力.     

Purification and properties of a nicotinamide adenine dinucleotide-linked cyclohexanol dehydrogenase from aNocardia species

An inducible pyridine nucleotide-linked cyclohexanol dehydrogenase activity was present in crude extracts from aNocardia species following growth on cyclohexane. The enzyme was purified 126-fold by affinity chromatography and has an oligomeric molecular weight of 145,000 ±5,000. There was an absolute requirement for NAD for activity and the products of the dehydrogenase reaction were stoichiometric amounts of NADH and cyclohexanone. The enzyme had a broad specificity for secondary alcohols including straight-chain secondary alcohols, cyclic and substituted cyclic alcohols, and cyclohexane diols. The apparentK _m values for cyclohexanol and NAD were 3.7×10⁻⁵ M and 2.4×10⁻⁵ M, respectively, and the optimal pH for cyclohexanol oxidation was 10.5. The enzyme was heat sensitive, losing about 50% activity after a 1-min incubation at 45°C. Enzyme activity was completely inhibited by the thiol agent,p-chloromercuribenzoate but not by metal chelating agents.     

25-Hydroxycholecalciferol-binding protein: Partial purification from rat duodenal mucosa cells

The 25-hydroxycholecalciferol-binding protein has been partially purified (purification factor: 37) from rat duodenal cytosol, using chromatographic procedures on gel and ionic exchange resin. This partially purified protein bound 25-hydroxycholecalciferol with high affinity (K_D = 5.7 × 10^?9M) and low binding capacity (23 × 10^?12 mole/mg of protein. Using a rabbit antiserum obtained against such partially purified protein, we demonstrated that 25-hydroxycholecalciferol cytosolic binder and 25-hydroxycholecalciferol plasmatic binding share common antigenic sites.     

A Combined Refolding Technique for Recombinant Human Interferon-γ Inclusion Bodies by Ion-exchange Chromatography with a Urea Gradient

Summary A refolding strategy was described for on-column refolding of recombinant human interferon-γ (rhIFN-γ) inclusion bodies by ion-exchange chromatography (IEC). The rhIFN-γ was expressed in E. colias inclusion bodies. Triton X-100 was used first to wash the rhIFN-γ inclusion bodies before chromatographic refolding. The refolding process was performed by gradually decreasing the concentration of urea in the column after the denatured rhIFN-γ protein had bound onto the ion-exchange gel SP-Sepharose Fast Flow. The refolding and purification process for the denatured rhIFN-γ was carried through simultaneously and the purity of the refolded rhIFN-γ was up to 95%. The effects of protein loading, flow rate, urea gradient length and final urea concentration on the refolding were investigated in detail. Under the optimum conditions, the specific activity of rhIFN-γ was up to 7.5 × 10⁵ IU mg⁻¹and active protein recovery was up to 54%.     

Effects of temperature, pH and additives on the activity of tannase produced by a co-culture of Rhizopus oryzae and Aspergillus foetidus

Summary Tannase was produced by modified solid-state fermentation (MSSF) of tannin rich substrates by a co-culture of the two filamentous fungi, Rhizopus oryzae and Aspergillus foetidus. The enzyme thus produced was then partially purified by solvent precipitation and DEAE-Sephadex column chromatography. A study on the effects of temperature and pH was made on the activity of tannase so purified. The optimum values of incubation time, reaction temperature and pH for tannase activity were 5 min, 40 °C and 5.0 respectively. The half-life period thermal stability and kinetic constants (K _m 0.21 mM, V _max 4.9×10⁻² M min^-1 at 40 °C) of this tannase were determined and the effects of different metal ions, surfactants, chelators, denaturants and inhibitors on the enzyme activity were also studied.     

Refolding of recombinant human interferon ��-2a from Escherichia coli by urea gradient size exclusion chromatography

Protein refolding is still a puzzle in the production of recombinant proteins expressed as inclusion bodies (IBs) in Escherichia coli. Gradient size exclusion chromatography (SEC) is a recently developed method for refolding of recombinant proteins in IBs. In this study, we used a decreasing urea gradient SEC for the refolding of recombinant human interferon ??-2a (rhIFN??-2a) which was overexpressed as IBs in E. coli. In chromatographic process, the denatured rhIFN??-2a would pass along the 8.0?C3.0 M urea gradient and refold gradually. Several operating conditions, such as final concentration of urea along the column, gradient length, the ratio of reduced to oxidized glutathione and flow rate were investigated, respectively. Under the optimum conditions, 1.2 × 10⁸ IU/mg of specific activity and 82% mass recovery were obtained from the loaded 10 ml of 1.75 mg/ml denatured protein, and rhIFN??-2a was also purified during this process with the purity of higher than 92%. Compared with dilution method, urea gradient SEC was more efficient for the rhIFN??-2a refolding in terms of specific activity and mass recovery.     

Effect of latitude and acclimation on the lethal temperatures of the peach‐potato aphid Myzus persicae

• 1 Aphids, similar to all insects, are ectothermic and, consequently, are greatly affected by environmental conditions. The peach potato aphid Myzus persicae (Sulzer) has a global distribution, although it is not known whether populations display regional adaptations to distinct climatic zones along its distribution and vary in their ability to withstand and acclimate to temperature extremes. In the present study, lethal temperatures were measured in nine anholocyclic clones of M. persicae collected along a latitudinal cline of its European distribution from Sweden to Spain. The effects of collection origin and intra‐ and intergenerational acclimation on cold and heat tolerance, as determined by upper and lower lethal temperatures (ULT₅₀ and LLT₅₀, respectively), were investigated.
• 2 Lethal temperatures of M. persicae were shown to be plastic and could be altered after acclimation over just one generation. Lower lethal temperatures were significantly depressed in eight of nine clones after acclimation for one generation at 10°C (range: ?13.3 to ?16.2°C) and raised after acclimation at 25°C (range: ?10.7 to ?11.6°C) compared with constant 20°C (range: ?11.9 to ?12.9°C). Upper lethal temperatures were less plastic, although significantly increased after one generation at 25°C (range: 41.8–42.4°C) and in five of nine clones after acclimation at 10°C. There was no evidence of intergenerational acclimation over three generations.
• 3 Thermal tolerance ranges were expanded after acclimation at 10 and 25°C compared with constant 20°C, resulting in aphids reared at 10°C surviving over a temperature range that was approximately 2–6°C greater than those reared at 25°C.
• 4 There was no clear relationship between lethal temperatures and latitude. Large scale mixing of clones may occur across Europe, thus limiting local adaption in thermal tolerance. Clonal type, as identified by microsatellite analysis, did show a relationship with thermal tolerance, notably with Type O clones being the most thermal tolerant. Clonal types may respond independently to climate change, affecting the relative proportions of clones within populations, with consequent implications for biodiversity and agriculture.