Synthesis of thermo-sensitive polyacrylamide derivatives for affinity precipitation and its application in purification of lysozyme

The thermo-sensitive N-alkyl substituted polyacrylamide polymer was synthesized by radical polymerization and its lower critical solution temperature (LCST) was controlled to be 28 °C. The thermo-sensitive recovery of polymer was over 95% in the presence of 0.05 M NaClO₄. Cibacron Blue F3GA was covalently immobilized onto the polymer via the nucleophilic reaction between the active chlorine atom of its triazine ring and the hydroxyl group of the polymer. The ligands density was 30 μmol g⁻¹ polymer. The adsorption capacity of lysozyme on the polymer was 3.4 mg g⁻¹polymer in affinity precipitation process. And over 90% of adsorbed lysozyme was eluted by 0.5 M KSCN at pH 8.0. When the affinity polymer was applied in the purification of lysozyme from egg white, the purification factor was 28 and lysozyme yield was 80% or so.     

Development response of Spodoptera exigua to eight constant temperatures: Linear and nonlinear modeling

Temperature-dependent development of Spodoptera exigua (Hübner) were evaluated at eight constant temperatures of 12, 15, 20, 25, 30, 33, 34 and 36 °C with a variation of 0.5 °C on sugar beet leaves. No development occurred at 12 °C and 36 °C. Total developmental time varied from 120.50 days at 15 °C to 14.50 days at 33 °C. As temperature increased from 15 °C to 33 °C, developmental rate (1/developmental time) of S. exigua increased but declined at 34 °C. The lower temperature threshold (T_min) was estimated to be 12.98 °C and 12.45 °C, and the thermal constant (K) was 294.99 DD and 311.76 DD, using the traditional and Ikemoto–Takai linear models, respectively. The slopes of the Ikemoto–Takai linear model for different immature stages were different, violating the assumption of rate isomorphy. Data were fitted to three nonlinear models to predict the developmental rate and estimate the critical temperatures. The T_min values estimated by Lactin-2 (12.90 °C) and SSI (13.35 °C) were higher than the value estimated by Briere-2 (8.67 °C). The estimated fastest development temperatures (T_fast) by the Briere-2, Lactin-2 and SSI models for overall immature stages development of S. exigua were 33.4 °C, 33.9 °C and 32.4 °C, respectively. The intrinsic optimum temperature (T_Φ) estimated from the SSI model was 28.5 °C, in which the probability of enzyme being in its native state is maximal. The upper temperature threshold (T_max) values estimated by these three nonlinear models varied from 34.00 °C to 34.69 °C. These findings on thermal requirements can be used to predict the occurrence, number of generations and population dynamics of S. exigua.     

Copper,zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692 bp with an open reading frame of 459 bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with K_m of 0.047 ± 0.008 μM and V_max of 1250 ± 24 units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80 °C with temperature optima at 20 °C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80 °C for 180 min (k_d, 6.54 ± 0.17 × 10⁻³ min⁻¹; t_1/2, 106.07 ± 2.68 min), and had midpoint of thermal transition (T_m) of 70.45 °C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.     

Spontaneously reduced body temperature and gasping ability as a mechanism of extreme tolerance to asphyxia in neonatal rats

The aim of the investigation was to verify our hypothesis that extreme tolerance of newborn rodents to anoxia is determined by their ability to maintain reduced body temperature and to keep on gasping.Newborn Wistar rats were used. In separate experiments we checked (1) effect of extreme thermal conditions on rectal temperature (T_re) of the newborns in their nests; (2) effect of ambient temperature (T_a) on oxygen consumption; (3) effects of controlled changes in T_re on thermoregulatory and respiratory responses to anoxia and on anoxia tolerance.In their nests rat pups controlled T_re at 32–36 °C while the T_re–T_a difference changed within a range of 1–20 °C. The lowest oxygen consumption of ∼24 ml O₂ kg⁻¹ min⁻¹ was recorded at T_a of 32 °C. Pups, exposed to anoxia at their normal T_re of 33 °C, were able to decrease T_re by another 1.7 °C and they kept on extremely slow and quiescent gasping for scheduled 25 min. In contrast, rats at T_re of 37 °C and 39 °C reached a critical phase of accelerated and shallow gasping after 14.95±0.40 min and 9.25±0.30 min, respectively.In conclusion, reduced T_re and unique gasping ability make newborn rats extremely tolerant to asphyxia.     

The discovery of novel and selective fatty acid binding protein 4 inhibitors by virtual screening and biological evaluation

Adipocyte fatty acid binding protein (AFABP, FABP4) has been proven to be a potential therapeutic target for diabetes, atherosclerosis and inflammation-related diseases. In this study, a series of new scaffolds of small molecule inhibitors of FABP4 were identified by virtual screening and were validated by a bioassay. Fifty selected compounds were tested, which led to the discovery of seven hits. Structural similarity-based searches were then performed based on the hits and led to the identification of one high affinity compound 33b (K_i = 0.29 ± 0.07 μM, ΔT_m = 8.5 °C). This compound’s effective blockade of inflammatory response was further validated by its ability to suppress pro-inflammatory cytokines induced by lipopolysaccharide (LPS) stimulation. Molecular dynamics simulation (MD) and mutagenesis studies validated key residues for its inhibitory potency and thus provide an important clue for the further development of drugs.     

Application of seaweeds for the removal of lead from aqueous solution

Ten different seaweed species were compared on the basis of lead uptake at different pH conditions. The brown seaweed, Turbinaria conoides, exhibited maximum lead uptake (at pH 4.5) and hence was selected for further studies. Sorption isotherms, obtained at different pH (4–5) and temperature (25–35 °C) conditions were fitted using Langmuir and Sips models. According to the Langmuir model, the maximum lead uptake of 439.4 mg/g was obtained at optimum pH (4.5) and temperature (30 °C). The Sips model better described the sorption isotherms with high correlation coefficients at all conditions examined. Various thermodynamic parameters such as ΔG°, ΔH° and ΔS° were calculated indicating that the present system was a spontaneous and endothermic process. Through potentiometric titrations, number of binding sites (carboxyl groups) and pK₁ were determined as 4.1 mmol/g and 4.4, respectively. The influence of co-ions (Na⁺, K⁺, Mg²⁺ and Ca²⁺) on lead uptake was well pronounced in the case of divalent ions compared to monovalent ions. The solution of 0.1 M HCl successfully eluted all lead ions from lead-loaded T. conoides biomass. The regeneration experiments revealed that the alga could be successfully reused for five cycles without any loss in lead biosorption capacity. A glass column (2 cm i.d. and 35 cm height) was used to study the continuous lead biosorption performance of T. conoides. At 25 cm (bed height), 5 ml/min (flow rate) and 100 mg/l (initial lead concentration), T. conoides exhibited lead uptake of 220.1 mg/g. The column was successfully eluted using 0.1 M HCl, with elution efficiency of 99.7%.     

Effects of rapid temperature fluctuations prior to breeding on reproductive efficiency in replacement gilts

Rapidly cooling pigs after heat stress (HS) results in a pathophysiological condition, and because rapid temperature fluctuations may be associated with reduced reproductive success in sows, it lends itself to the hypothesis that these conditions may be linked. Objectives were to determine the effects of rapid cooling on thermal response and future reproductive success in pigs. Thirty-six replacement gilts (137.8±0.9 kg BW) were estrus synchronized and then 14.1±0.4 d after estrus confirmation, pigs were exposed to thermoneutral conditions (TN; n=12; 19.7±0.9°C) for 6 h, or HS (36.3±0.5°C) for 3 h, followed by 3 h of rapid cooling (HSRC; n=12; immediate TN exposure and water dousing) or gradual cooling (HSGC; n=12; gradual decrease to TN conditions) repeated over 2 d. Vaginal (T_V) and gastrointestinal tract temperatures (T_GI) were obtained every 15 min, and blood was collected on d 1 and d 2 during the HS and recovery periods at 180 and 60 min, respectively. Pigs were bred 8.3±0.8 d after thermal treatments over 2 d. Reproductive tracts were collected and total fetus number and viability were recorded 28.0±0.8 d after insemination. HS increased T_V and T_GI (P=0.01; 0.98 °C) in HSRC and HSGC compared to TN pigs. During recovery, T_V was reduced from 15 to 105 min (P=0.01; 0.33 °C) in HSRC compared to HSGC pigs, but no overall differences in T_GI were detected (P<0.05; 39.67 °C). Rapid cooling increased (P<0.05) TNFα compared to HSGC and TN pigs during recovery-d 1 (55.2%), HS-d 2 (35.1%), and recovery-d 2 (64.9%). Viable fetuses tended to be reduced (P=0.08; 10.5%) and moribund fetuses tended to be increased (P=0.09; 159.3%) in HSRC compared to HSGC and TN pigs. In summary, rapid cooling prior to breeding may contribute to reduced fetal viability and reproductive success in pigs.     

Partial nitrification under limited dissolved oxygen conditions

Partial nitrification to nitrite is technically feasible and economically favourable, especially when wastewaters contained high ammonium concentrations or low C/N ratios. Partial nitrification can be obtained by selectively inhibiting nitrite-oxidizing bacteria (NOB) through appropriate regulation of the pH, temperature and dissolved oxygen (DO) concentrations. The effect of pH, DO levels and temperature on ammonia oxidation rate and nitrite accumulation was investigated in order to determine the optimal conditions for partial nitrification of synthetic wastewater with high ammonia concentration. The experiments performed at low DO levels to lower the total oxygen needed in the nitrification step, which means great saving in aeration. During the start-up stage pH and DO were set at 7.0–7.4 and 0.5 mg/l, respectively. The reactor was operated until complete partial nitrification was achieved. The effect of pH, DO on partial nitrification was studied, as pH was kept at 6.5, 7.5, 8.5, 9.5 and DO at 0.5±0.2, 1.5±0.2 and 2.5±0.2 mg/l, and temperature at 30 °C. The influence of temperature on k_a value was studied by keeping pH=7.5, DO=1.5 mg/l and temperature was controlled at 12, 20 and 30 °C, respectively. The results showed that partial nitrification to nitrite was steadily obtained and the optimal operational parameters were pH=7.5, DO=1.5 mg/l, T=30 °C based on ammonia oxidation rate and nitrite accumulation rate. The maximum k_a was achieved and to be 115.1×10⁻³ mg NH₄⁺–N (mg VSS h)⁻¹ under this condition.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Temperature selection by juvenile tuatara (Sphenodon punctatus) is not influenced by temperatures experienced as embryos

Most reptiles thermoregulate to achieve body temperatures needed for biological processes, such as digestion and growth. Temperatures experienced during embryogenesis may also influence post-hatching growth rate, potentially through influencing post-hatching choice of temperatures. We investigated in laboratory settings whether embryonic temperatures (constant 18 °C, 21 °C and 22 °C) influence selected body temperatures (T_sel) of juvenile tuatara (Sphenodon punctatus), providing a possible mechanism for differences in growth rates. We found that incubation temperature does not influence T_sel. Although the average daily mean T_sel was 21.6 ± 0.3 °C, we recorded individual T_sel values up to 33.5 °C in juvenile tuatara, which is higher than expected and above the panting threshold of 31–33 °C reported for adults. We found diel patterns of T_sel of juvenile tuatara, observing a general pattern of two apparent peaks and troughs per day, with T_sel being significantly lower around dawn and at 1500 h than any other time. When comparing our results with other studies on tuatara there is a remarkable consistency in mean T_sel of ~ 21 °C across tuatara of different ages, sizes and acclimatization histories. The ability of juvenile tuatara to withstand a wide range of temperatures supports their former widespread distribution throughout New Zealand and warrants further investigation into their plasticity to withstand climate warming, particularly where they have choices of habitat and the ability to thermoregulate.     

Discovery and characterization of a thermostable d-lactate dehydrogenase from Lactobacillus jensenii through genome mining

The demand on thermostable d-lactate dehydrogenases (d-LDH) has been increased for d-lactic acid production but thermostable d-DLHs with industrially applicable activity were not much explored. To identify a thermostable d-LDH, three d-LDHs from different Lactobacillus jensenii strains were screened by genome mining and then expressed in Escherichia coli. One of the three d-LDHs (d-LDH3) exhibited higher optimal reaction temperature (50 °C) than the others. The T₅₀¹⁰ value of this thermostable d-LDH3 was 48.3 °C, much higher than the T₅₀¹⁰ values of the others (42.7 and 42.9 °C) and that of a commercial d-lactate dehydrogenase (41.2 °C). The T_m values were 48.6, 45.7 and 55.7 °C for the three d-LDHs, respectively. In addition, kinetic parameter (k_cat/K_m) of d-LDH3 for pyruvate reduction was estimated to be almost 150 times higher than that for lactate oxidation at pH 8.0 and 25 °C, implying that d-lactate production from pyruvate is highly favored. These superior thermal and kinetic features would make the d-LDH3 characterized in this study a good candidate for the microbial production of d-lactate at high temperature from glucose if it is genetically introduced to lactate producing microbial.     

Disproportionate thermophysiological strain between intensively- and extensively-managed goats during summer

Goat keeping is feasible for smallholder farmers in many world regions especially those best suited for extensive management. However, summertime grazing in arid zones entails major challenges to animal thermoregulation and well-being. An experiment was conducted to evaluate the thermoregulatory performance and selected hemogramic parameters in intensively (INT) or extensively (EXT) managed goat kids (N = 14). We applied a previously established technique to evaluate body thermal state of freely ranging animals, in which contemporaneous temperatures of the core (T_c) and periphery (T_p) are chronically recorded. Animals were initially kept for 12 days under INT management. Subsequently, seven animals were transferred to a grazing pasture and gradually transitioned over a four-day acclimatization period, then kept for the last 22 days under EXT conditions. Water drinking was limited to twice daily in both groups. Excessive solar radiation-induced heat load – with daytime black globe temperatures (T_bg) often exceeding 40 °C – under EXT was primarily responsible (r² = 0.49; P < 0.05) for 0.57 and 1.72 °C rises in T_c and T_p, respectively, over INT kids. Unlike the typically biphasic pattern noticed for daily temperatures of both body sites in INT goats, that of EXT counterparts became rather polyphasic, whereby water drinking had drastic and prolonged thermolytic effect, inducing 0.40–0.41 and 0.79–1.45 °C declines in T_c and T_p, on midday and afternoon watering bouts, respectively. Despite indication for added daytime heat load, EXT goats displayed lower early morning T_c than INT. All animals exhibited hypohydration, as reflected by rises in hematocrit, serum osmolality, albumin, potassium, and sodium, being more pronounced in EXT conditions. Results emphasize the excessive thermophysiological strain facing grazing animals in arid zones during the summer.     

Blocking interaction of viral gp120 and CD4-expressing T cells by single-stranded DNA aptamers

To investigate the potential clinical application of aptamers to prevention of HIV infection, single-stranded DNA (ssDNA) aptamers specific for CD4 were developed using the systematic evolution of ligands by exponential enrichment approach and next generation sequencing. In contrast to RNA-based aptamers, the developed ssDNA aptamers were stable in human serum up to 12 h. Cell binding assays revealed that the aptamers specifically targeted CD4-expressing cells with high binding affinity (K_d = 1.59 nM), a concentration within the range required for therapeutic application. Importantly, the aptamers selectively bound CD4 on human cells and disrupted the interaction of viral gp120 to CD4 receptors, which is a prerequisite step of HIV-1 infection. Functional studies showed that the aptamer polymers significantly blocked binding of viral gp120 to CD4-expressing cells by up to 70% inhibition. These findings provide a new approach to prevent HIV-1 transmission using oligonucleotide aptamers.     

Affinity ligands for immunoglobulins based on the multicomponent Ugi reaction

This report describes a novel use of the four-component Ugi reaction to generate a solid-phase library suitable for the purification of immunoglobulins and their fragments by affinity chromatography. An aldehyde-functionalised Sepharose? solid-support constituted one component in the four-component reaction, whereas the other three components (a carboxylic acid, a primary or secondary amine and an isonitrile) were varied in a combinatorial fashion to generate a tri-substituted peptoidal scaffold structure which provides a degree of rigidity and functionality suitable for rational investigation of immunoglobulin binding. The Ugi ligand library was initially screened chromatographically against whole human IgG and its fragments (Fc and Fab) to yield a Fab-specific lead ligand based on its ability to bind Fab differentially over Fc. Preparative chromatography of IgG from human serum showed 100% of IgG was adsorbed from the 20 mg/ml crude stock and subsequently eluted with a purity of 81.0% as determined by SDS-PAGE analysis under non-optimised conditions. High purity Fab and IgG isolation was achieved from both yeast and E. coli host cell proteins according to silver-stained SDS-PAGE lane densitometry. The ligand density and spacer-arm chemistry of the immobilised ligand was optimised to define an affinity adsorbent which binds 73.06 mg IgG/ml moist gel (dynamic binding capacity at 10% breakthrough) and a static binding capacity of 16.1 ± 0.25 mg Fab/ml moist resin displaying an affinity constant K_d = (2.6 ± 0.3) × 10^?6 M. The lead candidate was modelled in silico and docked into a human Fab fragment (PDB: 1AQK) to suggest a putative binding interface to the constant CH₁-CL Fab terminal through six defined hydrogen bond interactions together with putative hydrophobic interactions.     

Effect of enzymatic and hydrothermal treatments of rapeseeds on quality of the pressed rapeseed oils: part II. Oil yield and oxidative stability

Response surface methodology was used to evaluate the quantitative effects of three independent variables: rapeseed moisture content (MC), enzymes dosage (ED) and conditioning temperature (T) on rapeseed oil yield (OY), efficiency of pressing (EP), and oxidative stability (OS). The highest OY (16.4%) and EP (42.8%) were obtained from pectolytic enzyme (0.1%) treated seeds (MC = 9%, T = 90 °C). The highest OS (12.6 h) was found for oil pressed from rapeseeds heated at 120 °C (MC = 11%), after the cellulolytic enzyme treatment. Results of OY, EP and OS determinations correlate with the predicted values calculated from the partial cubic models (PCMs) equations (R² = 0.9995, 0.9994, 0.9974 for the cellulolytic enzyme-treated oils and 0.9900, 0.9900, 0.9990 for the pectolytic enzyme-treated oils). The predicted optimum MC = 9.5% and 8.6%, ED = 0.06% and 0.1%, T = 91.2 °C and 90.1 °C resulted in OY = 15.5% and 16.5%, EP = 40.4% and 43.0% for rapeseed oils from seeds treated with cellulolytic and pectolytic enzymes. OS values (12.6 h and 11.8 h) at the optimum conditions of MC = 11.0% and 10.1%, ED = 0.04% and 0.08%, T = 120.0 °C and 119.9 °C for the cellulolytic and pectolytic enzyme-treated oils were also calculated using PCM. Moreover, scanning electron microscopy revealed structural changes in the rapeseed after enzymatic treatment.     

Ways to measure body temperature in the field

Body temperature (T_b) represents one of the key parameters in ecophysiological studies with focus on energy saving strategies. In this study we therefore comparatively evaluated the usefulness of two types of temperature-sensitive passive transponders (LifeChips and IPTT-300) and one data logger (iButton, DS1922L) mounted onto a collar to measure T_b in the field. First we tested the accuracy of all three devices in a water bath with water temperature ranging from 0 to 40 °C. Second, we evaluated the usefulness of the LifeChips and the modified iButtons for measuring T_b of small heterothermic mammals under field conditions. For this work we subcutaneously implanted 14 male edible dormice (Glis glis) with transponders, and equipped another 14 males with data loggers to simultaneously record T_b and oxygen consumption with a portable oxygen analyzer (Oxbox). In one individual we recorded T_b with both devices and analyzed recorded T_b patterns.LifeChips are able to measure temperature within the smallest range from 25 to 40 °C with an accuracy of 0.07±0.12 °C. IPTT-300 transponders measured temperature between 10 and 40 °C, but accuracy decreased considerably at values below 30 °C, with maximal deviations of nearly 7 °C. An individual calibration of each transponder is therefore needed, before using it at low T_bs. The accuracy of the data logger was comparatively good (0.12±0.25 °C) and stable over the whole temperature range tested (0–40 °C). In all three devices, the repeatability of measurements was high.LifeChip transponders as well as modified iButtons measured T_b reliably under field conditions. Simultaneous T_b-recordings in one edible dormouse with an implanted LifeChip and a collar-mounted iButton revealed that values of both measurements were closely correlated. Taken together, we conclude that implanted temperature-sensitive transponders represent an appropriate and largely non-invasive method to measure T_b also under field conditions.     

Cloning,expression, purification and characterization of an oligomeric His-tagged thermophilic esterase from Thermus thermophilus HB27

The esterase E34Tt (YP_004875.1) from Thermus thermophilus HB27 was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The gene sequence was subcloned into a T-vector, released with the restriction enzymes BamHI and HindIII, ligated to a pET-21d(+) vector, and transferred to E. coli BL21 (DE3) cells. Inducer concentration (isopropyl β-d-1-thiogalactopyranoside, IPTG) and cultivation time before and after induction were optimized. Best results were obtained by adding 0.25 mM IPTG after 8 h of cultivation and maintaining the induction during 4 extra hours. Most of the enzyme (94%) remained membrane-associated and had to be extracted with a detergent. From the membrane crude extract, the His-tagged E34Tt was purified as a dimer (71.8 kDa) in a single purification step by using metal affinity chromatography. The Rosso's model was used to optimize the reaction conditions. E34Tt-His₆ was active in a wide temperature (19.7–79.4 °C) and pH range (4.0–9.3), and maximal activity was determined at pH 6.3 and 58.2 °C, which is 10–18 °C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His₆ preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9 min at 85 °C), suggesting that E34Tt-His₆ has potential for industrial applications.     

Estimation of the core temperature control during ambient temperature changes and the influence of circadian rhythm and metabolic conditions in mice

It has been speculated that the control of core temperature is modulated by physiological demands. We could not prove the modulation because we did not have a good method to evaluate the control. In the present study, the control of core temperature in mice was assessed by exposing them to various ambient temperatures (T_a), and the influence of circadian rhythm and feeding condition was evaluated. Male ICR mice (n=20) were placed in a box where T_a was increased or decreased from 27 °C to 40 °C or to −4 °C (0.15 °C/min) at 0800 and 2000 (daytime and nighttime, respectively). Intra-abdominal temperature (T_core) was monitored by telemetry. The relationship between T_core and T_a was assessed. The range of T_a where T_core was relatively stable (range of normothermia, RNT) and T_core corresponding to the RNT median (regulated T_core) were estimated by model analysis. In fed mice, the regression slope within the RNT was smaller in the nighttime than in the daytime (0.02 and 0.06, respectively), and the regulated T_core was higher in the nighttime than in the daytime (37.5 °C and 36.0 °C, respectively). In the fasted mice, the slope remained unchanged, and the regulated T_core decreased in the nighttime (0.05 and 35.9 °C, respectively), while the slopes in the daytime became greater (0.13). Without the estimating individual thermoregulatory response such as metabolic heat production and skin vasodilation, the analysis of the T_a–T_core relationship could describe the character of the core temperature control. The present results show that the character of the system changes depending on time of day and feeding conditions.     

Cloning,purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10

A novel β-galactosidase gene (Tnap1577) from the hyperthermophilic bacterium Thermotoga naphthophila RUK-10 was cloned and expressed in Escherichia coli BL21 (DE3) cells to produce β-galactosidase. The recombinant β-galactosidase was purified in three steps: heat treatment to deactivate E. coli proteins, Ni-NTA affinity chromatography and Q-sepharose chromatography. The optimum temperatures for the hydrolysis of o-nitrophenyl-β-d-galactoside (o-NPG) and lactose with the recombinant β-galactosidase were found to be 90 °C and 70 °C, respectively. The corresponding optimum pH values were 6.8 and 5.8, respectively. The molecular mass of the enzyme was estimated to be 70 kDa by SDS-PAGE analysis. Thermostability studies showed that the half-lives of the recombinant enzyme at 75 °C, 80 °C, 85 °C and 90 °C were 10.5, 4, 1, and 0.3 h, respectively. Kinetic studies on the recombinant β-galactosidase revealed K_m values for the hydrolysis of o-NPG and lactose of 1.31 mM and 1.43 mM, respectively. These values are considerably lower than those reported for other hyperthermophilic β-galactosidases, indicating high intrinsic affinity for these substrates. The recombinant β-galactosidase from Thermotoga naphthophila RUK-10 also showed transglycosylation activity in the synthesis of alkyl galactopyranoside. This additional activity suggests the enzyme has potential for broader biotechnological applications beyond the degradation of lactose.     

Expanded bed adsorption of an alkaline lipase from Pseudomona cepacia

An extracellular lipase was isolated from Pseudomona cepacia by expanded bed adsorption on an Amberlite 410 ion-exchange resin. Enzyme characterization and hydrodynamic study of a chromatography column were done. Enzyme purification was done at three condition of expanded bed height (H): at one and half (6 cm), at two (8 cm) and at three (12 cm) times the fixed bed height (H₀ = 4 cm). The results showed that the experimental data was fitted to the Richardson and Zaki equation, and the comparison between the experimental and calculated terminal velocities showed low relative error. In enzyme purification for better condition, a purification factor of about 80 times was found at 6 cm of expanded bed height, or 1.5 times of expansion degree. Purified lipase had an optimal pH and a temperature of 8 and 37 °C, respectively.