Conjugation of α-chymotrypsin on a polymeric hydrophilic nanolayer covering magnetic nanoparticles

Magnetic nanoparticles, covered by a polymeric hydrophilic nanolayer containing reactive amino groups, were obtained via Hoffman degradation of the polyacrylamide-coated Fe₃O₄ nanoparticles synthesized by photochemical in situ polymerization, and then conjugated the model enzyme––α-chymotrypsin (CT) by use of EDC· HCl and NHS at room temperatures. The mechanism of photochemical in situ polymerization was briefly proposed in this paper. Superparamagnetic properties were retained for Fe₃O₄ after enzyme immobilization while slightly reducing the value of saturation magnetization. Crystalline structure of Fe₃O₄ after CT immobilization was consistent with that of the freshly prepared Fe₃O₄ by X-ray diffraction (XRD) analysis. The binding capacity was 69 and 61 mg enzyme/g nanogel determined by thermogravimetric (TG) analysis and by standard BCA protein assay, respectively. Specific activity of the immobilized CT was 0.93 U/(mg min), only 59.3% as that of free CT. Thermal stability of CT was improved after being bound to the amine-functionalized magnetic nanogel.     

Behaviour of water bound in bone marrow cells affected by organic solvents of different polarity

The behaviour of intracellular water affected by organic solvents of different polarity in partially dehydrated marrow cells obtained from tubular bones of broiler chickens was studied using ¹H NMR spectroscopy at 210–290 K. The ¹H NMR spectra of intracellular water include two signals which can be assigned to strongly (SAW, chemical shift of the proton resonance δ_H = 4–5 ppm) and weakly (WAW, δ_H = 1.2–1.7 ppm) associated waters which can be also divided into weakly (WBW, frozen at 250 < T < 273 K and changes in the Gibbs free energy ΔG > −0.8 kJ/mol) and strongly (SBW, unfrozen at T < 250 K, ΔG < −0.8 kJ/mol) bound intracellular waters. Solvents of different polarity such as dimethylsulfoxide-d₆ (Me₂SO-d₆), acetonitrile-d₃, and chloroform-d differently affect structure, Gibbs free energy, and molecular mobility of intracellular water. A maximal fraction of SBW in WAW and a minimal fraction of SBW in SAW are observed on absorption of acetonitrile (0.8 g/g) by cells. The opposite results are on addition of Me₂SO (0.8 g/g) which strongly changes organisation of intracellular water and enhances the freezing point depression of SBW.     

l-Dopa synthesis using tyrosinase immobilized on magnetic beads

Magnetic beads were prepared via suspension polymerization of glycidyl methacrylate (GMA) and methyl methacrylate (MMA) in the presence of ferric ions. Following polymerization, thermal co-precipitation of the Fe(III) ions in the beads with Fe(II) ions under alkaline condition resulted in encapsulation of Fe₃O₄ nano-crystals within the polymer matrix. The magnetic beads were activated with glutaraldehyde, and tyrosinase enzyme was covalently immobilized on the support via reaction of amino groups under mild conditions. The immobilized enzyme was used for the synthesis of l-Dopa (1-3,4-dihydroxy phenylalanine) which is a precursor of dopamine. The immobilized enzyme was characterized by temperature, pH, operational and storage stability experiments. Kinetic parameters, maximum velocity of the enzyme (V_max) and Michaelis–Menten constant (K_m) values were determined as 1.05 U/mg protein and 1.0 mM for 50–75 μm and 2.00 U/mg protein and 4.0 mM for 75–150 μm beads fractions, respectively. Efficiency factor and catalytic efficiency were found to be 1.39 and 0.91 for 75–150 μm beads and 0.73 and 0.75 for 50–75 μm beads fractions, respectively. The catalytic efficiency of the soluble tyrosinase was 0.37. The amounts of immobilized protein were on the 50–75 μm and 75–150 μm fractions were 2.7 and 2.8 mg protein/g magnetic beads, respectively.     

Toxicity of Carum copticum essential oil‐loaded nanogel against Sitophilus granarius and Tribolium confusum

Myristic acid–chitosan (MA‐chitosan) nanogels were synthesized with the technique of self‐assembly and loaded with Carum copticum (L.) essential oil. Scanning electron microscopy (SEM) study indicated nanogels with smooth surfaces and near spherical appearance, and according to transmission electron microscopy (TEM) image, core–shell stricter of nanogels was reported. Subsequently, fumigant toxicity of C. copticum oil and oil‐loaded nanogels was assessed against Sitophilus granarius (L.) and Tribolium confusum Jacquelin du Val. Adults were exposed to different concentrations of the oil and oil‐loaded nanogels at 27 ± 1°C and 55 ± 5% r.h. in continuous darkness. The mortality was counted after 3, 6, 10, 24 and 48 h of exposure. Mortality increased with increasing concentration levels. Fumigant toxicity of oil‐loaded nanogels was significantly more than the oil even after 48 h. According to the findings, oil‐loaded nanogels were 8.9‐ and 3.7‐fold more toxic than the oil against S. granarius and T. confusum, respectively. The persistence of oil and oil‐loaded nanogels was also investigated over period of time. Oil‐loaded nanogels lost the insecticidal effectiveness after 21 days post‐application for S. granarius and 33 days in the case of T. confusum, while the efficacy of oil decreased in the early days of application. Chemical composition of C. copticum essential oil was measured, and six components were identified. Thymol, ρ‐cymene and γ‐terpinolene were the major components of the oil. The essential oil‐loaded nanogels may have potential to be used as an alternative to synthetic insecticides for stored products protection. However, more research is necessary to improve the findings.     

Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus

Fructose-1,6-bisphosphate (FBP) aldolase, is a glycolytic enzyme that catalyzes the reversible condensation reaction of FBP to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). The aldolase gene from Aquifex aeolicus was subcloned, overexpressed in E. coli and purified to 95% homogeneity. The purified enzyme was activated by high concentrations of NH₄⁺ and low concentrations of Co²⁺. The native molecular weight of the purified FBP aldolase was identified as 67 kDa (dimer) by gel filtration chromatography. The enzyme exhibits optimum pH at 6.5 and temperature at 90 °C. Based on the kinetic characterizations, the apparent K_m was calculated to be 4.4 ± 0.07 mM, while V_max was found to be 100 ± 0.02 μM min⁻¹ mg protein⁻¹. The recombinant protein showed extreme heat stability; no activity loss was observed even at 100 °C for 2 h. In addition, the thermophilic enzyme also showed higher stability against several organic solvents viz. acetonitrile, 1,4-dioxane, and methanol. With higher stability against both heat and organic solvents than any other class II aldolase, the A. aeolicus FBP aldolase is an attractive enzyme for use as a biocatalyst for industrial applications.     

Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification

Candida rugosa lipase was immobilized on magnetic nanoparticles supported ionic liquids having different cation chain length (C₁, C₄ and C₈) and anions (Cl⁻, BF₄⁻ and PF₆⁻). Magnetic nanoparticles supported ionic liquids were obtained by covalent bonding of ionic liquids–silane on magnetic silica nanoparticles. The particles are superparamagnetic with diameter of about 55 nm. Large amount of lipase (63.89 mg/(100 mg carrier)) was loaded on the support through ionic adsorption. Activity of the immobilized lipase was examined by the catalysis of esterification between oleic acid and butanol. The activity of bound lipase was 118.3% compared to that of the native lipase. Immobilized lipase maintained 60% of its initial activity even when the temperature was up to 80 °C. In addition, immobilized lipase retained 60% of its initial activity after 8 repeated batches reaction, while no activity was detected after 6 cycles for the free enzyme.     

Water relations and CO2 exchange of the terrestrial lichen Teloschistes capensis in the Namib fog desert: Measurements during two seasons in the field and under controlled conditions

Although the coastal zone of the Central Namib Desert (Namibia) has negligible rainfall, frequent fog, dew and high air humidity support a luxurious lichen flora. Large areas of soil crust communities are dominated by the multibranched, fruticose Teloschistes capensis interspersed by a (still indeterminable) Ramalina species. In earlier communications, based on field measurements in autumn, we began the analysis of functional mechanisms that allow these lichens to exist under the special conditions of a fog desert. We have extended this work by monitoring lichen CO₂ exchange and water relations in spring and by experiments under controlled conditions.In both seasons, nocturnal hydration, by fog and/or dew, activated dark respiration of the lichens which was followed, after sunrise, by a short period of positive net photosynthesis (NP) that continued until metabolic inactivation occurred from desiccation. Dry thalli of T. capensis were able to reactivate NP through water vapour uptake alone, beginning at an air relative humidity of 82%, i.e. at a water potential of −26.3 MPa; the moisture compensation point during desiccation was at 13% thallus water content (WC, dry weight related). Optimal WC for photosynthesis was around 100%, and both species showed a large and extended suprasaturation depression of CO₂ assimilation. Light response showed “sun-plant” characteristics with saturation >1000 μmol m⁻² s⁻¹ photosynthetically active photon flux density (PPFD). However, due to rapid desiccation, the combination of light saturation with optimal WC very rarely occurred under field conditions. Light compensation point after sunrise was highly dependent on actual WC: at low hydration, it amounted to only ca. 10 μmol m⁻² s⁻¹ PPFD so that even the smallest levels of hydration could be used for carbon gain before desiccation took place again. This phenomenon was probably due to a hydration gradient in the thallus branches during transient moistening so that the outer photobiont layer was favoured in contrast to the internal mycobiont which remained dry longer and did not contribute respiratory CO₂ loss. Fully hydrated thalli had light compensation points around 50 μmol m⁻² s⁻¹ PPFD. Extended desiccation of 1–3 days had no impact on the magnitude and recovery of photosynthesis but, imposed desiccation of 10 days reduced NP in lab and field experiments and caused an extended period of recovery. “Resaturation respiration” was not detected in the field data, although it was present after experimental moistening of dry thalli.In spring, the higher fog frequency and intensity increased maximal nocturnal WC, maximal attained NP as well as integrated daily carbon income (ΣNP) compared to the autumn measurements. NP_max and ΣNP depended on maximal nocturnal WC with a saturation-type response. In terms of carbon gain both species seem to be optimally adapted to nocturnal moistening up to 160% WC and were not able to make use of higher degrees of hydration, a feature that might well influence their habitat selection.Maximal daily carbon-related ΣNP for T. capensis was 4.6 mg_C (g_C)⁻¹ day⁻¹. A rough estimate of the annual (projected) area-related carbon balance (photosynthetic income minus respiratory losses) based on published fog and dew frequencies and personal observations was 15–34 mg_C m⁻² yr⁻¹.     

Protective activity of plicatin B against human LDL oxidation induced in metal ion-dependent and -independent processes. Experimental and theoretical studies

Oxidation of low-density lipoproteins (LDL) is thought to be a major factor in the pathophysiology of atherosclerosis. Natural antioxidants have been shown to protect LDL from oxidation and to inhibit atherogenic developments in animals. Structurally related prenylated pterocarpans, erybraedin C and bitucarpin A, and the prenylchalcone plicatin B were examined for their ability to inhibit LDL oxidation in vitro. The kinetic profile of peroxidation is characterized by the lag time of oxidation (t_lag), the maximal rate of oxidation (V_max) and the maximal accumulation of oxidation products (OD_max). Specific variation of the set of kinetic parameters by antioxidants may provide important information about the mechanism of inhibitory action of a given compound. At equimolar concentrations (1 μM) the prenylated derivatives tested were found to inhibit 1 μM copper sulphate-induced oxidation of LDL (50 μg protein/ml) in accordance with the following order of activity: plicatin B>erybraedin Cbitucarpin A. Structural aspects, such as hydrogen-donating substituents, their number and arrangement in the aromatic ring moieties, and the prenyl and methoxy substituents, were investigated in order to explain the findings obtained. It is well known that the antioxidant activity of flavonoids is believed to be caused by a combination of transition metal chelation and free-radical-scavenging activities. To investigate these differences we comparatively studied the protective mechanism of plicatin B in copper-dependent or -independent LDL oxidation. The latter was mediated by 2,2’-azo-bis-(2-amidinopropane) dihydrochloride (ABAP). We measured the formation of conjugated dienes (OD_234 nm). Plicatin B (0.2-1.5 μM) delayed the Cu²⁺ (1 μM) promoted oxidation as conjugate diene formation (t_lag) of the LDL by 45.2-123.5 min and reduced V_max by 0.46-0.29 μM/min. In the ABAP (0.2 mM) promoted LDL oxidation t_lag increased by 67.2-110.2 min through plicatin B (0.5-2.5 μM). In experiments in which Cu²⁺ concentrations increased (0.5 - 3 μM) and the amount of plicatin B (1 μM) was maintained constant, a significant decrease in t_lag and an increase in V_max was observed. In this study plicatin B appeared to exhibit a mixed mechanism, interfering with the formation of the radicals by chelating copper involved in the initiation/propagation reaction, but also by scavenging free hydroperoxyl radicals resulting from ABAP thermolysis. In addition, theoretical analysis indicated that plicatin B preferentially established the chelating complex with Cu²⁺, because its affinity value is notably higher (by a factor of 5) than that for Cu⁺.     

Binding of estrogens by α-fetoprotein in rat amniotic fluid

Amniotic fluid from 15–17-day rat fetuses bound estrone and 17β-estradiol specifically. Related steroids such as estriol, 6-ketoestradiol, 17α-estradiol and testosterone were not bound to any significant extent. The apparent K_a for 17β-estradiol was 2.6·10⁸ M⁻ at 4°C; 6 nmoles of 17β-estradiol were bound per ml of amniotic fluid. The binding component appears to be α-fetoprotein in that it migrates as an α₁-globulin upon polyacrylamide gel electrophoresis and has an isoelectric pH of 4.7 as determined by isoelectric focusing. Furthermore, binding activity was precipitated by antiserum which was shown by immuno-electrophoresis to be specific for α-fetoprotein. Binding activity, partially purified by isoelectric focusing of amniotic fluid, was associated with one of two bands seen by polyacrylamide gel electrophoresis. This band migrated as an α₁-globulin.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

Immobilization of soybean (Glycine max) urease on alginate and chitosan beads showing improved stability: Analytical applications

The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol⁻¹, 5.02 kcal mol⁻¹, 6.45 kcal mol⁻¹ for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t_1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in K_m value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of V_max for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, 2.65 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein and 2.85 × 10² μmol NH₃ min⁻¹ mg⁻¹ protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t_1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer^®. The immobilization studies have a potential role in haemodialysis machines.     

Methane emission by goats consuming diets with different levels of condensed tannins from lespedeza

Twenty-four yearling Boer × Spanish wethers (7/8 Boer; initial body weight (BW) of 34.1 ± 1.02 kg) were used to determine effects on methane (CH₄) emission of dietary levels of a condensed tannin (CT)-containing forage, Kobe lespedeza (Lespedeza striata; K), and a forage very low in CT, sorghum-sudangrass (Sorghum bicolor; G). Treatments were dietary K levels (dry matter (DM) basis) of 1.00, 0.67, 0.33, and 0 (100, 67, 33, and 0 K, respectively). Forages were harvested daily and fed at approximately 1.3 times maintenance metabolizable energy requirement. The experiment lasted 21 days, with most measures on the last 8 days. The CT concentration was 0.3 and 151 g/kg DM in G and K, respectively. DM intake was similar among treatments (i.e., 682, 675, 654, and 648 g/day; S.E. = 30.0) and gross energy (GE) digestibility increased linearly (P<0.05) with decreasing K (0.472, 0.522, 0.606, and 0.666 for 100, 67, 33, and 0 K, respectively). CH₄ emission changed quadratically (P<0.05) with decreasing K (10.9, 13.8, 17.6, and 26.2 l/day; 32, 42, 57, and 88 kJ/MJ GE; 69, 81, 94, and 133 kJ/MJ digestible energy for 100, 67, 33, and 0 K, respectively). In vitro CH₄ emission by incubation of ruminal fluid for 3 weeks with a medium for methanogenic bacteria and other conditions promoting activity by methanogens also was affected quadratically (P<0.05) by K level (7.0, 8.1, 9.2, and 16.1 ml for 100, 67, 33, and 0 K, respectively). The total bacterial count of ruminal samples was similar among K levels, but the number of total protozoa increased linearly (P<0.05) as K declined (8.3, 11.8, 15.6, and 27.1 × 10⁵ ml⁻¹ for 100, 67, 33, and 0 K, respectively). The CT-containing forage K decreased CH₄ emission by goats regardless of its feeding level, although the effect per unit of K increased with decreasing K. Forage type (i.e., legume versus grass) may have contributed to the effect of K on CH₄ emission, but most of the change appeared attributable to CT, which appeared to directly impact activity of methanogenic bacteria, although alterations of protozoal activity could have been involved. These findings suggest that relatively low dietary levels of CT could be employed to lessen CH₄ emission without a marked detrimental effect on other conditions such as total tract protein digestion.     

Feasibility study of exponential feeding strategy in fed-batch cultures for phenol degradation using Cupriavidus taiwanensis

An indigenous phenol-degrading bacterial isolate Cupriavidus taiwanensis R186 was used to degrade phenol from an aqueous solution under fed-batch operation. An exponential feeding strategy combined with dissolved oxygen control was applied based on kinetic characteristics of cell growth and phenol degradation to meet sufficient metabolic needs for cellular growth and achieve the best phenol removal efficiency. Without the stress of phenol inhibition, the optimal set point of specific growth rate of exponential feeding for fed-batch phenol degradation was found to be 0.50–0.55μ_max (μ_max denotes the maximum specific growth rate from Monod model). Meanwhile, the sufficient set point of dissolved oxygen for maximal phenol degradation efficiency was approximately at 10–55% air saturation. With the optimal operation conditions, the best phenol degradation rate was 0.213 g phenol h⁻¹, while a shortest treatment time of 15 h was achieved for complete degradation of 11.35 mM (ca. 3.20 g) of phenol.     

Investigation of subcellular acidic compartments using alpha-aminophosphonate 31P nuclear magnetic resonance probes

The ³¹P nuclear magnetic resonance (NMR) characteristics, toxicity, and cellular penetration of five linear or cyclic α-aminophosphonate highly sensitive pH probes were investigated in Dictyostelium discoideum cells and isolated rat hearts and were compared with three phosphonic acid derivatives. The line width broadening at pH pK_a, which was satisfactorily modelized for all compounds, was significantly limited in biological milieu for the new markers, affording a four- to sixfold better accuracy in pH determination. Cellular uptake or washout of nontoxic concentrations (<15 mM) of α-aminophosphonates occurred by rapid passive permeation, whereas standard probes required a much slower fluid-phase pinocytosis and transport processes that could ultimately lead to trapping. Using mild concentrations (<4 mM) three α-aminophosphonates having 6 < pK_a < 7 allowed an easy and simultaneous ³¹P NMR determination of cytosolic, acidic, and extracellular compartments in anoxic–reoxygenated or starving D. discoideum.     

Elderberry flavonoids bind to and prevent H1N1 infection in vitro

A ionization technique in mass spectrometry called Direct Analysis in Real Time Mass Spectrometry (DART TOF-MS) coupled with a Direct Binding Assay was used to identify and characterize anti-viral components of an elderberry fruit (Sambucus nigra L.) extract without either derivatization or separation by standard chromatographic techniques. The elderberry extract inhibited Human Influenza A (H1N1) infection in vitro with an IC₅₀ value of 252 ± 34 μg/mL. The Direct Binding Assay established that flavonoids from the elderberry extract bind to H1N1 virions and, when bound, block the ability of the viruses to infect host cells. Two compounds were identified, 5,7,3′,4′-tetra-O-methylquercetin (1) and 5,7-dihydroxy-4-oxo-2-(3,4,5-trihydroxyphenyl)chroman-3-yl-3,4,5-trihydroxycyclohexanecarboxylate (2), as H1N1-bound chemical species. Compound 1 and dihydromyricetin (3), the corresponding 3-hydroxyflavonone of 2, were synthesized and shown to inhibit H1N1 infection in vitro by binding to H1N1 virions, blocking host cell entry and/or recognition. Compound 1 gave an IC₅₀ of 0.13 μg/mL (0.36 μM) for H1N1 infection inhibition, while dihydromyricetin (3) achieved an IC₅₀ of 2.8 μg/mL (8.7 μM). The H1N1 inhibition activities of the elderberry flavonoids compare favorably to the known anti-influenza activities of Oseltamivir (Tamiflu^®; 0.32 μM) and Amantadine (27 μM).     

The influence of N-glycosylation on biochemical properties of Amy1, an α-amylase from the yeast Cryptococcus flavus

The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL⁻¹ affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL⁻¹ TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The K_M values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL⁻¹, and V_max values were 0.038 and 0.047 mg min⁻¹, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg²⁺ and Cu²⁺ suggesting that N-glycosylation is involved in the folding of Amy1.     

Delayed parturition in cloned calves associated with persistently elevated placentomal TGF-β1 expression

The objective of this study was to investigate hormonal and TGF-β₁ characterizations of delayed parturition in the SCNT recipients (Korean native beef cattle: Hanwoo). The SCNT blastocysts produced by Hanwoo fetal fibroblast cells were transferred into the synchronized Hanwoo recipients. The artificially inseminated Hanwoo recipients (AI-R) were used as control. All AI-R were labored by natural delivery. The SCNT recipients (SCNT-R) with no signs of delivery were operated by Caesarean section. The blood and placentomes were collected during parturition. The weight of placentomes in SCNT-R (n = 12, 301 ± 41.22 g) was significantly higher than that of AI-R (n = 10, 204.8 ± 24.89 g) (p < 0.05). There were significantly lower E2 (p < 0.05) or higher P4 (p < 0.01) and TGF-β₁ (p < 0.01) levels in the SCNT-R compared to that of AI-R, respectively. The SCNT-R showed a higher placentomal TGF-β₁ protein level compared to that of AI-R (p < 0.01). Interestingly, the TGF-β₁ protein level in SCNT-R with normal delivery was dramatically decreased as same as AI-R, but it was highly maintained in C-sec at days 250 of pregnancy in AI-R. These results suggest that delayed parturition in clone calving may be associated with persistence of elevated TGF-beta-1 expression in late pregnancy.     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.     

Incorporation of Cestrum diurnum leaf improves intestinal Ca transport in broilers

The economy of Ca utilization is under the control of vitamin D₃, particularly its active metabolite 1,25-dihydroxy cholecalciferol [1,25(OH)₂D₃]. In sufficient Ca absorption leads to tibial dyschondroplasia resulting in not attaining optimum body weight. Our earlier studies [T.P. Prema, N. Raghuramulu, Phytochemistry 37 (1994) 167] have shown that the Cestrum diurnum (CD) leaves contain vitamin D₃ metabolites. It was felt whether incorporation of CD as a source of 1,25(OH)₂D₃ could improve the Ca absorption in broilers. Four groups of 60 birds each were fed with either normal diet or normal diet + 0.25% CD or normal diet without vitamin D₃ or normal diet without vitamin D₃ + 0.25% CD leaf powder for 45 days. In subsample of six birds it was observed that incorporation of CD leaves in the feed had the maximal effect on all the parameters studied. The results indicate that the intestinal Ca transport as represented by Serosa/Mucosa (S/M) ratio was found to be significantly (p < 0.01) higher in broilers fed diet with CD leaf powder and the 1α hydroxylase activity in kidney is significantly (p < 0.001) higher in negative controls. On the other hand the supplementation of CD leaves enhanced the serum Ca, body weight, tibia weight, density and strength resulting in the disappearance of tibial dyschondroplasia. No lesions of toxicity were observed in any of the soft tissue examined. The results suggest that the incorporation of CD leaf powder in poultry feed could be beneficial to the poultry.