An acid stable trypsin-chymotrypsin inhibitor from horse gram (Dolichos biflorus)

An inhibitor of trypsin and chymotrypsin was purified from horse gram (Dolichos biflorus) beans. The concentration of the inhibitor which provided total inhibition was 0.27 Μg/Μg tryptic enzyme and 0.46 Μg/Μg chymotryptic enzyme. The inhibitor was stable at 37‡C between pH of 3 to 11 and at 97‡C, upto pH 5.0 only. While the activities were rapidly lost in 0.1N NaO H the loss was only 5 0% in 0.1N HCl when kept for 2 h at 97‡C. On heating at pH 7.8, it remained stable upto 80‡C with a gradual loss in activities at 97‡C and a total loss occurring by autoclaving at 15 psi for 10 min. Reduction of disulphide bonds by 2-mercapto-ethanol, pronase digestion and boiling in the presence of 1 M NaCl led to reduction in the activities. However, the inhibitor was resistant to the action of pepsin and subtilisin and to urea at 37‡C.     

Effects of ultraviolet-B enhanced radiation and temperature on growth and photochemical activities inVigna unguiculata

Changes in growth characteristics and photochemical activities inVigna unguiculata L. Walp seedlings maintained at constant temperature of 10, 20, 30 and 40 ‡C under control and ultraviolet-B enhanced radiation (UV-B) were investigated. UV-B retarded the shoot elongation and also leaf expansion to a great extent at 30 ‡C but produced only marginal changes at 20 and 40 ‡C. Similar response was also observed with respect to changes in leaf fresh and dry masses and total chlorophyll (Chl) content under these temperatures. At 10 ‡C the total Chl content was 3-fold higher under the treatment than under control conditions. In seedlings growing at 20 and 30 ‡C the overall photosynthetic electron transport (H₂O -> methyl viologen) showed a significant enhancement during the 36-h UV-B treatment and thereafter a gradual reduction. Although a similar trend was found in photosystem 1 (PS1), the inhibition even after 60 h of UV-B treatment was not statistically significant. Photosystem 2 (PS2) activity was inhibited in seedlings treated for 60 h by UV-B at 20 and 30 ‡C. However, no inhibition was observed at 40 ‡C. No detectable photochemical activity was found in seedlings grown at 10 ‡C under either control or UV-B enhanced irradiation although the chloroplasts contained Chl. This work was supported by a Research Associateship to N.N. from the Council of Scientific and Industrial Research (India) and by a grant from the Ministerio de Education y Ciencia (ref. 5894- AM086772).     

Β-Galactosidase in immobilized cells ofPapaver somniferum

Cell suspension culture of poppy was permeabilized by Tween 80 and immobilized by glutaraldehyde. Β-Galactosidase showed optimum pH 4.8 and temperature of 50 ‡C. The enzyme hydrolysis was linear during 3 h reaching 68 % conversion. The cells characterized by high Β-galactosidase activity and stability in long-term storage showed convenient physico-mechanical properties.     

Cell-killing efficiency and number of platinum atoms binding to DNA,RNA, and protein molecules of hela cells treated with combinations of hyperthermia and cisdiamine (glycolato)platinum(II)

HeLa S-3 cells were treated with^195mPt-radiolabeledcisdiamine(glylato)platinum(II) (254-S) for 60 min at various temperatures, and the relationship between the lethal effect and the number of Pt atoms binding to DNA, RNA, and proteins was examined. The mean lethal concentration (Do) of 254-S for a 60-min treatment at 0‡C, 25‡C, 37‡C, 40‡C, 42‡C, and 44‡C was 233,132, 61.1, 42.7, 25.6, and 9.9 ΜM, respectively. By using identically treated cells, the numbers of Pt atoms combined with DNA, RNA, and protein molecules were determined in the subcellular fractions. Thus, the D₀ values given as drug concentrations were replaced with the number of Pt atoms combined in each fraction. Then, the cell-killing efficiency of the Pt atom was expressed as the reciprocal of the number of Pt atoms combined and was calculated for each molecule. The efficiency for the DNA molecule was 0.61X10⁴, 1.09xl0⁴, 1.88xl0⁴, 1.90xl0⁴, 2.66xl0⁴, and 5.88xl0⁴ nucleotides, respectively, for the conditions described. From 0‡C to 44‡C, the cell-killing efficiency of Pt atoms increased by a factor of 9.6.     

The rate of calcium extraction during EDTA decalcification from thin bone slices as assessed with atomic absorption spectrophotometry

Summary The rate of calcium extraction with EDTA (ethylenediamine tetraacetic acid) from thin bone slices (300 m-2mm thick) was determined by aid of an atomic absorption spectrophotometer. A 0.5 mm thick bone slice was completely decalcified with 15% (0.40 M), 8% (0.22 M), and 4% (0.11 M) EDTA in 24 h, 3 days, and 5 days, respectively (vol. 15 ml, temp. 4° C, pH 7.4). At 37 and 60° C the speed of demineralization was slightly increased as compared with that at 20° C, while no difference was observed between 4 and 20° C. Bone slices with a thickness of 0.3, 0.5, 1 and 2 mm were decalcified-in the same order-in 24 h, 2, 3, and 5 days (8% EDTA, 4° C, pH 7.4). At pH 7.4, the decalcification rate was a little slower than at pH 5.0 and 8.5. Agitation did not affect the decalcifying velocity, nor did the volume of the agent, except when the volume was very small. The demineralization of ordinary bone, containing both compact and spongy bone, was found to be more rapid than that of homogeneous bone reported earlier. The acidic buffers and New Decalc^®, which served as reference substances, exerted a more vigorous decalcifying effect than EDTA. K formate/formic acid buffer, pH 3.15, demineralized a 1 mm thick bone slice in 24 h, and 2 days was needed with Na lactate/lactic acid buffer, pH 3.70. With New Decalc^®, pH 0.9, the corresponding demineralization was accomplished in 1.5 h. Atomic absorption spectrophotometer proved to be a useful tool in the evaluation of calcium extraction velocity from bone slices.     

Effect of pH on sublingual absorption of oxycodone hydrochloride

The purpose of this study was to develop a sublingual spray drug delivery formulation of oxycodone and evaluate the effect of formulation pH on sublingual absorption of oxycodone for acute pain management using rabbit as the animal model. Using a new, sensitive, and specific liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization detector assay, the absorption bioavailability of sublingual oxycodone was determined in rabbits by comparing plasma concentration after sublingual spray delivery with equivalent intravenous dose. The effect of formulation pH on sublingual absorption of oxycodone was also tested on rabbits that had received oxycodone sublingually at a dose of 0.1 mg/0.1 mL (pH 4.0 and 9.0). Blood samples were collected at different time points, and plasma oxycodone concentrations were determined by LC/MS. Following administration of a 0.1 mg dose, the average C_max values were found to be 64.9±12.1 and 95.2±10.1 ng/mL, for pH 4.0 and 9.0, respectively. The area under the curve (AUC) values were found to be 5807.0, and 8965.3 ng.min/mL for formulation pH 4.0 and 9.0, respectively. The mean sublingual bioavailability of oxycodone was 45.4%±20.1% and 70.1%±17.9%, for pH 4.0 and 9.0, respectively. the formulation pH had no significant influence on oxycodone bioavailability (P<.05). A sublingual spray dosage form of oxycodone hydrochloride would be a good alternative for fast onset pain management, especially in children. Published: March 10, 2006     

Silicone-immobilized cells ofGeotrichum sp. G38 used in biotransformation

The conditions for the reduction of dibenzoyl by silicone-immobilizedGeotrichum sp. G38 were examined, and optimal concentrations of stannous octanoate, ethyl silicate, water and entrapped biomass for the reaction were established. The optimum pH and temperature of the reaction medium were 7.0, and 35‡C (free cells) or 40‡C (immobilized cells), respectively. The immobilized cells showed higher activity than free cells under optimum conditions. The extent of conversion remained greater than 90% even after immobilized cells had been recycled 28 times. When silicone-immobilizedGeotrichum sp. G38 was used in the reduction of ethyl benzoylacetate, the (R)-enantiomer was obtained in an 81% enantiomeric excess compared with 49% enantiomeric excess using free cells.     

Effect of digestive site acidity and compatibility on the species, lipopily and bioavailability of iron, manganese and zinc in Prunus persica Batsch and Carthamus tinctorus

The effects of compatibility, that is combination of Prunus persica Batsch (L.) and Carthamus tinctorus (L.), and different acidity of digestive site on the species, lipopily and bioavailability of coordinated complex of iron, manganese, and zinc in medical decoction were studied. In view of octanol, a long-chain alkanol, resembled as the configuration of carbohydrate and adipose in human body, the octanol- and water-solubility were used to define the species of trace element in phytomedicine, to identify the lipopily and bioavailability of trace element, and octanol-water system was adopted to study the distribution of trace element in decoction of P. persica Batsch (L.) (A), C. tinctorus (L.) (B), and combination of medicine A and B (C) in stomach and intestine. The total concentration, water- and octanol-solubility concentration of iron, manganese, and zinc in medicinal material A, B and C or its decoction under gastric and intestinal acidity, were determined respectively by flame atomic absorption spectrometry, analyzed and compared. The compatibility of medicine A and B enhances the extract percent, octanol-solubility concentration, and stability of coordinated complex of iron, manganese, and zinc. Different acidity of digestive site and compatibility of medicines impact on the ligands of iron, manganese, and zinc, then greatly affect the species and its quantification, the lipopily and bioavailability of trace element. Such influence is quite different for different trace element. Such factors, especially the concentration of octanol-solubility trace element, could be the basis of the dosage to avoid trace element overload.     

Laboratory Study Of Some Biological Potentialities Of Pullus Mediterraneus (Col., Coccinelidae)

Some biological potentialities of Pullus mediterranem F. (Coleoptera, Coccinellidae) were studied under controlled conditions. Eggs were laid under carapaces of Saissetia oleae (bdHomoptera, Coccidae), which is an important pest of olive trees. The eclosion of eggs occurred after more than 36 days at 7‡C, but no more than 1, 7 days at 30‡C. The eclosion rate of eggs exceeded 80% between 20 and 30‡C. In homogeneous juvenile population fed with aphids, 4 instars were distinguished on the basis of the width of the cephalic capsule. Mortality rate was low at all temperature tested. It did not exceed 25% at 20‡C and decreased considerably at 30‡C when P. mediterraneus fed on aphids.     

The directional absorption properties of rhodopsin and its photoproducts

The experimental data on the absorption of a plane polarised light by a solution of cattle rhodopsin at −196‡ C have been theoretically analysed to model the directional absorption properties of rhodopsin and its photoproducts. It is seen that these molecules behave like planar absorbers having a ratio of about 100∶7 between the extinction coefficients along the long axis and perpendicular to it. Using this result and the experimental observations on absorption and dichroism in the retina in situ, a model for the configuration of chromophores in the disc membranes has been derived. In this model the plane of the chromophore is perpendicular to that of the disc and the long axis of the chromophore makes an angle of 6.6‡ with the plane of the disc. The solution of the problem depends on the assumption that the absorption axes are the same for the rhodopsin, prelumirhodopsin and isorhodopsin. Work partially supported by Department of Science and Technology (India)     

Chromium in plants

Chromium is an essential trace element and is associated with some biological pathways, especially with glucose tolerance. For these reasons, we decided to determine the concentration of chromium in two sets of Brazilian medicinal plants. The first group consisted of plants that are considered as antidiabetic, whereas the second included plants that do not have this therapeutic property. The concentration of chromium was determined by flameless atomic absorption. All the plants analyzed contain chromium in the normal range for this element, but the hypoglycemic plants contain more chromium than the others (1–4 μg/g compared to 0.5–1.5 μg/g).     

Effect of temperature on endocytosis and degradation of sulphated proteoglycans by cultured skin fibroblasts

Temperature up to 16‡C reduced endocytosis of [³⁵S]-proteoglycans by human skin fibroblasts to less than 15% of that at 37‡C. At temperatures between 20–26‡C endocytosis was more than 50%. At temperatures below 26‡C, the relative rate of degradation of endocytosed [³⁵S]-proteoglycans was several fold less than the rate of endocytosis. Codistribution of endocytosed [³⁵S]-proteoglycans and the lysosomal marker enzyme Β-hexosaminidase upon subcellular fractionation indicated that endocytotic vesicles containing [³⁵S]-proteoglycans had fused with lysosomes at 37‡C and at 16‡C. The prolonged halflives of endocytosed [³⁵S]-proteoglycans at 16–26‡C could not be explained merely by a temperature dependent reduction of catalytic activity of lysosomal enzymes participating in the degradation of sulphated proteoglycans.     

Renal aluminum excretion

Urinary aluminum (Al) excretion was studied in humans with normal and impaired renal function. Al was measured by atomic absorption spectrometry. In healthy volunteers (n=50), renal Al excretion was 12.2±8.5 μg/24 h. Two patients on plasma exchange therapy with normal renal function and an inadvertent load of 870 and 388 μg Al/treatment showed a 23 and 14% positive balance until next treatment.     

Correlation of Toxicity with Lead Content in Root Tip Cells (Allium cepa L.)

The present study determines lead content in onion root tip cells (Allium cepa L.), correlating it with its toxicity. The treatment was carried at 25 ± 0.5°C using aqueous solutions of lead chloride at 0.1, 0.25, 0.50, 0.75, and 1 ppm for 12, 24, 48, and 72 h. For each treatment, a control where the lead solution was substituted by distilled water was included. After treatment, the meristems were fixed with a mixture of alcohol–acetic acid (3:1) and colored according to the technique of Feulgen. Lead content was quantified by graphite furnace absorption atomic spectrometry. The lead content in the roots ranged from 3.25 to 244.72 μg/g dry weight, with a direct relation with the concentration and time of exposure. A significant negative correlation was presented (r = −0.3629; p < 0.01) among lead content and root growth increment, and a positive correlation (r = 0.7750; p < 0.01) with the induction of chromosomic aberrations. In conclusion, lead is able to induce a toxic effect in the exposed roots, correlated with its content.     

Serum Selenium Concentrations Correlate Significantly with Inflammatory Biomarker High-sensitive CRP Levels in Hungarian Gestational Diabetic and Healthy Pregnant Women at Mid-pregnancy

Selenium is an essential trace element and a component of various enzymes with antioxidant functions. High-sensitive C-reactive protein (hsCRP) is an early indicator of increased lipid peroxidation. The serum selenium concentration, lipid parameters, and hsCRP values of gestational diabetic pregnant women (GD), control pregnant women (CP), and healthy nonpregnant controls (HC) were compared. Blood was taken between the 24th and the 28th week of pregnancy when the oral glucose tolerance test was performed. Selenium concentration was determined by atomic absorption spectrometry after hydride generation. HsCRP was measured by immunturbidimetry. HC had significantly higher serum selenium concentrations than GD and CP women (HC = 77.4 ± 14.82, GD = 51.7 ± 11.62, and CP = 40.5 ± 8.03 μg/l, respectively). HsCRP values of both GD and nondiabetic pregnant women were significantly higher compared to controls. Significant negative correlations were found between serum selenium and total cholesterol, low-density lipoprotein cholesterol, and hsCRP values indicating that low selenium levels are associated with increased lipid peroxidation. Serum selenium concentrations of Hungarian pregnant women are low compared to internationally published data.     

Isolation and characterization of lysosomal alpha-mannosidase of placental tissue

The acidic α-mannosidase was purified 4400-fold by affinity chromatography on Concavalin A-Sepharose and heat treatment at 65‡C in the presence of 1 mM zinc ion. The enzyme did not resolve into multiple forms as in the case of enzymes from human liver and human kidney. The pH optimum of the enzyme was 4.2 in citrate-phosphate buffer. The K_m value for p-nitrophenyl-α-D-mannose was 1.9 mM. The molecular weight of the enzyme determined by gel filtration was 300,000. The enzyme contained 10.6% neutral sugars.     

Indication of silicon essentiality in humans

Serum silicon concentrations were determined in Belgian healthy children and adults, including pregnant women, by electrothermal atomic absorption spectrometry. Serum levels appeared to be significantly higher in healthy children (1–18 yr) than in healthy adults (19–60 yr). Especially, levels in infants (<1 yr) were higher than in any other group. Compared to age-matched nonpregnants, the serum silicon content was very low in pregnant women. In addition to the fact that this study for the first time provides serum silicon values in adults and children in Belgium, the most important observation is that these serum profiles might be an indication of silicon essentiality in man.     

Structure and stability of glucoamylase II fromAspergillus niger: A circular dichroism study

Glucoamylase II (EC 3.2.1.3) fromAspergillus niger has 31 % α-helix, 36 %Β- structure and rest aperiodic structure at pH 4.8 as analysed by the method of Provencher and Glockner (1981,Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4.8 than at pH 7.0, were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures and storage at 30‡C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the ordered structure.     

Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum

Benzyl alcohol and starch-free commercial wheat bran were effective inducers of the laccase activity in cultures of Fusarium proliferatum (MUCL 31970). Initial pH value in the cultures was also an overriding factor for increasing its production. By gel permeation high-performance liquid chromatography, the enzyme eluted as an apparently homogeneous peak with a molecular mass of 54 kDa, but by isoelectrofocusing, two proteins with pI values of 5.17 and 5.07 were revealed. Two different phenoloxidase activities were also detected after nondenaturing polyacrylamide gel electrophoresis. By matrix-assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOF-MS), both proteins showed unique fingerprints, so they were classifiable as isozymes, and were named laccase 1 (Lac1, pI 5.17) and laccase 2 (Lac2, pI 5.07). No clear matches were found when compared with other proteins. The tandem mass spectrometry of some peptides from both isozymes reanalyzed by nanoelectron ionization–ion trap–mass spectrometry (nESI-IT-MS) confirmed their unique character. The following interesting properties, particularly its stability at alkaline pH, make this laccase a promising industrial enzyme for biotechnological applications: maximum activity at 60°C, thermal stability for 2 h at 40°C, optimum pH 3.5 (km=62 μM) measured on 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonate), and pH stability 4–8 (75% stability at pH levels 2.2 and 9) for 2 h at 25°C.     

Tissue digestion for aluminum determination in experimental animal studies

Four different procedures for the determination of aluminum in tissues by atomic absorption spectrometry (AAS) were investigated. They consisted of conventional acid digestion carried out before and after sample drying, associated or not with fat extraction. Drying was carried out in a conventional oven at 65 °C for 24 h. For fat extraction, different solvents and solvent mixtures were investigated considering both extraction yield and sample adequacy for further AAS measurement. Acid digestion was carried out with pure HNO₃ or with its mixture with HClO₄. After digestion, aluminum was measured by graphite furnace atomic absorption spectrometry. Tissues were collected from Al-exposed and nonexposed mice. The results indicated that drying the sample prior to digestion is advantageous as the amount of acid necessary can be significantly reduced. This procedure does not contribute to increase the aluminum level in the samples providing that careful measures to avoid contamination are taken, as the same procedures carried out without taking any precautions to avoid contamination produced imprecise results. Finally, aluminum was not found in the fatty fraction of any sample, even in exposed mice, demonstrating that aluminum does not accumulate in this part of the tissues.