Cryogenically cured hydroxyapatite-gelatin nanobiocomposite for bovine serum albumin protein adsorption and release

This research paper presents the first results on the protein adsorption and release kinetics and in vitro biodegradability of cryogenically cured hydroxyapatite-gelatin based micro/macroporous scaffolds (CHAMPS). While the adsorption and release of bovine serum albumin (BSA) protein exhibits steady state behavior over an incubation period of up to 10 days, Fourier transform infrared (FT-IR) analysis importantly confirms the absence of any change in the secondary structure of BSA proteins due to interaction with the CHAMPS scaffold. The compression properties of the CHAMPS scaffold with interconnected porosity (pore size similar to 50-200 mm) is characterized by a non-linear stress-strain response with a strength close to 5 MPa and a maximum strain of up to 24%. The slow but systematic increase in weight loss over a period of 7 days as well as apatite layer formation indicates its good bioactivity. The extensive micro-computed tomography (micro-CT) analysis establishes cancellous bone-like highly interconnected and complex porous architecture of the CHAMPS scaffold. Importantly, the excellent adsorption (up to 50%) and release (up to 60% of adsorbed protein) of BSA has been uniquely attributed to the inherent porous microstructure of the CHAMPS scaffold. Overall, the present study provides an assessment of the interaction of protein with the gelatin-hydroxyapatite macroporous scaffold in vitro, as well as reporting for the first time the efficacy of such scaffolds to release 60% of BSA loaded onto the scaffold in vitro, which is significantly higher than earlier literature reports.

Photoinduced DNA and Protein Cleavage Activity of Ferrocene-Appended L-Methionine Reduced Schiff Base Copper(II) Complexes of Phenanthroline Bases

Ferrocene-appended ternary copper(H) complexes of phenanthroline bases having CuN3OS coordination with an axial Cu-S bond derived from L-methionine reduced Schiff base shows red light induced oxidative DNA cleavage activity following a hydroxyl radical pathway. The dipyridophenazine complex, in addition, displays photoinduced oxidative cleavage of bovine serum albumin protein in UV-A light.

Hormonal induction of riboflavin carrier protein in the chicken oviduct and liver: a comparison of kinetics and modulation

Estrogen (E) induction of riboflavin carrier protein (RCP) in the chicken oviduct and liver was investigated to compare and contrast the kinetics, hormonal specificity and modulation of its elaboration in the 2 steroid-responsive tissues. During primary stimulation, continued daily E administration to immature female chicks elicited, after an initial lag, rapid growth and RCP content of the oviduct; neither progesterone (P) nor testosterone (T) could substitute for E in this respect. Furthermore, P given along with E curtailed tissue growth and its RCP content, whereas E + T had a synergistic effect on tissue growth only. During secondary stimulation, E administration steeply enhanced both tissue weight and RCP content without any lag. Interestingly, P (but not T) could substitute for E in augmenting magnum RCP concentration to a comparable extent while a concomitant effect on tissue growth was less marked. In contrast, hepatic induction of RCP was absolutely E-specific during both primary and secondary stimulations. Secondary stimulation with either E or P of E-primed birds enhanced the rates of RCP synthesis in the oviduct relative to that of total protein, whereas in the liver only E was effective in this regard. The absolute rate of E-induced RCP synthesis in both the steroid-stimulated tissues was significantly higher than that of general protein elaboration.

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.

Purification and properties of synephrinase from Arthrobacter synephrinum

Synephrinase, an enzyme catalyzing the conversion of (−)-synephrine into p-hydroxyphenylacetaldehyde and methylamine, was purified to apparent homogeneity from the cell-free extracts of Arthrobacter synephrinum grown on (±)-synephrine as the sole source of carbon and nitrogen. A 40-fold purification was sufficient to produce synephrinase that is apparently homogeneous as judged by native polyacrylamide gel electrophoresis and has a specific activity of 1.8 μmol product formed /min/mg protein. Thus, the enzyme is a relatively abundant enzyme, perhaps comprising as much as 2.5% of the total protein. The enzyme essentially required a sulfhydryl compound for its activity. Metal ions like Mg2+, Ca2+, and Mn2+ stimulated the enzyme activity. Metal chelating agents, thiol reagents, denaturing agents, and metal ions like Zn2+, Hg2+, Ag1+, and Cu2+ inhibited synephrinase activity. Apart from (−)-synephrine, the enzyme acted upon (±)-octopamine and β-methoxysynephrine. Molecular oxygen was not utilized during the course of the reaction. The molecular mass of the enzyme as determined by Sephadex G-200 chromatography, was around 156,000. The enzyme was made up of four identical subunits with a molecular mass of 42,000.

On the Specificity of Carbohydrate-Lectin Recognition The Interaction of a Lectin from Ricinus communis Beans with Simple Saccharides and Concanavalin A

A galactose-specific protein (RC1) isolated from Ricinus communis beans was found to give a precipitin reaction with concanavalin A. Its carbohydrate content amounted to 8–9% of the total protein and was found to be rich in mannose. The interaction of RC1 with galactose and lactose was measured in 0.05 M phosphate buffer containing 0.2 M NaCl (pH 6.8) by the method of conventional equilibrium dialysis. From the analysis of the binding data according to Scatchard method the association constant (Ka) at 5°C was calculated as 3.8 mM−1 and 1.2 mM−1 for lactose and galactose, respectively. In both cases the number of binding sites per molecule of RC1 with molecular weight of 120000 was found to be 2. From the temperature-dependent Ka values for the binding of lactose, the values of –5.7 kcal/mol and –4.3 cal × mol−1× K−1 were calculated for ΔH and ΔS, respectively. The addition of concanavalin A to RC1 or vice versa led to the formation of the insoluble complex RC1· ConA4 containing one molecule of RC1 and one molecule of tetrameric concanavalin A (ConA4) which could be dissociated upon addition of concanavalin A-specific sugars. The complex formation results in a time-dependent appearance of turbidity in the time range from 10s to 10 min. From the measurement of the time-dependent appearance and disappearance of the turbidity the formation (kf) and dissociation (kd) rate constants were calculated as 3 mM−1× s−1 and 0.07 ks−1 respectively. The ratio kf/kd (43μM −1), that corresponds to the association constant of complex RC1· ConA4, is higher than that of mannoside · ConA4 and thereby suggests that protein-protein interaction contributes significantly in stabilising glycoprotein · lectin complexes. The relevance of this finding to the understanding of the chemical specificities that are involved in a model cell-lectin interaction is discussed.

Early biochemical events during adventitious root initiation in the hypocotyl of Phaseolus vulgaris

Indole butyric acid (IBA) initiates roots in the hypocotyl tissue of Phaseolus vulgaris (French bean). The response is dependent on the concentration of IBA and the duration of exposure to the hormone. IBA enhances the rate of total protein synthesis in ca 30 min after exposure of the hypocotyl segments to the hormone. There is no detectable change in total or poly(A)-containing RNA synthesis in this period although significant increases are seen 2 hr after hormone pre-treatment. The early IBA-mediated increase in protein synthesis (30 min) is not sensitive to Actinomycin D but the antibiotic blocks the increase manifested 2 hr after hormone pre-treatment. Inhibition of early protein synthesis by cycloheximide depresses and delays root initiation. Cytosol prepared from IBA-treated hypocotyl tissue stimulates protein synthesis in vitro to a greater extent than that of the control.

Cobalt(II) complexes of terpyridine bases as photochemotherapeutic agents showing cellular uptake and photocytotoxicity in visible light

Cobalt(II) complexes of terpyridine bases Co(L)(2)](ClO4)(2) (1-3), where L is 4'-phenyl-2,2':6',2''-terpyridine (ph-tpy in 1), 4'-(9-anthracenyl)-2,2':6',2''-terpyridine (an-tpy in 2) and 4'-(1-pyrenyl)-2,2':6',2''-terpyridine (py-tpy in 3), are prepared and their photo-induced DNA and protein cleavage activity and photocytotoxic property in HeLa cells studied. The 1 : 2 electrolytic and three-electron paramagnetic complexes show a visible band near 550 nm in DMF-Tris-HCl buffer. The complexes 1-3 show emission spectral bands at 355, 421 and 454 nm, respectively, when excited at 287, 368 and 335 nm. The quantum yield values for 1-3 in DMF-H2O (2 : 1 v/v) are 0.025, 0.060 and 0.28, respectively. The complexes are redox active in DMF-0.1 M TBAP. The Co(III)-Co(II) and Co(II)-Co(I) couples appear as quasi-reversible cyclic voltammetric responses near 0.2 and -0.7 V vs. SCE, respectively. Complexes 2 and 3 are avid binders to calf thymus DNA giving K-b value of similar to 10(6) M-1. The complexes show chemical nuclease activity. Complexes 2 and 3 exhibit oxidative cleavage of pUC19 DNA in UV-A and visible light. The DNA photocleavage reaction of 3 at 365 nm shows formation of singlet oxygen and hydroxyl radical species, while only hydroxyl radical formation is evidenced in visible light. Complexes 2 and 3 show non-specific photo-induced bovine serum albumin protein cleavage activity at 365 nm. The an-tpy and py-tpy complexes exhibit significant photocytotoxicity in HeLa cervical cancer cells on exposure to visible light giving IC50 values of 24.2 and 7.6 mu M, respectively. Live cell imaging study shows accumulation of the complexes in the cytosol of HeLa cancer cells.

Characterization of a variant glucose-6-phosphate dehydrogenase from the south Indian population.

Glucose-6-phosphate dehydrogenase (G6PD) is coded by a gene on the X-chromosome. Earlier studies have shown that the South Indian population has a high incidence of this enzyme deficiency. The electrophoretic mobility, pH optimum and the K-m values for G6PD from normal and variant individuals were identical. However, the specific activity of the variant enzyme was 8 times less compared to the value of the normal enzyme. Western blot analysis of partially purified G6PD from normal and variant individuals performed using equal amounts of total protein showed that the variant protein was 3 times less in concentration. Similar analysis performed using protein corresponding to equal enzyme activity units in the normal and variant samples showed that the variant enzyme was 2.25 times less efficient compared to the normal enzyme. RNA dot blot analysis using full length G6PD cDNA probe (PGDT5B, a kind gift from Prof. L Luzzatto) revealed that lymphocytes from normal and variant individuals had equal amounts of G6PD specific mRNA.

278. Studies on ass's milk: Composition

The total solids of samples of ass's milk ranged from 7·80 to 9·10, the solids-not-fat from 7·14 to 8·50, and the fat from 0·54 to 0·71%. The nitrogen distribution in ass's milk is: casein 39·5, albumin 35·0, globulin 2·7 and non-protein nitrogen 22·8% of the total nitrogen. Ass's milk contains: casein 0·70, albumin 0·62 and globulin 0·07%. The total protein content is 1·39%. Ass's milk is therefore characterized by a low casein, a low globulin and a high albumin content. The non-protein nitrogen consists of amino nitrogen 8·1, urea nitrogen 24·3 and uric acid 0·7 mg./100 ml. of milk. The urea content is twice that present in cow's milk. The mean chloride and lactose contents of the milk samples are 0·037 and 6·1% respectively. The average calcium and phosphorus content of ass's milk are 0·081 and 0·059% respectively. Half the calcium is ionic, and half is in colloidal form. The phosphorus distribution is: total acid soluble 84·0, acid soluble organic 38·5, easily hydrolysable ester 27·4, inorganic 46·0, and colloidal inorganic 23·0 % of the total phosphorus. The ratio of CaO: P2O5 is 1:1. 46 % of the total phosphorus is in ester form; this is high when compared with only 12 % in cow's milk; most of the phosphoric ester forms soluble barium salts, which is a distinguishing feature of ass's milk. The total sulphur content is 15·8 mg./100 ml. The fat has a penetrating odour and is coloured orange-yellow. It has an iodine value of about 86, which is much higher than that for human milk fat. The Reichert (9·5) and Kirschner values (5·7) are low. In general, the composition of ass's milk resembles that of human rather than of cow's milk.

A possible connection between antidiabetic and antilipemic properties of psoralea corylifolia seeds and the trace elements present: a LIBS based study

Psoralea corylifolia (PC), a medicinal plant, is used in traditional medicine to treat diabetes. Purpose of the research was to examine the antidiabetic and antilipemic potential of PC and to determine the relationship between its antidiabetic potential and the trace elements present. Wistar rats (150-200 g) with fasting blood glucose (FBG) of 80-110 mg dl(-1)(sub-diabetic) and 150-200 mg dl(-1)(mild diabetic) were selected for the short term antidiabetic studies and severely diabetic rats (FBG > 300 mg dl(-1)) were chosen for the long term antidiabetic and hypolipemic studies of PC seed extract. Laser induced breakdown spectroscopy (LIBS) was used to detect trace elements in the PC extract and the intensity ratios of trace elements were estimated. The dose of 250 mg kg(-1) of PC extract was found to be the most effective in lowering blood glucose level (BGL) of normal, sub, mild and severely diabetic rats during FBG and glucose tolerance test (GTT) studies. Lipid profile studies on severely diabetic rats showed substantial reduction in total cholesterol, triglycerides, very low density lipoprotein, and low density lipoprotein and an increase in the total protein, body weight, high density lipoprotein, and hemoglobin after 28 days of treatment. Significant reduction in urine sugar and protein levels was also observed. LIBS analysis of the PC extract revealed the presence of Mg, Si, Na, K, Ca, Zn and Cl. The study validates the traditional use of PC in the treatment of diabetes and confirms its antilipemic potential. The antidiabetic activity of PC extract may partly be due to the presence of appreciable amounts of insulin potentiating elements like Mg, Ca, and K.

Differential expression of estrogen receptor alpha in the embryonic adrenal-kidney-gonadal complex of the oviparous lizard, Calotes versicolor (Daud.)

Estrogen signalling is critical for ovarian differentiation in reptiles with temperature-dependent sex determination (TSD). To elucidate the involvement of estrogen in this process, adrenal-kidney-gonadal (AKG) expression of estrogen receptor (ER alpha) was studied at female-producing temperature (FPT) in the developing embryos of the lizard, Calotes versicolor which exhibits a distinct pattern of TSD. The eggs of this lizard were incubated at 31.5 +/- 0.5 degrees C (100% FPT). The torso of embryos containing adrenal-kidney-gonadal complex (AKG) was collected during different stages of development and subjected to Western blotting and immunohistochemistry analysis. The ER alpha, antibody recognized two protein bands with apparent molecular weight similar to 55 and similar to 45 kDa in the total protein extracts of embryonic AKG complex of C. versicolor. The observed results suggest the occurrence of isoforms of ER alpha. The differential expression of two different protein isoforms may reveal their distinct role in cell proliferation during gonadal differentiation. This is the first report to reveal two isoforms of the ER alpha in a reptile during development. Immunohistochemical studies reveal a weak, but specific, cytoplasmic ER alpha immunostaining exclusively in the AKG during late thermo-sensitive period suggesting the responsiveness of AKG to estrogens before gonadal differentiation at FPT. Further, cytoplasmic as well as nuclear expression of ER alpha in the medulla and in oogonia of the cortex (faint activity) at gonadal differentiation stage suggests that the onset of gonadal estrogen activity coincides with sexual differentiation of gonad. Intensity and pattern of the immunoreactions of ER alpha in the medullary region at FPT suggest endogenous production of estrogen which may act in a paracrine fashion to induce neighboring cells into ovarian differentiation pathway. (C) 2014 Elsevier Inc. All rights reserved.

Isolation and characterization of riboflavin-binding protein from pregnant-rat serum

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.

Purification of goat serum retinol-binding protein and preparation of its antibodies

The method for the purification of goat serum retinol-binding protein consists of DEAE-cellulose chromatography of the serum followed by preparative polyacrylamide disc gel electrophoresis. After electrophoresis, the retinol-binding protein containing zone is identified by the specific fluorescence of retinol. For raising the antibodies, the portion of the gel containing retinol binding protein is homogenized and injected intradermally and intramuscularly to rabbits. The availability of this simple method for the isolation of retinol-binding protein and production of its antibodies enables the development of a radioimmunoassay for this protein.

Antigenic determinants of human serum retinol binding protein as probed with monoclonal antibodies

Monoclonal antibodies raised against human serum retinol-binding protein (hRBP) were used as probes for the study of the antigenic determinants of hRBP and those shared with the same protein from other species. The antibodies could be classified into four distinct groups and react with the homologous proteins from the rat as well as the rabbit sera. Three of these antibodies recognize sequential or continuous epitopes while the remaining antibody is directed against a discontinuous or conformational epitope. By chemical cleavage with cyanogen bromide, the domains recognized by the monoclonal antibodies could be delineated. By solid-phase synthetic approach, the core sequences recognized by two of these monoclonal antibodies were identified to amino acid sequences 45–51 and 128–131 of the primary amino acid sequence of hRBP.