Diamine oxidase ofLathyrus sativus seedlings. Purification and properties

Diamine oxidase (EC 1.4.3.6) was purified from 5-day-old etiolated seedlings ofLathyrus sativus by MnCl₂ treatment, (NH₄)₂SO₄ and acetone fractionations, DEAE-Sephadex chromatography followed by gel filtration on Sephadex G-200. A single step purification of the enzyme was achieved by using an immunoaffinity column, wherein rabbit antibodies to the homogeneous diamine oxidase were coupled to CNBr-activated Sepharose. The enzyme thus obtained was homogeneous by electrophoretic, immunological and ultracentrifugal criteria. It had anM _r of 148,000 (6.46S) and was a dimer with similar sub-units (M _r 75,000). Amino acid analysis showed the absence of cysteine residues although it contained five disulphide bonds. The enzyme had copper (2.7 g atom/mol enzyme) but was not a glycoprotein. No absorption maximum in the visible region was detectable. Ethylenediamine 1,3-diaminopropane and histamine were potent competitive inhibitors for the substrate putrescine. The addition of monospecific antibodies to the enzyme increased the K_m for benzyl amine without any change in the V_max Diamine oxidase from pea seedling, partially purified, exhibited complete crossreactivity with the antibodies to theL. sativus enzyme.     

Diamine oxidase of Lathyrus sativus seedlings

A diamine oxidase has been purified (ca 40-fold) from 5-day old etiolated seedlings of L. sativus by MnCl₂ treatment, (NH₄)₂SO₄ and Me₂CO fractionations, positive adsorption on alumina Cγ-gel followed by column chromatography on DEAE-Sephadex. This cytosol enzyme oxidatively deaminates a number of aliphatic and aryl alkylamines but not histamine. NSD-1055, semicarbazide and other carbonyl reagents, α,α'bipyridyl, 1,10-phenanthroline and 8-hydroxyquinoline inhibit the enzyme. Pargyline, SKF trans-385, atabrine were without effect on the enzyme.     

A binary segmentation approach for boxing ribosome particles in cryo EM micrographs

Three-dimensional reconstruction of ribosome particles from electron micrographs requires selection of many single-particle images. Roughly 100,000 particles are required to achieve approximately 10 A resolution. Manual selection of particles, by visual observation of the micrographs on a computer screen, is recognized as a bottleneck in automated single-particle reconstruction. This paper describes an efficient approach for automated boxing of ribosome particles in micrographs. Use of a fast, anisotropic non-linear reaction-diffusion method to pre-process micrographs and rank-leveling to enhance the contrast between particles and the background, followed by binary and morphological segmentation constitute the core of this technique. Modifying the shape of the particles to facilitate segmentation of individual particles within clusters and boxing the isolated particles is successfully attempted. Tests on a limited number of micrographs have shown that over 80% success is achieved in automatic particle picking.     

Nodavirus infection causes mortalities in hatchery produced larvae of Lates calcarifer: first report from India

Larvae (15 to 21 d post hatch, dph) of the Asian sea bass Lates calcarifer (Bloch) suffered heavy mortalities (60 to 90%) during the hatchery-rearing phase. Darkened and moribund larvae showed no evidence of bacterial or parasitic infections. Tissue sections of brain and spinal cord showed clear necrotic vacuolation. Electron microscopy revealed membrane-bound viral particles in the cytoplasm of the nerve cells. The viral particles measured 28 to 30 nm in diameter. Primer sets, designed for the amplification of the RNA2 segment of the piscine nodavirus coat protein gene, were used in the RT-PCR analysis of moribund larvae of 20 and 21 dph which produced the amplified product of 430 bp. The clinical manifestations, pathology and electron microscopy observations supported by the RT-PCR analysis suggest that the nerve necrosis was due to nodavirus infection in the larvae. This is the first report of piscine nodavirus infection from the Indian sub-continent.     

Enzyme-based sensor arrays for rapid characterization of complex disaccharide solutions

An enzyme-based sensor array has been developed to detect multiple disaccharides in aqueous solutions. Porous agarose beads, derivatized with enzymes for assaying disaccharides, are localized within wells etched into a silicon chip in a regular 5 x 7 array. Each well is individually addressable and acts as a microanalysis chamber where sample solution passes through the agarose matrix and is exposed to the enzymes. Detection is achieved by observing the increase in absorbance of a quinoneimine dye produced during the reaction. This technique is used to quantify the disaccharides lactose, sucrose, and maltose and the monosaccharide glucose. Preexisting glucose in the sample complicates multicomponent sensing but can be accounted for by including a glucose sensor in the array. This detection strategy is applied to the simultaneous analysis of these sugars in several beverages.     

Helix stabilization in the C-terminal peptide of chicken riboflavin carrier protein enhances immunogenicity and prolongs contraceptive potential as an epitope-based vaccine in female rats

Earlier investigations have shown that (a) antibodies against a carrier-coupled 20-residue synthetic peptide (C-20), (200)HACQKKLLKFEALQQEEGEE(219), corresponding to the C-terminal partially helical sequence of chicken riboflavin carrier protein (RCP; 219 AA) curtail pregnancy in mammals and (b) helix stabilization by introducing appropriately spaced salt bridges in the flanking sequences of its B-cell epitopic structure enhances RCP antigenicity to peptide antibodies. Among such engineered C-20 analogs, HE-20 (HAEQKKLLKFEALEQEKGKE) exhibited maximum helical propensity. Since C-20 per se, i.e., without carrier conjugation, elicits RCP-reactive neutralizing antibodies in rodents, we mapped its T-cell epitope which overlaps its B-cell epitope, both of which remain unmodified in HE-20. Comparative evaluation of immunogenicity of the two epitope-based peptide vaccines showed that HE-20 was far superior to C-20 in generating RCP-reactive antibodies in terms of both affinity and titer. With regard to bioefficacy, passive immunoneutralization of RCP in pregnant rats by administering purified IgG from either of the antipeptide sera terminated pregnancy. Similarly, active immunization of fertile female rats with the individual peptide analogs curtailed pregnancy. However, HE-20 was more efficient in eliciting higher affinity, longer-lasting, RCP-crossreactive antibodies with consequently more prolonged immunocontraceptive efficacy.     

Computational model for monitoring cholesterol metabolism

A non-deterministic finite automaton is designed to observe the cholesterol metabolism with the states of acceptance and rejection. The acceptance state of the automaton depicts the normal level of metabolism and production of good cholesterol as an end product. The rejection state of this machine shows the inhibition of enzymatic activity in cholesterol synthesis and removal of free fatty acids. The deficiency in human cholesterol metabolism pathway results in abnormal accumulation of cholesterol in plasma, arterial tissues leading to diseases such as hypercholesterolemia, atherosclerosis respectively and formation of gallstones. The designed machine can be used to monitor the cholesterol metabolism at molecular level through regulation of enzymes involved in the biosynthesis and metabolism of cholesterol for the treatment of diseases incident due to the respective metabolic disorder. In addition, an algorithm for this machine has been developed to compare the programmed string with the given string. This study demonstrates the construction of a machine that is used for the development of molecular targeted therapy for the disorders in cholesterol metabolism.     

Isolation and characterisation of a biotin-binding protein from the pregnant-rat serum and comparison with that from the chicken egg-yolk

A biotin-binding protein exhibiting partial immunological cross-reactivity with the purified chicken egg-yolk biotin-binding protein has been detected, for the first time, in the sera of pregnant/estrogenised female rats but not of the normal males. This protein, purified by affinity chromatography on a biotin-AH-Sepharose was homogeneous by electrophoretic and immunological criteria. It was a glycoprotein of Mr 66,000 without any detectable subunites, had a pI 4.1, and specifically bound [14C]biotin. Several structural and functional features of the biotin-binding protein of rat and chicken were found to be similar. These included immunological cross-reactivity, acidic and glycoprotein nature, ability to tightly bind [14C]biotin, estrogen stimulation for their appearance in circulation, and the pattern of distribution of radioiodinated peptides upon proteolysis with trypsin.     

Modulation of arginine decarboxylase activity in cucumber (Cucumis sativus) cotyledons in short-term organ culture

Among the various amines administered to excisedCucumis sativus cotyledons in short-term organ culture, agmatine (AGM) inhibited arginine decarboxylase (ADC) activity to around 50%, and putrescine was the most potent entity in this regard. Homoarginine (HARG) dramatically stimulated (3- to 4-fold) the enzyme activity. Both AGM inhibition and HARG stimulation of ADC were transient, the maximum response being elicited at 12 h of culture. Mixing experiments ruled out involvement of a macromolecular effector in the observed modulation of ADC. HARG-stimulated ADC activity was completely abolished by cycloheximide, whereas AGM-mediated inhibition was unaffected. Half-life of the enzyme did not alter on treatment with either HARG or AGM. The observed alterations in ADC activity are accompanied by change in K_m of the enzyme. HARG-stimulated ADC activity is additive to that induced by benzyladenine (BA) whereas in presence of KCl, HARG failed to enhance ADC activity, thus demonstrating the overriding influence of K⁺ on amine metabolism.     

Cissampelos pareira Linn: Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes

Background

Dengue, a mosquito-borne viral disease, poses a significant global public health risk. In tropical countries such as India where periodic dengue outbreaks can be correlated to the high prevalence of the mosquito vector, circulation of all four dengue viruses (DENVs) and the high population density, a drug for dengue is being increasingly recognized as an unmet public health need.

Methodology/Principal findings

Using the knowledge of traditional Indian medicine, Ayurveda, we developed a systematic bioassay-guided screening approach to explore the indigenous herbal bio-resource to identify plants with pan-DENV inhibitory activity. Our results show that the alcoholic extract of Cissampelos pariera Linn (Cipa extract) was a potent inhibitor of all four DENVs in cell-based assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral replication, based on plaque assays. Virus yield reduction assays showed that Cipa extract could decrease viral titers by an order of magnitude. The extract conferred statistically significant protection against DENV infection using the AG129 mouse model. A preliminary evaluation of the clinical relevance of Cipa extract showed that it had no adverse effects on platelet counts and RBC viability. In addition to inherent antipyretic activity in Wistar rats, it possessed the ability to down-regulate the production of TNF-α, a cytokine implicated in severe dengue disease. Importantly, it showed no evidence of toxicity in Wistar rats, when administered at doses as high as 2g/Kg body weight for up to 1 week.

Conclusions/Significance

Our findings above, taken in the context of the human safety of Cipa, based on its use in Indian traditional medicine, warrant further work to explore Cipa as a source for the development of an inexpensive herbal formulation for dengue therapy. This may be of practical relevance to a dengue-endemic resource-poor country such as India.