Isolation, macromolecular properties, and combining site of a chito-oligosaccharide-specific lectin from the exudate of ridge gourd (Luffa acutangula)

A lectin specific for chito-oligosaccharides from the exudate of ridge gourd (Luffa acutangula) fruits has been purified to homogeneity by affinity chromatography. The lectin has a molecular weight of 48,000, an S(0)20,w of 4.06 S and a Stokes radius of 2.9 nm. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single band corresponding to Mr of 24,000 was observed both in the presence and absence of beta-mercaptoethanol. The subunits in this dimeric lectin are, therefore, held together solely by noncovalent interactions. The lectin is not a glycoprotein, and secondary structure analysis by CD measurements showed 31% alpha-helix. The hemagglutinating activity of L. acutangula agglutinin was not inhibited by any of the monosaccharides tested. Among the disaccharides only di-N-acetylchitobiose was inhibitory. The inhibitory potency of chito-oligosaccharides increased dramatically with their size up to penta-N-acetylchitopentaose. The lectin has two binding sites for saccharides. The affinity of chito-oligosaccharides for L. acutangula lectin, as monitored by titrating the changes in the near UV-CD spectra and intrinsic fluorescence, increased strikingly with the number of GlcNAc units in them. The values of delta G, delta H, and delta S for the binding process showed a pronounced dependence on the size of the chito-oligosaccharides, indicating that the binding of higher oligomers is progressively more favored thermodynamically than di-N-acetylchitobiose. The thermodynamic data are consistent with an extended binding site in this lectin, which accommodates a tetrasaccharide.     

Identification and sequence characterization of a 1.3 Kb EcoRI repeat fragment that harbors a DNA repair site of rat pachytene spermatocytes

Introduction of well-programmed nicks and gaps and the associated DNA repair activity in the genome at the pachytene interval is a characteristic feature of the meiotic prophase in organisms as varied as lilium and mouse. In the present study we have shown that the DNA synthetic activity in rat pachytene spermatocytes is insensitive to aphidicolin, a specific inhibitor of DNA polymerase , and , suggesting DNA -polymerase-mediated repair synthesis in these cells. We have developed a novel approach for the isolation of the DNA repair sites by combining two independent techniques. Following incorporation of BrdUrd into pachytene spermatocytes in the presence of aphidicolin, the repair sites were released as ssDNA fragments by treatment of nuclei with 30 mM NaOH. Subsequently, the BrdUrd containing ssDNA fragments were specifically isolated using polyclonal anti-BrdUrd antibodies. The DNA fragments released were of two size classes, namely 4–7S (major) and 9–12S (minor) and constituted approximately 1.75% of the pachytene genomic DNA. These DNA repair fragments were distinct from Okazaki fragments and other replicative intermediates isolated from rat bone marrow cells as evidenced by (a) their different size distribution and (b) little cross-hybridization. Southern hybridization of restriction enzyme digests of rat genomic DNA with probes made against BrdUrd-ssDNA fragments revealed that although the repair sites were distributed throughout the genome, strong hybridization signals were observed in EcoRI, (1.3 kb and 2.4 kb), BamH1 (9 kb) and HindIII (5 kb) repetetive DNA fragments. The EcoRI 1.3 kb family were cloned into M13 mp19, and a repair positive (1.3 A) and a repair negative (1.3 B) were identified and sequenced. The repair positive clone contained (a) (CA)₂₂ repeat, (b) a (CAGA)₆ repeat and (c) 4 sequences sharing high homology with various hypervariable minisatellite (HVMS) sequences. One of the HVMS sequence contained a GGCAGG motif known to be responsible for germline instability. The repair negative clone had (a) (CA)₆ repeat and (b) a HVMS like sequence without GGCAGG. The significance of these motifs and their relevance to the events of DNA metabolism at pachytene interval have been discussed.     

Hybridization and introgression of the genomes of Drosophila nasuta and Drosophila albomicans: evolution of new karyotypes.

Drosophila nasuta (2n = 8) and Drosophila albomicans (2n = 6) are cross-fertile allopatric sibling chromosomal races of the nasuta subgroup of Drosophila. Hybrids of these races can be maintained for any number of generations. Some of the introgressed hybrid lineages of D. nasuta and D. albomicans, after passing through a transient phase of karyotypic polymorphism, ended up with a stable karyotype whose composition is different from those of the parental races. Such hybrid populations were called cytoraces, in which the chromosomes of D. nasuta and D. albomicans are represented in different combinations. The karyotypic composition of 16 such cytoraces have been presented and discussed with reference to evolutionary strategies such as balancing selection, directional selection, and sex-specific effect on different components of the evolving karyotypes.     

Characterization of Upstream Sequences of the <Emphasis Type=Italic>LOJ</Emphasis> Gene Leads to Identification of a Novel Enhancer Element Conferring Lateral Organ Junction-Specific Expression in <Emphasis Type=Italic>Arabidopsis thaliana</Emphasis>

Isolation and characterization of promoters are important in understanding gene regulation and genetic engineering of crop plants. Earlier, a pentatricopeptide repeat protein (PPR) encoding gene (At2g39230), designated as Lateral Organ Junction (LOJ) gene, was identified through T-DNA promoter trapping in Arabidopsis thaliana. The upstream sequence of the LOJ gene conferred on the reporter gene a novel LOJ-specific expression. The present study was aimed at identifying and characterizing the cis-regulatory motifs responsible for tissue-specific expression in the −673 and +90 bases upstream of the LOJ gene recognized as LOJ promoter. In silico analysis of the LOJ promoter revealed the presence of a few relevant regulatory motifs and a unique feature like AT-rich inverted repeat. Deletion analysis of the LOJ promoter confirmed the presence of an enhancer-like element in the distal region (−673/−214), which stimulates a minimal promoter-like sequence in the −424/−214 region in a position and orientation autonomous manner. The −136/+90 region of the LOJ promoter was efficient in driving reporter gene expression in tissues like developing anthers and seeds of Arabidopsis. A positive regulation for the seed- and anther-specific expression module was contemplated within the 5′ untranslated region of the LOJ gene. However, this function was repressed in the native context by the lateral organ junction-specific expression. The present study has led to the identification of a novel lateral organ junction-specific element and an enhancer sequence in Arabidopsis with potential applications in plant genetic engineering.     

A tetracopper(II) complex with N,N′-bis(picolinoyl)hydrazine

Synthesis, physical properties and X-ray structure of a hydrated tetranuclear copper(II) complex [Cu₄(μ-diph)₂(μ-H₂O)₂(O₂CCH₃)₄(H₂O)₂]·4H₂O with N,N′-bis(picolinoyl)hydrazine (H₂diph) are reported. The centrosymmetric complex has two types of copper(II) centres with distorted square-pyramidal N₂O₃ coordination spheres. The dinucleating trans planar diph²⁻ ligands are parallel to each other and act as N₂O-donor to one metal centre and N₂-donor to the other metal centre. The complex has a rectangular {Cu₄(μ-N-N)₂(μ-OH₂)₂} core with Cu···Cu distances as 4.834(1) and 3.762(1) Å. Solid state as well as solution electronic spectra show several transitions in the wavelength range 700-280 nm. The room temperature (298 K) solid state magnetic moment is 3.55 μ_B. The powder EPR spectra at 298 and 130 K are very similar and axial (g_∥ = 2.25 and g_⊥ = 2.08) in character.     

A Mass Spectrometric Study for Comparative Analysis and Evaluation of Metabolite Recovery from Plasma by Various Solvent Systems

A solvent system that extracts a maximum number of metabolites belonging to diverse chemical classes from complex biofluids, such as plasma, may offer useful inputs to understand the metabolic and physiological state of an individual. The present study compared seven solvent systems for extraction of metabolites from plasma. The extracts were analyzed by mass spectrometry (MS) and MS/MS (MS2) using a quadrupole time-of-flight liquid chromatography/MS system in positive and negative modes of ionization. Metabolites with molecular mass below 400 were identified using Human Metabolome Database MS2 and MS search interfaces. The acetone/isopropanol (2:1) system yielded promising results in positive ionization mode, as the maximum number of MS and MS2 features was detected in the extract. It was found to be superior in extraction of various classes of metabolites, especially organic acids, nucleosides and nucleoside derivatives, and heterocyclic molecules. Glycerophosphocholines in the mass range of 400–700 were found to be efficiently extracted by the methanol/chloroform/water (8:1:1) system. In negative mode as well, the maximum number of MS2 features was detected in methanol/chloroform/water and acetone/isopropanol extracts. The fingerprints of molecular features obtained in the negative and positive modes differed from each other to a significant extent.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450BM3–F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Amplification of phytochrome induced morphogenesis in plants by the cryptic red light signal (CRS)

The regulation of endogenous levels of ascorbic acid in soybean by far-red absorbing form of phytochrome (Pfr) and by cryptic red light signal (CRS) was studied. Cryptic red light signal is produced by red light pre-irradiation of a photoreceptor other than far-red absorbing form of phytochrome (Pfr) and CRS amplifies the action of phytochrome. The endogenous level of ascorbic acid levels enhanced by phytochrome was amplified by CRS. The lifetime of CRS was from 0 to 2 h and the peak of enhancement of ascorbic acid due to CRS was between 16 to 24 h of dark incubation after the end of the treatment. CRS was found to be ineffective on UV-B enhanced endogenous levels of ascorbic acid.Key words: ascorbic acid, cryptic red light signal, glycine max, phytochrome, ultraviolet-BThe phytochrome mediated morphogenesis involves the conversion of Pr [red absorbing form] to Pfr [far-red absorbing form] and the magnitude of the response is dependent on Pfr/P tot ratio established at the end of the irradiation.¹ In broom Sorghum anthocyanin synthesis induced by red light [R₁] is reversible with far-red light. But a second red pulse [R₂] given after the reversal resulted in increased anthocyanin production compared to the first pulse [R₁]. When the red pulse was repeatedly given after every reversal with far-red, the anthocyanin production increased proportionately to the number of previously given pulses.² Thus red pre-treatment induced a change in the cellular physiological state or change in content of a relevant substance[s] which is designated as Cryptic Red Light Signal [CRS] associated with red signal transduction.² CRS was first characterized in detail in Broom Sorghum as Pfr amplifying signal produced by red pre-irradiation. CRS is inactive in the absence of Pfr but enhances the action of Pfr. CRS escapes reversal when the plants are exposed to far-red and is probably produced by a different species of phytochrome, distinct from the conventional reversible phytochrome.³We have investigated whether CRS influences other phytochrome regulated processes in plants in addition to anthocyanin synthesis. We chose another process, the synthesis of endogenous ascorbic acid, which is also regulated by conventional phytochrome.⁴ In soybean, the endogenous level of ascorbic acid is enhanced by conventional far-red reversible form of phytochrome. In addition, an independent UV-B photoreceptor [non reversible with far-red light] also enhances the endogenous synthesis of ascorbic acid in soybean. By using repeated pulses of red light, we have demonstrated that the Cryptic Red Signal is operative in soybean also and it amplifies the red light induced enhancement in the level of ascorbic acid. That CRS is active only in the presence of Pfr is demonstrated by the fact that pre-irradiation with red light is ineffective in amplifying UV-B induced enhancement of ascorbic acid levels. A similar observation on UV-B induced anthocyanin synthesis has been made in Broom Sorghum.² A separate UV-B photoreceptor independent of phytochrome operates in the plants.⁵ Although CRS is presumably produced by pre-irradiation with red light, it does not enhance UV-B induced anthocyanin synthesis or ascorbic acid synthesis in the absence of formation of Pfr by the second red pulse.The life-time of CRS was determined as 6 h in 20°C and 3 h in 24°C grown seedlings of Broom Sorghum with reference to anthocyanin synthesis.² The life-time of CRS determined in soybean seedlings grown at 25°C was upto 1 h.⁶ Since growing seedlings at a low temperature enhanced the effectiveness of CRS in Broom Sorghum, it was concluded that low temperature may either extend the lifetime of CRS or generate higher amount of CRS.² Although the exact nature of CRS is yet to be analyzed, work in our laboratory has established the universal nature of this signal and evidences have been obtained for CRS effect in promoting red light induced hypocotyls inhibition in Cucumber seedlings and also red light induced synthesis of betacyanins in Amaranthus seedlings (submitted for publication).     

Non-enzymatic antioxidative defence in drought-stressed mulberry (<Emphasis Type="Italic">Morus indica</Emphasis> L.) genotypes

The present study investigated drought-induced responses of non-enzymatic antioxidants in four diverse mulberry genotypes (Morus indica L. S-36, M-5, MR-2 and V-1). Inside the glasshouse, potted plants were subjected to four water regimes for 75 days: (a) control: pots maintained at 100% pot water holding capacity (PC) (b) low water stress: 75% PC (c) medium water stress: 50% PC and (d) high water stress: 25% PC. Photosynthetic leaf gas exchange and non-enzymatic antioxidants including α-tocopherol, ascorbic acid (AA), glutathione, proline and total carotenoids were measured in leaves at regular intervals. Amongst all, V-1 was relatively drought tolerant and showed exceeded accumulation of α-tocopherol and AA-glutathione pool in association with higher carotenoids and proline contents. Susceptible S-36, M-5 and MR-2 could not induce any significant up-regulation in AA-glutathione pool leading to endogenous loss of α-tocopherol and more lipid peroxidation. Reactive oxygen species (ROS) like hydrogen peroxide (H₂O₂) and superoxide (O₂ ^{· ?}) showed apparent accumulation in water-stressed leaves and significantly contributed to lipid peroxidation in susceptible genotypes when compared to V-1. Our study demonstrated that proline, AA and glutathione were the major non-enzymatic antioxidants in mulberry with α-tocopherol and carotenoids as good additional indicators for drought stress tolerance. These non-enzymatic antioxidants can cumulatively render effective protection against oxidative damage and can be considered as reliable markers for screening drought-tolerant mulberry genotypes.