Isolation and characterization of thiamin-binding protein from chicken egg white.

A thiamin-binding protein was isolated and characterized from chicken egg white by affinity chromatography on thiamin pyrophosphate coupled to aminoethyl-Sepharose. The high specificity of interaction between the thiamin-binding protein and the riboflavin-binding protein of the egg white, with a protein/protein molar ratio of 1.0, led to the development of an alternative procedure that used the riboflavin-binding protein immobilized on CNBr-activated Sepharose as the affinity matrix. The thiamin-binding protein thus isolated was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, double immunodiffusion and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, had a mol.wt. of 38,000 +/- 2000 and was not a glycoprotein. The protein bound [14C]thiamin was a molar ratio of 1.0, with dissociation constant (Kd) 0.3 micrometer.     

Oestrogen-induced synthesis of thiamin-binding protein in immature chicks. Kinetics of induction, hormonal specificity and modulation

A specific radioimmunoassay procedure was developed to monitor the plasma concentrations of thiamin-binding protein, a minor yolk constituent of the chicken egg. By using this sensitive assay, the kinetics of oestrogen-induced elaboration of this specific protein in immature chicks was investigated. After a single injection of the steroid hormone, with an initial lag period of 4–5h the thiamin-binding protein rapidly accumulated in the plasma, attaining peak concentrations around 75h and declining thereafter. A 4-fold amplification of the response was noticed during the secondary stimulation, and this increased to 9-fold during the tertiary stimulation with the steroid hormone. The magnitude of the response was dependent on the hormone dose, and the initial latent period and the duration of the ascending phase of induction were unchanged for the hormonal doses tested during both the primary and secondary stimulations. The circulatory half-life of the protein was 6h as calculated from the measurement of the rate of disappearance of the exogenously administered ¹²⁵I-labelled protein. Simultaneous administration of progesterone, dihydrotestosterone or corticosterone did not alter the pattern of induction. On the other hand, hyperthyroidism markedly decreased the oestrogenic response, whereas propylthiouracil-induced hypothyroidism had the opposite effect. The anti-oestrogen E- and Z-clomiphene citrates, administered 30min before oestrogen, effectively blocked the hormonal induction. α-Amanitin and cycloheximide administered along with or shortly after the sex steroid severely curtailed the protein elaboration. A comparison of the kinetics of induction of thiamin- and riboflavin-binding proteins by oestrogen revealed that, beneath an apparent similarity, a clear-cut difference exists between the two vitamin-binding proteins, particularly with regard to hormonal dose-dependent sensitivity of induction and the half-life in circulation. The steroid-mediated elaboration of the two yolk proteins thus appears to be not strictly co-ordinated, despite several common regulatory features underlying their induction.     

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

The presence of O-acetyl-5-methoxytryptophol in the pineal glands of rats kept in the dark for 8 h, but not in the light, has been shown by means of g.l.c.-mass spectrometry. It is suggested that this compound may be the biologically active precursor of circulating methoxytryptophol.     

Saccharomyces cerevisiae Mre11 is a high-affinity G4 DNA-binding protein and a G-rich DNA-specific endonuclease: implications for replication of telomeric DNA

In Saccharomyces cerevisiae, Mre11p/Rad50p/Xrs2p (MRX) complex plays a vital role in several nuclear processes including cellular response to DNA damage, telomere length maintenance, cell cycle checkpoint control and meiotic recombination. Telomeres are comprised of tandem repeats of G-rich DNA and are incorporated into non-nucleosomal chromatin. Although the structure of the yeast telomeric DNA is poorly understood, it has been suggested that the G-rich sequences can fold into G4 DNA, which has been shown to inhibit DNA synthesis by telomerase. However, little is known about the factors and mechanistic aspects of the generation of appropriate termini for DNA synthesis by telomerase. Here, we show that S.cerevisiae Mre11 protein (ScMre11p) possesses substantially higher binding affinity for G4 DNA, over single- or double-stranded DNA, and binding was inhibited by poly(dG) or porphyrin. Binding of ScMre11p to G4 DNA was most robust, compared with G2′ DNA and the resulting protein–DNA complexes were strikingly very resistant to dissociation by NaCl. Remarkably, binding of ScMre11p to G4 DNA and G-rich single-stranded DNA was accompanied by the endonucleolytic cleavage at sites flanking the array of G residues and G-quartets in Mn²⁺-dependent manner. Collectively, these results suggest that ScMre11p is likely to play a major role in generating appropriate substrates for DNA synthesis by telomerase and telomere-binding proteins. We discuss the implications of these findings with regard to telomere length maintenance by telomerase-dependent and independent mechanisms.     

Statin decreases endothelial microparticle release from human coronary artery endothelial cells: implication for the Rho-kinase pathway

OBJECTIVE: Elevated plasma levels of endothelial microparticles (EMPs) are associated with the presence of clinical atherosclerosis. Considering the anti-inflammatory properties of HMG-CoA reductase inhibitors on the endothelium, we studied the effect of fluvastatin on the release of EMPs in cultured human coronary artery endothelial cells (HCAEC). METHODS AND RESULTS: EMPs were generated in TNF-alpha-activated HCAECs. The absolute number of EMPs was enumerated using a novel two-color flow cytometric immunostaining technique with TruCount beads as an internal reference. EMPs are defined as EC membrane vesicles (1-2 microm in size) with a characteristic immunophenotype. The addition of fluvastatin to TNF-alpha-activated HCAECs significantly suppressed EMP release. Fluvastatin suppressed TNF-alpha-induced Rho activation.The Rho-kinase inhibitor, Y-27632, reproduced the effect of statin. CONCLUSION: EMP release from TNF-alpha-activated HCAECs is suppressed by fluvastatin. In addition, the Rho/Rho-kinase may play an important role in modulating EMP release.     

Purification and Characterization of Indian Cassava Mosaic Virus

The Indian cassava mosaic virus (ICMV) was transmitted by the whitefly Bemisia tabaci and sap inoculation. ICMV was purified from cassava and from systemically infected Nicotiana benthamiana leaves. Geminate particles of 16–18 × 30 nm in size were observed by electron microscopy. The particles contained a single major protein of an estimated molecular weight of 34,000. Specific antiserum trapped geminate particles from the extracts of infected cassava and N. benthamiana plants in ISEM test. The virus was detected in crude extracts of infected cassava, ceara rubber, TV. benthamiana and N. tabacum cv. Jayasri plants by ELISA. ICMV appeared serologically related to the gemini viruses of Acalypha yellow mosaic, bhendi yellow vein mosaic, Croton yellow vein mosaic, Dolichos yellow mosaic, horsegram yellow mosaic, Malvastrum yellow vein mosaic and tobacco leaf curl.     

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein,a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

Mycobacterium leprae recA harbors an in‐frame insertion sequence that encodes an intein homing endonuclease (PI‐MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG‐type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI‐MleI is distinctive from other members of the family of LAGLIDADG‐type HEases for its modular structure with functionally separable domains for DNA‐binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI‐MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active‐site residues essential for DNA target site recognition and double‐stranded DNA cleavage, we performed site‐directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild‐type PI‐MleI and its variants disclosed that the two amino acid residues, Asp¹²² (in Block C) and Asp¹⁹³ (in functional Block E), are crucial to the double‐stranded DNA endonuclease activity, whereas Asp²¹⁸ (in pseudo‐Block E) is not. However, despite the reduced catalytic activity, the PI‐MleI variants, like the wild‐type PI‐MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA‐binding affinities, but abolished the double‐stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double‐stranded DNA cleavage activity, compared with the wild‐type PI‐MleI. These results provide compelling evidence that Asp¹²² and Asp¹⁹³ in DOD motif I and II, respectively, are bona fide active‐site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.     

Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.     

Weed and crop plants as reservoirs of peanut bud necrosis tospovirus and its occurrence in South India

Survey was carried out on occurrence of peanut bud necrosis disease (PBND) in Karnataka and Andhra Pradesh (AP) the major groundnut growing states in South India. PBND incidence was observed on crops, viz., greengram, blackgram, tomato, watermelon, cowpea, chilli, cucumber and sesame besides groundnut. The PBND incidence was higher in rainy season crops than in post-rainy season. Its proportion varied from place to place, and it was high in the vegetable and pulses growing areas. The weed plant species Achyranthus aspera, Acalypha indica, Alysicarpus rugosus, A. longifolia, Commelina bengalensis, Commelina jacobi, Corchorus trilocularis, Cyanoptis cuculetta, Eclipta alba, Euphorbia geniculata, Lochnera pusilla, Physalis minima and Sesbania rostrata were recorded for the first time as alternate hosts of PBND. Infection was also noticed for the first time on cucumber, muskmelon and sesame crops. Five weed species, viz., Achyranthus aspera, Ageratum conyzoides, Alysicarpus rugosus, Commelina bengalensis and Vigna trilobata were found abundant in AP and Karnataka. More infection was observed on Ageratum conyzoides (17.56%) compared with other weeds.     

Managament of peanut bud necrosis disease in groundnut by botanical pesticides

In the management of PBND in groundnut neem seed extract (10%), we recorded significantly lower PBND incidence, AUDPC, apparent infection rate (20.41, 19.5% PBND; 956, 756 AUDPC; 0.0026, 0.002 apparent infection rate) followed by neem cake extract 10% spray (2747, 23.36% PBND; 1275, 1316 AUDPC; 0.0034, 0.0025 apparent rate of infection in 2000–2001 and 2001–2002, respectively). Weekly spray of monocrotophos reduced the PBND (26.77%) and recorded significantly higher pod yields (2547, 2465 kg/ha) and lower AUDPC (861, 1373), and apparent infection rate (0.0021, 0.0019 in 2000–2001 and 2001–2002, respectively) compared to other treatments.