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D V Reddy

Publications and source records attributed to D V Reddy.

At least 19 recordsLinked to original sources
PubMed2001

Phage-displayed peptides that mimic aflatoxin B1 in serological reactivity.

AIMS: To test phage-displayed random peptide libraries as sources of peptides that mimic the binding of aflatoxin B1 to monoclonal antibodies raised against the toxin. METHODS AND RESULTS: For two of the three MAbs tested, clones were obtained by panning, producing phage that bound specifically to MAb 13D1-1D9 (MAb 24; specific for aflatoxins B1 and G1) and MAb 6E12-1E9 (MAb 13; specific for aflatoxins B1, G1 and B2) in ELISA. The amino acid sequences of the binding peptides varied. Those binding to MAb 24 contained the sequence of '...YMD...', and those that bound to MAb 13 contained the dipeptide 'PW'. Mimotope phage was used in a competition ELISA format for assaying aflatoxin concentrations. CONCLUSION: The results show that mimotope preparations are effective substitutes for pure toxin in these ELISA procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: These results should contribute significantly to enhancing the safety and diminishing the costs of aflatoxin assays.

Aflatoxin B1↗
PubMed2001

Occurrence of ochratoxin A in black pepper, coriander, ginger and turmeric in India.

Ochratoxin A (OA) contamination of black pepper, coriander seeds, powdered ginger and turmeric powder was estimated using indirect competitive ELISA. Samples (1 g) were extracted with 0.5% potassium chloride (KCl) in 70% methanol (5 ml) and diluted subsequently to give two-fold to ten-fold step-wise dilutions in phosphate-buffered saline containing 0.05% Tween 20 and 0.2% bovine serum albumin (PBS-T BSA). For extracts from the spices analysed, ELISA estimates of OA concentrations were compared with those made by HPLC. All estimates were within 1-2 standard deviation of the ELISA values. More than 90% of OA added to spice samples was recovered from samples containing between 5 and 100 microg/kg OA. Extracts of OA-free spice samples contained substances that interfered with ELISA, presumably because of non-specific reactions. This effect was avoided by preparing all the test solutions in extracts of OA-free spice samples. In 126 samples obtained from retail shops, OA was found to exceed 10 microg/kg in 14 (in the range of 15-69 microg/kg) of 26 black pepper samples, 20 (in the range of 10-51 microg/kg) of 50 coriander samples, two (23 microg/kg and 80 microg/kg) of 25 ginger samples and nine (in the range of 11-102 microg/kg) of 25 turmeric samples. This is the first record in India of the occurrence of OA in what are some of the most widely used spices in Indian cooking.

Apiaceae↗
PubMed2001

Aflatoxins B1 in different grades of chillies (Capsicum annum L.) in India as determined by indirect competitive-ELISA.

Samples of the three grades of chilli pod (grades 1 to 3) were collected during surveys in 1998 and 1999 from the principal market yards and cold storage facilities of the major chilli-growing areas of Andhra Pradesh (AP), India. Chilli powders were collected from different supermarkets in Hyderabad, AP. They were analysed for aflatoxin B1 (AFB1) content by an indirect competitive ELISA. To avoid the influence of interfering substances present in chilli extracts, it was necessary to prepare the aflatoxin standards in methanol extracts of chillies free from aflatoxins. For nine representative samples there was good agreement between ELISA and HPLC estimations of AFB1 and the results suggested that the ELISA procedure adopted was dependable. Of the 182 chilli samples tested, 59% of the samples were contaminated with AFB1 and 18% contained the toxin at non-permissible levels. The highest AFB1 concentration of 969 microg/kg was found in one sample representing grade 3. Overall the maximum percentage of chilli pods showing AFB1 levels higher than 30 microg/kg (non-permissible levels) was in grade 3. Chilli pods stored in refrigerated rooms showed the lowest proportion of samples containing aflatoxin. Nearly 9% of the chilli powders sold in supermarkets contained non-permissible aflatoxin levels. This report highlights the importance of using grade 1 chilli pods to minimize aflatoxin contamination.

Aflatoxin B1↗
PubMed2000

High resolution solution structure of the 1.3S subunit of transcarboxylase from Propionibacterium shermanii.

Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and oxalacetate. Within the multi-subunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier and also binds the other two subunits to assist in the overall assembly of the enzyme. The 1.3S subunit is a 123 amino acid polypeptide (12.6 kDa) to which biotin is covalently attached at Lys 89. The three-dimensional solution structure of the full-length holo-1.3S subunit of TC has been solved by multidimensional heteronuclear NMR spectroscopy. The C-terminal half of the protein (51-123) is folded into a compact all-beta-domain comprising of two four-stranded antiparallel beta-sheets connected by short loops and turns. The fold exhibits a high 2-fold internal symmetry and is similar to that of the biotin carboxyl carrier protein (BCCP) of acetyl-CoA carboxylase, but lacks an extension that has been termed "protruding thumb" in BCCP. The first 50 residues, which have been shown to be involved in intersubunit interactions in the intact enzyme, appear to be disordered in the isolated 1.3S subunit. The molecular surface of the folded domain has two distinct surfaces: one side is highly charged, while the other comprises mainly hydrophobic, highly conserved residues.

Acetyl-CoA Carboxylase↗
PubMed2000

Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA.

Polyclonal antibodies were produced for Ochratoxin A (OA) by injecting OA-bovine serum albumin (BSA) conjugate subcutaneously at multiple sites into a New Zealand White inbred rabbit. Antiserum could be used at a dilution exceeding 1:100 000 in an indirect competitive enzyme-linked immunosorbent assay (ELISA), and detected OA concentrations up to 0.1 ng/mL. The 50% inhibition binding (I(50)) of OA was 5 ng/mL. Antibodies did not react with ochratoxin B, coumarin, 4-hydroxycoumarin, L-phenylalanine, and aflatoxin B1. OA contamination in chilies (Capsicum annum L.) collected from commercial markets and cold storage units was determined. The mean recoveries from OA-free chilies spiked with 1 to100 microg of OA per kg of chili sample were 90-110% with a standard deviation of <10%. Of 100 chili samples tested, 26 were found to contain over 10 microg/kg of OA. In 12 samples the OA concentration varied from 10 to 30 microg/kg, in 10 samples from 30 to 50 microg/kg, in 3 samples from 50 to100 microg/kg, and in one sample it was 120 microg/kg. This is the first record in India of OA in chilies, a major component of cooked foods in this country, and it is noteworthy that OA contamination exceeded the permissible limit for human consumption of less than 20 microg/kg in over 26% of the market samples tested.

Antibodies↗
PubMed1999

Production and characterization of monoclonal antibodies for aflatoxin B1.

Hybridomas that secreted antibodies for aflatoxin B1 were selected using two immunization protocols referred to as A and B. Protocol A is a standard immunization method and resulted in the selection of only two clones that produced monoclonal antibodies against aflatoxin B1. In protocol B a unique immunization schedule which resulted in the generation of 10 hybridomas is described. Of the 10, one antibody was highly specific to B1, four antibodies reacted equally strongly with B1, G1 and weakly with B2. Another four reacted strongly with B1 and weakly with B2 and G1. One clone reacted equally strongly with B1, G1 and B2. Interestingly all the 10 antibodies showed little or no cross-reaction with G2.

Aflatoxin B1↗
PubMed1998

Structural characterization of the entire 1.3S subunit of transcarboxylase from Propionibacterium shermanii.

Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier. The 1.3S is a 123-amino acid polypeptide (12.6 kDa), to which biotin is covalently attached at Lys 89. We have expressed 1.3S in Escherichia coli with uniform 15N labeling. The backbone structure and dynamics of the protein have been characterized in aqueous solution by three-dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. The secondary structure elements in the protein were identified based on NOE information, secondary chemical shifts, homonuclear 3J(HNHalpha) coupling constants, and amide proton exchange data. The protein contains a predominantly disordered N-terminal half, while the C-terminal half is folded into a compact domain comprising eight beta-strands connected by short loops and turns. The topology of the C-terminal domain is consistent with the fold found in both carboxyl carrier and lipoyl domains, to which this domain has approximately 26-30% sequence similarity.

Amino Acid Sequence↗
PubMed1998

Peanut yellow spot virus is a member of a new serogroup of Tospovirus genus based on small (S) RNA sequence and organization.

Peanut yellow spot virus (PYSV) represents a distinct tospovirus species based on serology and nucleic acid hybridization. THe sequence of the S RNA was 2970 nucleotides with 22 nucleotide long inverted repeats (with three mismatches) at the termini. The coding was ambisense with a long open reading frame (ORF) in each strand. The 5'-large ORF (1,400 nucleotides in the viral sense (v) strand) encoded a protein with a predicted size of 53.2 kDa that was identified as the nonstructural (NSs) protein based on 16-21% sequence identity and 42-48% sequence similarity with other tospoviruses. A 3' ORF (741 nucleotides) in the virus complementary (vc) sense encoded a 28.0 kDa protein that was identified as the nucleocapsid (N) gene based on immuno-blot analysis of the in vitro expressed protein with PYSV polyclonal antiserum. The predicted N protein had 24-28% amino acid sequence identity and 44-51% sequence similarity with the members of other serogroups. In contrast to other tospoviruses, a third ORF (204 nucleotides) occurred in the vc strand, which could encode a protein with a predicted size of 7.5 kDa with two strong hydrophobic regions. The low degree of homology of N and NSs protein sequences with other serogroup members coupled with an additional ORF suggests that PYSV should be classified as a distinct species of the Tospovirus genus. This conclusion also is supported by the absence of serological cross reaction with other serogroups, and biological characteristics including thrips transmission, symptoms and host range.

Amino Acid Sequence↗
PubMed1998

Evidence that whitefly-transmitted cowpea mild mottle virus belongs to the genus Carlavirus.

Two strains of whitefly-transmitted cowpea mild mottle virus (CPMMV) causing severe (CPMMV-S) and mild (CPMMV-M) disease symptoms in peanuts were collected from two distinct agro-ecological zones in India. The host-range of these strains was restricted to Leguminosae and Chenopodiaceae, and each could be distinguished on the basis of symptoms incited in different hosts. The 3'-terminal 2500 nucleotide sequence of the genomic RNA of both the strains was 70% identical and contains five open reading frames (ORFs). The first three (P25, P12 and P7) overlap to form a triple gene block of proteins, P32 encodes the coat protein, followed by P12 protein located at the 3' end of the genome. Genome organization and pair-wise comparisons of amino acid sequences of proteins encoded by these ORFs with corresponding proteins of known carlaviruses and potexviruses suggest that CPMMV-S and CPMMV-M are closely related to viruses in the genus Carlavirus. Based on the data, it is concluded that CPMMV is a distinct species in the genus Carlavirus.

Amino Acid Sequence↗
PubMed1998

Characterization of the large (L) RNA of peanut bud necrosis tospovirus.

The nucleocapsids purified from peanut plants systemically infected with peanut bud necrosis virus (PBNV), a member of the genus Tospovirus, contained both viral(v) and viral complementary(vc) sense L RNAs. Defective forms of L RNA containing 'core polymerase region' were observed. The full length L RNA of PBNV was sequenced using overlapping cDNA clones. The 8911 nucleotide L RNA contains a single open reading frame (ORF) in the vc strand, and encodes a protein of 330 kDa. At the 5' and 3' termini of the v sense RNA there were 247 and 32 nt untranslated regions, respectively, containing an 18 nt complementary sequence with one mismatch. Comparisons of the predicted amino acid sequence of the L protein of PBNV with other members of Bunyaviridae suggest that the L protein of PBNV is a viral polymerase. The L protein had highest identity in the 'core-polymerase domain' with the corresponding regions of other tospoviruses, tomato spotted wilt virus and impatiens necrotic spot virus.

Amino Acid Sequence↗
PubMed1997

Absence of observable biotin-protein interactions in the 1.3S subunit of transcarboxylase: an NMR study.

Transcarboxylase (TC) is a biotin-containing enzyme catalyzing the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and oxalacetate. The transfer is achieved via carboxylated biotin bound to a 1.3S subunit within the multisubunit enzyme complex. The 1.3S subunit of TC is a 123 amino acid polypeptide, to which biotin is covalently attached at Lys 89. We have overexpressed 1.3S in Escherichia coli and characterized the biotinylated and apo-forms by 1D- and 2D-NMR spectroscopy. To search for protein-biotin interactions, which could modulate the reactivity of the biotin ring on the 1.3S subunit, we have compared the chemical shifts, relaxation parameters, and NH exchange rates of the ureido ring protons of free and 1.3S-bound biotin. These properties are similar for both forms of the biotin. Further, NOE experiments on 1.3S revealed no detectable cross peaks between biotin and the protein. Consistent with these findings, the 2D NMR data for holo- and apo-1.3S are essentially identical indicating little or no changes in conformation between the two forms of the protein. The conclusion that strong protein-biotin interactions do not exist in 1.3S contrasts with the findings for the biotin carboxylase carrier protein from E. coli acetyl-CoA carboxylase, which reveal significant biotin-protein contacts [Athappilly, F. K., and Hendrickson, W. A. (1995) Structure 3, 1407-1419]. Further, the biotin NH1' exchange rates determined for 1.3S show that in the region of optimal activity for TC (pH 5.5-6.5) acid-catalyzed exchange predominates. In this pH range the base-catalyzed rate is too small (< 1 s-1) to account for the turnover rate of the enzyme. Thus, the means by which the N1' atom is activated for nucleophilic attack of the carboxyl group in methylmalonyl-CoA does not appear to depend on interactions within the 1.3S subunit alone; rather activation must occur at the interfaces of the subunits in the holoenzyme.

Acetyl-CoA Carboxylase↗
PubMed1996

NMR characterization of side chain flexibility and backbone structure in the type I antifreeze protein at near freezing temperatures.

The flexibility of the polar side chains in the alpha-helical Type I antifreeze protein (AFP) near the solution freezing temperature was investigated by two-dimensional nuclear magnetic resonance spectroscopy. These experiments were conducted to define the rotameric conformations of the proposed ice-binding groups, threonines and asparagines, in order to probe the molecular mechanism for ice binding. On the basis of the 3J alpha beta 2 NMR coupling constant values of 7.1, 8.5, 8.5, and 6.8 Hz for residues T2, T13, T24, and T35, respectively, it can be calculated that the regularly spaced ice-binding threonines sample many possible rotameric states prior to ice binding. The lack of a dominant side chain rotamer is further corroborated by nuclear Overhauser distance measurements for T13 and T24. N16 and N27, both with 3J alpha beta 2 and 3J alpha beta 3 coupling constants of 8.4 and 4.5 Hz, respectively, show a slight preference for the side chain conformation with a chi 1 of -60 degrees. These data suggest that prior to ice binding the threonine and asparagine side chains are free to rotate and that a unique preformed ice-binding structure in solution is not apparent. These observations do not support the rigid side chain model proposed recently by an X-ray study [Sicheri, F., & Yang, D. S. C. (1995) Nature 375, 427-431].

Amino Acid Sequence↗
PubMed1996

NMR study of dideoxynucleotides with anti-human immunodeficiency virus (HIV) activity.

The molecular structures of 3'-azido-2',3'-dideoxyribosylthymine 5'-triphosphate (AZTTP), 2',3'-dideoxyribosylinosine 5'-triphosphate (ddlTP), 3'-azido-2',3'-dideoxyribosylthymine 5'-monophosphate (AZTMP) and 2',3'-dideoxyribosyladenine 5'-monophosphate (ddAMP) have been studied by NMR to understand their anti-HIV activity. For ddAMP and ddITP, conformations are almost identical with their nucleoside analogues with sugar ring pucker equilibriating between C3'-endo (approximately 75%) and C2'-endo (approximately 25%). AZTMP and AZTTP on the other hand show significant variations in the conformational behaviour compared with 3'-azido-2',3'-dideoxyribosylthymine (AZT). The sugar rings for these nucleotides have a much larger population of C2'-endo (approximately 75%) conformers, like those observed for natural 2'-deoxynucleosides and nucleotides. The major conformers around C5'-O5', C4'-C5' and the glycosidic bonds are the beta 1, gamma + and anti, respectively.

Antiviral Agents↗
PubMed1996

Peanut bud necrosis tospovirus S RNA: complete nucleotide sequence, genome organization and homology to other tospoviruses.

The complete nucleotide sequence of the S RNA of peanut bud necrosis virus (PBNV) has been determined. The RNA is 3 057 nucleotides in length, contains inverted repeats and two open reading frames (ORFs) with an ambisense coding strategy that are separated by an A+U-rich intergenic region. One ORF (1 320 nucleotides in the viral sense strand) encodes a Mr 49.5 kDa protein, identified as the nonstructural (NSs) protein based on similarity to published tospovirus sequences. The second ORF (831 nucleotides in virus complementary strand) encodes a Mr 30.6 kDa protein. This protein was identified as the nucleocapsid (N) protein based on sequence similarities. Amino acid sequence comparison of N and NSs proteins revealed identities of 22-34% with the reported tospovirus isolates of serogroups I, II, and III, whereas it had 82-86% identity with viruses in serogroup IV, watermelon silver mottle virus (WSMV) and tomato isolate of peanut bud necrosis (PBNV-To). Two subgenomic RNA species detected in PBNV infected tissue corresponded to the predicted sizes (1.65 and 1.4 kb) of the NSs and N mRNAs. The data presented show conclusively that PBNV should be included in serogroup IV, along with WSMV and PBNV-To.

Amino Acid Sequence↗
PubMed1996

The nucleotide sequence of RNA-1 of Indian peanut clump furovius.

The nucleotide sequence of RNA-1 of an isolate of the H serotype of Indian peanut clump virus (IPCV-H) was shown to comprise 5,841 nucleotides. The RNA contains three open reading frames (ORF) which are between nucleotides 133 and 3,522, nucleotides 3,526 and 5,103 (assuming expression by suppression of the ORF 1 termination codon) and nucleotides 5,168 and 5,539. The encoded polypeptides have M(r), of 129,687 (p130), 60,188 (p60) and 14,281 (p14). ORF 2 is thought to be expressed by suppression of the termination codon of ORF 1 to produce a M(r) 189,975 product (p190). p130 contains sequences characteristic of proteins with methyl transferase and NTP-binding properties and p190 contains these and sequences characteristic of RNA-dependent RNA polymerases. The nucleotide sequence of IPCV RNA-1 is similar to that of peanut clump virus (PCV) and corresponding encoded polypeptides are 88% (p130), 95% p60 and 75% (p14) identical. The sequences of the translation products are also similar to those of soil-borne wheat mosaic virus and barley stripe mosaic virus. Oligonucleotide primers, designed on the basis of the sequences of RNA-1 of IPCV and PCV, were effective in reverse transcription-PCR amplification of these RNAs and that of IPCV isolates of the serologically distinct L and T serotypes.

Amino Acid Sequence↗
PubMed1996

The complete nucleotide sequence and genome organization of the M RNA segment of peanut bud necrosis tospovirus and comparison with other tospoviruses.

The M RNA of peanut bud necrosis virus (PBNV; synonym groundnut bud necrosis virus) is 4801 nucleotides in length. It comprised two ORFs in an ambisense organization and terminal inverted repeats. The 3' large ORF (3363 nucleotides in the virus-complementary strand) encoded a protein with a predicted size of 127.2 kDa which was identified as the glycoprotein precursor (GP) of the G1 and G2 glycoproteins. A comparison of the deduced amino acid sequence of GP revealed 37% identity and 58-59% similarity with that of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), and 21-23% identity and 44-47% similarity with those of other members of the genus Bunyavirus. The 5' small ORF (924 nucleotides in the virussense strand) encoded a 34.2 kDa protein which was identified as the non-structural (NSm) protein based on 41-43% identity and 60-63% similarity with that of TSWV and INSV. Defective RNA molecules derived from the genomic M RNA were detected during continuous passage of the virus by sap inoculations.

Amino Acid Sequence↗
PubMed1995

NMR studies on the structure of some cyclic and linear antagonists of luteinizing hormone-releasing hormone (LHRH).

The structure of cyclic antagonists of luteinizing hormone-releasing hormone (LHRH), Ac-D-Phe(p-Cl)1-D-Phe(p-Cl)2-D-Trp3-Ser4-c(Asp5-D-Arg6-Leu7- Lys8)-Pro9- D-Ala10-NH2 (I), Ac-D-Phe(p-Cl)1-D-Phe(p-Cl)2-D-Trp3-Ser4-c(Glu5-D-Arg6-Leu7- Lys8)-Pro9-D-Ala10-NH2 (II) and their linear analogues, Ac-D-Phe(p-Cl)1-D- Phe(p-Cl)2-D-Trp3-Ser4-Asp5-D-Arg6-Leu7-Lys8-Pro9-D-++ +Ala10-NH2 (III) and Ac-D-Phe(p-Cl)1-D-Phe(p-Cl)2-D-Trp3-Ser4-Glu5-D-Arg6-Leu7-++ +Lys8-Pro9-D-Ala10-NH2 (IV), have been studied by NMR spectroscopy. The cyclic peptides I and II are more potent antagonists than the corresponding linear peptides in an in vivo assay. All the peptides show propensity of an unusual type II' beta-turn involving residues 3-6. Cyclic analogues also show some additional structure around residues 7 and 8 which is absent in the linear peptides. This additional structure in the cyclic peptides may be due to a minor conformation with a beta-turn between residues 5 and 8.

Amino Acid Sequence↗