Biomedical subjects

S Muthukrishnan

Publications and source records attributed to S Muthukrishnan.

At least 19 recordsLinked to original sources

PubMed2005

Effects of calibration on L-THIA GIS runoff and pollutant estimation.

Urbanization can result in alteration of a watershed's hydrologic response and water quality. To simulate hydrologic and water quality impacts of land use changes, the Long-Term Hydrologic Impact Assessment (L-THIA) system has been used. The L-THIA system estimates pollutant loading based on direct runoff quantity and land use based pollutant coefficients. The accurate estimation of direct runoff is important in assessing water quality impacts of land use changes. An automated program was developed to calibrate the L-THIA model using the millions of curve number (CN) combinations associated with land uses and hydrologic soil groups. L-THIA calibration for the Little Eagle Creek (LEC) watershed near Indianapolis, Indiana was performed using land use data for 1991 and daily rainfall data for six months of 1991 (January 1-June 30) to minimize errors associated with use of different temporal land use data and rainfall data. For the calibration period, the Nash-Sutcliffe coefficient was 0.60 for estimated and observed direct runoff. The calibrated CN values were used for validation of the model for the same year (July 1-December 31), and the Nash-Sutcliffe coefficient was 0.60 for estimated and observed direct runoff. The Nash-Sutcliffe coefficient was 0.52 for January 1, 1991 to December 31, 1991 using uncalibrated CN values. As shown in this study, the use of better input parameters for the L-THIA model can improve accuracy. The effects on direct runoff and pollutant estimation of the calibrated CN values in the L-THIA model were investigated for the LEC. Following calibration, the estimated average annual direct runoff for the LEC watershed increased by 34%, total nitrogen by 24%, total phosphorus by 22%, and total lead by 43%. This study demonstrates that the L-THIA model should be calibrated and validated prior to application in a particular watershed to more accurately assess the effects of land use changes on hydrology and water quality.

Calibration↗

PubMed2005

Regulation of chitin synthesis in the larval midgut of Manduca sexta.

In insects, chitin is not only synthesized by ectodermal cells that form chitinous cuticles, but also by endodermal cells of the midgut that secrete a chitinous peritrophic matrix. Using anti-chitin synthase (CHS) antibodies, we previously demonstrated that in the midgut of Manduca sexta, CHS is expressed by two cell types, tracheal cells forming a basal tracheal network and columnar cells forming the apical brush border [Zimoch and Merzendorfer, 2002, Cell Tissue Res. 308, 287-297]. Now, we show that two different genes, MsCHS1 and MsCHS2, encode CHSs of midgut tracheae and columnar cells, respectively. To investigate MsCHS2 expression and activity in the course of the larval development, we monitored chitin synthesis, enzyme levels as well as mRNA amounts. All of the tested parameters were significantly reduced during molting and in the wandering stage when compared to the values obtained from intermolt feeding larvae. By contrast, MsCHS1 appeared to be inversely regulated because its mRNA was detectable only during the molt at the time when tracheal growth occurs at the basal site of the midgut. To further examine midgut chitin synthesis, we measured enzyme activity in crude midgut extracts and different membrane fractions. When we analysed trypsin-mediated proteolytic activation, a phenomenon previously reported for insect and fungal systems, we recognized that midgut chitin synthesis was only activated in crude extracts, but not in the 12,000 g membrane fraction. However, proteolytic activation by trypsin in the 12,000 g membrane fraction could be reconstituted by re-adding a soluble fraction, indicating that limited proteolysis affects an unknown soluble factor, a process that in turn activates chitin synthesis.

Animals↗

PubMed2005

Detoxification of oxalic acid by pseudomonas fluorescens strain pfMDU2: implications for the biological control of rice sheath blight caused by Rhizoctonia solani.

Rhizoctonia solani isolates varying in their virulence were tested for their ability to produce oxalic acid (OA) in vitro. The results indicated that the virulent isolates produced more OA than the less virulent isolates. In order to isolate OA-detoxifying strains of Pseudomonas fluorescens, rhizosphere soil of rice was drenched with 100 mM OA and fluorescent pseudomonads were isolated from the OA-amended soil by using King's medium B. These isolates were tested for their antagonistic effect towards growth of R. solani in vitro. Among them P. fluorescens PfMDU2 was the most effective in inhibiting the mycelial growth of R. solani. P. fluorescens PfMDU2 was capable of detoxifying OA and several proteins were detected in the culture filtrate of PfMDU2 when it was grown in medium containing OA. To investigate whether the gene(s) involved in OA-detoxification resides on the plasmids in P. fluorescens PfMDU2, a plasmid-deficient strain of P. fluorescens was generated by plasmid curing. The plasmid-deficient strain (PfMDU2P-) failed to grow in medium containing OA and did not inhibit the growth of R. solani. Both PfMDU2 and PfMDU2P- were tested for their efficacy in controlling sheath blight of rice under greenhouse conditions. Seed treatment followed by soil application of rice with P. fluorescens strain, PfMDU2, reduced the severity of sheath blight by 75% compared with the control, whereas PfMDU2P- failed to control sheath blight disease.

Antibiosis↗

PubMed2005

The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix.

Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1-specific RNA interference (RNAi) disrupted all three types of moult (larval-larval, larval-pupal and pupal-adult) and greatly reduced whole-body chitin content. Exon-specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval-pupal and pupal-adult moults, whereas splice variant 8b was required only for the latter. TcCHS2-specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi-mediated down-regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut.

Animals↗

PubMed2005

Efficient genetic transformation of Sorghum using a visual screening marker.

To transform grain sorghum (Sorghum bicolor (L.) Moench) with a visual reporter gene (gfp) and a target gene (tlp), three genotypes (two inbreds, Tx 430 and C401, and a commercial hybrid, Pioneer 8505) were used. We obtained a total of 1011 fertile transgenic plants from 61 independent callus lines, which were produced from 2463 zygotic immature embryos via Agrobacterium-mediated transformation. The reporter gene, gfp, encoding green fluorescent protein (GFP), was used as a visual screening marker, and the target gene, tlp, encoding thaumatin-like protein (TLP), was chosen for enhancing resistance to fungal diseases and drought. Both genes were under the control of the maize ubi 1 promoter in the binary vector pPZP201. A total of 320 plants showing GFP expression, derived from 45 calli, were selected and analyzed by Southern blot analysis. There was a 100% correlation between the GFP expression and the presence of the target gene, tlp, in these plants. Transgenic plants showing strong TLP expression were confirmed by Western blotting with antiserum specific for TLP. The transgene segregated in various ratios among progeny, which was confirmed by examining seedlings showing GFP fluorescence. The progeny also showed different copy numbers of transgenics. This report describes the successful use of GFP screening for efficient production of stably transformed sorghum plants without using antibiotics or herbicides as selection agents.

Agrobacterium tumefaciens↗

PubMed2002

Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers.

Early and reliable detection of plant transformation events is essential for establishing efficient transformation protocols. We have compared the effectiveness of using the gene encoding a green fluorescent protein (GFP) and a beta-glucuronidase (gus) as reporter genes for early detection of transgene expression in explants subjected to biolistic bombardment and Agrobacterium-mediated transformation. The results indicate that gfp gene is superior to gus gene in following transgene expression in transiently transformed materials in both methods of transformation. Using GFP as the screenable marker, we have optimized sorghum transformation with respect to the conditions for transformation, type of explants, promoters, and inbreds. These optimized conditions have been used to obtain stably transformed explants for subsequent regeneration.

Biolistics↗

PubMed2001

Properties of Manduca sexta chitinase and its C-terminal deletions.

Manduca sexta (tobacco hornworm) chitinase is a molting enzyme that contains several domains including a catalytic domain, a serine/threonine-rich region, and a C-terminal cysteine-rich domain. Previously we showed that this chitinase acts as a biopesticide in transgenic plants where it disrupts gut physiology. To delineate the role of these domains further and to identify and characterize some of the multiple forms produced in molting fluid and in transgenic plants, three different forms with variable lengths of C-terminal deletions were generated. Appropriately truncated forms of the M. sexta chitinase cDNA were generated, introduced into a baculovirus vector, and expressed in insect cells. Two of the truncated chitinases (Chi 1-407 and Chi 1-477) were secreted into the medium, whereas the one with the longest deletion (Chi 1-376) was retained inside the insect cells. The two larger truncated chitinases and the full-length enzyme (Chi 1-535) were purified and their properties were compared. Differences in carbohydrate compositions, pH-activity profiles, and kinetic constants were observed among the different forms of chitinases. All three of these chitinases had some affinity for chitin, and they also exhibited differences in their ability to hydrolyze colloidal chitin. The results support the hypothesis that multiple forms of this enzyme occur in vivo due to proteolytic processing at the C-terminal end and differential glycosylation.

Animals↗

PubMed2001

Enhanced resistance to sheath blight by constitutive expression of infection-related rice chitinase in transgenic elite indica rice cultivars.

Genetic transformation has been attempted for management of rice sheath blight disease, caused by Rhizoctonia solani. We introduced a PR-3 rice chitinase gene (RC7), isolated from R. solani-infected rice plants, into indica rice cultivars IR72, IR64, IR68899B, MH63, and Chinsurah Boro II by the biolistic and PEG-mediated transformation system. Inheritance was studied up to the T(2) generation by Southern blot analysis. Western blot analysis of transgenic plants with polyclonal antibody revealed the presence of chitinase protein with a molecular weight of 35 kDa that reacts with chitinase antibody. The transformants synthesized different levels of chitinase proteins constitutively and progeny from the plants containing the chitinase gene showed different levels of enhanced resistance when challenged with the sheath blight pathogen R. solani.

Journal Article↗

PubMed2001

Antifungal proteins and other mechanisms in the control of sorghum stalk rot and grain mold.

Research on antifungal proteins and other mechanisms that provide the biochemical basis for host-plant resistance to stalk rot and grain molds is reviewed in this paper. Stalk rot caused by Fusarium species leads to substantial yield loss due to poor grain filling and/or lodging. A transgenic sorghum expressing high levels of chitinase exhibited less stalk rot development when exposed to conidia of F. thapsinum. Grain mold of sorghum is associated with warm humid environments and results from colonization by several fungi (F. thapsinum, Curvularia lunata, and Alternaria alternata) of the developing caryopsis. The roles of several biochemical mechanisms (tannins, phenolic compounds, red pericarp, proteins, hard endosperm, and antifungal proteins) on grain mold resistance are discussed. Resistance mechanisms related to these compounds appear to be additive, and pyramiding of genes is a feasible approach to limit grain deterioration. Several experimental approaches are proposed to extend current findings.

Antifungal Agents↗

PubMed2000

Homology modeling of the insect chitinase catalytic domain--oligosaccharide complex and the role of a putative active site tryptophan in catalysis.

Knowledge-based protein modeling and substrate docking experiments as well as structural and sequence comparisons were performed to identify potential active-site residues in chitinase, a molting enzyme from the tobacco hornworm, Munduca sexta. We report here the identification of an active-site amino acid residue, W145. Several mutated forms of the gene encoding this protein were generated by site-directed mutagenesis, expressed in a baculovirus-insect cell-line system, and the corresponding mutant proteins were purified and characterized for their catalytic and substrate-binding properties. W145, which is present in the presumptive catalytic site, was selected for mutation to phenylalanine (F) and glycine (G), and the resulting mutant enzymes were characterized to evaluate the mechanistic role of this residue. The wild-type and W145F mutant proteins exhibited similar hydrolytic activities towards a tri-GlcNAc oligosaccharide substrate, but the former was approximately twofold more active towards a polymeric chitin-modified substrate. The W145G mutant protein was inactive towards both substrates, although it still retained its ability to bind chitin. Therefore, W145 is required for optimal catalytic activity but is not essential for binding to chitin. Measurement of kinetic constants of the wild-type and mutant proteins suggests that W145 increases the affinity of the enzyme for the polymeric substrate and also extends the alkaline pH range in which the enzyme is active.

Amino Acid Sequence↗

PubMed2000

Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene.

Creeping bentgrass (Agrostis palustris Huds.) is a cool season grass widely used on putting greens in golf courses. Transformation of creeping bentgrass has been conducted using microprojectile bombardment and protoplast electroporation. The objective of our study is to develop an alternative and more efficient approach in transforming the grass using Agrobacterium (strain EHA 101). This technique was effective in transforming 40-day old calli derived from mature seeds cultured on MS medium supplemented with 2,4-D, kinetin, and sucrose. Dozens of transgenic plants have been produced from two independent transformed calli. Presence of functional green fluorescence protein (GFP) was detected in leaves, stems, and roots of transgenic seedlings. Four putative transgenic plants and two control plants were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the GFP gene were clearly shown in transgenic plants. These results indicated that Agrobacterium transformation can successfully be applied to creeping bentgrass.

Adenine↗

PubMed1998

The corn inhibitor of activated Hageman factor: purification and properties of two recombinant forms of the protein.

A cDNA clone that encodes the 14-kDa bifunctional inhibitor from corn seeds (L. Wen et al., Plant Mol. Biol. 18, 813-814, 1992) has been expressed in Escherichia coli after being incorporated into the pT7 expression vector. This inhibitor protein, referred to as CHFI (for the corn inhibitor of activated Hageman factor) or as the popcorn inhibitor, is an important tool for specific inhibition of human activated Hageman factor (activated forms of coagulation Factor XII) and has been well characterized as isolated from corn seeds. Recombinant CHFI was expressed in E. coli in high levels but was insoluble. We solubilized the expressed protein by sonication in 5 M urea and 1% Triton X-100. Several steps of purification, culminating with reversed-phase HPLC, yielded pure, recombinant corn inhibitor in about 5% yield (about 1 mg per liter of culture). The form with which we have worked most, 7N-CHFI, contains 7 amino acid residues at its N-terminus that are encoded by the expression vector. Physical properties of this recombinant protein indicate it has the expected mass and is properly folded. Functionally, 7N-CHFI is indistinguishable from the inhibitor isolated from corn seeds in its inhibition of porcine trypsin, human beta-Factor XIIa, failure to inhibit human plasma kallikrein, and its inhibition of an insect alpha-amylase. A second recombinant form, (4N-11)-CHFI, which lacks 11 residues from the corn inhibitor's N-terminus, is indistinguishable from 7N-CHFI in its pattern of inhibition of the three test proteinases but is inactive against the insect alpha-amylase. This suggests that the N-terminal region of 7N-CHFI forms at least part of the protein's site of interaction with alpha-amylase.

Base Sequence↗

PubMed1998

Insect resistance of transgenic tobacco expressing an insect chitinase gene.

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control.

Animals↗

PubMed1997

Isolation and characterization of a genomic clone for the gene of an insect molting enzyme, chitinase.

Genomic clones for a chitinolytic enzyme were isolated from a library of Sau 3A digested DNA from the tobacco hornworm, Manduca sexta, using a previously isolated chitinase cDNA clone as a probe [Kramer et al., Insect Biochem. Molec. Biol. 23, 691-701 (1993)]. Restriction enzyme mapping and Southern blot analysis of four genomic clones suggested that these are overlapping clones. Sequence analysis of the genomic clones and Southern blot analysis of total genomic DNA also suggest that the M. sexta genome has only one chitinase gene detectable by the cDNA probe. This gene is organized into at least 11 exons in a region spanning > 11 kb. The sequenced M. sexta chitinase gene has a series of exons corresponding to identifiable structural/functional regions of the protein. Similarities in structure and organization between the M. sexta chitinase gene and chitinase genes from other sources are described.

Amino Acid Sequence↗

PubMed1997

Insect chitinases: molecular biology and potential use as biopesticides.

Chitin, an insoluble structural polysaccharide that occurs in the exoskeletal and gut linings of insects, is a metabolic target of selective pest control agents. One potential biopesticide is the insect molting enzyme, chitinase, which degrades chitin to low molecular weight, soluble and insoluble oligosaccharides. For several years, our laboratories have been characterizing this enzyme and its gene. Most recently, we have been developing chitinase for use as a biopesticide to control insect and also fungal pests. Chitinases have been isolated from the tobacco hornworm, Manduca sexta, and several other insect species, and some of their chemical, physical, and kinetic properties have been determined. Also, cDNA and genomic clones for the chitinase from the hornworm have been isolated and characterized. Transgenic plants that express hornworm chitinase constitutively have been generated and found to exhibit host plant resistance. A transformed entomopathogenic virus that produces the enzyme displayed enhanced insecticidal activity. Chitinase also potentiated the efficacy of the toxin from the microbial insecticide, Bacillus thuringiensis. Insect chitinase and its gene are now available for biopesticidal applications in integrated pest management programs. Current knowledge regarding the molecular biology and biopesticidal action of insect and several other types of chitinases is described in this mini-review.

Amino Acid Sequence↗

PubMed1997

Local rules for protein folding on a triangular lattice and generalized hydrophobicity in the HP model.

We consider the problem of determining the three-dimensional folding of a protein given its one-dimensional amino acid sequence. We use the HP model for protein folding proposed by Dill (1985), which models protein as a chain of amino acid residues that are either hydrophobic or polar, and hydrophobic interactions are the dominant initial driving force for the protein folding. Hart and Istrail (1996a) gave approximation algorithms for folding proteins on the cubic lattice under the HP model. In this paper, we examine the choice of a lattice by considering its algorithmic and geometric implications and argue that the triangular lattice is a more reasonable choice. We present a set of folding rules for a triangular lattice and analyze the approximation ratio they achieve. In addition, we introduce a generalization of the HP model to account for residues having different levels of hydrophobicity. After describing the biological foundation for this generalization, we show that in the new model we are able to achieve similar constant factor approximation guarantees on the triangular lattice as were achieved in the standard HP model. While the structures derived from our folding rules are probably still far from biological reality, we hope that having a set of folding rules with different properties will yield more interesting folds when combined.

Algorithms↗

PubMed1996

Cloning, expression, and hormonal regulation of an insect beta-N-acetylglucosaminidase gene.

Chitinolytic enzymes such as beta-N-acetylglucosaminidases are major hydrolases involved in insect molting. By screening a Manduca sexta (tobacco hornworm) cDNA library with an antibody against beta-N-acetylglucosaminidase from molting fluid of M. sexta pharate pupae, several putative cDNA clones for this enzyme were isolated. The longest of the cDNA clones has an insert of approximately 3 kb, and the complete nucleotide sequence was determined. Because this clone is missing the initiation codon and nucleotides corresponding to the leader peptide, the mRNA 5'-end sequence was determined by PCR (polymerase chain reaction) amplification and cycle sequencing. The sequence of the encoded protein from positions 23 to 35 is identical to the NH2-terminal sequence of one of the beta-N-acetylglucosaminidases isolated from pharate pupal molting fluid. The amino acid sequence is similar to those of silkworm, human, mouse, bacterial, and several other beta-N-acetylglucosaminidases. Two highly conserved regions in the amino acid sequence were found in all members of this family. Southern blot analysis suggested that the number of genes in the Manduca genome closely related to the cDNA clone may be as few as one. The beta-N-acetylglucosaminidase gene is expressed most abundantly in epidermal and gut tissues on days 6 and 7 of fifth instar larvae. Injection of 20-hydroxyecdysone induced expression of the beta-N-acetylglucosaminidase gene, whereas topical application of the juvenile hormone analog, fenoxycarb, suppressed the inductive effect of molting hormone.

Acetylglucosaminidase↗

PubMed1993

A chloroplast DNA deletion located in RNA polymerase gene rpoC2 in CMS lines of sorghum.

Fertile lines of sorghum (Sorghum bicolor) were shown to differ from cytoplasmic male sterile (CMS) lines by the presence of a 3.8 kb HindIII chloroplast DNA fragment in the former and a smaller (3.7 kb) fragment in the latter. DNA/DNA hybridization studies showed that these two fragments are homologous. Fertile plants from S. versicolor, S. almum, S. halepense, and Sorghastrum nutans (Yellow Indiangrass) also have the 3.8 kb fragment, and CMS lines studied containing A1, A2 and A3 cytoplasms have the 3.7 kb fragment. The size difference between the two fragments was localized to a 1.0 kb SacI-HindIII fragment by restriction mapping. A 165 bp deletion, which is flanked by a 51 bp tandem repeat, was identified in the CMS lines by sequencing the clones. Comparison of the two sequences with those from maize, rice, tobacco, spinach, pea, and liverwort revealed that the deleted sequence is located in the middle of the RNA polymerase beta" subunit encoded by the gene rpoC2. The amino acid sequence deleted in the CMS lines is in a monocot-specific region which contains two protein motifs that are characteristic of several transcriptional activation factors, namely, a leucine zipper motif and an acidic domain capable of forming an amphipathic alpha-helix. Further studies designed to determine whether or not the deletion is involved in CMS of sorghum are underway.

Amino Acid Sequence↗