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A Sanyal

Publications and source records attributed to A Sanyal.

At least 55 records · Page 3Linked to original sources
PubMed2001

Genetic and antigenic analysis of type A foot-and-mouth disease viruses isolated in India during 1987-1996.

Twenty-three foot-and-mouth disease virus (FMDV) type A field isolates, recovered from different outbreaks during 1987-1996 in India, were subjected to antigenic and genetic analysis. The isolates showed a close antigenic relationship to the current vaccine strain (IND 17/77) in micro-neutralization test conducted using a vaccine strain (IND 17/77) antiserum and a peptide (aa 136-151 of VP1 protein of the A22/Azerbaijan/65 strain) antiserum. However, the isolates revealed minor antigenic differences in their reactivity with three neutralizing monoclonal antibodies (MAbs) recognizing trypsin-sensitive conformation-independent epitopes of the vaccine virus strains. Phylogenetic relationship between the isolates was carried out employing a part of the 1D gene (168 nucleotides at the 3'-end). Additional seven type A Indian field isolates reported earlier were included in the analysis. The percent similarity among the Indian isolates varied from 82.7% to 99.4% at nucleotide level, and from 83.9% to 100% at amino acid level. These observations clearly demonstrate genetic heterogeneity of the field isolates. The current vaccine strain IND 17/77 showed divergence of 9.7% at nucleotide level and 5.6% at amino acid level from the A22 Iraq 24/64 isolate. The field strains were divergent from the vaccine strain IND 17/77 by 5.6%-14.6% and 3.7% 13.7% at nucleotide and amino acid level, respectively. In the phylogenetic tree, the isolates were distributed into 21 genetic groups. The clustering pattern of the isolates in the phylogenetic tree revealed no specific distribution pattern of the foot-and-mouth disease (FMD) outbreaks in relation to their geographical locations, caused by unrestricted animal movement and endemic nature of the disease.

Amino Acid Sequence↗
PubMed2001

Sequence analysis of recent Indian isolates of foot-and-mouth disease virus serotypes O, A and Asia 1 from clinical materials.

Partial nucleotide sequences of 1D gene of 38 isolates of foot-and-mouth disease virus (FMDV) of serotypes O, A and Asia 1 originating from various parts of India were determined. Field materials were subjected straight to RNA extraction, reverse transcription - PCR (RT-PCR) and sequencing. Also 3 FMDV vaccine strains, IND R2/75 (serotype O), IND 63/72 (serotype Asia 1) and IND 17/77 (serotype A) were included in the analysis. The seqences were compared mutually as well as with available corresponding sequences of other FMDV isolates, and their phylogenetic relationships were calculated. The deduced amino acid sequences showed that the serotype O isolates were relatively conserved as compared to serotype Asia 1 or A isolates from India. In phylogenetic analysis, the serotype O viruses clustered in two genotypes, one including the European vaccine strain (O1/K) and the other represented by the isolates from Bangladesh, India, Nepal and Turkey. The serotype Asia 1 viruses clustered in two groups of single genotype where the prototype strain from Pakistan (PAK 1/54) formed one group and the other was formed by the isolates from Bangladesh, Bhutan, India, Israel and Nepal. In serotype A viruses three well-differentiated genotypes were observed. The isolates from Azerbaijan, Bangladesh, Malaysia and India formed the first genotype. The second genotype was formed by isolates from Iran, Saudi Arabia and Turkey, while two recent Iranian isolates represented the third genotype. In India, the prevalence of at least one genotype could be identified in each serotype. This evolutionary clustering of isolates from the neighbor countries is not surprising, since these countries share border with India. The genetic relatedness between sequences of isolates from India and those from distant places is indicative of spread of the virus between the countries. Of importance is the fact that clinical materials proved useful for rapid generation of sequences and subsequent studying of molecular epidemiology of the disease.

Amino Acid Sequence↗
PubMed2000

Stereoselective diels-alder reactions of chiral anthracenes

Various chiral (9-anthryl)carbinol templates undergo Diels-Alder cycloadditions with a variety of symmetric and nonsymmetric dienophiles with excellent pi-facial selectivity and regioselectivity, under both thermal and Lewis acid catalyzed conditions.

Journal Article↗
PubMed2000

Genetic analysis of foot-and-mouth disease virus type O isolates responsible for field outbreaks in India between 1993 and 1999.

Partial nucleotide sequence at the 3' end of ID (VP1-encoding) gene of 90 foot-and-mouth disease virus type O isolates recovered from field outbreaks in India between 1993-9 were determined. The sequences were compared with each other and reference viruses. The published sequences of 15 type O isolates recovered from different parts of Asia and one isolate (O1BFS) from Europe and one from Egypt (O1/Sharquia/Egypt/72) were also included in the analysis for comparison. On the basis of phylogenetic analysis the viruses could be grouped into four distinct genotypes (genotypes I-IV). All 90 isolates from India were genotype-I, as were the reference isolates from Bangladesh, China, Egypt, Iran, Saudi Arabia, Syria and Turkey. Genotype-I isolates were further subdivided into 16 sub-genotypes. The Indian isolates were found to be extremely heterogeneous in nature and clustered into 12 different genetic groups. In genotype-I, the nucleotide sequence difference seen between the isolates was 0-11.6%, while among the Indian isolates it is 0-8.8%. Viruses of similar genetic groups are circulating in India, Bangladesh and countries of the Middle East. Genotype-II and -III are represented by isolates from Lebanon (O1/South Lebanon) and Europe (O1-BFS), respectively. Genotype-IV is formed by isolates from China, Hong Kong and Taiwan. The present study reveals the occurrence of viruses belonging to multiple genetic groups over a short period of time and persistence of single genetic group in the same geographical area over several years. This is consistent with the endemic nature of the disease in the country.

Animals↗
PubMed2000

Nucleotide sequence of the structural protein-encoding region of foot-and-mouth disease virus A22-India.

Nucleotide sequence of the structural protein-encoding region of foot-and-mouth disease virus (FMDV) A22-India 17/77 was determined using non-radioisotopic technique. Comparison of nucleotide and deduced amino acid sequence with A22-Iraq 24/64 revealed 175 synonymous (silent) and 42 non-synonymous nucleotide changes resulting in 34 amino acid substitutions along the capsid proteins (VP1-VP4). Out of the 4 structural proteins VP4 is highly conserved. The highly variable and immunodominant protein VP1 showed 47% of the total amino acid substitutions. VP2 and VP3 contain 38.2% and 14.7% of the amino acid substitutions, respectively. The VP1-based phylogenetic analysis of 18 different type A viruses including A22-India 17/77 divided them in to two broad genetic groups (Asian and European/South American), and each group is further subdivided in to two separate genotypes. A22-India 17/77, A22-Iraq 24/64 and A22-Azerbaijan/65 formed one genotype and the 4 Chinese strains formed a separate genotype in the Asian group of viruses. In the European/South American group, A-Argentina/87 represents one genotype and the remaining 10 strains formed the second genotype in this group.

Amino Acid Sequence↗
PubMed2000

Isolation of a cDNA sequence of rabbit GDF5 (mature form) and pattern of its mRNA expression during periosteal chondrogenesis.

Articular cartilage has a limited ability for repair and/or regeneration. Periosteal grafts, having chondrogenic potential, are used clinically and in experimental models to study the repair and regeneration of cartilage. Growth/differentiation factor 5 (GDF5), recently shown to be involved in chondrogenesis and normal skeletal development, is a bioactive candidate for augmenting the repair of damaged cartilage. In order to investigate the role of GDF5 during periosteal chondrogenesis, the rabbit sequence must be known, as most experimental models involve rabbit tissues. For this purpose, the complete rabbit-specific cDNA sequence of the mature form of GDF5 was determined. Mature rabbit GDF5 was found to be 100% identical to that of human GDF5 at the amino acid level. Using the cDNA sequence, specific primers for PCR were designed. Quantitative RT-PCR, using rabbit-specific primers, showed up-regulation of GDF5 mRNAs early during periosteal chondrogenesis suggesting its potential involvement in this process. The timing and magnitude of this expression was markedly stimulated by TGF-beta 1, which has already been shown to be a potent inducer of periosteal chondrogenesis.

Amino Acid Sequence↗
PubMed2000

Comparison of amino acid sequences at the amino acid 130-160 region of VP1 polypeptide of Indian field isolates of foot-and-mouth disease virus serotype Asia 1.

The nucleotide and deduced amino acid sequences in the amino acid (aa) 130-160 region of VP1 polypeptide of 65 field isolates of foot- and mouth disease virus (FMDV) serotype Asia 1 were determined and the consensus sequences were deduced. Comparison of amino acid sequences revealed conservation of NGK (130-132), TYG (134-136), RGD (142-144), and LPTSF (156-160) motifs and aa 148 (L) while variation was observed at the rest of the region (variability index (VI) of 2.06 to 16.85). Synonymous and non-synonymous mutations at the nucleotide level were well correlated with those of the corresponding amino acids. Comparison of the aa 130-160 sequence of Asia 1 serotype with those of other serotypes of FMDV revealed conservation of aa 135, 148-149, 157 and 160. Amino acids 133-138 and 148-154 were unique for Asia 1 serotype and are presumably responsible for its distinct antigenic nature. The present study revealed that the FMDV isolates of serotype Asia 1 causing outbreaks in India are very much heterogeneous in the aa 130-160 region of VP1.

Amino Acid Motifs↗
PubMed1999

Increased gene transfer into human cord blood cells by centrifugation-enhanced transduction in fibronectin fragment-coated tubes.

We investigated whether transduction of human cord blood progenitor cells can be increased by spinoculation in fibronectin fragment CH-296 (FN)-coated tubes. Bicistronic vectors PA317/LgEIN, containing the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (neo) genes, and PG13/LgDIN, containing the dihydrofolate reductase and neo genes, were used to transduce CD34-enriched human cord blood cells. Transduction by spinoculation in FN-coated tubes (spin/FN+) was compared with spinoculation in noncoated tubes (spin/FN-) and transduction in plates coated with FN (plate/FN+). Antibody to TGF-beta was added to spin/FN+ to evaluate its impact on transduction. Using producer cell line PA317/LgEIN for transduction of CD34+ cord blood cells, FACS analysis for expression of EGFP revealed mean transduction of 30.6+/-4.3, 9.1+/-1.6, and 21.1+/-6.5% of CD34+ cells in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. Transduction of CD+CD38low cells was also higher in the spin/FN+ arm as compared with transduction in the spin/FN- arm. These results were corroborated by colony-forming assays. Antibody to TGF-beta did not further increase transduction. Using a different producer cell line, PG13/pLgDIN, a higher number of G418-resistant CFU-GM was observed in the spin/FN+ as compared with the plate/FN+ and spin/FN-arms. NOD/SCID mice were transplanted with transduced, CD34-enriched human cord blood cells, and persistence of transduced human cells was analyzed in the mice marrows after 6-8 weeks: 32.8, 6.0, and 23.9% human G418-resistant CFU-GM colonies were observed in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. These results suggest that spinoculation in FN-coated tubes increases transduction of early human cord blood progenitor cells as compared with spinoculation in noncoated tubes.

Animals↗
PubMed1999

Periosteum responds to dynamic fluid pressure by proliferating in vitro.

Periosteum provides a source of undifferentiated chondrocyte precursor cells for fracture healing that can also be used for cartilage repair. The quantity of cartilage that can be produced, which is a determining factor in fracture healing and cartilage repair, is related to the number of available stem cells in the cambium layer. Cartilage formation during both of these processes is enhanced by motion of the fracture or joint in which periosteum has been transplanted. The effect of dynamic fluid pressure on cell proliferation in periosteal tissue cultures was determined in 452 explants from 60 immature (2-month-old) New Zealand White rabbits. The explants were cultured in agarose suspension for 1-14 days. One group was subjected to cyclic hydrostatic pressure, which is referred to as dynamic fluid pressure, at 13 kPa and a frequency of 0.3 Hz. Control explants were cultured in similar chambers without application of pressure. DNA synthesis ([3H]thymidine uptake) and total DNA were measured. The temporal pattern and distribution of cell proliferation in periosteum were evaluated with autoradiography and immunostaining with proliferating cell nuclear antigen. Dynamic fluid pressure increased proliferation of periosteal cells significantly, as indicated by a significant increase in [3H]thymidine uptake at all time points and a higher amount of total DNA compared with control values. On day 3, when DNA synthesis reached a peak in periosteal explants, [3H]thymidine uptake was 97,000+/-5,700 dpm/microg DNA in the group exposed to dynamic fluid pressure and 46,000+/-6,000 dpm/microg in the controls (p < 0.001). Aphidicolin, which blocks DNA polymerase alpha, inhibited [3H]thymidine uptake in a dose-dependent manner in the group subjected to dynamic fluid pressure as well as in the positive control (treated with 10 ng/ml of transforming growth factor-beta1) and negative control (no added growth factors) groups, confirming that [3H]thymidine measurements represent proliferation and dynamic fluid pressure stimulates DNA synthesis. Total DNA was also significantly higher in the group exposed to dynamic fluid pressure (5,700+/-720 ng/mg wet weight) than in the controls (3,700+/-630) on day 3 (p < 0.01). Autoradiographs with [3H]thymidine revealed that one or two cell cycles of proliferation took place in the fibrous layer prior to proliferation in the cambium layer (where chondrocyte precursors reside). Proliferating cell nuclear antigen immunophotomicrographs confirmed the increased proliferative activity due to dynamic fluid pressure. These findings suggest either a paracrine signaling mechanism between the cells in these two layers of the periosteum or recruitment/migration of proliferating cells from the fibrous to the cambium layer. On the basis of the data presented in this study, we postulate that cells in the fibrous layer respond initially to mechanical stimulation by releasing growth factors that induce undifferentiated cells in the cambium layer to divide and differentiate into chondrocytes. These data indicate that cell proliferation in the early stages of chondrogenesis is stimulated by mechanical factors. These findings are important because they provide a possible explanation for the increase in cartilage repair tissue seen in joints subjected to continuous passive motion. The model of in vitro periosteal chondrogenesis under dynamic fluid pressure is valuable for studying the mechanisms by which mechanical factors might be involved in the formation of cartilage in the early fracture callus and during cartilage repair.

Aging↗
PubMed1999

Initial evidence for the involvement of bone morphogenetic protein-2 early during periosteal chondrogenesis.

The potential of periosteum to form cartilage makes periosteal transplantation a viable approach to repairing defects in articular cartilage, which has a limited potential for repair. However, cartilage repair, including that by periosteal chondrogenesis, is poorly understood. Consequently, a thorough understanding of its molecular mechanisms will help to achieve the quality of neocartilage required for its clinical application in damaged joints. An in vitro model was used to study the early molecular events of periosteal chondrogenesis. During the search for the expression of transforming growth factor-beta-related mRNAs in this model system, bone morphogenetic protein-2 mRNA expression was found to be upregulated 20-fold within the first 12 hours of culture. This stimulation was dependent on the explants being suspended in agarose and did not occur with explants cultured in liquid medium. The upregulation of bone morphogenetic protein-2 mRNA expression was also enhanced by exogenously added transforming growth factor-beta1 in the presence of fetal calf serum. The upregulation, however, was not transient; rather, it persisted over a prolonged period in both transforming growth factor-beta1-treated and untreated explants. Further data indicate that this stimulation of bone morphogenetic protein-2 mRNA expression was regulated at the transcriptional level and that no new protein synthesis was required for this. Bone morphogenetic protein-2 is known to influence developmental chondrogenesis; therefore, these observations direct our attention toward an important potential role of it as a regulator of the early events in cartilage repair. Furthermore, because periosteum produces fracture (cartilage) callus, these findings may be important in defining the molecular mechanisms of fracture healing.

Animals↗
PubMed1999

Monoclonal antibodies to an Indian strain of type A foot-and-mouth disease virus.

A set of five neutralizing monoclonal antibodies (MAbs) to an Indian strain (IND17/77) of type A (subtype A22) foot-and-mouth disease (FMD) virus (FMDV) was used in the study. Four of the MAbs (27S, 37S, 85S, and 143S) identified a trypsin-sensitive (TS) epitope(s) and were specific for VP1, while the remaining MAb (145S) reacted with a trypsin-resistant (TR) epitope and was specific for VP3 in Western blot analysis. Both the epitopes (TS and TR) were conformation-independent in nature. Results obtained in MAb-competition enzyme-linked immunosorbent assay (ELISA), and profiling of the (MAb) neutralization-escape mutants in ELISA and cross-neutralization test revealed two overlapping TS epitopes (27S/37S and 85S/143S) on the virus. Variation at both these epitopes was observed in some field isolates of serotype A. Comparison of deduced amino acid sequence in the VP1 region (aa 140-213) between the parent virus and the mutants identified Gly148 and Arg153 as critical for the formation of both the TS epitopes. Substitution of R153 by Gly or Ser was observed in mutants with no reactivity for the MAbs 85S/143S. However, these mutants maintained partial reactivity with MAbs 27S/37S, and substitution of Gly148 by Glu eliminated both the epitopes. No amino acid substitution was observed in the VP1 region of aa 200-213. Efficient neutralization of the MAb neutralization escape mutants (MAb-resistant (MAR) mutants) by bovine vaccinate serum (BVS) indicated involvement of other epitopes on the virion surface in eliciting neutralizing antibodies following vaccination.

Amino Acid Substitution↗
PubMed1998

Gene digging. A method for obtaining species-specific sequence based on conserved segments of nucleotides in open reading frames.

A method termed "gene digging" has been developed based on our observation of stretches of highly conserved nucleotide sequence in the coding region of many genes across related species. Rabbit-specific nucleotide sequences corresponding to desired coding segments of 14 different genes were obtained with primers that were designed based on conserved nucleotide stretches. Our success in gene digging could be attributable to the method's inherent ability to reduce the degeneracy of primers by more than two orders of magnitude (sometimes by more than three orders of magnitude) compared to primers designed from conserved amino acids. Our results not only demonstrate the value of the method, but also hint at a thus far unknown functional significance of conserved nucleotide stretches in the coding region of various genes. In our hands the method worked 14 out of 14 times indicating generality of the concept.

Activin Receptors, Type I↗
PubMed1998

Genetic heterogeneity of Indian field isolates of foot-and-mouth disease virus serotype O as revealed by partial sequencing of 1D gene.

The sequence of 165 nucleotides at the 3' end of the 1D gene, determined from RT PCR amplified cDNA fragments, of 25 type O strains isolated from different parts/regions of India during 1987 1995 and the vaccine strain (R2/75) currently in use in India were subjected to phylogenetic analysis. One isolate from the neighbouring country Nepal was also included in the study. The virus/ field strains showed high degree of genetic heterogeneity among themselves with % divergence in nucleotide sequence ranging from 1.2 to 19.4%. The Indian strains were much away (13.3 20.6%) from the exotic type O strains of O1BFS, O1K, and O1Campos. The type O strains analyzed were classified into three genotypes basing on level of divergence observed in nucleotide sequence. The type O vaccine virus (R2/75) was > 71% divergent (7.3-15.2%) from the field strains which revealed significant ( > 5%) genetic heterogeneity between the two. The phylogenetic analysis identified three distinct lineages, viz., (i) lineage 1 represented by the exotic strains, (ii) lineage 2 represented by 25 of the field strains which clustered into seven subgroups/sublines (2a-2g), and (iii) lineage 3 represented by a unique field isolate which shared the branching/origin with the vaccine strain. The lineage 2 which comprised of 25 of the 26 type O field strains analyzed, was placed almost at equidistance from the lineages 1 and 3 in the phylogenetic tree. The vaccine strain was closer to the viruses in lineage 2. Though there was no specific distribution pattern of sequences in different geographical regions of India, the viruses/ sequences in subgroup 2f appeared to be restricted to the southern states. Comparison of deduced amino acid sequence in the immunodominant regions 133-160 and 200-208 of the 1D gene product (VP1) showed that the two viruses in lineage 3 had unique amino acid residues at the positions 138 (D), 139 (G), 144 (I), and 158 (A) compared to rest of the strains including the exotic ones. Comparison of amino acid residues at critical positions 144, 148, 149, 151, 153, 154, and 208 revealed similarity between the type O strains analyzed. The virus strains showed variation (V/L/I) at position 144. One field strain showed replacement from Q149-->E and another from P208-->L. Thus, the study revealed that the type O FMD virus populations circulating in India and causing disease outbreaks are genetically much heterogeneous but related at the immunodominant region of VP1 polypeptide, and there are more than one genetically distinct virus populations in almost every region of the country which is possible due to unrestricted animal movement in the country. The involvement of vaccine virus in disease outbreaks was ruled out as the field strains (excluding the one in lineage 3) were phylogenetically distinct from it.

Amino Acid Sequence↗
PubMed1997

Antigenic features of foot-and-mouth disease virus serotype Asia1 as revealed by monoclonal antibodies and neutralization-escape mutants.

Neutralizable antigenic sites/epitopes of serotype Asial foot-and-mouth disease virus (strain IND63/72) were identified using monoclonal antibodies (mabs) and their neutralization-escape mutants. Relative affinity/reactivity of the mabs for viral (both native and trypsin-cleaved) and subviral antigens in enzyme-linked immunosorbent assay (ELISA) showed dominance of trypsin-sensitive and conformation-dependent neutralizable antigenic sites. Characterization of neutralization escape mutants identified at least four independent trypsin-sensitive neutralizable antigenic sites on Asial FMD virus. One site was identified by mabs B3, 1A, 24, 2A, 40 and 63, second site by mabs 34 and 81, third site by mab 72 and fourth site by mab 89. The reaction profile of the mabs with selected field isolates in ELISA identified four different neutralization epitopes within the site B3/1A/24/2A/40/63.

Animals↗
PubMed1997

One-tube and one-buffer system of RT-PCR amplification of 1D gene of foot-and-mouth disease virus field isolates.

A method of reverse transcription (RT) and polymerase chain reaction (PCR) amplification of 1D (VP1) gene of foot-and-mouth disease (FMD) virus using one reaction mixture containing both avian myeloblastosis virus (AMV) reverse transcriptase (RTase) and Tfl DNA polymerase is described. The procedure was time saving, made use of a single buffer for both RT and subsequent amplification and performed better than the two-step procedure usually conducted with Moloney murine leukemia virus (MMLV) RTase and Taq DNA polymerase for amplification of the VP1 gene of field isolates of FMD virus serotypes O,-A, C and Asia 1. The failure to amplify the VP1 gene of many type O and Asia 1 viruses using MMLV RTase-Taq polymerase enzyme system could be overcome by performing RT of the viral genome at a higher temperature (48 degrees C) with AMV RTase which is not possible with MMLV RTase.

Animals↗
PubMed1997

Evaluation of primers for PCR amplification of RNA polymerase gene sequences of foot-and-mouth disease virus.

Eight oligonucleotide primers in 7 different combinations were used to amplify 3D gene sequences of foot-and-mouth disease virus (FMDV) by reverse transcription-polymerase chain reaction (RT-PCR). Six of the primers were designed at this laboratory. All the primer combinations could specifically amplify 3D gene sequences of FMDV serotypes O, A, and C. The largest fragment amplified was of 1,393 bp and the smallest was of 208 bp in size. The 1,393 bp fragment included sequences from the preceeding P18 region of FMDV genome. The second largest fragment of 734 bp included sequences from the 3'-extracistronic region of viral genome. The remaining fragments were amplified either from the 3'- or 5'-half of the 3D gene. Specific amplification of the entire 3D gene in fragments of different size showed sequence conservation in the 3D genomic region of FMDV and usefulness of the primers reported in detecting inapparent or persistent FMDV infection in susceptible animals by RT-PCR.

Animals↗