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Biomedical subjects

M B Singh

Publications and source records attributed to M B Singh.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of a Ca2+ binding protein as a new Bermuda grass pollen allergen Cyn d 7: IgE cross-reactivity with oilseed rape pollen allergen Bra r 1.

cDNA clones encoding two isoforms of an allergen from pollen of Bermuda grass (Cynodon dactylon) have been isolated using IgE from allergic patients. Homologous transcripts are present in pollen of 15 other grasses tested. This allergen, tentatively designated as Cyn d 7, contains two calcium binding domains and shows significant sequence similarity with other Ca2+ binding pollen allergens, namely Bet v 4 from birch and Bra r 1 from oilseed rape. Approximately 10% of allergic sera tested showed IgE reactivity to this allergen. IgE cross-reactivity was observed between this allergen and Bra r 1 of oilseed rape. IgE reactivity of this allergen requires protein-bound Ca2+. Using IgE affinity-purified from the recombinant allergen to probe Western blots of pollen extracts Cyn d 7 has been identified as a 12 kDA protein.

Allergens↗
PubMed1997

Knowledge and attitudes of pregnant women regarding AIDS in a semi arid area of Rajasthan.

A total of 792 pregnant women from three hospitals of Jaipur were surveyed to assess and compare their knowledge and attitudes regarding various aspects of AIDS. Observations revealed that level of knowledge was significantly higher in pregnant women from upper income group than low income group (P < 0.05). The misconceptions regarding various aspects of AIDS viz. symptoms and signs, transmission and prevention and opinion towards the AIDS patients, indicate that there is strong need of AIDS education compaign in pregnant women too.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Infant feeding and weaning practices in some semi-arid rural areas of Rajasthan.

The paper contains information on infant feeding and weaning practices in the rural communities of a semi-arid district of Rajasthan. The findings have been drawn from a recent survey of 328 rural mothers. The rural women of this area are found to believe in old deep seated beliefs and customs, which in turn deprive their infants from advantages of colostrum (77% discarded colostrum) and nutritionally rich supplements, which otherwise should be supplemented to them at any cost in order to keep them healthy. The practices of prolonged breast feeding and delayed supplementation to infants are rampant in this area. Mean age at weaning (27.1 months) again not only affects the health status of mothers and their children but also leads to the undernutrition among both. The findings of the study necessitate to evolve an exhaustive educative programme dealing with various aspects of infant feeding and weaning practices, keeping in view their traditions, so that the useful practices can be encouraged and harmful ones be prohibited.

Breast Feeding↗
PubMed1996

Recombinant expression and epitope mapping of grass pollen allergens.

We have studied the expression of recombinant forms of Group 1 allergens from rye-grass and Bermuda grass pollens. Recombinant Lol p 1 expressed in bacteria bound serum IgE from allergic patients. Based on analysis of fragments of the Lol p 1 cDNA clone, the major IgE-reactive epitope has been mapped to the C-terminus. However, although SDS-denatured natural Cyn d 1 (from Bermuda grass) bound IgE, the full or partial recombinant proteins expressed in bacteria did not bind IgE. We have since expressed Cyn d 1 in the yeast Pichia pastoris and restored IgE binding. cDNA clones encoding two isoforms of Lol p 5, Lol p 5A and Lol p 5B, have been expressed in bacteria and resulting polypeptides show IgE-binding. Random fragments of these clones have been generated and when expressed as partial recombinant proteins in bacteria, allowed us to identify the major IgE-binding epitopes. The allergenic epitopes were localised towards the C-terminal half of the molecule. Although both isoforms shared similar IgE-reactive epitopes, Lol p 5B did not recognise the Lol p 5A-specific monoclonal antibody A7. At sequence level, there appear to be several amino acid differences between the antigenic epitopes of these two isoallergens. These results aid in the design of diagnostics and in grass pollen immunotherapy.

Allergens↗
PubMed1996

Generative cells of Lilium longiflorum possess translatable mRNA and functional protein synthesis machinery.

Metabolic labelling with [35S]-methionine demonstrated that generative cells of Lilium longiflorum possess their own set of mRNA and are capable of synthesising proteins independently from the vegetative cell. The isolated generative cells synthesised ten proteins, of which six were unique to these specialised cells. Isolation of generative cells from pollen grains after [35S]-methionine labelling resulted in an identical protein profile, therefore the synthesis of these proteins was not due to isolation shock. Addition of cycloheximide, abolished TCA-precipitable counts, whilst actinomycin D had no qualitative effect on the observed protein profile, indicating active translation of pre-existing mRNAs by the generative cells.

Cell Separation↗
PubMed1996

Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen.

BACKGROUND: Pollen of grasses, such as Bermuda grass (Cynodon dactylon), represent a major cause of type I allergy. OBJECTIVE: In this report we attempted to clone and express a biologically active form of recombinant Cyn d 1, the major allergen of Bermuda grass pollen, in the yeast Pichia pastoris. METHODS: Clones encoding Cyn d 1 were isolated by screening a Bermuda grass pollen complementary DNA library with specific monoclonal antibodies and by polymerase chain reaction amplification. Recombinant Cyn d 1 was expressed in Escherichia coli and yeast. The expressed proteins were analyzed by Western blotting to assess binding to Cyn d 1-specific monoclonal antibodies and IgE from sera of patients allergic to Bermuda grass pollen. RESULTS: Two isoforms of Cyn d 1 were cloned. Recombinant Cyn d 1 expressed in bacteria bound two monoclonal antibodies raised against Cyn d 1 but was not recognized by IgE from sera of patients allergic to Bermuda grass pollen. Cyn d 1 expressed in yeast bound both the monoclonal antibodies and human IgE. CONCLUSION: An IgE-reactive Cyn d 1 was expressed in yeast but not in bacteria, suggesting that posttranslational modifications (e.g., glycosylation), which occur in eukaryotic cells such as yeast, are necessary for the production of a biologically active allergen.

Allergens↗
PubMed1996

Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen.

T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

Allergens↗
PubMed1996

Nutritional status of school age children of salt-workers in Rajasthan.

Nutritional status of 1044 school age children (6-18 yr) of salt workers in various salt manufacturing sites of Rajasthan, was assessed using anthropometry. Heights and weights of girls were observed to be better than in boys in the menarcheal age groups. Children of the present study showed reduced growth in comparison to National Centre for Health Statistics (NCHS), World Health Organisation (WHO), well-to-do Indian children and Rajasthan reference values of the same age groups. The prevalence of stunted with severe wasting was 4.5 per cent in the children of salt workers. Higher prevalence of severe wasting was observed in girls than the boys (P < 0.01).

Adolescent↗
PubMed1995

Cloning, expression and immunological characterization of Ory s 1, the major allergen of rice pollen.

We have isolated and characterized a cDNA clone, Ory s 1, encoding a group-1 allergen of rice pollen. The Ory s 1 protein shows significant sequence identity to the major allergen of rye-grass pollen, Lol p 1. RNA gel blot analysis shows that the Ory s 1 gene is expressed in mature anthers, but not in vegetative or other floral tissues tested. Southern blot analysis indicates that this clone represents a member of a small gene family in rice. Western blot analyses of total rice pollen proteins with the group-1 allergen-specific monoclonal 3A2 and IgE antibodies from grass pollen-allergic patients, revealed the presence of cross-reactive antigenic and allergenic epitopes in Ory s 1.

Allergens↗
PubMed1995

Bcp1, a gene required for male fertility in Arabidopsis.

Male fertility in flowering plants is dependent on production of viable pollen grains within the anther. Genes expressed exclusively in the anther are likely to include those that control male fertility. On the basis of their tissue specificity, such genes have been isolated, yet in none of them has this function been demonstrated. Here we report that one such gene, Bcp1, is active in both diploid tapetum and haploid microspores and is required for pollen fertility. Perturbation of this gene in either tapetum or microspores prevents production of fertile pollen in transgenic Arabidopsis plants. When tapetum expression of this gene is perturbed, mature anthers contain dead shriveled pollen. On the other hand, when microspore expression is perturbed, anthers show 1:1 segregation of viable/aborted pollen. These findings identify a class of sporophytic/gametophytic genes controlling male fertility and, hence, reproduction in flowering plants.

Amino Acid Sequence↗
PubMed1995

A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca(2+)-binding proteins.

Thirteen cDNA clones encoding IgE-binding proteins were isolated from expression libraries of anthers of Brassica rapa L. and B. napus L. using serum IgE from a patient who was specifically allergic to Brassica pollen. These clones were divided into two groups, I and II, based on the sequence similarity. All the group I cDNAs predicted the same protein of 79 amino acids, while the group II predicted a protein of 83 amino acids with microheterogeneity. Both of the deduced amino acid sequences contained two regions with sequence similarity to Ca(2+)-binding sites of Ca(2+)-binding proteins such as calmodulin. However flanking sequences were distinct from that of calmodulin or other Ca(2+)-binding proteins. RNA-gel blot analysis showed the genes of group I and II were preferentially expressed in anthers at the later developmental stage and in mature pollen. The recombinant proteins produced in Escherichia coli was recognized in immunoblot analysis by the IgE of a Brassica pollen allergic patient, but not by the Ige of a non-allergic patient. The cDNA clones reported here, therefore, represent pollen allergens of Brassica species.

Amino Acid Sequence↗
PubMed1995

Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation.

The recombinant proteins of Lol p VA and Lol p VB expressed in E. coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1. Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants. Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB. When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not. The data suggest that there are at least two IgE binding determinants in Lol p VB. In addition, only fragment Met1-Val196 reacted with mAb PpV1. The localization of these determinants was further resolved using random fragment expression libraries. The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule. The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254). These IgE binding determinants are conserved in Lol p VA.

Allergens↗
PubMed1995

Cloning, sequencing and expression in Escherichia coli of Pha a 1 and four isoforms of Pha a 5, the major allergens of canary grass pollen.

BACKGROUND: The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen source in the external environment of southern Australia. This study was performed to characterize the major recombinant allergens of canary grass pollen. It is anticipated that recombinant allergens may be useful in diagnosis and immunotherapy of grass pollen induced allergies. OBJECTIVE: To clone major canary grass pollen allergens and assess their nucleotide and amino acid sequence homologies with other grass pollen allergens. This sequence information may then be useful in T and B cell epitope mapping studies. METHODS: A canary grass pollen lambda gt11 cDNA expression library was constructed and screened with sera of grass-pollen-sensitive patients. IgE-reactive clones were isolated, sub-cloned into Escherichia coli, sequenced and, along with the deduced amino acid sequences, compared with other sequences in nucleotide and amino acid databases. RESULTS: One of the clones encoded the group 1 allergen of canary grass pollen, Pha a 1, with a deduced amino acid sequence identity of 88.8% with Lol p 1, from rye-grass pollen, 88.1% with Hol l 1, from velvet grass pollen and 86.6% with Phl p 1, from timothy grass pollen. The other clones (e.g. clones, 5, 14, 28, 29) encoded polymorphic forms of Pha a 5. These polymorphic forms showed between 60.6-95.5% nucleotide and 40.1-81.7% deduced amino acid sequence identities with each other. Moreover, they shared significant sequence identity with other group 5 allergens from rye-grass, timothy and Kentucky bluegrass pollens. CONCLUSIONS: Group 1 and four isoforms of group 5 allergens of canary grass pollen have been cloned and upon sequencing demonstrated strong nucleotide and amino acid sequence identities with other group 1 and 5 grass pollen allergens.

Allergens↗
PubMed1995

Morbidity profile of desert population engaged in salt production in Rajasthan.

A total 2120 salt workers were examined in 8 camps organised at different salt sites in Rajasthan. These camps were organised to find out the morbidity conditions among salt workers and to provide preventive and curative treatment for the ailments. The majority of salt workers who attended the camps were males (79.0%) in comparison to females (21.0%). The majority of workers belonged to Scheduled Castes followed by Backward Class and Scheduled Tribes at all sites. The major morbidity disorders observed during these camps were dermatological, respiratory, musculoskeletal, gastro-intestinal and ophthalmological in both the sexes. The prevalence of hypertension was 4.3% among both sexes. The morbidity pattern in male workers was agreeing among Sujangarh and Kuchaman and after that Pokran and Didwana were agreeing with highest rank correlation, whereas in female workers it was agreeing among Nawa and Sujangarh and after that Phalodi and Pokran were agreeing with highest correlation.

Adolescent↗
PubMed1994

Localization of the two major allergens in rye-grass pollen using specific monoclonal antibodies and quantitative analysis of immunogold labelling.

The intracellular localization of the two major allergens, Lol p I and Lol p IX, in rye-grass anthers was examined using monoclonal antibodies FMCA1 (specific for Lol p I) and FMCA7 (specific for Lol p IX) with immunocytochemical techniques and quantitative analysis. A newly developed anhydrous fixation technique in a mixture of glutaraldehyde, paraformaldehyde and 2,2-dimethoxypropane followed by embedding in LR Gold resin resulted in both improved infiltration of pollen grains compared with existing techniques and the localization of these water-soluble antigens in their original sites compared with diffusion artefacts following aqueous methods. After anhydrous fixation, Lol p I was predominantly located in the electron-opaque regions of the cytosol of the vegetative cell of the tricellular pollen grains (24 counts microns-2), whereas Lol p IX was detected mainly within starch granules (16 counts microns-2). For both Lol p I and Lol p IX, similar labelling was detected in the cells of the endothecium and middle layer (18 counts microns-2), but none was found in the tapetal cells or orbicules.

Allergens↗
PubMed1994

Immunological relationships among group I and group V allergens from grass pollen.

Specific IgE antibodies have been affinity-purified from recombinant grass pollen allergens, and used to identify isoforms of the two major allergens of rye-grass pollen, Lol p I and Lol p V and cross-reactive allergens in other grasses. Lol p I-specific IgE (affinity-purified from the recombinant protein expressed by clone 13R which encodes amino acids 96-240 of Lol p I) identified four isoforms of the allergen. The same probe recognized cross-reactive epitopes in pollen proteins from 14 out of 16 grasses. The allergens identified by Lol p V-specific IgE (affinity-purified from the recombinant protein expressed by clones 12R or 19R which encode the full Lol p V protein) varied more in their physicochemical characteristics than the Group I isoforms. At least eight isoforms of Lol p V were identified by the Lol p V-specific IgE. The same probe recognized cross-reactive epitopes in pollen protein from 13 out of 16 grasses. Group I proteins were identified in grasses from two sub-families of the Poaceae, while the Group V allergens were only identified in pollen of grasses from one sub-family, the Pooideae.

Allergens↗
PubMed1994

Isolation and characterization of group-I isoallergens from Bermuda grass pollen.

Two isoallergens of Cyn d I were isolated using preparative isoelectric focussing, and were designated Cyn d Ia and b. These isoallergens differ in their pI, molecular weight (Cyn d Ia, 32 kD and Cyn d Ib, 31 kD) and their NH2-terminal sequence. Four monoclonal antibodies (Mabs) were produced using Cyn d Ia as antigen. These Mabs recognized both Cyn d Ia and b. One of the Mabs recognized four different pI forms of Cyn d I on 2D gels. The Mabs also recognized cross-reactive epitopes on proteins from eight other grasses including rye grass, timothy grass, Kentucky bluegrass and Johnson grass.

Allergens↗