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Biomedical subjects

T Chandra

Publications and source records attributed to T Chandra.

At least 37 records · Page 2Linked to original sources

Work of adhesion of respiratory tract mucus.

A method was devised to measure the work of adhesion (WA) to a substrate of mucus, a viscoelastic gel, from the measured contact angle of glycerol on a mucus substrate and the known physical properties of a Teflon surface. Fifteen sputum samples from cystic fibrosis (CF) patients were compared with 25 mucus samples from canine tracheal pouches (CP), studied in the hydrated and partially dehydrated states. Apparent viscosity (eta A) and recoverable shear strain (SR) were measured by fluxgate magnetometry, and water content was inferred from vapor pressure osmometry. Na+, K+, and Ca2+ concentrations were measured with specific ion electrodes and Cl- with a chloridimeter. The Cl- concentration of the CP mucus was inversely proportional to its osmolality, and the Cl- concentration of the CP mucus was 102.5 +/- 1.6 meq/l compared with 55.6 +/- 2.5 meq/l for CF sputum. When CP mucus osmolality was increased from 316.0 +/- 5.5 to 430.0 +/- 7.5 mosmol/kg, WA increased from 25.1 +/- 1.8 to 31.1 +/- 1.2 ergs/cm2 and eta A increased from 391 +/- 55 to 622 +/- 121 P, respectively. CF sputum WA was 30.2 +/- 0.6 ergs/cm2, eta A was 1,110 +/- 316 P, and osmolality was 466.0 +/- 14.0 mosmol/kg. The increased WA and eta A of mucus in CF patients may thus be dependent on the hydration of mucus, which is related to the documented Cl- transport defect.

Adolescent↗

The effect of diacylglycerols on fibronectin release and its reversal by retinoic acid in cell culture.

Previous work from our laboratory showed that tumor promoters such as phorbol ester (TPA) stimulated the release of fibronectin (FN) from the surface of several cell types in culture, and that this stimulation was counteracted by retinoic acid. Diacylglycerols (DAGs) are the endogenous ligands of the TPA receptor and can activate and translocate protein kinase C (PKC) in a manner similar to TPA. To show that the release of FN is related to activation of PKC, we tested the action of DAGs on FN release from human lung fibroblasts and its counteraction by retinoic acid. We found that DAGs stimulated the release of FN in a concentration- and time-dependent manner. The stimulation of the release of FN correlated with the translocation-activation of PKC by DAG. Retinoic acid reversed the action of DAG with respect to stimulation of FN release and inhibited this release even in the absence of DAG. These results suggest that the release of FN is in some way related to translocation-activation of PKC.

Cell Membrane↗

Haptoglobin phenotypes in diabetes mellitus and diabetic retinopathy.

An analysis of haptoglobin (HP) phenotypes in 81 cases of diabetes mellitus (DM) without retinopathy and 122 cases with diabetic retinopathy (DR) were studied in relation to 180 normal and healthy controls matched for age and sex. A significant decrease in HP 2-1 frequency was found, suggesting protection for heterozygotes in both DM and DR (with a relative risk of about 0.31). As an acute-phase reactant HP may be functionally involved in the etiology of DM and DR, which are associated with immunologic and inflammatory processes, respectively. No significant differences were found with respect to sex, age at onset, duration of DR, types of DM and DR, and family history.

Alleles↗

The anti-inflammatory activity of Enicostemma littorale and Mollugo cerviana.

The anti-inflammatory activity of E. littorale and M. cerviana was assessed by carrageenan-induced inflammation and cotton pellet granuloma method in rats. E. littorale and M. cerviana exerted 54 and 26% anti-inflammatory activity for a dose of 100 mg/100 g body wt, respectively, in carrageenan-induced acute inflammation. In chronic inflammation of cotton pellet granuloma, E. littorale and M. cerviana exerted 30 and 46% anti-inflammatory activity at the above dosage, respectively. The optimal dose for these drugs was determined in carrageenan inflammation. The effect of the alcoholic extract of these drugs on human erythrocyte membrane stabilization and inhibition of cobra venom phospholipase A2 was studied in vitro and the drugs were found to be effective. Further, these drugs were found to inhibit the levels of lipid peroxides, acid phosphatase, and gamma-glutamyl transpeptidase activity in the exudate of cotton pellet granuloma. The effects were compared with those of standard anti-inflammatory drug, hydrocortisone. A possible mode of action of these drugs is suggested.

Acid Phosphatase↗

Complete sequence of the cDNA for human alpha 1-antitrypsin and the gene for the S variant.

A 1434 base pair human liver cDNA coding for the entire alpha 1-antitrypsin protein has been isolated and sequenced. Translation of the coding region into amino acids reveals a precursor molecule which contains a 24 amino acid signal peptide and 394 amino acids present in the mature polypeptide chain. The human gene for the S variant of alpha 1-antitrypsin has also been subcloned and sequenced. The gene is composed of 10226 nucleotide bases and is approximately equimolar for all 4 nucleotides. The gene contains four intervening sequences (introns) and 5' and 3' noncoding regions which are 54 and 79 nucleotides in length, respectively. A 5.3-kilobase intron exists in the 5' noncoding region and contains a 143 amino acid open reading frame, an Alu family sequence, and a pseudo transcription initiation region. No significant differences in base composition are seen between the introns and those regions corresponding to coding regions of the corresponding mRNA (exons). A sequence of 1951 nucleotides flanking the 5' end of the gene has also been determined and contains a "TATA" box sequence (TTAAA-TA) 21 nucleotides upstream from the proposed transcription start site. Comparison of the gene sequence with the cDNA sequence reveals a single base substitution (A----T), which results in a Glu----Val substitution at position 264 in the S variant protein. The position and size of introns, the overall base composition, and the codon preference for the alpha 1-anti-trypsin gene differ from those for the chicken ovalbumin gene even though the two proteins belong to a common protein family, as judged by amino acid sequence homology.

Amino Acid Sequence↗

Sequence homology between human alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III.

alpha 1-Antichymotrypsin mRNA was isolated by specific polysome immunoprecipitation from turpentine-treated baboon liver. The highly enriched mRNA was used for synthesis and cloning of the corresponding cDNA. Baboon alpha 1-antichymotrypsin cDNA clones were identified by hybrid-selected translation, and the insert DNA fragment from one of the putative clones was used as a probe to screen a human liver cDNA library comprised of 40 000 independent transformants. One of the human cDNA clones was unambiguously identified to contain alpha 1-antichymotrypsin DNA sequences by comparison of its 5'-terminal nucleotide sequence with the N-terminal amino acid sequence of the protein. This cDNA clone, designated phACT235, contains 1524 base pairs of human DNA, which was sequenced in its entirety. The inserted DNA codes for a 25 amino acid signal peptide sequence and the entire mature alpha 1-antichymotrypsin of 408 amino acid residues. Comparison of the amino acid sequence of alpha 1-antichymotrypsin with that of the human alpha 1-antitrypsin has revealed a homology level similar to that between chymotrypsin and trypsin.

Amino Acid Sequence↗

Identification of multiple species of calmodulin messenger RNA using a full length complementary DNA.

Poly(A) RNA from eel electroplax was used to construct a full length cDNA complementary to calmodulin (CaM) mRNA which was cloned in the PstI site of pBR322 DNA. Recombinant plasmids containing sequences complementary to CaM mRNA were identified by hybridization using a 32P-labeled CaM cDNA (pCM109). Nucleotide sequence analysis reveals that the clone from the plasmid pCM116 contains a 5' nontranslated region of 26 nucleotides, the entire coding region, and a 3' nontranslated region of 408 nucleotides. The amino acid sequence deduced from the nucleotide sequence is similar to those previously reported for CaM from other species. Comparison between the nucleotide sequence of the functional domains of the protein shows extensive homology between all four domains. pCM116 was utilized to determine and compare the populations of RNA present in different tissues. In eel electroplax, three species of cytoplasmic RNAs at 820, 1100, and 2000 nucleotides hybridize to the cDNA probe. The nucleus contains an additional CaM RNA molecule of 5500 nucleotides which may represent a primary transcript of the calmodulin gene. Sequence analysis of the 3' noncoding region of pCM116 reveals 3 possible polyadenylation sites (AATAAA) at positions 573, 580, and 855. The mRNA of 820 nucleotides was derived by polyadenylation at the first site whereas the mRNA of 1100 nucleotides was derived by poly(A) addition at position 855. These data are compatible with the idea that at least 2 of the 3 CaM mRNAs in eel electroplax tissue are derived from a single nuclear transcript by differential polyadenylation during processing.

Amino Acid Sequence↗

Purification of antithrombin III mRNA and cloning of its cDNA.

Antithrombin III mRNA was enriched from a baboon liver by specific polysome immunoprecipitation. The partially purified antithrombin III mRNA preparation was used for cDNA synthesis and cloning. Candidate antithrombin III cDNA clones were identified by differential hybridization using as probes [32P]cDNAs synthesized from the polysome-enriched and -depleted RNA fractions, respectively. The candidate clones were further analyzed by hybrid-selected translation. The authenticity of a cDNA clone positive to both tests was unambiguously confirmed by matching its nucleotide sequence with the known amino acid sequence of human antithrombin III. The baboon antithrombin III cDNA clone hybridized well with human antithrombin III mRNA and can be used as a probe to isolate the corresponding human gene.

Animals↗

Construction and identification of mouse amelogenin cDNA clones.

The determination of the biochemical phenotype of tooth epithelium requires specification by the dental mesenchyme. This is a general feature of epithelial-mesenchymal interaction in a number of different epidermal organ systems (e.g., salivary gland, mammary gland, feather, skin, and hair morphogenesis). To investigate these developmental processes, we have identified a cDNA clone representing the major group of gene products associated with enamel extracellular matrix formation. The mRNAs for mouse amelogenins, representing approximately equal to 90% of the total enamel proteins, have been isolated and partially characterized by specific immunoprecipitation. The poly(A)-containing RNAs were used for the synthesis and cloning of the mouse amelogenin cDNA. Recombinant plasmids containing amelogenin cDNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analyses. A cloned sequence was used to identify the expression of amelogenins during tooth development. The mouse cDNA sequence hybridized to genomic mouse and human DNAs. This amelogenin cDNA probe now enables molecular investigations of a number of classical problems in developmental biology.

Amelogenin↗

Isolation and sequence characterization of a cDNA clone of human antithrombin III.

A human liver cDNA library was constructed by using poly(A)-containing RNA isolated from a human liver biopsy specimen. This library is comprised of 40,000 independent transformants with an average inserted DNA length of 1,200 base pairs. By using the previously cloned baboon antithrombin III cDNA as a specific hybridization probe, greater than 30 human antithrombin III cDNA clones were identified from this library. The clone with the longest DNA insert was selected for sequence analysis. This antithrombin III cDNA clone contains 1,479 base pairs of inserted human DNA and was designated phATIII 113. It contains DNA sequences that code for a signal peptide and the entire mature antithrombin III protein which is comprised of 432 amino acid residues.

Amino Acid Sequence↗

Sequence homology and structural comparison between the chromosomal human alpha 1-antitrypsin and chicken ovalbumin genes.

The human chromosomal alpha 1-antitrypsin gene has been cloned. This gene is approximately 5 kilobase pairs long and contains three intervening sequences in the peptide-coding region. DNA sequences coding for the amino and carboxyl termini of a alpha 1-antitrypsin have been identified. Human alpha 1-antitrypsin and chicken ovalbumin show significant sequences homology and belong to a common protein super-family. Yet the number, position and size of intervening sequences reveal that the two genes are dissimilar.

Animals↗

Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

The mRNA for phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been purified from total rat liver mRNAs, of which it constitutes less than 0.25%, to greater than 10% purity in a single step by specific polysome immunoprecipitation. The purified mRNA was used for synthesis and cloning of its cDNA. Recombinant colonies containing phenylalanine hydroxylase DNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analysis. The rat cDNA clone was capable of hybridizing with human phenylalanine hydroxylase mRNA, which will permit the isolation of the corresponding human gene for analysis of phenylketonuria, a hereditary disorder in phenylalanine metabolism that causes permanent mental retardation in humans.

Animals↗

Transcriptional activity of the uteroglobin gene in rabbit endometrial nuclei during early pregnancy.

In studies on the regulation of rabbit uteroglobin production by progesterone, we have shown previously that the increase in uteroglobin secretion by the uterus in early pregnancy is preceded by an increase in uteroglobin synthesis and a parallel increase in uteroglobin messenger (m) RNA activity, which is accounted for by a rise in the steady state level of this specific mRNA in uterine epithelial cells. To investigate whether similar changes in the transcriptional activity of the uteroglobin gene occur during this period, we have transcribed endometrial nuclei in a "run-off" endogenous transcription system with [32P]uridine triphosphate as the precursor, given as a short pulse over 7 min at 25 C in vitro. Specific uteroglobin RNA transcripts were quantitated by hybridization of [32P]RNA to nitrocellulose filters containing cloned, full length uteroglobin complementary DNA. The percentage of uteroglobin RNA transcripts rose from day 0 (nonpregnant rabbits) to a peak on day 4 and declined on day 6 of pregnancy. No differences in RNA degradation and no preferential degradation of newly transcribed uteroglobin RNA were found during incubation of nuclei from the different days of pregnancy. The pattern of change in rate of transcription of the uteroglobin gene corresponds to the pattern of change in level of uteroglobin mRNA, suggesting that transcriptional controls are important in regulating the production of uteroglobin in pregnant rabbit uterus.

Animals↗

Mosaic structure and mRNA precursors of uteroglobin, a hormone-regulated mammalian gene.

The synthesis of uteroglobin in the rabbit uterus is induced by progesterone and is repressed by estrogen which has an over-riding effect over the inducer. The dual hormonal control system offers an excellent model for studying hormonal regulation of mammalian gene expression. Using a full-length uteroglobin cDNA clone as a specific hybridization probe, recombinant lambda phages containing the entire chromosomal uteroglobin gene have been isolated from a rabbit genomic DNA library. Electronmicroscopic analysis of hybrid molecules formed between the chromosomal uteroglobin gene and uteroglobin mRNA indicated the presence of 2 intervening sequences within this gene. The mosaic structure of the uteroglobin gene has been substantiated by detailed restriction mapping and Southern hybridization. The gene is 3.0 kilobases in length to code for a mature mRNA of 465 nucleotides. Northern hybridization of poly(A)-containing RNA from 4-day-pregnant rabbit uterus with the full-length cDNA clone revealed the presence of uteroglobin mRNA precursors. The size of the largest precursor RNA species detected by the cDNA clone is the same as the entire chromosomal uteroglobin gene. The fidelity of the precursor RNAs was established by their ability to hybridize with specific intervening sequence probes. Thus the entire uteroglobin gene is expressed into primary RNA transcripts, which are subsequently processed into mature mRNA molecules by splicing.

Animals↗

Cloning and sequence of cDNA coding for alpha 1-antitrypsin.

Recombinant plasmids containing human and baboon cDNA have been screened for alpha 1-antitrypsin, a major serine protease inhibitor present in blood. One plasmid, designated pBa alpha 1a2, was found to contain a cDNA insert of 1352 base pairs coding for the baboon inhibitor. It included 45 nucleotides that code for 15 amino acids present in the amino-terminal signal sequence of the protein, 1182 nucleotides that code for 394 amino acids in the mature protein, a stop codon, and a noncoding region of 76 nucleotides. Comparison of the amino acid sequences of baboon alpha 1-antitrypsin, human antithrombin III, and chicken ovalbumin indicated that these three proteins are about 230% homologous. A second plasmid, designated pH alpha 1a1, was found to contain a human cDNA insert of 306 base pairs. This plasmid coded for 69 amino acids present in the carboxyl-terminal region of human alpha 1-antitrypsin. The human and baboon cDNAs and their amino acid sequences are greater than 96% homologous.

Amino Acid Sequence↗

A cloned calmodulin structural gene probe is complementary to DNA sequences from diverse species.

Calmodulin mRNA has been partially purified from a total nucleic acid extract of the electroplax of Electrophorus electricus by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation. A 9- to 10S fraction was determined to contain 39% calmodulin mRNA by translation in a reticulocyte lysate followed by immunoprecipitation with antibodies to calmodulin. Double-stranded cDNA was synthesized from the RNA fraction by using reverse transcriptase from avian myeloblastosis virus. The double-stranded cDNA was joined to pBR322 linearized by restriction endonuclease Pst I and used to transform Escherichia coli RRI. DNAs from 60 tetracycline-resistant cloned hybridized to [32P]cDNA synthesized from the partially purified calmodulin mRNA fraction. By direct DNA sequence analysis, one of these clones, pCM109, was shown to contain calmodulin-specific sequences corresponding to amino acid residues 93--148 of calmodulin or approximately 38% of the peptide-coding region of the calmodulin structural gene sequence. pCM109 was hybridized to DNA isolated from three vertebrate and one plant species by the procedure of Southern. Positive hybridization bands were noted regardless of the DNA source. These data suggest thaat calmodulin gene sequences are evolutionarily conserved, as has been shown to be the case for the primary amino acid sequence.

Animals↗