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Biomedical subjects

B Jayaram

Publications and source records attributed to B Jayaram.

At least 37 records · Page 2Linked to original sources
PubMed1994

Identification of peptides within the base binding domains of the GTP- and ATP-specific binding sites of tubulin.

Using gamma-32P-labeled 8-azidopurine nucleotide photoaffinity probes of GTP and ATP, the respective purine ring binding domain peptides of tubulin have been identified. First, the location of the GTP-specific binding site was shown to be on the beta-subunit, whereas the major ATP-specific binding site was on the alpha-subunit. Using a combination of anion-exclusion and immobilized Al3+ column chromatography, the respective photolabeled tryptic peptides of both nucleotide binding sites were isolated, further purified by reverse phase high performance liquid chromatography (HPLC) and sequenced. Chymotryptic peptides were also generated for the GTP binding site. High retention of the photoinserted radiolabel was observed with many of the peptides on reverse phase HPLC at low flow rates. The stability of the photoinserted radiolabel to HPLC varied with different peptides. However, certain peptides were easily distinguished as being within the base binding domains of the GTP and ATP binding sites of tubulin. Two beta-tubulin peptides containing the majority of photoinserted [gamma-32P]8-azidoguanosine 5'-triphosphate corresponded to N-terminal beta-tubulin amino acid residues 3EIVHIQAGQCGNQIGAK19 and 20FWEVISDEHGIDPTGS35. The peptide containing the majority of photoinserted [gamma-32P]8-azidoadenosine 5'-triphosphate corresponded to the C-terminal alpha-tubulin sequence 431DYEEVGVDSVEGEGEEEGEE450.

Adenosine Triphosphate↗
PubMed1994

Use of isotope-dilution phenomenon to advantage in the determination of kinetic constants Km and Kcat for BamHI restriction endonuclease: an empirical and iterative approach.

An assay using a very small amount of 35S-labeled deoxyoligonucleotide as a substrate for the determination of Km and Kcat for the restriction enzyme BamHI is described. Two synthetic deoxyoligonucleotides, ATGGCGGATCCGC and ATGGCGGAGCCGC, containing the cognate and a mismatch BamHI sequence, respectively, were labeled by an end-filling reaction using the Klenow fragment of DNA polymerase and [35S]dATP to generate the labeled self-complementary substrates. The dependence of BamHI hydrolysis on substrate concentration was investigated using mixtures of a fixed amount of radiolabeled substrate and varying amounts of cold-labeled substrate over a wide range. The apparent competitive inhibition observed due to the phenomenon of carrier dilution was analytically corrected by an empirical as well as an iterative approach to give Km values comparable to those reported in the literature. We have found that the values obtained using the empirical formula are very close to the precise values obtained through iteration. Our procedure has used isotopic dilution to advantage to make the assay less expensive and can be applied effectively to any enzyme-substrate reaction in which the substrate and the product have radioactive labels. The method would be especially useful for a rapid analysis and comparison of kinetic constants of various mutant enzymes or substrates.

Base Sequence↗
PubMed1989

Purification of human interleukin-4 produced in Escherichia coli.

An interleukin-4 (IL4)-encoding cDNA isolated from human splenocytes was used to construct an expression plasmid that directs a high-level synthesis of mature IL4 protein in Escherichia coli. The expression was under the control of the major leftward promoter, pL, of phage lambda and the phage Mu ribosome-binding site. The IL4 protein was present as insoluble inclusion bodies in the bacterial extract. The IL4 could be solubilized in 5 M MgCl2 and was purified to homogeneity by several chromatographic steps. The yield of protein from bacteria ranged between 3 and 5 mg of IL4 protein per gram of wet cells. The specific activity of the recombinant human IL4 was about the same as that of the natural product.

Animals↗
PubMed1989

The electrostatic potential of B-DNA.

Electrostatic potentials around DNA are obtained by solving the nonlinear Poisson-Boltzmann (PB) equation. The detailed charge distribution of the DNA and the different polarizabilities of the macromolecule and solvent are included explicitly in the calculations. The PB equation is solved using extensions of a finite difference approach applied previously to proteins. Electrical potentials and ion concentrations are compared to those obtained with simpler models. It is found that the shape of the dielectric boundary between the macromolecule and solvent has significant effects on the calculated potentials near the surface, particularly in the grooves. Sequence-specific patterns are found, the most surprising result being the existence of positive regions of potential near the bases in both the major and minor grooves. The effect of solvent and ionic atmosphere screening of phosphate-phosphate repulsions is studied, and an effective dielectric function, appropriate for molecular mechanics simulations, is derived.

DNA↗
PubMed1988

Interleukin 1 alpha acts as an autocrine growth factor for RPMI 1788, an Epstein-Barr virus-transformed human B cell line.

The Epstein-Barr virus-transformed B cell line RPMI 1788 constitutively produces autocrine growth factors with molecular masses of 17 kDa, 24 kDa and 35 kDa. All three molecular forms were completely neutralized with anti-interleukin (IL) 1 alpha antiserum. Although IL 1 alpha and IL 1 beta mRNA were both equally detectable by Northern blotting, no IL 1 beta activity was found in partially purified RPMI 1788 supernatant. The growth of low density-seeded RPMI 1788 cells is specifically dependent on the presence of either IL 1 alpha or IL 1 beta. Since no other cytokine was found to be capable of sustaining proliferation, this cell line is suitable for the identification and quantification of IL 1, even in the presence of other cytokines.

B-Lymphocytes↗
PubMed1986

Classification and surgical correction of postburn axillary contractures.

Postburn axillary contractures should be surgically corrected as soon as the diagnosis has been established in order to avoid deeper tissue involvement. We have classified axillary contractures based upon local anatomic conditions and present procedures for correction of each type that we find most suitable. Type I contractures are characterized by a linear web at either axillary fold with minimal adjacent scarring. A double Z-plasty with Y-V advancement is the procedure of choice. Type II contractures involve either axillary fold, with adjacent skin scarring. A double incisional release on both sides of the hair-bearing area is the procedure of choice. Type III contractures are characterized by linear webs at both axillary folds without involvement of the adjacent skin. The procedure of choice in these cases is a double incisional release. Type IV axillary contractures involve the hair-bearing area and the periaxillary region. This diffuse scar contracture is best treated by a single incisional release.

Axilla↗
PubMed1985

A retrospective study of treatment of orbital floor fractures with the maxillary sinus approach.

The Cook County Hospital, Chicago, Illinois, protocol for the management of orbital fractures is presented. Diplopia which fails to resolve in 1 week or appears after oedema subsides, enophthalmos or radiographic depression of the orbital floor, associated tripod fracture deformity, or purulent maxillary sinusitis are indications for surgery. The surgical procedure is a modification of the maxillary sinus approach, which includes the use of iodoform gauze packing, nasal antrostomy, a double layer oral closure and prophylactic antibiotics. A review of 102 patients treated surgically over a 6-year period is presented. Post-operative diplopia was 9% and there were minor complications in 3%, which were significantly lower than previously reported series of orbital fractures treated either non-operatively, or through an orbital exploration approach. No patient treated non-operatively required surgery in the future. The protocol presented can eliminate unnecessary exploration and yields excellent results with a simple surgical procedure.

Diplopia↗
PubMed1985

Recombinant interleukin 2 induces immunoglobulin secretion in Staphylococcus aureus Cowan strain I activated human B-cells.

Human B-cells, exhaustively depleted for T-cells, were activated with Staphylococcus aureus Cowan strain I (SAC) and responded to recombinant human interleukin 2 (rIL2) by secretion of immunoglobulin (Ig), as measured by a protein A hemolytic plaque assay. The rIL2, however, had to be present early, since addition later than 24 h after SAC-activation of the B-cells reduced the response to background levels. No clear dose response was observed and Ig-secreting cells (ISC) could be induced even with rIL2 at 0.5 U/ml. The monoclonal antibody anti-TAC prevented the rIL2-promoted induction of ISC. Ig production could be induced in SAC-activated cultures with supernatants of Xenopus laevis oocytes injected with sucrose-gradient-fractionated poly(A+) RNA derived from a stimulated human spleen cell culture. This activity coincided with the IL2 mRNA activity and was well separated from the interferon-gamma mRNA activity. Our results suggest that IL2 is not only a B-cell growth factor but also promotes the differentiation of activated human B-cells towards Ig secretion.

Antibody-Producing Cells↗
PubMed1984

Aqueous hydration of nucleic acid constituents: Monte Carlo computer simulation studies.

Monte Carlo computer simulations were performed on dilute aqueous solutions of thymine, cytosine, uracil, adenine, guanine, the dimethyl phosphate anion in the gauche-gauche conformation and a ribose and deoxyribose derivative. The aqueous hydration of each molecule was analysed in terms of quasi-component distribution functions based on the Proximity Criterion, and partitioned into hydrophobic, hydrophilic and ionic contributions. Color stereo views of selected hydration complexes are also presented. A preliminary discussion of the transferability of functional group coordination numbers is given. The results enable to comment on two current problems related to the hydration of nucleic acids: a) the theory of Dickerson and coworkers on the role of water in the relative stability of the A and B form of DNA and b) the idea of water bridges and filaments emerging from the computer simulation results on the hydration of DNA fragments by Clementi.

Carbohydrates↗
PubMed1981

Interferons, double-stranded RNA, and RNA degradation. Isolation and characterization of homogeneous human (2'-5')(a)n synthetase.

(2'-5')(A)n synthetase is one of the mediators of interferon action. If activated by double-stranded RNA it converts ATP into pyrophosphate and (2'-5')(A)n. In turn, (2'-5')(A)n activates a latent endoribonuclease (RNase L) which cleaves single-stranded RNA. We report here the isolation and characterization of a homogeneous human (2'-5')(A)n synthetase. The enzyme was purified from interferon-treated HeLA S3 cells by chromatography of a ribosomal salt wash fraction on DEAE-cellulose, poly(I) . poly(C) agarose, and CM-cellulose. The purified (2'-5')(A)n synthetase can convert over 90% of ATP into (2'-5')(A)n. The enzyme is unstable but can be stabilized by certain nonionic detergents (e.g. Triton X-100). Its apparent Mr = 100,000, as determined by gel electrophoresis in sodium dodecyl sulfate, and about 80,000, as determined by centrifugation through a glycerol gradient. The human (2'-5')(A)n synthetase is similar to the corresponding enzyme from mouse Ehrlich ascites tumor cells, but differs from the latter in size (100,000 versus 105,000 daltons) and in ionic conditions required for maximal activity.

2',5'-Oligoadenylate Synthetase↗