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L Everitt

Publications and source records attributed to L Everitt.

10 recordsLinked to original sources
PubMed1995

A side chain at position 48 of the human immunodeficiency virus type-1 protease flap provides an additional specificity determinant.

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.

Amino Acid Sequence↗
PubMed1994

Identification of temperature-sensitive mutants of the human immunodeficiency virus type 1 protease through saturation mutagenesis. Amino acid side chain requirements for temperature sensitivity.

Human immunodeficiency virus type 1 encodes a protease whose activity is required for the production of infectious virus. An Escherichia coli expression and processing assay system was used to screen 285 protease mutants for temperature-sensitive activity. Fourteen protease mutants had a temperature-sensitive phenotype, and approximately half resulted from conservative amino acid substitutions. Of the 14 substitutions that conferred a temperature-sensitive phenotype, 11 substitutions occurred at 6 positions that represent 3 pairs of residues in the protease that contact each other in the three-dimensional structure. These mutants assist in pinpointing regions of the protease that are important for enzyme activity and stability.

Amino Acids↗
PubMed1991

Analysis of retroviral protease cleavage sites reveals two types of cleavage sites and the structural requirements of the P1 amino acid.

Retroviruses encode a protease which cleaves the viral Gag and Gag/Pol protein precursors into mature products. To understand the target sequence specificity of the viral protease, the amino acid sequences from 46 known processing sites from 10 diverse retroviruses were compared. Sequence preference was evident in positions P4 through P3' when compared to flanking sequences. Approximately 80% of all cleavage site sequences could be grouped into two classes based on the sequence composition flanking the scissile bond. The sequences at the amino-terminal cleavage site of the major capsid protein of Gag is always a member of one of the two classes while the carboxyl-terminal cleavage site is of the other class, suggesting a biological role for the two classes. Known processing site sequences proved useful in a motif searching strategy to identify processing sites in retroviral protein sequences, particularly in Gag. In all known cleavage sites, the P1 amino acid is hydrophobic and unbranched at the beta-carbon. The sequence requirements of the P1 position were tested by site-directed mutagenesis of the P1 Phe codon in an HIV-1 Pol cleavage site. Mutations were tested for protease-mediated cleavage of the Pol precursor expressed in Escherichia coli.

Amino Acid Sequence↗
PubMed1990

The role of peptide growth factors in epithelial ovarian cancer.

We studied the effect of epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, and transforming growth factor-beta on proliferation of four epithelial ovarian cancer cell lines (OVCA 420, OVCA 429, OVCA 432, and OVCA 433). Epidermal growth factor stimulated growth of OVCA 429 cells (P = .0001) and OVCA 433 cells (P = .0002). Platelet-derived growth factor did not stimulate growth of any of the cell lines. Fibroblast growth factor stimulated growth of OVCA 420 cells (P = .003). Transforming growth factor-beta inhibited growth of OVCA 420 cells (P = .0001), OVCA 432 cells (P = .003), and OVCA 433 cells (P = .004). To detect production of known growth factors by the cancer cell lines, we tested the effect of cancer cell-conditioned media on proliferation of cell lines known to respond to growth factors. Only media exposed to OVCA 433 cells were found to contain activity that mimicked one of the known growth factors (transforming growth factor-beta). These results suggest that individual ovarian cancers vary widely in their response to and production of known peptide growth factors. Finally, we found that OVCA 429-conditioned medium significantly inhibited proliferation of mitogen-stimulated lymphocytes (P less than .0001). The characteristics of this immunosuppressive factor were distinct from those of transforming growth factor-beta. Production of this factor by an immortalized cell line provides a unique opportunity to identify an immunosuppressive substance associated with ovarian cancer.

Cell Division↗
PubMed1989

Complete mutagenesis of the HIV-1 protease.

Retroviruses encode a protease which needs to be active for the production of infectious virions. A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease. Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein. Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation. These results show that random mutagenesis can be used to identify functionally important regions within a protein. Mutants with conditional phenotypes have also been identified within this collection.

Amino Acid Sequence↗
PubMed1989

Heterogeneity of antigen expression in benign and malignant breast and ovarian epithelial cells.

Expression of II distinct antigen families has been studied in normal and malignant epithelial cells from breast and ovary by avidin-biotin immunoperoxidase staining of frozen tissue sections. Each of the antigens was expressed in a fraction of the normal and malignant breast tissues from different individuals. Among breast and ovarian cancers, a similar fraction of tumors expressed each of the determinants. An epitope on a 42-kDa glycoprotein was expressed significantly more often by ovarian carcinomas than by benign ovarian epithelium. Several determinants on 66-, 240-kDa or high-molecular-weight proteins or glycoproteins were expressed significantly more often on benign breast epithelium than on normal ovarian epithelium. Substantial heterogeneity in antigen expression was observed among different tumor specimens. Each of 14 epithelial ovarian cancers expressed a distinctive combination of antigens. Among 18 breast cancers, 16 distinct antigenic phenotypes were observed. Interestingly, comparable heterogeneity was observed in the expression of epitopes on benign breast and ovarian epithelium, compatible with the possibility that the monoclonal reagents recognized polymorphic determinants. Substantial variations in antigen expression were also observed among cells within each neoplasm. However, when antibodies against 5 different determinants were used in combination, a majority of cells could be stained intensely in all 13 breast carcinomas tested and more than 95% of tumor cells could be stained in 10 of the 13.

Antigens, Neoplasm↗
PubMed1981

Wholeheartedly.

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Cardiac Surgical Procedures↗