David F. Hickok

Molecular dynamics simulations in solvent of the glucocorticoid receptor protein in complex with a glucocorticoid response element DNA sequence.

We investigated protein/DNA interactions, using molecular dynamics simulations computed in solvent, between the glucocorticoid receptor (GR) DNA binding domain (DBD) amino acids and DNA of a glucocorticoid receptor response element (GRE). We compared findings obtained from a fully solvated 80 Angstrom water droplet GR DBD/GRE model with those from a 10 Angstrom water layer GR DBD/GRE model. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Molecular dynamics simulations from both models yielded similar findings; amino acids of the GR DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon/anticodon nucleotide base sites within the GRE right major groove halfsite. Likewise GR DBD amino acids in a beta strand structure adjacent to the DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon/anticodon nucleotide base and backbone sites. We also investigated protein/DNA interactions with a 10 Angstrom water layer model consisting of the same GR DBD as above but with a predicted alpha helix attached to the carboxyl terminus of the GR DBD docked at the same GRE as above with additional flanking nucleotides. In this model, the interactions between amino acids of the DNA recognition helix and beta strand and nucleotides within the GRE right major groove halfsite were at cognate codon/anticodon nucleotide sites as found in the two models above. In addition, amino acids within the predicted alpha helix located on the carboxyl terminus of the GR DBD interacted at codon/anticodon nucleotide sites on the DNA backbone of the GRE flanking nucleotides. These interactions together induced breakage of Watson-Crick nucleotide base pairing hydrogen bonds, resulting in bending of the DNA, strand elongation and unwinding events similar to those described for helicases.

Laser laparoscopic vagotomy and pyloromyotomy

We performed bilateral truncal vagotomy and gastric drainage procedure using standard laparoscopic instruments in five mongrel dogs. The procedure consisted of a transthoracic thoracoscopic bilateral truncal vagotomy and transperitoneal laparoscopic pyloromyotomy. A contact Nd:YAG laser fiber was used. There was no mortality, minimal morbidity, and post-operative gastric emptying was satisfactory. Pathologic studies indicated vagotomy was complete. We believe that this may be the initial step in the development of a simple, safe, and effective endoscopic procedure for the treatment of peptic ulcer disease.

Genetic sequences of hormone response elements share similarity with predicted alpha helices within DNA binding domains of steroid receptor proteins: A basis for site-specific recognition

Abstract The 150 amino acid sequence comprising the DNA binding region of rat glucocorticoid receptor protein, RGRDBR, was compared to amino acid sequences of members of the superfamily of eukaryotic DNA regulatory proteins. Maximal similarity fell within the 86 amino acid sequence of RGRDBR reported to contain both DNA binding and transcription regulating properties and within the reported DNA binding regions of those proteins to which it was compared. Chou-Fasman secondary structure predictions within these DNA binding domains revealed a conserved alpha helix-beta turn-alpha helix motif. The 450 nucleotide sequence comprising the complementary DNA (cDNA) of amino acids making up RGRDBR was compared to a nucleotide sequence (−312 to −38) from mouse mammary tumor virus 5′ long terminal repeat, MMTV5LTR, known to contain glucocorticoid response elements (GREs). The maximally similar subsequence was found within the coding region for predicted alpha helix B of RGRDBR (nucleotides 1376 to 1412) and within a reported GRE of MMTV5LTR (nucleotides −199 to −131). This MMTV5LTR GRE sequence contains an imperfect palindrome of TGTTCT which is the specific recognition motif for DNA binding by both glucocorticoid and progesterone receptors. Since there are multiple coding possibilities for the majority of the 20 known amino acids, the exceptions being methionine and tryptophan which have a single codon, to thoroughly investigate the extent of genetic information conserved between RGRDBR and GRE, we converted this MMTV5LTR GRE nucleotide subsequence (−199 to −131) to amino acids in all three reading frames reading rightward and leftward in both strands. This procedure revealed all coding possibilities within the MMTV5LTR nucleotide subsequence, as well as the location of the codon sites. A comparison of these MMTV5LTR amino acid coding possibilities to RGRDBR predicted helix B amino acids revealed highly conserved genetic information localized within the GRE half-sites, predominantly in the right half-site containing the TGTTCT sequence. In the absence of atomic coordinates for eukaryotic DNA regulatory proteins, a computer model of a eukaryotic/procaryotic hybrid protein was created with RGRDBR predicted helix B replacing helix of F of E. coli cAMP-dependent regulatory protein (CRP) for which coordinates from X-ray crystallography were available. This hybrid protein was docked onto MMTV5LTR at the region of maximal similarity to helix B. Our computer model shows that the side chains of amino acids within RGRDBR helix B are oriented toward, and appear to be capable of interacting with, nucleotides on both strands of their respective codons within a functional GRE. Calculations of H-bonding in this model indicate that amino acids of helix B are forming H-bonds with nucleotides of their cognate codon/anti-codon sites within the major grooves of the GRE half-sites.

Experimental transperitoneal laparoscopic pyloroplasty.

Interest in laparoscopic abdominal surgery continues to grow, which has persuaded a number of centers to pursue actively laparoscopic techniques that will allow surgeons to perform additional operative procedures in a less invasive manner. Peptic ulcer surgery, because of the morbidity associated with gastric surgery as well as the pain and discomfort associated with any major abdominal operation, has been largely replaced by pharmacologic therapy. As a result, patients are often advised to continue drug therapy indefinitely. This form of therapy, however, often only partially relieves the symptoms associated with peptic ulcer disease and leaves the patient at risk to develop life-threatening complications such as bleeding and perforation. Therefore, the rapid advances occurring in the field of laparoscopic surgery provide a fertile area for the development of simple, safe, and effective procedures to treat peptic ulcer disease in selected patients. A variety of different peptic ulcer operations have already been successfully performed under laparoscopic guidance. This report describes an experimental technique of transperitoneal stapled laparoscopic pyloroplasty using a modified end-to-end anastomotic stapling device (EEA Stapler; United States Surgical Corporation, Norwalk, CT, U.S.A.). The feasibility of this procedure was documented by detailed histologic evaluation of the pyloroplasty and revealed that the pyloric musculature had been excised, resulting in a true gastroduodenostomy. Pyloroplasty, coupled with either transabdominal or transthoracic vagotomy, could be a simple alternative to more extensive open abdominal surgery. This procedure represents one additional step in providing the practicing surgeon with the ability to perform a variety of different ulcer operations in a minimally invasive (laparoscopic) fashion.

EVIDENCE FOR TUMOR-SPECIFIC ANTIGENS IN FILTERED TISSUE CULTURE MEDIA FROM HUMAN CANCER CELLS

SummaryThe tissue culture medium taken from 7-day-old cultures of human cancer cells was filtered and the filtrate (TCTA) was used to activate autologous lymphocytes. The activated lymphocytes were found to inhibit the growth of autologous human cancer cells in vitro but not the growth of normal autologous cells, allogeneic tumor cells, or xenogeneic tumor cells.Autologous serum blocked activation of the lymphocytes. Further, autologous serum, when incubated with autologous tumor cells, prevented the inhibition of tumor cell growth by activated lymphocytes. Marked specificity was noted in these studies, which suggests that these reactions may be of value in isolating human cancer antigens. Thus, TCTA may be a source of soluble tumor-specific antigens.

A One Nanosecond Molecular Dynamics Simulation of the Glucocorticoid Receptor Protein in Complex With a Glucocorticoid Response Element DNA Sequence in a 10 Angstrom Water Layer

We investigated protein/DNA interactions, using molecular dynamics simulations computed for one nanosecond, between a 10 Angstom water layer model of the glucocorticoid receptor (GR) DNA binding domain (DBD) amino acids and DNA of a glucocorticoid receptor response element (GRE) consisting of 29 nucleotide base pairs. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the GR DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the GRE DNA right major groove halfsite. Likewise amino acids in a beta strand structure adjacent to the DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within both the GRE right and left major groove halfsites. In addition, amino acids within a predicted alpha helix located on the carboxyl terminus of the GR DBD interacted at codon-anticodon nucleotide sites on the DNA backbone of the GRE right major groove flanking nucleotides. These interactions together induced breakage of Watson-Crick nucleotide base pairing hydrogen bonds, resulting in significant structural changes and bending of the DNA into the protein.