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.

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.

Conservation of genetic information: a code for site-specific DNA recognition.

We present findings of genetic information conservation between the glucocorticoid response element (GRE) DNA and the cDNA encoding the glucocorticoid receptor (GR) DNA-binding domain (DBD). The regions of nucleotide sub-sequence similarity to the GRE in the GR DBD occur specifically at nucleotide sequences on the ends of exons 3,4, and 5 at their splice junction sites. These sequences encode the DNA recognition helix on exon 3, a beta-strand on exon 4, and a putative alpha-helix on exon 5, respectively. The nucleotide sequence of exon 5 that encodes the putative alpha-helix located on the carboxyl terminus of the GR DBD shares sequence similarity with the flanking nucleotide regions of the GRE. We generated a computer model of the GR DBD using atomic coordinates derived from nuclear magnetic resonance spectroscopy to which we attached the exon 5-encoded putative alpha-helix. We docked this GR DBD structure at the 39-base-pair nucleotide sequence containing the GRE binding site and flanking nucleotides, which contained conserved genetic information. We observed that amino acids of the DNA recognition helix, the beta-strand, and the putative alpha-helix are spatially aligned with trinucleotides identical to their cognate codons within the GRE and its flanking nucleotides.

Laparolithic cholecystectomy: laboratory data and first clinical case.

A method of laparoscopic cholecystectomy is described. After control of the cystic duct and artery, gallstones are emulsified with a laparoscopic lithotriptor and the debris aspirated from the gallbladder. The free wall of the gallbladder is excised and the remaining gallbladder mucosa ablated with holmium:yttrium-aluminum-garnet (Ho:YAG) laser. This technique eliminates the need for dissection of the gallbladder from the liver, thereby reducing the possibility of hemorrhage from the gallbladder fossa. At the same time stones and bile are aspirated so that the excised portion of gallbladder can be easily removed through an access port without spillage of contaminated bilious debris into the abdominal cavity or puncture wound that could cause infection. Acute and chronic animal studies confirm the feasibility of this technique. A clinical case is described.

An experimental technique of laparoscopic bowel resection and reanastomosis.

With the development of laparoscopic cholecystectomy, surgeons have been stimulated to develop techniques that allow many open surgical procedures to be performed laparoscopically. Appendectomy, hernia repair, and vagotomy have already been introduced clinically. Laparoscopic bowel resection, however, is somewhat more complicated. Bowel transection, mass tissue removal, and reanastomosis in the proper geometric fashion are critical to the success of this type of operation. The introduction of the Endo-GIA stapler (United States Surgical Corp., Norwalk, CT) will make this procedure feasible on a large-scale basis. The major problem with bowel resection is not transection or tissue removal, but, rather, reanastomosis. With intracorporeal anastomosis, manipulation of the bowel with proper orientation becomes difficult. This is less of a problem when performing low-anterior resection, however, because one of the bowel limbs is fixed. The purpose of this study was to develop a technique in the laboratory that would ensure proper orientation of the two bowel limbs, with minimal manipulation prior to performance of the anastomosis. The technique that we developed and describe herein does not require manual orientation during anastomosis. Improper bowel alignment with kinking and twisting is thereby avoided. The technique appears to be useful for small- and large-bowel resections, but not for low-anterior resection. For this technique to become a reality clinically, longer endoscopic staplers with taller staple height will be required.

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.

Complications of laparoscopic cholecystectomy: a prospective review of an initial 100 consecutive cases.

In order to identify problems in concept or technique with laparoscopic cholecystectomy, a prospective analysis of the initial consecutive 100 procedures was accomplished. Ongoing review of the results led to modifications in order to improve operative outcome. Minor complications such as nausea for more than 12 h (20%) and right shoulder pain (29%) were self-limiting. There were no deaths, two bile duct injuries, two abscesses, two retained common duct stones, and one case requiring transfusion, totaling a 7% major complication rate. In the subsequent 200 laparoscopic cholecystectomies, there was a 1.5% rate of major complications. Specific measures and modifications in technique that account for this improvement are detailed. Complications of laparoscopic cholecystectomy are more frequent in initial cases but can be minimized by observing specific intraoperative principles.

Cholecystcholangiography during laparoscopic cholecystectomy: cholecystcholangiography or cystic duct cholangiography.

Laparoscopic cholecystectomy is being used more frequently in the treatment of symptomatic cholelithiasis. The procedure as originally described was performed with cystic duct cholangiography. An alternate technique of performing cholangiography is cholecystcholangiography. Because of the objections that have been voiced concerning direct gallbladder injections namely, reliability of the technique, quality of the studies, and the risk of forcing stones into the common bile duct this study was performed. Subjects were 25 consecutive patients who underwent cholecystcholangiography during laparoscopic cholecystectomy. A standard technique was developed and used. Studies were graded from 0 to 5 depending upon quality with 5 being the best and 0 the worst. A 5 consisted of visualization of all of the biliary tract structures and the duodenum and a 0 consisted of visualization of only the gallbladder. Acceptable studies (graded 3, 4, or 5) were obtained in 20 patients (80%). An inability to obtain an acceptable study could usually be determined prior to contrast injection. Accordingly there would be no time delay in proceeding directly to cystic duct cholangiography. In our patients, 48% had stones in the gallbladder smaller than the caliber of the cystic duct. Based upon the results of this study we believe that cholecystcholangiography is the technique of choice for intraoperative cholangiography during laparoscopic cholecystectomy. In patients in whom this technique is not feasible the surgeon should proceed directly to cystic duct cholangiography. There was no added risk to the patient when cholecystcholangiography was performed. There was a benefit in terms of the ease of the procedure and the performance of the procedure over cystic duct cholangiography. The determination of ductal anatomy prior to cystic duct dissection may be important in minimizing the risk of ductal injury during laparoscopic cholecystectomy.

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.

Reactivity of serum-armed xenogeneic macrophages to breast cancer antigens.

Sera from breast cancer patients contained cytophilic antibody which armed guinea pig peritoneal macrophages. These macrophages then exhibited specific adherence inhibition in the presence of tissue culture tumor antigens. These antigens were obtained from primary cultures of autologous or allogeneic breast cancer cells. Sera from control subjects for the most part did not induce macrophage adherence inhibition in the presence of these same antigens.

Regional hyperplastic lymph nodes in breast cancer: the role of lymphocytes and nodal macrophages. An immunological study with a five-year follow-up.

Five stage I and stage II breast cancer patients with sinus histiocytosis in two or more enlarged regional lymph nodes were studied. Peripheral lymphocytes, serum, and nodal lymphocytes were tested in vitro for cytotoxicity against autologous normal and tumor cells. Nodal macrophages were incubated with autologous peripheral lymphocytes and these "activated" lymphocytes then were tested in vitro for cytotoxicity against autologous normal and tumor cells. Peripheral lymphocytes (L) were not cytotoxic to autologous tumor (T) cells at 25:1 L/T ratios. Nodal lymphocytes were specifically cytotoxic to autologous tumor cells. Macrophages from hyperplastic regional lymph nodes transferred tumor specific inmunity to peripheral lymphocytes. Macrophages from small, nonhyperplastic regional lymph nodes did not transfer tumor specific immunity. With the advent of adjuvant chemotherapy and its attack on systemic immunity, a quantitative, immunopathological classification of breast cancer patients is needed in order to properly select patients for further therapy.

Adoptive transfer of tumor immunity between twins by in vitro immunized nodal macrophages and lymphocytes: reversal of serum enhancement.

The lymphocytes and serum of a nonidentical twin male with advanced colon cancer were evaluated in vitro for their ability to inhibit the growth of autologous tumor cells. The patient's serum enhanced autologous tumor cell growth. Three siblings including an HL-A matched grade A female twin underwent similar studies. Benign lymph nodes of the twins and one sibling were evaluated for the ability of nodal macrophages to be sensitized to the patient's tumor antigen. The male twin patient received intralymphatically in vitro sensitized nodal macrophages and lymphocytes from the female twin donor. Thirty days after treatment the patient's lymphocytes and serum inhibited the growth of autologous tumor cells.