Transport defects of rabbit medullary thick ascending limb cells in obstructive nephropathy.

To characterize the sodium transport defect responsible for salt wasting in obstructive nephropathy, the major sodium transporters in the medullary thick ascending limb (mTAL), the apical Na-K-2Cl cotransporter and the basolateral Na-K-ATPase, were studied in fresh suspensions of mTAL cells and outer medulla plasma membranes prepared from obstructed and untreated kidneys. Oxygen consumption (QO2) studies in intact cells revealed marked reductions in the inhibitory effects of both furosemide and ouabain on QO2 in cells from obstructed, as compared with control animals, indicating a reduction in activities of both the Na-K-2Cl cotransporter and the Na-K-ATPase. Saturable [3H]bumetanide binding was reduced in membranes isolated from obstructed kidneys, but the Kd for [3H]bumetanide was unchanged, indicating a decrease in the number of functional luminal Na-K-2Cl cotransporters in obstructed mTAL. Ouabain sensitive Na-K-ATPase activity in plasma membranes was also reduced, and immunoblots using specific monoclonal antibodies directed against the alpha and beta subunits of rabbit Na-K-ATPase showed decreased amounts of both subunits in outer medullas of obstructed kidney. A significant decrease in [3H]bumetanide binding was detected after 4 h of ureteral obstruction, whereas Na-K-ATPase activity at this time was still not different from control. We conclude that ureteral obstruction reduces the amounts of both luminal Na-K-2Cl cotransporter and basolateral Na-K-ATPase in mTAL of obstructed kidney and that these reductions contribute to the salt wasting observed after release of obstruction.

Molecular dynamics simulation in solvent of the bacteriophage 434 cI repressor protein DNA binding domain amino acids (R1-69) in complex with its cognate operator (OR1) DNA sequence.

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the 434 cI Repressor protein DNA binding domain (DBD) amino acids (R1-69) and DNA of operator (OR1) and its flanks consisting of 28 nucleotide base pairs. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the 434 cI repressor DNA recognition helix 3 formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the OR1 DNA major groove halfsites and flanking regions. In addition, hydrophilic amino acids within the loop between helix 3 and helix 4 have strong electrostatic attraction to codon-anticodon nucleotides located within the central nucleotides of the minor groove between the OR1 major groove halfsites. These interactions together induced significant structural changes in the operator DNA manifested by overtwisting of the central nucleotide base pairs and narrowing of the minor groove between the DNA major groove halfsites. Finally, these findings offer a code for site specific DNA recognition by the 434 cI repressor protein.

Molecular dynamics simulation in solvent of the estrogen receptor protein DNA binding domain in complex with a non-consensus estrogen response element DNA sequence.

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the estrogen receptor (ER) protein DNA binding domain (DBD) amino acids and DNA of a non-consensus estrogen response element (ERE) consisting of 29 nucleotide base pairs. This ERE nucleotide sequence occurs naturally upstream of the Xenopus laevis Vitelligenin A1 gene. The ER DBD is encoded by three exons. Namely, exons 2 and 3 which encode the two zinc binding motifs and a sequence of exon 4 which encodes a predicted alpha helix. We generated a computer model of the ER DBD using atomic coordinates derived from the average of 30 nuclear magnetic resonance (NMR) spectroscopy coordinate sets. Amino acids on the carboxyl end of the ER DBD were disordered in both X-ray crystallography and NMR determinations and no coordinates were reported. This disordered region includes 10 amino acids of a predicted alpha helix encoded in exon 4 at the exon 3/4 splice junction. These amino acids are known to be important in DNA binding and are also believed to function as a nuclear translocation signal sequence for the ER protein. We generated a computer model of the predicted alpha helix consisting of the 10 amino acids encoded in exon 4 and attached this helix to the carboxyl end of the ER DBD at the exon 3/4 splice junction site. We docked the ER DBD model within the DNA major groove halfsites of the 29 base pair non-consensus ERE and flanking nucleotides. We constructed a solvated model with the ER DBD/ERE complex surrounded by a ten Angstrom water layer and conducted molecular dynamics simulations. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the ER DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ERE DNA right major groove halfsite. Amino acids of the ER DBD exon 4 encoded predicted alpha helix formed direct and water mediated H-bonds with base and backbone sites of their cognate codon-anticodon nucleotides within the minor grooves flanking the ERE DNA major groove halfsites. These interactions together induced bending of the DNA into the protein.

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.

Cotrel-dubousset instrumentation in children using simultaneous motor and somatosensory evoked potential monitoring.

STUDY DESIGN: To record prospectively combined motor- and somatosensory-evoked potentials in children during scoliosis surgery using Cotrel-Dubousset instrumentation, without using special anesthetic or muscle relaxant regimens. OBJECTIVE: To determine the outcome of scoliosis surgery guided by a new technique of monitoring motor- and somatosensory-evoked potentials simultaneously. SUMMARY OF BACKGROUND DATA: Other techniques used to assess cord function generally are limited by special anesthetic requirements or assess only a limited part of the cord or monitor motor function separately from somatosensory function. METHODS: Spinal cord function was monitored using epidural leads to record simultaneously the descending motor volley (by transcranial electrical stimulation) and the ascending somatosensory volley (by tibial nerve stimulation) at two spinal levels. RESULTS: Combined motor- and sensory-evoked potentials were recorded successfully in 138 of 160 children (81%). Changes in evoked potential waveforms were seen in eight patients (5%), but resolved or lessened in response to appropriate measures. Curve correction was satisfactory, and there were no new postoperative deficits or worsening of preexisting deficits in any patient. CONCLUSION: A spinal cord monitoring system is described that is safe, reliable, accurate, and makes it unnecessary to resort to the "wake-up" test.

The effects of equine somatotropin (eST) on follicular development and circulating plasma hormone profiles in cyclic mares treated during different stages of the estrous cycle.

The effects of exogenous equine somatotropin (eST) administration on ovarian activity and plasma hormone levels were evaluated on horse and pony mares. The objectives of this study were to determine the effects of eST on follicular development and circulating concentrations of leutinizing hormone (LH), estradiol, progesterone, and insulin-like growth factor I (IGF-I) in cyclic horse and pony mares. Sixteen mares received daily injections (i.m.) of eST at a concentration of 25 micrograms/kg body weight on either Days 6 through 12 (Treatment A) or 13 through 19 (Treatment B) postovulation. In addition, contemporary mares were similarly given the carrier vehicle and served as controls (Treatments C and D). Blood samples were collected at 24-hr intervals and ultrasonographic evaluations were performed on the ovaries of each mare at 48-hr intervals beginning on the first day of treatment and ending either on the day of ovulation or 5 d postovulation. Circulating levels of insulin-like growth factor-I (IGF-I) were increased in treated mares by Day 3 post-treatment (P < 0.05). Also, mares in Treatment B exhibited a decrease in plasma estradiol concentrations (P < 0.05) when compared with control mares on Days 1 through 5 postovulation of the post-treated estrous cycle. In addition, circulating leutinizing hormone levels were different for mares in Treatment A compared with controls on Days--8 through--1 pre-ovulation (P < 0.05). All follicles present on the ovaries of each mare were measured and placed into one of five categories based on their diameter. Neither the mean number of follicles per size category > or = 8 mm in diameter nor the mean follicular diameter within each size category differed among treatment and control mares. However, eST treatment significantly increased the number of follicles < or = 7 mm on the ovaries of mares treated early in the estrous cycle when compared with control mares on Days 3 and 7 post-treatment and at the onset of standing estrus.

Unusual stress fractures of the proximal phalanx of the great toe: a report of two cases.

Stress fractures of the toes are rare. Most reported fractures of the proximal phalanx of the great toe have been associated with halux valgus deformity. Two cases are presented that illustrate several unique features of this rare injury which have not been reported before. One of the cases went on to non-union, requiring bone grafting and internal fixation.

Evaluation of cranberry tablets for the prevention of urinary tract infections in spinal cord injured patients with neurogenic bladder.

STUDY DESIGN: Randomized, double blind, placebo-controlled trial with a crossover design. OBJECTIVE: To evaluate cranberry tablets for the prevention of urinary tract infection (UTI) in spinal cord injured (SCI) patients. SETTING: Spinal Cord Injury Unit of a Veterans Administration Hospital, MA, USA. METHODS: Subjects with spinal cord injury and documentation of neurogenic bladder were randomized to receive 6 months of cranberry extract tablet or placebo, followed by the alternate preparation for an additional 6 months. The primary outcome was the incidence of UTI. RESULTS: Forty-seven subjects completed the trial. We found a reduction in the likelihood of UTI and symptoms for any month while receiving the cranberry tablet (P<0.05 for all). During the cranberry period, 6 subjects had 7 UTI, compared with 16 subjects and 21 UTI in the placebo period (P<0.05 for both number of subjects and incidence). The frequency of UTI was reduced to 0.3 UTI per year vs 1.0 UTI per year while receiving placebo. Subjects with a glomerular filtration rate (GFR) greater than 75 ml min(-1) received the most benefit. CONCLUSION: Cranberry extract tablets should be considered for the prevention of UTI in SCI patients with neurogenic bladder. Patients with a high GFR may receive the most benefit. SPONSORSHIP: Spinal Cord Research Foundation, sponsored by the Paralyzed Veterans of America.

Directed molecular evolution.

We propose the existence of a relationship of stereochemical complementarity between gene sequences that code for interacting components: nucleic acid-nucleic acid, protein-protein and protein-nucleic acid. Such a relationship would impose evolutionary constraints on the DNA sequences themselves, thus retaining these sequences and governing the direction of the evolutionary process. Therefore, we propose that prebiotic, template-directed autocatalytic synthesis of mutally cognate peptides and polynucleotides resulted in their amplification and evolutionary conservation in contemporary prokaryotic and eukaryotic organisms as a genetic regulatory apparatus. If this proposal is correct, then the relationships between the sequences in DNA coding for these interactions constitute a life code of which the genetic code is only one aspect of the many related interactions encoded in DNA.

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.

An endoscopic method for recording DC potentials in the upper gastrointestinal tract.

An endoscopic sensing electrode for measuiring mucosal potential differences along the duodenum, stomach and esophagus is described. Potential profiles were plotted for 31 humans. The profiles of 11 patients with documented carcinoma differed in shape from the profiles of 20 reference subjects with no such documentiation. The profiles of all the reference subjects demonstrated a distinct concave shape with a single pronounced dip. On the other hand, carcinoma profiles did not manifest such a dip but were flattened out and/or manifested an undulatory trend.

Dermatofibrosarcoma protuberans arising on the hand.

Dermatofibrosarcoma is a relatively common, locally aggressive, soft tissue fibrous histiocytoma. Ignorance of its marked propensity for locally invasive growth can lead to inadequate excision, and patients often suffer multiple recurrences. We present a case of dermatofibrosarcoma arising on the hand, and discuss its natural history and proper surgical therapy.

Transport defects of rabbit inner medullary collecting duct cells in obstructive nephropathy.

Urinary obstruction markedly reduces collecting duct Na+ reabsorption. To define the cellular mechanisms of this derangement in Na+ reabsorption in inner medullary collecting duct (IMCD) of obstructed kidneys, suspensions of intact IMCD cells and inner medulla plasma membranes (IMPM) were prepared from 24 h obstructed and untreated control kidneys. Oxygen consumption (QO2) studies revealed marked reductions in both amiloride-sensitive and ouabain-sensitive QO2 but not ouabain-insensitive QO2 in intact IMCD cells from obstructed, compared with control animals, indicating a reduction in oxygen-dependent transport activities of both the Na+ channel and the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Amiloride-sensitive conductive 22Na+ uptake in intact IMCD cells from obstructed kidneys was significantly decreased by 45% at 10 s, 30 s, and 1-5 min (10 s: 2.42 +/- 0.63 vs. 4.49 +/- 0.64 nmol Na+ flux/mg protein, n = 7, P < 0.05; 1 min: 4.65 +/- 0.7 vs. 8.27 +/- 0.98 nmol Na+ flux/mg protein, n = 7, P < 0.05), indicating decreased activity of amiloride-sensitive Na+ channels in these cells. However, immunoblots of IMPM with antibodies to Na+ channel proteins did not show significant differences in content of Na+ channel proteins between membranes from obstructed and control groups. Ouabain-sensitive Na(+)-K(+)-ATPase activity in IMPM of obstructed kidneys was also reduced (61.1 +/- 18.1 vs. 152.6 +/- 25.8 nmol ATP degradation.min-1.mg protein-1, n = 6, P < 0.02), and immunoblots with monoclonal antibodies against the alpha 1- and beta-subunits of rabbit Na(+)-K(+)-ATPase showed a 51 +/- 7% reduction of both subunits in IMPM from obstructed kidneys (n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)