Restriction enzyme fingerprinting of enterobacterial plasmids: a simple strategy with wide application

Restriction enzyme fingerprints were generated from purified plasmid DNA from 324 clinical isolates that belonged to 7 enterobacterial genera and 88 single plasmids in Escherichia coli K 12 according to the following strategy. Purified plasmid DNA was digested with PstI. The number of fragments detected in a 0.8 agarose gel was used to determine which 2 of 6 restriction enzymes including PstI was most likely to provide a fingerprint comprising sufficient fragments to ensure specificity but sufficiently few to allow easy visual assessment and minimize coincidental matching. When PstI produced greater than 20 fragments, EcoRI and HindIII were used; when PstI generated less than 6 fragments Bsp 1286 and AvaII were used and SmaI was employed when between 6 and 20 fragments were obtained from PstI digests. Using a minimum of 12 fragments from a combination of 2 enzymes as the criterion for characterizing a strain/plasmid, satisfactory 2-enzyme fingerprints were obtained from 87% of the strains and plasmids studied using PstI and no more than two additional enzymes per strain. Of the remaining 54 strains, 51 harboured only small plasmids (less than 10 kb) and 3 produced satisfactory fingerprints when digested with a fourth enzyme.     

An assessment of intrarenal hydrostatic pressure measurements in the diagnosis of acute renal allograft rejection

Postoperative intrarenal pressure measurements may be an aid to the diagnosis of acute renal transplant rejection, especially in patients treated with cyclosporine. Serial measurements of intrarenal pressure were made in 38 recipients using a fine-needle technique. Thirty-two intraoperative and 207 postoperative measurements were made, and 39 clinical rejection episodes (23 confirmed by biopsy) monitored. Intraoperative pressures in grafts with immediate function (37.4 +/- 4.0 mmHg, mean +/- SEM) were not significantly different from those with delayed function (30.9 +/- 4.8 mmHg), whereas postoperative pressures were greater (P less than 0.01) in kidneys with acute tubular necrosis (29.4 +/- 1.9 mmHg) than in functioning grafts (20.4 +/- 0.9 mmHg). Pressures recorded during clinical rejection episodes (44.3 +/- 2.3 mmHg) exceeded (P less than 0.001) those during quiescent periods (23.6 +/- 1.0 mmHg). During rejection episodes, higher pressures (P less than 0.01) were recorded from tender or palpably enlarged grafts (52.5 +/- 3.0 mmHg) than in the absence of these signs (36.3 +/- 3.1 mmHg), and patients whose transplants biopsies showed cellular rejection tended to have greater pressures (50.1 +/- 4.1 mmHg) than those with concomitant vasculopathy (36.4 +/- 3.9 mmHg), but the latter did not reach statistical significance. In 7 cases of cyclosporine toxicity the intrarenal pressure was 17.8 +/- 4.2 mmHg. Using a diagnostic cut off point of 40 mmHg, the investigation failed to recognize 26% of acute rejection episodes--and, in the presence of acute tubular necrosis, it wrongly categorized 21% of nonrejectors. While its predictive capacity was limited, the test may occasionally be helpful in the differentiation of cyclosporine toxicity and rejection in functioning kidneys.     

Coronary blood flow and cardiac adenine nucleotides in E. coli endotoxemia in dogs: effects of oxygen radical scavengers

The purposes of this study were to determine the effects of E. coli endotoxin shock on coronary blood flow (CBF) and myocardial adenine nucleotides and to determine if reactive oxygen species are major causal factors in these effects of endotoxin. Twenty-three pentobarbital-anesthetized Beagle dogs were instrumented for recording cardiorespiratory parameters, injected i.v. with saline (time-matched controls; n = 6) or endotoxin (1.5 mg/kg; n = 17), and studied for 4 h. Endotoxin dogs also received either i.v. saline (shock controls; n = 6) or i.v. treatment with either deferoxamine (30 mg/kg; n = 5) or triple therapy (n = 6) with a combination of allopurinol (150 mg/kg), superoxide dismutase (SOD) (5 mg/kg), and catalase (CAT) (5 mg/kg). Cardiorespiratory and tissue blood flow variables were constant in sham-shock controls during the study, whereas endotoxin dogs developed typical canine endotoxemia with decreased left ventricular (LV) function. CBF was decreased by approximately 40% (P less than or equal to 0.5) in all endotoxin groups throughout the 4 h study period. However, based on hemodynamic estimates of myocardial O2 demand and endocardial/epicardial blood flow ratios, it seemed that coronary flow was matched to metabolic rate in all endotoxin groups. Endotoxin significantly lowered LV myocardial concentrations of ADP, AMP, NADH, and NADPH (range = 37 to 54%, P less than or equal to 0.05), but ATP, NAD, and NADP concentrations were not changed. The adenylate charge of the myocardium was between 0.91 and 0.95 in all endotoxin groups, suggesting that adequate energy was available in the myocardium during endotoxin shock. The lack of influence of deferoxamine, allopurinol, SOD, and CAT is indirect evidence that oxygen radicals are not primary pathophysiologic mediators in the cardiac response to gram-negative endotoxemia in this endotoxin model.     

Impact of the revision of DUI legislation in Alabama

On May 19, 1980, a major revision in the Alabama DUI laws went into effect which gave judges greater discretion in sentencing. This revision resulted in an increase in the proportion of DUI convictions, a reduction in the number of DUI citations reduced to reckless driving, a reduction in the proportion of offenders acquitted and/or dismissed, an increase in the proportion of revocations, and an increase in court referrals to an educational program on the first offense. However, the 1980 revision was accompanied by a significant increase in the percentage of alcohol-related accidents. Consequently, the Alabama legislature revised the 1980 law on July 29, 1983, the revision taking effect immediately. The more stringent penalties in the new law apparently had a positive effect on all six alcohol-related measures cited above. Most importantly, the latest revision was accompanied by a significant decrease (2.80%) in the proportion of alcohol-related accidents.     

Transformation of immortal, non-tumorigenic osteoblast-like human osteosarcoma cells to the tumorigenic phenotype by nickel sulfate

Epidemiological studies have indirectly linked compounds ofchromium, nickel and arsenic to human carcinogenesis. However,there is no evidence that metal compounds can transform humancells to the tumorigenic phenotype in culture. We show herethat exposure to 36 µM NiS0₄ for 48–96 h resultsin transformation of an immortal, non-tumorigenic, osteoblast-likecell line, HOS TE85, to the tumorigenic phenotype. Continuouspassaging following treatment leads to the formation of a fewdense foci. The cells isolated and expanded from the foci aremorphologically transformed, and form anchorage-independentcolonies of the size and abundance comparable to that formedby Kirsten murine sarcoma virus transformed HOS TE85 cells.The transformed cells from tumors in nude mice, have enhancedlevels of plasminogen activators and have lost the ability toform model bone matrix on extended culture in the presence ofascorbic acid and ß-glycerophosphate. A number ofcell lines have been established from nude mouse tumors. Cytogeneticanalysis reveals 16 marker chromosomes and an aberrant chromosome16. This is the first report of the transformation of a humancell line to tumorigenic phenotype by a metal carcinogen.     

Alterations in erythrocyte Na+,K+-cotransport in normal and hypertensive human pregnancy

Many physiological variables known or thought to affect erythrocyte Na+,K+-cotransport are altered in pregnancy. The interrelationships of Na+,K+-cotransport and pregnancy were therefore examined. Values were elevated by more than 30% in both second and third trimesters with a return towards non-pregnant levels in the postpartum period. Although pregnancy was also associated with elevated plasma cholesterol, renin activity and aldosterone, there was no significant relationship within the pregnant group between Na+,K+-cotransport and any of these factors. No change could be demonstrated in Na+,K+-cotransport values after 7 days of either high (greater than 250 mmol/day) or low (less than 50 mmol/day) sodium intake and values for those who developed pregnancy-associated hypertension (PAH, pre-eclampsia) were not significantly different from those in continuously normotensive women in either the second or the third trimesters of pregnancy.     

Enzymatically controlled drug delivery.

An approach for providing feedback control for polypeptide drugs in a polymeric controlled-release system uses a trigger molecule and a polymer-bound enzyme that, in the presence of that trigger molecule, will cause an acid or a base to form. When the pH inside the polymer system changes, the solubility of the drug shifts dramatically, which changes the diffusion or dissolution driving force, and hence the release rate changes correspondingly. This concept was tested using a controlled-release system of ethylene/vinyl acetate copolymer containing insulin and immobilized glucose oxidase. The enzymatic reaction of glucose to gluconic acid reduces the pH in the polymer microenvironment. Since insulin solubility increases with decreasing pH (at physiologic pH, this is true for an insulin with an isoelectric point of 7.4 or higher), the release of insulin increases in response to glucose concentration. The feasibility of this concept has been shown using trilysyl insulin with an isoelectric point of 7.4. Multiple exposures to buffered glucose solutions over several weeks caused insulin release to reversibly increase during each exposure. Polymer-implanted diabetic rats infused with glucose solutions showed a significant increase in insulin concentration in 30 min-an effect not observed in three different sets of control rats.     

Surgical division of Wolff-Parkinson-White pathways utilizing the closed-heart technique: a 2-year experience in 47 patients

Kent bundle interruption for ventricular preexcitation has been successfully accomplished utilizing several different surgical techniques. The external closed-heart technique of Guiraudon combining surgical dissection and cryoablation has been used to interrupt 52 accessory pathways in 47 consecutive patients since May, 1985. The 35 male and 12 female patients ranged in age from 10 to 67 years (mean, 30 years). There were 25 left free wall, 13 right free wall, 13 posterior septal, and 1 anterior septal accessory pathways. Preoperative and intraoperative electrophysiological studies were performed in all patients to induce the arrhythmia and localize all accessory pathways. The operation consisted of dissection of the atrioventricular fat pad. Following this, the delta wave and retrograde accessory pathway conduction disappeared, thereby indicating successful pathway ablation. In 4 patients with right-sided accessory pathways, interruption of the pathway required cryoablation. Cryolesions (made with cryoprobe at -60 degrees C for two minutes) were created in the region of the accessory pathway insertion. All accessory pathways were successfully ablated without any deaths or heart block. Concomitant surgical procedures were performed in 4 patients. Two patients required a second operation the next day for an accessory pathway not found at the initial operation. Three patients had postpericardiotomy syndrome, and 4 had recurrent atrial fibrillation requiring therapy. The remaining patients have had no arrhythmia recurrence and have remained drug free after a follow-up of 1 month to 22 months (mean, 12.5 months). We conclude that the closed-heart technique of accessory pathway ablation is safe and reproducible, obviates the necessity for aortic cross-clamping and cardioplegic arrest, and allows instantaneous monitoring of conduction over the pathway.