Rapid and efficient purification of actin from nonmuscle sources.

The need for biochemical quantities of nonmuscle actin has been increased by observations that actin isoform composition of a cell influences the cell's motile and structural properties. In addition, the number of actin binding proteins that exhibit different binding interactions with beta- and gamma-actin compared to alpha-actin from skeletal muscle is growing. We report a procedure designed to purify actin from nonmuscle tissues employing extraction of monomeric actin from tissues with high concentrations of Tris, chromatography on DE-53 cellulose, and affinity chromatography of DNase I-agarose. The preparation is easy to perform and yields quantities of nonmuscle actin sufficient for biochemical and cell biological assays. Actin from bovine erythrocytes and from brains of adult and embryonic chickens was obtained using this method, which can be readily used with other sources of tissue. Coomassie-Blue-stained SDS gels of the purified actin show no contaminants; capping protein, a common contaminant of actin preparations, is absent by immunoblotting. This method for purifying nonmuscle actin will be useful to investigate functional differences in the biology of actin isoforms or their regulating proteins.

Dynamics of capping protein and actin assembly in vitro: uncapping barbed ends by polyphosphoinositides.

Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, we studied the kinetics of capping protein, the major barbed end capper, binding to barbed ends in vitro. First, the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. However, we find that phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo. Second, the on-rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime. Third, capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded. Fourth, the proposed capping protein regulators, Hsc70 and S100, had no effect on capping protein binding to actin in vitro.

U.S. military working dogs with Vietnam service: definition and characteristics of the cohort.

We verified and corrected inaccuracies in descriptive profile information on military working dogs (MWDs) that died from 1965 to 1980 and were reported in the Armed Forces Institute of Pathology Registry of Veterinary Pathology. Using other available military records, we determined which dogs served in Vietnam. Identified were 3,895 MWDs with a unique identity tattoo that served in Vietnam, of which 2,389 served exclusively with U.S. military forces and died in Vietnam. Another 479 Vietnam veteran MWDs completed their service elsewhere. This overall effort resulted in signalment corrections, generally age at death, in 21% of the Registry MWD accessions during the study period. The improved definition and characterization of the Vietnam cohort will lead to greater precision in epidemiologic investigations of the health effects from the Vietnam experience in U.S. military working dogs.

Idiopathic renal hematuria in a military working dog.

A 1.5-year-old male Belgian Malinosis Military Working Dog presented with a 1-month history of intermittent hematuria. Diagnostic ultrasound and contrast radiography demonstrated large blood clots in the urinary bladder and a filling defect in the right renal pelvis. At surgery, clotted blood was present in the right ureter and bladder. Following right nephrectomy, the dog returned to training. One month later, elevations in urea nitrogen and creatinine were noted. Hematuria recurred at 3 months and the dog was found dead in its kennel. Necropsy showed a blood-filled left renal pelvis and ureter.

Epidemiology of gastric dilatation-volvulus in the military working dog program.

Examination of case records of 914 military working dogs which died during the period January 1, 1987-December 31, 1989, revealed 31 deaths (3.4%) due to gastric dilatation-volvulus (GDV) or its complications. The fata episode of GDV was the only reported occurrence of this condition in 81% of the dogs. Male German shepherd dogs, 6-10 years of age, that were fed a medicated high-energy ration once per day, predominated. Dogs in this study that had a prior history of acute gastric dilatation (AGD) or GDV, and that were decompressed by non-surgical means, eventually died of GDV. A comparison with dogs surviving an AGD or GDV episode from the same time period showed most to have had surgical decompression and gastropexy.

Intermittent gastric dilatation after gastropexy in a dog.

Gastroperitoneal adhesions, which developed after tube gastrostomy in a 3-year-old dog, caused an inverted L configuration of the pyloric antrum and duodenum, resulting in periodic episodes of gastric dilatation. The dog had undergone tube gastrostomy for treatment of gastric dilatation/volvulus, but gastropexy adhesions broke down 27 months later, necessitating a second pexy procedure. Adhesions then developed, constricting gastric outflow and trapping gas in the stomach and proximal duodenum. When the ventral row of adhesions was surgically dissected, the angle between the pyloric antrum and the duodenum was straightened, facilitating flow of digesta. Gastropexy rarely causes the degree of adhesion formation and the complications reported in this dog.

A comparison of surgical training with live anesthetized dogs and cadavers.

Cadavers were compared with live anesthetized dogs for their effectiveness as models for surgical training of veterinary medical students. One group of students was trained using cadavers, and a peer group was trained using live anesthetized dogs. Both groups then performed an intestinal anastomosis using a live subject. The time to completion of the procedure was recorded. The anastomoses and celiotomy closures were evaluated. Each anastomosis was isolated and pressure tested. Reviewers blindly scored each surgical team's performance based on actual inspection of the surgical site and on viewing videotapes of the procedure. The participants' attitudes toward the use of live animals in teaching and research were documented before and after training. No statistically significant differences could be detected between the two groups. The results suggest that some substitution of cadavers for live dogs in surgical training might be feasible.

Veterinary care of the Belgian Malinois military working dog.

The Belgian Malinois dog was introduced into the Department of Defense Military Working Dog Program in 1984. All dogs are purchased in Europe and have adapted well to the rigorous training requirements of the program. Most veterinary problems seen in this breed are related to kennel confinement and include traumatic dental disease, tail lesions, a variety of dermatologic conditions, and an inability to gain weight. Epilepsy is a breed problem and has been seen in this population, as has gastric dilatation/volvulus. Both elbow and hip dysplasia exist in the Malinois breed.

Serum zinc levels in sheep with experimental pancreatic abnormalities.

Serum zinc levels were measured in sheep before and after total pancreatectomy, pancreatic duct ligation, alloxan treatment, and pancreatic biopsy. Values were elevated after each procedure, but only total pancreatectomy and pancreatic duct ligation caused a significant increase (t = 3.5, p less than 0.05; t = 2.8, p less than 0.05, respectively). These effects were not due to changes in serum proteins. The results are consistent with a model in which the pancreas removes zinc from blood and subsequently eliminates it via the pancreatic duct. Facilitation of intestinal absorption of zinc by pancreatic products and effects of insulin on serum zinc are less significant.

Intracoronary streptokinase therapy in the coronary care unit for acute myocardial infarction.

Intracoronary streptokinase was offered and preliminary coronary angiography performed in 14 patients who were seen with the clinical diagnosis of acute myocardial infarction within 4 h of onset of symptoms. The procedure was performed in the Coronary Care Unit (CCU) of St. Peter's Medical Center with the use of a portable C-arm fluoroscope. Angiography was recorded on video tape. Service was provided by an "on-call" team consisting of two physicians, a CCU nurse, and a radiology technician, on a 24-h service basis. Adequate visualization of coronary anatomy was obtained in all patients. Patency of occluded vessels was achieved in 10 of 11 patients who received intracoronary streptokinase. The initial streptokinase bolus was administered at a mean interval of 4.1 h from onset of symptoms. It is concluded that speedy and effective coronary thrombolytic therapy can be provided in the CCU on a 24-h service basis by an on-call team. The use of CCU for this purpose will make this therapy widely available across the country, without the need for Cardiac Catheterization Laboratory.

Tumoricidal effects of Mycobacterium ulcerans toxin on murine adenocarcinoma (C3HBA).

Fresh homogenized mammary gland adenocarcinoma (C3HBA) material was subcutaneously inoculated in the anterior right thigh of transplant host mice (C3H/HeJ). When tumors were palpable, the mice were separated into three groups. The test group received biweekly intratumoral injections of Mycobacterium ulcerans filtrate containing 40 mg of protein per ml. One control group received intratumoral injections of normal physiological saline, and the remaining group received no treatment at all. The survival rate of toxin-treated animals was 13.5% at 15 weeks when compared with the control groups. Additionally, tumor metastasis was apparently abrogated in test animals when compared to the control animals. Marked necrosis of the tumors in the test animals was noted by histological examination. Controls showed slight to moderate degrees of tumor necrosis. In this model Mycobacterium ulcerans toxin appears to have therapeutic value as an antitumor agent.

Absence of circulating HL-A lymphocytotoxic antibodies in men 21 to 44 months after vasectomy.

Nineteen vasectomized men were followed for 21 to 42 months after surgery, and their sera were tested for the presence of HL-A lymphocytoxic antibodies. In a previous study, the sera of two of these men had shown a definite increase in serum reactivity 6 to 12 months after surgery. Only one of the nineteen tested in the study demonstrated, a single, weakly positive, reaction 24 months after surgery. It was considered that the initial stimulus for lymphocytotoxic antibody production was related to surgery. There was no evidence of antibody stimulation 21 to 44 months postoperatively.