Diabetes mellitus in a black-footed ferret.

Diabetes mellitus was tentatively diagnosed in a black-footed ferret with polyuria, polydipsia, polyphagia, dehydration, and weight loss. Laboratory findings (marked hyperglycemia (724 mg/100 ml), glycosuria, and ketonuria) and the subsequent favorable response to insulin therapy confirmed the diagnosis. Although lesions were not observed in the pancreas, gross and histologic findings concomitant with diabetes mellitus included arteriosclerosis, with calcification of the aorta and other major vessels; mild necrotizing hepatitis; and mild proliferative glomerulonephritis. A perineal adenocarcinoma, with metastasis to an internal iliac lymph node, was an incidental finding. Special stains demonstrated adequate numbers of beta cell granules in the islets of Langerhans. Thus, the diabetes was apparently due to a lack of release of the synthesized insulin or to diminished effectiveness of the secreted insulin.


Tiletamine and zolazepam for immobilization of wild lions and leopards.

A 1:1 mixture of tiletamine and zolazepam was used for the immobilization of lions on 26 occasions and of leopards on 22 occasions. There was a significant (P less than 0.001) positive linear relationship between duration of anesthesia and dosage for all animals in which the duration was recorded (n = 36). This response could be divided into 4 separate regressions according to species and sex; lions being more susceptible to the drug than leopards and males more so than females. When dosage was expressed in terms of metabolic weight, the duration of anesthesia depended on the sex of the animal rather than the species, males being anesthetized 15 minutes longer than females for a given dosage. The threshold dosage was higher in leopards than in lions. The amount of time that could be spent working on the animal immobilized by the drug, compared with the total time committed to its induction, anesthesia, and recovery was 35 to 55%, a proportion that is similar to that associated with the use of other irreversible intramuscular anesthetics.


Hematologic effects of xylazine when used for restraint of Bactrian camels.

Xylazine was found to be a safe and reliable drug for chemical restraint of Bactrian camels (Camelus bactrianus). Dosages of 0.27 to 0.51 mg/kg of body weight provided adequate sedation for the performance of various procedures (e.g., tuberculin testing, lymph node biopsy, and electroejaculation). Hematologic and serum biochemical values for camels restrained manually were compared with those for camels restrained with xylazine. Xylazine-treated camels had significantly lower values for RBC, hemoglobin, and packed cell volume, and significantly higher blood glucose concentrations. Venous blood gas analyses did not reveal any major acid-base disturbances resulting from the use of xylazine. Rapid arousal from the sedative effects of xylazine occurred after the intravenous administration of doxapram hydrochloride in dosages of 0.05 to 0.13 mg/kg of body weight.


Capture myopathy in pronghorns in Alberta, Canada.

From 1971 to 1975, inclusive, 594 pronghorns (Antilocapra americana) were drive trapped in Alberta; subsequently, 74 of these animals were transported to enclosures. Deaths attributable to capture myopathy (CM) occurred in 20 of the trapped pronghorns and in 17 of the pronghorns that were trapped and subsequently transported. Two neonatal pronghorns that had been pursued died with signs and lesions similar to those seen in adult pronghorns affected with CM. The diagnosis of CM was based on history, clinical signs, clinico-pathologic findings, and gross and histologic lesions. Clinical signs of CM included depression, stiffness, weakness, incoordination, recumbency, and paralysis; myoglobinuria was observed in 1 neonatal animal. Of the pronghorns that died, fewer than one-half died acutely during processing, whereas the remainder died from 1 to 13 days following their release. Gross and histologic lesions were observed in the large muscles of the hindlimbs of most affected pronghorns. Values for serum enzymes, potassium, glucose, and creatinine, were higher for drive-trapped pronghorns than for tame, hand-reared pronghorns. These findings indicated extensive damage to muscle and to other organs as a result of the capture procedures.


Capture myopathy in a moose.

Of 18 moose captured by dart immobilization from a helicopter, 1 became recumbent after release. Blood samples were collected at the time of capture and on the following 2 days. Serum creatine phosphokinase and glutamic-oxalacetic transaminase activities increased, and serum potassium concentrations decreased. Necropsy revealed extensive myopathy.


Capture myopathy in Elk in Alberta, Canada: a report of three cases.

Acute capture myopathy was diagnosed in 3 elk (Cervus canadensis). In 2 of the elk, hindlimb lameness developed within 10 days of capture. Clinical signs were not observed in the third elk, which was found dead 7 days after its capture. Elks 1 and 2 were euthanatized and all 3 animals were necropsied. In elks 1 and 2, rupture of the semi-membranosus muscle was associated with massive hemorrhage and swelling. Microscopic findings included hemorrhage, edema, myofibril fragmentation, and evidence of attempted muscular regeneration. In elk 3, several of the large muscles of the hindlimbs as well as the biceps brachii muscles of the forelimbs appeared pale, dry, and friable. Except for lack of hemorrhage and edema, microscopic findings in this elk were similar to those of elks 1 and 2.


Prevalence and distribution of Setaria yehi in southeastern white-tailed deer.

Filarial worms, Setaria yehi, were found in 286 of 1,045 white-tailed deer (Odocoileus virginianus) from 13 of 16 southeastern states. Infections were widespread in the piedmont and coastal plain physiographic provinces, but rarely occurred in deer of the mountains or in southern Florida. Infections were significantly more prevalent among fawn and yearling deer. Infections with S yehi largely accounted for a mild fibrinous peritonitis commonly observed among southeastern deer.


Fascioloides magna infection in white-tailed deer of southeastern United States.

Livers of 4,023 white-tailed deer (Odocoileus virginianus) from 13 southeastern states were examined for flukes; 514 (12.8%) harbored Fascioloides magna. Other species of liver flukes were not found. Forty-two nidi of infection were found, encompassing 56 counties in 9 states. As many as 125 flukes were recovered from a single deer. Although F magna was widely distributed throughout southeastern United States, most enzootic areas were confined to the coastal plain physiographic province. Since deer serve as reservoirs for F magna infections in domestic livestock, it was concluded that every precaution should be exercised to prevent introduction of liver flukes into areas now free of this helminth.


Agglutinins to Coxiella burnetii and Brucella spp, with particular reference to Brucella canis, in wild animals of southern Texas.

The prevalence of agglutinins to Coxiella burnetii and Brucella spp, particularly Brucella canis, was determined in 269 wild animals (14 species) in southern Texas. Serologic evidence of coxiellosis and brucellosis, including B canis infection, was shown for coyotes, raccoons, opossums, badgers, jackrabbits, and feral hogs. Using the microagglutination test, the seroprevalence of C burnetii, phases I and II (titer greater than or equal to 4) was 4.1 and 27.9%, respectively. For brucella agglutinins, prevalence rates were 7.1, 8.9, and 6.7%, as determined by the brucellosis card test, the rapid slide agglutination test, and the salt 2-mercaptoethanol tube agglutination (titer greater than or equal to 50) test, respectively.


Type-A influenza viruses in the feces of migratory waterfowl.

Type-A influenza viruses were commonly recovered from cloacal swab specimens from migratory waterfowl. Experiments indicated that the the virus replicated in various cells of the digestive tract.


Isolation of type A influenza and Newcastle disease viruses from migratory waterfowl in the Mississippi flyway.

Twenty-seven chicken red blood cell agglutinating agents were isolated from 187 tracheal swabbings of apparently healthy migratory mallard ducks (Anas platyrhynchos) in the Mississippi flyway. Twenty-four of the isolants were type A influenza virus; 3 lentogenic Newcastle disease viruses were isolated. Isolations were not made from either 65 giant Canada geese (Branta canadensis) or 60 Franklins' gulls (Larus pipixcan).


Suspected lead toxicosis in a bald eagle.

An immature bald eagle (Haliaeetus leucocephalus) was submitted to the University of Maryland, College Park, for clinical examination. The bird was thin, had green watery feces, and was unable to maintain itself in upright posture. Following radiography, the bird went into respiratory distress and died. Numerous lead shot were recovered from the gizzard, and chemical analysis of liver and kidney tissue revealed 22.9 and 11.3 ppm lead, respectively. The clinical signs, necropsy findings, and chemical analysis of the eagle were compatible with lead toxicosis.


Iodine-induced goiter in penguins.

Goiter caused by excessive exposure to an iodine-containing disinfectant was an incidental finding at necropsy of captive penguins dying from renal failure, believed due to nutritional secondary hyperparathyroidism. The disease was reproduced experimentally in ducklings.


Chronic cutaneous candidiasis in bottle-nosed dolphins.

In a 20-month period, generalized chronic cutaneous candidiasis developed in 3 performing bottle-nosed dolphins kept in an indoor pool. Extensive esophagogastric ulcerations were observed in 2 of the dolphins, each of which died, presumably because of these lesions. The 3rd dolphin died during a surgical procedure and did not have any esophagogastric ulcerations. Candida albicans was the only organism isolated from skin lesions but was not isolated from adjacent normal skin of dolphins. Treatment with antifungal drugs was unsuccessful. Subsequently, immunopotentiating treatment with levamisole phosphate resulted in formation of granulation tissue and healing of the skin lesions.


Cephalosporins. I. Cephaloglycin analogs with six-membered heterocycles in the C-3 side chain.

Cephaloglycin analogs with six-membered heterocycles in the C-3 side chain have been prepared by nucleophilic substitution of 7-aminocephalosporanic acid with appropriate azine thiols followed by 7-N-acylation with phenylglycine by the mixed anhydride method. Seventeen thiols of non-substituted or substituted pyridines, pyridazines, pyrimidines, pyrazines and triazines were used as the S-nucleophiles. In general, pyridazine thiols gave cephalosporins processing good antimicrobial activity against both gram-positive and gram-negative bacteria. Among them 6-hydroxypyridazine-3-thiol gave the most active compound of this series, BB-S 118 (1f), which was significantly more active than cephalexin and cephaloglycin in vitro against gram-positive and gram negative bacteria.


Cephalosporins. II. 7-(O-Aminomethyl-phnylacetamido) cephalosporanic acids with six membered heterocycles in the C-3 side chain.

7-(o-Aminomethylphenylacetamido)cephalosporanic acids with six-membered heterocycles in the C-3 side chain were prepared by nucleophillic substitution of 7-ACA at the C-3 acetoxy group followed by N-acylation of the 7-amino group. The 7-side chain acid, o-aminomethylphenylacetic acid (5), was prepared by two new convenient routes, which involved Schmidt reaction of indanone (2) followed by cleavage of the lactam ring or reduction of o-cyanophenylacetic acid (10) starting from o-nitrotoluene. The antibacterial activity of the cephalosporins in this series depends on the heterocycle in the C-3 side chain. In general pyridazines gave cephalosporin derivatives possessing better activity than those with a pyridine or pyrimidine ring. The most active member of the new cephalosporins was 7-(o-aminomethylphenylacetamido)-3-(6-hydroxypyridazin-3-ylthilmethyl)-3-cephem-4-carboxylic acid (BB-S 150) (1g) which has in vitro antibacterial activity superior to cephalothin and cefazolin against both gram-negative and gram-positive organisms. The in vitro activity of BB-S 150 determined in mice was superior to cephalothin and comparable to cefazolin.


Cephalosporins. III. 7-(O-Aminomethylphenylacetamido)cephalosporanic acids with bicyclic heteroaromatics in the C-3 side chain.

Bicyclic heteroaromatic thiols with a bridge-head nitrogen atom were used for nucleophilic substitution of 7-ACA at the C-3 acetoxy function followed by N-acylation of the 7-amino group with o-aminomethylphenylacetic acid to afford a series of new cephalosporins (24) with potent antibacterial activity against gram-positive and gram-negative organisms. The most active member of this series was 7-(o-aminomethylphenylacetamido)-3-(tetrazolo-[4,5-b]pyridazin-6-ylthiomethyl)-3-cephem-4-carboxylic acid (BB-S 226) (24e) with antibacterial activity superior activity superior to cephalothin and cefazolin.


Structure of aplasmomycin.

A new antibiotic, aplasmomycin, was isolated from a broth cultivated with a marine isolate of actinomycete, and inhibits Gram-positive bacteria in vitro and Plasmodium berghei in vivo. It is a natural ionophore and the structure of the Ag-salt was solved by an X-ray crystallographic analysis. It has symmetric structure having boron in the centre of the molecule.


Ribostamycin, as an intermediate in the biosynthesis of neomycin.

A mutant of a neomycin-producting Streptomyces fradiae was found which synthesizes ribostamycin instead of neomycin. After a reverse mutation new colonies were obtained producting neomycin again. Ribostamycin might thus be considered as an intermediate in the biosynthesis of neomycin.


A new pencillin with anti-klebsiella activity: 3-(5-tetrazolyl) penam.

The in vitro activity of a new semi-synthetic penicillin, CP-35,587, 3-(5-tetrazolyl) penam, was investigated against 496 clinical isolates of gram-negative bacilli and 113 clinical isolates of gram-positive cocci. All of the gram-positive cocci were sensitive to CP-35,587 except penicillin G resistant isolates of Staphylococcus aureus. This antibiotic inhibited a majority of isolates of Escherichia coli, Klebsiella spp. and Proteus mirabilis at a concentration of 6.25 microgram/ml. Also, approximately half of the isolates of Serratia marcescens and Enterobacter spp. were inhibited at a concentration of 12.5 microgram/ml. CP-35,587 was inactive when high concentrations of organisms were used as inocula. CP-35,587 was more active than mezlocillin, azlocillin, amoxicillin, ticarcillin and carbenicillin against isolates of K. pneumoniae, but other penicillins were more acte than CP-35,587 against other species of gram-negative bacilli.


In vitro studies with cefazaflur and other parenteral cephalosporins.

Cefazaflur has a broad-spectrum of in vitro antibacterial activity equal to or greater than that of the commercially-available cephalosporins. In addition, cefazaflur has activity against isolates of Enterobacter, Citrobacter and indole-positive Proteus; however, this activity decreased markedly when the MIC determinations were carried out with a large inoculum size. A similar inoculum effect was observed with cefamandole, however, cefoxitin's activity was relatively unchanged at increased inoculum sizes. Human serum had a relatively small effect on the in vitro activity of cefazaflur.


The effect of the absence of close supervision on the use of response cost in a prison token economy.

A naturally occurring experiment, in which direct supervision of a token economy in a penal system was removed and re-instated, is reported. A retrospective analysis revealed that in the absence of close supervision the use of response cost rose dramatically, both in terms of categories of behaviors for which response costs were levied and in the frequency of their use. The return of direct supervision led to a decreased use and and an end to the growth of categories of behaviors punished.


Acquisition, discriminative stimulus control, and retention of increases/decreases in blood pressure of normotensive human subjects.

The aims of this study of 24 normotensive subjects were: to compare a free-operant with a discrete-trials training format; to determine the most effective training procedure by comparing instrumental conditioning with instructional set and a control; to see if both increases and decreases in blood pressure could be brought under discriminative control, and to examine the maintenance of acquired self-control of blood pressure. A 2 X 3 design was employed in which two trial formats (free operant and discrete trials) were factorially compared with three training conditions (instrumental conditioning, instructional set, and control). Instrumental conditioning was found superior to both the instructional set and control conditions in producing increases and decreases in mean diastolic blood pressure. The free-operant format led to a greater degree of learned BP control in that subjects were able to increase and decrease their blood pressure by 10% to 15% of basal value and to maintain the blood-pressure operant after contingent auditory feedback/reinforcement was removed. Training was discontinued when subjects in the other five groups failed to reach criterion after 10 consecutive acquisition sessions.


Temporal and climatic variables in naturalistic observation.

Home-observation data on 5- to 7-yr-old boys collected over 2 yr were examined for systematic variations in rates of desirable and undesirable behaviors associated with several temporal and climatic variables. Significant effects associated with time of day, day of the week, precipitation, and temperature were found. No significant effects on the naturalistic observation data were found for environmental factors associated with lunar phase. It was noted that the correlational nature of the findings did not obviate the necessity for control of the influence of temporal and climatic variables. Several methodological strategies for such control were discussed.


Stimulus control in the classroom as a function of the behavior reinforced.

Eight fifth- and sixth-grade children with behavior problems performed in a classroom under three conditions: (1) unreinforced baseline, (2) reinforcement for being on task, and (3) reinforcement for the accuracy and rate of math problems solved. The teacher was absent for a portion of the class session under each of these conditions. In the teacher's absence, on-task behavior declined markedly and disruption markedly increased, regardless of the reinforcement condition in operation. In addition, the teacher's absence resulted in fewer problems attempted and decreased accuracy. However, the extent to which the children became disruptive was reduced and the number of problems attempted increased when reinforcement was contingent on academic accuracy and rate, instead of being contingent on being on task. The results suggest that by providing contingencies for the products of a child's classroom activities, rather than for being on task, the child will become more independent of the teacher's presence, and more under the control of the academic materials.


Attention training: the use of overcorrection avoidance to increase the eye contact of autistic and retarded children.

A crucial first step in teaching and training the retarded and autistic is to develop and maintain eye contact with the therapist. Functional movement training (an overcorrection procedure) plus edibles and praise were compared with edibles and praise alone as a method of developing eye contact in three such children. In both conditions, the child was given food and praise when eye contact occurred within 5 sec of the therapist's verbal prompt: "Look at me." Functional movement training avoidance plus edibles and praise produced about 90% attention for the three children, while edibles and praise alone were less effective (eye contact never exceeded 55%). Functional movement training avoidance combined with edibles and praise appears to be an effective method of teaching eye contact and possibly other forms of instruction-following to behaviorally disordered children who are not always responsive to positive consequences.


Issues and implications of establishing guidelines for the use of behavioral techniques.

Several guidelines surrounding the use of behavioral procedures have recently appeared, the best of which is that of the National Association of Retarded Citizens (NARC). Some issues and implications of the establishment of guidelines are briefly reviewed in the context of the NARC guidelines. Issues include the factual versus opinion bases for guidelines and the need to continue the development of explicit behavioral criteria for assessing staff competence. Implications for programs include the impact of guidelines on the professional boundaries, administrative decision-making, and budgeting, together with the dangers of expanding the regulatory bureaucracy. Several miscellaneous impacts are noted, including a potential for curbing innovative behavioral technology.


Why no guidelines for behavior modification?

This paper reviews the guidelines for behavioral programs published by the National Association of Retarded Children. The review discusses a number of reasons why guidelines should not be enunciated for behavior modification, e.g., the procedures of behavior modification appear to be no more or less subject to abuse and no more or less in need of ethical regulation than intervention procedures derived from any other set of principles and called by other terms. The review recommends alternative methods for protecting the rights of clients who participate in behavioral programs. Specifically, behavioral clinicians, like other therapists, should be governed by the ethics codes of their professions; also, the ethics of all intervention programs should be evaluated in terms of a number of critical issues.


What types of sex-role behavior should behavior modifiers promote?

Rekers and Lovaas (1974) reported a study using reinforcement procedures aimed at modifying the behavior of a 5-yr-old who engaged in cross-gender behavior. The use of traditional sex-role concepts in forming target behaviors is criticized and androgynous behavior is suggested as an empirically based alternative. The pathological nature of the behavior that the study was designed to prevent is also questioned, as is the ability to predict sex-role behavior from a 5-yr-old child's current behavior. These issues are raised in the context of the more general question, whom should the therapist serve?


Implications of the stereotyping and modification of sex role.

This article discusses and questions a study by Rekers and Lovaas (1974), which sought to "normalize" a young boy's sex-role behavior. The reasons given for treatment and the treatment itself are questioned. The ethical implications of the treatment and its outcome are then discussed, and alternative treatment procedures are suggested. Finally, the experimenters' description of the feminine sex-role is criticized.


Automated analysis of thiamine, ascorbic acid, and vitamin A.

Methods currently used in most nutrient analysis laboratories are the classical AOAC methods. One of the major challenges facing the analytical chemist is the need for new or modified quantitative and reliable nutrient assay methods, which are rapid and less labor intensive. Recently, numerous vitamin assays have been published which use continuous flow and high pressure liquid chromatographic techniques. This paper deals with some of the recent methods reported for analysis of thiamine and ascorbic acid, using continuous flow wet chemistry, and high pressure liquid chromatography for vitamin A in food products.


Automated determination of protein-nitrogen in cereals and grains.

This paper presents the design, performance, and operational characteristics of a new direct reading instrument for determining nitrogen in cereal and grain. Precision, accuracy, range, speed, ease of operation, sensitivity, sample size, and flexibility of the instrument are presented. Results on various cereals and grains are presented and compared with those obtained by the Kjeldahl technique. The apparatus is the result of research for a rapid method for determining nitrogen in organic material. The instrument uses a combination of a unique high temperature sample oxidation, a sampling valve for separating combustion products, and thermal conductivity detection for the liberated nitrogen in a helium carrier gas.


Review on computerized prediction of nutrients in thermally processed canned food.

Basic principles are briefly discussed for mathematical evaluation of nutrient concentration in thermally processed food. This evaluation is based on the use of equations for predicting the temperature of food subjected to a thermal process and for evaluating the rates of thermal degradation of a nutrient at different temperatures. Several computer programs were prepared by various researchers to minimize manual calculations required for estimating nutrient retention in thermally processed food. The computational characteristics of these programs are critically examined.


Assuring reliable performance of antibiotic assay media.

The Microbiological Assay Branch of the National Center for Antibiotics Analysis assays over 100,000 samples of antibiotic products annually, using more than 1000 Ib dehydrated media. The media must be consistently dependable to produce accurate, reliable test results. To assure that the supply of media will meet the established requirements, each lot before purchase is subjected to a series of trials designed to examine growth support, sensitivity, and behavioral and physical factors. Actual antibiotic assays are conducted with the test medium, and performance is rated against a control medium. Controls on the system reduce the variables to allow appraisal of the medium itself.


Effects of threonine supplementation on the slope assay for protein quality.

Several diets considered to be marginally deficient in threonine were fed to young male rats at protein levels ranging from 3 to 9%. Protein sources included lactalbumin (reference standard), soy protein with added methionine, pea protein with added methionine, rice-casein, and peanut-sesame-fish. Chemical scores and plasma amino acid scores indicated that these diets were limiting in threonine at the 3--4% protein level; increased growth resulted from supplementation with threonine. After threonine supplementation, all protein sources except pea were limiting in lysine. Increased growth at low protein levels with little or no extra growth at high protein levels due to threonine supplementation resulted in decreased slopes in the relative protein value (RPV) assay. Consequently, threonine supplementation apparently decreased RPV values for these samples but the protein efficiency ratio was not affected. It was concluded that the RPV assay underestimates the protein quality of lysine-deficient proteins.


Gas-liquid chromatographic determination of cholesterol and other sterols in foods.

A gas-liquid chromatographic (GLC) method, using the butyryl esters of sterols, has been developed for the measurement of cholesterol, stigmasterol, sitosterol, and campesterol in foods. An immobile phase of 1% SE-30 coated on 100-120 mesh Gas-Chrom Q packed in a 6 inch X 4 mm id glass column operated at 255 degrees C was the most satisfactory of 7 column packings evaluated. Extraction with chloroform-methanol gave 98.7% recovery with a coefficient of variation of 1.8% for cholesterol added to a variety of foods. When cholesteryl palmitate was added to vegetable oil and the butyryl derivative was prepared, followed by GLC analysis, the recovery was 99.3% with a coefficient of variation of 0.9%. Amounts as low as 1 mg/100 g food can be detected with a precision of 2.5%. The results of the analysis of a variety of foods for cholesterol, campesterol, sitosterol, and stigmasterol are given.


Gas-liquid chromatographic determination of total inorganic iodine in milk.

Total inorganic iodine in milk is determined by conversion to iodobutanone, which is quantitated by gas-liquid chromatography and electron capture detection. As little as 10 microgram/L can be determined. The thyroid-active iodine content of milk can be determined rapidly with a relative standard deviation of 1.9%. Average recoveries for added iodide and iodine were 95.5 and 94.6%, respectively.


Polarographic determination of robenidine residues in chicken tissues, eggs, litter, soil, and plant tissue.

Polarographic residue methods have been developed for determining robenidine (Robenz), 1,3-bis[p-chlorobenzylidene)amino]-guanidine monohydrochloride, in chicken tissues, eggs, litter, soil, and plants. The compound is extracted from chicken fat, skin, muscle, liver, and eggs with ethyl acetate; from blood with acetone; from plant tissue, litter, and kidney with acidic acetone; and from soil with basic methanol. After extraction by high-speed blending or overnight shaking, the extract is cleaned up by evaporation, solvent partition and/or elution from CG-50 ion exchange resin. Robenidine is quantitated by differential cathode ray polarography, using acidic aqueous methanol or acetic acid (1+1) supporting electrolyte. Recoveries ranged from 64 to 125% with an average overall recovery of 90%. The validated sensitivity is 0.1 ppm for chicken tissues, soil, and plants, 0.01 ppm for eggs, and 1 ppm for litter.


High pressure liquid chromatographic determination of saccharin in alcoholic products.

After preliminary cleanup, sodium saccharin can be quantitatively determined in wines and proprietary drugs by using high pressure liquid chromatography. The preliminary cleanup removes materials that cause column deterioration, loss of baseline stability, and interfering peaks. The average experimental error found in analyses of 6 wines for sodium saccharin content was 2.3% and the standard deviation was 0.01. In the analysis of 10 cough medicines and vitamin preparations, the difference between declared and experimental values for sodium saccharin averaged 5.7%. The recovery for the extraction process ranged from 97.8 to 101.6%.


Isolation of chlorophenoxy herbicides on columns of glass beads.

A method for the analysis of chlorophenoxy herbicide residues has been developed based on the observed adsorptive affinity of chlorophenoxy acids for glass surfaces. Extracted residues of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) herbicides are adsorbed onto a column of fine mesh glass beads, and subsequently esterified with diazomethane for gas-liquid chromatographic quantitation, using an electron capture detector. Prior to injection, the esterified sample is cleaned up by passing through a Florisil column. The method is sensitive to about 0.5 ppm 2,4-D and 0.01 ppm 2,4,5-T. Recoveries averaged from 81 to 101%, depending on the level added and the substrate.


Simultaneous determination of captan and captafol in apples and potatoes by thin layer chromatography and in situ fluorometry.

A simple method is described for the simultaneous quantitative determination of captan [N-(trichloromethylthio)-3a,4,7,7a-tetrahydrophthalimide] and captafol [N-(1,1,2,2-tetrachloroethylthio) -3a,4,7,7a - tetrahydrophthalimide] in apples and potatoes. The fungicide residues are extracted with acetonitrile, partitioned into a solution of methylene chloride in petroleum ether, separated from each other and the co-extractives on a thin layer chromatographic plate impregnated with aluminum chloride, sprayed with sodium chlorate, and heated. The fluorescence is then measured directly from the chromatogram. Recoveries were greater than 90% in the test crops at the 0.2 ppm level.


Gas-liquid chromatographic determination of isosorbide dinitrate in tablets.

A gas-liquid chromatographic (GLC) method is described for determining isosorbide dinitrate (ISDN) in tablets. ISDN is extracted from tablet excipients by a 2-phase system composed of water-ethyl ether, and detected and quantitated with a flame ionization detector after separation from the internal standard (glyceryl tributyrate) on a 3% OV-210 column. Ten replicate assays on 2 different batches of tablets, each containing 5 mg ISDN, gave coefficients of variation of 1.16 and 1.48%, respectively. Comparison of results obtained for tablets containing 5, 10, and 40 mg ISDN, and for diluted isosorbide dinitrate raw material containing 25% ISDN, showed good correlation among the GLC, USP colorimetric, and USP polarographic procedures. Assay of synthetic mixtures containing ISDN, isosorbide-2-mononitrate, and isosorbide-5-mononitrate demonstrated that the GLC procedure is specific for ISDN, whereas the USP polarographic procedure is subject to interference from the mononitrates.


Comparison of methods for determining probenecid in tablets and flavored oral suspensions containing ampicillin.

Several methods for quantitating probenecid were compared: USP XIX method for probenecid in tablets, a modified USP extraction method for probenecid in tablets, a new column extraction method for probenecid in tablets and oral suspensions containing ampicillin, and a high pressure liquid chromatographic (HPLC) method for probenecid in tablets and oral suspensions containing antibiotics. The first 3 methods were satisfactory for probenecid in bulks and tablets but proved unsatisfactory for oral suspeonsions containing flavors. The flavors gave positive interference for probenecid in oral suspensions. The HPLC method, although more time-consuming, separated probenecid from excipients, thus eliminating positive interference from flavors.


Collaborative study of a spectrophotometric determination of ferric, ferrous, and total iron in drugs by reaction with  alpha,alpha'-dipyridyl.

A spectrophotometric method for determining Fe2+, Fe3+, and total Fe in drugs has been developed, using alpha,alpha'-dipyridyl as the colorimetric reagent. The method is applicable to tablets elixirs, injectables, and bulk powder. Eight collaborates analyzed a synthetic iron preparation and 7 commercial samples. For the synthetic iron preparation, the average recovery was 100.3% and the coefficient of variation was 1.02%. For 7 commercial samples, the average coefficient of variation was 1.44%. The method has been adopted as official first action.


The variability of AOAC methods of analysis as used in analytical pharmaceutical chemistry.

Pharmaceutical analytical chemistry, which ordinarily deals with the analysis of formulations containing from 0.1 to 100% of active ingredient, uses methods with a reproducibility (between-laboratory variability) of about 2.5% and a repeatability (within-laboratory variability) of about half that amount. The best between-laboratory precision attainable appears to be about 1.0% and within-laboratory precision, about 0.5%. On the basis of the results available, automated methods do not appear to be any more precise than manual methods, although the studies show fewer outlying data points. Replicates (preferably blind ones) should always be conducted in a collaborative interlaboratory study in order to obtain the important information as to whether efforts should be concentrated on improving the method itself or on the performance of laboratories and analysis in applying it.


Screening method for the detection of aflatoxins, ochratoxin, patulin, sterigmatocystin, and zearalenone in cereals.

A screening method has been developed for the detection of aflatoxins, ochratoxin A, patulin, sterigmatocystin, and zearalenone in cereals. After extraction, the sample is cleaned up by gel filtration. The mycotoxins are separated by thin layer chromatography. The limits of detection are about 5 microgram aflatoxins, 10 microgram ochratoxin A, 50 microgram patulin, 10 microgram sterigmatocystin, and 35 microgram zearalenone/kg.


Application of linear discriminant analysis to the differentiation of pure milk from different species and mixtures.

Discriminant analysis is used to identify different milk samples on the basis of the gas chromatographic data for fatty acids in 20 samples each of milk fat from cows, sheep, and goats. The method can differentiate mixtures and pure milks with a high degree of correct classifications. A good discrimination can also be obtained by using a reduced set of variables. The method is useful for the interpretation of gas chromatographic data and should allow a higher proportion of correct classifications than is possible by visual inspection of the chromatograms.


Rapid determination of alkaline phosphatase reactivation.

A study was conducted to determine the feasibility of using the qualitative screening method specifying phenolphthalein monophosphate for differentiating reactivated and residual alkaline phosphatase activity. The relative increase in activity of the enzyme in the presence of MgCl2 serves to distinguish reactivated and residual alkaline phosphatase activity. Ten samples each of pasteurized (172 degrees F for 24 sec), sterilized (about 300 degrees F for a minimum of 2 sec) half-and-half and heavy cream were analyzed. Most samples yielded negative results initially but demonstrated activity after incubation 1 hr at 34 degrees C. The average values in terms of + marks, for the half and half and heavy cream in samples without MgCl2 were less than 1 and 2.4, respectively; for samples treated with MgCl2, the values were 2.18 and 4.6, respectively, indicating reactivated phosphatase activity. In samples containing various levels of raw milk, the activity observed in the diluted, Mg2+-containing samples was less than in the undiluted samples containing no Mg, indicating residual phosphatase activity.


Rapid determination of alkaline phosphatase activity in cheese.

A visual screening method specifying phenolphthalein monophosphate as the substrate was applied to cheese by analyzing the extract obtained with 7.5% butanol. In the extraction step, all of the alcohol was added at one time, and the mixture was filtered after mixing. Analysis of 10 samples of various types of cheese resulted in data identical to that obtained by the Scharer rapid method. Quantitative data obtained by dialysis of the butanol filtrate and spectrophotometric measurement of the phenolphthalein compared favorably (r = 0.96) with data obtained by the Scharer spectrophotometric method on 13 samples of cheese.


Acid digestion, hydride evolution atomic absorption spectrophotometric method for determining arsenic and selenium in foods: collaborative study. tpart I.

The hydride evolution atomic absorption spectrophotometric (AAS) method for determining As and Se in foods developed and evaluated by the Food and Drug Administration and Agriculture Canada laboratories was subjected to collaborative study, Twenty-four laboratories provided results for As and 23 provided results for Se levels in 13 different samples consisting of tuna, swordfish, flounder, oyster, liver, flour, skim milk poweder, spinach, kale, and apple containing natural levels of As and Se in the ranges 0-15,000 and 0-4000 ng/g, respectively. Reference materials formed a substantial segment of samples, and a number of other laboratories using fluorometry, colorimetry, neutron activation, spark source mass spectrometry, and graphite furnace AAS provided confirmative reference values for the remaining samples. A variety of hydride generation instruments were used, ranging from commercially available devices to semiautomated and fully automated custom-made instruments. Although the accuracy of the method was fairly good, between-laboratory and between-determination (hydride evolution AAS measurement) precisions were not favorable. The main advantage of the hydride AAS method is the rapidity of the determinative step.


Microtubular structures in a stable staphylococcal L-form.

Microtubular structures, which were demonstrated as straight, dense-walled cylinders attached to cell membranes, were found in a stable staphylococcal L-form grown in the absence of antibiotic.


Energy-dependent inactivation of citrate lyase in Enterobacter aerogenes.

Enterobacter aerogenes was grown in continous culture with ammonia as the growth-limiting substrate, and changes in citrate lyase and citrate synthase activities were monitored after growth shifts from anaerobic growth on citrate to aerobic growth on citrate, aerobic growth on glucose, anaerobic growth on glucose, and anaerobic growth on glucose plus nitrate. Citrate lyase was inactivated during aerobic growth on glucose and during anaerobic growth with glucose plus nitrate. Inactivation did not occur during anaerobic growth on glucose, and as a result of the simultaneous presence of citrate lyase and citrate synthase, growth difficulties were observed. Citrate lyase inactivation consisted of deacetylation of the enzyme. The corresponding deacetylase could not be demonstrated in cell extracts, and it is concluded that, as in a number of other inactivations, electron transport to oxygen or nitrate was required for inactivation.


Phycocyanin synthesis and degradation in the blue-green bacterium Anacystis nidulans.

Cellular content and rates of synthesis of the apoprotein subunits of phycocyanin in Anacystis nidulans cultures undergoing, and recovering from, nitrate starvation were measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total and immunoprecipitable soluble proteins. Results indicated that (i) nitrate starvation provokes coordinate degradation of apoprotein subunits: (ii) de novo synthesis of these subunits is selectively depressed during starvation; (iii) nitrate restoration provokes coordinate increases in the rates of synthesis of these subunits, although maximal rates are not achieved for 6 to 10 h after readdition of nitrate; and (iv) illumination affects both relative and absolute rates of apoprotein formation.


Plasmid content and tumor initiation complementation by Agrobacterium tumefaciens IIBNV6.

Avirulent strains IIBNV6 and NT1, derived from virulent strains of Agrobacterium tumefaciens, were tested for their ability to enhance tumor initiation (complement) on coinoculation with tumorigenic strains. Strain NT1, cured of the Agrobacterium virulence plasmid, failed to complement when inoculated with its virulent parental strain or with other virulent strains. Strain IIBNV6, however, complemented with all virulent strains tested. Attachment to host wound sites by both strain IIBNV6 and the virulent strain was essential for this effect. Inoculation of the tumorigenic strain at different times on leaves previously inoculated with IIBNV6 showed that the capacity to complement is lost during the period between 4 and 8 h after IIBNV6 inoculation. The rate of tumor appearance obtained with an inoculum containing IIBNV6 and a virulent auxotrophic strain was characteristic of the appearance rate obtained with prototrophic bacteria. Evidence is summarized which suggests that strain IIBNV6 can induce tumors when supplied with a substance produced or induced by a virulent bacterium at a separate site. A deoxyribonucleic acid plasmid about 40% the size of the Agrobacterium virulence plasmid was obtained from strain IIBNV6. We propose that this plasmid accounts for the ability of strain IIBNV6 to complement and that it contains part of the genetic information necessary for tumor initiation.


Donor strains of the soft-rot bacterium Erwinia chrysanthemi and conjugational transfer of the pectolytic capacity.

Donor strains of Erwinia chrysanthemi ICPB EC16, a member of the soft-rot (pectolytic) section of the enterobacterial genus Erwinia, were obtained by chromosomal integration of an F'lac(+) plasmid originating from Escherichia coli. These stable donor strains, selected from an unstable F'lac(+) heterogenote by repeated platings of single Lac(+) colonies on lactose minimal agar, do not segregate (as does the parent F'lac(+) heterogenote) into Lac(-) or F(-) clones, in either the presence or absence of acridine orange. One representative donor strain (from the 12 that have been selected) has been examined in more detail; it can transfer ade(+), gal(+), gtu(+) (utilization of galacturonate), his(+), lac(+), leu(+), lys(+), mcu(+) (multiple carbohydrate utilization), pat(+) (production of polygalacturonic acid trans-eliminase), thr(+), and trp(+) in a polarized manner to appropriate recipient strains of E. chrysanthemi; the frequencies of ade(+), leu(+), and thr(+) transfer were higher than those of the other markers tested to date. This donor strain transfers lac(+) genes during a 6-h mating on membranes; most of the Lac(+) recombinants are donors of chromosomal markers. The kinetics of entry as well as the frequencies of transfer of chromosomal markers indicate that thr(+) and leu(+) enter the recipient as proximal markers and that lac(+) enters as a distal marker. Analysis of the recombinants demonstrates close linkage between thr and leu, ade and thr, his and pat, and his and trp loci. The results suggest that the integration of F'lac(+) into the chromosome of E. chrysanthemi has occurred at a region adjacent to the leu-thr loci, and that the chromosome is transferred in the following sequence: origin----leu--thr--ade--lys--mcu--pat--his--trp--gal--gtu--lac--F. Plant-tissue maceration occurs in Pat(+) recombinants and not in Pat(-) recombinants, even though both form another pectolytic enzyme, hydrolytic polygalacturonase. This genetic evidence supports the idea that the E. chrysanthemi polygalacturonic acid trans-eliminase plays an essential role in bringing about plant-tissue maceration.


Host-dependent, thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae.

The thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae, was studied. pJY5 consisted of 61 million daltons of covalently closed circular (CCC) deoxyribonucleic acid (DNA) with a buoyant density of 1.714 g/cm3 (55 mol % guanine plus cytosine). In Escherichia coli, this plasmid replicated stringently at 32 degrees C, but ceased its CCC DNA replication after a short incubation at 42 degrees C, resulting in production of R- segregants. The thermosensitive replication of pJY5 was not overcome by the coexistence of non-thermosensitive R plasmids. The plasmid manifested an inhibitory effect on host bacterial cell growth at 42 degrees C, although the effect was less prominent than that of R plasmids belonging to the T-incompatibility group, Rts1, R401, and R402. When the pJY5 plasmid was transferred into E. cloacae, however, no R- segregants were detected at any culture temperature, even 42 degrees C. Alkaline sucrose gradient analysis revealed that a significant amount of pJY5 CCC DNA was synthesized in E. cloacae at the high temperature but not in E. coli. Furthermore, the growth-inhibitory effect of pJY5 on hosts at 42 degrees C was not observed in E. cloacae. On the other hand, Rts1 and R401 were found to be thermosensitive in E. cloacae as well as in E. coli.


Novel bacterium infecting an African snail.

Nodules found in the superficial tissues of laboratory-maintained snails (Bulinus jousseaumei) contained a bacterium of two forms. This nonmotile microorganism occurred in intracellular packets as a simple gram-negative rod that appeared to undergo intrapacket transition to a cephalotrichous form. The latter is characterized by a "head" from which emerge long, thick, rigid, flagella-like, helically constituted filamentous organelles with a core and an outer component that is not an extension of the bacterial envelope. Neither form was successfully cultured, but clean snails derived from eggs removed before hatching developed nodules within 1 to 3 months of exposure to infected snails. The infectivity was specific for the host snail, and no transmission occurred to snails of 5 other genera tested. The presence of nodules did not interfere with longevity or reproduction of infected snails. Details of infectivity, transition, and taxonomic position of the bacterium remain to be explored, but it is reported because of unique morphological and ultrastructural features not previously known.


Acetylcholine receptor of a myogenic cell line, L6.

The biochemical characters of acetylcholine receptor (AChR) in a myogenic cell line, L6, are as follows. 1. AChR extracted with Triton X-100 has a molecular weight of 5.5 X 10(5) determined by gel-chromatography, an apparent S value of 9.7 determined by sucrose density gradient centrifugation, and an isoelectric point of 5.3. AChR is shown to interact with concanavalin A. 2. The association rate constant of the binding reaction between AChR and alpha-bungarotoxin (alpha-BGT) labelled with 125I is 1 X 10(6) M-1 with S-1 at 35 degrees C. Preincubation with d-tubocurarine blocks the binding. The half-life of the AChR-toxin complex exceeds 180 h.


Translocation reaction promoted by polypeptide chain elongation factor-2 from pig liver.

Translocation of peptidyl-tRNA from the ribosomal A- to the P-site in the eukaryotic system was extensively investigated using a model system, in which translocation of Phe-tRNA from the A- to the P-site was examined by using the puromycin reaction, and the following results were obtained. 1) The puromycin reaction but not the translocation reaction proceeded at 0 degrees C. Since the latter could be demonstrated at 30 degrees C, it was possible to analyze translocation per se separately from the puromycin reaction. 2) Translocation was completely dependent on the elongation factor-2 (EF-2) and required the presence of GTP, which could be replaced by GMP-P(NH)P provided that the stoichiometric amount of EF-2 with respect to the amount or ribosomes was present. It was further demonstrated that translocation observed in the presence of GTP was catalytic, while that in the presence of GMP-P(NH)P was stoichiometric, indicating that hydrolysis of GTP was required for the catalytic reutilization of EF-2. 3) Translocation promoted by EF-2 in the presence of GMP-P(NH)P could be reversed, which suggests that hydrolysis of GTP is indispensable of the translocation reaction to proceed  catalytically and unidirectionally forward.


Effects of gastrointestinal and related hormones on glycogenolysis and gluconeogenesis in cultured liver cells.

When isolated rat liver cells were incubated for 15 min in the presence of vasoactive intestinal peptide, secretin, gastrin, caerulein or glucagon at concentrations ranging from 0.2 microgram to 2 microgram per ml, glycogenolysis was stimulated. Among the gastrointestinal hormones or peptides tested, vasoactive intestinal peptide had the highest stimulatory activity. However, somatostatin was inhibitory for liver glycogenolysis. Combination experiments showed that somatostatin also inhibited the stimulatory effects of vasoactive intestinal peptide and secretin, but not that of glucagon, while glucagon and vasoactive intestinal peptide, or glucagon and secretin showed additive effects on glycogenolysis, but secretin and vasoactive intestinal peptide did not. The results suffest that the receptor site of glucagon is different from those of secretin and vasoactive intestinal peptide. Slight but significant stimulation of gluconeogenesis was also observed by vasoactive intestinal peptide and secretin. The evidence presented in this paper indicates that the so-called enterohepatic axis, in which a part of serum glucose levels is regulated directly by gastrointestinal hormones, exists and that the axis is inhibited by somatostatin.


Metabolism of putrescine in neuroblastoma and glioma cells during culture.

The metabolism of putrescine in neuroblastoma and glioma cells was analyzed during culture. In the former cells the formation of gamma-aminobutyric acid from putrescine was low and constant during the logarithmic phase and increased dramatically during the stationary phase or in cultures with low serum concentrations, while in the latter cells it was low and constant throughout culture. The formation of spermidine from putrescine in both cell lines was active at days 1-2 of culture and decreased gradually during culture. These findings indicate that spermidine formation is closely related to proliferation of the cells, and gamma-aminobutyric acid formation to differentiation or maturation of the neuroblastoma cells.


A circular dichroic spectral study on disulfide-reduced pancreatic ribonuclease A and its renaturation to the active enzyme.

Disulfide-reduced RNase A, which could be reoxidized to give the native enzyme, was shown to have a CD spectrum quite different from that of the native enzyme or a random coil. Disulfide-reduced and fully cysteine-S-carboxamidomethylated RNase A because the derivative was stable and gave a spectrum identical to that of reduced RNase A. Curve-fitting analyses showed the presence of 14% alpha-helix and 25% beta-structure in this open chain derivative of RNase A. The time dependence of CD spectra during the oxidative renaturation of reduced RNase A was analyzed and changes in alpha-helical and beta-structure contents during the reaction were estimated. It was shown that the change in the content of beta-structure was slower than that of alpha-helix content and approximately paralleled the appearance of the enzymatic activity.


Purification and some properties of rabbit C1 inactivator.

C1 inactivator (C1 INA) was highly purified from rabbit serum. C1 INA thus purified was a single polypeptide chain with a molecular weight of 105,000 or 140,000, as estimated by SDS-polyacrylamide gel electrophoresis or gel filtration on Sephadex G-200, respectively. It inhibited rabbit and also human C1, when the C1 activities were measured in terms of hydrolyses of acetylglycyl-L-lysine methylester, N-alpha-acetyl-L-arginine methylester and N-alpha-acetyl-L-tyrosine ethylester. These properties showed that rabbit C1 INA bears a marked structural similarity to human C1 INA. Furthermore, rabbit C1 INA was capable of inhibiting similarly both rabbit C1s and its active fragment lacking a half of the H chain of C1s, indicating that deletion of a half of the H chain did not affect the susceptibility of C1s to the inhibitory activity of C1 INA.


The effect of formycin on the processing of transfer RNA precursors in the posterior silk gland of Bombyx mori.

Formycin, an adenosine analog, was used in studies of RNA synthesis by silk glands of silkworms in organ culture. Formycin has been shown to inhibit preferentially the incorporation of [3H]uridine into low molecular weight RNA in the cytoplasm. When silk glands were pulse-chase labeled with [3H]uridine in the presence of formycin, the synthesis of 4.5S tRNA precursors was not affected, as determined by gel electrophoresis. However, the accumulation of mature 4S tRNA was greatly reduced in the formycin-treated system, although the precursors synthesized disappeared from the 4.5S region on chase at the same rate as in the control system. [14C]Formycin was incorporated into the 4.5S precursors but not into 4S tRNA. These findings suggest that the inhibition of tRNA synthesis by formycin is due to the existence of some degradation mechanisms of formycin-containing 4.5S precursor RNA's and the failure to process them into normal 4S tRNA.


Light-induced formation of the 410 nm intermediate from reconstituted bacteriorhodopsin.

Light-induced formation of the 410 nm intermediate was investigated with reconstituted bacteriorhodopsin. Only bacteriorhodopsin with all-trans retinal formed the 410 nm intermediate in response to light excitation. In equilibrium in the dark, both native and reconstituted bacteriorhodopsin contained about equal amounts of the all-trans and 13-cis forms over a wide range of temperature.


Thermotropic transition in the states of proteins in sarcoplasmic reticulum vesicles.

Interaction between protein and phospholipid molecules in sarcoplasmic reticulum (SR) was studied by measurement of the conformational fluctuation of the protein using the kinetics of the hydrogen-deuterium exchange reaction. It was revealed that the state of SR ATPase undergoes a phase transition at about 18 degrees C with boundary lipids, corresponding to a discrete change in the activation energy of the Ca2+, Mg2+-ATPase reaction.


The stoichiometry of inhibition and binding of a protein proteinase inhibitor from Streptomyces (Streptomyces subtilisin inhibitor) against subtilisin BPN'1.

The stoichiometry of inhibition and binding of Streptomyces subtilisin inhibitor, a protein proteinase inhibitor produced by Streptomyces albogriseolus S-3253 (Sato, S. and Murao, S. (1973) Agric. Biol. Chem. 37, 1067) against subtilison BPN' [EC 3.4.21.14] was studied. The inhibition of the hydrolysis of p-nitrophenyl acetate by subtilisin BPN' was measured both at the pre-steady state with a stopped-flow apparatus and at the steady state. The stopped-flow study demonstrated the disappearance of the initial burst of the enzyme reaction. The ultraviolet absorption difference spectra observed on mixing the inhibitor and the enzyme suggested changes in the environment of tryptophyl and tyrosyl residues in the proteins. Titration by means either of the degree of inhibition at the steady state or of the magnitude of the ultraviolet difference absorbance revealed that the inhibitor (dimer, MW: 23,000) bound and inhibited two molecules of subtilisin BPN'. The inhibitor constant, Ki, against subtilisin BPN' was estimated to be less than 10(-9)M at pH 8.50. The type of inhibition of this inhibitor is discussed.


Amino acid sequence of an active fragment of potato proteinase inhibitor IIb.

The amino acid sequence of an active fragment of potato proteinase inhibitor IIb was determined by the Edman degradation procedure and the carboxypeptidase technique. Analyses were carried out on peptides derived from the reduced and carboxymethylated active fragment by digestion with trypsin and chymotrypsin. The active fragment consisted of a single polypeptide chain of 40 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by trypsin and subtilisin at pH 6.3--6.4 yielded 3 cystine-containing peptides, and sequence analyses of these peptides revealed that the three disulfide linkages are located between Cys(2) and Cys(16), Cys(6) and Cys(28), and Cys(12) and Cys(38). The reactive site peptide bond of inhibitor IIb, a Lys-Ser bond, is located between positions 26 and 27. The overall sequence of the active fragment is compared with that of an active fragment of inhibitor IIa and the "structure-specificity" relationships of both are discussed. Correlation of the fragments to a naturally-occurring low molecular weight inhibitor is also discussed.


Role of the carbohydrate moiety in determining the survival of interferon in the circulation.

The role of the carbohydrate moiety in influencing the survival time of human lymphoblastoid interferon in the rat circulation was monitored at varying time intervals after intravenous injection. The serum survival time of native interferon was drastically reduced by prior enzymatic cleavage of the terminal sialic acid residues to yield the galactose-terminal product, asialo-interferon. This phenomenon was completely reversed by further extensive digestion with a mixture of glycosidases which effectively removed over 85% of the total carbohydrate residues. These results suggest that the intact carbohydrate structure is neither essential for normal circulation time in vivo nor for biologic activity in vitro.


Biotin carboxylations--concerted or not concerted? That is the question!

When beta-fluoropropionyl coenzyme A is used as substrate, propionyl-CoA carboxylase catalyzes the formation of ADP and the elimination of fluoride ion. No F- release occurs in the absence of ATP or in the presence of avidin. ADP formation occurs as rapidly as in the presence of propionyl-CoA, but the rate of F- release is 6 times that of ADP formation. The rate of F- release is indicative of the minimal rate of abstraction of the alpha proton, and the rate of ADP formation is equivalent to the rate of formation of biotin-CO2. The results, therefore, show that hydrogen abstraction can occur without concomitant CO2 transfer from biotin-CO2 to the substrate. Therefore, the concerted mechanism which has been proposed for this, and other biotin enzymes, is not applicable when propionyl-CoA carboxylase acts on beta-fluoropropionyl-CoA. We believe the concerted mechanism is also not involved in the carboxylation of the normal substrate, propionyl-CoA.


Purification and reconstitution of the adipocyte plasma membrane D-glucose transport system.

Rat adipocyte plasma membranes have previously been shown to retain stereospecific transport activity for D-glucose following extraction of extrinsic proteins with dimethylmaleic anhydride (Shanahan, M. F., and Czech, M. P. (1977) J. Biol. Chem. 252, 6554-6561). When these extracted plasma membranes were incubated in 2% sodium cholate and centrifuged, the resultant supernatant contained only one major glycoprotein fraction of 94,000 daltons, as determined by dodecyl sulfate-polyacrylamide gel electrophoresis. This protein fraction was combined with cholate-dispersed, exogenous phospholipids. The detergent was removed by gel filtration and vesicles composed of phospholipid and membrane protein were formed which exhibited preferential, time-dependent uptake of D- versus L-glucose when measured by a rapid filtration method. In addition, D-glucose uptake was inhibited by cytochalasin B, phlorizin, phloretin, and dipyridamole. These results suggest the direct involvement of the 94,000-dalton glycoprotein fraction in fat cell hexose transport.


Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by xylulose 1,5-bisphosphate.

Xylulose 1,5-bisphosphate is a potent inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase. The enzyme was inhibited 50% at a xylulose 1,5-bisphosphate concentration of 0.56 micrometer and an enzyme concentration of 0.14 micrometer. When both ribulose 1,5-bisphosphate and xylulose 1,5-bisphosphate were added simultaneously to the enzyme, this inhibition appeared to be competitive. A preincubation of 20 to 30 min was needed for maximum inhibition. The inhibitory effect of this compound is probably exerted through its binding at the ribulose 1,5-bisphosphate active site, since both the carboxylase and oxygenase activities were inhibited similarly.


Chloroplast biogenesis. Net synthesis of protochlorophyllide from protoporphyrin IX by developing chloroplasts.

Developing chloroplasts were isolated from greening Cucumis cotyledons in a co-factor-enriched medium and were incubated in the dark with 14C-labeled and unlabeled protoporphyrin IX. The metabolic pools between protoporphyrin and protochlorophyllide were monitored spectrofluorometrically. The incorporation of the 14C label into protochlorophyllide was also determined. It was shown that protoporphyrin IX, a postulated intermediate of the chlorophyll biosynthetic pathway, was convertible into protochlorophyllide. Since protochlorophyllide is the immediate precursor of chlorophyll a it was concluded that protoporphyrin IX was indeed an intermediate of the chlorophyll biosynthetic pathway.


Messenger RNA for renal phosphoenolpyruvate carboxykinase (GTP). Its translation in a heterologous cell-free system and its regulation by glucocorticoids and by changes in acid-base balance.

A translational assay was used to measure the level of mRNA coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in the rat kidney in various conditions in which the enzyme is induced. RNA extracted from whole kidneys was chromatographed on oligo(dT)-cellulose to select poly(A)-containing RNA. This crude mRNA preparation was able to stimulate amino acid incorporation into protein in a cell-free system containing an extract of wheat germ. Phosphoenolpyruvate carboxykinase could be detected among the polypeptides synthesized and quantitated by immunoprecipitation with a monospecific antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of enzyme synthesized was proportional to the quantity of RNA added. The level of mRNA coding for phosphoenolpyruvate carboxykinase is increased 3-fold 6 h after triamcinolone injection. Translatable enzyme mRNA also increases 3-fold within 6 h of the onset of metabolic acidosis caused by an ammonium chloride load. In both cases, the increase in functional mRNA is commensurate with the stimulation of enzyme synthesis measured in vivo. Glucocorticoid administration and acidosis cause additive increases in the level of translatable phosphoenolpyruvate carboxykinase mRNA. The inductive effect of acidosis is preserved in the absence of the adrenals, hypophysis, thyroid, and parathyroids.


Residual esterase activity of lima bean inhibitor-binding anhydrochymotrypsin preparations.

A search for the source of the residual esterase activity of crude lima bean protease inhibitor-binding anhydrochymotrypsin preparations was undertaken. The preparations were found to contain about 40% of protein that possesses 1% (kc/Km) to 12% (kc) of the esterase activity of alpha-chymotrypsin. The active protein was isolated by affinity chromatography on soybean trypsin inhibitor-Sepharose. It appears to be an anhydroenzyme or a mixture of a limited number of anhydroenzymes in which a serine other than the catalytically essential serine-195 of the native enzyme has been converted to dehydroalanine.


Importance of initiation factor preparations in the translation of reovirus and globin mRNAs lacking a 5'-terminal 7-methylguanosine.

Reovirus and globin mRNAs which lack a 5'-terminal 7-methylguanosine are translated in a fractionated cell-free protein-synthesizing system. Efficient translation occurs only at optimal concentrations of reticulocyte initiation factor preparations and does not occur at optimal concentrations of ascites initiation factor preparations or at suboptimal concentrations of reticulocyte initiation factor preparations. The translation of "uncapped" mRNA in vitro, therefore, appears to be related to the efficiency of initiation of protein synthesis. At optimal concentrations of reticulocyte initiation factors, mRNA containing a 5'-terminal 7-methylguanosine is preferentially translated in the presence of mRNA which lacks a "cap." These results indicate that the 5'-terminal 7-methylguanosine on mRNA has a facilitatory rather than obligatory role in translation.


Priming of deoxyribonucleic acid synthesis on phage fd and phiX174 templates by oligoribonucleotides contaminating nucleoside 5'-triphosphates.

Commercial preparations of guanosine 5'-triphosphate and deoxyguanosine 5'-triphosphate are contaminated with oligoribonucleotides 4 to 6 residues in length. The oligoribonucleotides can be separated from the nucleoside 5'-triphosphates by chromatography on DEAE-Sephadex A-25 or by gel filtration through Sephadex G-25. The oligoribonucleotides are effective primers for the DNA polymerase of bacteriophage T7 on the single-stranded circular DNAs of phage fd and phiX174; they are covalently attached to the 5' terminus of the newly synthesized DNA. The priming activity is specific; the oligoribonucleotides do not serve as primers for DNA polymerase I of Escherichia coli or for the DNA polymerase induced by phage T4.


Dissociation equilibrium constant of beta nerve growth factor.

The beta nerve growth factor (NGF) isolated from the high molecular weight 7 S NGF complex from mouse submaxillary gland is a protein of molecular weight 26,500 which contains two noncovalently associated peptide chains. A variety of techniques were employed to determine whether the NGF dimer dissociates at the low concentrations at which it is biologically active. No dissociation was detected in 48 h at low NGF concentrations using the techniques and minimum concentrations which follow: sedimentation equilibrium of NGF at 200 nM and of 125I-NGF at 2.5 nM; gel filtration chromatography of NGF at 5 nM, of 125I-NGF at 5 pM, and of 125I-NGF from which COOH-terminal arginyl residues, or NH2-terminal octapeptide had been cleaved, at 10 pM; and sucrose gradient centrifugation of 125I-NGF at 10 pM. Dissociation of heavily succinylated 125I-NGF was detected by gel filtration chromatography, and the equilibrium dissociation constant for this material is estimated to be 10 pM.


Schiff base adducts of hemoglobin. Modifications that inhibit erythrocyte sickling.

Normal and sickle erythrocytes were exposed in vitro to millimolar concentrations of 31 different carbonyl compounds. Schiff base (imine) linkages were formed with amino groups of intracellular hemoglobin. Adducts were isolated by gel electrofocusing and could be dissociated by dialysis. Aromatic aldehydes proved more reactive than aliphatic aldehydes, and ketones were unreactive. The influence of various ring substituents on the reactivity of aromatic aldehydes was found to conform closely to traditional concepts regarding electronic and steric effects. Several of the aromatic aldehydes were shown to markedly increase the oxygen affinity of hemoglobins A and S. In particular, 2,4-dihydroxybenzaldehyde and o-vanillin, at concentrations of 5 mM, produced 2- to 3-fold reductions in the P50 (partial pressure of oxygen at half-saturation) of sickle hemoglobin in whole blood. Since low degrees of oxygen saturation promote erythrocyte sickling, compounds of this type significantly inhibit sickling at reduced partial pressures of oxygen.


A novel ketone monooxygenase from Pseudomonas cepacia. Purification and properties.

A ketone monooxygenase was purified from cells of Pseudomonas cepacia grown on 2-tridecanone as sole carbon source. Enzyme stability is maintained by the addition of ethanol, EDTA, and dithiothreitol. Stoichiometric studies show that for 1 mol of undecyl acetate formed, 1 mol of O2 is consumed and 1 mol of NADPH is oxidized. The monooxygenase, purified to homogeneity, has a molecular weight of approximately 123,000 and consists of two equal subunits with molecular weights of 55,000. The enzyme contains FAD and exhibits absorption maxima at 375 and 488 nm. Enzyme activity is inhibited by thiol-active reagents and the inhibition by the cations, cadmium, copper, zinc, and mercury, is reversed by dithiothreitol, indicating the presence of essential sulfhydryl groups. Substrate specificity tests show that acetate esters are formed from methyl ketones from C-7 through C-14. The oxygenase is also active on isomers of 2-tridecanone forming esters from 3- through 7-tridecanone. With 6-tridecanone, two esters are formed, heptyl hexanoate and pentyl octanoate, indicating that oxygen is inserted on either side of the carbonyl group. In addition, the enzyme catalyzes the lactonization of the cyclic ketone, cyclopentanone, with the formation of 5-valerolactone.


Subunit structure of the acetylcholine receptor from denervated rat skeletal muscle.

Acetylcholine receptor from denervated rat leg muscle was purified 48,000-fold by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose. A control preparation containing only contaminants was made by performing a parallel purification in which alpha-bungarotoxin was added to the preparation prior to the cobrotoxin/Sepharose step. Comparison of the receptor was greater than 90% pure. Examination of the purified receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two major polypeptide chains with apparent weights of 45,000 and 51,000, along with minor components of 49,000, 56,000, 62,000, and 110,000. Polypeptides of the same molecular weight were found when purified acetylcholine receptor was radioiodinated and further purified by sucrose gradient sedimentation. Analysis of receptor purified from denervated muscles that had been incubated with [35S]methionine in organ culture showed that all of the identified polypeptide chains were radioactive, indicating that they had been synthesized de novo after denervation.


Studies on the metabolism of the renal glomerular basement membrane. Turnover measurements in the rat with the use of radiolabeled amino acids.

The synthesis and degradation of the renal glomerular basement membrane have been investigated in the rat with the aid of injected tracer doses of various tritiated amino acids including L-proline, L-lysine, L-phenylalanine, L-leucine, and glycine. After incorporation into the basement membrane the turnover times of these amino acid constituents, as well as of hydroxyproline and hydroxylysine, were determined from the decay in their specific radioactivities. The loss of radioactivity from the proline and hydroxyproline of the glomerular basement membrane was as slow as that from tail tendon collagen in the same animals (turnover time of more than 100 days) and contrasted with the radiodecay of the proline in other glomerular proteins (turnover time of 9 days). The glycine of the membrane similarly turned over at this very slow rate. The total replacement times of the leucine, hydroxylysine, lysine, and phenylalanine constituents of the basement membrane were determined to be somewhat shorter with a range of 65 to 23 days. The nonuniform turnover of these membrane components may be a function of the polydispersity of the peptide subunits of the basement membrane observed by polyacrylamide gel electrophoresis and may reflect a subtle morphological and functional heterogeneity. The amino acid and saccharide composition of the rat glomerular basement membrane used in these studies are also reported.


alpha-Bungarotoxin binding and cholinergic receptor function on a rat sympathetic nerve line.

A clonal rat sympathetic nerve cell line, PC12, binds iodinated alpha-bungarotoxin. The binding is saturable and is inhibited by a variety of cholinergic agonists and antagonists. The pseudo-first order rate constant for binding is 2.1 x 10(5) M-1 S-1 at 22 degrees. In contrast to the alpha-bungarotoxin binding reaction found with muscle, the binding to PC12 is reversible with a first order rate constant of 4.9 x 10(-5) S-1 at 37 degrees. Toxin binds to an integral membrane component which sediments in sucrose gradients containing Triton X-100 with an apparent sedimentation coefficient of 10.5 S. The nicotinic acetylcholine receptor of PC12 was assayed by determining the agonist-induced increase in permeability to sodium ions. Using this assay, we determined the apparent binding constants for a variety of cholinergic ligands and found no correlation between their ability to affect cholinergic function and to inhibit binding of alpha-bungarotoxin. Therefore, the site at which cholinergic ligands affect receptor function is different than the site at which cholinergic ligands inhibit toxin binding.


Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle.

Two populations of mitochondria were observed upon ultrastructural examination of cardiac muscle tissue, one located directly beneath the sarcolemma (subsarcolemmal mitochondria) and another between the myofibrils (interfibrillar mitochondria). Subsarcolemmal mitochondria were released by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by nagarse digestion of the remaining tissue. These results were supported by electron microscopy of Polytron-treated heart tissue showing rupture and loss of sarcolemma with release of the underlying mitochondria but with retention of intact mitochondria between the myofibrils. Electron microscopy of the isolated mitochondria indicated that both mitochondrial types maintained their structural integrity throughout the isolation procedure. Specific activities of succinate dehydrogenase and citrate synthase were higher in the interfibrillar mitochondria as compared to the subsarcolemmal mitochondria, while those of carnitine palmitoyltransferase and alpha-glycerophosphate dehydrogenase were nearly the same in both. Interfibrillar mitochondria oxidized all substrates tested approximately 1.5 times faster than did the subsarcolemmal mitochondria. Thus the two mitochondrial types differed not only in their respective locations in the cell, but also in certain biochemical properties.


A sensitive endonuclease probe for lesions in deoxyribonucleic acid helix structure produced by carcinogenic or mutagenic agents.

The highly single strand-specific extracellular nuclease of Pseudomonas BAL 31 is shown to cleave non-supercoiled closed circular duplex PM2 bacteriophage DNA containing regions of altered helix structure produced in vitro by irradiation with ultraviolet light or by treatment with the carcinogen, N-acetoxy-N-2-acetylaminofluorene. Untreated samples of this DNA are affected very little by the nuclease. The unwinding of the DNA helix associated with the above treatment renders the closed circular DNA positively supercoiled compared to untreated samples. The extent of unwinding can be accurately measured and correlated with the average number of lesions per molecule of DNA by monitoring the alterations of the electrophoretic patterns, relative to those observed for untreated DNA, of such DNA in agarose gels. Interstrand cross-links and mismatched base pairs produced by treatment of non-supercoilded circular duplex DNA with the mutagen, nitrous acid, do not detectably unwind the DNA helix. The nitrous acid-treated DNA provides substrates for cleavage by the Pseudomonas nuclease which are likely to be the interstrand cross-links rather than the mismatched base pairs. Use of the Pseudomonas nuclease in conjunction with agarose gel electrophoresis can provide a powerful method for the detection of damage in duplex DNA such as that introduced by carcinogenic and mutagenic agents.


Prevention of NH2-terminal acetylation of proteins synthesized in cell-free systems.

The NH2 terminus of ovalbumin is acetylated in cell-free protein-synthesizing systems as it is in vivo. The acetyl group is derived from acetyl-CoA and it is incorporated during translation. Acetylation can be prevented by metabolizing the available acetyl-CoA to citrate with the addition of citrate synthase and oxalacetate to the translation system. The NH2 terminus of ovalbumin synthesized under these conditions can be sequenced by automated Edman degradation. This procedure has also been applied to the sequencing of Pr 76gag, the viral core protein precursor synthesized from 35 S Rous sarcoma virus RNA.


Can an antibody-combining site be mimicked synthetically? The possible surface simulation synthesis of two antibody-combining sites complementary to two antigenic sites of lysozyme.

Our recent determination of the complete antigenic structure of native lysozyme was made possible by our introduction of the "surface simulation" synthetic concept. The remarkable success afforded by this strategy in the synthetic construction of antigenic sites prompted us to investigate whether it can be applied to mimic antibody-combining sites. Two peptides, designed to be complementary to antigenic sites 2 and 3 of lysozyme, were synthesized and their immunochemistry was studied. Each of the two complementary peptides exhibited an appreciable inhibitory activity towards the reaction of lysozyme with its antisera. Peptide immunoadsorbents bound only lysozyme and not antibody or myoglobin. Neither of the two peptides had any immunochemical activity in the myoglobin or bovine serum albumin immune systems. Furthermore, it was shown that three control synthetic peptides of myoglobin, of similar charge but different sequence, had no inhibitory effect on the lysozyme immune reaction. The evidence indicates that the antibody-combining sites sgainst antigenic sites 2 and 3 of native lysozyme were successfully mimicked synthetically, at least in terms of binding function.


Production, purification, and characterization of excitability-inducing molecule.

Excitability-inducing molecule (EIM) is a high molecular weight polymeric protein. It is a channel-forming ionophore which can induce action potential in lipid bilayers and lyse red blood cells. It is also a potent mitogen for mouse B lymphocytes. EIM is produced and secreted into a chemically defined medium during the early growth period by Enterobacter cloacae ATCC 961. It is now 6000-fold purified as compared to the original egg white EIM. Production of the active material requires calcium ion. EIM contains 10 to 20% lipid by weight which is primarily composed of fatty acids, with an unsaturated 18-carbon chain. Amino acid analysis shows 18 common amino acids. The maximum specific activity of 18 common amino acids. The maximum specific activity of EIM is associated with the molecular weight of 3.6 x 10(5).


Inhibition of sterol biosynthesis in L cells and mouse liver cells by 15-oxygenated sterols.

Described herein are the chemical syntheses of 5alpha-cholest-8(14-en-3beta-ol-15-one, 14alpha-methyl-5alpha-cholest-7-en-3beta-ol-15-one, 3beta-methoxy-14alpha-methyl-5alpha-cholest-7-en-15-one, 3beta-methoxy-14alpha-methyl-5alpha-cholest-7-en-15beta-ol, 3beta-methoxy-14alpha-methyl-15alpha-ol, and 5alpha-cholest-8(14)-en-3beta, 7xi,15xi-triol. The effects of these compounds and of 5alpha-cholest-8(14)-en-3beta,15beta-diol, 5alpha-cholest-8(14)-en-3beta,15alpha-diol, 5alpha,14beta-cholest-7-en-3beta,15beta-diol, 5alpha,14beta-cholest-7-en-3beta, 15alpha-diol, 14alpha-methyl-5alpha-cholest-7-en-3beta,15beta-diol, and 14alpha-methyl-5alpha-cholest-7-en-3beta,15alpha-diol on sterol synthesis in L cells and primary cultures of fetal mouse liver cells grown in serum-free media have been studied. With the exception of 3beta-methoxy-5alpha-cholest-7-en-15-one, all of the compounds were found to be potent inhibitors of sterol synthesis. With a few exceptions, the concentrations required to cause a 50% reduction in sterol synthesis were similar to those required to cause a 50% reduction in the level of HMG-CoA reductase activity.


Studies of low molecular weight RNA from cells infected with adenovirus 2. II. Heterogeneity at the 5' end of VA-RNA I.

The 5'-terminal sequences of VA-RNA I produced in cells infected with adenovirus 2 were analyzed. The RNA contained a mixture of mono-, di-, and triphosphate ends. The largest part of the RNA has 5'-terminal mono-, di-, or triphosphoguanylic acid, but a portion of the RNA has a 5'-terminal mono-, di-, or triphosphoadenylic acid. An RNA with 5'-terminal adenylic acid was purified and gave the same oligonucleotide map as the major form of VA-RNA I, except for the 5'-terminal products. We therefore conclude that transcription of VA-RNA I can initiate at two different nearby sites.


Evolutionary microdivergence of chick and rat liver ribosomal proteins.

We have set out to establish a nomenclature for ribosomal proteins which transcends, as far as is possible, the species from which the ribosomes were isolated. Initally, we identified individual proteins of rat liver by two-dimensional polyacrylamide gel electrophoresis and comparison with the classification system established by others (Sherton, C.C., and Wool, I.G. (1972) J. Biol. Chem. 247, 4460-4467). This was possible with minor modifications. We carried out a detailed comparison of the rat liver proteins with those of chick liver; parallel separate and mixed preparations of ribosomal subunits from the two species were made. Spots on the two-dimensional gels were carefully scrutinized for possible species differences. Proteins which gave congruent spots for the separate and the mixed preparations were tentatively assumed to be identical between the two species. For these proteins, a species-independent nomenclature was adopted. Contrary to earlier reports, a number of proteins were found which were not identical between the two species. These were assigned a species-specific nomenclature. In all, 7 proteins in the small subunit and 17 from the large subunit of the rat liver ribosome were found to be significantly different in the chick. The evolutionary implications of this are discussed.


Isolation of eukaryotic ribosomal proteins. Purification and characterization of the 40 S ribosomal subunit proteins Sa, Sc, S3a, S3b, S5', S9, S10, S11, S12, S14, S15, S15', S16, S17, S18, S19, S20, S21, S26, S27', and S29.

The proteins of the small subunit of rat liver ribosomes were separated into five main groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Twenty-one proteins (Sa, Sc, S3a, S3b, S5', S9, S10, S11, S12, S14, S15, S15', S16, S17, S18, S19, S20, S21, S26, S27', and S29) were isolated from three groups (A40, C40, and D40) by ion exchange chromatography on DEAE-cellulose, carboxymethylcellulose, and phosphocellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.1 to 11 mg. Six of the proteins (S5', S10, S11, S18, S19, and S27') had no detectable contamination; the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.


Conversion of fused hips to low friction arthroplasty.

Fifty-four hips converted to low friction arthroplasty between 1965 and 1975 have been reviewed one to eleven years after operation. In many cases malposition had led to degenerative changes in the opposite hip, the lumbar spine or the knee, often with severe loss of function due to pain. It was found that total replacement could give useful relief of pain and improved function, though the range of movement obtained was not as good as in primary replacement. An outstanding feature was the correction of inequality of leg length. In general, the results were much better in cases of ankylosis acquired in adult life than in cases of spontaneous fusion after sepsis in childhood. The most important complication was a single case of sciatic palsy.


Arthroplasty of the hip in ankylosing spondylitis.

In fifty-six patients with ankylosing spondylitis three types of arthroplasty had been performed in ninety-nine hips. Forty-one of the patients were men and fifteen were women, their average age at operation being forty-two years. Primary pseudarthrosis produced well-satisfied patients, but only a fair result in five hips, whereas cup arthroplasty resulted in a poor outcome for eight hips, all of which needed revision. Total replacement of eighty-six hips, however, led to 73 per cent being graded as good or excellent up to ten years later. The main complications were deep infection of five hips, para-articular ossification around nine hips (six leading to bony ankylosis), and fibrous ankylosis of six hips.


Systemic disturbance from Thompson's arthroplasty: a age-matched and sex-matched controlled retrospective survey.

The records of 107 patients with displaced subcaptial hip fractures treated by Thompson's femoral head replacements have been reviewed to determine the mortality and certain aspects of systemic morbidity. Comparison was made with a group, matched exactly for age and sex, in which intertrochanteric fractures were treated by nail-plate fixation. The patients treated by Thompson's arthroplasty were further subdivided into two groups: one in which methylacrylic cement was used, and one in which it was not used. We found no difference in the mortality of the patients undergoing replacement arthroplasty and nail-plate fixation. Similarly there was no difference in the incidence of cerebrovascular incidents and myocardial infarction after operation. There was a higher incidence of transient cardiac failure following Thompson's arthroplasty. Hypotension during the operation occurred irrespective of whether cement was used; nor was the use of cement associated with increased mortality. However, it seems that methylacrylic cement does contribute to the cardiac failure that may occur after Thompson's arthroplasty.


Anterior dislocation of the radial head in children: aetiology, natural history and management.

The results of operation for traumatic anterior dislocation of the head of the radius in eight children have been reviewed. We are satisfied on the basis of the results obtained and the outcome in one untreated patient that operative reduction is fully justified, provided that the annular ligament is reconstructed and internal fixation employed. We have not seen unilateral congenital dislocation and doubt its existence. Secondary subluxation of the distal radio-ulnar joint has been noted in an untreated patient.


Surgical management of traumatic non-infective tenosynovitis of the wrist extensors.

Traumatic tenosynovitis of the wrist extensors is a common and disabling condition associated with overuse. It has been found to be associated with hypertrophy of the bellies of abductor pollicis longus and extensor pollicis brevis where they overlie the radial extensor tendons in the forearm, compressing these tendons and their enveloping paratenons against the deep structures beneath. Simple surgical decompression of the sheath of these overlying muscles has been found to give quick relief of symptoms and to allow a more rapid return to strenuous work than the conservative treatments usually employed.


The cast-brace treatment of femoral shaft fractures.

Ninety-eight fractures of the shaft of the femur were seen in one unit over the two years 1974 and 1975, and the results have been assessed in sixty-nine. Of these, thirty-eight were treated by skeletal traction in a Thomas's splint followed by skin traction, and thirty-one by skeletal traction followed by a cast-brace. The technique of application is described in some detail. The average time for application of the cast-brace was six weeks after the injury, the time in hospital eight weeks and the time till removal fifteen weeks. The patients selected for a cast-brace were in hospital for just over half the time of the others and their fractures on average united more quickly, though with some trouble from angulation of fractures of the uppermost third of the shaft. It is concluded that when used with all the judgment and skill it demands, the cast-brace method is a great advance in conservative treatment.


Complete brachial plexus lesions: a ten-year follow-up of twenty cases.

Twenty patients with complete brachial plexus lesions were reviewed approximately nine and a half years after injury. Thirteen were amputees and seven had received no surgical treatment. Amputation did not alleviate pain and a prosthesis was frequently of no greater use of the patient than the useless limb it replaced: only two of the thirteen amputees were true prosthetic users and they both had dominant limb involvement, the rest adapting easily to being one-handed. Initial treatment should therefore be conservative, with intensive rehabilitation and retraining. It is recommended that amputation should not be considered until a year after injury and only if the flail limb causes repulsion, prevents sporting activities or if the patient has difficulty in converting to the non-dominant limb. In no instance should smputation be done for relief of pain.


The treatment of flexion contracture of the knee in myelomeningocele.

A follow-up study, on average four years after operation, of seventy-six cildren with myelomeningocele who had surgical treatment for flexion contracture of the knee, is reported. After a single operation sixty-eight knees (55 per cent) had a contracture of less than 20 degrees, which was the main criterion of success. About half of the cases of transplantation of the hamstrings to the femur, division or elongation of the hamstrings, or supracondylar osteotomy had successful results. Transfer of the hamstring tendons to the patella was successful in nine of twelve instances, so giving the lowest rate of failure. Forty-five patients were active walkers at follow-up as compared to eighteen before operation. However, only one of the eleven patients over twelve years of age with a motor level above T.12 was an active walker at review. Several conclusions are drawn. A flexion contracture of the hip greater than 30 degress should be corrected before operation of the knee. Supracondylar osteotomy as a primary procedure should only be used to correct flexion contracture of the knee secondary to malunited fractures. Faradic stimulation is useful in the evaluation of muslce function before operation. An intelligence quotient below 65 and weakness of the arms are relative contraindications for correction of the deformity of the knee.


Clinical assessment of a new weight-relieving brace.

A new cosmetic weight-relieving brace which utilises stainless steel and light alloy in its structure is described. A clinical assessment of thirty-six patients (four bilateral cases) has shown the Salford Cosmetic brace to be suitable for over 80 per cent of patients attending for assessment. Five patients rejected the brace, and the reasons are discussed. Contra-indications which emerged during the assessment included limb shortening of more than 5 centimetres; fixed equinus of more than 10 degrees; and fixed deformity of the knee of more than 10 degrees. The safety and durability of the brace, first demonstrated in laboratory tests, are confirmed. Further possible development is outlined.


Joint surface incongruity and its maintenance: an experimental study.

The paper reports an experimental investigation into the mechanisms whereby the joints of living animals maintain their precise contours. The normal patter of joint incongruity in the rabbit's hip having first been established, three groups of experimental animals underwent operative procedures designed to reduce the joint pressure to a level unrealistic in normal life. In one group the hip was dislocated by manipulation; in a second group the muscles around the hip joint were divided; and in a third group the hind limb was disarticulated through the knee. When the animals were killed at intervals of a few weeks after the operations the experimental hip joint showed increased congruity in all cases. It is concluded that in immature animals there is a mechanism in joints to control the modelling of their shapes towards an ideal incongruity, and that that mechanisms is pressure sensitive.


Bacterial infection and acrylic cement in the rat.

In thirty-one rat tibiae, plugs of plain acrylic cement were inoculated with Staphylococcus aureus; these all remained contaminated at the end of two weeks when the animals were killed. Inoculation with known strains of Pseudomonas, Proteus and Gp. G Streptococcus resulted in 70 to 93 per cent persisting contamination. Gentamicin, to which the organisms were fully sensitive, was efficacious in controlling the infection (90 per cent plugs proving sterile after two weeks). Fucidin was less successful against Staphylococcus aureus although effective in vitro. Intravenous inoculation with a suspension of Staphylococcus aureus succeeded in contaminanting 70 per cent of sixty plain cement plugs when injected into the tail vein half an hour after closure of the leg wounds. Only 11 per cent of sixty-four plugs were so contaminanted when the injection was delayed for two weeks. This animal model is submitted as a possible future means of testing different antibiotic-cement combinations against infection.


The muscles in club foot--a histological histochemical and electron microscopic study.

In talipes equino-varus the diminished bulk of the calf muscle suggests a neuromuscular defect. Accordingly, biopsies were taken from the postero-medial and peroneal muscle groups, and occasionally from abductor hallucis, in sixty patients mostly under the age of five years; 111 were studied histochemically and histologically, and a further fifty-three by electron-microscopy. Histochemical anomalies were revealed in ninety-two specimens; the muscle fibres in the other nineteen varied in size but were abnormal at the ultramicroscopic level, as were all specimens examined with the electron microscope. Evidence of neurogenic disease was seen in most instances and was more obvious in the older patients. The pattern of abnormality was similar in both muscle groups. It is thought that shortening of the postero-medial muscles may result from a small increase of fibrosis due to minor innervation changes occurring in intra-uterine life. There is evidence that immobilisation, stretching or relaxation of muscles does not account for the anomalies observed. This study of the extrinsic muscles in talipes equino-varus indicates a dominant neurogenic factor in its causation.


Analytical radiography of club feet.

A radiographic method is described for making the diagnosis of talo-navicular subluxation before ossification of the navicular bone occurs. Seven basic combinations of deformities are thought to occur in the club foot. Radiography before operation enables the surgeon to determine which of these combinations exists in a particular foot, and radiography during operation enables  him to determine wheter or not all the existing deformities have been corrected, and therefore whether or not the operation must be extended.


Spondylolisthesis in children and adolescents.

A study of spondylolysis and spondylolisthesis in 142 children and adolescents is reported. In twelve of the seventy-nine patients followed for over a year the affected vertebra slipped further by 10 per cent or more. Increasing slip occurred mainly during the adolescent growth spurt, and was greater when spinal bifida or other vertebral anomalies were present. If at presentation the slip is less than 30 per cent then further slip beyond 30 per cent is unlikely. Decompression posteriorly is advised when signs of nerve pressure are present. Indications for spinal fusion are suggested; the intertransverse method of fusion was used in sixty-nine patients.


Gray-scale ultrasound of billary tract disease. A correlative study with percutaneous transhepatic cholangiography.

Gray-scale ultrasound can be useful in the diagnosis of extrahepatic obstructive jaundice by the demonstration of dilatation of the biliary tree. The present study was undertaken to test the specificity and sensitivity of gray-scale ultrasound (US) in the detection of dilation of intrahepatic bile ducts, as compared to percutaneous transhepatic cholangiography (PTC). Forty-seven patients were studied prospectively by both techniques. In 23 patients demonstrated by PTC to have dilated intrahepatic ducts, 18 were detected by US. Of the 11 patients shown to have non-dilated ducts by PTC, all were accurately predicted by US. In 13 cases in which PTC was unsuccessful, the US study showed 12 to be non-dilated and one to be mildly dilated. Ultrasound as a non-invasive technique, should be the primary imaging technique in the evaluation of possible extrahepatic obstructive jaundice.