Long term tissue responses to catgut and collagen sutures. Reaction of catgut and collagen sutures in both wounded and nonwounded bladder, kidney, muscle, and liver tissue was studied in guinea pigs up to 130 days after implantation. A minimal amount of degradation of ths so-called absorbable sutures was observed. There was little difference in absorption rates in the tissues studied although there seemed to be slightly greater absorption in the kidney than in other tissues. Tissue responses were characterized by the formation of a bilaminar connective tissue capsule. The inner zone contained primarily fibroblasts and histiocytes, while lymphocytes, neutrophils, and eosinophils were scattered throughout both layers. Multinucleated giant cells were often observed in the inner layer, and aggregates of lymphocytes and mast cells were occasionally found in the pericapsular areas. Calculi formed on those suture loops that were loosely tied and thus projected into the bladder lumen.


Investigation of urine transport in the ureter of the pig by x-ray videodensitometry. I. The effect of increasing diuresis on velocity and rhythm of contractions: relationship between pressure wave and urine bolus. The flow rate is the decisive parameter of ureteric function. The frequency and the velocity of the contraction waves can be measured by X-ray videodensitometry without having to touch the ureter. The relationship between flow rate (milliliters per minute) and wave velocity (millimeters per second) is investigated in the pig ureter. It can be demonstrated that the wave velocity is a quadratic function of the perfusion. The graphic representation of this function corresponds ot a parabolic curve. The relationship between the bolus and the pressure wave is analyzed and it is shown that the time lag between the rear end of the bolus and the pressure peak is independent of the wave velocity. The theoretical ureter model of Weinberg is experimentally confirmed by simultaneous manometry and videodensitometry.


Investigation of urine transport in the ureter of the pig by x-ray videodensitometry. II. The effect of an increasing obstruction on velocity, rhythm, and efficiency of the contraction waves. The effect on an increasing acute obstruction on the motility of the ureter was analyzed by means of X-ray videodensitometry. The mean wave velocity decreases and the efficiency of the individual contraction is reduced. The lumen of the ureter is no longer closed completely and an increasing fraction of the urine bolus flows backward. The regurgitation can be seen cineradiographically and assessed semiquantitatively by densitometry.


Effect of chlorothiazide and furosemide diuresis on in vivo growth of Staphylococcus aureus in kidneys of mice. In mice subcutaneous administration of the diuretics chlorothiazide or furosemide resulted in a prompt diuresis characterized by production of dilute urine within 30 min and lasting for 6 hr. In vivo proliferation or clearance of Staphylococcus aureus from the kidneys was only temporarily impaired during the time of maximum diuresis. When treatment was delayed until after phagocytosis was initiated, there was no effect on clearance. Clinically it would appear that bacterial proliferations in the renal parenchyma would not be promoted as a consequence of diuretic treatment.


The effect of radiation on prostatic growth. Radiation is increasingly important in the treatment of prostatic carcinoma. The effect and mechanism of action of radiation on prostatic growth was studied using the ventral prostate of the testosterone-treated rat. Prostatic radiation blocked testosterone-stimulated prostatic growth. Electron microscopy showed this inhibition to be associated with specific morphologic alterations. While radiation caused profound inhibition of prostatic DNA synthesis, only modest effects on nuclear uptake of dihydrotestosterone and on prostatic 5alpha-reductase were observed. Based on these animal studies, it is concluded that a major mechanism whereby radiation blocks testosterone-stimulated prostatic growth operates through inhibition of DNA synthesis.


A new concept of the anatomy of the anal sphincter mechanism and the physiology of defecation. IV. Anatomy of the perianal spaces. A study of the perianal spaces was performed in 22 cadaveric specimens using dissection and microscopic examination. Six spaces could be recognized: subcutaneous, central, intersphincteric, ischiorectal, pelvirectal, and submucous. A mention of the central and intersphincteric spaces could not be traced in the literature. The anatomic relations and communications of the perianal spaces are discussed. The central space occupies the area between the longitudinal muscle termination and the base loop of the external anal sphincter. It is the headquarters of all of the perianal spaces as it communicates with all of them. The intersphincteric spaces lie along the longitudinal anal muscle. They constitute the main tracks for extension of pus along the anal canal. The ischiorectal space communicates directly with main tracks for extension of pus along the anal canal. The ischiorectal space communicates directly with the subcutaneous space without the intervention of a septum. The pelvirectal space communicates with the perianal skin through the intersphincteric spaces. The author failed to demonstrate the musculus submucous space; in its place a collagenous internal anal septum could be identified. Furthermore, no trace for the suspensory ligament of Parks was detected; the lower anal lining was found to be pulled by the medial central septum derived from the central tendon.


Flutamide. Mechanism of action of a new nonsteroidal antiandrogen. Flutamide is a new nonsteroidal antiandrogen useful in the treatment  of prostatic carcinoma. The mechanism of action of this compound was investigated using the sex accessory tissue of the male rat. Flutamide was found to block the action of both endogenous and exogenous testosterone and, in addition, to be a potent inhibitor of testosterone-stimulated prostatic DNA synthesis. Moreover, it is capable of inhibiting prostatic nuclear uptake of androgen. Electron microscopic observations further confirm the antiandrogenic properties of flutamide.


Viscoelastic properties of bladder wall strips at constant elongation. The experimental and data processing methods used were improved in several respects compared with those described in previous publications. One hundred twenty-two stress decrease curves for bladder wall strips were measured at constant elongation; reproducibility was tested. Consideration of the results led to the addition of two elements (a dashpot and an active element) to our model of the process involved. The influence of the dashpot on the measurements is mathematically analyzed in terms of an increase in rest length during stretch. The model could be successfully fitted to the experimental data and the parameters turned out to be in the physiologic range. Some recent models used to describe muscular stress relaxation were tested and criticized.


Compensatory renal adaptation after progressive renal ablation. A quantitative study of compensatory adaptation in renal growth, function, and blood flow after progressive renal ablation was performed in the rat. Four weeks after surgery, renal mass increased 31% in control animals (Group A), 81% in nephrectomized rats with 50% ablation of total renal mass (Group B), and 168% in rats subjected to 75% ablation of total renal mass (Group C). Whole animal glomerular filtration rate (GFR) was reduced to 68% of control levels after nephrectomy (Group B), while renal blood flow (RBF) was 81% of control. Similarly, RBF was 68% of control in Group C with ablation of 75% of renal mass, while GFR was reduced to 49% of control. Since the adaptive increases in RBF exceeded those of GRF, the filtration fraction fell from 0.39 in Group A to 0.35 in Group B, and to 0.29 in Group C. Mean nephron GFR (whole animal GFR per total number of glomeruli) was 47 nl per min in Group A and increased 60% to 76 nl per min in Group B and 136% to 112 nl per min in Group C. Compensatory changes in mean nephron blood flow (RBF per total number of glomeruli) exceeded those of nephron filtration rate. Mean nephron blood flow increased from 215 nl per min in Group A to 404 nl per min in Group B (88%) and to 724 nl per min in Group C (237% over contol). These data indicate that the compensatory changes in growth, function, and blood flow after resection of renal mass correlate with the amount of tissue removed. Changes in RBF parallel but exceed those of mass and function, especially after extensive renal ablation. No apparent limitation to the compensatory response was seen under the conditions of this study.


Intravenous pyelographic visualization after complete ureteral obstruction with hypertonic solute loading. Intravenous pyelography was performed after complete unilateral ureteral obstruction in dogs under conditions of mannitol diuresis and 3% NaCl solute loading. Pyelographic visualization with mannitol diuresis was uniformly poor. Satisfactory intravenous pyelograms were obtained in the 3% NaCl solute loaded group during the first 2 weeks, with nephrogram visualization noted up to 45 days postocclusion. The clinical use of pyelography, boosted by hypertonic solute loading during the early period after complete ureteral obstruction, may yield unexpected good quality studies.


[The lymphocyte transformation test in dermatology]. At first, immunologie and methodic basies of the lymphocyte transformation test are discussed. Then the results gained by this test in several dermatologic diseases are summarized. Finally, practice of the lymphocyte transformation test is critically reviewed.


[Round-headed spermatozoa: a rare andrologic finding ("globe-headed spermatozoa", "globozoospermia")]. The case of an infertile patient is reported who had 100% round-headed ("globe-headed") spermatozoa without acrosomes in his ejaculate. The spermatozoa were studied by light and phase contrast microscopy, as well as by transmission and--for the first time--by scanning electron microscopy. Determination of acrosin showed no enzyme activity. We propose the term "globozoospermia" for this condition which was first described in 1971. It is discussed whether it may be considered as a symptom or a distinct disease.


[Elastoma perforans serpigiosum].

[Elastoma perforans serpigiosum]. After a survey of the literature a case of "elastoma perforans serpiginosum" - characteristic from the clinical and histological viewpoint - is discussed. Our findings reveal that it deals with an "elastica-degeneration" within a zone of inflammation. Its catabolic products are discharged as necrotic masses through the epithelium or the follicles to the outside. Despite of the imposing clinical appearance it is not primarily a keratosis. The massive proliferation is secondary. The "forceps-like" enclosure of necrotic masses by proliferating epithelium may stimulate  a space within the follicle. Aetiopathogenesis and therapy are discussed.


[The radiophosphorus (32P)-test in precanceroses and malignant tumors of the skin]. In 21 patients with a variety of skin tumors (squamous cell carcinomas, malignant melanomas, basal cell epitheliomas and mycosis fungoides) or pre-cancerous lesions (Bowen's disease, actinic keratosis, junctional nevus cell nevus) the radioactive phosphorus uptake test demonstrates a significantly increased concentration of P32 in those tumors. There were no false negative tests. The possibility of differentiation of malignant melanoma from benign nevus cell nevus and the early recognition of cutaneous metastases is described. Furthermore recurrence of previously irradiated or excised basal cell epitheliomas can be detected without a biopsy. No hematological side-effects were observed.


The number of personnel needed to perform examinations. The Association of State and Territorial Public Health Laboratory Directors and the Center for Disease Control jointly developed a relative value system for measuring laboratory workload and productivity. This system has also served as a basis for determining the cost of tests. An additional usage, estimating both the number and grade levels of personnel needed by a laboratory to perform examinations, is explained in this article.


Effects of pooled samples.

Effects of pooled samples. Pooling of specimens before testing may reduce the costs of expensive tests for rare conditions. This paper presents models of how test performance is degraded by such pools, and of the financial savings that pooling allows. The method of computing optimal pool size is demonstrated on a screening test and on the test for gastrin. Test characteristics that make pooling especially effective are listed.


Mycoplasma in biological systems: induction of interferon. Mycoplasmal infection leads to a variety of effects on host cells both in vivo and in vitro. Among these is the induction of interferon, which has been demonstrated in mycoplasma-infected human and sheep lymphoreticular cell cultures and in mice. Different species and strains of mycoplasma vary in their ability to induce interferon in these experimental models. Several studies have shown that mycoplasmas do not elicit the production of interferon in murine cells in vitro or in non-lymphoreticular cells of other animal species. This may be related to the ability of host cells to take up mycoplasmas or to mycoplasma-associated products, such as mycoplasmal viruses. Initial studies of mixed infections with mycoplasmas and animal viruses in vivo have demonstrated both an inhibition and an enhancement of viral disease, indicating the complex nature of mycoplasma-animal virus interactions.


Mycoplasmas in humans: significance of Ureaplasma urealyticum. Isolation of mycoplasmas from clinical specimens has resulted in compelling evidence that in addition to M. pneumoniae, the documented pathogen, the T strain mycoplasmas are not always benign commensals colonizing the mucous membranes of the genitourinary tract of men and women, but may be actively involved in infection with sequelae expressed as nongonococcal urethritis and some form of reproductive wastage such as infertility, spontaneous abortion and premature births.


Infertility of cattle caused by mycoplasmas. Mycoplasmas have been well established as pathogens of the bovine urogenital tract, and produce pathologic lesions resulting in infertility. Serologic examination of cattle with infertility problems with Mycoplasma bovigenitalium and Mycoplasma agalactiae subsp. bovis show a high incidence of positive reactors suggesting that mycoplasmas play an important role in bovine infertility. The similarities of pathologic lesions in the urogenital tract of cattle and women with infertility problems and the frequency of isolation of mycoplasmas from human females suggest closer examination for mycoplasmas in human infertility. Studies with bulls suggest that mycoplasmas as a cause of human male infertility should not be ignored.


[Regulation factors of stomach emptying in dogs]. Gastric emptying of organic acids was studied in 6 healthy mongrel dogs. After chronic esophagostomies were performed according to the method of Komarov, a total of 340 test meals were instilled. Each test meal consisted of 300 ml of 6 different organic acids with decreasing molecular weight and different concentrations. After the experiments were achieved, each dog underwent a proximal gastric vagotomy according to the method of Amdrup, and experiments with citric acid were repeated. The results may be summarized as follows: multiple stepwise regression analysis of the data is consistent with a model in which gastric emptying of organic acids is regulated by 3 receptors. The receptors respond to concentration of the organic acid, the actual volume, and the type of acid. The volume receptor is located in the corpus of the stomach because the effect of volume accelerates the emptying rate after proximal gastric vagotomy while the effects of concentration and type of acid remain unchanged.


[A fixation apparatus for differential indication on fibular band lesions]. Description of a new radiological technique for examination of lesions of the lateral ligaments of the ankle. With this method it is possible to make differential diagnosis. The drawer sign of the talus is measured. In contrast with conventional stress a.-p. roentgenograms it is also possible to detect isolated tears of the anterior talo-fibular ligament. Additional advantages of the method are no X-ray exposure for the examiner, local anaesthesia is not required and the results are reproducible independent of the examiner.


[Therapeutic hazard of supracondylar humeral fracture in a child]. A case of a supracondylar fracture in a 4-year-old female is presented. The risks of treatment are discussed. The question is raised whether the complication which resulted could have been avoided.


[Experiences with AO-semitubular plate in the stabilization of ankle fractures]. Anatomical restoration of the lateral malleolus in ankle dislocation fractures is known to be very important. With the AO-small semitubular plate an armamentarium is available which accomplishes this task very well. Exercising stability nearly always can be attained. In case of postoperative infection, healing generally takes place without plate removing and, therefore, under good fracture position.


[Valve of plastic repair of the deep femoral artery in chronic circulatory disease of the lower extremities]. It is reported about indication, surgical technique, early and late postoperative results of the plastic of the profunda femoris artery. On 17, mainly old patients with chronic circulatory disease of the lower limbs was this operation carried out, because a longer angioplastic operative procedure would have been a great risk. The statement, that 47% of our patients were practically without symptoms after four years is promising for the plastic of the profunda femoris artery with chronic ischemia syndrome of the lower limbs.


[Appendiceal carcinoma].

[Appendiceal carcinoma]. Three cases of carcinoma of the vermiform appendix are reported. The rarity of the disease has been emphasized as well as the resulting lack of clinical experience. Medical literature on the subject has been reviewed.


[Complications in bronchoplastic surgery of benign and malignant lung neoplasms]. Out of 1629 patients who were hospitalised 1963 to 1973 for diagnosis and treatment of bronchogenic tumors, 731 underwent resection. 20 patients could be operated upon by lobectomy or bilobectomy and bronchial resection (Sleeve-resection). In two other patients the tumor could be resected through a bronchotomy. Postoperative complications were frequent (n = 6) on patients with malignant tumors (n= 15). We saw 3 times a bronchopleural fistula (2 patients had to be reoperated, 1 death), once a fatal empyema, once a fatal pneumonia of the contralateral side and once a bronchial stenosis of 50% of the lumen. On the patients with a benign tumor (n = 7) we saw in one patient a bronchopleural fistula and in another endobronchialgranulomas due to non resorbable suture material. Methods to prevent or cure these complications are presented consisting in the use of absorbable suture material and long lasting intrathoracic suction.


[Indicatory and technical faults as cause of postoperative complications in arterial surgery]. In all vascular surgery the technic and quality of the first operation is a factor of decisive importance for the long-term results. The need to perform a second or third operation considerably reduces the chances of success. Routine use of an intra-operative control method like arteriography or vascular endoscopy, makes a decisive contribution in the peripheral circulation of the limbs to the immediate detection of errors of operative technic and their correction during the same operation. A change of indication for occlusive processes in the femoropopliteal branch is evident: patients with claudication should be treated with profunda  revascularisation combined with lumbal sympathectomy and patients with rest-pain and necrosis should be treated with venous autografts using a microsurgical technic. To avoid deep wound infections the selection of an incision line which does not damage the lymphatic channels, particularly in the groin, the short duration of operations and the use of a purely instrumental operative technic with minimal damage to the tissues are of decisive importance. Examples are used to illustrate errors of indication and technic and the post-operative sequelae of such errors.


Fundus photography in neonates, an approach to obtain indirect information on cerebral blood flow. In view of the possibility to obtain indirect information on the interdependency between brain tissue metabolish and the diameter of the intraparenchymatous arterioles through measurement of systemic blood gases and blood pressure on one hand and the diameter of the central retinal artery on the other, we describe a modified method of fundus photography which is practical for neonatologists owing to the simplification brought about by the use of a special contact lens. In an appendix the relevant optical data are analyzed. Fundus photographs of neonates suffering from cardiac malformation or asphyxia are shown.


[Plasma aldosterone in childhood].

[Plasma aldosterone in childhood]. According to a radioimmunological method modified in our laboratory we determined plasma aldosterone levels under standardized conditions in children of different age from 2 weeks to 14 years, in women undergoing uncomplicated vaginal delivery, and in the cord blood of the corresponding newborn babies. Furthermore, the effect of different stimulating factors (ACTH, salt restriction, severe vomiting, fever) in children was studied. In mothers and cord blood we found 68.6 +/- 39.3 ng/100 ml and 75.5 +/- 37.4 ng/100 ml, respectively. In the first two years of life there is a decrease in plasma aldosterone levels to the relatively constant concentration at the age of 2 to 14 years (7.3 +/- 4.7 ng/100 ml). The age groups 2 weeks to 1 year, 1 to 2 years and 2 to 14 years all show significant differences. A correlation of age (first two years of life) and plasma aldosterone level could be shown (correlation coefficient -0.50 according to Pearson). The stimulation results are comparable with literature data concerning adults. The mechanism of stimulation by vomiting and fever is discussed.


Urinary hydroxyproline values following continuous and intermittent administration of prednisolone in children. The administration of 2 mg/kg body weight of prednisolone daily in divided doses to 5 healthy children aged 5-7 years resulted in a significant decrease in the amount of total urinary hydroxyproline (HOP) which was restored to pretreatment levels two weeks after discontinuation of the drug. On the other hand, the administration of 2 mg/kg body weight of prednisolone in a single dose on alternate days (every-other-day) to two children and for a period of 10 days caused insignificant alterations in the amount of urinary HOP.


Reye's syndrome without fatty liver.

Reye's syndrome without fatty liver. A 2 1/2-year-old child died after an illness of three days duration, characterized by a sudden onset, vomiting, coma and a striking hyperammonemia. At autopsy, the liver was practically normal. This is a further example of Reye's syndrome with only minimal fatty infiltration of the liver. It appears that the classical massive and diffuse fatty degeneration of the liver is not an essential feature of the syndrome. The evidence for this is discussed.


Pancreatic ascites in the first year of life. A case of pancreatic ascites in a 4-month-old baby girl is reported. To the best of our knowledge only 4 previous cases occurring in the first year of life have been reported in the pediatric literature. In patients with unexplained ascites, amylase determination should be performed on ascitic fluid and serum. The baby was first treated by external drainage and later by internal drainage of the pseudocyst which had formed along the fistula.


Congenital neuroblastoma with paraplegia. Case report. A case of congenital neuroblastoma presenting with paraplegia in a newborn baby is described. The tumor was removed and chemotherapy was given. The child is now 2 years old, without relapse, but still suffers from flaccid paralysis and anaesthesia of both legs.


Haematological studies in a new variant of glucosephosphate isomerase deficiency (GPI Utrecht). The haematological data in a 9-year-old Dutch child suffering from an unusual new variant of glucose-6-phosphate isomerase deficiency (GPI Utrecht) are discussed. Symptoms and signs differ in many respects from those generally observed in GPI deficiency, as mental retardation, drug sensitivity and increased susceptibility to infections were present. Extensive studies on mechanisms involved in drug-sensitive haemolytic anaemia did not reveal its causes. Though the defect was generalized, no disturbance in granulocyte and thrombocyte functions were detected.


An inherited defect in granulocyte function: impaired chemotaxis, phagocytosis and intracellular killing of microorganisms. The neutrophil granulocytes of a 1-year-old boy with severe recurrent infections were found to have almost no chemotactic responsiveness, impaired phagocytosis and reduced intracellular killing of Candida albicans "in vitro". During the course of a febrile illness of unknown etiology which was accompanied by a leukemoid reaction, phagocytic activity became normal; the chemotactic response was also increased but still remained slightly subnormal. Family studies suggested that the defect was an inherited autosomal recessive one.


Epidermolysis bullosa hereditaria letalis: report of a case and probable ultrastructural defects. A case of epidermolysis bullosa hereditaria letalis (Herlitz) is described. In accordance with findings in other cases of Herlitz disease blister formation occurs in form of junctional separation. Electron microscopic study revealed scarcity and abnormal structure of hemidesmosomes, which may play an important role in the pathomorphogenesis of junctional blistering.


The role of the blood-brain barrier in perfusion fixation of the brain for electron microscopy. Regions of the brain vascularized by capillaries of the blood-brain barrier (BBB) type require a different fixative from regions which have capillaries of the endocrine type. Fixative with isotonic buffer gives excellent ultrastructural preservation in the BBB regions, but cause severe shrinkage of cells in the endocrine regions. This is evidently due to the difference in the permeability of the capillary walls to solutes in the fixative. In the BBB regions in less permeable capillaries do not allow outflow of osmotically active particles to a harmful extent, whereas in the endocrine regions osomotic imbalances are created between the intra- and extracellular compartments. The diffusion rate of the fixative and the final volume of the fixed brain depend on the balance between the intravascular and intersitial hydrostatic and oncotic pressures across the capillary wall during the perfusion fixation, as those pressures regulate the amount of perfusate that will enter the parenchyma. Generally, as high a perfusion pressure as possible is recommended to obtain effective wash-out of blood and rapid diffusion of fixative into the tissue. Addition of macromolecules (2% PVP, mol. wt. 40,000) into the fixative slightly improved the ultrastructural preservation in the BBB regions of the central nervous system.


Some quantitative aspects of Acridine Orange fluorescence in unfixed, sucrose-isolated mammalian nuclei. Nuclei were isolated from mouse liver and central nervous system (CNS). These nuclei were fluorochromed without fixation in a 0.25 M sucrose medium containing 5-5 X 10(-5) M Acridin Orange and measured with an incident microfluorometric system. In the case of mouse CNS nuclei, the major and minor axes of the nuclei were measured with a filar micrometer. Three modal values were obtained from the hepatocyte suspension corresponding to 2C, 4C, and 8C nuclei, respectively. While the CNS nuclei displayed substantial variability in size, the Acridine Orange emission values at 530 nm were nearly constant. The data suggest that under these conditions, Acridine Orange fuorescence reflects DNA content. Further, the 530 nm fluorescence emission is not affected by chromatin condensation or proteins complexed with DNA.


Zonal rotor analysis of the subcellular localization of alpha-glycerphosphate dehydrogenase, alpha-naphthyl palmitate and beta-naphthyl laurate hydrolases in the mucosa of the guinea-pig small intestine. Whole homogenate of guinea-pig small intestine mucosa was analysed by centrifugation in a zonal rotor. The results indicate that FAD-linked alpha-glycerophosphate dehydrogenase is localized in the mitochondria and that NAD-linked alpha-glycerophosphate dehydrogeanse is a soluble phase enzyme. An enzyme hydrolysing alpha-naphthyl palmitate at an acid pH was localized in the lysosomes and was activated by 0.1% Triton X-100 and by freezing and thawing. An alkaline hydrolase acting on beta-naphthyl laurate was localized in the 'microsomes'. The possibility of this enzyme being different from alpha-naphthyl acetate hydrolase is discussed.


Bulla curettage for chronic otitis media and interna in dogs. In a series of 10 clinically affected and 6 clinically normal dogs, the technique of bulla curettage via the external ear canal, after lateral ear resection, was evaluated. In 5 of the 10 dogs with chronic otitis media and interna, the head carriage became normal 2 to 8 weeks after surgery and remained so for at least 18 months; 4 other dogs improved, but the improvement was either temporary or incomplete. In the clinically normal dogs, postsurgical complications were minimal; the tympanic membrane healed completely in 4 of the 6 dogs.


Ascites associated with intrahepatic arteriovenous fistula in a cat. A 1 1/2-year-old male domestic short-haired cat with ascites was found to have an intrahepatic fistula between the hepatic artery and portal vein. The diagnosis was made by means of angiography. The ascites was attributed to portal hypertension caused by the fistula.


Evaluation of blood urea nitrogen and serum creatinine concentrations as indicators of renal dysfunction: a study of 111 cases and a review of related literature. Blood urea nitrogen concentration (BUN), serum creatinine concentration (SC), and BUN/SC ratios, as recorded for 111 dogs and cats with azotemia, were evaluated to determine their usefulness in evaluation of renal dysfunction. Cases were categorized into prerenal, renal, and post-renal causes of azotemia, on the basis of histologic and clinical criteria. The severity of azotemia varied within groups, but the mean value for degree of azotemia was lowest in the prerenal group. The BUN/SC ratios were highest when azotemia was mild, regardless of cause. The BUN/SC ratios in the 3 groups were not significantly different when the degree of azotemia was considered, indicating that differentiation of renal from extrarenal azotemia was not possible in the dog and cat, on the basis of relative values of BUN and SC. Comparison of BUN/SC ratios in acute and chronic azotemia of comparable magnitude revealed no significant differences between groups, indicating that this ratio cannot be used to differentiate acute and chronic azotemia. Following supportive therapy, BUN decreased significantly (P less than 0.05) more than SC, suggesting that extrarenal factors contributed to the increased BUN and that proportionately more urea than creatinine was excreted by the kidneys during therapy. The BUN/SC ratios varied widely and to a similar degree in all groups examined. Inasmuch as the BUN and SC were not correlated with more accurate measurements of renal function, conclusions could not be drawn concerning the superiority of either factor as a measure of renal function. Many nonrenal factors, previously identified, influence serum concentration of urea and creatinine. In recent studies involving azotemic human beings and rats, it was suggested that up to 25% of the urea and 65% of the creatinine produced in the body is degraded by enteric bacteria rather than excreted directly by the kidneys. These data indicate  that neither BUN or SC can be used as precise tests of renal function, although SC is subject to alteration by fewer nonrenal factors than is BUN. In older medical and veterinary medical literature, the use of SC as a prognostic indicator had been advocated. Newer findings on the pathophysiology of creatininemia and retrospective case studies do not support this view. In the present study, severe creatininemia was documented in cases in which primary renal dysfunction did not exist. It was concluded that BUN and SC should continue to be regarded as crude indexes of renal function. Clinical value lies in the relative ease of their determination. Because of their lack of sensitivity, more specific evaluation of renal function (urine concentrating ability, phenolsulfonphthalein excretion) may be indicated when BUN and SC are normal or only slightly elevated. Because extrarenal factors may alter BUN and SC, it is necessary to correlate these values with clinical and other laboratory data to differentiate renal from extra-renal azotemia. Single determinations of BUN or SC provide no basis for prognosis.


Tyzzer's disease in a dog.

Tyzzer's disease in a dog. A 5-week-old mixed-breed dog was examined because of emaciation and depression associated with chronic anorexia, diarrhea, and vomiting. Its rectal temperature was subnormal and it died on the day of admission. At necropsy, small focal lesions were distributed through the liver. Enteric alterations included catarrhal enteritis with fluid contents, excess production of mucus, and mucosal hyperemia. Microscopically, the hepatic lesions were disseminated foci of coagulative necrosis, with little or no associated inflammatory cell response. Numerous organisms morphologically consistent with Bacillus piliformis were demonstrated within viable hepatocytes at the periphery of the necrotic foci and in the intestinal mucosa. Numerous coccidial forms were found within the epithelial cells of the intestinal mucosa, which was focally necrotic.


Determination of melengestrol acetate in bovine tissue: collaborative study. Seven laboratories collaboratively studied a method for the assay of melengestrol acetate at the 0, 10, and 20 ppb levels in bovine fat, liver, muscle, and kidney. The study included fortification of tissue by each laboratory and analysis of fat samples taken from treated heifers which had endogenous levels of 0, 10, and 20 ppb melengestrol acetate. The multistep cleanup procedure used included extraction, solvent partition, column chromatography, and electron capture gas-liquid chromatographic, determination. Results of the study for muscle, liver, kidney, and fat showed that the method gave satisfactory recoveries and accuracy. In fat, the most critical tissue, recovery was greater than 93%. A statistical comparison of the results reported for fat tissue from treated heifers demonstrated that 5 of the 7 laboratories obtained similar results. The results produced by the method can be expected to be repeatable within and among laboratories. On the basis of the collaborative results the method has been adopted as official first action.


Appraisal of existing methodology in aspiration toxicity testing. New methods or modifications of currently used methods in aspiration toxicology must be capable of accurately assessing the hazard and toxicity potentials of those products which are capable of being aspirated. The methods evaluated in the rat were intratracheal injections,intravenous injections, and modifications of the Gerarde technique. A product containing petroleum distillates and a standard viscosity petroleum distillate sample were administered to Osborne-Mendel rats, rabbits, and guinea pigs in ml/kg doses. The utility of the methods was determined by comparing lung weight increases, lung-body weight ratios, gross pathological lung changes, and mortality. The modified Gerarde technique showed the best potential for predicting aspiration hazard and toxicity based on the severity of the reactions which, using a radiolabeled oil, was shown to be a function of the amount of product which enters and reacts with the lungs. The influence of preventing the swallowing reflex to assure aspiration of the oil was assessed in the rat. The results show that less than 10% of the dose is aspirated when the swallowing reflex occurs. Methods of sacrifice were investigated for an effect on rat lung weight. No tested method except stunning with cervical dislocation produced an effect. Tests were conducted on rats and rabbits to determine differences in lung reactivity to the influence of inhalation anesthetics, pentothal sodium, or no anesthesia in response to petroleum distillate administration and to determine which test species is the better animal model. The resuls indicate that the rat is the better test model because of degree of lung reaction, size, and economy, and that inhalation anesthesia with ether is preferable over the other choices.


Survey of food products for volatile N-nitrosamines. A variety of food products containing nitrite were analyzed for 14 volatile N-nitrosamines by using a method demonstrated to be sensitive to 10 ppb. A total of 121 food samples were screened for volatile N-nitrosamine content. N-Nitrosopyrrolidine was confirmed in fried bacon at levels up to 139 ppb. N-Dimethylnitrosamine, N-nitrosopyrrolidine, and N-nitrosopiperidine were also confirmed in spice-cure mixtures at levels ranging from 50 to 2000 ppb.


Determination of bergapten in fragrance preparations by thin layer chromatography and spectrophotofluorometry. A method has been developed for the determination of bergapten (5-methoxypsoralen), a known phototoxin, in perfumes,colognes, and toilet waters. The bergapten and other lactonic compounds were first isolated from the sample by a series of extractions. The extract containing the bergapten was diluted to a known volume and a aliquot was spotted on a thin layer chromatographic (TLC) plate coated with silica gel G. After 2-dimensional development with hexane - carbon tetrachloride - tert - butylamine (180+12+9) and hexane-toluene-ethyl acetate-acetic acid (100+10+15+0.5), the TLC plate was dried and the emitted fluorescence of bergapten was measured, using a spectrophotofluorometer equipped with a TLC accessory and coupled to an automatic digital integrator. The amount of bergapten was determined by comparing its peak area to those of bergapten standards. The average recovery for levels of 0.001, 0.005, and 0.01% bergapten was 88%.


Determination of polychlorinated dibenzo-p-dioxins and dibenzofurans in commercial pentachlorophenols by combined gas chromatography-mass spectrometry. Samples of commercial pentachlorophenol (PCP) and its sodium salt (PCP-Na) were examined for the presence of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), using a rapid, highly specific method of analysis. Phenolic compounds are removed by alkaline extraction, and the neutral components are fractionated on an alumina minicolumn. After gas chromatographic separation, individual PCDDs and PCDFs are detected by mass fragmentography and their presence is confirmed by complete mass spectral analysis. While some samples had only low amounts of PCDDs and PCDFs, others contained much higher amounts of these components. PCP-Na samples showed the unexpected presence of a tetrachlorodibenzo-p-dioxin. Re-analysis of PCP and PCP-Na samples with high PCDD contents on a high-resolution glass capillary column showed the presence of 3 hexa- and the 2 heptachlorodibenzo-p-dioxins with nearly constant isomeric ratios.


Qualitative tests for added coloring matter in meat products. The rapid qualitative detection of coal tar dyes, chemicals added to color meat products, and some natural coloring materials was investigated through extraction and subsequent identification by spectrophotometric, paper chromatographic, and specific reaction techniques. These techniques will detect the presence of coal tar dyes, imidazole, histamine, histidine, cochineal, beet powder, annatto, alkanet, carotene, paprika, saffron, turmeric, and materials causing discoloration in meats.


Determination of vitamin B12 in dry feeds by atomic absorption spectrophotometry. Vitamin B12 was determined in dry feeds by atomic absorption spectrophotometry (AAS). Samples containing B12 were extracted with an assay solution, 5 g EDTA was added to the filtrate, the pH was adjusted to 7 with NH4OH, and 5 g charcoal was added. The charcoal was removed by filtering through ashless paper which was then placed in a beaker and ashed at 600 degrees C. After dissolving the cobalt oxide from the ash in 5N HNO3, cobalt content was determined by using AAS. To determine mg B12/lb feed, ppm cobalt in the feed is multiplied by 10.43. The sensitivity of the proposed procedure is 1 mg vitamin B12/lb. The procedure is rapid and precise, and results compare favorably with AOAC method 43.109.


Relationship between the biological availability and solubility rate of reduced iron. Solubility rate determinations were conducted on electrolytic iron and hydrogen reduced iron and carbonyl iron samples to study the correlation of biological availability and solubility. There was excellent correlation for the hydrogen reduced iron and electrolytic iron samples,. The carbonyl iron samples showed a direct relationship between solubility and bioavailability, but these data were not numerically equivalent to data from other types.


A screening method for protein characterization and differentiation. A circular paper chromatographic method was developed for the separation of the amino acids in proteins into 7 subgroups. Butanol-acetic acid water (4+1+1) was used as the developing solvent. Eluted ninhydrin-stained aminograms gave rise to graphic profiles or numerical indexes based on absorbance percentages. The profiles can be used to compare protein-containing samples. Twenty different samples were studied through 190 comparisons of graphic profiles and coefficients of correlation, with only 4% misleading results. The method showed excellent reproducibility for the identification or differentiation of proteins and has the advantage of being performed with low-priced apparatus and reagents.


Semiautomated ring carrier to facilitate and expedite disinfectant testing by the AOAC use-dilution method. The AOAC use-dilution test requires the replicate examination of a number of contaminated metal rings. Ten metal rings may be tested within the 20 min structure of the test. From a statistical and economical viewpoint, it would be highly desirable to increase the number of rings examined in a single test. A semiautomated ring carrier permits testing 200 rings within a 20 min test.


Phosphorus pentoxide as a drying agent for bacterial culture extracts analyzed by gas-liquid chromatography. The procedure for gas chromatographic analysis of metabolic products of microbial fermentation includes solvent extraction of the aqueous growth media, drying of the extract, and direct chromatographic analysis of the solvent. In this study, 2 drying agents, magnesium sulfate and phosphorus pentoxide, were compared. Both were effective in removing water; however, phosphorus pentoxide removed water more completely and at a faster rate than magnesium sulfate. When a thermal conductivity detector is used, it is important to completely remove water from the solvent to prevent interference with volatile acids and alcohols. When  water is present, short-chain alcohols (C2-C5) are eluted together with the water, causing peak overlap and shoulder separations. Phosphorus pentoxide quickly and effectively removed water so that a baseline was established following the solvent front on the chromatogram. The use of phosphorus pentoxide is particularly advantageous for identification or fermentation studies on Clostridium and Propionibacterium when rapid identification is desired or when large numbers of cultures are to be tested.


Lactic and succinic acid levels and refractive indices in the determinations of the age of eggs. The increase in the concentration of lactic and succinic acid during storage was measured in an attempt to determine the age of eggs. The increase in succinic acid alone is not sufficient to indicate egg age. Although lactic acid concentrations increase more rapidly, the levels are still very low, the increase is not linear, and concentrations in fresh eggs vary widely. On the other hand, refractive indices showed a nearly linear correlation with age and very little variation between eggs. These measurements are reproducible and easy to perform and, with further study, should provide an alternative to the AOAC method.


Gas-liquid chromatographic analysis of ethephon and fenoprop residues in apples and their decline before and after harvest. Ethephon (2-chloroethylphosphonic acid) and fenoprop (2-(2,4,5-trichlorophenoxy) propionic acid) may be determined in the same apple sample. After extraction with methanol, 2 separate methylation procedures were required to quantitatively convert each compound. Ethephon was esterified with diazomethane and analyzed by a flame photometric detector in the P-mode. Fenoprop was esterified with boron trifluoride/methanol and analyzed by electron capture gas chromatography. Average recoveries were about 95% at 0.05 ppm for both compounds. The limit of detection was 0.05 ppm for ethephon and 0.01 ppm for fenoprop in a 1 g sample. The persistence of both compounds before and after harvest was studied. Ethephon and fenoprop were applied simultaneously to apple trees at the recommended concentrations of 300 and 20 ppm, respectively. Ethephon residues averaged 1.6, 0.75, and 0.4 ppm at 2 hr, 10 days, and after washing at 13 days, respectively. The corresponding fenoprop residues were 0.70, 0.025, and 0.024 ppm.


Mass spectra of sodium N-alkyl and N,N-dialkyl dithiocarbamates and some related compounds. Electron impact mass spectra of 8 sodium alkyl and dialkyl dithiocarbamate salts are reported with the spectra of 2 dithiocarbamate zinc salts and N',N'-dimethyl-N,N-dimethylthiocarbamylsulfenamide. The investigated compounds all gave molecular ions, and generalized descriptions of their fragmentation behavior are presented.


Analysis pesticides by chemical derivatization. III. Gas chromatographic characteristics and conditions for the formation of pentafluorobenzyl derivatives of ten herbicidal acids. The reaction conditions to form pentafluorobenzyl derivatives of 10 herbicidal acids, at pesticide residue levels, were studied. The optimum conditions were reaction at room temperature for at least 5 hr (or overnight), using potassium carbonate solution as catalyst. The gas chromatographic characteristics of these derivatives were also studied. Nine of 10 of these derivatives can be separated on an OV-101/OV-210 gas chromatographic column.


Gas-liquid chromatographic analysis and chemical confirmation of azodrin (monocrotophos) residues in strawberries. A gas-liquid chromatographic (GLC) method is described for the analysis and confirmation of azodrin (monocrotophos, 3-(dimethoxyphosphinyl)-N-methyl-cis-crotonamide) residues in strawberrires. The strawberries are extracted with acetone, and the filtrate is partioned with a mixture of methylene chloride and petroleum ether followed by further extraction with methylene chloride. The organic phases are combined, dried with anhydrous sodium sulfate, and concentrated to a small volume for GLC analysis on a 3% OV-210 column with flame photometric detection. Identity of the compound is confirmed by chromatography on the same column after trifluoroacetylation of an aliquot of the strawberry extract. The detection limit is about 2 ppb. The types of strawberry samples analyzed were fresh, frozen, pureed, and jam.


Thin layer chromatography of parathion as paraoxon with cholinesterase inhibition detection. A simple, sensitive, and rapid method is described for the quantitative estimation of ng amounts of parathion (O,O-diethyl O-p-nitrophenyl phosphorothioate) as paraoxon (O,O-diethyl O-p-nitrophenyl phosphate) on thin layer chromatograms. Paraoxon is detected by inhibition, using p-nitrobenzenediazonium fluoroborate as the chromogenic reagent. This chromogenic reagent is more sensitive than Fast Blue B or indoxyl acetate; 0.1 ng may be detected and amounts from 5 to 50 ng may be estimated. The method is a viable alternative to gas chromatographic analysis for parathion.


Determination of arsenic and selenium in foods by electroanalytical techniques. Arsenic and selsnium are determined in foods be differential pulse polarography and cathodic stripping voltammetry. The sample is digested with nitric acid and magnesium nitrate and then dissolved in dilute hydrochloric acid. An aliquot is removed, the arsenic is chemically reduced to the trivalent state, and interferences are removed by ion exchange before polarography. Selenium is determined in a second aliquot by cathodic stripping voltammetry. Recoveries for both elements in several foods were from 90 to 110%. The relative standard deviations for arsenic at 5 ppm and selenium at 0.48 ppm were 5.8 and 7.3%, respectively.


Digestion of fish samples for mercury determination by flameless atomic absorption spectrophotometry. Methods of digestion of fish samples for mercury determination by flameless atomic absorption spectrophotometry have been investigated. Digestion in Teflon bombs, Kjeldahl flasks, or borosilicate test tubes gives comparable precisions and mercury recoveries. Because of their cost and effect in limiting productivity, Teflon bombs were considered unnecessary for analysis of fish samples. Experiments with cooked and uncooked fish samples have confirmed that no appreciable loss of mercury occurs during baking 30 min at 170 degrees C.


Determination of zearalenone in corn: collaborative study. Corn samples spiked at levels of 100, 300, 1000, and 2000 mug zearalenone/kg were sent to 22 collaborators for analysis by the Eppley method. All samples were yellow corn except one white corn sample spiked at 2000 mug/kg. Results from 16 collaborators were statistically analyzed. Only 4 of 16 collaborators detected zearalenone in the sample containing 100 mu/kg, but 11 detected the toxin in the sample containing 300 mug/kg. Average recoveries from all samples were 129% at 300 mug/kg, 101% at 1000 mug/kg, and 88% at 2000 mug/kg. The between-laboratory coefficients of variation were 53.0% at 300 mug/kg, 38.2% at 1000 mug/kg, and 27.0% at 2000 mug/kg. Five naturally contaminated corn samples, one in triplicate, were also provided. The mean level of zearalenone in the naturally contaminated samples ranged from 431 to 7622 mug/kg. The mean coefficient of variation for all samples was 40.5%. Two collaborators measured quantities of zearalenone on thin layer chromatographic plates densitometrically. Their results were not included in the statistical analysis, but the results indicated that densitometric measurement, given proper dilutions of solutions, could be used. The method has been adopted as official first action.


Gas-liquid chromatographic determination of beta-asarone in wines and flavors. Wine samples containing beta-asarone (cis-2,4,5-trimethoxy-l-propenylbenzene) are distilled; beta-asarone is extracted by hexane and then quantitatively determined by gas-liquid chromatography (GLC), using ethyl palmitate as the internal standard. The GLC procedure is rapid and yields precise and accurate results. Mass spectrometry confirmed the identity of the GLC peak as beta-asarone. The ultraviolet spectra of beta-asarone and its isomer were also determined.


Infrared identification of maleic acid in pharmaceutical maleate salts. Difficulties are encountered in the USP melting point identification test for maleic acid extracted from chlorpheniramine maleate. A general procedure is described for the isolation and infrared identification of the maleic acid moiety of pharmaceutical salts from drug substances or dosage forms. This procedure permits the concurrent infrared identification of the amine moiety.


Spectrophotometric analysis of alkaloids with picrolonic acid. The spectroscopic characteristics of the interaction of alkaloids with picrolonic acid were studied. In solvents of low and intermediate polarity, the presence of alkaloids caused a red shift of the 322 nm band of nonionized picrolonic acid to 355 nm, corresponding to the anionic resonance band. There was also a considerable increase in absorptivity, which was dependent on both the basicity (pKa) and molar concentration of the alkaloid present. Arylamines, aromatic N-heterocycles, and alkaloids lacking an aliphatic amine moiety did not show observable shifts. The interaction was developed into a spectrophotometric assay for the following alkaloids in pharmaceutical preparations: atropine, ephedrine, codeine, emetine, quinine, and strychnine. The method is sensitive to 2 mug alkaloid/ml, with an accuracy of not equal to 1.5% and a standard deviation of not equal to 1.05 - 1.31%.


Spectrophotometric determination of allylisothiocyanate in mustard seed oil. Allylisothiocyanate is determined spectrophotometrically after reaction with 2,3-dichloro-1,4-naphthoquinone. For pure samples, the color intensity is proportional to allylisothiocyanate content in the range 0.8-3.0 mg/ml reaction mixture. A modified procedure is used to estimate allylisothiocyanate content of mustard seed oil. The reaction is linear for allylisothiocyanate  concentrations in the range 40-240 mug/ml reaction mixture. Two mustard seed oil samples contained 0.995 not equal to 0.020 and 0.981 not equal to 0.019% allylisothiocyanate.


High-speed liquid chromatographic determination of phenothiazine in commercial pesticide formulations. Phenothiazine (thiodiphenylamine) in commercial pesticide formulations is analyzed by high-speed liquid chromatography and ultraviolet absorption detection. Phenothiazine is extracted from the formulation with methanol and injected into the liquid chromatograph. Methanol-water (1+1) is the mobile phase. The amount of phenothiazine is calculated from peak height ratios. The liquid chromatographic procedure is much faster than the infrared or colorimetric method and yields values in close agreement with both of these methods.


Derivatization procedure for identification of aflatoxin M1 on a thin layer chromatogram. A commodity extract containing presumptive aflatoxin M1 is placed on an origin spot of a thin layer chromatographic plate and overspotted with trifluoroacetic acid. The mixture is held in the dark 30 min at ambient temperature and then 30 min at 55 degrees C. The plate is developed with CHCL3-acetone-2-propanol (85+10+7). The Rf values of reacted and unreacted aflatoxin M1 are compared with authentic M1 similarly treated for identification. The lowest concentration that has been identified is 0.1 mug/kg.


Beta-glycosidases and diabetic microangiopathy. II. An insulin-dependent isozyme of beta-N-acetylglucosaminidase. Previously we reported that beta-glycosidase activities were markedly decreased in the kidney but increased in the serum of diabetic rats. To examine these changes, the isozymes of beta-N-acetylglucosaminidase [EC 3.2.1.30] of rats were examined by DEAE-cellulose column chromatography. At least 3 major isozymes were found in both the kidney and liver. The main isozyme was type II isozyme in normal rat kidney and type III in normal rat liver. The activity of the type II isozyme in the kidney was markedly lowered when the total activity was decreased in diabetes and its normal activity was restored on insulin treatment, in parallel with increase in the total activity in diabetes. No significant change was found in the chromatographic pattern of isozymes in the liver in diabetes. In diabetic rat serum, the increase of total activity was found to be due to increase of type I and II isozymes.


Wound-induced phenylalanine ammonia-lyase in potato tuber tissue. Development of enzyme activity and effects of antibiotics. Phenylalanine ammonia-lyase [EC 4.3.1.5.] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25 degrees. The activity reached a maxinum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.


Biosynthesis of sulfated glycoprotein in the endometrium of rabbit uterus. The endometrial scrapings obtained from the uteri of estrogen-treated rabbits were incubated with N-acetyl-d[1-3H]glucosamine and [35S]sulfate, and then the incubation medium (M-Fr) was separated from the tissue. The tissue was subsequently homogenized exhaustively in 0.25m sucrose, and the insoluble residue (R-Fr) was separate. The supernatant at 8,500Xg for 10 min of the homogenate was subjected to subcelular fractionation by discontinuous sucrose gradient ultracentrifugation, and a thiamine pyrophosphatase-rich fraction (g-fr) was obtained. Complex carbohydrates were then separated from M-Fr, R-Fr, and G-Fr. The radioactivities incorporated into these complex carbohydrates suggested that sulfated glycoprotein synthesized in G-Fr was secreted into M-Fr. In order to confirm the above observation, labelled sulfated glycoprotein was isolated from the incubation medium. Subsequently, N-ACETYL-D[1-3H]glucosamine was incorporated into N-acetylglucosamine residues and [35S]sulfate into sulfates located most probably at the 6-position of N-acetylglucosamine residues of sulfated glycoprotein.


The role of C1 esterase inhibitor in the activation of C1r, a subcomponent of the first component of complement from human plasma. Clr was isolated from human serum by DEAE-cellulose column chromatography in the presence of EDTA. The isolated Clr did not hydrolyze N(alpha)-acetyl-L-arginine methyl ester, unless activated by brief treatment with trypsin [EC 3.4.21.4]. On thecolumn, the C1 esterase inhibitor activity was found to coincide with Clr but not C1s (another subcomponent of the first component)  C1r was isolated from the euglobulin fraction of human serum by DEAE-cellulose column chromatograph. On Sephadex G-200 column chromatography, Clr was eluted in the void volume, whereas Clr was eluted in a position corresponding to a molecular weight of 140,000-160,000. The results indicated that, on activation, Clr was converted to an enzyme of lower molecular weight...


Isolation and characterization of calcitonin from pericardium and esophagus of eel. Calcitonin was extracted from the pericardium and esophagus of eel in quantities sufficient to permit purification and chemical characterization. Homogeneous calcitonin could be isolated by a six-step fractionation starting from acetone powder of the organs. The fractionation procedure consisted of acid extraction, gel filtration on Sephadex G-75, chromatography on SP-Sephadex C-25, gel filtration on the Sephadex G-50, chromatography on carboxymethylcellulose, and gel filtration on Sephadex G-50. Fractionation of the hormone was monitored by assay of its biological activity and from its behaviour on thin layer chromatography and polyacrylamide gel disc electrophoresis. The hormone contained 32 amino acid residues, like calcitonins from other species of animals, but its amino acid composition was different from those of previously characterized hormones. Eel calcitonin possessed almost the same, or higher, biological activity as the salmon or chicken hormone, which show the highest specific activity among calcitonins so far isolated.


Binding of retinol-binding protein obtained from human urine with vitamin A derivatives and terpenoids. Retinol-binding protein(RBP) was purified from fresh urine of patients suffering from Itai-Itai disease. The purified preparation contained two types of apo-RBP(Apo I and II) in equal amounts as major components (about 85% of the total RBP). The corresponding two retinol-binding forms (Holo I and II) were present as minor components (about 15% of the total)...


Amino acid sequence of an active fragment of potato proteinase inhibitor IIa. The complete amino acid sequence of an active fragment of potato proteinase inhibitor IIa has been established by the Edman degradation procedure and the carboxypeptidase technique. Sequence analyses were carried out on the reduced and carboxymethylated active fragment and its tryptic peptides. To aid in the alignment of some tryptic peptides, the partial sequences of two fragments obtained by selective tryptic cleavage of the reactive site peptide bond of inhibitor IIa at acidic pH, with subsequent reduction and carboxymethylation, were also analyzed. The active fragment consisted of 45 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by subtilisin [EC 3.4.21.14.] at pH 6.5. yielded 3 cystine-containing peptides. Sequence analyses of these peptides revealed that the 3 disulfide linkages were located between Cys(10) and Cys(24), Cys(14) and Cys(35), and Cys(20) and Cys(43). The reactive site peptide bond of inhibitor IIa, a Lys-Ser bond, was located between positions 32 and 33 of the active fragment. The overall sequence of the active fragment was quite different from those of potato chymotrypsin inhibitor I (subunit A) and potato carboxypeptidase inhibitor.


Phospholipid metabolism in Ehrlich ascites tumor cells. I. Turnover rate of phosphatidylinositol. 1. Radioactive precursors, 32 PI, [1-14C]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fraction of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate of [1-14C]glycerol, a higher rate of incorporation of 32Pi into phosphatidylinositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour. 2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection 32Pi and [2-3H]inositol. The incorporation of 32Pi into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32Pi) of phosphatidylinositol only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr. 3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.


Changes in ornithine decarboxylase activity in normal tissues of tumor-bearing mice during tumor growth. The ornithine decarboxylase [EC 4.1.1.17] activities in the liver and spleen of tumor-bearing mice increased remarkably, reaching a peak 4 to 6 days after inoculation of tumor cells. On the contrary, the enzyme activity in the kidney decreased during tumor growth and had almost disappeared on day 6 after tumor inoculation. Injection of cell-free tumor homogenate also raised the enzyme activities in the liver and spleen, but did not change the activity in the kidney. No increase in enzyme activity in the liver of mice was observed on injection of homogenates of normal tissues, such as liver, spleen, kidney, and muscle.


Subunit structure of AMP-deaminase from chicken and rabbit skeletal muscle. The AMP-deaminases from chicken and rabbit muscle have been investigated by techniques which include sedimentation equilibrium, sodium dodecyl sulfate gel electrophoresis, amino acid analysis, NH2- and COOH-terminal analyses, and tryptic peptide mapping. The molecular weights of the native chicken (276,000) and rabbit (271,000) enzymes obtained by sedimentation equilibrium studies are in good agreement with values of 276,000 (chicken) and 275,000 (rabbit) calculated from amino acid analyses. The enzymes were reduced, carboxymethylated, and treated with either maleic or succinic anhydride in the presence of 6 M guanidine hydrochloride. Sodium dodecyl sulfate gel electrophoresis of the chemically modified enzymes resulted in a single electrophoretic species having an apparent molecular weight of 85,000. This observation is consistent with previous studies on the nonacylated enzymes and suggests that the muscle AMP-deaminases from chicken and rabbit do not contain noncovalent linkages which are readily disrupted by a large increase in negative charge. NH2-terminal analyses by the method of Stark and Amyth as well as the dansyl technique, indicate that the NH2-terminal positions of these enzymes are blocked. The enzymes are also resistant to digestion with carboxypeptidases A or B (or both) in the presence of sodium dodecyl sulfate. The most distinctive feature of the amino acid compositions of both the chicken and rabbit AMP-deaminases is the presende of eight half-cystine residues per 69,000 g of protein. Tryptic digests of the S-14C-carboxymethylated proteins were fractionated by ion exchange chromatography and high voltage electrophoresis. Six and five radioactiviely labeled peptides were detected in the electrophoretograms of the chicken and rabbit enzymes, respectively. This observation and the number of ninhydrinposition spots, together with the physical data on the molecular weights of the native enzymes and their subunits, suggest that the AMP-deaminases from chidken and rabbit muscle consist of four identical or very similar polypeptide chains.


Leucine 2,3-aminomutase, an enzyme of leucine catabolism. The initial step in the fermentation of leucine to acetate, isobutyrate, and ammonia by Clostridium sporogenes is the B12 coenzyme-dependent conversion of alpha-leucine to beta-leucine (3-amino-4-methylpentanoate). The amino group migration reaction, catalyzed by leucine 2,3-aminomutase, is reversible and is inhibited by intrinsic factor. The enzyme activity has been found in several clostridia, in rat, sheep, rhesus, and African green monkey livers, and in human leukocytes.


Primary structure of streptococcal proteinase. I Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragments 1 to 4. Tryptic and chymotroptic peptides were isolated and characterized from cyanogen bromide fragments 1 to 4 of streptococcal proteinase and subjected to sequence analysis by the Edman degradation, carboxy-peptidase digestion, and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. The results, together with the sequence data of the cyanogen bromide fragment 5 reported in the accompanying papers, provide the structural formula of streptococcal proteinase.


Primary structure of streptococcal proteinase. II. Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragment 5. The amino acid sequence of the cyanogen bromide fragment 5 of streptococcal proteinase has been determined. This fragment comprises residues 130 to 253 of the proteinase chain. Six tryptic peptides were isolated from maleylated cyanogen bromide fragment 5, and their alignment was obtained by the overlap of chymotryptic peptides. Sequence analysis of tryptic, chymotryptic, and thermolysin peptides was performed by the 5-deimethylaminoaphthalene-1-sulfonyl technique and carboxypeptidases digestion.


Primary structure of streptococcal proteinase. III. Isolation of cyanogen bromide peptides: complete covalent structure of the polypeptide chain. The following sequence has been derived for streptococcal proteinase. (See article). The sequence permits the assignment of the single cysteine residue essential for catalytic action at position 47 from the NH2 terminus of the protein. The tryptophan residue at the binding site of the enzyme is at position 214. A histidine residue at position 195 has been assigned as the catalytically important entity in the molecule. Streptococcal proteinase and papain, an enzyme with similar properties, are compared with respect to structure and function.


Bovine secretory component. Isolation, molecular size and shape,  composition, and NH2-terminal amino acid sequence. Bovine free secretory component was purified from whey by salt precipitation, gel filtration, DEAE-cellulose and phosphocellulose chromatography, and immunoadsorption. It was obtained in immunologically pure form and in 56% yield. The Stokes radius of pure free secretory component was found to be 4.3 nm by gel filtration, and an (see article) of 4.1 S was determined by the ultracentrifuge. The molecular weight was 79,000 by sodium dodecyl sulfate gel electrophoresis and by sedimentation dquilibrium in the ultracentrifuge, using a v of 0.73 determined by ultracentrifugation in D2O and H2O. A minimal axial ratio of approximately 5 was calculated. Amino acid analysis of bovine free secretory component showed remarkable similarity to that of human, dog, and rabbit but carbohydrate analysis showed significant differences. In contrast to the human, bovine free secretory compoennt has 2 methionine residues/mol. The NH2-terminal sequence was found to be Lys-Ser-Pro-Ile-PPHE-Gly-Pro-Glu-Glu-Val-Asp-Ser-Val. This sequence is identical with that the human and dog. However, the poor immunological cross-reactivity between the dog, human, and bovine proteins suggests that significant structural differences will be found in other regions of the molecule.


Purification and characterization of a prokaryotic glycoprotein from the cell envelope of Halobacterium salinarium. The glycoprotein which accounts for approximately 50% of the protein and all of the nonlipid carbohydrate of the cell envelope of Halobacterium salinarium (Mescher, M. F., Strominger, J. L., and Watson S. W. (1974) J. Bacteriol. 120, 945-954) has been purified and partially characterized. The glycoprotein has an apparent molecular weight of 200,000, is extremely acidic, and has a carbohydrate content of approximately 10 to 12%. The carbohydrate included neutral hexoses, amino sugar, and uronic acid. Information regarding the number, composition, and mode of attachment of the carbohydrate chains was obtained by isolation and examination of the glycopeptides derived from degradation of cell envelope protein with trypsin and pronase. Trypsin digestion resulted in two glycopeptides. One of these was large (approximately 55,000 daltons) and had most of the neutral hexose linked to it. The carbohydrate moieties consisted of di- and trisaccharides of glucosylgalactose and (uronic acid, glucose)-galactose attached via O-glycosidic linkages between galactose and threonine. The other tryptic glycopeptide had a relatively large heterosaccharide attached to it via an alkaline-stable linkage. The heterosaccharide contained 1 glucose, 8 to 9 galactose, 1 mannose, and 10 to 11 glucosamine residues, and approximately 6 residues of an unidentified amino augar. The alkaline stability of the linkage and the amino acid composition of glycopeptides resulting from Pronase digestion of the tryptic glycopeptide showed that the heterosaccharide was attached to an asparagine residue, presumably via an N-glycosylamine bond to the amide group. The intact glycoprotein has a single N-linked heterosaccharide, 22 to 24 O-linked disaccharides, and 12 to 14 O-linked trisaccharides per molecule. N- and O-glycosidic linkages are the most common carbohydrate-protein linkages in mammalian glycoproteins but, to our knowledge, this is the first report of either type of linkage in a prokaryotic cell envelope protein.


Analysis of ribonuclease-nucleotide interactions by quantitative affinity chromatography. Quantitative affinity chromatography on uridine-5'-(Sepharose-4-aminophenylphosphoryl)-2'(3')-phosphate was developed for the study of binding of ribonuclease species to nucleotide ligands. Elution of the native species ribonuclease-A and -S on the afffinity matrix in 0.4 M ammonium acetate, pH 5.2, containing various amounts of the soluble competing ligand 2'-cytidine monophosphate, reveals an inverse response of elution volume to concentration of soluble ligand. This response conforms to behavior expected for the competing binding equilibria enzyme-soluble ligand and enzyme-insoluble ligand. A-NALYSIS OF ELUTION DATA ALLOWS CALCULATION OF KI and KIM, the dissociation constants, respectively, for the soluble and insoluble protein-ligand complexes. The values of these chromatographically derived constants are similar to values of dissocation constants determined in solution by kinetics of inhibition by 2'-cytidine monophosphate and uridine-5'-(j-aminophenylphosphoryl)-2'(3')-phosphate. Successful competitive elution experiments with [p-F-Phe8]semisynthetic ribonuclease-S' and individual elution trials for [4-F-His12]semisynthetic ribonuclease-S' indicate the utility of the quantitative affinity chromatographic technique for determination of ligand binding properties of ribonuclease derivatives, including inactive species. Nonbiospecific aspects of the interaction of ribonuclease with the affinity matrix in ammonium acetate buffers of concentrations 0.1 M and below were noted, delinating limits of conditions allowing the biospecificity needed for ligand-binding analyses by competitive elution. The dependence of ribonuclease competitive elution behavior on the amount of protein eluted also was examined and related to theoretical considerations in the quantitative application of affinity chromatography.


Accumulation of analgo of phosphocreatine in muscle of chicks fed 1-carboxymethyl-2-iminoimidazolidine (cyclocreatine). Newly hatched chicks fed a commercial diet containing 1% cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) rapidly accumulate in breast muscle a derivative of cyclocreatine; within a few days concentrations up to 35 mumol of this derivative/g fresh weight of muscle are attained. The following evidence suggests that this derivative is N-phosphorylated cyclocreatine. (a) The derivative is adsorbed by Dowex 1 resin and eluted near phosphocreatine. (b) The derivative present in muscle is converted to free cyclocreatine either by homogenization of muscle in water at room temperature, conditions under which endogenous creatine kinase is active, or by heating a cold acidic muscle extract at 65 degrees for 45 min. (c) The isolated derivative reacts in vitro with the specific reagents crystalline creatine kinase and MgADP to give cyclocreatine. Essentially all of breast muscle cyclocreatine appears to be in the form of P-cyclocreatine, which persists in muscle long after cyclocreatine is removed from the diet. Long term conservation of P-cyclocreatine in muscle is aided by the fact that, unlike P-creatine, P-cyclocreatine is not continuously degraded to an inactive cyclic lactam. It is suggested that the maximal concentrations of P-cyclocreatine2- (and P-creatine2-) attained in sarcoplasm not only affect the phosphorylation potential of muscle cells, but also can account for more than half of the normal inorganic cation concentration of muscle sarcoplasm, and hence play an important role in muscle function. Other chick tissues active in accumulation of cyclocreatine are heart (up to 20 mumol/g fresh weight within 11 days on the diet) and brain (up to 10 mumol/g fresh weight after 30 days on the diet). Addition of 1% creatine to the diet of cyclocreatine-fed chicks does not prevent accumulation of cyclocreatine in muscle. Chicks fed cyclocreatine do not grow as rapidly as those on control diets, but they appear healthy, and mortality is very low when oxytetracycline is added to the drinking water. Cyclocreatine is also taken up by rat muscle, heart, and brain. A sensitive and specific assay for cyclocreatine has been developed. Cyclocreatine reacts with an aged aqueous solution of Na3[Fe(CN)8NH3] under alkaline conditions to give a blue product with a molar absorption coefficient (epsilon) of 4,400 M(-1) cm(¿ at 605 nm. The following compounds give an epsilon605  in this assay of less than 4 M%¿ cm(% N-phosphorylcyclocreatine, creatine, P-creatine, creatinine, 1-carboxymethyl-2-iminohexahydropyrimidine, guanidinoacetate, 3-guanidinopropionate, and arginine. Cyclocreatine does not interfere with the diacetyl-alpha-naphthol assay for creatine.


Fragments formed by the side chain cleavage of a 20-aryl analog of 20alpha-hydroxycholesterol by adrenal mitochondria. An analog of 20alpha-hydroxycholesterol, (20R)-20-phenyl-5-pregnene-3beta,20-diol, which is completely substituted at C-22 was prepared with radioisotopes at various positions. The analog labeled with 3H at C-M and 14C at C-4 and C-IU was converted into radioactive pregnenolone by an enzyme preparation derived from adrenal mitochondria. Cleavage of the phenyl analog labeled with 3H in the aromatic ring by the same enzyme preparation led to the formation of [3H]phenol. Using the substrate doubly labeled with 14C at C-4 and 3H in the aromatic ring, it appeared that the products of the reactions, pregnenolone and phenol, were formed in equal amounts. During incubation of the side chain labeled substrate, another labeled fragment was formed. It was identified as acetophenone, a product resulting from cleavage of the C17,20 bond. The steroidal fragment corresponding to this C8 ketone was traced using nuclear label analog. From its nonpolar chromatographic properties it appears to be a C-17-deoxy-C19 steroid.


Transmembrane asymmetry of vesicle lipids. The role of fatty acyl chain unsaturation in promoting asymmetry in phospholipid vesicle bilayers was investigated in mixed lipid systems with differing acyl chains and a constant phosphatidylcholine headgroup. Ratios of outside to inside components were determined by nuclear magnetic resonance spectroscopy of 13C-enriched egg phosphatidylcholine. An asymmetry or disproportionation ratio is defined and used to express quantitatively how a mixture of two lipids distributes in the outer and inner vesicle surfaces. In mixed systems with 13C-enriched egg phosphatidylcholine as one component, increasing fatty acyl unsaturation in the other component results in an increasing preference of the unsaturated chains for the outer surface.


Self-association of cytochrome b5 in aqueous solution. Gel filtration and ultracentirfugational studies. The aggregation state of detergent-extracted cytochrome b5 was examined by gel filtration and sedimentation equilibrium ultracentrifugation. Both techniques indicated the presence of a mixture of monomer and octomer. The proportion of monomer was decreased as the buffer salt concentration was raised and was approximately 1 muM in 10 mM Tris acetate/0.1 mM EDTA. The monomeric cytochrome b5 was isolated by gel filtration and showed a decreased tendency to aggregate in dilute buffer, although its other physical properties were identical to those of the original cytochrome b5. The monomer was found to have a Stokes radius of approximately 26 A, as determined by classical gel filtration and by an equilibrium saturation method where the sample was monitored in the gel by measuring the absorbance of the gel column directly in a dual wavelength spectrophotometer. The gel filtration and ultracentrifugation data together with the studied on the monomeric cytochrome b5 suggest the octomer is not a rapid equilibrium with monomer and this observation should be taken into account in lipid binding experiments with cytochrome b5.


Purification and physical characterization of nucleic acid helix-unwinding proteins from calf thymus. We have devised a general protein fractionation procedure which selects for eukaryotic DNA-binding proteins, some of which resemble DNA-unwinding proteins from prokaryotes. Proteins were selected which (a) pass through a native DNA-cellulose column, (b) bind to a denatured DNA-cellulose column, and (c) remain bound to the latter column during a rinse with a dilute solution of the sodium salt of the polyanion dextran sulfate. When this fractionation was applied to the soluble proteins fo calf thymus, three major protein species were recovered. The predominant one has an apparent molecular weight of about 24,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is isoelectric near neutrality, and elutes as a monomer from denatured DNA-cellulose at moderate NaCl concentrations. This protein, designated calf-unwinding protein 1 (UP1), has been purified to homogeneity. However, isoelectric focusing reveals four or five subspecies (apparently separated by single-charge differences) which differ appreciably in their affinities for DNA. Two other major proteins are obtained which have apparent molecular weights in sodium dodecyl sulfate of 33,000: the first, which elutes with low salt from DNA-cellulose as a homogeneous preparation, appears to be a basic protein (although it is clearly not a histone); the other, which elutes from DNA-cellulose as the major component of a "high salt eluting fraction," is an acidic protein which co-purifies with less prominent species of higher molecular weights. Proteins similar to each of these three major calf thymus proteins have been observed by us and others in tissue culture cells of mouse, hamster, monkey, and humans, suggesting their wide occurrence among eukaryotes.


Nucleic acid helix-coil transitions mediated by helix-unwinding proteins from calf thymus. We have studied nucleic acid double helix destabilization mediated by purified calf helix-unwinding proteins, measuring ultraviolet hyperchromicity to detect helix melting. Both calf unwinding protein 1 (UP1) and a high salt eluting protein fraction are found to depress strongly the helix melting temperature (Tm) of the synthetic alternating copolymers poly[d(AT)] and poly[r(AU)], indicating that both DNA and RNA are recognized by these proteins. UP1 also destabilizes natural, GC-containing DNA helices, but to a smaller extent than observed with the above polymers. A simple model is presented to aid in the qualitative interpretation of the data, outlining the expected effect on the helix-coil transition of a protein ligand with differential affinity for the helix or coil form of nucleic acid. The observed helix-destabilizing effect of UP1 is dependent on the protein to nucleic acid ratio in an expected manner. Competition studies demonstrate a low, but appreciable affinity of UP1 for native DNA, opening the possibility that protein-mediated denaturation might be initiated by protein binding to the double helix. "Hairpin" helical regions of denatured DNA are strongly destabilized by UP1. Despite the fact that removal of these hairpin helices might greatly facilitate DNA renaturation, we failed to observe renaturation from the UP1-DNA complex after a switch to helix-stabilizing conditions. Thus, UP1 shows an important difference from its presumed prokaryotic analogue, T4 gene 32-protein. Possible in vivo functions of the calf proteins are discussed in light of these observations.


Single-stranded DNA structure and DNA polymerase activity in the presence of nucleic acid helix-unwinding proteins from calf thymus. In the preceding articles we have described the isolation and some of the properties of two calf thymus proteins which bind selectively to single-stranded DNA and which appear analogous to previously isolated prokaryotic DNA-unwinding proteins. In the present work we demonstrate two further points of analogy. First, both the calf UP1 and the high salt eluting proteins form protein-rich complexes with single-stranded DNA, and hold this DNA in a rigid, extended conformation. Second, these proteins stimulate the calf thymus DNA polymerase-alpha; phage T4 gene 32-protein does not. The stimulation of a homologous DNA polymerase is characteristic of several prokaryotic DNA-unwinding proteins and is assumed to reflect their in vivo role in DNA synthesis.


Phosphorylation of axonemal proteins in Chlamydomonas reinhardtii. In order to determine whether microtubular proteins of flagellar axonemes were phosphorylated, cells of Chlamydomonas reinhardtii were grown in medium containing [32P]orthophosphate for several generations. Only one (alpha subunit) of the two tubulin polypeptides separated by Na dodecyl-SO4-polyacrylamide gel electrophoresis appeared labeled, as detected by autoradiography of the dried gel. 3H- and 32P-labeled alpha tubulin subunit purified by preparative Na dodecyl-SO4-polyacrylamide gel electrophoresis and Na dodecyl-SO4-hydroxyapatite chromatography contained about 0.2 mol of phosphate per mol of polypeptide. Upon partial acid hydrolysis, radioactivity could be accounted for as serine and threonine phosphate. By altering the conditions of the Na dodecyl-SO4-polyacrylamide gel electrophoresis is was possible to resolve the purified alpha-tubulin subunit into five or more components: a major band comprising approximately 65% of the total mass, not phosphorylated, and four or more minor bands comprising together 35% of the mass. Among the minor components at least two were phosphorylated.


Exchange and stability of HeLa ribosomal proteins in vivo. The relative stabilities of individual HeLa ribosomal proteins and their capacity for exchange between ribosome-bound and -free states in the cytoplasm were examined. Most ribosomal proteins on cytoplasmic ribosomes were found to have uniform, high stability as measured by comparing the short term (12-hour) to steady state (3-day) labeling ratios determined for each ribosomal protein. This would be expected if the proteins in ribosomes either were all stable or were all degraded as a unit. The data do not rule out the possibility that individual proteins have different stabilities prior to their assembly into ribosomes. Four proteins labeled atypically. One large subunit protein (L5) had a lower than average ratio. We interpret this low ratio as being due to a large free pool of this protein. Three proteins (L10, L28, S2) had higher than average ratios, interpreted as being due to reduced protein stability. Two of these proteins (L10, L28) with high ratios were also found to exchange in vivo. The exchangeable proteins may be subject to increased degradation during the time that they spend in the exchangeable free pool. The third protein (S2) with an atypically high ratio is thought to be degraded or altered while on the ribosome, or slowly lost as ribosomes age, because exchange of this protein was not detected. These interpretations and some alternate interpretations are explained. The exchange of three large subunit proteins (L10, L19, L28) was detected by labeling of protein after ribosome synthesis had been inhibited with actinomycin D. Autoradiography of two-dimensional polyacrylamide gels showed labeling of these spots.


Relative stoichiometry in ribosomal proteins in HeLa cell nucleoli. Total protein was released from isolated HeLa cell nucleoli by guanidine hydrochloride, purified by cesium chloride density gradient centrifugation, and analyzed by two-dimensional polyacrylamide gel electrophoresis. Conditions of electrophoresis restricted attention to proteins that are positively charged at pH 8.6. Most of the major nucleolar protein spots co-electrophoresed with ribosomal proteins; the majority of ribosomal proteins from both the large and small ribosomal subunits were represented. Several proteins found in association with polysomes but not on ribosomal subunits and several proteins unique to the nucleolus were also identified in these nucleolar protein patterns. In order to determine whether the ribosomal proteins found in the nucleolus represented sizable pools of ribosomal proteins, or merely ribosomal proteins contained in the preribosomal particles, [35S]methionine-labeled nucleoli were mixed with [3H]methionine-labeled polysomes. From analysis of isotopic ratios in individual protein spots it was possible to determine the stoidchiometry of individual ribosomal proteins in the nucleolus relative to their complement on cytoplasmic ribosomes. All but a few proteins exhibited relative nucleolar stoichiometry values of approximately one, indicating that there are not significant pools of most ribosomal proteins in isolated nucleoli.


Role of changes in protein degradation in the growth of regenerating livers. The significance of changes in rates of synthesis, export, and degradation of proteins during liver regeneration was assessed. (a) Proteins were pulse labeled by the intravenous injection of radioactive leucine and, 5 min later, pactamycin (an inhibitor of the initiation of protein synthesis). One-half of the protein radioactivity was lost from the normal liver within 3 hours. From the radioactivity of the plasma proteins at that time and a study of the disappearance of these proteins from the circulation, it was calculated that 28% of the newly synthesized proteins were exported. Serum albumin accounted for a third of the exported proteins. Thirty-six hours after partial hepatectomy the proportion of albumin to total protein synthesis remained constant, while that of the other plasma proteins increased by 50%. The fraction of the newly synthesized proteins retained by the liver after 3 hours decreased by 20%. (b) During the first 36 hours of liver regeneration the average rates of protein degradation slowed down to one-half the normal values. This was determined either by the loss of radioactivity from total protein (or the guanidino-C of protein-bound arginine) in livers labeled with [14C]bicarbonate, or calculated as the balance between protein synthesis and net protein gain. (c) From these results, and those of our previous study of the protein synthetic machinery of normal and regenerating livers (Scornik, O.A. (1974)J. Biol. Chem. 249, 3876-3883), we conclude that changes in the rate of protein degradation are the single most important factor determining the increase in protein content during liver compensatory growth.


Interaction of anions and divalent metal ions with phosphoenolpyruvate carboxykinase. The catalytic activity of phosphoenolpyruvate carboxykinase in rat liver cytosol is stimulated by incubating with Fe2+, Mn2+, Co2+, and Cd2+. When purified, the enzyme no longer responds to Fe2+, Co2+, or Cd2+ but retains a response to Mn2+. Low concentrations of SO4(2-) in the incubation medium with enzyme and divalent transition metal allow stimulation by Fe2+ and Co2+ and enhance the response to Mn2+. Under identical conditions, orthophosphate with Fe2+ is a potent inhibitor of the enzyme (half-maximal inhibition at 50 muM). A thiol is required in the incubation medium for the effects of Fe2+ plus sulfate or orthophosphate to be expressed. The magnitude of these effects depends on the thiol concentration. Dithiothreitol is more effective than GSH and activation by sulfate plus Fe2+ appears to require the reduced form of dithiothreitol. Sulfate ion is not considered to be the physiological Fe2+-activator of P-enolpyruvate carboxykinase in rat liver cytosol, as this function is fulfilled by a newly discovered liver protein. Knowledge concerning the interaction of Fe2+ and sulfate with the enzyme may be useful in examining their interaction between the enzyme, ferrous ion, and this activator protein.


A protein factor required for activation of phosphoenolpyruvate carboxykinase by ferrous ions. When rat liver cytosolic P-enolpyruvate carboxykinase is purified, its activity is no longer enhanced by incubation with 30 muM Fe2+. Ferrous ion stimulation of the purified enzyme is restored by the addition of rat liver cytosol. The agent responsible is a cytosolic protein, named P-enolpyruvate carboxykinase ferroactivator, that was readily separated from the enzyme during purification of the latter. A quantitative assay for P-enolpyruvate carboxykinase ferroactivator is described. Subcellular fractionation of livers from fasted rats shows that 98% of the combined mitochondrial and cytosolic P-enolpyruvate carboxykinase ferroactivator activity resides in the cytosol. Fasting does not produce significant change in this cytosolic activity when compared to that of fed animals. Examination of various tissue homogenates shows that the ferroactivator is found in liver, kidney, erythrocytes, adipose tissue, and brain. No activity was detected in blood serum or skeletal muscle. The ability to enhance the activity of purified rat liver cytosolic P-enolpyruvate carboxykinase in the presence of Fe2+ is not species specific. P-enolpyruvate carboxykinase ferroactivator may have an important function in regulating enzyme activity in vivo.


Lipophosphonoglycan of the plasma membrane of A canthamoeba castellanii. Inositol and phytosphingosine content and general structural features. Lipophosphonoglycan, a major component of the plasma membrane of Acanthamoeba castellanii, has now been shown to contain 8% inositol and 13% C25- and C24-phytosphingosines in addition to the previously identified content of neutral sugars (26%), amino sugars (3%), aminophosphonates (10%), acidhydrolyzable phosphate (3%), and long chain fatty acids (14%). The fatty acids and phytosphingosines are in ceramide groups. Lipophosphonoglycan can be separated by dodecyl sulfate-polyacrylamide electrophoresis into two major components that are similar in composition except for different oligosaccharide groups. A tentative structural model incorporating these features is proposed in which each of the two components of lipophosphonoglycan is conceived as an oligomeric inositol-containing glycosphingolipid.


Induction of amino acid transport in primary cultures of adult rat liver parenchymal cells by insulin. Amino acid transport was studied in primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion technique and maintained as a monolayer in a serum-free culture medium. Amino acid transport was assayed by measuring the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid. Rat liver parenchymal cells transported alpha-aminoisobutyric acid by an energy-dependent Na+-requiring system which displayed Michaelis-Menten kinetics. Addition of insulin to cultured rat liver parenchymal cells resulted in an increased influx of alpha-aminoisobutyric acid which was reflected in a higher initial rate of alpha-aminoisobutyric acid transport as well as an increased accumulation of alpha-aminoisobutyric acid at later time points. Cycloheximide effectively blocked the increase while results with actinomycin D were equivocal. Insulin at concentrations as low as 50 pM was effective in stimulating alpha-aminoisobutyric acid transport while the maximal response was observed at 80 nM.


A new vitamin K-dependent protein. A phospholipid-binding zymogen of a serine esterase. Conclusive evidence is presented that a recently purified (Stenflo, J. (1976) J. Biol. Chem. 251, 355-363) vitamin K-dependent protein (arbitrarily referred to as Protein C) which is not related to prothrombin, Factors IX or X is also unrelated to Factor VII. It therefore appears to be a new, previously unrecognized vitamin K-dependent protein. In contrast to prothrombin, which binds to negatively charged phospholipid only in the presence of Ca2+ ions, Protein C, like the other vitamin K-dependent proteins, is a precursor of a serine esterase, presumably a protease, but it does not seem to be necessary for blood coagulation. Although the lipid-binding properties of Protein C may suggest that it is associated with membrane structures, its biological function remains unknown.


In vitro translation and estradiol-17beta induction of Xenopus laevis vitellogenin messenger RNA. Administration of estradiol-17beta to male Xenopus laevis induces synthesis and secretion by the liver of the egg yolk precursor protein vitellogenin. RNA extracted from livers of estradiol-17beta-treated Xenopus laevis directs the synthesis of the entire 200,000-dalton vitellogenin monomer in a cell-free protein synthesizing system derived from rabbit reticulocytes. Vitellogenin synthesized in vitro was isolated and quantitated by indirect immunoprecipitation and identified by comparison to authentic [14C]vitellogenin. The in vitro product and [14C]vitellogenin co-migrate on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and they exhibit identical immunoprecipitation curves. Xenopus laevis vitellogenin messenger RNA has a sedimentation coefficient of approximately 30 S in sucrose density gradients. It can be purified approximately 60-fold from cell RNA by poly(U)-Sepharose chromatography and therefore appears to contain a polyadenylate sequence. Vitellogenin mRNA and vitellogenin synthesis in vivo could not be detected in unstimulated male Xenopus laevis. The relative rate of vitellogenin synthesis and the level of vitellogenin mRNA were determined at various times following the administration of estradiol-17beta. Vitellogenin synthesis is maximal 12 days after estradiol-17beta administration when it comprises approximately 70% of cell protein synthesis. The level of vitellogenin mRNA and the intracellular rate of vitellogenin synthesis exhibit a close correspondence from 4 to 16 days after administration of estradiol-17beta.