[Characterization of radioactivity distribution in the organism during constant intravenous infusion of tracer amino acids and calculation of the rate of tissue protein synthesis in rats]. Male wistar rats (100 g live mass) were given infusions into the tail vein of 14C-leucine and 14C-lysine simultaneously for 0.5; 1.0; 2.0; 3.0; 4.5; 6.0 and 7.0 hours. At the end of the infusion the specific radioactivity of the free leucine and lysine in the blood plasma, liver, M. gastrocnemius, small intestines and colon were ascertained as well as after the 6-and-7-hour infusion that of the protein-bound leucine and lysine. In all tested tissues the specific radioactivity of the free amino acids attained a plateau during the 6-and-7-hour infusion. The rate constants for the increase were calculated for each organ tested. The two amino acids used are suitable for the calculation of the fractional rate of protein synthesis in tissues. The values of the fractional rate of protein synthesis calculated on the basis of the 6-and-7-hour infusion were: 54 +/- 7.7%/day for the liver, 9.4 +/- 1.2%/day for muscles, 89 +/- 12.2%/day for the small intestines and 42 +/- 5.9%/day for the colon. The simultaneous application of two tracer amino acids is recommendable for the estimation of the precursor pool for protein synthesis and the more accurate calculation of the rate of protein synthesis.


[Assessment of the proteins in crill meal and lucerne meal extract]. In a feed test with rats lucerne meal extract and crill meal were used as protein sources in order to test their quality in comparison to casein. On the basis of the applied criteria, feed conversion, protein efficiency ratio productive protein value and efficiency of the gross energy we can conclude that lucerne meal extract alone is unsuitable to cover the protein requirement of growing rats. Lucerne meal extract can be supplied particularly on a higher protein level if casein is given in addition. Crill meal as protein source proves to be superior to casein concerning feed conversion and inferior concerning protein accumulation. In this case, complementary doses of casein did not lead to better results.


[Glucose content of lambs' blood. 1. Effect of administration of various carbohydrates]. After the oral administration of lactose or starch the blood sugar content was proofed as an indicator for the carbohydrate digestion and resorption of lambs at the age of 7 to 35 days. With increasing age the blood sugar level during fasting decreased, on an average of all lambs, from 65 to 50 mg glucose/100 ml blood. The administration of lactose resulted in an increase of the blood glucose content on all days. In contrast to this, the administration of starch only resulted in an increase of the blood glucose content of lambs that were at least three weeks old.


[Glucose content of lambs' blood. 2. Blood glucose content during several hours after feeding]. During a 16-hour fasting period, the development of the blood glucose level of ewelessly reared lambs of 4 to 30 days of age was analysed. While the blood glucose level of lambs during their first week of life reacted with a considerable increase and a relatively quick decrease on the administration of milk substitutes, the course of this curve became smoother with increasing age. After a five-hour fasting period, the blood glucose level--after 4 days of the lambs' life--amounted to 101 mg, after 9 days to 96 mg, after 16 days to 91 mg, after 23 days to 64 mg, after 30 days to 71 mg per 100 ml blood. The significance of these results for the establishment of a suitable drinking technology is being discussed.


[2,6-Diaminopimelic acid (DPA) content and the DPA:N ratio in rumen bacteria in relation to time after feeding]. The interrelation between the time of the withdrawal of rumen fluid and the DPA: N-relation in the fraction of mixed rumen bacteria was ascertained in a test with two cows with rumen fistulae. At the moment of feeding the DPA-content of 63 mg per g bacteria was very high. In the period of 3 to 9 hours after feeding it remained relatively constant (50 mg). A mean conversion relation of 20.4 of DPA to bacteria N was ascertained. The amino acid model proved to be the same in all fractions, the content values were comparable to those found in technical literature.


[Interaction among the trace elements zinc, copper and iron after depletion and repletion of dairy cows with zinc]. Imbalances in the supply with trace elements may be caused by the excessive administration of one or several elements or the insufficient administration in relation to other trace elements. This article deals with the interactions between the trace elements zinc and copper resp. zinc and iron under the conditions of the insufficient supply with Zn (6 mg per kg dry matter of the fodder) and the supply according to the demand with other trace elements (14 mg copper resp. 83 mg iron per dry matter of the fodder). For this purpose we investigated the copper, iron and zinc content of the milk and the serum of cows that were first depleted of zinc through a semi-synthetic zinc deficiency diet and then repleted with extra allowances of zinc. The closest connections exist between the copper and zinc content of the milk. Thus extreme Zn-deficiency feeding conditions the decreased Zn-content on the one hand and increased Cu-content on the other. In contrast to this, the cows' Zn-excretion in the milk increases after Zn-repletion whereas the Cu-content decreases. This shows a distinctly negative correlation. A loose connection could only be detected for the Cu- and Zn-content of the serum. Though the Zn-content changed considerably in dependence on the Zn-supply, the Cu-content remained largely uninfluenced. The Fe-content of both milk and serum shows no interaction with the nutritive Zn-supply. Only after 19 test weeks of extreme Zn-deficiency could a slight increase of the Fe-concentration be indicated.


[Effect of fasting on metabolism of free amino acids of various broiler tissues]. 16 male broiler chicken fed with commercial-grade broiler fattening feed were killed in groups of four in intervals of 3, 6, 12 and 25 hours after the last food intake. The content of free amino acids was quantitatively determined in the small intestines, the liver and the complete muscular system. The highest concentration of free amino acids per gram of tissue could be detected in the small intestines between 3 and 6 hours and--phase shifted--in the liver between 6 and 12 hours after the last food intake. The changes of the concentration in the muscular system between the four measuring points were in most cases not statistically significant. The content of free amino acids in the three proofed tissues of fattening hybrids with a high demand of amino acids and a high protein synthesis performance was considerably above the values for rats as they are given in technical literature. The rhythmic changes of the concentration of free amino acids in the tissues in dependence on the interval between the last food intake and the killing require standardized conditions for metabolism-kinetic investigations of which the calculation of the content of free amino acids is part.


[New method of checking the quality of food proteins required for maintenance. 2. 15N excretion in feces of test rats labelled with 15N after feeding with different protein sources]. For 7 days 37 test rats received a casein diet with an extra of 6.6 mg 15N-excess in the form of ammonium acetate. From the eighth test day onwards 4 resp. 5 rats each received various protein sources under maintenance conditions (115 kcal/kg body mass0,75). The atom-% 15N-excess was determined in feces, blood liver and muscles (urine cf. 1st information). The endogenous quota of N in the feces was calculated as follows: (formula: see text). The numerical value of the TCA-soluble fraction of N in the total blood was corrected by the decrease of the atom-%15N' in the last 12 hours (time for the passage of the fecel matter from small intestines to excretion). Since the endogenously excreted N-amount varied greatly according to different feed, a scale is proposed as biologic value of food proteins, which exclusively refers to the metabolic fecal nitrogen (MFN) under conditions of maintenance (abbr. MFN-BV). A proposal for its definition is: (formula: see text). Above that, a total BV is suggested which also refers to maintenance metabolism. The total BV is calculated as follows: (formula: see text). The following values were ascertained for MFN-BV and total BV: casein = 80 and 82; complete egg=68 and 67; fish meal=61 and 86; Torula yeast=31 and 46; peas=41 and 43; soya (assay protein)=73 and 61; wheat=47 and 71; gelatin=64 and 42. Finally, the recommendation is given to include in feed tables real digestibility values for food proteins ascertained with the 15N method. In the above mentioned order the following values of the real digestibility of proteins were ascertained with the 15N method and classical methods: casein=98.2 and 97.2; complete egg=100.0 and 98.7; fish meal=96.9 and 93.4; Torula yeast=83.0 and 67.6; peas=97.1 and 85.6; soya (assay protein)=98.3 and 96.4; wheat=95.7 and 87.3; gelatin=99.1 and 96.0. *cf. 1st information Bergner et al. (1978)


[Nitrogen and amino acid resorption by swine. 4. Amino acid resorption]. The seeming and the actual absorption of amino acids of three pigs with a body mass of 50 kg was determined by inserting re-entrant cannulas at the upper small intestine and its end. The test ration contained dry curds labelled with 15N. The content of N and of amino acids as well as the 15N-excess in the amino acids were determined in the food and the chyme. This served the ascertainment of the actual absorption of amino acids in the small intestines.


[Metabolism-oriented lysine requirement of mature rats based on the catabolism rate for 14C- and 15N-labelled lysine]. Mature male albino rats (ca. 300 g body mass) received 10 diets with a varying lysine content (1.6 to 8.4 g/16 g N). In one partial test the animals were fed ad libitum and in another partial test they were kept in the state of maintenance by limiting the amount of the diet. After 7 feeding days the catabolisation of 14C-lysine into 14CO2 was measured and after 8 feeding days the 15N-excretion in urine after 15N-lysine doses was ascertained. Based on these characteristics typical of metabolism, which show increased catabolisation of the amino acid after the lysine requirement was met, the lysine requirement of a mature rat was determined as 3.5 to 4.0 g/16 g N in the diet. In conclusion, this shows that the lysine requirement in the state of maintenenace and of mature animals is considerably lower than during the period of growth.


[Effect of fasting on N-metabolism of broilers]. Four male broilers each were killed 3, 6, 12 and 24 hours after the last food intake and divided into nine parts, The significant diminishing of the fresh liver mass in the test period amounted to 31%. With the increasing fasting time the N-content per g dry matter increased in liver, pancreas, muscles and the rest of the carcass. Because of the decrease in mass a statistically proved diminishing of the total N mass could be registered. The most significant changes were established in the total content of N soluble in TCA in the organs. The decrease in % from the first to the fourth measuring point amounted to 44% (liver), 40% (kidneys) and 28% (small intestines). Concerning these three organs we can maintain that the changes in the total N-content registered in the 24 hours fasting are chiefly limited to the TCA-soluble quota.


[Modeling cerebral dropsy (hydrocephalus) in animals by using angiotensin II]. The effect of a synthetic polypeptic hormone angiotensin-II on the structure of the brain ventricular system was studied in rats. A single injection of angiotensin-II, firm "Ciba", was given to rats in a dose of 0.05 mg/kg of body weight on the 1, 3, 7, 14 and 21st days. The brain was extirpated 15 min after the last injection, fixed in formalin and embedded in celloidin. Intact animals served as controls. A single injection of angiotensin-II was found to be accompanied by a slight dilatation of the brain lateral ventricles. Seven injections resulted in further accumulation of the fluid in the ventricles, and the first symptoms of the corpus callosum atrophy appeared. After the 21st injection of angiotensin-II a typical picture of hydrocephalus demonstrating further accumulation of the fluid in the brain ventricles was seen. The atrophic changes were especially pronounced in the corpus callosum, in white and grey substances of the frontal lobes of the cerebral hemispheres. Besides the atrophic changes, angiotensin-II-induced hydrocephalus resulted in subependymic edema, exfoliation of ependymal cells--the features characteristic for other types of hydrocephalus. Thus, injections of the synthetic hormone angiotensin-II can facilitate in producing an experimental atraumatic hydrocephalus. By means of angiotensin-II a desirable degree of hydrocephalus can be obtained by changing the dose of the preparation.


[Comparative analysis of the neuronal and synaptic organization of the mesencephalic visual center of Greek and steppe turtles]. The stratification, neuronal structure and synaptic organization of tectum opticum have been studied in two species of tortoises (Testudo graeca and Testudo horsfieldi, Gray) by means of Golgi methods and electron microscopy. The midbrain tectum in these closely related animals has some common properties, but at the same time it possesses certain species specific features. Their properties in common appear to be connected with the common ecology and closely systematic place of Testudo graeca and Testudo horsfieldi. The specific features appear to be connected with the species differences in visual behavior of these species.


[Effect of electrostimulation of the medial portion of the hypothalamus of cats on features of sciatic nerve fiber restructuring in experimental neuritis]. In order to study the processes of rearrangement in nerve fibers of the ischiatic nerve and its nervi nervorum at experimentally induced neuritis, the middle part of the hypothalamus was electrically stimulated in 74 mature cats. Twenty three cats were electrically stimulated with alternating current of a rectangular form, 50 Hz 1 m/sec, 1.5 V, for 30 min on each side of the hypothalamic subtubercle. Seven days after electrode implantation, the experimental neuritis was produced by inserting aseptic mica plates subepineurally into the ischiatic nerve. Samples of the nerve stem were taken from the traumatized area and histological sections and film preparations were made. The material was treated after Foot, Ramon y Cajal, Rasskazova, Bielschowsky-Gross-Kampos, Sokoliansky, McManus. As demonstrated the analysis of the preparations, at early stages of the experiments the nerve fibers were preserved better under the electrostimulation than in the intact hypothalamus. However, by the 60th day, resulting from the pathology of the diencephalon, some distrophic changes developed in the peripheral nerve. Lateral branching processes were forming on the axonal cylinders. Nervi nervorum were spreading and forming long and dense wrappings around the endoneural sheaths where they terminated with loops simulating Perroncito's spirals.


[Effect of hydrocortisone on differentiation of white rat embryonal submandibular gland epithelium in organ culture]. At organic cultivation of the submandibular rudiments in 17, 18 and 19-day-old white rat embryos in the nutritional medium without the hormone, the processes of the gland morphogenesis are similar to those in vivo. However, the time for formation of the terminal buds, acini, system of of deferent ducts differs from that in vivo, as it is necessary 1-3 days for the germ to adopt to the cultural conditions. When hydrocortisone is injected into the nutritional medium, the processess in the submandibular gland accelerate, acinar portions differentiate, the secrete is forming, the ducts resembling by their structure salivary tubes in adult animals are constructing. Hydrocortisone injection inhibits proliferative activity in the epithelial cells, decreasing the index of the labeled nuclei from 17.05% to 0.06% after 3 days of cultivation. Possible mechanisms of the hydrocortisone effect on morphogenesis of epithelium in the white rat submandibular gland in organic culture are discussed.


[Comparative study of the activities of dehydrogenases and diaphorases. Basis of the technic]. In order to reveal dehydrogenase and diaphorase in spinal ganglia neurons of 12-day-old chick embryos in cryostat sections, the following modifications of the medium were used: for dehydrogenase - sodium salt substrate 50 mM, NAD or NADPh 0.75 mM, nitro-BT 0.61 mM, phosphate buffer pH 7.2 15 mM, NaCl 50 mM, MgCL2 5 mM, for diaphorase - NAD-N2 or NADHh-N2 0.78-0.66 mM, NaCl 100 mM. To compare relative activity of the enzymes (optic density of histochemical preparations determined cytophotometrically) it is suggested to calculate the values obtained during proportional development of the staining regarding the time unit (hour). The possibility to compare the data obtained with the results of biochemical investigations is discussed, as well as an attempt is made to represent graphically metabolic peculiarities of various cell types.


[Use of gas laser LG-55 to determine the optical density of biological objects]. In order to registrate optic density of such subjects as vessels, tissues, roentgenogram and electrophoregram images, a universal device is suggested. It consists of a) a gas laser lg-55 supplying a parallel bundle of light with small angular deflection and wave length of 0,6328 mc; b) mounting for attachment the reversive motor pd-9 with transmission to change the speed of the subject table, photo resistance fCK-1, focussing lens triple-edged prism and diaphragm, c) double coordinative recorder (endim 620.01). Repeatedly performed registration of the same arterioroentgenogram (or any other investigated) has demonstrated a high accuracy of the registered curves.


[Methods for working with semi-thin epoxide sections in histological practice]. Since ordinary histological laboratories have no special equipment, semi-thin epoxide embedded sections, to perfectly suitable to realize the resolving capacity of the light optic are not widely used in histological practice. However, even in an ordinary histological laboratory it is possible to master the method of their production. A special device is suggested to fix glass knives in the rotational microtome MPC-2. Simultaneously, a number of suggestions and recommendations are given as to certain specificities for obtaining serial semi-thin sections and the method for their staining. Besides, an original method is offered for producing film preparations by means of mild tissue compression in the process of polymerization in epoxide resin. This method allows not only to study some features in the composition of complex three-dimentional objects, but to obtain a series of semi-thin sections from the film preparation to reveal intimal interconnections between certain structural components.


[Problem of toxoplasmosis].

[Problem of toxoplasmosis]. The paper presents brief characteristics of the causative agent and methods of its detection in tissues. The time course of the development of morphological changes in the central nervous system (CNS) is characterized on the basis of the authors own studies. In particular, the possibility of formation of congenital CNS defect upon intrauterine infection with toxoplasma has been demonstrated. A direct correlation between the results of morphological and biochemical examinations of the CNS has been found. A case of toxoplasmosis developing postnatally as a result of exogenous infection with a description of the primary focus in the lungs is presented.


[Allergic orchitis in animals caused by streptococcal sensitization]. Guinea pigs and mice develop orchitis after immunization with killed streptococcus groups A (type 12) cells in complete adjuvant. In guinea pigs the process is characterized by the atrophy of spermatogenous epithelium of the testicles, and in mice by the disappearance of mature spermia from convoluted seminiferous tubules. Globulin fixed on spermia was detected in testicles of the immunized mice. Guinea pigs and mouse sera contained antibodies reacting with spermatid and spermatozoid structures of normal testicles. Immunized guinea pigs showed positive reactions of blast-transformation of lymphocytes and skin reactions of the delayed type to testicle antigens. The development of orchitis in guinea pigs and mice correlates in time with the appearance of antibodies and the development of the delayed type hyperseusitivity to the testicle antigens.


[Cardiomyopathy in influenza and staphylococcal infection]. The myocardium in influenza-staphylococcal infection was studied experimentally in 43 newborn white mice. Lesions and focal necrosis of muscle fibers with perifocal cellular reaction terminating in the development of cardiosclerosis were found. Outside of the zone of necrosis, diffuse involvement of the myocardium with signs of destructive changes was detected ultrastructurally. Myocytes recovered by means of intracellular regeneration.


[Immunomorphological aspects of nonspecific bronchitis in pulmonary tuberculosis]. Bronchi of the lungs resected for tuberculosis were examined histotopographically, morphometrically and immunohistologically. The greatest number of cells in the mucous membrane of the bronchi and the greatest extent of hypertrophy of bronchial glands were found in the stage of progression of lung tuberculosis, while normalization of the quantitative values-in the stage of remission. In the stage of progression the cellular infiltrate was characterized by marked pyroninophilia, abundance of blast cells and antigen specificity; when the process stabilises, plasmocytes are predominant in the infiltrate. In chronic tuberculosis, lymphonodules are frequently found in the bronchial wall. The above morphological signs of hypertrophic bronchitis are regarded as manifestations of immunity reactions (cellular, humoral, secretory) induced by penetration into the bronchi of the antigen-containing detritis from destructive foci.


[Morphology of the microcirculatory bed of the lungs and visceral pleura in liver cirrhosis]. Morphological examinations of the microcirculatory bed of the lungs and pulmonary pleura in 30 fatal cases of cirrhosis of the liver revealed generalized involvement of the blood and lymph vessels. Most significant changes were found in venular and lymphomicrocirculatory parts of the pulmonary pleura as a manifestation of compensatory-adaptative processes directed at retention of the hemodynamic homeostasis of the pulmonary circulation. In the author's opinion, the most important factors causing reactions of the microcirculatory bed of the pulmonary pleura include venous hypervolemia of the pulmonary circulation followed by hypoxia. It is suggested that changes in the vessels of the microcirculatory bed of the lungs and pulmonary pleura in patients with cirrhosis of the liver are an important part of the compensatory-adaptative mechanisms; their disorders cause the development of the right ventricle insufficiency.


[Significance of the chronic hypoxia and neurohormonal activity in the pathogenesis of ischemic heart disease]. Two kinds of changes in the myocardium were found in the heart beyond the focus of necrosis in people of two age groups (senescent and senile) dying of miocardium infarction. Changes of the first kind (hypoxic) reflect the influence of chronic hypoxia of coronoarogenic origin, are always regional and characterized by fields of optically "empty" muscle fibers with reduced enzymatic activity under sacrolemma and frequently by the presence of non-oriented glycogen granules. Those of the second kind correspond to cardiotoxic effect of high doses of catecholamines and are represented by numerous disseminated micronecroses, focal or diffuse lipid infiltration with myocytes against the background of stromal edema with cell reaction and hemorrhages in it. Cases are considered in which hypoxic and cardiotoxic changes were predominant. Their comparison with the activity of the hypothalamo-hypophysis-adrenal system determined by morphological and histochemical tests (very high and moderately high) revealed some correlation.


[Spindle-cell thymoma (a histological and electron microscopic study)]. The paper describes 12 cases of spindle cell thymoma without myasthenia or any other syndromes such as hypogammaglobulinemia, hypoplasia of the red sprout of the bone marrow. In most patients the tumours were detected roentgenologically. Microscopic diagnosis of spindle cell thymoma is based on the detection in it of epithelial cell structures. In those cases where the tumours were represented by spindle cells alone, verification of the tumor was possible only by electron microscopic studies (the presence in the cells of tonofilaments, desmosomes and basal membrane).


[Kidney morphology in the hemolytic-uremic syndrome]. A case of hemolytic-uremic syndrome (Gasser syndrome) in a boy of 7 is described. The disease was accompanied by thrombocytopenia, anemia, hemorrhagic syndrome and acute renal insufficiency (ARI). Morphological lesions consisted in the predominant involvement of the kidneys presenting thrombosis of the glomerular capillaries and reglomerular arterioles, fine cortical necroses, and considerable degenerative changes in the tubular epithelium. The peculiar features of the disease included low intensity of hemolysis and the lack of the oligoanuric phase of ARI.


[2 cases of pulmonary histoplasmosis in combination with tuberculosis]. Two observations by the authors of a chronic cavernous histoplasmosis in a patient of 13 and a histoplasmoma of the lungs in a patient of 53 are described. The diagnosis was established by the examination of the operation material. Clinically, these patients were diagnosed as pulmonary tuberculosis cases. The attention of pulmonologists is drawn to the features and difficulties of differential diagnosis of histoplasmosis and tuberculosis.


[Reproduction of amyloidosis in mice by contact sensitization with dinitrochlorobenzene]. Induction of murine amyloidosis by contact sensitization with hapten--dinitrochlorobenzene is described. Three to five applications of 15--25% dinitrochlorobenzene solution in acetone during two weeks are necessary for this. Within 3 weeks after the 1st application amyloidosis develops involving consecutively the spleen, liver, kidney and heart. Such consequence of the organ involvement and tinctorial pecularities are typical of murine amyloidosis.


[Method of microroentgenography using a laser-plasma source of soft x-ray radiation in medicine and biology]. The paper analyses and illustrates experimentally the possibilities of using the method of microroentgenography for the analysis of the structure and element composition of thin sections of biological tissues. Soft roentgen radiation of laser plasma formed by focusing of a sufficiently powerful laser impulse on the surface of a solid-body target is used. The method requires no preliminary treatment of the specimens.


High flux hemofiltration.

High flux hemofiltration. Experiments were performed using a new hollow fiber hemofilter. Ultrafiltration rates and whole blood urea clearances were measured in post-dilution hemofiltration. High ultrafiltration rates were obtained with the new hemofilter. No adverse effects were detected despite filtration fractions above 45%. These experiments suggest that it is possible in post-dilution hemofiltration to obtain small solute clearances comparable to those of hemodialysis without apparent deleterious effects.


Sorbent regeneration of ultrafiltrate as a long-term treatment of end-stage renal failure. A sorbent system (Redy D 11 cartridge) capable of on-line regeneration of ultrafiltrate during hemofiltration (Amicon 0.5 m2) has been developed and applied on a 3 X 4 hr/week schedule to three patients with end-stage renal failure previously treated for up to six months with hemodialysis. Total experience, to date, is sixteen patient months (three to seven months). Tolerance to fluid removal improved with the new system. Patient well-being and rehabilitation have been maintained. The system offers the potential of hemofiltration without sterile replacement fluid or expensive fluid-balancing machines.


Hemodynamic studies, acid-base status and osmolality in different hemodialysis procedures. Three patients on maintenance dialysis were each treated with three different procedures: A) twenty-liter recirculation dialysis with bicarbonate buffering, B) recirculation-adsorption dialysis and C) single-pass dialysis. Hemodynamic parameters were measured invasively and procedures A, B and C were compared for each patient. In this intraindividual comparative study, the authors attempted to establish a relationship between the varying hemodynamic parameters and the changes in osmolality and acid-base status. There were indications of some causal relationship to circulatory stability: in A and B, there were peripheral resistance increases of 24.5% and 38.4%, respectively, with stable circulation; in C, there was a 6.1% increase and unstable circulation. Additionally, the influence of acidosis is shown in B, with disproportionately strong reductions of cardiac output (21.9%) and pulmonary artery pressure (44.9%); In spite of a decrease of osmolality (A: 15.6 mOsm/L), stable circulation could be achieved if the peripheral resistance was substantially increased and acid-base status was equalized.


A 1978 perspective of hemoperfusion.

A 1978 perspective of hemoperfusion. The present status of adsorbent hemoperfusion is introduced with emphasis on available systems and their roles in uremia, acute intoxication, fulminant hepatic failure and chronic schizophrenia.


Adsorptive capacities of hemoperfusion devices in clinical use. Five different hemoperfusion devices have been used in the treatment of patients, intoxicated with barbiturates. The changes in drug clearance values which occurred during hemoperfusion varied according to the device used. Even after very long periods of hemoperfusion, however, drug clearance values never fell to zero. Using data collected from comparable cases, it has been shown that the rate of uptake of drug from the blood is independent of the amount of adsorbent in the columns, those containing 100 gm charcoal having the same efficiency as the conventional 300 gm. The prime factor which determines the rate of drug uptake has been shown to be the arterial plasma drug concentrations. Other factors, such as the deposition of cellular debris and proteins as hemoperfusion progresses, are also thought to influence the efficiency of drug removal.


Sorbent membranes: device designs, evaluations and potential applications. The Enka sorbent membranes are designed to meet the requirements for the chronic extracorporeal treatment of body fluids with sorbents. The various configurations allow the construction of devices employing sorption alone, sorption plus filtration, or sorption plus filtration and dialysis. While conventional device designs can be employed, novel schemes are being explored to take advantage of the special structural features of the sorbent membranes. A general scheme for the evaluation of sorbents and, in particular, the sorbent membranes allows optimal use of sorbents in practical device designs. While the potential applications include those of sorbents in general, the unique configuration of the sorbent membranes with the possibilities of using multiple sorbents and of combining sorption with other transport processes should expand the therapeutic capabilities.


Sorbent membranes used in a conventional dialyzer format. In vitro and clinical evaluation. The possibility of installing sorbent membranes into proven dialyzer configurations could result in certain advantages over dialyzers with conventional membranes. A sorbent membrane dialyzer might prove superior in that it allows a higher clearance in the middle molecule molecular weight range, thereby shortening dialysis time and possibly allowing a reduction in the amount of dialysate fluid required. Finally, this device could be used without dialysate if the technician's goal is to eliminate a single substance readily adsorbed by charcoal. For example, this would apply to the treatment of certain intoxications. In vitro data were established for six substances in the molecular weight range of 60 to 5200 daltons and compared to corresponding results for conventional Cuprophan dialyzers. Clinical evaluation of sorbent membrane dialyzers for patients with uremia and intoxication, both with and without use of dialysate, has proven effective with no adverse reactions.


Preparative isolation of middle molecular weight fractions from the hemofiltrate of patients with chronic uremia. Fractions in the molecular weight range of the so-called middle molecules (500--3000 daltons) were isolated on a preparative scale from hemofiltrate of patients. Hemofiltration was performed with an RP-6 dialyzer using a predilution technique. The hemofiltrate was concentrated and desalinated by reverse osmosis with membranes with a normal cut-off of 500 daltons. The retentate was freeze-dried, redissolved in volatile buffer and fractionated on Sephadex G-15 macrocolumns. The middle molecule molecular weight range in the elution profiles (detected at 206 and 280 nm simultaneously) was marked with peptides. Isolated fractions in the middle molecule molecular weight range showed strong inhibitory activity upon 3H-thymidine incorporation into rat bone-marrow cells in vitro. The described combination of hemofiltration, reverse osmosis and gel chromatography is suggested as a useful approach to the isolation of these ninhydrin-positive substances which, according to their proved heterogeneity, are to be subfractionated by further procedures.


Influence of middle molecules on the anemia of uremic patients. To evaluate their toxicity at the cellular level, middle molecules from uremic serum were incubated with erythrocytes from healthy subjects and the activity of the enzyme Delta-aminolevulinic acid dehydrase (D-ALA-D) and peroxidative hemolysis were investigated. Uremic middle molecules caused a significant decrease of the D-ALA-D activity of normal erythrocytes which was not due to differences in the concentrations of Pb, Cd or Zn. The decreased enzyme activity could be restored by adding reduced glutathione (GSH; 5 mmol/L) together with the middle molecules to the assay system. Uremic middle molecules caused a significant increase of peroxidative hemolysis in normal erythrocytes. Uremic middle molecules contribute to the anemia of uremic patients by impeding hemoglobin synthesis and by increasing peroxidative hemolysis, possibly by affecting SH-groups. H2O2-producing compounds should be avoided in uremic patients.


Renin, growth hormone and insulin in hemodialyzed uremic diabetics. In order to detect metabolic aberrations which could account for the poor therapeutic response of the diabetic to dialysis, a comparative study of nondiabetic and diabetic uremia patients treated with hemodialysis was undertaken. Plasma renin activity and levels of growth hormone, glucose and insulin were investigated.


Chronic hemofiltration. A critical evaluation of a new method for the treatment of blood. The experiences which have been compiled in more than 2400 hemofiltrations confirm that this method represents an alternative way of treating uremic patients. The main advantages of chronic hemofiltration are the comfort of the patient and the ease in handling excess overhydration without extending treatment time, which is less than 3 X 3 hours/week if adequate hemofilters are used. With regard to the improvement of such typical uremic complications as severe hypertension, hypertriglyceridemia or neuropathy, hemofiltration does not seem to be superior to hemodialysis. However, since most hemofiltration patients do not require phosphate binders and, additionally, remarkable amounts of parathyroid hormone are removed during one hemofiltration, it appears possible that hemofiltration might be an important therapeutic alternative for those renal patients who suffer from severe hyperphosphatemia and secondary hyperparathyroidism.


Clearance of meprobamate by hemoperfusion over columns of charcoal and Amberlite resin. Studies in a patient. Perfusion of the blood of a patient with toxic levels of meprobamate through an activated charcoal cartridge resulted in efficient early clearance of the drug, then a decline in extraction. Perfusion through a resin column resulted in total drug extraction without a decline in clearance over four hours. Both procedures were stable with minimal disturbance in hematological values or blood chemistries. This is the first report of in vivo hemoperfusion over resin for meprobamate poisoning. The efficacy and safety of the procedure need emphasis.


Digoxin removal from an anephric patient by hemoperfusion over XAD-4. Removal of digoxin by hemoperfusion over Amberlite XAD-4 was determined in a functionally anephric patient. During four hours of hemoperfusion, 50.45 microgram of digoxin were removed by the column and serum digoxin concentrations decreased by 0.2 ng/ml after the post-hemoperfusion reequilibration was complete.


Evolution of the solenoid-actuated left ventricular assist system: integration with a pusher-plate pump for intra-abdominal implantation in the calf. The performance of an implantable left ventricular assist system (LVAS) utilizing a pulsed solenoid energy converter and a pusher-plate blood pump has been characterized in vitro and in vivo. A microprocessor-based electronic control system makes the LVAS completely self-regulating over the range of operating conditions and provides considerable flexibility in various assist modalities. Over forty thousand hours of in vitro and in vivo operating experience has been accumulated with current systems, and significant progress has been acheived in system durability and reliability. A new toggle latch has provided nearly a year of failure-free operation on the bench, without measurable wear. Energy converter efficiencies of 50% have been demonstrated. In vivo evaluation has been highlighted by an animal experiment still in progress after nearly four months of fault-free, continuous synchronous pumping.


Treatment of metabolic alkalosis with peritoneal dialysis in a patient with renal failure. Peritoneal dialysis using a chloride-rich dialysate succeeded in correcting systemic metabolic alkalosis in a patient with renal failure. The present approach may have a place in the therapy of metabolic alkalosis.


Infectious diseases of the newborn.

Infectious diseases of the newborn. Despite the advent of antibiotics, infectious diseases remain a significant cause of mortality and morbidity in the neonatal period. Infection is the third commonest cause of perinatal mortality after hypoxia and malformations. Neonatal mortality rates from infection are of the order of 1:1000 live births. Although infections are theoretically preventable, there has been no significant change in incidence over the last 30 years.


Immunization for the common communicable diseases. Infectious diseases have dropped from the limelight in developed countries with the advent of immunization and antibiotics, but their worldwide significance in the terms of morbidity and mortality is still considerable. Whereas there are grounds for substantial differences in immunization schedules between the developed and developing countries, the acceptance of simplified immunization schedules in the former remains a crucial factor for gaining significant cover of young children and success in control of the target diseases. In the developed countries, continuing surveillance and re-evaluation of programmes is necessary to ensure that the benefits so far obtained are maintained. This paper summarizes the approach to immunization programmes both in developing countries and in Australia, and outlines the schedule recommended by the National Health and Medical Research Council at its 85th Session in June, 1978.


The special problem of hospital infections. One in twenty patients admitted to hospital will develop an infection. Hospital-associated infections have a significant effect on patient morbidity, with consequent economic implications. Both indigenous flora and cross infection represent significant sources of infection. It is of prime importance to identify the patient at risk and institute preventative measures.


Antibiotic treatment of common bacterial respiratory tract infection in general practice. Respiratory tract infections, particularly in children, are some of the most common conditions seen in general practice. It would seem reasonable, therefore, that these conditions would be treated well. However, analysis of the figures taken from the Australian Morbidity Survey conducted by The Royal Australian College of General Practitioners shows that whilst most general practitioners use antibiotics satisfactorily, there are a few occasions in which it is fairly obvious that there is a lack of knowledge of the action of a particular antibiotic against a specific organism. This article is not intended to be a treatise on the microbiology of different respiratory tract infections and antibiotic use, but it is intended to be a guide to the treatment of common respiratory infections as seen in general practice.


Rheumatic diseases and the eye.

Rheumatic diseases and the eye. The ocular complications of the rheumatic diseases are common, frequently insidious and sight threatening, and deserve review to heighten awareness and facilitate earlier diagnosis and treatment.


Sex offences.

Sex offences. The investigation of offences involving sexual assaults and the examination of the victims of such crimes, are matters of concern to the community, to medical people and to law enforcement agencies. Throughout the world, much thought has been given to these matters during the last few years. The subject has stimulated anger and frustration as well as sympathy. The public conscience has been stirred and professional groups--doctors, lawyers, legislators and law enforcement agencies--goaded on many occasions by feminist groups, have deliberated the various aspects of this problem. A deal of practical advice has been given on this subject and throughout the world there has been a number of different approaches to the manner in which the medical examination of these victims is handled.


Diagnosis and management of acute diarrhoea in adults. Acute diarrhoea is a frequent, worldwide complaint. On any given day, 200 million people suffering from gastroenteritis will pass a volume of diarrhoeal water comparable with the flow of water over the Victoria Falls in one minute. In most attacks, routine microbiological techniques will fail to demonstrate the cause.


Step-by-step management of female victims of sexual assault. Various authors have published advice on the management of victims of sexual assault--male and female. Most published accounts provide general advice, ordered according to theoretical considerations rather than to the chronological sequence of tasks to be performed. Our experience has been that a task list sequenced in chronological fashion is very useful in helping the attending doctor to remember what to do and how to do it. The step-by-step list used for women seen at Perth's Sexual Assault Referral Centre (SARC) is presented here.


Hyperlipaemia in ponies.

Hyperlipaemia in ponies. The case histories and clinical findings of 15 ponies with hyperlipaemia are presented. The disease was characterised by hyperlipidaemia with inappetance, progressing somnolence, muscle fasciculation, diarrhoea, and ventral oedema as the predominant clinical findings. Post mortem examinations of 12 ponies showed extensive lipidosis and vascular thrombosis with widespread secondary changes. Most cases occurred in late pregnant and early lactating mares in the summer months and it is postulated that the disease was initiated in this group by a falling nutritional plane in the face of high nutrient requirements.


Breed differences in abnormalities of the reproductive organs of young beef bulls. A total of 702 examinations were conducted on young (16 to 31 months) beef bulls of 3 breeds and 3 breed crosses (Brahman cross--100; Africander cross--93; Shorthorn cross--109; Brahman--81; Africander--89, and Hereford--230). A high occurrence of both increased size (overall 11.7%) and consistency (overall 40.2%) of seminal vesicles was observed. The Hereford breed had most enlarged (19.1%, P less than .01) and firm (20.9%, P less than .01) seminal vesicles. The significance of these findings is discussed. Testicular hypoplasia was diagnosed in 2.7% of examinations. The Brahman breed had most testicular hypoplasia (8.6%, P less than .01). The implications of this finding are discussed and the need is stressed for a wider investigation of gonad development in the Brahman breed in Australia. Other findings included: small seminal vesicles in 1.6% of examinations (Brahman breed highest with 8.6%, P less than .001), enlarged ampullae, 1.9%, 'soft' testicles, 3.4% and small testicles, 7.5%. Abnormal carriage of the testicles was detected in 0.3% of examinations. Ulceration of the prepuce was observed in 6.8% of examinations. The highest occurrences were in the SH (13.8%, P less than .01) and Africander (13.5%, P less than .05) breed groups. Varying degrees of preputial eversion or prolapse were seen in 0.7% of examinations with the Bx breed group having highest representation (3.0%, P less than .05).


Efficacy of oxfendazole against an ovine isolate of benzimidazole resistant Haemonchus contortus. Eighteen parasite-free sheep were each infected with 6000 +/- 25 infective larvae of the AH-2, benzimidazole-resistant strain of Haemonchus contortus on each of experimental days -27, -15, -8 and -1. The sheep were subsequently randomized into 3 groups of 6 sheep each. Group 1 served as untreated controls; Groups 2 and 3 were treated orally with oxfendazole at dose levels of 5 mg/kg and 10 mg/kg body weight, respectively. At both dosage levels, oxfendazole was 100% effective against third, fourth, early fifth, and adult stages of the worms.


The efficiency of cypermethrin (NRDC 149) for the treatment and eradication of the sheep louse Damalinia ovis. A synthetic pyrethroid, cypermethrin (NRDC 149), showed activity in killing lice (Damalinia ovis) in the fleece of sheep at concentrations as low as 1 ppm in a dip. In addition concentrations of 5 and 10 ppm showed a persistent effect and prevented reinfestation from contact challenge sheep for 7 and 19 weeks respectively.


Testicular pathology of Merino rams.

Testicular pathology of Merino rams. The scrotal contents of 2,281 Merino rams of wide age range examined at 3 abattoirs in Perth, Western Australia, showed a 40% prevalence of rams with 1 or more gross lesions. The percentage prevalence of gross lesions found on autopsy were: adhesions 21, testicular atrophy/hypoplasia 14, testicular calcification 13, congenital cyst of the epididymis 6, cryptorchidism 4, chronic epididymitis/scrotal abscesses 2, varicocoele 2, testicular aplasia less than 1, seminoma less than 1. The prevalence of lesions increased with age of rams. Testicular atrophy was often present when varicocoele, chronic epididymitis and scrotal abscess affected that side of the scrotal sac. Corynebacterium spp was the most frequent isolate from chronic epididymitis. Actinobacillus seminis was isolated once and Brucella ovis was not isolated. About 20% of the rams showed lesions compatible with a diagnosis of reproductive unsoundness. A large proportion of lesions were revealed only after removal of the scrotum and most of these did not appear to affect normal spermatogenesis except for an estimated 9% of rams, which showed mild atrophy/hypoplasia. Samples of these testes showed mild to moderate changes in spermatogenesis on histopathology. It was postulated that the latter group of rams might represent those ill-defined clinical cases which show equivocal results on appraisal of semen samples despite the presence of palpably normal testes.


Leucoencephalomyelitis of goat kids.

Leucoencephalomyelitis of goat kids. A leucoencephalomyelitis in 6 goat kids 2 to 5 months old is described. The disease was characterised by fever, ataxia, posterior paresis, circling and hyperaesthesia progressing to prostration. The neural lesion was confined to the white matter of the cerebellum and posterior brain stem in 4 kids, but in 2 others the cervical spinal cord was the main site affected. The lesion was characterised microscopically by dense perivascular cuffing with mononuclear cells, infiltration of the parenchyma with macrophages and a proliferation of glial cells and by a marked primary demyelination. In more advanced lesions, areas of the neurophil were replaced by a loose glial scar. There were associated pulmonary lesions of interstitial pneumonitis and hyperplasia of the peribronchiolar lymphoid tissue. Attempts to isolate an aetiological agent and to transmit the disease to young goat kids and lambs were unsuccessful. The disease has not been reported before in Australia but has distinct similarities to an infectious leucoencephalomyelitis of young goats which has been described in North America.


Administration of a vaccine prepared from the Australian V4 strain of Newcastle disease virus by aerosol and drinking water. An experimental vaccine containing the avirulent Australian V4 strain of Newcastle disease virus was used to vaccinate 3-or 6-week-old chickens by aerosol and drinking water application. The chickens lacked maternally derived antibody to Newcastle disease virus. When the vaccine virus was diluted in tap water more than 90% of the infectivity was destroyed immediately. The addition of 0.25% skim milk prevented this loss and there was no loss in distilled water. Rates of inactivation at 37 degrees C were similar in tap water and distilled water and were unaffected by the addition of skim milk. Both methods of vaccination resulted in the production of haemagglutination-inhibition antibodies which persisted for at least 8 to 12 weeks. The antibody response to aerosol vaccination was significantly better than that following drinking water vaccination. No clinical disease was induced by exposure to vaccine virus. Serum neutralisation antibodies paralleled those detected by haemagglutination-inhibition in chicks vaccinate once by drinking water. After revaccination through the drinking water, haemagglutination-inhibition antibodies were boosted temporarily while neutralising antibodies were maintained at an enhanced level. From chickens vaccinated by aerosol, Newcastle disease virus was recovered for 10 days from lungs and for 7 days from tracheas and caecal tonsils. Peak viraemia was detected 2 and 3 days after vaccination while both neutralising and haemagglutination-inhibition antibodies became detectable 5 days after vaccination.


The establishment of a laboratory colony of Ixodes holocyclus. A laboratory colony capable of producing annually 3000 female Ixodes holocyclus was maintained for 3 years. Guinea-pigs were used for larval feeding and bandicoots for nymphs and adults. The methods and equipment employed are described in detail.


Experimental infection of 35, 50 and 60 day old pig foetuses with porcine parvovirus. Foetuses of six seronegative gilts, two of which each respectively 35, 50 and 60 days pregnant, were inoculated intrauterinely with porcine parvovirus (PPV) and examined 7 and 11 days after inoculation. HI antibody was not detected in any of the foetuses although all but one gilt developed low levels of antibody. All but one of the foetuses inoculated with PPV died in utero prior to examination at 11 days after inoculation. Infection also spread to non-inoculated litter mates. Histological changes were mild in the gilts but there was widespread tissue necrosis in infected foetuses, and intranuclear inclusion bodies were observed in cells of the liver, lung, kidney and cerebellum. The increased survival of foetuses infected at later stages of gestation appeared to be related to increased numbers of mononuclear cells then present in many tissues.


Serological comparison between Histophilus ovis, Actinobacillus seminis and Brucella ovis. The serological relationships between 4 strains of Histophilus ovis, the neotype strain of Actinobacillus seminis and Brucella ovis were examined using a cross-absorption complement-fixation technique. It was found that the 4 strains of H. ovis were serologically homologous and that an incomplete relationship existed between these organisms and A. seminis. Anteriserums prepared against one strain of H. ovis and the strain of A. seminis gave a weak, apparently non-specific cross-reaction with Br. ovis antigen. The practical significance of these results is discussed.


Neonatal lamb losses due to feral pig predation. An investigation into causes of low lamb marking percentages was made on a property in north-west New South Wales from 1971 to 1975. Investigations revealed that from 11 to 70% of the ewes in lamb were losing all their lambs. Observations suggested that feral pig predation was a factor in the perinatal loss. In 1975, 2 groups of ewes were placed in adjoining paddocks prior to lambing. Feral pigs were excluded from one paddock for most of lambing by means of an electric fence. In this paddock, 117% of lambs were marked compared with 80% in the adjoining paddock. It was estimated that in 1975 over 600 lambs were killed by feral pigs from 1,422 ewes lambing in paddocks with feral pigs. The problems involved in the diagnosis of feral pig predation are discussed.


Snake bite in cats.

Snake bite in cats. In 6 years snake bite was diagnosed in 41 cats, with an average age of 20 months. The commonest presenting signs were dilated pupils, absence of the pupillary light reflex, depression and generalised muscle weakness. Other frequent findings were vomiting, dyspnoea, hindlimb ataxia and complete flaccid paralysis. Thirty-seven cases (90%) occurred in the 6 warmer months of the year. Tiger snakes were positively identified in 7 cases. A recovery rate of 89% was obtained in cases receiving 3000 units Tiger snake antivenene, fluid therapy and nursing. Cases presenting with a complete flaccid paralysis and sub-normal temperatures were poor prognostic risks.


Histiocytic ulcerative colitis in a Boxer dog. A 2-year-old male Boxer dog had passed loose faeces mixed with fresh blood and mucus for 8 months. Tenesmus after defaecation was a feature. Colitis was diagnosed from the proctoscopic appearance of the recto-colonic mucosa and confirmed from a biopsy. The disorder proved unresponsive to sulphasalazine therapy, but oral chloramphenicol, betamethasone and prednisolone enemas administered over a 6-week period produced a satisfactory clinical improvement, which persisted for a further 3 weeks without treatment. However, follow-up proctoscopy showed only a marginal improvement in the appearance of the mucosa and appeared to exacerbate further bloody diarrhoea, which persisted. The dog was destroyed and histiocytic ulcerative colitis confirmed at autopsy.


Glossy mutants of maize. VIII. Accumulation of fatty aldehydes in surface waxes of gl5 maize seedlings. In corn seedlings (Zea mays L.) homozygous for the mutation gl5, the surface waxes are characteristically altered. In this mutant the main wax constituents (83.5%) are aldehydes while in the normal waxes alcohols predominate (62.7%). Moreover, in the normal waxes aldehydes and alcohols are made up mainly of the C32 term (99%), whereas in gl5 waxes the principal aldehyde is still C32 (90.7%) but the free alcohol composition pattern is noticeably modified. Here the predominant terms are C24, C26, and C28, with C32 representing only 16.6% of the total. The results indicate that the mutant induces a block in the synthesis of fatty alcohols while accumulating fatty aldehydes, the substrates from which the alcohols originate.


Genetic control of nucleoside transport in sheep erythrocytes. Nucleoside transport in sheep erythrocytes is under the genetic control of two allelomorphic genes (NuI and Nui), where NuI codes for the functional absence of a high-affinity nucleoside transport system and is dominant to the gene (Nui) coding for the presence of the transport system. Kinetic and inhibitor experiments show that the high-affinity transport system is not present in heterozygous erythrocytes, demonstrating that the NuI gene is completely dominant over the Nui gene. It is suggested that the Nu locus may not represent the structural gene locus of the nucleoside transport system. Instead, it may be a regulator gene locus.


Interaction of the opaque-2 gene with starch-forming mutant genes on the synthesis of zein in maize endosperm. The combination of opaque-2 with starch-modified or starch-deficient mutants produced a cumulative and synergistic effect, respectively in regulating zein synthesis. The double mutant, brittle-2 opaque-2, which almost completely prevented the synthesis of Z1 and Z2, had high RNase activity. The possible involvement of RNase in effecting zein synthesis is discussed.


Segregation of genetic determinants for murine glucuronidase synthesis and loss in CXB recombinant-inbred strains. A set of recombinant-inbred strains developed from mouse strains BALB/c and C57BL/6 includes two beta-glucuronidase phenotypes that are not seen in either of the progenitor strains. These new recombinant phenotypes indicate that glucuronidase levels are regulated by genes additional to the Gur locus, which is closely linked to the glucuronidase structural gene (Gus) and is known to regulate the rate of glucuronidase synthesis. In this study, induced rates of glucuronidase synthesis were determined for these recombinant-inbred strains, and rate constants for enzyme loss were calculated. The rate of synthesis was found to segregate with the Gus gene in all of the strains, and only the determinants for rate of enzyme loss recombined to give new phenotypes. It was concluded that at least two genes affect the rate of enzyme loss, that these genes are not closely linked to each other or to the Gur-Gus region on chromosome 5, and that no major determinants of glucuronidase synthesis segregate independently of Gur.


Role of adenine phosphoribosyltransferase in adenine uptake in wild-type and APRT- mutants of CHO. Adenine uptake in cultured Chinese hamster fibroblasts showed biphasic saturation kinetics. The transport system was highly specific for adenine and was competitively inhibited by adenosine. Utilizing mutant clones of Chinese hamster fibroblasts that have either reduced or negligible adenine phosphoribosyltransferase (APRT) activity, we found that (1) adenine was not accumulated against a concentration gradient in the absence of APRT activity and (2) after rapid initial uptake equal to that of the parent the rates of adenine accumulation found for the mutants correlated strongly with their residual APRT activities. Furthermore, using either artificially depressed phosphoribosylpyrophosphate pool size and APRT activities or the mutants with decreased APRT activity, we found that adenine transport was independent of phosphorylation by APRT. These studies suggest that adenine is transported as the free base by facilitated diffusion and is subsequently phosphorylated by APRT.


Species identification in protozoa: glucosephosphate isomerase variation in the Paramecium aurelia group. Results are presented for intra- and interspecies variation in electrophoretic mobility of the enzyme glucosephosphate isomerase in the Paramecium aurelia species complex. Three new observations have been made: (1) the hitherto indistinguishable species 1 and 5 can be distinguished on the basis of GPI electrophoretic mobility, (2) the degree of intraspecies variation is much higher for GPI than for the previously studied mitochondrial dehydrogenases and esterases, and (3) several of the enzymatic variants observed in one species are apparently indistinguishable from some found in other species. The intraspecies variants found have been shown to be allelic, and, on the basis of the enzyme patterns of the heterozygotes, it is proposed that GPI is a dimeric enzyme determined by two loci. In view of the use of enzyme variation as a means of species identification in protozoa, these results suggest that the use of such methods can lead to underestimating the number of species and possibly to misclassification. The implications of these findings together with the results obtained with Tetrahymena are discussed.


Analysis of plant genomes. V. Comparative study of molecular properties of DNAs of seven Allium species. The genomes of seven plant species belonging to the genus Allium and exhibiting a threefold variation in their nuclear DNA content were analyzed by studying their reassociation kinetics, equilibrium centrifugation behavior in neutral CsCl gradients, and melting properties. The reassociation kinetics experiments revealed the presence of 44-65% repeated DNA sequences. A comparison between DNA contents and the proportion of repeated DNA sequences indicated that, in Allium, increase in the genome size is not exclusively due to variations in the proportions of repetitive DNA. The total DNA as well as the various repetitive DNA fractions in all the Allium species examined exhibited, in spite of a few differences, a gross similarity in their behavior in neutral CsCl gradients and in their melting properties.


Relationship between subunit size and number of rare electrophoretic alleles in human enzymes. Data from published sources were used to compare the numbers of different electrophoretic alleles of 29 monomeric and dimeric human enzymes to their respective subunit molecular weights. Only those human enzymes were considered for which the total sample sizes were in excess of 2000 individuals. Correlations between these two variables were determined within sample size ranges of 2000 less than or equal to n less than or equal to 3000 and 4000 less than or equal to n less than or equal to 5000 individuals, and separately by quaternary class. There was no statistically significant correlation observed for the smaller sample size range in monomers; however, the correlations for the larger sample size range in monomers and both ranges in dimers were significant. Since there is no relationship between subunit size and heterozygosity, the relationships are due primarily to the incidence of rare alleles. These findings demonstrate the effect of locus-specific mutation rates, expected as a consequence of variation of cistron sizes, and imply that other forces are responsible for the relative frequencies of common alleles at some of the loci.


Chloroperoxidase-catalysed oxidation of 4-chloroaniline to 4-chloronitrosobenze. The incubation of 4-chloroaniline with chloroperoxidase and H2O2 resulted in a rapid formation of 4-chloronitrosobenzene. This enzymic oxidation displayed a pH optimum at 4.4 with a Km of 8.1x10(-4)M and catalytic-centre activity of 312. The initial rate of the reaction was strongly affected by the presence of halide ions. 4-Chlorophenylhydroxylamine was even more rapidly converted into the nitroso compound. A reaction mechanism is proposed on the basis of currently accepted theory for the catalytic action of chloroperoxidae. A noteworthy aspect of this new reaction is the difference in the products previously reported for the action of classical peroxidases on anilines and the single nitroso product resulting from chloroperoxidase oxidation.


A new intermediate of the aldolase reaction, the pyruvaldehyde-aldolase-orthophosphate complex. Fructose 1,6-bisphosphate aldolase from rabbit muscle forms by reaction with dihydroxyacetone phosphate a pyruvaldehyde-aldolase-orthophosphate complex that is in equilibrium with the eneamine intermediate. The new intermediate accumulates in two phases. The first one is practically complete in 40ms, and the second occurs with an apparent first-order rate constant of 4.6 +/- 0.5s-1. The new intermediate breaks down slowly with the release into the medium of pyruvaldehyde and Pi. The rate of the spontaneous release is higher at acidic than at neutral pH.


Purification and properties of the native form of rabbit liver aldolase. Evidence for proteolytic modification after tissue extraction. Aldolase was purified from rabbit liver by affinity-elution chromatography. By taking precautions to avoid rupture of lysosomes during the isolation procedure, a stable form of liver aldolase was obtained. The stable form of the enzyme had a specific activity with respect to fructose 1,6-bisphosphate cleavage of 20-28 mumol/min per mg of protein and a fructose 1,6-bisphosphate cleavage of 20-28mumol/min per mg of protein and a frutose 1,6-bisphosphate/fructose 1-phosphate activity ratio of 4. It was distinguishable from rabbit muscle aldolase, as previously isolated, on the basis of its electrophoretic mobility and N-terminal analysis. Muscle and liver aldolases were immunologically distinct. The stable liver aldolase was degraded with a lysosomal extract to a form with catalytic properties resembling those reported for aldolase B4. It is postulated that liver aldolase prepared by previously described methods has been modified by proteolysis and does not constitute the native form of the enzyme.


Activation of the first component of human complement (C1) by antibody-antigen aggregates. The activation of subcomponents C1r and C1s in the first component of complement, C1, when bound to antibody-antigen complexes was investigated. Activation was followed both by the splitting of the peptide chains of subcomponents C1r and C1s and by the development of proteolytic activity. For the maximum rate of activation to occur, all components must be present in approximate molar proportions of antibody: C1q:C1r:C1s of 13:1:5:5. For activation of subcomponent C1s, subcomponents C1r or C1r, but not C1r inactivated with iPr2P-F (di-isopropyl phosphorofluorideate), are effective. For activation of subcomponent C1r, subcomponents C1s, C1s or C1s inactivated with iPr2P-F are effective. Subcomponent C1s is activated by C1r, and C1r is activated autocatalytically, probably through the formation of an intermediary C1r. in which the peptide chain is unsplit but a conformational change caused by interaction with the other components has led to the formation of a catalytic site able to split subcomponent C1r to C1r.


Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties. Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.


Topography of the rhodopsin molecule. Identification of the domain phosphorylated. Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.


Purification and properties of arginase from human liver and erythrocytes. Arginase was isolated from human liver and erythrocytes. The purification procedure used acetone precipitation, heat-treatment, (NH4)2SO4 precipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-200 in the presence of 2-mercaptoethanol. Both enzymes migrated to the anode at pH8.3 on polyacrylamide-gel electrophoresis. After incubation at pH8.0 and 37 degrees C the purified anionic liver arginase migrated to the cathode on polyacrylamide-gel electrophoresis. It is assumed that the multiple forms of the enzyme reported in the literature are partly artifacts of the purification procedure. The liver arginase showed a mol.wt. of 107000 determined by gel filtration and a sedimentation coefficient of 5.9S. Treatment of the liver enzyme with 0.25% sodium dodecyl sulphate at pH10 demonstrated an oligomeric structure of the enzyme with a mol.wt. of the subunit of 35000. The kinetic properties determined for the purified liver arginase showed an optimum pH of 9.3 and an optimal MnCl2 concentration of 2mM. The Km for L-arginine was 10.5 mM and for L-canavanine 50mM, and L-lysine exhibited a competitive type of inhibition with a Ki of 4.4mM. L-Homoarginine was not a substrate for liver arginase.


Phosphonate analogues of aminoacyl adenylates. Phosphonomethyl analogues of glycyl phosphate and valyl phosphate, i.e. NH2-CHR-CO-CH2-PO(OH)2, were synthesized and esterified with adenosine to give analogues of aminoacyl adenylates. The interaction of these adenylate analogues with valyl-tRNA synthetase from Escherichia coli was studied by fluorescence titration. The analogue of valyl phosphate has an affinity for the enzyme comparable with that of valine, but that of valyl adenylate is bound much less tightly than either valyl adenylate or corresponding derivative of valinol. The affinity of the analogue of glycyl adenylate was too low to be measured. We conclude that this enzyme interacts specifically with both the side chain and the anhydride linkage of the adenylate intermediate.


Purification and characterization of a galactan-reactive agglutinin from the clam Tridacna maxima (Röding) and a study of its combining site. 1. A beta-galactosyl-binding lectin was purified from the haemolymph of the clam Tridacna maxima by affinity chromatography using polylecyl larch galactan, D-galactosamine coupled to epoxy-activated Sepharose or acid-treated Sepharose. Elution with N-acetyl-D-galactosamine or lactose displaced the bound lectin, which appeared homogeneous by sedimentation analysis. On immunoelectrophoresis at pH8.6 and against rabbit antisera to crude T. maxima haemolymph, the lectin gave one precipitin arc in the alpha-region. 2. On a alkaline polyacrylamide disc gels, one lightly stained band and a broad diffuse band were seen close to the cathode. Ioselectric focusing in solution revealed two peaks of pI4.05 and 4.25 and a shoulder, pI4.0, whereas at least three bands close together (pI3.9-4.3) were seen after electrofusing in gel. 3. The agglutinin is a glycoprotein with a mol.wt. of 470300 +/- 20000. Amino acid analysis revealed no methionine and a significant amount of half-cystine residues. 4. Tridacna lectin is a metalloprotein requiring Ca2+ for its haemagglutinating and precipitating activities. 5. In haemagglutination studies the agglutinin exhibited a broad pH optimum (4.8-10.6). 6. Polysaccharides and glycoproteins with terminal non-reducing beta-D-galactosyl residues reacted with the lectin to form precipitates both in gel and in solution. Inhibition experiments showed that N-acetyl-D-galactosamine was the best inhibitor of the agglutinin combining sites, followed by p-nitrophenyl beta-D-galactoside, methyl beta-D-galactoside, D-galactosamine and 60O-beta-D-galactopyranosyl-D-galactopyranose. On a molar basis, N-acetyl-D-galactosamine was 20-fold more active than D-galactose and nearly 10-fold more inhibitory than D-galactosamine. 7. Circular-dichroism studies showed that the lectin contains a relatively high proportion of beta-structure. 8. Mercaptoethanol treatment of the agglutinin followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed subunits with approx. mol.wts. of 10000, 20000 and 40000.


Three neurotoxins from the venom of a sea snake Astrotia stokesii, including two long-chain neurotoxic proteins with amidated C-termini. From the venom of a sea snake Astrotia stokesii three neurotoxic components, toxins Astrotia stokesii a, b and c were isolated in 40, 15 and 5% yield by weight respectively of the whole venom. Their LD50 values for 20g mice were 0.13, 0.096 and 0.098 microgram/g body wt. respectively and accounted for almost all the lethal activity of the venom. Their amino acid sequences were determined. Astrotia stokesii a was composed of 60 amino acid residues with nine half-cystine residues and was quite homologous to other sea-snake short-chain neurotoxins in its amino acid sequence. Toxins Astrotia stokesii b and c were composed of 70 and 72 amino acid residues respectively with 10 half-cystine residues. They are the first long-chain neurotoxins with high activity isolated from sea-snake venoms. The C-terminal carboxy groups of toxins b and c were found to be amidated; the amidation is known for some polypeptides, but is novel for a protein. The amide group may make a hydrogen-bond with glutamic acid-39, which replaces a lysine that has so far been found invariably in long-chain neutrotoxins. Astrotia stokesii b and c are also novel in having phenylalanine-25 and isoleucine- or valine-42. The ordinary Tyr-Glu pair, which is observed in X-ray structure [Low, Preston, Sato, Rosen, Searl, Rudko & Richardson (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2991-2994] and n.m.r.study [Inagaki, Tatsumi, Miyazawa, Hori & Tamiya (1977) Abstr. Int. Congr. Pure Appl. Chem. 26th, p. 336] on erabutoxins may be replaced by a hydrophobic pair. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 5009o (30 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B1, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.


Gel-filtration analysis of soluble adenylate cyclase from bovine corpus luteum. 1. Sepharose 6B gel-filtration analysis of soluble adenylate cyclase from bovine corpus luteum is described. Both zonal and frontal techniques of analysis were used. 2. Under conditions of zonal analysis recoveries of activity were low. It was concluded that dissociation of two or more components of the adenylate cyclase complex was occurring on the column and that the maintenance of the complex was essential for the high-activity state of the catalytic unit. Two peaks of adenylate cyclase activity, of approximate mol. wts. 45,000 and 160,000 were detected. 3. The theory of frontal analysis (or steady-state gel filtration), applied to the study of the interacting components of the adenylate cyclase complex is discussed, and activity profiles are predicted. Activity profiles obtained experimentally be frontal analysis compared well with the theoretically predicted profile and provide evidence that dissociation of a high-activity complex, with concomitant loss of activity, does occur. Recoveries of activity under conditions of frontal analysis were higher than with zonal analysis. 4. The effects of concentration and removal of detergent on the activity of the soluble enzyme are discussed.


Extraction and biochemical characterization of a nuclear deoxyribonucleic acid polymerase activity in bull spermatozoa. Bull spermatozoa heads were separated from cytoplasmic contaminants, especially mitochondria-rich middle pieces, by centrifugation through 2.4M-sucrose. DNA polymerase activity was demonstrated by incubating nuclear heads for 1 h at 37 degrees C or for 20 h at room temperature in a medium containing detergent and dithiothreitol or 2-mercaptoethanol. Optimal DNA polymerase activity was detected after extraction in a medium containing 50 mM-borate, pH9, 1 mg of soya-bean trypsin inhibitor/ml and supplemented with either 20 mM-dithiothreitol and 4% Tween 80 or 100mM-2-mercaptoethanol and 10% Tween 80. The DNA polymerase reaction was Mg2+-dependent; Mn2+ or Ca2+ could not replace Mg2+ and all four deoxynucleoside triphosphates were required for optimal activity. The polymerase activity was pH-dependent (optimum between 8.2 and 10.5) and was a function of buffer composition and also of pH values. Optimal activity was obtained with 50 mM-Na+ or 150mM-K+ and was partially lowered by N-ethylmaleimide; it was inhibited by spermidine and by salmon protamines, but was greatly stimulated by calf thymus histones. It was also resistant to actinomycin D, netropsin and ethidium bromide. The present results suggest that bull spermatozoa heads contain a beta-type DNA polymerase activity.


Presence of two deoxyribonucleic acid polymerases in bull spermatozoa. A DNA polymerase-endogenous template complex was isolated from nuclear heads of bull spermatozoa. The buoyant density of the complex was 1.15 g/cm 3. The sedimentation coefficient of the nuclear DNA polymerase isolated from the complex was higher at low ionic strength, but approached 3.4S when centrifuged in a medium containing 2M-KCl. Activated exogenous DNA increased polymerase activity. Only very low activities were detected with synthetic templates such as poly(A).(dT)12-18 and poly(dT).poly(A). The nuclear reaction was stimulated by 150mM-KCl and was slightly inhibited by N-ethylmaleimide; it was resistant to actinomycin D, netropsin and ethidium bromide. Another DNA polymerase, highly sensitive to ethidium bromide, was extracted from the mitochondira-rich middle-piece fraction. Its sedimentation coefficient was close to 9S, but fell to approx. 4S in high-ionic-strength medium.


Modification by liposomes of the adenosine triphosphate-activating effect on adenylate deaminase from pig heart. Adenylate deaminase (AMP deaminase, EC 3.5.4.6) of a high substrate specificity was purified from pig heart by chromatography on cellulose phosphate. The enzyme shows a co-operative binding of AMP [h (Hill coefficient) 2.35, with SO.5 (half-saturating substrate concentration) 5mM]. ATP and ADP act as positive effectors, lowering h to 1.55 and SO.5 to 1 mM. The addition of liposomes (phospholipid bilayers) to ATP-activated or ADP-activated enzyme causes a further shift of the h value to 1.04 and SO.5 to 0.5 mM. For ATP-activated enzyme the addition of liposomes increases Vmax. by about 100%, and for ADP-activated enzyme by 50%. Liposomes have no effect on the kinetics of AMP deaminase in the absence of ATP and ADP, and neither do they influence the inhibitory effect of orthophosphate on heart muscle AMP deaminase. Metabolic implications of these findings are discussed.


The N-terminal sequences of blood coagulation factor X1 and X2 light chains. Mass-spectrometric identification of twelve residues of gamma-carboxyglutamic acid in their vitamin K-dependent domains. Peptides (residues 1-42) (bovine prothrombin numbering) from bovine Factor X1 and X2 have been separately purified and digested before mass-spectrometric sequence assignment and identification of gamma-carboxyglutamic acid. N-terminal sequence was found to be identical, and 12 residues of gamma-carboxyglutamic acid were unambiguously identified. The new data give conclusive evidence for the N-terminal primary structure of bovine Factor X and extend present knowledge to show (i) unambiguous assignment of gamma-carboxyglutamic acid residues, including a previously unreported residue of gamma-carboxyglutamic acid at position 40 in both Factor X1 and X2, (ii) the physical difference between Factors X1 and X2 is not due to either different amino acid sequences or different gamma-carboxyglutamic acid contents of the N-terminal 42 residues.


P-cresol and 3,5-xylenol methylhydroxylases in Pseudomonas putida N.C.I.B. 9896. Pseudomonas putida N.C.I.B. 9869, when grown on 3,5-xylenol, hydroxylates the methyl groups on 3,5-xylenol and on p-cresol by two different enzymes. 3,5-Xylenol methylhydroxylase, studied only in relatively crude extracts, requires NADH, is not active with p-cresol and is inhibited by cyanide, but not by CO. The p-cresol methylhydroxylase requires an electron acceptor and will act under anaerobic conditions. It was purified and is a flavocytochrome c of mol.wt. approx. 114,000 consisting of two subunits of equal size. The enzyme catalyses the hydroxylation of p-cresol (Km 16 micron) and the further oxidation of product, p-hydroxybenzyl alcohol (Km 27 micron) to p-hydroxybenzaldehyde. A different p-cresol methylhydroxylase of the flavocytochrome c type is induced by growth on p-cresol. It too was purified and has mol.wt. approx. 100,000, and again consisted of two equal-size subunits. The Km for p=cresol 3.6 micron and for p=hydroxybenzyl alcohol, 15 micron.


The aromatic alcohol dehydrogenases in Pseudomonas putida N.C.I.B. 9869 grown on 3,5-xylenol and p-cresol. Whole cells of Pseudomonas putida N.C.I.B 9869, when grown on either 3,5-xylenol or p-cresol, oxidized both m- and p-hydroxybenzyl alcohols. Two distinct NAD+-dependent m-hydroxybenzyl alcohol dehydrogenases were purified from cells grown on 3,5-xylenol. Each is active with a range of aromatic alcohols, including both m- and p-hydroxybenzyl alcohol, but differ in their relative rates with the various substrates. An NAD+-dependent alcohol dehydrogenase was also partially purified from p-cresol grown cells. This too was active with m- and p-hydroxybenzyl alcohol and other aromatic alcohols, but was not identical with either of the other two dehydrogenases. All three enzymes were unstable, but were stabilized by dithiothreitol and all were inhibited with p-chloromercuribenzoate. All were specific for NAD+ and each was shown to catalyse conversion of alcohol into aldehyde.


The assembly of early components of complement on antibody-antigen aggregates and on antibody-coated erythrocytes. Radioimmune assays were developed to assay the binding of complement components C1q, C1s and C4 to antibody aggregates and to cell-bound antibody. The binding of the components was compared with the haemolytic activity and with the capacity to form the C3 convertase activity in the presence of excess C2. The destruction of whole complement and of C4 activity is similar per 1,000 molecules of antibody in aggregates and cell-bound antibody, as is the binding of C1g and C1s, the latter being in a 1:2 molar ratio. The binding of C4 is about 12 times greater, per 1,000 molecules of antibody, on cells than in aggregates. However, the effective C4 molecules, as judged by the formation of C3 convertase activity, are much more similar on cells and aggregates. An assembly mechanism of the early components of complement on antibody-coated cells, which is compatible with these results, is suggested.


A radioimmunoassay for chicken avidin. Comparison with a [14C]biotin-binding method. A double-antibody solid-phase radioimmunoassay for chicken avidin is reported. Avidin was labelled with 125I by the chloramine-T method. The bound and free avidin were separated with a second antibody bound to a solid matrix. In the logit-log scale the standard curve was linear from 1-2 to 100-200ng of avidin/ml. Cross-reaction of ovalbumin was less than 0.015%. Saturation of biotin-binding sites of avidin with an excess of biotin decreased radioimmunoassay values by about 15%. Recovery studies indicated that avidin can be assayed from all chicken tissues studied with radioimmunoassay, whereas the [14C]biotin/bentonite method gave poor recoveries for avidin in the liver and kidney. Radioimmunoassay and the [14C]biotin/bentonite method gave similar concentrations for oviduct avidin.


The molecular-weight-dependence of the anti-coagulant activity of heparin. It is proposed that the anti-coagulant activity of heparin is related to the probability of finding, in a random distribution of different disaccharides, a dodecasaccharide with the sequence required for binding to antithrombin. It is shown that this probability is a function of the degree of polymerization of heparin. The hypothesis has been been tested with a series of narrow-molecular-weight-range fractions ranging from 5,600 to 36,000. The fractions having mol.wts. below 18,000 (comprising 85% of the original preparation) followed the predicted probability relationship as expressed by the proportion of molecules capable of binding to antithrombin. The probability that any randomly chosen dodecasaccharide sequence in heparin should bind to antithrombin was calculated to 0.022. The fraction with mol.wt. 36,000 contained proteoglycan link-region fragments, which may explain the deviation of the high-molecular-weight fractions from the hypothetical relationship. The relationship between anti-coagulant activity and molecular weight cannot be explained solely on the basis of availability of binding sites for antithrombin. The activity of high-affinity heparin (i.e. molecules containing high-affinity binding sites for antithrombin), determined either by a whole-blood clotting procedure or by thrombin inactivation in the presence of antithrombin, thus remained dependent on molecular weight. Possible explanations of this finding are discussed. One explanation could be a requirement for binding of thrombin to the heparin chain adjacent to antithrombin.


An estimate of the enthalpic contribution to the interaction between dextran and albumin. It is demonstrated that exclusion phenomena appear to dominate the interaction of dextran with albumin in aqueous solution. The enthalpic contribution to the interaction coefficient describing dextran/albumin mixtures is small, although its determination was subject to considerable error. These results support the earlier assumptions of the type of interaction between the two polymers. The conclusions are primarily based on the interpretation of the temperature-dependence of the interaction coefficient, as measured by light-scattering in the temperature range 6--33 degrees C. Enthalpy of dilution measurements of dextran/albumin mixtures by microcalorimetry were in qualitative agreement with the light-scattering data.


Resonance Raman-spectroscopic studies of the hapten features involved in the binding of 2,4-dinitrophenyl haptens by the mouse myeloma proteins MOPC 315 and MOPC 460. The binding of four dinitrophenyl haptens to the mouse myeloma proteins MOPC 315 IgA (immunoglobulin A) and MOPC 460IgA was studied by resonance Raman spectroscopy. Isotopic substitution with 15N and 2H was used to assign features in the resonance Raman spectra of the free haptens. Changes in each of these features on binding to the proteins could then be attributed to interactions of the proteins' binding sites with either the p-NO2 or the o-NO2/amine regions of the haptens. The interactions between a given hapten and MOPC 315 IgA are often quite distinct from those between the same hapten and MOPC 460 IgA. Moreover, for both antibodies the nature of the R side chain in a Dnp-NHR (Dnp, 2,4-dinitrophenyl) compound appears to modify the interactions between the Dnp chromophore and the protein. Thus, with the haptens studied, there is no unique set of contacts between the Dnp group and the binding site. The contacts expected between epsilon-2,4-dinitrophenyl-L-lysine and the site on MOPC 315 IgA, on the basis of a recent model for this site [Dwek, Wain-Hobson, Dower, Gettins, Sutton, Perkins & Givol (1977) Nature (London) 266, 31--37] were not detected. However, the contacts between this hapten and the site on MOPC 460 IgA were closer to those predicted by the model for MOPC 315 IgA.


Further characterization of galactosylhydroxylysyl glucosyltransferase from chick embryos. Amino acid composition and acceptor specificity. A modified purification procedure, consisting of affinity chromatographies on concanavalin A-agarose, collagen-agarose and UDP-glucose-derivative-agarose and one gel filtration, is reported for galactosylhydroxylysyl glucosyltransferase. The enzyme obtained is entirely pure when studied by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The enzyme protein was rich in glutamic acid + glutamine, aspartic acid + asparagine, glycine and alanine. The enzyme catalysed no significant glucose transfer to any of the glycoproteins tested, except for collagens. This included all the glycoproteins that have previously served as glucosyl acceptors for impure enzyme preparations, thus indicating a high degree of specificity of the enzyme for galactosylhydroxylysine. Galactosylsphingosine would act as a glucosyl acceptor, however. This compound has a close structural similarity to galactosylhydroxylysine in that they both have an unsubstituted amino group next to the hydroxy group to which the galactose is attached.


Mechanism of the reaction of papain with substrate-derived diazomethyl ketones. Implications for the difference in site specificity of halomethyl ketones for serine proteinases and cysteine proteinases and for stereoelectronic requirements in the papain catalytic mechanism. The reactions of papain (EC 3.4.22.2) with substrate-derived diazomethyl ketones reported by Leary, Larsen, Watanabe & Shaw [Biochemistry (1977) 16, 5857--5861] are unusual in that (i) these reagents fail to react with low-molecular-weight thiols and (ii) the rate of reaction with the papain thiol group does not decrease to near-zero values across a pKa of 4 as the pH is decreased. Existing data are shown to suggest an interpretation involving neighbouring-group participation via transient thiohemiketal formation, rate-determining protonation by imidazolium ion and alkylation on sulphur via a three-membered cyclic transition state. Implications for (a) the difference in site-specificity exhibited by halomethyl ketones in their reactions with serine proteinases and cysteine proteinases and (b) stereoelectronic requirements in the mechanism of papain-catalysed hydrolysis are discussed. The possibility of two tetrahedral intermediates between adsorptive complex and acyl-enzyme is indicated.


Purification and properties of peroxisomal pyruvate (glyoxylate) aminotransferase from rat liver. Pyruvate (glyoxylate) aminotransferase from rat liver peroxisomes was highly purified and characterized. The enzyme preparation has a mol.wt. of approx. 80,000 with two identical subunits, and isoelectric point of 8.0 and a pH optimum between 8.0 and 8.5. The enzyme catalysed transamination between a number of L-amino acids and pyruvate or glyoxylate. The effective amino acceptors were pyruvate, phenylpyruvate and glyoxylate with serine, and glyoxylate and phenylpyruvate with alanine as amino donor. These properties and kinetic parameters of the enzyme are remarkably similar to those previously described for mitochondrial alanine-glyoxylate aminotransferase isoenzyme 1 from glucagon-injected rat liver [Noguchi, Okuno, Takada, Minatogawa, Okai & Kido (1978, Biochem. J. 169, 113-122].


Characterization of the mutant alpha-mannosidase in bovine mannosidosis. Residual acidic alpha-mannosidase, varying in amount up to approx. 15% of normal values, can be measured in various organs of a calf with mannosidosis. The highest specific activity and relative proportion of residual activity were found in the liver. Chromatography on DEAE-cellulose showed that the residual activity was associated with two components, which were eluted at comparable positions with those found in normal tissues. The residual activity had a lower thermal stability and a higher K(m) value for a synthetic substrate than did the normal enzyme. No differences in molecular weight or electrophoretic mobility between normal acidic alpha-mannosidase and the residual activity were observed by gel filtration and electrophoresis on cellulose acetate respectively. The isoelectric focusing profiles for the alpha-mannosidase in the normal and pathological livers were very similar. It is suggested that a mutant enzyme, resulting from a mutation in a structural gene, accounts for the residual acidic alpha-mannosidase in mannosidosis. The mutant enzyme, which cross-reacts with antiserum raised against normal bovine acidic alpha-mannosidase, is present at a decreased concentration compared with the normal enzyme. There is a correlation between the concentrations of residual activity and cross-reacting material in mannosidosis. alpha-Mannosidase with a pH optimum of 5.75 and which is activated by Zn(2+) was also detected in the liver of the calf with mannosidosis. However, it is probably not a product of the defective gene because addition of Zn(2+) indicated that it was also present in normal tissues.


p-NN'-phenylenebismaleimide, a specific cross-linking agent for F-actin. Covalent cross-links can be inserted between the subunits of F-actin by using p-NN'-phenylenebismaleimide. Cross-linking reaches its maximum value when one molecule of reagent has reacted with each actin subunit. p-NN'-Phenylenebismaleimide reacts initially with a cysteine residue on one subunit, the slower cross-linking reaction involving a lysine residue on a neighbouring subunit. Hydrolysis of the actin-bound reagent limits the extent of cross-linking. Quantitative analysis of the amounts of cross-linked oligomers seen on polyacrylamide gels containing sodium dodecyl sulphate suggests that neither the binding of the reagent to actin nor the formation of cross-links introduces strain into the structure. The cross-links do not join together different F-actin filaments, and evidence is presented that suggests that the cross-links join subunits of the same long-pitched helix.


Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues. Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.


Structure revision of disaccharidic conjugates of bilirubin-IX alpha in human bile and identification of phenylazo derivatives B4, B5, and B6 as 2-, 3- and 4-O-acylglucuronides. Aniline azopigments B4, B5 and B6, derived from conjugates of bilirubin-IX alpha in human bile, and previously characterized as disaccharidic esters [Kuenzle (1970) Biochem. J. 119, 387-394 and 411-435], were analysed by using t.l.c. and mass spectrometry. The compounds were identified as partially separated mixtures of 2-, 3- and 4-O-acylglucuronide positional isomers. The 1-O-acylglucuronide was not detected in the mixtures and was the only compound hydrolysed with beta-glucuronidase. Further scrutiny of structural assignments made by Kuenzle [(1970) Biochem. J. 119, 411-435] led to identification of the lactone and hexuronic acid derivatives that were obtained from azopigment B5 along with glucuronolactone and glucuronic acid. A branched-chain structure, i.e. 3-C-hydroxy-methyl-D-riburonic acid, was assigned previously, but the derivatives have now been identified as various incompletely silylated forms of glucuronolactone and glucuronic acid. Several trimethylsilyl derivatives glucuronolactone were isolated and characterized by n.m.r. and mass spectrometry.