The role of right heart angiography in the management of selected cases. Three cases of Wilms' tumor with inferior vena cava involvement are presented. All three cases were shown to have nonvisualization of the inferior vena cava on the initial intravenous pyelogram obtained by internal saphenous vein injection. The first case ended fatally, due primarily to delay in diagnosing extension of the tumor into the right atrium. There was no cardiac involvement in the other two cases, both of which were managed successfully. A right heart angiogram demonstrated intraatrial tumor in the first case and helped to identify the presence and extent of caval involvment in the other two. It is recommended that right heart angiography be performed in those cases of Wilms' tumor where the inferior vena cavagram shows non-visualization of the cava or suggests the presences of intraluminal tumor. The immediate availability of two separate surgical teams in the management of such cases is suggested.


[The fixing complement antibodies to respiratory viruses--influenza A1, B, C, parainfluenza 1 (Sendai), syncytial respiratory, Adenovirus, Echo 11--and "Mycoplasma pneumoniae" in healthy persons (author's transl)]. Serological research of complement fixation test (Kolmer micromethod) into 163 young healthy subjects (18-22 years old) in order to point out the contaminations effects of respiratory viruses and of Mycoplasma pneumoniae. In is pointed out serological simultaneous reactions on some subjects, expression of multiple infections; the different frequency of serological tests for each antigen, pointing out the rôll played by the singles diseases, the different titre distribution of the antibodies to 1:4 to 1:128. It is pointed out the value of the antibodies patrimony in existence in healthy persons, in order to prevent the diseases caused by virus and Mycoplasma pneumoniae. It is stressed the rôll of some mammalia and birds as hosts of human influenza virus, subjects of mutation in such organismes. The new virus attack the man with the virus pertinent to the animals, in special way to equins and birds.


Grand rounds: autoimmune hemolytic anemia. Autoimmune hemolytic anemia (AHA) may be either primary (ie, "idiopathic," one third of all patients) or secondary (ie, associated with underlying illness, two thirds of all patients). A positive Coombs antiglobulin test is the most important criterion for diagnosis of AHA, and characterization of RBC coating (as to whether it is by IgG alone, by IgG plus complement, or by complement alone) may be valuable in ruling out certain underlying illnesses as causative in selected patients. Many limitations to the antiglobulin test must be kept in mind. As routinely performed, a positive result requires greater than 500 molecules of IgG per RBC. Red blood cells from normal subjects have less than 35 molecules of IgG per RBC. Up to 2% to 5% of patients with AHA will have RBC coating in the 50 to 500 molecules per cell range and may therefore have "false"-negative antiglobulin tests. Apparent failures of strength of antiglobulin reactions to correlate with severity of in vivo RBC destruction may result from technical factors or may reflect intrinsic differences among autoantibodies (eg, IgG subclass, affinity). A likely mechanism of in vivo RBC destruction in AHA has been demonstrated in immune-mediated in vitro RBC "rosette" formation about macrophages and lymphocytes bearing specific receptors for subclasses of IgG and for subcomponents of complement. Increased avidity of macrophages for coated RBCs in response to such stimulus as infection may play an additional role. Treatment involves control of underlying disease and the judicious use of adrenal steroids, splenectomy, and immunosuppressive agents. Transfusions, while life-saving in life-threatening anemia, carry substantially increased risks in AHA patients. Their use should be strictly limited.


Focal nodular hyperplasia of the liver associated with high-dosage estrogens. A patient developed benign nodular hyperplasia of the liver after long-term, high-dose estrogen therapy. A 10.5-cm benign liver nodule appeared after three years of high-dose estrogen therapy and was resected. Fourteen months later, while still receiving estrogen, a 4.5-cm nodule and multiple small nodules were seen on angiography. Four months after estrogen was discontinued, the larger liver nodule disappeared and the smaller ones decreased.


Sulfonylurea-induced factitious hypoglycemia. A growing problem. Two patients had sulfonylurea-induced factitious hypoglycemia. Both patients demonstrated hyperinsulinism during hypoglycemia suggesting the presence of an insulin-secreting tumor. One patient underwent an exploratory laparotomy with subtotal pancreatectomy before the etiology of the hypoglycemia was discovered. Inboth patients, the diagnosis was made by detecting sulfonylurea agents in blood. A survey of Portland, Ore metropolitan hospitals suggests that factitious hypoglycemia occurs with a frequency similar to the insulinoma syndrome. The biochemical similarity of these disorders and the apparent increasing incidence of factitious hypoglycemia suggests that blood determinations of sulfonylurea agents should be performed prior to exploratory laparotomy for an insulinoma.


A mathematical model for the mechanism of rapid eye movements induced by an anticholinesterase in the decerebrate cat. The oculomotor pattern which appears in intact preparations during desynchronized sleep is characterized by the irregular occurrence of isolated ocular movements and bursts of rapid eye movements (REM). This complex oculomotor pattern results from the activity of two premotor structures which influence the extraocular motoneurons during this phase of sleep: one is located in the pontine reticular formation, the other in the vestibular nuclei. In the decerebrate preparation the intravenous injection of an anticholinesterase leads to the appearance of a typical pattern of oculomotor activity, which differs from that occurring during physiological sleep in so far as it consists quite exclusively of bursts of REM which appear at very regular intervals. Lesion experiments as well as unit recordings have shown that these bursts of REM depend in particular upon rhythmic discharges of the vestibular nuclear neurons. The underlying anatomical structures responsible for these bursts of REM are therefore the vestibular nuclei, the oculomotor nuclei and the oculo-orbital system. This mechanism is under the influence of cholinergic reticular neurons which generate the oculomotor rhythm. We have postulated the existence of a self-excitatory cholinergic system, located in the pontine reticular formation, whose steady discharge impinges upon an oscillatory neuronal system located in the dorso-lateral pontine tegmentum, which transforms the tonic input into a sinusoidal final output. We have assumed also that the periodic increases in the discharge frequency of this oscillatory system trigger a fast phase generator acting on the different components of the REM system, and that the behavior of each component follows a first-order differential equation. The state of excitation of the components of the system is defined as proportional to frequency of nerve impulses. Assuming ipsilateral and crossed connections, a pattern of oculomotor activity is obtained that simulates the experimental oculomotor output fairly well. The repetition of the eye jerks is described by a Fourier series. The model proposed in this paper may be taken as a first approach in describing the generation mechanism of REM, and as a theoretical guide to new experimental researches and the development of other more realistic models.


Selective discharge of pontine neurons during the postural atonia produced by an anticholinesterase in the decerebrate cat. I. Episodes of postural atonia associated with bursts of REM similar to those which occur spontaneously either in the intact preparation during desynchronized sleep, or in the chronic decorticate or decerebrate preparations, can be elicited in acute decerebrate cats following intravenous injection of small doses of an anticholinesterase. The present experiments were performed in precollicular decerebrate animals in order to identify the pontine neurons which show increases in their firing rate related in time with the appearance of the cataplectic episodes. In particular long-term recordings of single units were obtained before, during and after the episodes of postural atonia produced by i.v. injection of 0.03-0.1 mg/kg of eserine sulphate. Spontaneous discharge rates were used to measure the selectivity of each individual unit, i.e., the tendency of the unit to discharge more during the cataplectic episode than during the postural rigidity. The physiological data obtained from neurons histologically localized in different nuclear groups were then averaged. 2. Neurons localized in the pontine reticular formation as well as in the region of the locus coeruleus and the raphe system showed low rates of discharge when rigidity was present. The same units, however, showed a remarkable increase in firing rate which preceded by several tenths of seconds the onset of postural atonia and lasted throughout the cataplectic episodes. 3. The neurons of the pontine reticular formation had a selectivity which was higher than that of the neurons located in the locus coeruleus-raphe system; moreover the cells of the gigantocellular tegmental field (FTG) had the highest selectivity of all pontine reticular structures studied. 4. The relation of the discharge rate curves to the occurrence of the cataplectic episodes suggests that these neurons constitute output elements of a generator system for postural atonia. It is postulated that these pontine reticular neurons are directly involved in the activation of the bulbospinal inhibitory system, which is finally responsible for the abolition of the decerebrate rigidity. 5. During cataplectic episodes these pontine neurons showed some clustered discharges which appeared in association with bursts of eye movements. In most instances, however, there was no constant relationship of the unit activity to individual eye movements. Moreover large phasic increases in firing rate appeared also during the intervals between successive bursts of REM. 6. The striking increase in firing rate of the FTG neurons observed during the cataplectic episodes cannot be attributed to an increased excitatory input to these neurons. In fact excitatory influences following intense somatic stimulation are unlikely to occur during the cataplectic episodes; moreover the response of these neurons to intense somatosensory stimulations did not reach rates comparable with those occurring spontaneously during the induced cataplectic episodes...


The oscillatory system responsible for the oculomotor activity during the bursts of REM. 1. In precollicular decerebrate cats the electrical activity of single pontine neurons was recorded before, during and after the episodes of postural atonia produced by i.v. injection of 0.03-0.1 mg/kg of eserine sulphate. These episodes were characterized by the regular occurrence of horizontal conjugate eye movements, which were mainly grouped in bursts of REM; moreover, a burst of REM in one direction was generally followed by a burst of REM in the opposite direction. 2. Among the recorded units, 32 showed an increase in their discharge rate during these cataplectic episodes. However, while these units fired at regular frequency when postural rigidity was present, they showed periodic changes in their discharge rate as soon as the bursts of REM appeared in the electrooculogram. In particular a nearly sinusoidal increase in the discharge rate was related to the appearance of an ocular burst in one direction, while a decrease in the unit discharge occurred during an ocular burst in the opposite direction. In some instances neighbouring pontine units located within each side of the brain stem showed reciprocal rate profiles during REM bursts oriented in a given direction, making it likely that the cyclic alternation of their activity depended upon their reciprocal interaction. 3. The alternative hypothesis, i.e., that these periodic changes in unit discharge depend upon the proprioceptive feedback due to the eye movements was excluded by the fact that these changes started before the occurrence of the bursts of REM and began to decline before the end of the burst. Moreover no variation in their firing rate was observed during the positional nystagmus induced by tilting the animal in the control period, i.e., when postural rigidity had reappeared following the end of the cataplectic episode. 4. Most of the neurons showing periodic changes in their discharge frequency during the bursts of REM were located in the pontine reticular formation. Scattered units were also found within the region of the locus coeruleus and the raphe system, close to the surrounding reticular structures. 5. In addition to these neurons, 60 pontine units were recorded, which did not show any changes in their discharge rate during transition from the control period to the cataplectic episode. However, phsiic increases or phasic decreases in their discharge rate appeared synchronously with the individual eye movements. Since in most instances these phasic changes in unit activity coincided with the appearance of the individual monophasic potentials recorded from the ascending MLB, which immediately preceded the rapid eye movements, these units could be attributed either to the premotor neurons responsible for these REM or to the closely related structures which generate their rhythmic discharge. In only a few instances did the discharge of these units not precede but follow the individual eye movements, indicating that they resulted from a proprioceptive feedback originating during the eye movements. 6...


Sequential discharges of phasic activities (PGO waves) during paradoxical sleep after selective cortical lesions in the cat. We investigated the role that cortical areas have upon the sequences of PGO waves and associated phasic activity in the lateral rectus muscles of the eyes (PALRE) during PS. We performed several combined type of lesions (bilateral ablations of frontal lobes carried out in two sessions, or occipital lesions combined with bifrontla lobotomies). We used statistical methods previously described for the normal cat (4). We showed that frontal lesions need to be bilateral in order to be effective, that thereafter there is a recovery in the complexity of the patterns in the survival period. We showed that occipital lobectomies "per se" do not affect the PGO pattern, but their combination with bifrontal lobotomies produce the most dramatic changes which lasted throughout the survival period. These findings are discussed on the light of the well known anatomical cortical projections to brain stem structures which participate in the determinism of PGO waves during paradoxical sleep.


Polyhedral inclusion bodies in cells of Nitrosomonas spec. Polyhedral inclusion bodies were observed in cells of a Nitrosomonas species. They were present in growing cells as well as in resting cells. In thin sections their size was about 130 nm in growing cells and about 185 nm in diameter in resting cells. The bodies were commonly located in the nucleoplasm. They appeared to be bounded by a nonunit membrane and had a granular substructure. In thin sections about 70% of the exponentially grown cells and about 20% of the resting cells of the investigated strain showed 1-7 respectively 1-3 inclusion bodies.


[Various physical and chemical properties of the 73s unit of the foot-and-mouth disease virus]. At all 3 studied FMD-viruses typs O2, A5 and C we could show the 73S unit in the analytical ultracentrifuge and in the electron microscope. 73S unit is found in the normal cycle of purification of virus and by density gradient centrifugation separated and purified. In CsCl pH 7.6 its density is 1.308 +/- 0,005 g/ml. Its sedimentation coefficient has a value of 72.7 +/- 1,5S. In electron microscope it show itself as a empty virus capsid. Its diameter is in partial purified preparations with 25 +/- 1 nm the same as of the virion. Its wall diameter is 2 to 3 nm. Further purification induced defiguration of particles and increase of its diameter. 73S unit dissociates in 19S and 12S units and shows a typical protein-UV-absorption spectrum with a maximum at 276 to 278 nm and a minimum at 250 nm. Emax/Emin is 2.3. Extinction coefficient E276nm is 1,4 mg/cm2. By sucrose density gradient centrifugation and titration of fractions in the complement fixation test it was detected, that croude virus solution contained already the 73S unit.


[Experimental reproduction of necrotic enteritis in the chicken. 1. Mono- and polyinfections with Clostridium perfringens and coccidia with reference to cage managemen]. Experimental mono-infection and poly-infection, using vegetative germs of a CL. perfringens Type A strain 1663 and its toxin as well as E. acervulina, necatrix or mitis oocysts, were applied to 100 SPF chicken aged seven days. While clostridial or coccidial mono-infection did not cause any loss and only unspecific pathologic-anatomic changes, polyinfection was accompanied by diarrhoea and slower weight increase, with 55.4 per cent of the chicken having been lost three to nine days from the outbreak of the infection. The pathologic-anatomic findings recorded from the chicken with poly-infections included haemorrhagic, ulcerative, and necrotic intestinal inflammations, primarily in the small intestin. The results of these experimental infections indicated a possible correlation between CL. perfringens and coccidia in the pathogenesis of necrotic enteritis.


[Experimental reproduction of necrotic enteritis in the chicken. 2. Further mono- and polyinfections with CL. perfringens and coccidia with special reference to ground-kept chickens]. Coccidial oocysts and/or vegetative germs, spores, and toxins of Cl. perfringens Type A were used in mono-infection and poly-infection experiments on SPF chicken aged seven and 56 days and kept under different conditions. Necrotic enteritis was regularly reproduced in all experimental groups with polyinfections. In chicken aged seven days necrotic enteritis was reproduced even after repeated mono-infections with 4 X 10(9) gerus and toxin of Cl. perfringens. The loss figures recorded from ground-kept infected groups were higher than those established from the cage-kept animals. The pathologica-anatomic findings recorded from the dead chicken included necrotic, ulcerative, and catarrhal to haemorrhagic intestinal inflammations, with necrotic enteritis being most strongly pronounced in the ileum and jejunum. All infected groups lost not only animals but weight as well. Their average weight on the 21 st day from the onset of infection was up to 26.4 per cent lower than that of the control groups.


Temporal effects of drug and placebo in delaying relapse in schizophrenic outpatients. The potential for unwanted side effects, particularly tardive dyskinesia, following long-term after care maintenance with antipsychotic medication has led to serious questioning of its continued use for schizophrenic patients. Does the risk of relapse decline with the passage of time? If so, is the advantage of drug therapy sustained and large enough to justify continued treatment beyond one or two years? More appropriate methods for analyzing the after care experience of 374 schizophrenic patients treated with drug or placebo reveals that while the risk of relapse does decline substantially, it remains twice as high for placebo-treated patients than drug-treated patients even after two years of treatment. The prophylactic effect of maintenance chemotherapy appears to be one to two times larger than generally estimated by the less precise "cumulative percentage" method.


Short vs long hospitalization: a prospective controlled study. VI Two-year follow-up results for schizophrenics. A controlled, prospective study examined the relative effectiveness of short-term versus long-term psychiatric hospitalization. The results of a two-year follow-up of a sample of 141 schizophrenic patients are reported here. The differences favoring long-term subjects that were apparent at one year postadmission had decreased by two years postadmission. However, there appears to be an interaction between prehospital functioning and length of hospital stay, with subjects who had good prehospital functioning doing better at two years when assigned to long-term hospitalization. Subjects with poor prehospital functioning did about equally well, regardless of length of stay, and may even have showed some tendency to do better with a shorter hospital stay. This reversal of effect was more prominent for women, although this sex difference was not statistically significant.


[The vehaviour of monoaminooxydase (MAO) activity in tumours. I. MAO activity measurement in experimental tumours (author's transl)]. The activity of MAO (EC 1.4.3.4) was measured in liver homogenates of mice with experimental tumours Sarcoma S-180 and Leukemia L-1210. The enzyme activity was determined by two methods: spectrophotometric--with benzylamine as a substrate and the second with the application of oxygen electrode and adrenaline as a substrate. An increase of the enzyme activity was observed in liver homogenates of mice with Sarcoma S-180 as compared with the controls. High activity of MAO with both substrates was also observed in Sarcoma ascites. In Leukemia L-1210 changes of activity in relation to adrenaline as a substrate were very small, but towards benzylamine the affinity of enzyme was higher (the increase of activity was about 50%).


Liver tumors induced by 4-dimethylaminoazobenzene: experimental basis for a chemical carcinogenesis concept. In animals fed 4-dimethylaminoazobenzene there are modifications in the distribution of extra- and intracellular cations in liver. The correlation between the cell ionic permeability changes and the induction of cholangiocarcinoma or hepatoma is analyzed at the light of the histological and biochemical findings. A new concept of chemical carcinogenesis is stated in which the increase of passive Ca2+-influx produces mitochondria damage associated with permanent modifications of the structural and functional characteristics of the cell membranes as well as of the genetic mechanisms controlling cell division.


[Tumorigenicity of TTT dissolved in DMSO (author's transl)]. In short-term experiments TTT (trinitroso-trimethylene-triamine) dissolved in DMSO (dimethylsulfoxide) caused, when orally or intraperitoneally applied, a significant enhancement of the mitotic rate in the adrenal cortex. By long-term studies the carcinogenic action of TTT in DMSO was demonstrated. In female Wistar rats a 100% liver tumor yield was observed following chronic application. After 362 days of treatment cholangiomas had developed, hepatomas and liver carcinomas occurred after 470 days of administration of TTT dissolved in DMSO.


Cytomegalovirus endolabyrinthitis.

Cytomegalovirus endolabyrinthitis. A premature male infant, who died 22 days after birth with hyaline membrane disease, was found to have had cytomegalic inclusion disease at autopsy. Histopathologic examination of the temporal bones showed cytomegalovirus (CMV) infection of the entire endolabyrinth without involvement of the neural and sensory structures. These findings support the thesis that late gestational or perinatal fetal CMV infection results in an endolymphatic labyrinthitis. We hypothesize that blood-borne virus passes from the stria vascularis into the endolymphatic spaces and infects the nonneurosensory epithelium. This pattern of infection differs from the perilabyrinthitis of human varicellazoster and experimentally produced mouse CMV.


Selective deprivation of sleep in pycnoleptic children. Effects of deprivation of slow-wave and REM sleep on the frequency and duration of petit mal attacks. 1. Selective deprivation of slow-wave and paradoxical sleep was performed in 10 children with pycnoleptic attacks (8 of them before anticonvulsive treatment, 2 of them while under medication). The frequency and duration of petit mal attacks were intraindividually compared during night sleep and after waking for a 5-h period. 2. After deprivation of slow-wave sleep with reduction of EEG stages 3 and 4 to about one-third of the baseline but normal duration of sleep, petit mal attacks are more frequent and long-lasting than after normal sleep or selective deprivation of REM sleep. 3. Although total sleep time is significantly diminished after selective deprivation of paradoxical sleep the frequency of attacks during the waking state was lower than after normal sleep and deprivation of slow wave sleep. This observation shows a clear i nfluence of the quality of sleep on the frequency of epileptic attacks. 4. During sleep petit mal seizures were mainly found during stages 2 and paradoxical sleep. Single spike and irregular spike were discharges, however, occurred more frequently during slow-wave sleep. Their frequency was not significantly different in the deprivation conditions. 5. In contrast to experimental data in animals, REM deprivation is less provoking to epileptic attacks outside sleep than deprivation of stages 3 and 4 sleep. Therefore a sufficient amount of slow-wave should be preserved for pycnoleptic children.


The value of healing by secondary intention for sizeable defects of the face. Sizeable facial wounds from trauma or tumor excision were allowed to heal by secondary intention in 20 patients. Because of its many advantages, including simplicity and satisfactory outcome as well as absence of donor site scarring, this most ancient mode of wound repair may be the procedure of choice under certain circumstances. The surgeon should not always feel compelled to use a flap or graft on the face when simple closure is not possible and when "nature" is available.


Paraganglioma simulating primary rib tumor. We report an unusual case of a nonchromaffin paraganglioma, 7 cm in diameter, presenting clinically within the thorax of a 7-year-old girl as a primary rib tumor. It is believed to be the first report of such a location for this tumor. The rib of origin and parts of the two adjacent ones were removed, along with the involved middle lobe of the lung. The possibility is proposed that the ganglion cells of origin were carried to the tumor site during development of the intercostal nerve. The defect in the patient's chest wall was repaired with Marlex mesh and there is no evidence of difficulty, nor of recurrence, four and one-half years later.


[Ultrastructural changes in the central nervous system of monkeys in poliomyelitis]. The ultrastructure of cells of the central nervous system of Rhesus monkeys in experimental poliomyelitis caused by virus of poliomyelitis Type 3 was studied. It was found out that the virus of poliomyelitis was localized in nerve cells, astocytes, oligodendrocytes, the endothelium of capillaries and macrophages. This justifies the conclusion that pathological changes in the cells referred to the above result from reproduction of the virus therein. Ultrastructural changes in infected cells are divided into three stages: the first stage corresponded to eclipse-phase of infection; the second -- to the period of reproduction of the virus and composition of viral particles; and the third -- to the period following the escape of the virus from the cell. A definite combination of nonspecific changes in organoids of various cells with specific viral inclusions makes the ultrastructural picture of cells in poliomyelitis sufficiently characteristic for differential diagnosis. Pathogenesis of the developing lesions is discussed.


[Cytospectrophotometric study of DNA content in dysplasia and breast cancer]. In displasia nuclei of the epithelial cells have diploid and in some cases also tetraploid set of chromosomes, the modal class of cells--paradiploid. In cancer there were noted poly- and heteroploidy changes in the modal class, which formed most often paratetraploid cells and more seldom--paraoctoploid ones. Comparison of intraductus cancer with invasive forms of carcinoma showed a displacement of the peak on the histogram towards greater and intermediate (within 4n and 8n) values of DNA, which was apparently associated with an increase in the number of aneuploid cells of the modal class as the tumour progressed. The average content of DNA in dysplasia was about 3n. In all forms of cancer it was significantly above (greater than 4n). Such average content of DNA and increase in the standard deviation (above 1.5) which reflects the degree of heteroplody, surve as objective indices of the onset of malignization.


[Problems of histogenesis and classification of nephroblastoma (Wilms' tumor) in children]. Nephroblastoma, a tumour of the kidney, is most often observed in children; it always includes the nephrogenous tissue in any variant of its differentiation. Histogenesis of this neoplasm is multiform and complex. Its development from very immature pluripotential (polypotent) cells (possibly, at the level of blastodermic vesicle) is not ruled out. The histological classification of nephroblastomas, offered by the author, is based on the principles of differentiation of the tumour tissue and on its histogenesis and opens new perspectives for further investigation of neoplasms and for clinico-anatomic collations.


[Ultrastructural changes in the liver in experimental hypothyroidism]. Electron microscopic studies of the liver of thyroidectomized rabbits were carried out. Ultrastructural changes in hepatocytes at various periods after the operation were established. Morphological changes in hepatic cells occurred already at early stages of hypothyrosis (1 month) and led to considerable dystrophyc processes at later stages of the experiment. In the hepatic tissue there was noted a marked myxedema, as well as a considerable intracellular edema which led to destruction of hepatocytes. The studies conducted revealed lesion of the morphological structure of the liver, impairment of general metabolic processes and of the hepatic function.


[So-called pseudolymphomatosis of the stomach]. An observation of a polymorphnocellular variant of pseudolymphomatosis of the stomach diagnosed retrospectively is described. Initially, the case was treated as lymphogranulomatosis of the stomach, and in connection with the latter the patient was given long-term immunodepressive therapy. At autopsy (6 years later) atrophy of the lymphoreticular tissue and mucinous carcinoma of the stomach were revealed. The authors hold that the second tumour developed against the background of the immuno-depressive therapy, whereas at the beginning the condition was pseudolymphomatosis.


Meningoencephalopathy secondary to infectious mononucleosis. Unusual presentation with stupor and chorea. We report a case of infectious mononucleosis in which central nervous system involvement was the presenting and sole manifestation of the disorder. The major symptomatology consisted of stupor, chorea, and signs of aseptic meningitis. We also discuss the clinical and laboratory features of the neurological manifestations of infectious mononucleosis and the theories as to its pathogenesis.


Reversible subacute peripheral neuropathy induced by phenytoin. Dysesthesia and sensory and reflex loss in the legs developed in a patient treated for seizures with phenytoin (diphenylhydantoin) for one year. Her serum drug levels had been in the therapeutic range, and no other clinical toxic reactions had been noted. The peripheral neuropathy resolved when phenytoin therapy was discontinued.


Polypeptides from serum low-density lipoproteins of pigs (Sus domesticus). Low-density lipoproteins floating between densities 1-006 and 1-063 g cm-3 were isolated by centrifugation of blood serum obtained from 24-h fasted pigs  (Sus domesticus). This lipoprotein fraction contained two components with Sf 1-063 values of 3-4 and 2-3 at 20 degrees C when examined by analytical ultracentrifugation. Delipidation of the lipoprotein yielded 15% recovery of soluble protein. Chromatography on Sephadex G100 in 8 M urea of these delipidation products yielded three fractions of different sizes which were present in both native and succinylated apoproteins. These fractions from the succinylated apolipoproteins were further characterized. A polypeptide fraction comprising 70% of the total protein had an apparent molecular weight of 34000 and contained greater amounts of amino acids with hydrophobic side chains than did the second fraction of apparent molecular weight 22000 which contained 15% of the protein. The third fraction of apparent molecular weight 12500 contained 15% of the protein.


Comparison of collagenase activity in eosinophil and neutrophil fractions from rat peritoneal exudates. The collagenase activity had been compared in extracts of eosinophils and of neutrophilss from peritoneal exudates in two groups of rats, one of which had been treated to augment the numbers of eosinophils and the other the numbers of neutrophils. The proportion of granulocytes to other cells in each preparation was increased by differential centrifugation over a continuous gradient. Collagenase was extracted from the fractions in which granulocytes were concentrated and the activity assayed by the radioactive fibril method. There was at least as much collagenase in the eosinophil-enriched extracts as in the neutrophil-enriched extracts. It is postulated that eosinophil collagenase may have a function in the remodelling of newly-synthesised collagen during the post-inflammatory phase of healing, since eosinophil leucocytes appear in significant numbers within the connective tissue during this phase. This suggests a different role for eosinophil collagenase than that for neutrophil collagenase, since neutrophil are present only in the early stages of inflammation, when collagen is being degraded.


Avian adenoviruses isolated from poultry in Queensland. Adenoviruses isolated from Queensland poultry flocks with respiratory disease and/or egg production problems have been serotyped by comparison with Japanese and Irish types. Two serological types were recognized among 14 isolates examined; 4 were type 112 (Celo type) and the remaining 10 type 506. Isolates B1363 and B4015 were further characterized by immunodiffusion. Both isolates were shown to contain the avian adenovirus group antigen and B1363 also shared a second (type) antigen with Celo (Phelps) strain. Immuno-electron microscopy revealed the presence of adenovirus group antigen and freedom from contamination with other viruses.


A mixed tumour and an adenoma both of ceruminous gland origin in a dog. Ceruminous gland tumours are uncommon in the dog and this paper reports a case of two such tumours which arose in either ear of a 12 year old male long-haired Dachshund. Histologically, one tumour was a ceruminous adenoma and the other a rare ceruminous mixed tumour of borderline malignancy.


The causal role of hypoalbuminemia in human nephrotic hyperlipidemia. Serum lipids and their lipoprotein fractions were measured in 16 nephrotic syndrome patients. All component of lipids and beta-lipoprotein fractions (LDL) showed an increase in all uncomplicated patients. The increase in serum lipids were inversely proportional to the albumen level in these patients. In 3 patients, lipids and beta-lipoprotein fractions returned to near normal after treatment, the proteinuria diminished and serum albumen became normal. Most of these patients did not need any treatment for hyperlipidemia.


A nonproducer T lymphoblastoid cell line from Marek's disease transplantable tumor (JMV). A continuous lymphoblastoid cell line was established from a JMV tumor transplant related to Marek's disease (MD). It is designated RPL1 (JMV) lymphoblastoid cell line. This cell line contains DNA sequences complementary to MD virus DNA and has an antigen similar to MD-tumor-associated surface antigen (MATSA). However, it lacks any MD virus (MDV) rescuable in vivo or in vitro. The cell line has surface antigens typical of chicken thymus cells (T cells) and histocompatability antigens different from those of the host chicken.


Pathogenesis of abnormal feathers in chickens inoculated with reticuloendotheliosis virus. Abnormal feathers, characterized by thinness and increased transparency of the calamus and rachis, and loss of barbs, were induced at a high frequency by inoculating day-old chicks with reticuloendotheliosis virus (REV) propagated in chicken-embryo fibroblast (CEF) cultures. The few birds that survived inoculation with oncogenic stock of REV derived from liver tissue of an infected chick developed similar abnormalities. Lesions of an inflammatory-degenerative type were observed in close association with the presence of viral antigen and numerous c-type virus particles, characteristic of REV, in the intermediate and cylindrical cell layers of all abnormal feathers examined. These findings, first detected in the intermediate and cylindrical cell layers of developing feathers 6--9 days after infection, suggest that degeneration and necrosis of feather-forming cells result from productive infection of REV, resulting in the development of the abnormal feathers.


Effect of maternal antibody on experimental infections of chickens with a type-8 avian adenovirus. Mortality was 60% when chickens without detectable maternal antibody to avian adenoviruses were inoculated intra-abdominally with 10(6) plaque-forming units of AMG 5(2a), a type-8 avian adenovirus. Other results were macroscopic and microscopic lesions in a wide range of organs, statistically significant depression of body weights, AMG 5(2a) virus in the liver and feces, and high virus-neutralizing antibody titers to AMG 5(2a). The disease produced was similar to that described in a previous report of AMG 5(2a) infection of chickens, and similar to inclusion body hepatitis as described in the literature. In contrast, similar inoculation of chickens with maternal antibody to type-8 avian adenovirus resulted in no mortality, lesions in the liver only, no depression of body weight, AMG 5(2a) virus in the feces only, and relatively low virus-neutralizing antibody titers. During this study a hemorrhagic-aplastic anemia syndrome occurred in both AMG 5(2a)-inoculated and control chickens in one trial. Pathologic, virologic, and serologic findings indicated that the spontaneously occurring disease was not caused by an avian adenovirus.


Involvement of a type-8 avian adenovirus in the etiology of inclusion body hepatitis. Type-8 avian adenoviruses were isolated from chickens in a commerical flock suffering an outbreak of inclusion body hepatitis. Serum-neutralizing titer to this type, but not to 7 other types of avian adenovirus, was more than 4 times as high in convalescing chickens as in chickens from the flock bled 2 weeks previously, during the disease outbreak. A disease similar to that in the commercial flock and to inclusion body hepatitis as described in the literature was produced by intra-abdominal inoculation of a type-8 isolant, AMG 5 (2a), into 1-day-old specific-pathogen-free chicks. Pathologic features of the disease included necrotizing hepatitis, pancreatitis, and severe lymphoid depletion of the bursa of Fabricius, thymus, and spleen. It was concluded that type-8 avian adenoviruses were involved in the etiology of the naturally occurring outbreak of inclusion body hepatitis.


[Chemotherapeutic activity of ifosfamide in 6 different isologously transplanted experimental tumours of BD-rats (author's transl)]. The chemotherapeutic activity of ifosfamide, applied i.v. at a high single dose, was tested on some transplantable tumours of BD-rats. As standard substance cyclophosphamide was used. The margin of safety of ifosfamide in DS-carcinosarcoma and TA-nephroblastoma was found to be wider than that of cyclophosphamide. Therefore, using massive single doses of ifosfamide, rats could be cured of DS-carcinosarcoma in higher percentage than by an analogous treatment with cyclophosphamide. Further, with a single i.v. dose of 280 mg/kg ifosfamide was it possible to cure all TA-nephroblastoma-bearing animals. The ANH-nephroblastoma, however, could not be cured even with the highest tolerable doses of ifosfamide. It was, nevertheless, possible to attain higher increase of life span over the controls than by cyclophosphamide-treatment. In some other isologous tumours ifosfamide proved to be less effective or without any activity, respectively. Pretreatment by repeated small doses of ifosfamide resulted in resistance of DS-carcinosarcoma against curative action of subsequent high single doses of this drug.


Tropine derivatives with central activities. Part II: Solvolysis of N-substituted nortropine methanesulfonates. The preparation and the reaction kinetical properties in solvolysis of a series of N-substituted nortropine methanesulfonates 1-8 are described. It was found that the rate of solvolysis depends on the nature of the N-substituents considerably. The solvolysis rates can be brought into correlation with the electron donating and withdrawing and steric properties of the N-substituents. Methane-sulfonates of other two bicyclic aminoalcohols 10, 11 were also prepared and kinetically studied.


[Novel Bis-sulfonic Acid Esters with Cytostatic Activity (author's transl)]. The introduction of electron-withdrawing groups into the group R of piposulfan analogs R--SO2--O--X--O--SO2--R substantially increases their antileukemic activity in animal tests (leukemia L 1210). The central moiety X can be varied considerably (provided it contains amide groups) without appreciable loss of activity. A number of busulfan analogs as well as busulfan itself proved to be practically inactive against murine leukemia L 1210. Among a total of 122 bis-sulfonic acid esters tested 35 gave a survival time index of 150% or more after i.p. application and 5 after oral application.


[Cardioprotection by oral application of the calcium antagonist fendiline. Prevention of isoproterenol-induced myocardial necroses in rats (author's transl)]. 1. N-(2-Benzhydryl-ethyl)-N-(1-phenyl-ethyl)-amine hydrochloride (fendiline, Sensit) -- in accordance with its characteristics as a Ca++-antagonist -- shows a strong cardioprotective effect in rats against isoproterenol-induced myocardial necroses. 2. Fendiline is effective via oral administration. 3. The protective effect of fendiline on the myocardium is demonstrated by means of histopathological and histoenzymatic techniques. 4. A particularly reliable method of quantifying the cardio-protective effect of a drug has proven to be the measurement of mitochondrial enzyme activity (cytochrome oxidase, succinate dehydrogenase) using histoenzymatic microphotometry.


Biochemical effects of ethyl apovincaminate. Some cerebrobiochemical effects of a new cerebrovasodilatory agent, ethyl apovincaminate (RGH-4405, Cavinton) have been studied. Changes of biogenic amines, 5-hydroxyindole acetic acid (5-HIAA) levels, serotonin (5-HT) turnover rate, furthermore effect on 3',5'-cyclic nucleotide phosphodiesterase (PDE; E.C.3.1.4.c) activity, isolated from different tissues, were determined. Lasting increase of cerebral 5-HIAA level was observed after treatment with the compound, 5-HT levels were transitorily enhanced 2 h following i.p. treatment. At later periods (4-6h) after treatment catecholamine levels were significantly raised. 5-HT turnover was practically uninfluenced by the compound. Activities of PDE preparations isolated from cerebral tissues were markedly inhibited. Various hypotheses are suggested in order to explain the biochemical mechanism of action of the compound.


Preliminary studies of nonvirion antigens associated with herpes simplex virus 1 and 2 (HSV1 - HSV2). Biochemical and radioimmunological studies have indicated the presence of a nonvirion antigen associated with herpes virus. Guinea pig kidney cells were grown in culture and infected (I-cells) for 3h with herpes virus. Uninfected cells (N-cells) served as control. The cells were collected and sonicated to obtain, after centrifugation, "soluble extract" (SI, SN). The pellets were extracted with KCl (ISP, NSP). SN, SI were submitted to gel-filtration on Sephadex G-200. Extracts from I and N cells gave similar patterns. After labelling with 125I, greater resolution was obtained in a second gel-filtration with a much smaller load. ISP contained protein species not present in NSP. Polyacrylamide gel electrophoresis of peaks from SN and SI showed that SI contained some radioactive bands not found in SN. Labelled materials from SI and SN were incubated with sera from cancer patients and normal subjects. No differences in binding were seen with SN, but SI showed 14-30% more 125I binding with sera of cancer patients than with sera from normal subjects. No such difference was seen between ISP and NSP.


Adenosine triphosphatase activity in myxoviruses. Sendai and PR 8 viral particles show Na+-K+-ATPase activity, an enzymatic activity which is assumed to be a typical plasma membrane marker and which probably derives from the host cell membrane. Attention has been paid to the peculiar behaviour of Na+-K+-ATPase activity in the two viral strains upon hypoosmotic treatment: Sendai viral particles show enzymatic activity only after swelling, when for PR 8 particles the reverse is true. The results obtained are discussed as suggesting a different organization in the envelope of the viral strains. This approach is supported also by morphological evidence concerned with the osmotic response of viral particles.


Sialic acid content in four cell strains transformed by adenoviruses and/or SV40. The sialic acid content in hamster cells primarily transformed by adenovirus was found lower than in cells transformed by papovavirus SV40. Intermediate concentrations were shown in a cell strain transformed by an adenovirus-SV40 hybrid. An adenovirus 19 - transformed cell strain derived from the NIL-2 hamster cell line, adapted to unlimited growth in vitro prior to the exposure to the adenovirus, revealed a high content of sialic acid. The concentration of sialic acid in cells transformed by DNA-viruses may depend both on the transforming virus and on the preceding history of the cell line. The tumors revealed more sialic acid than the in vitro cultures derived from the same cell strains. This finding is discussed.


[Action of emetine hydrochloride in some experimental viral infections]. The Authors tested the virulicidal effect of emetine hydrochloride (EH) observed by Fusillo in clinical practice. By adding EH to HE p-2 cellular system, in a concentration such as to cause some inhibition of cellular growth, there is no meaningfull virostatic or virulicidal action against poliovirus, Mahoney's strain. EH, in the same proportion as that employed by Fusillo in human therapy, damages in the albino mouse liver. By halving the concentration of EH such a damage is not evident but no beneficial effect on the pattern of the subacute hepatitis, caused by 5 LD50 of MHV3 ensues.


[Intrathoracic tumors in children].

[Intrathoracic tumors in children]. Intrathoracic tumors in pediatric ages are unfrequent. A review of 138 cases in patients under 15 years of age showed in the first place, the same as in adults, malignant neoplasms represented by pulmonary metastases. However, in those of primary localization, the histologically benign are more frequent. Out of 69 cases, such tumors came up to 78.2% and 47% of them were cystic malformations. Among malignant primary neoplasms, out of 30.4%, neuroblastoma showed 8.6%, diverse sarcomas were 8.6% and the epithelial type added up to 4.3%. The main symptoms were: frequent bronchopulmonary infections, obstructive respiratory insufficiency and vascular stasis. Radiological examinations are of great diagnostic aid, but a full clinical evaluation is always necessary. A surgical treatment removing the mass leads to radical recovery leaving limited sequelae. The comparative group of 69 cases with pulmonary metastases showed that neoplasms considered of embryonal type are practically always multiple and appear during the final stage of the disease. Surgery may be attempted when they are single and the primary has been removed; however, chemotherapy and radiotherapy are always necessary.


[Human seroepidemiology Polyoma virus BK in Umbria]. A serological study on the antibody level against BK virus in healthy population and respiratory patients in Umbria was performed. Serum samples of 465 healthy people and 155 patients grouped by age, were tested using HAI. 47% of all sera was positive. The epidemiological implications are discussed.


Prevalence in the Rome healthy population of antibodies to a human Polyoma - virus (BK - strain). A serological study of determine the prevalence of infections caused by human Polyoma-virus (BK strain) among the healthy population of Rome has been performed. Such virus was originally isolated from a patient undergoing a kidney transplantation, and later found in patients undergoing immunosuppressive therapy. Previous serological studies showed high incidences of infections, caused by this virus, in human populations. Other viruses, antigenically related, were found in patients suffering from progressive multifocal leukoencephalopathy. Serum samples coming from 482 healthy subjects classified by age and 100 cord blood serum samples were analyzed for antibody titers using haemoagglutination-inhibition and complement fixation tests. High incidence of infection caused by BK virus in the Roman population was found: specifically the haeomagglutination-inhibition test resulted positive in 65.2% of the samples. A remarkable similar trend between the results obtained with both the techniques was observed, although differences in antibody serum levels were present. The infections, which are not transmitted during the intrauterine life, are first noted at about two years of age. The Authors finally briefly discuss the possible epidemiological meaning of the spreading of this infection.


Ultrastructural studies of epidermal lesions in pityriasis lichenoides chronica: occurrence of tubular aggregates and intracytoplasmic desmosomes. Very early skin lesions from four patients with pityriasis lichenoides chronica were examined by electron microscopy. Aggregates of tubular structures, which resembled those reported in endothelium in lupus erythematosus and are currently being observed in an increasing variety of conditions, were noticed in the epidermal cells of all patients. These cells also showed intracytoplasmic desmosomes, and isolated mitochondria and myelin-like figures in intercellular spaces, in addition to the occurrence of vacuoles and lysosomes in the cytoplasm, dilatation of endoplasmic reticulum and alteration of mitochondria. Occasionally, epidermal cells were seen to be individually filled with fibrils. These ultrastructural findings seem to indicate that in pityriasis lichenoides chronica some injury to epidermal cells occurs at the early stage of pathological processes, as suggested by light microscope studies.


ACTH levels in maternal, fetal and neonatal plasma after short-term prenatal dexamethasone therapy. The effect of prenatal dexamethasone therapy (12, 8 and 4 mg doses given intramuscularly on three consecutive days) on ACTH levels in maternal plasma (n=33), mixed umbilical cord plasma (n=31) and plasma from the newborn (n=29) was studied, and the results were compared with those obtained in 56 healthy parturients and 50 of their newborn. Maternal ACTH after delivery was significantly lower in the mothers treated with dexamethasone than in the control group. Cord ACTH values were similar in the two groups. ACTH levels fell during the early neonatal period, but only at 12 to 24 hours were the ACTH levels significantly lower in the dexamethasone group than in the controls. Gestational age, birth weight and the interval between the dexamethasone therapy and delivery had no significant effect on cord ACTH levels. Short-term prenatal dexamethasone therapy seemed to have very little effect on ACTH secretion in the mother, in the fetus and in the newborn.


Epidemic keratoconjunctivitis and chronic papillary conjunctivitis in London due to adenovirus type 19. Since July 1973 cases of keratoconjunctivitis resembling epidemic keratoconjunctivitis were observed in the External Eye Disease Clinic at Moorfields Eye Hospital; City Road, London. Adenovirus type 19 was isolated in human embryonic kidney cells from 21 patients. The majority were males between 20 and 40 years old. A small hospital outbreak involving six patients occurred. Clinical features of the disease, consisting of moderate to severe follicular conjunctivitis, major subepithelial punctate keratitis, sometimes with pseudomembrane and scarring, were closely similar to those of epidemic keratoconjunctivitis caused by adenovirus type 8. This similarity, as well as the ability of the agent to cause hospital outbreaks, indicates that adenovirus type 19 is a cause of epidemic keratoconjunctivitis. A case of bilateral chronic papillary conjunctivitis that persisted for 16 months following an acute onset was described. Adeno 19 was isolated from the eye of the patient after 12 months of recrudescent or recurrent illness. Chronic adenovirus infection lacking the usual clinical picture of an acute follicular reaction has not hitherto been described. Such cases are probably important because of the obvious danger of continuing the carriage and shedding of infective adeno 19 from one outbreak to another, by presenting subsequently in eye clinics, and providing an unrecognised source of infection to initiate further outbreaks of hospital transmission.


Pleomorphic adenoma of the nasal septum. A brief review of the world literature on pleomorphic adenoma of the nasal septum is presented. One further case is reported, treated by surgical excision via lateral rhinotomy. Alternative surgical approaches are considered.


The enhancement by dietary zinc deficiency of the susceptibility of the rat duodenum to colchicine. 1. The incidence of colchicine-induced lesions in the germinal epithelium oof the rat duodenum was studied in young rats in an early stage of zinc deficiency and in their pair-fed controls. At both dose levels of colchicine used, a marked increase in the amount of cell damage was observed in the duodenum of Zn-deficient rats as compared with the pair-fed, control (Zn-supplemented) rats. 2. No statistical interaction between Zn and colchicine was demonstrable, and no lesions were found in the duodenum of animals that had not been treated with colchicine. 3. The results are discussed in relation to the effects of Zn deficiency in animals and the possible involvement of Zn in the maintenance of the integrity of microtubular structures.


Analysis of the defect structure of gel-phase lipid. The partitioning of the spin label 2,2,6,6-tetramethylpiperidinyl-1-oxy (Tempo) into phosphatidylcholine bilayers and the monomer-aggregate equilibrium for chlorophyll a incorporated into phosphatidylcholine bilayers have been interpreted in terms of the formation of defects in the gel-phase lipid, starting some 20 degrees C below the temperature of the main gel to liquid crystalline phase transition. By contrast, defects seem to be largely absent from bilayers of dipalmitoylphosphatidylethanolamine in the gel phase. The defect structure accounts for the continuous nature of the phase transition for phosphatidylcholines, and also for the increase in width of the transition caused by the addition of alcohols.


Fluidity of the lipids next to the acetylcholine receptor protein of torpedo membrane fragments. Use of amphiphilic reversible spin-labels. Choline esters of spin-labeled fatty acids (long-chain acylcholines) were used to probe the hydrophobic environment of the acetylcholine receptor protein in membrane fragments from Torpedo marmorata. These spin-labels competitively inhibit the binding of [3H]acetylcholine to the receptor site. Their inhibition constants (KI) were close to 200 nM. At the high membrane concentration required for electron spin resonance (ESR) experiments, the apparent inhibition constants (KIapp) differed from KI determined by using dilute membrane concentration. This is due to the amphiphilic character of long-chain acylcholine. For most spin-labels used, only difference ESR spectroscopy provided reliable spectra corresponding to receptor-bound spin-labeled acylcholines. Acetylcholine receptor agonists and antagonists displaced the acylcholine from the receptor sites, whereas choline had only a weak effect. This produced a modification in the ESR spectra of the bound acylcholines and provided evidence that the acylcholines bound to the receptor sites in a specific manner. The interpretation of the spectra of receptor-bound spin-labels favored a strong barrier to the motion of the probe when attached to the middle of the acyl chain. However, when the probe was close to the methyl terminal of a stearoylcholine molecule a much greater fluidity was found. Short-range spin-spin interactions were created between spin-labels bound to the receptor site and spin-labels in a fluid phase. This indicates that lipids next to the receptor protein are not completely immobilized in spite of the semicrystalline organization of the proteins in the postsynaptic region.


Reaction of essential lysyl residues of pig heart diphosphyridine nucleotide dependent isocitrate dehydrogenase with 2,4-pentanedione. The rate of inactivation of pig heart DPN-specific isocitrate dehydrogenase by 2,4-pentanedione is pseudo-first-order and linearly dependent on reagent concentration. Isocitrate incombination with manganous ion can prevent inactivation, and a dissociation constant (KIC) for the enzyme-isocitrate complex can be calculated which is similar in magnitude to the Km for isocitrate under the same conditions. Although neither the cofactor,DPN, nor the allosteric activator, ADP, prevents inactivation by reagent, ADP lowers both KIC and Km to the same extent. These data suggest that the reagent may be reacting with residues within a binding site for manganeous-isocitrate. DPNH accelerates the inactivation and also enhances protection by isocitrate, lowering KIC by a factor of 20. Because ADP does not prevent the DPNH rate enhancement, it is unlikely that the two nucleotides compete for identical binding sites. Reaction with 2,4-pentanedione thus provides a probe of the mode of ligand interaction with the enzyme. Inactivation appears to result from the reaction of 2,4-pentanedione with lysyl residues to form enamines. The occurrence of a new absorbance band during inactivation and the isolation by gel filtration of enzyme with an absorbance peak at 312 nm are consistent with enamine formation. Hydroxylamine, which abolishes the 312-nm peak, also causes appreciable reactivation of the enzyme. By use of [2,4-14C]-2,4-pentanedione, it was established that reaction of an average of no more than 3 lysines of the 26 per peptide chain resulted in complete inactivation; and an average of only 2 lysines react when enzymatic activity is retained in the presence of 50 mM isocitrate. Reaction with arginine was excluded by the unchanged amino acid composition of modified enzyme. These data suggest that formation of an enamine of possibly 1, and certainly no more than 3, lysine residue(s) in the catalytic center of the enzyme is responsible for inactivation by 2,4-pentanedione.


Enzymic hydrolysis of phosphonate esters. Reaction mechanism of intestinal 5'-nucleotide phosphodiesterase. The mechanism of bovine intestinal 5'-nucleotide phosphodiesterase was investigated by determining kinetic constants of systematically varied substrates, with emphasis on esters of phosphonic acids (which have much higer Vmax values than conventional phosphodiester substrates), and by pre-steady-state kinetics using bis(4-nitrophenyl) phosphate as substrate. The results suggest a ping-pong type mechanism, with participation of a covalent enzyme intermediate.


Free radical and ionic reaction of bisulfite with reduced nicotinamide adenine dinucleotide and its analogues. 1,4-Dihydronicotinamide adenine dinucleotide (NADH) and its analogues undergo two reactions in sulfite buffers in the pH range 5.5-7.1: (1) an oxygen-mediated free-radical chain reaction which results in the oxidation of the dihydropyridine to the pyridinium salt, and (2) an ionic reaction which results in the hydration of the 5,6 double bond of the dihydropyridine. The free-radical reaction is inhibited by superoxide dismutase (indicating the involvement of superoxide radicals) and by free-radical inhibitors. The ionic reaction is not affected by free-radical inhibitors and follows the rate law: rate = [substrate] [HSO3-] (K + SIGMAK' [HA]), where HA is a general acid of hydronium ion. The occurrence of third-order terms of the type [substrate] X [HSO3-] [HA] is consistent with the formation of a reactive bisulfite-substrate complex, which undergoes general acid catalyzed hydration.


Complement-induced decrease in membrane mobility: introducing a more sensitive index of spin-label motion. We have used spin-labeling to investigate complement-induced changes in lipid organization of antibody-sensitized sheep erythrocyte membranes. The spectrum of methyl 5-doxylstearate incorporated into the lipid component of sheep erythrocyte membranes is typical of a membrane bilayer. The membranes from complement-lysed erythrocytes have a small, but statistically significant, reduction in fluidity when compared to membranes from osmotically-lysed erythrocytes, as indicated by a small increase in T'. In theory, measurements of the widths of the outer hyperfine extrema should be more sensitive to motion than the separation of the outer hyperfine extrema (2T'). Our results indicate that the half-width at half-height of the outer hyperfine extrema show a severalfold greater percentage change than T'. The sign and magnitude of these changes are in general agreement with previous predictions. Our results imply that motional corrections to the S formalism of Hubbell, Gaffney, and McConnell are necessary because spin-label motion appears to be explicitly represented in this type of electron spin resonance spectra.


Spin-labeled acyl atractyloside as a probe of the mitochondrial adenosine diphosphate carrier. Asymmetry of the carrier and direct lipid environment. A number of spin-labeled acyl derivatives of atractyloside, (m,n)acyl-ATR (general formula: CH3- (CH2)mCX(CH2)nCOO-ATR, where X is an o-azolidine ring containing a nitroxide), have been synthesized. As shown by electron spin resonance (ESR) spectra of spin-labeled acyl-ATR, the nitroxide placed on the acyl chain interacts with the diterpene residue of the atractyloside moiety when incorporated in liposomes. Spin-labeled acyl-ATRs were used to probe the ADP carrier in heart mitochondria. They inhibit ADP transport with the same efficiency as unlabeled acyl-ATRs. The inhibition is a mixed competitive and noncompetitive inhibition. The inhibitor constant is close to 10(-7) M. The long chain acyl-ATRs (10,3)- (7,6)-, (7,8)-, and (5,10)acyl-ATRs) and also the short chain (0,2)acyl-ATR, when added at low concentrations to heart mitochondria, give rise to more immobilized ESR spectra than when added to liposomes. Immobilization is stronger for the first three molecules of the series. The (1,14)acyl-ATR, which possesses a nitroxide almost at the end of the acyl chain near the terminal methyl, gives rise to a spectrum corresponding to a high degree of fluidity. Upon addition of atractyloside or of other specific ligands, spin-labeled long-chain acyl-ATRs bound to the ADP carrier are displaced from their binding site toward the lipid phase of the mitochondrial membrane and the short chain (0,2)acyl-ATR is released into the aqueous phase. Spin-labeled long-chain acyl-ATRs do not show any evidence of binding to a protein when incubated with "inside out" submitochondrial particles, in spite of the fact that these particles are able to transport ADP. These results are discussed with respect to the size and the asymmetry of the ADP carrier in the mitochondrial membrane and the mechanism of ADP transport.


Structural and dynamical aspects of membrane immunochemistry using model membranes. Three different phospholipid haptens have been synthesized, in which the haptenic group is the paramagnetic nitroxide (spin-label) group. These lipid haptens differ from one another in the length and composition of the molecular chain linking the 2,2,6,6-tetramethylpiperidinyl-N-oxy moiety to the phosphodiester group of the lipid. These lipid haptens have been incorporated at low molar concentrations (0.01 to 0.5 mol %) in liposomes containing various proportions of cholesterol and dipalmitoylphosphatidylcholine (DPPC). A study has been made of specific antinitroxide IgG (and Fab) binding to these liposomes, and the fixation of complement. From these studies we conclude: (a) For lipid haptens whose possible extension above the bilayer plane is limited (e.g., approximately 10-20 A), antibody binding and complement fixation depend strongly on the hapten structure and host lipid composition, because of steric limitations on the accessibility of lipid haptens to the binding sites in the protein. (b) Complement fixation by specific IgG antibodies directed against the nitroxide group as part of a lipid hapten depends strongly on the lateral mobility of the lipid hapten when its molar concentration in the plane of the membrane is of the order of 0.1 mol % or less. It is likely that this conclusion applies to many lipid haptens, and possibly other membrane components. (c) The inclusion of cholesterol in lipid membranes has at least two distinct effects on complement fixation involving lipid haptens. Through a steric effect on bilayer structure (probably involving lateral molecular ordering) cholesterol in phosphatidylcholine bilayers can enhance hapten exposure to antibody binding sites, enhance antibody binding, and thereby enhance complement fixation. It is likely that cholesterol also affects complement fixation at low hapten concentrations through a modification of membrane fluidity.


Recognition of different pools of phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii by phospholipase A2. Phospholipase A2 (EC 3.1.1.4) from pig pancreas hydrolyzes phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii. Complete degradation of phosphatidylglycerol in intact cells at 37 degrees C does not result in lysis as shown by the retention of intracellular K+ ions and the cytoplasmic glucose-6-phosphatase, as well as the inability to detect activity of membrane-bound intracellular NADH-oxidase. A. laidlawii was grown on linoleic acid. Phospholipase A2 treatment of these cells at 5 degrees C, at which temperature the lipids are still in the liquid-crystalline state, results in a rapid breakdown of 50% of the phosphatidylglycerol. The residual phosphatidylglycerol can be hydrolyzed only at elevated temperatures and at much smaller rates, depending strongly on the incubation temperature. When membranes isolated from these cells are incubated at 5 degrees C, 70% of the phosphatidylglycerol is hydrolyzed immediately. The hydrolysis of the residual 30% is again strongly temperature dependent. Cells were grown on palmitate, elaidate, or oleate to investigate possible effects of the lipid phase transition on the accessibility of phosphatidylglycerol for phospholipase A2. Under conditions in which all the lipid is in the solid state, no hydrolysis occurs. When solid and liquid-crystalline lipid phases coexist, a limited hydrolysis of phosphatidylglycerol can be observed. The results demonstrate the disposition of phosphatidylglycerol in three different pools in the membrane of A. laidlawii. Phospholipase A2 has been used to discriminate between these pools and to estimate the amount of phosphatidylglycerol which is present in the liquid-crystalline phase. The present data, however, do not allow a definite localization of the phosphatidylglycerol pools.


31P nuclear magnetic resonance evidence for polyphosphoinositide associated with the hydrophobic segment of glycophorin A. Glycophorin A, the major human erythrocyte sialoglycoprotein, contains a significant amount of phosphorus when isolated by the lithium diiodosalicylate-phenol procedure. Only a small percentage (approximately 1%) of this phosphorus is phosphoprotein. 31P nuclear magnetic resonance (NMR) analysis of glycophorin A has identified the remaining phosphorus content as phospholipid in origin. From the 31P chemical shifts, the phospholipid has been identified as diphosphoinositide. 31P NMR spectra of the peptides produced by trypsin hydrolysis of glycophorin A reveal that all the diphosphoinositide is closely associated with the hydrophobic region of the protein, suggesting that there is a specific affinity between this phospholipid and the intramembranous portion of glycophorin A.


Electron spin resonance evidence for vertical asymmetry in animal cell membranes. Two electron spin resonance (ESR) spin labels were used to monitor the physical state of bacterial and animal cell membranes: 5N10, a nitroxide derivative of decane, and 12NS-GA, a glucosamine derivative of 12-nitroxide stearic acid. Spectra were recorded at 1 degrees C intervals from approximately 5 to 45 degrees C. Arrhenius plots of log hH/hP vs. 1/K were obtained by measuring the amplitudes of the hydrocarbon and water signals, hH and hP, respectively. Two discontinuities in the Arrhenius plot (at characteristic temperatures t1 and th) were observed with bacterial cell membranes independent of the spin label employed. Analysis of sealed animal cell membrane samples revealed four characteristic temperatures when the hydrophobic spin lable 5N10 was used, but only two when the amphiphilic spin label 12NS-GA was used. The specific set of characteristic temperatures revealed with 12NS-GA depended on whether the membrane preparation was inside out (ISO) or right side out (RSO). Analysis of Newcastle disease virus, a source of RSO plasma membrane derived from host, revealed two characteristic temperatures at approximately 14 and 33 degrees C. Analysis of phagosomes, a source of ISO plasma membrane derived from LM cells, revealed two characteristic temperatures at approximately 23 and 38 degrees C. When unsealed or disrupted membrane preparations were spin labeled with 12NS-GA, both sets (RSO and ISO) of characteristic temperatures were revealed. The results indicate that the inner and outer monolayers of animal cell membranes are physically distinct and that the glycosylated spin label, 12NS-GA, is apparently restricted in its ability to flip across the membrane bilayer. In this study, characteristic temperatures were pinpointed by computer analysis of the ESR spectral data.


A method for gene enrichment based on the avidin-biotin interaction. Application to the Drosophila ribosomal RNA genes. A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure.


Primary structure of mouse, rat, and guinea pig cytochrome c. For immunochemical and evolutionary reasons we determined the primary structure of cytochrome c from two strains of laboratory mice. Thioacetylthioethane and thioacetylthioglycolic acid were used in addition to conventional reagents for sequence determinations. The sequence was found to be identical with that of the rabbit except for residues 44 and 89 and consistent with the peptide compositional data reported by Hennig (Hennig, B. (1975), Eur. J. Biochem. 55, 167-183). The rat cytochrome c cymotryptic peptides were identical with those of the mouse in amino acid composition and amino-terminal residues. Further, peptide maps of cytochromes c of the guinea pig and two strains of rat indicate that all these animals have the same cytochrome c as the laboratory mouse. It is concluded that rodent cytochromes c are evolutionarily conservative and that there is no evidence for a generation-time effect in cytochrome c evolution.


Benzo[a]yrenedione/benzo[a]pyrenediol oxidation-reduction couples and the generation of reactive reduced molecular oxygen. The ability of the isomeric quinone metabolites of benzo[a]pyrene, benzo[a]pyrene-6,12-dione, benzo[a]pyrene-1,6-dione, and benzo[a]pyrene-3,6-dione to undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo[a]pyrenediols and intermediate semiquinone radicals has been characterized. Under anaerobic conditions, all three benzo[a]pyrenediones are easily reduced to benzo[a]pyrenediols, even by mild biological agents such as NAD(P)H, cysteamine, and glutathione. The benzo[a]pyrenediols, in turn, are very rapidly autoxidized to the benzo[a]pyrenediones when exposed to air. Substantial amounts of hydrogen peroxide are produced during these autoxidations, and other reactive reduced oxygen species, such as the superoxide and hydroxyl radicals, are probably formed transiently as well. The benzo[a]pyrenediol-benzo[a]pyrenedione interconversions proceed by one-electron steps; the corresponsing semiquinone radicals can be monitored by electron spin resonance spectroscopy as inter mediates during these reactions carried out at high pH. Benzo[a]pyrenediones induce DNA strand scission when incubated with bacteriophage T7 DNA. This damage is modified by conditions which indicate that reduced oxygen species propagate the free-radical reactions responsible for the strand scission. Benzo[a]pyrenediones are electron-acceptor substrates for NADH dehydrogenase from Clostridium kluyveri. Catalytic amounds of these benzo[a]pyrene metabolites, together with this respiratory enzyme function as cyclic oxidation-reduction couples which link NADH and molecular oxygen in the continuous production of hydrogen peroxide. These data, together with preliminary results with cells in culture, indicate that benzo[a]pyrenediones are potentially harmful metabolites of benzo[a]pyrene, acting by processes which lead to their regeneration rather than depletion; nucleic acid and call damage is probably produced by the reactive reduced oxygen species resulting from such regenerative oxidation-reduction cycles.


Separation of oligo(adenosine diphosphate ribose) fractions with various chain lengths and terminal structures. Oligo(adenosine diphosphate ribose) preparations with chain lengths of 3 to 10 adenosine diphosphate ribose units were fractionated according to their chain lengths and their terminal structures by hydroxyapatite column chromatography and then polyacrylamide gel electrophoresis. The peak fractions from the hydroxyapatite column were each separated into two distinct subfractions by gel electrophoresis. The two subfractions were found to differ in chain length and terminal structure. A linear correlation was observed between the mobility and the logarithm of the chain length of oligo(adenosine diphosphate ribose) on gel electrophoresis, irrespective of the terminal structure.


Sedimentation of homogeneous double-strand DNA molecules. Sedimentation velocity studies have been carried out with isolated double-strand DNA fragments prepared by digestion of PM2 phage with the restriction endonuclease Hae III. The results show that DNA molecules shorter than about 200 base pairs behave almost exactly as rigid rods with a diameter of 27 A. The behavior of the larger fragments (up to 1735 base pairs) can be described very well by either the theory of Yamakawa and Fujii (Yamakawa, H., and Fujii, M. (1973), Macromolecules 6. 407) using the same diameter and a persistence length of 575 A, or the theory of Hearst and Stockmayer (Hearst, J.E., and Stockmayer, W.H. (1962), J. Chem. Phys. 37, 1425) using a diameter of 20 A and a persistence length of 525 A.


Novikoff hepatoma deoxyribonucleic acid polymerase. Identification of a stimulatory protein bound to the beta-polymerase. The Novikoff hepatoma DNA polymerase-beta sediments as a 7.3S form in crude extracts but during purification sediments as a 4.1S form (after diethylaminoethyl-Sephadex chromatography) or as a 3.3S form (after DNA-cellulose chromatography). If 0.25 M ammonium sulfate or 0.5 M NaCl is included in the sucrose gradients, the 7.3S form sediments at 3.3 S; after removal of the salt, it sediments again at 7.3 S, indicating the reversibility of the aggregation phenomenon. By careful adjustment of ionic strength in the gradient, four distinct and reproducible forms of the enzyme sedimenting at 7.3, 5.8, 4.1, and 3.3 S can be generated. The isoelectric point of the DNA polymerase also changes during purification; the 7.3S form has a pI of 7.5, while the 4.1S form isoelectrically focuses at a pH of 8.5. During DNA-cellulose chromatography, the Novikoff beta-polymerase is separated from a stimulatory factor designated as Novikoff factor IV. Factor IV is a protein as shown by its sensitivity to protease and resistance to nucleases. It is responsible for converting the 3.3S enzyme to the 4.1S form since the 3.3S homogeneous DNA polymerase-beta sediments at 4.1 S in the presence of factor IV. Factor IV confers stability to the polymerase in low ionic strength buffers as well as stability to heat denaturation. Factor IV has the ability to increase the activity of the 3.3S homogeneous polymerase by about fourfold.


Electron acceptors of photosynthetic bacterial reaction centers. Direct observation of oscillatory behaviour suggesting two closely equivalent ubiquinones. When reaction centers are illuminated by a series of single turnover flashes ubisemiquinone is formed and destroyed on alternate flashes. This oscillatory behaviour can be observed with both optical and electron spin resonance techniques. The oscillations are dependent upon the presence of excess ubiquinone in a manner which suggests that two molecules may act almost equivalently as metastable primary acceptors forming a two-electron gate between the one-electron primary photoact and a two-electron secondary acceptor pool.


Acetylation and phosphorylation of Drosophila histones. Distribution of acetate and phosphate groups in fractionated chromatin. The phosphorylation and acetylation patterns of Drosophila histones were studied after whole cell incubation with ortho [32P] phosphate or [3H] acetate. Radioactive phosphate associated mainly with H1, H3, and H4 and radioactive acetate was associated mainly with H3, H4, and H2B. H3 showed the highest specific activity of both labels. Two chromatin fractionation methods were employed to investigate the distribution of acetate and phosphate groups in histones form template-active and template-inactive regions. The levels of both acetate and phosphate groups were higher in histones from template-active regions.


Albumin synthesis in cultured hempatoma cells. Regulation by essential amino acids. The effects of essential amino acids on albumin synthesis by a mouse hepatoma cell line have been investigated. The amino acids tested were tryptophan, phenylalanine, histidine, isoleucine and leucine. Cellular rates of synthesis (molecules albumin/cell per min) were determined from rates of [3H]leucine incorporation into immunoprecipitable albumin in the culture medium. The effects of amino acids on albumin synthesis fall into three distinct groups. The concentration of tryptophan producing half-maximal synthesis is 4 micronM. The corresponding concentration for leucine is 100 micronM. Histidine, phenylalanine and isoleucine were very similar, the half-maximal concentrations being approximately 15 micronM. The concentrations of amino acids producing half-maximal synthesis correlate directly with the amino acid composition of albumin. The levels of these essential amino acids necessary to saturate albumin synthesis have been compared with amino acid levels in normal plasma.


Inhibition of simian virus 40 DNA synthesis in Yaba virus-preinfected cells. The effect of Yaba virus preinfection on DNA synthesis in SV40-infected Jinet cells was studied. Time-course synthesis studies were conducted using the incorporation of labeled thymidine. Yaba virus preinfection resulted in the inhibition of SV40 DNA synthesis when the elapsed time between Yaba virus and SV40 infections was three days. This inhibition was demonstrated by hybridization studies and sedimentation analysis. In addition, the usual stimulation of cellular DNA synthesis induced by SV40 infection was inhibited. This inhibition occurred at a time in Yaba virus infection when no cytoplasmic Yaba virus-specific DNA synthesis occurred.


The translation of globin messenger RNA with use of muscle initiation factors. The ability of embryonic chicken muscle initiation factors to translate rabbit globin messenger RNA in an efficient, fractionated cell-free system has been examined. Although muscle factors stimulate leucine incorporation to only 15--35% the levels achieved with rabbit reticulocyte initiation factors, they synthesize more than one globin chain per mRNA molecule and both alpha and beta globin are produced. Increasing the ribosome concentration and adding the polyamine spermidine to the system produce stimulatory effects which are quantitatively and qualitatively similar for both factor preparations. The lower efficiency of synthesis of muscle factors relative to reticulocyte factors is also apparent when mRNA from encephalomyocarditis virus or embryonic chicken muscle polysomes are used in the cell-free system. These results do not support a specific restriction in the capacity of muscle factors to translate globin mRNA. Furthermore, the similarity of the effects of presumed non-specific components on the activity of muscle and reticulocyte factors suggests that globin synthesis in the cell-free system may be controlled in a similar fashion for both preparations.


Organophosphate inhibitors. The stereospecificity of hydrolysis of methyl n-butyl p-nitrophenyl phosphate by serum phosphotriesterases (EC 3.1.1.2) and by acetylcholinesterases (EC 3.1.1.7). A chiral phosphotriester, methyl n-butyl p-nitrophenyl phosphate was used to determine the stereospecificity of hydrolysis catalysed by serum phosphotriesterases (aryl-ester hydrolases, EC 3.1.1.2) from horse, ox and rabbit. Each enzyme hydrolysed the (-)-enantiomer more quickly. The same phosphate was used to inhibit acetycholinesterase (acetycholine hydrolase, EC 3.1.1.7) from ox, rabbit and electric eel, and in each case, the (+)-enantiomer caused more rapid inhibition. Serum phosphotriesterase did not catalyse the dephosphorylation of dialkyphosphoryl-acetylcholinesterase or dialkylphosphoryl-carboxylesterase. Levels of serum phosphotriesterase in rabbits which received sub-lethal injections of the phosphotriester remained unchanged after one or several injections. In the same rabbits, the levels of blood acetylcholinesterase fell sharply following injections, but normal values were regained in 2-8 days. Serum phosphotriesterases seem incapable either of preventing acute phosphotriester poisoning or of regenerating active enzyme from phosphorylated acetylcholinesterase. However, phosphotriesterases would act in cases of chronic exposure by catalysing the hydrolysis of such organophosphate poisons as remain in the blood.


Purification and properties of a phosphoprotein phosphatase from rat liver. A phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been partially purified from rat liver homogenates by (NH4)2SO4 and ethanol precipitations followed by DEAE-cellulose and Sepharose 6B chromatography. The phosphoprotein phosphatase is capable of cleaving [32P]phosphate from radiolabelled phosphopyruvate kinase (type L) (EC 2.7.1.40), phosphohistones, and phosphoprotamine. However, it did not detectably dephosphorylate ATP, ADP, DL-phosphorylserine or beta-glycerophosphate. Dephosphorylation of [32P]phosphopyruvate kinase was stimulated by divalent cations and inhibited by ATP, ADP, Fru-1,6-P2, and orthophosphate. Divalene cations could reverse inhibition induced by ADP or ATP. At least one function of the phosphoprotein phosphatase may be to remove phosphate groups from the phosphorylated form of pyruvate kinase in the liver.


UDPglucose dehydrogenase. Kinetics and their mechanistic implications. Initial velocity and product inhibition studies were carried out on UDP-glucose dehydrogenase (UDPglucose: NAD+ 6-oxidoreductase, EC 1.1.1.22) from beef liver to determine if the kinetics of the reaction are compatible with the established mechanism. An intersecting initial velocity pattern was observed with NAD+ as the variable substrate and UDPG as the changing fixed substrate. UDPglucuronic acid gave competitive inhibition of UDPG and non-competitive inhibition of NAD+. Inhibition by NADH gave complex patterns.Lineweaver-Burk plots of 1/upsilon versus 1/NAD+ at varied levels of NADH gave highly non-linear curves. At levels of NAD+ below 0.05 mM, non-competitive inhibition patterns were observed giving parabolic curves. Extrapolation to saturation with NAD+ showed NADH gave linear uncompetitive inhibition of UDPG if NAD+ was saturating. However, at levels of NAD+ above 0.10 mM, NADH became a competitive inhibitor of NAD+ (parabolic curves) and when NAD+ was saturating NADH gave no inhibition of UDPG. NADH was non-competitive versus UDPG when NAD+ was not saturating. These results are compatible with a mechanism in which UDPG binds first, followed by NAD+, which is reduced and released. A second mol of NAD+ is then bound, reduced, and released. The irreversible step in the reaction must occur after the release of the second mol of NADH but before the release of UDPglucuronic acid. This is apparently caused by the hydrolysis of a thiol ester between UDPglucoronic acid and the essential thiol group of the enzyme. Examination of rate equations indicated that this hydrolysis is the rate-limiting step in the overall reaction. The discontinuity in the velocities observed at high NAD+ concentrations is apparently caused by the binding of NAD+ in the active site after the release of the second mol of NADH, eliminating the NADH inhibition when NAD+ becomes saturating.


Distinction between NAD- and NADH-binding forms of mitochondrial malate dehydrogenase as shown by inhibition with thenoyltrifuoroacetone. The inhibition of mitochondrial malate dehydrogenase (L-malate : NADH oxidoreductase, EC 1.1.1.37) by 2-thenoyltrifluoroacetone (TTFA) was investigated at pH 8.0 where both forward and backward reactions can be measured. The inhibition with respect to malate is non-competitive at finite NAD concentrations. Increasing the NAD concentrations lowers the slope of the double reciprocal plot so that at infinite NAD the inhibition is uncompetitive. The inhibition with respect to oxaloacetate is non-competitive. Increasing the NADH concentration lowers the slope and intercept of the double reciprocal plot so that at infinite NADH the inhibition is nil. The inhibition with respect to NADH is competitive, whatever the oxaloacetate concentrations are. The inhibition with respect to NAD, at all malate concentrations, is non-competitive. This pattern of inhibition is incompatible with any model assuming that NAD and NADH reacts with identical forms of the enzyme. On the other hand the reciprocating compulsory ordered mechanism, where the two subunits of the dimeric enzyme are working in concert, can account for all the experimental results. It is concluded that NAD and NADH bind to different forms of the enzyme separated by reversible steps. Only one form (see text), the one which binds NADH, can react to form the dead end complex (see text). The similarity between mechanism of inhibition by thenoyltrifluoroacetone and other hydrophobic inhibitors of malate dehydrogenase is discussed.


Regulation of glycogen synthetase and phosphorylase phosphatase activities in rat adipose tissue. Incubation of a rat adipose tissue homogenate causes a time and temperature dependent activation of glycogen synthetase (UDP glucose:glycogen 4-alpha-glucosyltransferase) and simultaneous inactivation of phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Activation of glycogen synthetase at 15 and 23 degrees C was preceded by a lag period. The duration of the lag period could not be correlated with significant changes in phosphorylase activity. Addition of glucose and methylxanthines caused an increase in the rates of glycogen synthetase activation and phosphorylase inactivation. The effect on glycogen synthetase activation was mainly on the linear phase. Addition of AMP inhibited phosphorylase inactivation and accelerated glycogen synthetase activation. Addition of muscle phosphorylase alpha caused a prolongation of the lag period which lasted until phosphorylase alpha activity had decreased to the level originally present in the preparation. It is concluded that in adipose tissue activation of glycogen synthetase is not dependent on prior inactivation of phosphorylase and that other factors should be looked for to explain the lag period preceding glycogen synthetase activation.


The EPR of low spin heme complexes. Relation of the t2g hole model to the directional properties of the g tensor, and a new method for calculating the ligand field parameters. A simple method is presented for calculating the parameters of the hole model for distorted octahedral, low spin (t2g)5 complexes. In the case of negligible covalent bonding explicit formulas for the coefficients of the Kramer's doublet, (formula: see text). The two numerically largest g values define the plane and orientation of the orbital with the largest coefficient, which in turn indicates the directions of maximal unpaired spin density. The energy of eta with respect to xi (in units of lambda, the spin-orbit coupling constant) is (formula: see text). The product gzgygx, independent of axes, and positive for free electrons, is shown to be positive for tetragonal and negative for nearly octahedral complexes. It is considered positive for hemes. In this method coefficients will only be normalized when there is no covalency. For the majority of published cases they are, to about 1%. Since this discrepancy is larger than can be caused by propagated errors, covalency must be the rule. For comparative purposes A and B, uncorrected for covalency, should still be useful. Examination of published complete g tensors for five hemes shows that the largest g value is nearly normal to the heme plane. If the g values are taken positive and labelled so that gz greater than gy greater than gx, then the proper tetragonal axis is roughly normal to plane of the ring in hemes, but not in chlorins.


Quantitative determination of human apolipoprotein D by electroimmunoassay and radial immunodiffusion. 1. An electroimmunoassay and a radial immunodiffusion procedure are described for the quantitative determination of human serum apolipoprotein D. Purified apolipoprotein D and antisera to both lipoprotein D and apolipoprotein D were used to standardize the assays. The assays are applicable to measurement of apolipoprotein D in serum and density classes. The electroimmunoassay is more sensitive (50 ng apolipoprotein D quantitatively detectable), rapid (time required for completion of assay is 5 h) and precise (the within- and between-assay coefficients of variation are 4 and 7%, respectively) than radial immunodiffusion. However, comparable results were obtained by both methods (r = 0.85). 2. Serum apolipoprotein D levels of normal subjects and hyperlipoproteinemic phenotypes IIa, IIb, III, IV and V were in the same range (10 to 12 mg/dl). In contrast, patients with hyperchylomicronemia (type I) had decreased apolipoprotein D levels (5 mg/dl; P less than 0.001). The apolipoprotein D in serum of normolipidemic subjects was detectable in all density classes but measurable only in HDL2 (21%), HDL3 (43%) and VHDL (36%). 3. Rocket electrophoresis is also a valuable tool for assessing the structural relationships among apolipoproteins or their constituent polypeptides. Interaction between serum and a mixture of antibodies to A-I, A-II and apolipoprotein D resulted in the formation of separate lipoprotein A and lipoprotein D rockets indicating that apolipoprotein D is not a constituent polypeptide of apolipoprotein A. This observation confirms the existence of lipoproteins A and D as separate lipoprotein families.


Low molecular weight angiotensin I converting enzyme from rat lung. A low molecular weight angiotensin I converting enzyme (light angiotensin enzyme) was isolated from a homogenate of rat lung subjected to dialysis against sodium acetate at pH 4.8. This enzyme has a molecular weight of 84 000 on Sephadex G-200 and a molecular weight of 91 000 on SDS-poly-acrylamide gel as compared with a molecular weight of 139 000 for angiotensin I converting enzyme on SDS-polyacrylamide. Light angiotensin enzyme was activated by NaCl  and inhibited by EDTA, angiotensin II, and bradykinin potentiating factor nonapeptide. Light angiotensin enzyme cross-reacted with antibody prepared against angiotensin I converting enzyme and stained with periodic acid-Schiff reagent as a glycoprotein. The evidence suggests that light angiotensin enzyme is a fragment of the higher molecular weight enzyme.


Effect of cartilage proteoglycans on human collagenase activities. No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.


The effect of antibiotics on the electrical polarisability of aqueous suspensions of bacteria. The orientation of washed Escherichia coli bacteria in suspension induced by applied a.c. electric fields has been monitored by observing the accompanying changes in the intensity of light scattered by the suspension. The data enable the anisotropy of electrical polarisability deltaalpha to be determined. Changes in deltaalpha due to the addition of various antibiotics to the suspension have been measured as a function of both the antibiotic concentration and the temperature of the suspension. The results are taken to indicate that there is an accumulation of the antibiotic molecules at the bacterial surface.


Action of cyclic nucleotide analogues in Chinese hamster ovary cells. Cyclic nucleotide analogues have been tested for their ability to cause the morphological conversion of Chinese hamster ovary cells in culture, as well as for effects on cyclic AMP-related enzymes. The ability of the analogues to inhibit the cyclic AMP phosphodiesterase activity and to activate the cyclic AMP-dependent protein kinase activity in cell extracts has been measured. Cell cultures were incubated with the analogues and the effects on morphology, intracellular level of cyclic AMP, and in vivo protein kinase activation were determined. All analogues which induced the morphological conversion also caused in vivo activation of the cyclic AMP-dependent protein kinase. Only N6,O2'-dibutyryl and N6-monobutyryl cyclic AMP caused on increase in intracellular cyclic AMP, presumabley through inhibition of the intracellular cyclic AMP phosphodiesterase activity. The increase in cyclic AMP appears to cause the protein kinase activation. However, analogues such a 8-bromo and 8-benzylthio cyclic AMP do not cause any change in intracellular cyclic AMP level and appear to activate the intracellular cyclic AMP-dependent protein kinase directly.


Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain. The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.