Inhibitor of Clostridium perfringens formed by heating sodium nitrite in a chemically defined medium. An inhibitor of Clostridium perfringens formed when low levels of nitrite were autoclaved with a defined chemical medium. A systematic study of the medium revealed that only amino acids and mineral salts were involved in the production of this inhibitor, which was proven to be a toxic compound formed from cysteine, ferrous sulfate, and sodium nitrite. The inhibitor was compared to several known compounds. S-nitrosocysteine inhibited the test organism, but would not form in the test system in amounts large enough to explain the observed inhibition. Roussin red salt was unstable in the test system and therefore was not the inhibitor. Roussin black salt, which was also inhibitory, could form in sufficient amounts to explain the inhibition. A complex of cysteine, iron, and nitric oxide was detected in the autoclaved solution of cysteine, ferrous sulfate, and sodium nitrite; this cysteine complex did not appear to be inhibitory, however, at levels which could form in the autoclaved medium. The observed inhibition may have been due to the combined effects of sublethal concentrations of each compound.


[Anomalies of corticotropin secretion in chromophobe adenomas]. Corticotrophic secretion has been studied in several groups of subjects during metopirone test and arginine L-Dopa infusion. In animals, a net plasma ACTH increase is observed under while it is inconstant after arginine L-Dopa. In hypopituitarism, metopirone ACTH levels are low and unchanged during the tests. In chromophobe adenoma, basal ACTH levels are high, ACTH increase is normal during test and significantly higher than in controls after L-Dopa injection. Significance of these metopirone corticotrophic abnormalities is discussed.


Repair of heat-injured Clostridium perfringens spores during outgrowth. Clostridium perfringens strain NCTC 8798 spores were injured by ultrahigh temperature treatment and were unable to outgrow in the presence of antibiotics used in selective enumeration media. Injured spores underwent repair in a nonselective laboratory medium and in foods.


Etiology of hyperuricemia.

Etiology of hyperuricemia. Hyperuricemia is a common laboratory finding with significant clinical implications. It is easily detected, but its mechanisms may not be clearly elucidated. A scheme of pathogenesis has been outlined and diagrammed but much is conjectural; therefore, the classification is merely tentative. About 45 diseases or categories of conditions, 20 drugs, and nine states of intoxication have been surveyed. Hyperuricemia can be a multifactorial genetic disorder or a discrete response to a specific stimulus. It may be governed by a complex interplay of biochemical disorders for a lifetime duration, or it may be determined by environmental forces for a very transient course. Some conditions have both increased production of uric acid as well as decreased renal outflow. For many patients, the underlying mechanisms have not yet been elucidated.


Acquired ichthyosis. A sign of nonlymphoproliferative malignant disorders. Acquired ichthyosis was recognized in four patients, in whom the associated malignant disorders were carcinoma of the lung, breast, and cervix. This skin lesion may be a valuable clinical sign in recognizing the existence of a malignant condition. Although the emergence of an ichthyosiform dermatosis may have serious prognostic implications, it may also be useful in monitoring the course of the malignant disorder and the patient's response to therapy. The relationship to nonlymphoproliferative malignant neoplasms may be more common than has been previously recognized.


Urinary 3'5' cyclic AMP. Diagnostic test in pseudohypoparathyroidism. Measurement of urinary cyclic AMP (adenosine 3'5'-cyclic phosphate) and examination of calcium and phosphorus metabolism was carried out in two children with pseudohypoparathyroidism. In both patients infusion of parathyroid hormone failed to elicit any change in urinary cyclic AMP, while a dose-dependent increase in urinary cyclic AMP occurred in a normal control. The findings agree with concept of unresponsiveness of renal cortical tissue to parathyroid hormone in pseudohypoparathyroidism and provide further evidence that measurement of urinary cyclic AMP during parathyroid hormone infusion may be the method of choice in the diagnosis of this disease.


Dense bodies and Langerhans granules after application of podophyllum resin. The application of an alcoholic solution of podophyllum resin at concentrations above 10% for seven hours on guinea-pig udders increases the number of dense intracytoplasmic bodies in the Langerhans cells localized in the epidermis. A close relationship can be established between these inclusions and the typical Langerhans granules in view of the numerous intermediate forms with features of one or the other. Serial sections of these structures confirm the irregularity not only of the lysosome-like bodies but also of the Langerhans granules, which may be fusiform or oblong or even present two vesicular dilatations, one at each end of the rod.


[Tumor- and drug-induced cutaneous axonal dystrophy. An electronmicrocopy proof of multiple lamellated bodies]. A marked cutaneous axonal dystrophy has been observed electronmicroscopically for the first time in the skin of three patients: (a) lesion of pityriasis lichenoides chronica in a patient with bronchogenic carcinoma, (b) non involved skin of a patient with malignant melanoma and (c) non involved skin of a patient with gout and retinal damage after prolonged use of chloroquine. The affected myelinated and non myelinated axons showed distinct alterations of mitochondria and multiple osmiophilic lamellated bodies (LK). These changes were interpreted as a (poly-)neuropathy, due to the influence of toxic systemic agents, such as malignant tumor and abuse of drugs. Chloroquine is known to induce neural damage. Moreover, some other compounds (ergotamine, ethaverine, analgetic preparations) may also be responsible for the drug-induced axonal dystrophy described in this study.


Sexual differentiation in Aspergillus nidulans: the requirement for manganese and the correlation between phosphoglucomutase and the synthesis of reserve material. Aspergillus nidulans was completely devoid of fruit bodies when grown on manganese deficient cultures. This result was shown earlier to be due to a lack of alpha-1,3 glucan in the cell wall. Several enzymes of carbon and nitrogen metabolism were investigated in an attempt to explain the absence of this reserve material. Synthesis of glucose-6-phosphate dehydrogenase, phosphoglucoisomerase and aldolase, were not strongly affected by manganese deficiency. However, phosphoglucomutase showed only 60% of the activity of the control cultures and it was argued that this was connected with the low amounts of alpha-1,3 glucan synthesized. Malate dehydrogenase was the enzyme the least affected by manganese deficiency and the two to threefold higher activity measured after glucose depletion might indicate the induction of the glyoxylate cycle. An impaired glutamine synthetase could explain the increase in activity observed for NAD-glutamine dehydrogenase.


Comparative studies on sporulation-promotive actions on cyclic AMP, theophylline and caffeine in Saccharomyces cerevisiae. Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylline did significantly when added to a presporulation medium containing glucose. Caffeine inhibited the vegatative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.


[Electron microscopy demonstration of fibrillar structures in the foot-and-mouth disease virus]. Purified FMD virus was heat treated and then examined by electron microscopy with the negative contrast technique. Fibrils of varying length and thickness were present; they were not present after ribonuclease treatment. They were similar to the fibrils described for other picornaviruses. Viral RNA was the essential component of these artificially arranged fibrils, which were composed of threads about 10 A wide, separated from one another by a gap of about 10 A.


Kinetics of neutralization of two strains of Myxovirus Parainfluenza 3 in the presence of complement and anti-gammaglobulin-serum. Using the kinetics of neutralization, it was established that two strains of Myxovirus Parainfluenza 3 of cattle and sheep origin which are immunologicaly identical have a different sensitivity to early neutralizing antibodies of reconvalescent calf sera. Complement-dependent neutralizing antibodies, supplied by adding 5% guinea pig serum to the calf serum, appear earlier than the usual neutralizing antibodies. Goat anti-cattle gammaglobulin serum decreases the neutralizing activity of early reconvalescent serum and release some of the neutralizing virus from the virus-antibody complex. It is believed that the lability of the virus-antibody complex depends not only on the activity of the antibody, but also on some properties of the strains. The addition of complement stabilizes this complex. The previous addition of adenovirus antigen to bivalent cattle serum against Myxovirus Parainfluenza 3 and cattle adenovirus, decreases the complement-dependent neutralizing antibody of the serum against Myxovirus Parainfluenza 3. It is suggested that this is the possible mechanism for the mutual activation of mixed virus infections.


[Haptoglobulin in domestic mammals. IV. Experimental influencing of the haptoglobulin level]. The effects of turpentine oil, vaccines, pyrogens, corticotrophin, prednisolone, adrenaline and autologous haemoglobin on the haptoglobin content of plasma was investigated in cattle and pigs. In comparison with rabbits, relatively small doses of turpentine oil led to a considerable increase in haptoglobin, a 50% increase occurring within 4 to 6 days in pigs and only 1.5-2 days in cattle. Bacterial products also increased haptoglobin. It was confirmed that pigs react very rapidly (after 8 hours) to injection of corticotrophin, prednisolone or adrenaline with an increase in plasma haptoglobin. Following complete saturation of infused haemoglobin by haptoglobin, new haptoglobin was rapidly formed, so that the initial haptoglobin concentration was regained about 24 hours after infusion of haemoglobin.


[Structure of the foot-and-mouth disease virus. 2. Various physical and chemical properties of the 12S components]. The 12S units were purified by heat treatment and acidification of purified, highly-concentrated virus, with separation of viral RNA by gel filtration. The following physical parameters were obtained: - partial specific volume 0.737 +/- 0.020 g/cm3; diffusion soefficient D020,W = 3.96 +/- 0.24 F; sedimentation coefficient S020,W = 12.1 +/- 0.5S; molecular weight 283,00 +/- 30,000 Dalton; refraction increment (determined by ultracentrifugation) was dn/dc = 0.189 +/- 0.010 cm3/g for white light of the "HBO 200". Examination of the 12S component by analytical ultracentrifugation and in sucrose and CsCl gradients showed that the product was uniform; density determined in CsCl was 1.302 +/- 0,00 s g/cm3. The ultraviolet absorption spectrum was characteristic of proteins; extinction values at 278 nm in a layer 1 cm thick and 10 mg/ml at pH was 13.9 +/- 1.9, while the ratio E278 nm/E250 = 2.2. Electron microscopy showed that the diameter was 11.0 +/- 1.6 nm. Morphology of 12S unit was discussed.


REM phase deprivation and schizophrenia II. Nine schizophrenic patients with active symptomatology were compared with seven patient controls in their response to two nights of rapid eye movement (REM) sleep deprivation. The control subjects demonstrate "normal" increases in total REM and percentage REM time increase on recovery nights compared to base line nights. The schizophrenic subjects differ substantially from the control subjects in both these measurements and show no perceptible change from base line nights on recovery nights. The effects of medication, anxiety, sleep loss, ceiling effects, and intensity change were not considered adequate to account for the above results. However, many questions, such as the specificity of this rebound failure to the schizophrenic patients and the possibility of a sleep disturbance factor operating independently of psychiatric diagnosis, remain to be answered.


[Morphogenesis of eleastotic transformation of breast cancer stroma (author's transl)]. Within the tissue proteins in stroma of breast cancer occured multifocal elastotic transformations. The transformations concern collagen bundles that are devoided of mucopolysaccharide substance. The fields of degenerated connective tissue take on the capacities to dye like elastic fibres. The authors suggest that the increase of elastotic material in stroma of breast cancer is bound with the collagen degradation that is caused by metabolites of cancer tissue. The phenomenon occured in all age groups and is not closely bound with the grade of histological malignancy of cancer.


[Experimental investigations on the accumulation of radioactive compounds in autochtonal tumors in the rat (author's transl)]. For the first time results of investigations on rats with radioactive compounds in autochtonal tumors of the central nervous system, induced via placenta by Ethyl-Nitrose-Urea (ENU), are reported. In contrast to transplantation tumors the tumors induced by ENU are comparable in regard to the szintigrafic results with human brain tumors. The radiopharmaceuticals 131I-HSA, 99mTc-Pertechnetate, 203Hg-Chlormerodrin and 113m/111In-DTPA are similar to human brain tumors taken up by the ENU-tumors. For these findings it may be important, that the ENU-tumors are of neurogenic origin and do not differ histologically from the human brain tumors. The accumulation of the radioactive substances in ENU-tumors can be explained with the lesion at the blood-brain-barrier and at the blood-nerv-barrier. Therefore, it is discussed that the mechanism of the uptake is similar in human brain neoplasms and in ENU-tumors of the brain. The ENU-tumor model is suitable for testing new radiopharmaceuticals before their application in men.


[Results of a controlled clinical trial for evaluation of intensive preoperative irradiation in operable bronchial cancer (author's transl)]. To examine the value of an intensive preoperative irradiation on the bronchial carcinoma, 196 operable patients with tumors being histologically confirmed were divided into two groups by drawing the lot: 99 patients for preoperative irradiation, 97 for primary operation. The correspondence of the following criteria was tested and confirmed: age, sex, no symptons, histological tumor type. The preoperative irradiation dose amounted to 5,500 RHD (CO-60). Out of both groups the same number of patients was resected (83 and 85 respectively); within the preoperative irradiated group, however, 11 patients were omitted before the operation (information on reasons), while within the not irradiated group the number of thoracotomized but not resected cases was adequately greater (13). There is no statistically significant difference within the annual survival rate, although the mortality of the primarily operated patients is a bit more favourable. Quite the same is valid for postoperative mortality and complications. The local recurrences (in all cases of necropsy), however, are statistically significant reduced after preoperative irradiation. Since there is no influence on the rate of surviving, the conclusion is drawn that the majority of the operable cases has already developed small distant metastatic formations, being decisive for the prognosis before any therapy was performed. That is why a new prospective series with a low irradiation dose is started, where an increase of complications is not to be expected, but the possibility exists to influence local micrometastatic formations, that means an influence only to those cases with the probability of a low degree of remote metastatic formations.


Subcellular distribution of phospholipids during liver damage induced by rare earths. After intravenous injection of praseodymium nitrate, female Wistar rats develop fatty livers. In contrast to the marked increase of triglycerides, the phospholipid content was only increased by 50%. The subcellular distribution of phospholipids showed that major changes occur in the microsomal fraction within the first 24 hrs. Among the individual phospholipids only phosphatidylcholine and phosphatidylethanolamine concentrations were elevated. Further subfractioning revealed that phospholipid concentration increased in the smooth endoplasmic reticulum, whereas it decreased in the rough endoplasmic reticulum. The individual phospholipids in the smooth endoplasmic reticulum increased to the same degree as did the total phospholipids. On the other hand, in the rough endoplasmic reticulum only the lecithin fraction decreased, while all other phospholipids remained unchanged. Cytochrome P450, cytochrome b5, and glucose 6-phosphatase activity were drastically reduced in the rough endoplasmic reticulum, while no changes could be observed in the smooth endoplasmic reticulum. In the serum, phospholipid concentration fell to half the normal value within the first 24 hrs after praseodymium intoxication.


Demonstration of infectious DNA in transformed cells. II. Characterization of uptake of SV40-transformed mouse cell DNA by simian cells. The initial steps in the DNA-transfer, or transfection, method of virus rescue were characterized using primary green monkey (GMK) cells exposed to SV40-transformed mouse (SV-3T3) cell DNA in the presence of 1 mg/ml DEAE-dextran. When large amounts (10-50mug) of high molecular weight DNA (greater than 10(7) daltons) were inoculated onto 10(6) GMK cells, usually less than 1 mug became cell-associated. DNA fragmented to a size of 1-3 X 10(6) daltons was bound more efficiently by the recipient cells, but generally only 5-10 per cent of the inoculum (representing 1-4 mug) was taken up. Approximately 50 per cent of the cell-associated DNA had penetrated to a DNase-resistant state by the end of the 30-minute incubation period. The effect of the size of thr transformed cell DNA molecule on the recovery of SV40 in transfection experiments was investigated. The trend appeared to be that rescue was more efficient with the larger molecular weight samples.


Specific cleavage of Sendai virus nucleocapsid protein subunits during virus storage. The alteration of whole Sendai virus and especially of its nucleocapsid polypeptides, during storage of the virus at 4 degree C in the allantoic fluids in which it was cultivated, has cultivated, has been studied by sodium dodecyl sulfate gel electrophoresis. During virus storage the nucleocapsid protein subunits with a molecular weight of 60,000 and the putative inner envelope protein with a molecular weight of 38,000 were mainly affected. Both virus components were partially degraded to smaller components. Examination of nucleocapsids isolated from "stored" virus showed that, in addition to the 60,000-molecular weight polypetide component, a smaller polypeptide component with a molecular weight of 46,000 appeared. The relative proportion of the small component increased with the storage period: a kind of specific conversion of large to small components occurred during storage. Since viruses kept in the absence of allantoic fluids revealed no similar modifications of their polypeptides, we concluded that a cellular component present in the allantoic fluids - very likely of enzymatic nature - is responsible for the observed cleavage of virus polypeptides.


Effect of tetraethyl thiuram disulfide (disulfiram) on the multiplication of enveloped viruses. Disulfiram at concentrations between 0.1 and 0.3 mM inhibits the multiplication of Semliki Forest virus (SFV), fowl plague virus (FPV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), and pseudorabies virus (PRV), when administered 1 hour before and during adsorption. There is, however, no inhibition of virus multiplication, when the drug is added after adsorption onto chick embryo cells. Disulfiram interferes neither with the receptors of the virus nor of erythrocytes, and it does not prevent virus adsorption. Possibly an early step in virus multiplication is affected by disculfiram. Infected cells once treated with the drug recover after some time of incubation in an ingibitor-free medium. The inhibitory state can be maintained, however, if relatively low doses of disulfiram are present in the culture medium also after adsorption. Disulfiram has no effect on macromolecular synthesis of the host cells. It has, however, a marked affect on membrane function. While virus multiplication is readily inhibited by disulfiram when chick embryo or BHK cells were investigated, virus multiplication in HeLa cells is almost resestant against the action of disulfiram.


SV40 induced polypeptides in infected and transformed cells. 5 structural (VP) and 3 non-structural (NSVP) SV40 induced polypeptides which display two kinetic patterns of synthesis are identified in infected cells. NSVP 1 and 2 are "early" functions of which only NSVP 1 is present in transformed cells; NSVP 3 is "late" function which is also present in a line (T-22) of transformed AGMK.


Inhibition of Newcastle disease virus-induced fever in rabbits by cycloheximide. Pretreatment with cycloheximide prevents virus-induced fever in rabbits. Compared to the febrile control animals, CH-pretreated afebrile animals exhibit similar concentrations of endogenous pyrogen in the blood and of prostaglandins of the E-group and adenosine 3', 5'-cycle monophosphate in the cerebrospinal fluid. The fever response seems to involve one or several additional so far unknown mediators which are sensitive to cycloheximide.


[Influence of hormones on the functional activity of the regenerating cortex of the enucleated adrenal in situ]. The functional activity of the regenerating cortex was studied in 90 female albino rats (150-200 g) after ablation of the right adrenal and enucleation of the left one. In the period of active growth of the regenerating tissue (10 days after operation) the functional activity of parenchymatous cells was low which is evidenced by poor content of both lipids and "ketosteroids". In parallel with reparation of the adrenal adrenocortical tissue mass the content of physiologically active substances was also restored (20 days after operation). After injection of hydrocortizone (daily dosage 2,5 mg) the growth and differentiation of the bundle-reticular zone in the regenerating area was inhibited. In the glomerular zone the reactions to lipids and "ketosteroids" were mainly similar to those in the glomerular zone of intact adrenal. After injection of ACTH (daily dosage 5 or 10 mg) during 10 days the regenerating area was functionally better developed than in the control since moderately pronounced reactions to "ketosteroids" and lipids appeared in it. Fairly high content of these substances in the regenerated cortex after 20 days of injections of ACTH (10 units) as well as presence of secondary necrobiotic changes pointed to functional overstrain of the newly formed organ.


[Histochemistry of the oxidation-reduction enzymes in the cells of neuroectodermal tumors]. The activity of 8 enzymes (dehydrogenase and diaphorase) in cells of 110 neuroectodermal tumours was studied. The overall assessment showed that the activity of these enzymes varied: the highest was noted for NAD-diaphorase and lactadehydrogenase, the lowest--for enzymes of Krebs' cycle. The activity of dehydrogenase and diaphorase was different in neuroectodermal tumours of different origin. Not infrequently, there was observed "enzymatic polymorphism" of the cells of one and the same tumour. A higher activity of these enzymes in tumour cells, as a rule, correlated with a greater amount of cytoplasma and with a shift to the latter of the nucleo-cytoplasmatic ratio. Metabolism of tumour and reactively hypertrophied astrocytes, as judged by some histochemical findings, showed traits of similarity.


Neostigmine methylsulfate. Does it have a chronic effect as well as a transient one? Three groups of 200-gm rats were injected subcutaneously with neostigmine methylsulfate (1 mg/kg/day) for 7, 30, and 100 days. Electrophysiological changes were assessed in vitro, using microelectrode techniques to examine diaphragm muscles of treated and untreated animals. Miniature end-plate potential (MEPP) amplitude decreased in neostigmine-treated preparations. Guanidine hydrochloride enhances transmitter release and increases MEPP frequency in control preparations. Neostigmine-treated animals examined between 6 to 72 hours after discontinuation of neostigmine therapy showed impaired response to the facilitating influence of guanidine. Recovery of response to guanidine was inversely related to length of treatment with neostigmine. Results of electron-microscopic examination of motor end-plates in treated animals revealed ultrastructural changes, including simplified end-plates, and, occasionally, multiple, separate, junctional regions. Therefore, the chronic administration of cholinesterase inhibitors in man may have a deleterious effect, as well as a transient beneficial one.


Myasthenic syndrome studied with single fiber electromyography. A patient with the myasthenic syndrome was investigated with single fiber electromyography (SFEMG). It was found that 35 of 36 potential pairs had increased "jitter" and that 29 of these demonstrated impulse blockings. There was improvement in neuromuscular transmission with higher rates of innervation. Transmission worsened following rest. The SFEMG findings at the motor end-plate level correlate well with the results of repetitive stimulation and in vitro studies.


Idoxuridine ocular insert therapy. Use in treatment of experimental Herpes simplex keratitis. Therapy of acute Herpes simplex keratitis in rabbits with idoxuridine-releasing ocular inserts showed that an application rate of 30mug/hr gave significantly better results than conventional treatment with idoxuridine drops and ointment while exposing the eye to 40% less drug. Delivery rates lower than this were equal or not as effective as drop and ointment therapy and rates up to 100 mug/hr did not produce significantly better results than rates of 30mug/hr. Serial viral cultures demonstrated the persistence of virus beyond the period of clinical resolution of disease in all treatment groups, indicating that therapy should be continued longer than apparent resolution of disease.


Bilateral synchronous mixed tumors of the parotid gland. A 62-year-old man had bilateral parotid masses. This case represents a rare occurrence of bilateral synchronous mixed tumors of the parotid gland. We discuss this case, including preoperative evaluation, reason for rarity, treatment, and a review of the literature.


Recurrent benign mixed tumor and the facial nerve. In surgical treatment of patients with recurrent benign mixed tumor, the surgeon strives to preserve the facial nerve. If there is inadvertent nerve injury, immediate repair is the treatment of choice. We report two patients with recurrent benign mixed tumor in whom the facial nerve was deliberately sacrificed. Repair of the nerve was accomplished by nerve grafts. Gross and microscopic tissue examination are presented in each instance, supporting the concept of en bloc removal of tumor and nerve. Such a surgical procedure is reserved only for a highly selected group of patients.


Encephalomyocarditis virus infection of pigs. 1. An outbreak in New South Wales. Substantial pig mortalities due to heart disease caused by EMC virus infection are described. The majority occurred in 1970 in conjunction with a rodent plague, but retrospective diagnosis of occasional similar mortalities in previous years was possible. Mortality rates in pigs aged 3 to 16 weeks ranged from a few percent to over 50% and the rate was generally highest in younger animals. It is likely that a considerable proportion of cases was sub-clinical. Focal or diffuse pale areas occurred in the myocardium, the wall of the conus arteriosus being most severely affected. In a few animals, resolving lesions were present. Encephalomyocarditis virus was consistently recovered from pigs with acute heart lesions, but not from those in which myocardial fibrosis had commenced. The virus was demonstrated in rodents, although the dependence of pig disease on rodent-derived virus could not be established. Because of similarities between EMC and FMD myocarditides in pigs, specific diagnosis by virus isolation and identification should be obtained whenever possible.


Encephalomyocarditis virus infection of pigs. 2. Experimental disease. Encephalomycarditis virus recovered from a pig mortality in New South Wales was used to produce experimental infections. Of 34 pigs exposed, 17 died and a further 7 were found to have severe heart lesions when killed. Deaths occurred from 2 to 11 days after exposure with a mode of approximately 3 days. Ten of 11 pigs exposed by intramuscular injection died and the remaining pig was killed after 28 days and found to have severe resolving heart lesions. Of 15 pigs exposed per os to various doses of virus, 6 died, 4 were killed and found to have severe heart lesions and 5 were apparently not infected. Intranasal exposure of 8 pigs resulted in 1 death and 4 pigs with mild to severe heart lesions. Different doses of virus and routes of exposure did not substantially influence the character of the lesions. Lesions were similar to those found previously in field cases, and virus was recovered from all of 19 animals examined with severe acute lesions of 10 days standing or less.


Investigation of equine infectious anaemia in Queensland using gel diffusion. An antigen for the gel diffusion test for equine infectious anaemia (EIA) was prepared from the spleen of a horse experimentally infected with the CQ strain of the virus. The antigen produced a single, distinct line of precipitation when tested against a range of known positive serums, and did not react with pre-inoculation and known negative serums. Extracts prepared from uninfected spleens displayed no reaction when similarly tested. Serum from 34 of 451 Queensland horses contained detectable levels of antibody to EIA virus. The positive serums were from horses in widely separated areas of the State.


Infectious tenosynovitis (viral arthritis): characterization of a Connecticut viral isolant as a reovirus and evidence of viral egg transmission by reovirus-infected broiler breeders. Connecticut isolant S1133 of tenosynovitis was characterized as a reovirus by physicochemical, biological, and morphologic studies. After inoculation of 35-week-old broiler breeders with tenosynovitis virus by subcutaneous route, virus was found in embryos of eggs laid by the breeders during a period of 8 to 12 days after inoculation.


Variation between electrophoretically identical alleles at the alcohol dehydrogenase locus in Drosophila melanogaster. A new variant of alcohol dehydrogenase (ADH 7lk) was found in a laboratory stock of Drosophila melanogaster. ADH in this stock had the same electrophoretic mobility as the F variant both on acrylamide and on agar. Activity levels were similar to the levels in F flies at temperature between 15 and 25 C. But while ADH F enzyme is inactivated rapidly at 40 C, ADH 7lk is still active. Also, ADH S is not inactivated at this temperature, but has a far lower activity per fly than ADH 7lk. Genetic analysis showed that the new variant is an allele of the Adh locus.


The roles of the infectious bursal agent and several avian adenoviruses in the hemorrhagic-aplastic-anemia syndrome and gangrenous dermatitis. The effect that breeder-flock immune status regarding the infectious bursal agent (IBA) and two avian adenoviruses (DPI-1 and DPI-2) has on the susceptibility of their commercially reared Delmarva broiler progeny to the hemorrhagic-aplastic-anemia syndrome and concurrent gangrenous dermatitis was determined. Lack of immunity to the IBA in breeder flocks was related to an increased susceptibility of progeny to anemia and dermatitis. Breeder-flock immunity to the two adenoviruses tested could not be related to the resistance or susceptibility of their progeny to the hemorrhagic-aplastic-anemia syndrome and gangrenous dermatitis.


Effects of increased mechanical work by isolated perfused rat heart during production or uptake of ketone bodies. Assessment of mitochondrial oxidized to reduced free nicotinamide-adenine dinucleotide ratios and oxaloacetate concentrations. Metabolic effects of increased mechanical work were studied by comparing isolated pumping rat hearts perfused by the atrial-filling technique with aortic-perfused non-pumping hearts perfused by the technique of Langendorff. The initial medium usually contained glucose (11 mm) and palmitate (0.6 mm bound to 0.1 mm albumin). During increased heart work (comparing pumping with non-pumping hearts) the uptake of oxygen and glucose increased threefold, but that of free fatty acids was unchanged. Tissue contents of alpha-oxoglutarate, NH4+, malate, lactate, pyruvate and Pi rose with increased heart work, but contents of ATP, phosphocreatine and citrate fell. Ketone bodies were produced with a ratio of beta-hydroxybutyrate/acetoacetate of about 3:1 in both pumping and non-pumping hearts but with higher net production rates in non-pumping hearts. When ketone bodies were added in relatively high concentrations (total 4 mm) to a glucose (11 mm) medium the medium, ratios of beta-hydroxybutyrate/acetoacetate were not steady even after 60 min of perfusion. The validity of calculating mitochondrial free NAD+/NADH ratios from the tissue contents of the reactants of the glutamate dehydrogenase system or the beta-hydroxybutyrate dehydrogenase system is assessed. The activities of these enzymes are considerably less in the rat heart than in the rat liver, introducing reservations into the application to the heart of the principles used by Williamson et al. (1967) for calculation of mitochondrial free NAD+/NADH ratios of liver mitochondria...


Relationship between elongation factor I- and elongation factor II- dependent guanosine triphosphatase activities of ribosomes. Inhibition of both activities by ricin. The elongation factor 1- and elongation factor 2-dependent GTPase (guanosine triphosphatase) activities of ribosomes are inhibited by ricin, a toxic protein known to inactivate the 60S ribosomal subunit. It is suggested that also in eukaryotic ribosomes a "GTPase site', located on the larger subunit, is common to the two elongation factors.


Hemagglutinating properties of CELO, an oncogenic avian adenovirus. Chicken-embryo-lethal-orphan (CELO) virus, Phelps strain, agglutinated erythrocytes at 37 C. The hemagglutinating activity, which is a function of complete and incomplete virus particles, was sensitive to heat but not to pH. The soluble components of the virus were similar in sedimentation characteristics to those obtained from human adenovirus type 1. The effects of chemical and physical agents on CELO hemagglutinin, CELO infectivity, and red-cell receptors suggested that the last were protein in nature and that cell-virus attachment was mediated by amino groups on the virion. The attachment of virus to red blood cells via the penton projection was demonstrated by electron microscopy.


Adenovirus respiratory infection in turkey poults. A virus from turkey poults with respiratory signs was isolated in specific-pathogen-free embryonated chicken eggs and subsequently adapted to chicken embryo fibroblast and turkey kidney cell cultures, where round cell formation was observed. The cloned virus was ether-resistant and incorporated tridiated thymidine. Intra-nuclear icosahedral virus particles of 80 nm were detected. These physicochemical characteristics place this isolant into the adenovirus group of viruses. The disease was experimentally reproduced by intratracheal inoculation of one-day-old turkey poults. Snicking occurred in 100% of the birds and mortality reached 50%. CELO (Phelps strain) antiserum neutralized uncloned and cloned CUA-2 in chicken embryos and uncloned virus in chicken embryo fibroblast cell cultures. Quail bronchitis virus antiserum neutralized cloned CUA-2 in TK cells. Agar-gel precipitin lines of identity were formed using CELO antiserum and postinoculation sera from experimentally infected turkeys. Serologically, this virus should be classified as a type-1 adenovirus.


Enhanced synthesis de novo of phosphatidylinositol in lymphocytes treated with cationic amphiphilic drugs. A variety of amphiphilic cations caused very large increases in the rates of incorporation of Pi and glycerol into phosphatidylinositol in pig mesenteric small lymphocytes. This synthesis de novo of phosphatidylinositol led to a doubling of the phosphatidylinositol concentration in the cells within 3.5 h. The increase in synthesis of phosphatidylinositol labelled with [3H]- or [14C]-glycerol was matched by an approximately equivalent decrease in incorporation of glycerol into phosphatidylcholine, phosphatidylethanolamine and triacylglycerol. Amphilic cations which produced these effects included, in order of decreasing effectiveness, trifluoperazine (half-maximal effect at about 70 mum) greater than chlorpromazine approximately promethazine approximately imipramine greater than cinchocaine greater than amethocaine approximately cetyltrimethylammonium greater than fenfluramine greater than amphetamine greater than 2-phenethylamine greater than cocaine approximately procaine; the most effective compounds were those with the largest and most hydrophobic non-polar substituents. The response to cations was not changed by varying the extracellular Ca2+ concentration in the range 10 nm-1mm. The active amphiphilic cations interacted with anionic phospholipids causing aggregation of aqueous dispersions and/or changes in chromatographic behaviour. These results indicate that amphiphilic cations redirect glycerolipid synthesis de novo, probably owing to inhibition of phosphatidate phosphohydrolase, so that phosphatidylinositol synthesis is increased at the expense of other glycerolipids.


The relationship of calcium to receptor-controlled stimulation of phosphatidylinositol turnover. Effects of acetylcholine, adrenaline, calcium ions, cinchocaine and a bivalent cation ionophore on rat parotid-gland fragments. The possibility that Ca2+ ions are involved in the control of the increased phosphatidylinositol turnover which is provoked by alpha-adrenergic or muscarinic cholinergic stimulation of rat parotid-gland fragments has been investigated. Both types of stimulation provoked phosphatidylinositol breakdown, which was detected either chemically or radiochemically, and provoked a compensatory synthesis of the lipid, detected as an increased rate of incorporation of 32Pi into phosphatidylinositol. Acetylcholine had little effect on the incorporation of labelled glycerol, whereas adrenaline stimulated it significantly, but to a much lower extent than 32P incorporation: this suggests that the response to acetylcholine was entirely accounted for by renewal of the phosphorylinositol head-group of the lipid, but that some synthesis de novo was involved in the response to adrenaline. The responses to both types of stimulation, whether measured as phosphatidylinositol breakdown or as phosphatidylinositol labelling, occurred equally well in incubation media containing 2.5 mm-Ca2+ or 0.2 mm-EGTA [ethanedioxybis(ethylamine)-tetra-acetic acid]. Incubation with a bivalent cation ionophore (A23187) led to a small and more variable increase in phosphatidylinositol labelling with 32Pi, which occurred whether or not Ca2+ was available in the extracellular medium: this was not accompanied by significant phosphatidylinositol breakdown. Cinchocaine, a local anaesthetic, produced parallel increases in the incorporation of Pi and glycerol into phosphatidylinositol. This is compatible with its known ability to inhibit phosphatidate phosphohydrolase (EC 3.1.3.4) and increase phosphatidylinositol synthesis de novo in other cells. These results indicate that the phosphatidylinositol turnover evoked by alpha-adrenergic or muscarinic cholinergic stimuli in rat parotid gland probably does not depend on an influx of Ca2+ into the cells in response to stimulation. This is in marked contrast with the K+ efflux from this tissue, which is controlled by the same receptors, but is strictly dependent on the presence of extracellular Ca2+. The Ca2+-independence of stimulated phosphatidylinositol metabolism may mean that it is controlled through a mode of receptor function different from that which controls other cell responses. Alternatively, it can be interpreted as indicating that stimulated phosphatidylinositol breakdown is intimately involved in the mechanisms of action of alpha-adrenergic and muscarinic cholinergic receptor systems.


An investigation of the involvement of adenosine 3':5'-cyclic monophosphate in steroidogenesis by using isolated adrenal cell column perfusion. The involvement of cyclic AMP in corticosteroidogenesis was investigated by using isolated adrenal cell column perfusion. Steroids were produced in response to 0.5, 1.0 and 5.0 mg of cyclic AMP/ml. Analysis of the shape of the response curves indicated an inverse relationship between rate of onset of steroid production and dose. A further increase in steroid production during the washout period after the 5 mg/ml dose was considered to indicate an intracellular inhibitory effect of cyclic AMP. Release of cyclic AMP into the perfusate only occurred in response to supramaximal steroidogenic doses of ACTH (adrenocorticotrophin). A connexion between dose and response was demonstrated over a narrow concentration range. Variation in the time-lag before cyclic AMP production and in the duration of the response was marked; further, no reproducible ratio of steroid output to cyclic AMP output was shown at any level of stimulation. These results are discussed together with those of other recent investigations. It is considered that these findings do not support an obligatory role for cyclic AMP as mediator of ACTH action in the adrenal.


Proportional activities of glycerol kinase and glycerol 3-phosphate dehydrogenase in rat hepatomas. The activities of glycerol 3-phosphate dehydrogenase (EC 1.1.1.8), glycerol kinase (EC 2.7.1.30), lactate dehydrogenase (EC 1.1.1.27), "malic' enzyme (L-malate-NADP+ oxidoreductase; EC 1.1.1.40) and the beta-oxoacyl-(acyl-carrier protein) reductase component of the fatty acid synthetase complex were measured in nine hepatoma lines (8 in rats, 1 in mouse) and in the livers of host animals. With the single exception of Morris hepatoma 16, which had unusually high glycerol 3-phosphate dehydrogenase activity, the activities of glycerol 3-phosphate dehydrogenase and glycerol kinase were highly correlated in normal livers and hepatomas (r = 0.97; P less than 0.01). The activities of these two enzymes were not strongly correlated with the activities of any of the other three enzymes. The primary function of hepatic glycerol 3-phosphate dehydrogenase appears to be in gluconeogenesis from glycerol.


The stimulation of the phospholipase A2-acylation system of synaptic membranes of brain by cyclic nucleotides. Hydrolysis of phosphatidylcholine by phospholipase A2 of synaptic membranes i n Tris-CHl buffer was stimulated by cyclic AMP, cyclic GMP, cyclic CMP, cyclic UMP and adenosine (0.1 mm). In the presence of 1 mm-NaF and cofactors, the same cyclic nucleotides and adenosine (10 mm) stimulated the incorporation of added oleate into the choline glycerophospholipids of synaptic membranes. Cyclic AMP and noradrenaline stimulated the incorporation of added oleate into position 2 of choline glycerophospholipid. Stimulation of net acylation was increased by preincubation in conditions which stimulated hydrolysis of phosphatidylcholine. Cyclic AMP only slightly stimulated the transfer of oleate from oleoyl-CoA into choline glycerophospholipid. The optimum concentration of CaCl2 for the stimulation of hydrolysis by phospholipase A2 by cyclic AMP was 1 mum. Stimulation of the incorporation of added oleate was maximal in the CaCl2 concentration range 1 mum-1mm. MgCl2 also enhanced stimulations, maximum effects being obtained with concentrations of 10 mum and 0.5 mm for hydrolysis by phospholipase A2 and incorporation of added oleate respectively. ATP enhanced the stimulation of incorporation of oleate but had no effect on the cyclic nucleotide stimulation of hydrolysis of added phosphatidylcholine by phospholipase A2. Adenosine, guanosine, ADP and 5'-AMP (all at 1 mm) inhibited the stimulation of incorporation of oleate by cyclic nucleotides and inhibited the transfer of oleate from oleoyl-CoA to phospholipid. They did not inhibit the stimulation of hydrolysis of added phosphatidylcholine (by phospholipase A2) by cyclic nucleotides, but inhibited the stimulation by noradrenaline, acetylcholine, 5-hydroxytryptamine, dopamine (3,4-dihydroxyphenethylamine) and histamine. Preincubation of synaptic membranes in the water or buffer increased the net activity of phospholipase A2. Preincubation with a mixture of ATP and MgCl2 increased the initial rate of acylation of membrane lipid.


Regulation of carbohydrate metabolism in lymphoid tissue. Quantitative aspects of [U-14C]glucose oxidation by rat spleen slices. When washed spleen slices from fed rats are incubated with 3 mm-[U-14C]glucose, the rate of glucose utilization (46.2 mumol/h per g dry wt.) is sufficient to account, theoretically, for 80% of the O2 consumption. Measurement of net lactate production, however, and the fate of the radioactive carbon, indicates that the contribution of glucose to the respiratory fuel of the tissue is only 25-30% whereas 60-70% of the glucose utilized is converted into lactate. At saturating glucose concentrations (above 5 mm) its contribution to the respiratory fuel of the slice is increased to a maximum value of 34-39%. Only 2% of the glucose utilized is metabolized via the oxidative steps of the pentose phosphate pathway. Starvation for 72 h marginally increases both the rate of glucose utilization (by 21%) and its net contribution to the respiratory fuel (by 29%). Insulin, glucagon, adrenaline and adenosine 3':5'-cyclic monophosphate have no significant effect on either the rate of glucose utilization or on the pattern of radioactive isotope distribution. The uptake of glucose is increased by only 20%, whereas the production of lactate doubles when slices are incubated under anaerobic conditions. In assessing the suitability of spleen slices for metabolic studies, the only serious major perturbation, compared with the freeze-clamped organ, is an elevated mitochondrial [NAD+]/[NADH] ratio (connected with increased endogenous NH3 production) that is partially restored to normal values on incubation with glucose. Equal proportions of erythrocytes and leucocytes are found in the washed spleen slice. Metabolic contributions of the constituent cell populations in the washed slice are calculated and it is concluded that lymphocytes account for the major part of the glycolytic metabolism (80-90%), whereas the contribution of erythrocytes is insignificant.


The effects of adenine nucleotides and guanine nucleotides on urate synthesis de novo by isolated chick liver and kidney cells. Isolated chick liver and kidney cells produce urate de novo from glycine, and this is partially inhibited by 1 mm-AMP and by 1 mm-GMP in liver cells but not in kidney cells. Azaserine fully inhibits this synthesis de novo, but attempts to isolate formylglycine amide ribonucleotide from azaserine-blocked cells were unsuccessful.


The phosphorylation of troponin I from cardiac muscle. 1. Troponin I isolated from fresh cardiac muscle by affinity chromatography contains about 1.9 mol of covalently bound phosphate/mol. Similar preparations of white-skeletal-muscle troponin I contain about 0.5 mol of phosphate/mol. 2. A 3':5'-cyclic AMP-dependent protein kinase and a protein phosphatase are associated with troponin isolated from cardiac muscle. 3. Bovine cardiac 3':5'-cyclic AMP-dependent protein kinase catalyses the phosphorylation of cardiac troponin I 30 times faster than white-skeletal-muscle troponin I. 4. Troponin I is the only component of cardiac troponin phosphorylated at a significant rate by the endogenous or a bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. 5. Phosphorylase kinase catalyses the phosphorylation of cardiac troponin I at similar or slightly faster rates than white-skeletal-muscle troponin I. 6. Troponin C inhibits the phosphorylation of cardiac and skeletal troponin I catalysed by phosphorylase kinase and the phosphorylation of white skeletal troponin I catalysed by 3':5'-cyclic AMP-dependent protein kinase; the phosphorylation of cardiac troponin I catalysed by the latter enzyme is not inhibited.


Electron-paramagnetic-resonance spectroscopy of iron-binding fragments of hen ovotransferrins. 1. It is confirmed that there are two e.p.r. (electron-paramagnetic-resonance) signals associated with fully loaded ovotransferrin, which has two iron-binding sites. 2. Through experiments in which either of the two sites of whole ovotransferrin is occupied, the other being empty, the first occupied site is shown to belong to the N-terminal region of the protein; the second occupied site is in the C-terminal region. 3. When the protein is cleaved with trypsin or subtilisin, the N-terminal fragments are spectroscopically similar to the monoferric ovotransferrin complexes in which the iron atom occupies the N-terminal or C-terminal site respectively. Each fragment displays the same two e.p.r. signals, though not in the same proportions. 4. Computer summations of the e.p.r. spectra confirm that there is no iron-iron interaction which affects the spin Hamiltonian parameters at the iron-binding sites.


Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of reduced nicotinamide-adenine dinucleotide in the presence of formylphenylacetic acid ethyl ester. The oxidase-peroxidase from Datura innoxia which catalyses the oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid was also found to catalyse the oxidation of NADH in the presence of Mn2+ and formylphenylacetic acid ethyl ester. NADH was not oxidized in the absence of formylphenylacetic acid ethyl ester, although formylphenylacetonitrile or phenylacetaldehyde could replace it in the reaction. The reaction appeared to be complex and for every mol of NADH oxidized 3-4 g-atoms of oxygen were utilized, with a concomitant formation of approx. 0.8 mol of H2O2, the latter being identified by the starch-iodide test and decomposition by catalase. Benzoylformic acid ethyl ester was also formed in the reaction, but in a nonlinear fashion, indicating a lag phase. In the absence of Mn2+, NADH oxidation was not only very low, but itself inhibited the formation of benzoylformic acid ethyl ester from formylphenylacetic acid ethyl ester. A reaction mechanism for the oxidation of NADH in the presence of formylphenylacetic acid ethyl ester is proposed.


Ox liver glutamate dehydrogenase. The role of lysine-126 reappraised in the light of studies of inhibition and inactivation by pyridoxal 5'-phosphate. The time-course of inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5'-phosphate was studied in the presence of varied amounts of 2-oxoglutarate or NADH. Pseudo-first-order analysis reveals that the protection by both these compounds is competitive with respect to the chemical modifier. The competition is only partial, however: saturation with either NADH or 2-oxoglutarate decreases the rate constant for inactivation to a finite minimum and not to zero. Similarly, the plot of activity at equilibrium as a function of the concentration of the protecting substrate or coenzyme reveals that neither NADH nor 2-oxoglutarate protects completely against inactivation. In initial-rate experiments, pyridoxal 5'-phosphate, used as an instantaneous inhibitor rather than a long-term inactivator, displayed non-competitive inhibition with respect to both 2-oxoglutarate and NADH. These results clearly indicate that, although there is mutual hindrance between the binding to the enzyme of pyridoxal 5'-phosphate, on the one hand, and 2-oxoglutarate or NADH on the other, binding is not mutually exclusive. These findings are discussed in terms of the two-step mechanism for inactivation by pyridoxal 5'-phosphate. It is concluded that lysine-126 cannot be solely responsible for binding either the substrate or the coenzyme, but could be essential for the catalytic step.


Horse liver alcohol dehydrogenase. A study of the essential lysine residue. 1. The inactivation of horse liver alcohol dehydrogenase by pyridoxal 5'-phosphate in phosphate buffer, pH8, at 10 degrees C was investigated. Activity declines to a minimum value determined by the pyridoxal 5'-phosphate concentration. The maximum inactivation in a single treatment is 75%. This limit appears to be set by the ratio of the first-order rate constants for interconversion of inactive covalently modified enzyme and a readily dissociable non-covalent enzyme-modifier complex. 2. Reactivation was virtually complete on 150-fold dilution: first-order analysis yielded an estimate of the rate constant (0.164min-1), which was then used in the kinetic analysis of the forward inactivation reaction. This provided estimates for the rate constant for conversion of non-covalent complex into inactive enzyme (0.465 min-1) and the dissociation constant of the non-covalent complex (2.8 mM). From the two first-order constants, the minimum attainable activity in a single cycle of treatment may be calculated as 24.5%, very close to the observed value. 3. Successive cycles of modification followed by reduction with NaBH4 each decreased activity by the same fraction, so that three cycles with 3.6 mM-pyridoxal 5'-phosphate decreased specific activity to about 1% of the original value. The absorption spectrum of the enzyme thus treated indicated incorporation of 2-3 mol of pyridoxal 5'-phosphate per mol of subunit, covalently bonded to lysine residues. 4. NAD+ and NADH protected the enzyme completely against inactivation by pyridoxal 5'-phosphate, but ethanol and acetaldehyde were without effect. 5. Pyridoxal 5'-phosphate used as an inhibitor in steady-state experiments, rather than as an inactivator, was non-competitive with respect to both NADH and acetaldehyde. 6. The partially modified enzyme (74% inactive) showed unaltered apparent Km values for NAD+ and ethanol, indicating that modified enzyme is completely inactive, and that the residual activity is due to enzyme that has not been covalently modified. 7. Activation by methylation with formaldehyde was confirmed, but this treatment does not prevent subsequent inactivation with pyridoxal 5'-phosphate. Presumably different lysine residues are involved. 8. It is likely that the essential lysine residue modified by pyridoxal 5'-phosphate is involved either in binding the coenzymes or in the catalytic step. 9. Less detailed studies of yeast alcohol dehydrogenase suggest that this enzyme also possesses an essential lysine residue.


Kinetic studies on (N-formyltryptophyl)cytochrome c. The formylation of the ring nitrogen atom of the tryptophan residue in cytochrome c was carried out and consequent changes in the kinetic properties of the protein were investigated. The reduction of formylated cytochrome c by Cr2+ was studied by stopped-flow techniques. At pH 6.5 the reduction process shows the presence of two phases. One phase (k = 4 X 10(4) M-1-s-1) is dependent on Cr2+ concentration and one phase (k = 5.0 s-1) is not. A study of the temperature dependence of the two phases yields values for their activation energies of 38.6kJ-mol-1 and 42.4kJ-mol-1 respectively. The reaction of the reduced formylated cytochrome c with CO was followed by means of both stopped-flow techniques and flash photolysis. The combination with CO at pH 6.8 measured in stopped-flow experiments shows two phases, both dependent on the concentration of CO (k1 = 1.8 X 10(2) M-1-s-1). If CO was dissociated from the protein by photolysis and then allowed to recombine with it, it was found to do so in a simple manner, at a rate which depended on the concentration of CO (k = 1.9 X 10(2) M-1-s-1). A tentative model which can accommodate these findings is proposed. The reaction of the oxidized form of formylated cytochrome c with NO was followed by means of stopped-flow techniques. The reaction was found to be biphasic with one phase dependent on the concentration of NO (k = 2.8 X 10(3) M-1-s-1) and one phase (k = 0.2x-1) independent of the concentration of NO. This behaviour is compared with that of the native molecule. A comparison of these kinetic observations with those on other tryptophan-specific modifications leads to the conclusion that the main alteration in kinetic properties is due, not to the nature of the modifying group, but rather to the disruption of the normal environment of the haem.


A pitfall in the interpretation of data on ligand-protein interaction. Binding reactions are usually investigated by separation of free and bound labelled ligand. Underestimation of the amount bound leads to inaccurate estimation of the dissociation constant and to wrong conclusions on the validity of the model of binding. The activation of cyclic AMP-dependent protein kinase is treated as an example.


Metabolic regulation of beta-galactosidase synthesis in Escherichia coli. A test for constitutive ribosome synthesis. The rate of differential synthesis of beta-galactosidase (alphalac) was measured in maximally induced cultures of Escherichia coli B/r with 0.01 M-inducer and 0.01 M-cyclic AMP. The value of alphalac decreases with growth rate (60% between 0.67 and 2.1 doublings/h) and after a nutritional shift-up. This decrease is presumed to reflect a decrease in the intracellular concentration of free active RNA polymerase after a shift-up, which implies that the increase in ribosome synthesis after a shift-up is due to an active induction of the ribosomal components.


Utilization of gluconate by Escherichia coli. A role of adenosine 3':5'-cyclic monophosphate in the induction of gluconate catabolism. 1. Cultures of Escherichia coli growing on gluconate use both gluconate and glucose when glucose is added. 2. Glycerol-grown cells adapt to gluconate utilization even in media containing glucose as well as gluconate. 3. The rates of gluconate utilization by cells growing on a mixture of glucose and gluconate, and the specific activities of the gluconate uptake system and of gluconate kinase, are greater if adenosine 3':5'-cyclic monophosphate (cyclic AMP) is present in the medium than in its absence. 4. Growth on media containing gluconate and cyclic AMP is accompanied by the formation of methyl glyoxal and pyruvate, and progressive inhibition of growth. 5. A mutant devoid of adenylate cyclase activity (cya) grew well on glucose in the absence of exogenous cyclic AMP but grew only poorly on gluconate; neither the gluconate uptake system nor gluconate kinase was adequately induced. The addition of cyclic AMP promoted growth on gluconate and facilitated the induction of proteins required for gluconate catabolism. 6. Phage Pl-mediated transduction of cya+ into the cya-mutant also restored the wild-type phenotype in its ability to adapt to gluconate utilization.


Regulation of citrate synthase activity in methylotrophs by reduced nicotinamide-adenine dinucleotide, adenine nucleotides and 2-oxoglutarate. Citrate synthase from two typical facultative methylotrophs, but not from four obligate methylotrophs or from two restricted facultative methylotrophs, is inhibited by 0.1 mM-NADH. ATP or ADP (both at 10mM) inhibits all the citrate synthases, whereas 2-oxoglutarate (10mM) inhibits those from only three of the eight methylotrophs.


Subcellular localization of superoxide dismutase in rat liver. The subcellular localization of superoxide dismutase was investigated in rat liver homogenates. Most of the superoxide dismutase activity is present in the soluble fraction (84%), the rest being associated with mitochondria. No indications for the occurrence of superoxide dismutase in other subcellular structures, particularly in peroxisomes, was found. Mitochondrial activity is not due to adsorption, since the sedimentable activity is essentially latent. Subfractionation of mitochondria by hypo-osmotic shock and sonication shows that half of the mitochondrial superoxide dismutase activity is localized in the intermembrane space, the rest of the enzyme being a component of the matrix space. In non-ionic media the matrix enzyme is, however, adsorbed to the inner membrane, from which it can be desorbed by low (0.04M) concentration of KCl. Superoxide dismutase activity was found in all rat organs investigated. Maximal activity of the enzyme is observed in liver, adrenals and kidney. In adrenals, the highest specific activity is associated with the medulla.


Influence of anti-oestrogens on the specific binding in vitro of (3H)oestradiol by cytosol of rat mammary tumours and human breast carcinomata. The anti-oestrogenic potential of two nitrogen-mustard-containing compounds, I.C.I. 79792 and I.C.I. 85966, was studied. I.C.I. 85966 usually did not decrease specific binding of [3H]oestradiol by breast-tumour cytoplasmic proteins. I.C.I. 79792 decreased specific [3H]oestradiol binding, but not to the same extent as similar concentrations of I.C.I. 46474, diethylstilboestrol or dibutyldihydrostilboestrol.


Stimulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) activity by low concentrations of circulating glucose in perfused rat liver. 1. After nicotinic acid treatment, rat liver glycogen is depleted and phosphoenolpyruvate carboxykinase activity increased, to about twice the initial value. 2. The increase in phosphoenolpyruvate carboxykinase activity promoted by nicotinic acid is prevented by cycloheximide or actinomycin D, suggesting that this effect is produced by synthesis of the enzyme de novo. 3. Despite the enhancement of phosphoenolpyruvate carboxykinase activity and glycogen depletion, which occurs 5h after the injection of nicotinic acid, the gluconeogenic capacity of liver is low and considerably less than the values found in rats starved for 48h. 4. When the livers of well-fed rats are perfused in the presence of low concentrations of glucose, the activity of phosphoenolpyruvate carboxykinase significantly increases compared with the control. 5. This increase is not related to the glycogen content, but seems to be also the result of synthesis of the enzyme de novo, since this effect is counteracted by previous treatment with cycloheximide or actinomycin D. 6. Phosphoenolpyruvate carboxykinase activity is not increased in the presence of low concentrations of circulating glucose when 40 mM-imidazole (an activator of phosphodiesterase) is added to the perfusion medium. 7. Addition of dibutyryl cyclic AMP to the perfusion medium results in an increase in phosphoenolpyruvate carboxykinase activity, in spite of the presence of normal concentrations of circulating glucose. On the other hand, the concentration of cyclic AMP in the liver increases when that of glucose in the medium is low. 8. These results suggest that, in the absence of hormonal factors, the regulation of phosphoenolpyruvate carboxykinase can be accomplished by glucose itself, inadequate concentrations of it resulting in the induction of the enzyme. The mediator in this regulation, as in hormonal regulation, seems to be cyclic AMP.


Recent advances in the identification of enzyme abnormalities underlying excessive purine synthesis in man. Work is continuing in the attempt to increase knowledge of the regulation of the rate of purine synthesis in man by means of an analysis of biochemical alterations leading to purine overproduction among patients with gout. The authors are now assessing the frequency of kinetic mutations in enzymes whose alterations already have been associated with increased purine synthesis. Efforts in this regard have been rewarded by the identification of a new form of alteration leading to partial deficiency of HGPRT and a kinetic variant of PRPP synthetase with increased affinity for ribose-5-phosphate. In order to identify new forms of enzyme abnormalities associated with excessive purine synthesis, the value of a proposed classification scheme requiring measurement of PRPP and ribose-5-phosphate concentration and generation is being assessed in cultured fibroblasts. It is hoped that the results of these measurements will lead to the identification of additional kinetic variants of presently known enzyme abnormalities and will help to identify new classes of mutants in the regulation of human purine metabolism. The excessive purine synthesis that underlies the hyperuricemia of a substantial number of patients with gouty arthritis reflects alterations in the normal mechanism regulating the rate of purine nucleotide synthesis. The study of such purine "overproducers" has provided insight into the nature of this regulatory mechanism and has underscored the diversity of specific genetic and biochemical aberrations affecting it. Despite these advances, however, less than 10% of all patients with gout and excessive purine production can presently be accounted for by known enzyme abnormalities (1). Recognition that current knowledge of the regulation of the rate of purine nucleotide synthesis in man is incomplete has provided the authors impetus leading to the studies described here, which are preceded by a brief review of background.


[Studies on the influence of anticoagulants on metastase formation of autochthonic hepatomas in the rat (author's transl)]. The frequency of metastases of malignant diethylnitrosamine-induced hepatomas and haemangioendotheliomas of the liver of female Sprague-Dawley rats (29%) was not influenced by application of acetyl-salicylic acid or phenprocoumon. Likewise the anticoagulants did not alter tumor yields and induction periods.


[Experimental results with systematically synthetized substances for antiviral chemotherapy / 4th communication: The role of physical binding in the synthesis of antiviral chemotherapeutics and its influence on potential mutagenic effects (author's transl)]. 13 antiviral substances containing specific hydrogen bridge linkage systems out of the classes of 2-substituted 4-phenylthiazoles, 4-phenylimidazoles and indandiones-(1,3) were tested for their mutagenic potency in the host-mediated assay, in bone marrow of rats, in spermatogonia of mice and in the micronucleus test of rats. Only one substance, N1-methyl-N2-[4-phenylthiazolyl-(2)]-urea, was found to be mutagenic. This fact substantiates the hypothesis that the antiviral effectiveness of the substances is not caused by a chemical change in the coding system but by forming "physical" linkages like hydrogen bridge complexes with coding structures. Altogether 159 compounds of very different structures were investigated for their antiviral chemotherapeutic potency in 893 tests in cell cultures and 461 animal tests. It can be stated that compounds with a low molecular weight containing the "effective structures" (see article) show a larger yield of antiviral compounds than do substances without these structures (about 50% positive results in cell cultures and 25% in animals 15% and 0%, respectively).


[Studies on the antigenicity of synthetic ACTH (beta1-24-corticotropin) in normal subjects (author's transl)]. 9 healthy volunteers were subjected to a 3-week treatment with synthetic ACTH. Antibody response against beta1-24-corticotropin (Synacthen) was tested by passive transfer in the Prausnitz-Küstner reaction, complement fixation, passive hemagglutination and agar-gel diffusion. Failure of the healthy persons to produce reaginic or non-reaginic antibodies is compared titerature.


Metabolism of lipoproteins in nonhuman primates. Reduced secretion of very low density lipoproteins in squirrel monkeys with diet-induced hypercholesterolemia. We have studied the effects of diet-induced hypercholesterolemia on the rates of secretion of triglycerides into the plasma of fasted squirrel monkeys. Two groups of monkeys were studied: control animals which were fed a semipurified diet not associated with hyperlipemia (plasma cholesterol 127 +/- 8 mg/100 ml), and animals made hypercholesterolemic (plasma cholesterol 307 +/- 31 mg/100 ml) by being fed a diet containing 25% butter and 0.5% cholesterol. After intravenous infusion of Triton WR 1339 (300 mg/kg body wt), plasma triglycerides increased almost linearly for 9-12 hours. Analysis of individual lipoproteins separated by ultracentrifugation showed that newly secreted triglycerides were present almost exclusively in the very low density lipoprotein fraction. The rates of triglyceride secretion in the hypercholesterolemic group of monkeys (5.15 +/- 0.86 mg/kg/hr) were less than half those of the control animals (10.96 +/- 2.15 mg/kg/hr). We suggest that in monkeys with diet-induced hypercholesterolemia high concentrations of plasma low density lipoproteins may inhibit the synthesis and/or secretion of their parent very low density lipoprotein molecules into the circulation.


[Determination of anti-cytomegalovirus antibodies by the immunoperoxidase method: comparison with the methods of complement fixation and immunofluorescence]. Indirect immunoperoxidase technique (IIP) has been employed for determination of IgG antibody titer to cytomegalovirus (CMV) in sera from 110 blood donors. Titers have been compared with complement fixation (CF) and indirect fluorescent antibody (IFA) technique titers, the latter being applied to determination of all specific antibody classes. Within variability range of the quality of reagents employed in different tests, it seems reasonable to conclude IIP technique is as sensitive as IFA and much more sensitive than CF test. Considering CF test is able to detect only IgG antibody, the advantage of using immunohistochemical techniques is obvious. Moreover IIP does not need fluorescence microscope and results reading is easier and can be delayed. Non-specific staining of Golgi area is shared by both techniques, but endpoint determination has to take into account only the nuclear virus-specific inclusion labelling.


[The immunoperoxidase technic in the rapid identification of human cytomegalovirus on primary isolation]. Immunoperoxidase technique has been applied to the rapid identification of human cytomegalovirus (CMV) strains on primary isolation. Negative and positive results have been constantly confirmed by indirect fluorescent antibody technique (IFA) and by electron microscopy. Using either direct or indirect method it was observed that problems of nonspecific staining of uninfected cells were encountered mostly with direct method, probably because of the lower working dilution of the conjugate (1:20) as compared with higher conjugate dilution used for indirect method (1:150). However, nonspecific cytoplasmic stain of Golgi area of infected cells was observed also with indirect method. Pathogenesis of nonspecific labelling of this area is discussed. Application of the immunoperoxidase technique directly into tubes on primary isolation allows to reduce identification time in comparison with IFA technique and is less cumbersome and time-consuming than complement fixation and neutralization tests.


Intranuclear inclusions in shope fibroma virus infected cells. The presence of nuclear inclusions consisting of accumulated filamentous material has been observed in Shope fibroma virus infected cells, analogously to what has been demonstrated in other poxvirus infected cells.


Chylomicron formation and composition in unanaesthetised rabbits. Emulsified lipid was infused steadily into the upper small intestine of unanaesthetised rabbits for 6 h and 24 h periods. Lymph was collected from a thoracic duct cannula, the output of infused lipids was measured and the lymph chylomicrons were isolated. Recovery of infused triacylglycerol was 63 +/- 6.4% and recovery of infused radioactive cholesterol was 24 +/- 4.2% during a 24 h period. There was considerable dilution of radioactive exogenous cholesterol with endogenous cholesterol. Provided that absorption was well-established most exogenous lipid was present in the lymph in the form of chylomicrons, and there was a close relationship between lipid content of the lymph and the presence of chylomicrons. During absorption of fats of differing fatty acid composition chylomicrons remained the predominant transport form in the lymph. Chylomicrons obtained during absorption of several fats have been analysed in detail. The protein, phospholipid, free and esterified cholesterol content of these chylomicrons varies within narrow limits. The fatty acid composition of chylomicron triacylglycerols reflects the type of fat in the test meal but the composition of chylomicron phospholipids and cholesteryl esters shows clear discrimination. During absorption of coconut oil there is a strong negative discrimination towards lauric acid both for cholesteryl esters and for phospholipids.


Properties of two pig low density lipoproteins prepared by zonal ultracentrifugation. Pig plasma lipoproteins were separted into four density classes (very low density, two low density and high density lipoproteins, VLDL, LDL1, LDL2 and HDL respectively) from 670 ml plasma by ultracentrifugation in a continuous density gradient using the Spinco Ti15 zonal rotor. LDL1 and LDL2 were partly characterised. LDL1 and LDL2 are beta-migrating lipoproteins of different size and hydrated density; they are similar to human LDL2 and LDL3 respectively. Pig plasma contains about twice as much LDL1 as LDL2. LDL1 migrates at Sf 4.9 (modal value), and has a mean diameter of 217 A and a modal density of 1.035 g/ml (range 1.03-1.04 g/ml). LDL2 migrates at Sf 1.8 and has a mean diameter of 195 A and a density of 1.050 g/ml. Both lipoproteins are precipitated by heparin and Mn++ or by dextran sulphate and Ca++. The apoproteins of LDL1 and LDL2 are both largely insoluble in 8 M urea solution. When dissolved in 1% sodium dodecyl sulphate solution and electrophoresed on polyacrylamide gel at pH 7.0, the apoproteins of LDL1 and LDL2 formed a pattern of multiple bands of high molecular weight similar to that obtained from the apoprotein of human LDL. Both LDL1 and LDL2 share a major antigen with each other and with VLDL; in this respect again they resemble human LDL. The amino acid compositions of LDL1 and LDL2 are very similar. We concluded that the apoprotein moieties of pig plasma LDL1 and LDL2 are probably identical, and similar to apoprotein B in human serum. Zonal ultracentifugation has proved to be a rapid and effective method for isolating large quantities of these two lipoprotein classes for further metabolic studies. This method allows rapid bulk preparation of lipoproteins, and provides a record of their distribution and quantity in a continuous density gradient.


Interaction of Sendai virus with human erythrocytes. III. Further investigation on the effects of pyridine on the virus envelope. The ultrastructure of the pyridine-treated Sendai virus has further been investigated by electron microscopy of chemically fixed and negatively stained virions. Marked changes in the ultrastructural appearance of the viral envelope have been detected. The extent of these changes depends on pyridine dose and ranges from minor extraction of the outer track components to multiple breaks and disintegration of the whole envelope. At 16% pyridine a fragmentation of viral nucleocapsid has also been noted. Both the nature of these changes and the fact that even fully burst viral particles can eventually retain regularly-arranged spikes, strongly suggest that pyridine primarily affects the organization of the double-track viral membrane, probably by sequential extraction of lipids and protein components. The results are discussed in the light of the selective property of pyridine in discriminating among the biological activities of the Sendai virus and give additional support to the idea that phospholipids and a glycoprotein spike play cooperative roles in the hemolytic activity of the virus.


The plasma and tissue turnover and distribution of two radio-iodine-labelled pig plasma low density lipoproteins. Two classes of pig plasma low density lipoprotein (LDL1 and LDL2) with different densities and molecular sizes were isolated by zonal ultracentrifugation and were further purified by flotation. The peptide component was iodinated with 125I, and the labelled lipoprotein was injected intravenously. 125I-LDL1 turnover studies were performed on 22 3-4 month old female Large White pigs, and 125I-LDL2 turnover studies on 4 similar pigs. A biological screening experiment confirmed that the shape of the plasma activity curve was not a function of protein denaturation. The pattern of radioactivity decline in plasma was not affected by the degree of LDL iodination. 125I-LDL1 turnover: The curve of plasma radioactivity plotted against time over the first 5 days after injection could be resolved into two exponentials. The plasma biological half-life (T 1/2) was calculated from the slower exponential predominant from the second day. The mean T 1/2 over 2-5 days was 22.9 hr (range 17.2-28.5 hr). Multicompartmental analysis of the plasma decay curve using an open mammillary model gave a mean fractional catabolic rate per day for LDL1 of 1.4 (range 0.9-1.9). The mean T 1/2 was 0.26-0.31 times and the fractional catabolic rate 3.0-3.9 times those values found in two studies on adult humans. The tissue distribution of 125I was analysed in a series of 20 animals killed from 1.0 to 33.8 days after 125I-LDL1 injection. Most tissue 125I (86-89%) was protein bound. An appropriate correction was made to the 125I counts for retained plasma in liver and spleen (using 131I-albumin); retained plasma in other tissues was negligible. Highest 125I tissue levels were found in the liver, supporting other evidence that the liver may be the major site of LDL1 catabolism. After 2.06 and 4.06 days the livers in two animals contained 1.6% and 0.7% respectively of the total injected 125I, equal to 33% and 54% of the total plasma 125I at those times. The skin contained about one-third to one-ninth the 125I in the liver at various times. Distribution in other organs was quantitatively minimal. Higher levels of radioactivity were found in the intima and inner media of the aorta than in the outer media. These results suggest that plasma LDL in the pig diffuses through the endothelial surface into the arterial wall. These findings are confirmed by autoradiography. 125I-LDL2 turnover: Parallel studies of plasma 125I-LDL2 turnover and tissue distribution were performed. The plasma biological decay curve was multi-exponential, suggesting that LDL2 metabolism is complex, and possibly more rapid than that of LDL1 (LDL2 is smaller and denser than LDL1). The tissue distribution of 125I-LDL2 in these pigs was very similar to that of 125I-LDL1. As LDL1 and LDL2 differ in the amount of lipid they contain, they may have different roles to play in lipid transport, and there may be interconversion of one into the other at different sites. This hypothesis remains conjectural.


[Effect of cyclic nucleotides on the transformation and proliferation of lymphocytes]. When the studies on the immunologic system have met with the "second messenger" hypothesis of the cyclic nucleotides, new ideas have arisen about mechanisms regulating the immunologic reactions. Several works sustain a specific action of cyclic AMP and cyclic GMP to modulate the immunologic system, but the more subtle actions must be discovered. A hypothesis which attributes the control-task of the cellular functions to the c-AMP/c-GMP relation is presented.


Characterization of the T antigen induced by SV40. 3 or 4 antigenic SV40 specific components were established in hamster tumors induced by SV40-transformed cells, using linear sucrose gradient centrifugation and Sephadex G-200 column chromatography. The lightest component, approximate molecular weight 21,000, did not inhibit the nuclear staining of SV40-transformed cells in the fluorescent antibody inhibition test.


Renal subcellular fractions producing angionecrosis and increased vascular permeability. In order to define the subcellular localization and characters of substances in the rat kidney which increase vascular permeability and produce angionecrosis, the following investigations have been undertaken: (1) subcellular fractionation by use of differential centrifugation and osmotic shock treatment with enzyme profile determination; (2) chromatographic separation of lysosomal contents with concanavalin A affinity column. Lysomal contents contained substances that induced an increase of vascular permeability of the rabbit skin and angionecrosis in the pancreas of the bilaterally nephrectomized rats and the spontaneously hypertensive rats. Lysosomal contents treated at 60 degrees for 30 min showed no renin activity and yet produced angionecrosis. Non-affinity fraction from concanavalin A column chromatography showed no renin activity but produced angionecrosis and an increase of vascular permeability of rabbit skin.


The occurrence of vacuolated neurons in the brains of hamsters affected with subacute sclerosing encephalitis following measles or Langat virus infection. Hamsters infected by the intranasal route with either hamster adapted Langat virus passaged at least 10 times in hamsters and the HNT strain of measles virus passaged 149 times in hamsters, developed subacute sclerotic lesions in the pyriform cortex, and beginning from 2 months after infection, were accompanied by neuronal vacuolation with ballooning of the cytoplasm, especially in parts of the brain in close proximity of the sclerotic lesions. The vacuolated neurons resembled those seen in scrapie, especially of sheep and goats, suggesting a similarity between the effects of slow and subacute viral infections.


Diverse mechanisms of hepatocellular injuries due to chemicals: evidence in rats administered carbon tetrachloride or dimethylnitrosamine. Differences in acute hepatotoxicity of carbon tetrachloride (CCl4) and dimethylnitrosamine (DMN) have been tested in normal foetal, newborn and adult rats, foetal, newborn and adult rats pretreated with phenobarbitone and partially hepatectomized adult rats. While CCl4 is non-toxic to the foetal and newborn liver, DMN induces identical necrosis at all ages. Prior dosing with phenobarbitone augments CCl4 toxicity only in the adult and the newborn but the foetus continues to be resistant. Such pretreatment, on the other hand, significantly reduces the effects of DMN on liver in all animals. Partial hepatectomy makes the liver less susceptible to CCl4 and more so to DMN. Such diversities of hepatic response to the two toxins can be accounted for by the levels of the respective toxifying enzymes in the liver cell in different situations.


Tubuloreticular structures in human lymphoid cell lines. A cell biological study. Human lymphoid cell lines were studied as an experimental model for the spontaneous or induced occurrence of tuburloreticular structures (TRS). It was possible to induce TRS after culturing the EB-3 cell line with 20 mug/ml bromodeoxyuridine (BrUdR) during 96 h. Starvation, culturing at lower temperature (32 degrees) or inhibition of DNA synthesis did not give rise to the production of TRS. The response to BrUdR could be blocked with 60 mug/ml thymidine but not with 60 mug/ml deoxycytidine. The addition of 5 mug/ml cytarabine or the removal of BrUdR at different times resulted in inhibition of TRS induction, indicating that BrUdR had to be incorporated into DNA during at least 48 h. After incorporation, neither the presence of BrUdR nor DNA synthesis was necessary for the production of TRS. These experiments and the finding that in the cell line IHTC-33, which does not produce Epstein-Barr virus associated antigens, TRS were spontaneously present, exclude a correlation between TRS and these antigens. However, the induction of TRS by BrUdR may be related to the activation of another (latent) virus.


Influence of carbon tetrachloride or riboflavin on liver carcinogenesis with a single dose of aflatoxin b1. Liver carcinogenesis with a single dose of aflatoxin B1 (7 mg/kg body weight) has been investigated in a group of female Wistar strain rats by repeated biopsies and necropsies. Another group received a subsequent intoxication with carbon tetrachloride by inhalation (approximately 200 doses) and another one was overloaded with riboflavin (25 parts/10(6) in drinking water). The frequency of hepatomata was almost equal in the aflatoxin and aflatoxin-carbon tetrachloride group. It was lowere in the riboflavin-aflatoxin group. In these 3 groups cirrhosis was never present in neoplastic livers. Megalocytosis was the first lesion observed. All tumoral livers had previous or concomitant megalocytosis. This modification was about as frequent, intense and widespread in aflatoxin-CCl4 and aflatoxin groups but appeared much earlier, as did the first hepatoma, in the aflatoxin-CCl4 group. It was less frequent, less intense and less widespread in the riboflavin-aflatoxin group than in the aflatoxin group. There was also a lower frequency of hepatomata in the riboflavin-aflatoxin group, but the difference was not significant due to the too small number of animals involved. The facts are not a proof of the existence of an obligatory link between megalocytosis and carcinogenesis since a slight megalocytosis was observed in the riboflavin group not affected by the neoplastic process. However, the simplest explanation of our results would be to consider that the potential tumour cells are located among the megalocytic cells, without admitting that every megalocyte is obligatorily a precancerous cell. CCl4 seems to act in shortening the time of appearance of megalocytosis. The protective effect of riboflavine should be regarded with more caution.


Ultrastructural localization and differentiation of membrane-bound ATP utilizing enzymes including adenyl cyclase in normal and psoriatic epidermis. The total membrane-bound ATP hydrolytic activity in human epidermis is due to the activities of at least three differently located enzymes, namely Mg++-activated ATPase, phosphomonoesterase and adenyl cyclase. Cytochemical studies on psoriatic epidermis with various inhibitory and stimulatory substances showed reduced activities of ATPase and phosphomonoesterase, and a lack of sensitivity of adenyl cyclase to specific stimulators such as isoproterenol and glucagon. Since no differences of basal adenyl cyclase activity were observed between normal and psoriatic human skin without stimulation, it seems likely that in psoriasis a latent defect of adenyl cyclase may exist, resulting in a deficient response of this enzyme to regulatory agents. In conclusion, the present study reveals that not a single enzyme but the entire membrane-bound nucleotide metabolism is altered in psoriatic keratinocytes, causing a disturbance of the membrane-bound energy utilization, similar to findings in proliferating tumour cells.


Cellular immunity and circulating antibody to herpes simplex virus in subjects with recurrent herpex simplex lesions and controls as measured by the mixed leukocyte migration inhibition test and complement fixation. Cellular immune responses and circulating antibody levels to herpes simplex virus were examined in patients with recurrent herpes simplex (HSV) infections and controls. Mixed leukocyte migration inhibition by herpes simplex antigen was less in affected patients than in controls but serum antibody levels were higher. There was no significant difference in leukocyte migration between patients with active or recent lesions and other herpes subjects, and the response in the mixed leukocyte migration test to phytohaemagglutinin (PHA) was similar in patients and controls. The data presented suggest that a localized defect in cell mediated immunity to herpes simplex virus may exist and be responsible for recurrent herpes simplex infections.


An epidemic of picornavirus and adenovirus conjunctivitis. A pandemic of acute haemorrhagic conjunctivitis occurred in south-east Asia during 1970 and 1971, and became epidemic in Lucknow in May 1971. From the conjunctival swabs adenovirus-like agents were isolated in monkey kidney tissue culture; one was typed as adenovirus 2. In paired sera rising antibody titres were found against an adeno-like agent isolated and the picornavirus (EC2/71) isolated in Singapore. The epidemics in south-east Asia were caused by a picornavirus while in Lucknow both adenovirus and picornavirus appeared to be implicated.


Studies on NO2-Tyr82and NH2-Tyr82 derivatives of adrenodoxin. Effects of chemical modification on electron transferring activity. Bovine apoadrenodoxin was treated with tetranitromethane to introduce a nitro group into the tyrosyl residue at position 82 of this protein. The degrees of nitration under the best conditions were estimated to be 90% and nearly 100% on the basis of amino acid analysis and the spectrophotometric method, respectively. An amino derivative was prepared by reducing the nitro group with sodium dithionite. The apoadrenodoxin derivatives could be reconstituted to have an iron-sulfur chromophore similar to the native adrenodoxin which contains a 1:1 molar ratio of labile sulfur to iron content and displays absorption peaks at 414 and 450 nm. The enzymatic acitivies of these reconstituted nitro and amino derivatives toward cytochrome c reduction in the presence of adrenodoxin reductase and NADPH were 19 and 7% of native adrenodoxin, respectively. We studied the kinetics of the direct reduction of the reconstituted amino derivative in the presence of NADPH and adrenodoxin reductase under anaerobic conditons. The initial rate of reduction for the amino derivative was 7% of the native adrenodoxin, which is in good agreement with its activity toward cytochrome c reduction. From these results, it is concluded that by modifying the tyrosyl residue at position 82 of the adrenodoxin polypeptide, the electron-transferring activity of the molecule is largely diminished.


Stimulation of cholesterol ester exchange by lipoprotein-free rabbit plasma. A fraction in normal and hypercholesterolemic rabbit plasma of density greater than 1.25 stimulates the exchange of cholesterol esters between very low density and low density lipoproteins from hypercholesterolemic rabbit plasma. The exchange does not result from lecithin:cholesterol acyltransferase activity. The active factor appears to be a high molecular weight globulin with an isolelectric point of 5.2.


Conformational changes of glyceraldehyde-3-phosphate dehydrogenase induced by the binding of NAD. A unified model for positive and negative cooperativity. The fluorescence of the natural coenzyme, NADH, is used to monitor the environment of the nicotinamide moiety at the active centre of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). Changes of the fluorescence quantum yield and polarization of a small amount of NADH, totally bound by an excess of enzyme, show that at half-saturation of the oligomer with NAD a conformational change is induced which affects the active centre regions of the remaining subunits. This conformational transition is not effected by adenosine diphosphoribose, suggesting that the binding of the nicotinamide moiety of NAD to two subunits is essential for the change of tertiary structure of the remaining subunits that causes the observed changes of the fluorescence properties of the ADH "tracer probe". It is suggested that this conformational transition of the oligomer is responsible for the major decrease of affinity for NAD which occurs at half-saturation, and possibly for the activation by NAD+ of the reductive dephosphorylation reaction catalysed by the enzyme. It is also suggested, by analogy with haemoglobin, that the molecular basis of the negative cooperativity may be the creation of additional intersubunit bonds during the binding of the first two NAD molecules to the tetramer, and a change from a "relaxed" quaternary structure to a "tense" structure at half-saturation.


Further studies on the activation of microsomal (Na+ + k+)-atpase by a leukocytic product. Previous studies showed that microsomal (Na+ + K+)ATPase (ATP phosphohydrolase, EC 3.6.1.3) is activated by a proteinaeous material released by polymorphonuclear leukocytes. Investigations on the mode of action of the activator have been conducted by the siolation of 32P-labeled phosphoenzyme intermediates formed in the reaction of ATP and (Na+ + K)-ATPase, which has been postulated to occur through the formation and hydrolysis of acyl phosphate intermediates. The activator caused a concentration-dependent decrease in the recovery of phosphoenzyme intermediates that was not quantitatively altered by the Na+ or K+ concentration of the reaction mixture of by the presence of 1 mM oubain. A decline in phosphoenzyme intermediate recovery was promoted by the addition of the activator to preformed phosphoenzyme intermediates but not by activator that had been pretreated with protease or phenol. In addition, the activator caused a concentration-dependent stimulation of the p-nitrophenyl phosphatase and acetyl phosphatase activities of microsomal (Na+ + K+)-ATPase. It was proposed that the activator stimulates the dephosphorylation step of the (Na+ + K+)-ATPase reaction sequence.


Inhibition of peptide chain initiation in lysates from ATP-depleted cells.I. Stages in the evolution of the lesion and its reversal by thiol compounds, cyclic AMP or purine derivatives and phosphorylated sugars. Anaerobic incubation of rabbit reticulocytes at 37 degrees C in Krebs-Ringer solution supplemented with hemin but devoid of glucose resulted at the end of 1-2h in a drastic decline of their ATP content and an attendant arrest of protein synthesis. Subsequent provision of glucose and reoxygenation of the cells was followed by a rapid replenishment of the ATP pool, while resumption of protein synthesis was markedly delayed. This lag period could be considerably reduced by addition of 5-10 mM adenine or 2,6-diaminopurine to the incubation medium. Lysates prepared from ATP-depleted cells exhibited disaggregation of the polysomes and an inhibition of the nedogenously coded protein synthesis, when tested in a cell-free system supplied with an adequate ATP generator. Both alterations increased in severity with the progressive decay of the intracellular ATP pool. The early phase of partial inhibition following a 40-70% decrease of the cellular ATP level was fully reversible by fortifying the cell-free preparation with dithiothreitol or a suitable NADPH-generating system. Aternative, the inhibition could be also overcome by millimolar amounts of adenine, 2,6-diaminopurine and a variety of other purine derivatives or cyclic AMP. The effect of these compounds was unrelated to the endogenous cyclic AMP pool. Joint addition of both dithiothreitol and cyclic AMP or adenine was necessary for relieving the initiation block in lysates derived from cells depleted of 80-90% of their ATP content. On further aggravating the conditions of energy starvation, an additional requirement for phosphorylated sugars, e.g. glucose 6-phosphate or fructose 1,6-diphosphate, became apparent. ATP depletion brought about by exposing the cells to Antimycin A or 2,4-dinitrophenol resulted in a lesion which was indistinguishable from that induced by anaerobic incubation. On the other hand, energy deprivation in cell-free lysates from untreated reticulocytes, preincubated in the absence of an ATP-generating system failed to duplicate the deleterious effect of intracellular ATP depletion. Some aspects bearing on the biochemical mechanism of the lesion and its reversal are discussed in the light of the available data.


Inhibition of peptide chain initiation in lysates from ATP-depleted cells. II. Studies on the mechanism of the lesion and its relation to similar alterations caused by oxidized glutathione and hemin deprivation. The impairment of peptide chain initiation in lysates from ATP-depleted rabbit reticulocytes is accompanied by a loss of their ability to form the 40 S methionyl-tRNAfMet complex with a resultant failure to promote the AUG codon-dependent combination of the complex with the 60 S ribosomal subunit. These partial initiation reactions, as well as the overall protein-synthetic activity of the defective lysates could be restored by addition of a 0.5 M KC1 ribosomal extract or normal postribosomal supernatant or a 40-70% (NH4)2-SO4 fraction derived from it. Alternatively, reactivation of the impaired lysates could be achieved by supplementation with millimolar amounts of cyclic AMP or certain purine derivatives. The same subcellular fractions, as well as cyclic AMP or purine derivatives were also capable of overcoming the inhibition caused by incubating reticulocyte lysates in the presence of oxidized glutathione or in the absence of hemin. Severe intracellular ATP deprivation resulted in accumulation of a soluble translational inhibitor in the postribosomal fraction, thus resembling the parallel phenomenon described in hemin-deprived lysates. The striking similarities between the three kinds of inhibition studied by us point to an identical site of the underlying biochemical lesion, despite the different mechanisms mediating their induction.


Protein synthesis in homologous and heterologous cell-free systems from chick embryo connective tissues. Two homologous systems for cell-free protein synthesis from chick embryo connective tissues are described. Both the skin polysomes and the wing-leg polysomes are active in collagen synthesis, but they have different requirements for optimum protein synthesis. Protein synthesis was not dependent on tissue-specific factors, since heterologous preparations of supernatant enzymes or initiation factors were able to stimulate maximum protein synthesis with each fraction of polysomes.