Dialysis in renal failure casued by amyloidosis of familial Mediterranean fever. A report of ten cases. Ten unselected patients with renal failure casued by amyloidosis associated with FMF were treated by regular hemodialysis therapy from 1969 to 1974. They were compared to age-matched control patients treated by hemodialysis in the same unit during the same period, who were suffering from renal failure caused by other disease. Mortality in FMF and control patients was 30% with no significant difference in mean survival, shunt life, serum albumin or hemoglobin levels between the two groups. There was no significant difference in blood pressure measured predialysis or postdialysis in patients with FMF or in controls. The synthetic ACTH stimulation test showed adequate or elevated adrenocortical function. It is concluded that life can be prolonged up to 3 1/2 years by hemodialysis in renal failure caused by amyloidosis complicating FMF, and that renal failure casued by FMF is not a contraindication to regular hemodialysis therapy.


[Pseudohypohyperparathyroidism or Frame's pseudohypoparathyroidism type I]. Case report of a girl with fibrous osteitis lesions associated with clinical and biological symptoms of pseudo-hypoparathyroidism: hypocalcemia, hyperphosphoremia, absence of increase of phosphaturia (urine phosphate level) and especially of cyclic AMP after parathormone injection, high serum parathormone level. Similar cases described in literature are shortly analyzed. The genetic, pathogenic and nosologic problems of such an association are reviewed.


Recorded and reported sleep in chronic primary insomnia. Sleep polygraph and questionnaire data of 18 chronic primary insomniacs were compared with those of 18 age- and sex-matched controls. The insomniacs had significantly longer sleep latencies, less total sleep, less sleep efficiency, more terminal wake time, and less delta sleep. There were significant discrepancies between the insomniacs' and controls' subjective assessments of their sleep and the sleep-polygraph data, but in opposite directions. The insomniacs' recorded sleep also showed more night-to-night variability than that of the controls. However, the controls, in contrast to the insomniacs, reported sleeping worse in the laboratory than at home. Significant differences between insomnia subtypes validly reflected the insomniacs' subjective complaints and were generally in accord with expectations based on them.


[The "greifswald-therapy-model" for carcinoma of the cervix uteri for clinical testing of the cancer-multistep-therapy after M. von Ardenne (author's transl)]. The results of a preliminary study of the cancer-Multistep-Therapy (C.M.T.) (so called "The Greifswald-Modell") are reported. This Greifswald-Therapy-Model has been developed by hitherto existing experiences in the clinical test of the C.M.T. 4 patients with carcinoma uteri T2bNOMO to T2bNOMO level epithelium-carcinomas histologically verified showed after a preoperative treatment with C.M.T. and cantactradiatio (60CO) under the employment of a reduced dose of radios (1/2 to 1/3 of the pretherapy used in this clinic or 1/4 to 1/5 of the tumor dose) a complete regression of the tumor-specific changes. In the surgical specimen won after 4-7 weeks after C.M.T., carcinoma could not be detected either macroscopically or microscopically. Patients with a good therapy -- compatibility show a liberty of relapse with an increase in weight and an excellent physical and psychical health two year after C.M.T. This preliminary study shows the capability of the well - chosen therapy-model for the clinical test of the therapy-effect of the C.M.T. and it urgently demands to carry on with adequate alternate rows by including other conventional therapy schemes and other kinds of tumor to estimate definitively the effect of the C.M.T. in the tumor therapy.


Human adenohypophysis in Nelson syndrome. Ultrastructural and clinical study. The manifestations that comprise the disease known as Nelson syndrome are pituitary hyperplasia and cutaneous hyperpigmentation, which sometimes follow bilateral adrenalectomy, in patients with hypercortisolism. We present a comprehensive endocrinologic, structural study of a patient in whom the evidence obtained supports the hypotheses that: (a) the primary disorder in this form of hypercortisolism is probably hypothalamic; (b) the hyperplasia of the adenohypophysis, following adrenalectomy, is closely associated with lowered plasma cortisol levels; and (c) the cillular hyperplasia in the adenohypophysis involves primarily the corticotroph, a cell believed to be associated with the secretion of adrenocorticotrophic hormone and melanocyte-stimulating hormone.


Mixed tumors of the salivary glands. Growth pattern and recurrence. In 112 patients with mixed tumors of the salivary glands, 94 tumors were located in parotid glands, 11 in submandibular salivary glands, and 7 in monor palatal salivary glands. Capsular ingrowth, a characteristic growth pattern, may explain the frequency of multilobulation and capsular defects in these tumors, particularly those of long standing. The capsular thickness, the extent of fibrosis with stromal hyalinization, and the degree of lymphocytic infiltration within and around the capsule, were associated with cellularity of the tumors. Thus, 69% of the hypocellular neoplasms had incomplete capsules, and only 13% showed moderate to substantial lymphocytic infiltration; corresponding figures for hypercellular tumors were 22% and 42%, respectively. Of the recurrent lesions, 61% were hypocellular; 11% of the hypercellular tumors recurred. Incomplete resection, hypocellularity, and incomplete encapsulation were considered to be the major factors in recurrence.


Gigantic cystosarcoma phyllodes in a man with gynecomastia. A 70-year-old man had a breast tumor for 50 years. The tumor, which weighed 8.6 kg, proved to be a malignant cystosarcoma phyllodes. It was successfully removed by a simple mastectomy with low axillary dissection, and a skin graft was performed. The lesion was associated with gynecomastia, and it is believed to have represented a cystosarcomatous transformation of a giant fibroadenoma.


[Histoautoradiographic and biochemical study of the transport of atherogenic lipoproteins in the vascular wall]. Transport of serous pre-beta and beta-lipopropeins (beta-LP) labeled by C14 protein and by C14 lipid into the wall of the aorta was studied histoautoradiographically and biochemically. The aortas of healthy rabbits and rabbits wuth experimental atherosclerosis were perfused in vitro with a medium containing rabbit pre-beta and beta-LP during 6 hours. After perfusion the mark was identified histoautoradiographically in all layers of the aorta. The presence of gradient in distribution of the mark from the inner layers of the aortas towards the outward part of the media indicated that the process of infiltration of the wall of the LP vessel was caused by their penetration from the lumen of the aorta. Nonuniformity in distribution of LP penetrated into the atherosclerotic aorta was noted, which was considered in connection with the changes occurred in the connective tissue of the wall of the aorta in the process of atherogenesis. Results of both morphological and biochemical investigations confirm an increased permeability of the aorta wall for LP in atherosclerosis and also substantiate the assumption about penetration of pre-beta and beta-LP into the wall of the vessel as a whole particle.


[Structural-metabolic characteristics of the myocardium in acute hemorrhage and hyperbaric oxygenation]. Histochemical and pathomorphological changes in the myocardium in acute loss of blood and hyperbaric oxygenation were investigated in experiments on 130 white rats. It was established that acute loss of blood brought about an activation of phosphorylase, a decrease in the content of glycogen, an inhibition of the activity of cytochrome oxidase and succinic dehydrogenase in the myocardium. Foci of dystrophy were formed in the subendocaridal zone of the two ventricles and septum. Oxygenobarotherapy contributed to normalization of the level of activity of enzymes, preservation of glycogen, reduced the extent of manifestation of dystrophic changes in myocardiocytes. Hyperbaric oxygenation of healthy animals led to changes in the enzymatic activity in the myocardium. Dystrophic changes were noted in individual myocardiocytes. The data obtained testify to a direct influence of oxygen on metabolism of the myocardial cells.


[The histoenzymology of arterial walls under normal conditions and during the development of atherosclerosis]. Investigation of enzymes of the vascular wall in atherosclerosis in man and in experiment should include biochemical studies in combination with histochemical control. It is most probable that the total activity of enzymes in the vascular wall in atherosclerosis depends upon the functional state of smooth-muscle cells. The character of changes in the activity of enzymes in the arteries susceptible to atherosclerosis makes it possible to prognose disturbances in metabolism of the vascular wall and partially to explain the sequency of the development of the atherosclerotic process.


Granular cell myoblastoma of the cervical trachea. Only five granular cell myoblastomas affecting the cervical trachea have been previously reported. Two of these tumors appear to be primary lesions of the trachea, while the remaining three appear to involve it only secondarily. We report a case of an intraluminal granular cell myoblastoma arising from the right tracheal wall in a 45-year-old woman. The tumor extended into the partition wall between trachea and esophagus. Treatment was by surgical excision of the tumor and the involved tracheal ring. The patient was free of recurrence one year after treatment.


Facial diplegia.

Facial diplegia. Sudden facial diplegia or recurrent facial palsy is a rare occurrence, and may be the only symptom indicative of a systemic illness. Two patients had this condition in the absence of Guillain-Barré-Strohl syndrome.


[The fructose induced "glycogenosis". II. Histochemical studies of glycogen metabolism in rat liver after fructose overload and similar diets (author's transl)]. Feeding of fructose for 7 days has been morphometrically shown to induce a SER-reduction and an accumulation of glycogen in rat liver cells. This hypothetical model "glycogenosis" is investigated with histochemical methods. Rats are given a solution of 60% fructose in water as only nutritional source. Controls are given a solution of 60% glucose in water, an isocaloric Altromin-R-standard diet and an Altromin-R-standard diet ad libitum. Reversion of fructose induced metabolic changes is investigated by a 7 days fructose diet followed by an 1-4 days Altromin-R-standard diet ad libitum. Glycogen and glycogen metabolizing enzymes are demonstrated after a 7 days diet and in the course of an 1-7 days fructose diet. Feeding of fructose leads to a high glycogen content, combined with a high activity of glycogen-phosphorylase and glucose-6-phosphatase in the liver parenchyma. Glycogen-synthetase activity increases during the first 4 days and then it drops to a low level. A pathological alteration of liver cell metabolism seems to be improbable, for all fructose induced changes are reversibel after 2 days of Altromin-R-standard diet. Glucose-6-phosphatase, as a marker-enzyme of the smooth endoplasmatic reticulum, is discussed to become activated by disruption of SER membranes due to fructose.


Is atheroma a reversible lesion?

Is atheroma a reversible lesion? In this review atherogenic factors are discussed in relation to the possibility of regression. Evidence for regression of human lesions comes mainly from postwar studies and observations on persons with chronic wasting diseases. The entry, exit and effects of lipids in the arterial wass are considered as important factors which might determine regression. A variety of experiments in different animals which have been done in order to study regression are described. Some involve cholesterol feeding and withdrawal, others are concerned with the effects of hyperoxia and drugs. It is concluded that certain forms of atheroma can be induced to regress.


Clofibrate-induced low density liporotein elevation. Therapeutic implications and treatment by colestipol resin. Plasma lipid and lipoprotein responses to clofibrate were assessed in fifteen hypertriglyceridemic patients for the purpose of ascertaining low-density lipoprotein (LDL) changes. Subjects were grouped into either Type IV (11) or IIB (4) subgroups according to initial LDL level. Clofibrate was without effect on LDL in the IIB group, but consistent, often large, elevations were noted in Type IV cases (mean increase, 37.6%, P less than 0.001). In the IIB subgroup, addition of the bile-acid sequestrant, colestipol, lowered LDL (27.8%, P less 0.02) and total cholesterol (21.3%, P less 0.01) below pre-treatment values. In the Type IV subgroup, LDL fell to 19.5% above baseline (P great than 0.05). Significant LDL elevations induced by clofibrate in three of six subjects were restored to initial levels. In both groups, triglycerides and very-low density lipoproteins (VLDL) were not affected. The efficacy of colestipol in reducing LDL levels, expressed as either absolute or percentage reductions, increased as a function of increasing post-clofibrate LDL concentration (r = 0.84, P less than 0.001). In these subjects the level of LDL after treatment with clofibrate depended upon their LDL level prior to drug therapy, the effect of clofibrate on this level, and lipoprotein phenotype. Thus colestipol was most effective in IIB subjects, Type IV subjects with the lowest baseline VLDL and hence reciprocally highest LDL, and Type IV individuals who exhibited the largest LDL induction by clofibrate. The reported ineffectiveness of clofibrate on mortality and morbidity in patients with established coronary heart disease might be related to elevations and infrequent reductions of LDL. From the perspective of lipoprotein lowering, the combination with colestipol appears more favorable.


Lipoprotein concentrations in serum and in biopsy samples of arterial intima: a quantitive comparison. Lipoprotein concentrations were compared in serum and in samples of arterial intima obtained during cardiovascular surgery. The interval between blood sampling and surgery did not exceed 48 hours. Apolipoproteins B and C, the major proteins of low density lipoprotein (LDL) and very low density lipoprotein (VLDL) respectivity, were detectable by immuno-assay in all arterial specimens. Highly significant positive correlations existed between the levels of lipoproteins in the intima and in serum. In patients with elevated serum VLDL levels (Type IV hyperlipoproteinaemia) arterial concentrations of apolipoproteins B and C were increased about two-fold; in those with raised LDL levels in serum (Type II), with or without concomitant elevation of VLDL levels, the concentration of apolipoprotein B was increased almost 5-fold in the arterial intima. The arterial wall therefore contains lipoproteins or their immunologically-similar metabolites at concentrations which are determined in part by serum lipoprotein levels.


Relation between extent of coronary artery disease and severity of hyperlipoproteinaemia. Lipoprotein analyses were performed in 133 male patients and were correlated with the coronary arteriographic findings. The prevalence of hyperlipoproteinaemia was significantly higher in those patients with coronary artery disease (P less than 0-02). In addition, the more extensive the degree of coronary artery pathology the higher were the plasma concentrations of total cholesterol, triglyceride, and low density lipoprotein (LDL) cholesterol. Hyperlipoproteinaemia was more prevalent in the younger patients with coronary artery disease (P less than 0-02).


Triglyceride accumulation in cultured human fibroblasts: the effects of hypertriglyceridemic serum. Cultures of human diploid fibroblasts were shown to accumulate elevated levels of intracellular triglyceride when grown in medium supplemented with sera from patients with endogenous hypertriglyceridemia (Type IV hyperlipoproteinemia). Time course studies indicated cell triglyceride levels reached maxium in 6 to 12 hours. Isotopic studies indicated that the source of the accumulated cell triglyceride was serum triglyceride, and that most of the triglyceride was taken up without hydrolysis. Triglyceride was not significantly removed by trypsinization or extensive washing. The accumulated triglyceride was also demonostrated to be metabolized, as indicated by conversion to phospholipids and free fatty acids. Increasing concentrations of hypertriglyceridemic serum in the medium resulted in increasing cell triglyceride levels. However, increasing amounts of normal serum did not result in comparable cell triglyceride accumulation. Intracellular triglyceride accumulation seemed to correlate with levels of hypertriglyceridemic serum triglyceride, whether this serum was present at equivalent concentrations or normalized to triglyceride levels equivalent to normal serum. It is concluded that elevated serum triglyceride levels can result in intracellular triglyceride accumulation even in the absence of lipolytic activity.


Relationships between plasma lipoprotein cholesterol concentrations and the pool size and metabolism of cholesterol in man. The plasma concentration of unesterified and esterified cholesterol within very low density (VLDL), low density (LDL) and high density (HDL) lipoproteins have been examined in relation to the metabolism and pool size of cholesterol in normal and hyperlipidaemic subjects. Cholesterol metabolism was assessed as faecal endogenous neutral and acidic steroid excretion, a 2-pool model of cholesterol turnover, and in vitro plasma cholesterol esterifying activity. VLDL total cholesterol (TC) concentration was positively correlated with cholesterol turnover, endogenous neutral steroid excretion, bile acid excretion and the absolute rate of plasma cholestrol esterification. The correlations with cholesterol turnover and neutral steroid excretion, but not that with bile acid excretion, remained significant when these were corrected for their relationships to body weight. LDL-TC was negatively correlated with the fractional rate of plasma cholesterol esterification and, in subjects with primary type IIa hyperlipoproteinaemia, also with the rate constant for cholesterol elimination from the rapidly exchanging cholesterol pool. No correlation was found between LDL-TC concentration and bile acid excretion. HDL-TC concentration was negatively correlated with both the rapidly and slowly exchanging pools of tissue cholesterol, after correction for their relationships to body weight and adiposity. In contrast, cholesterol pool sizes were not correlated with the concentration of VLDL or LDL-TC; nor was there any relationship to plasma cholesterol esterifying activity. No correlation was found between the relative proportions of unesterified cholesterol within any lipoprotein fraction and either the pool size or metabolism of cholesterol. These findings accord with previous reports of enhanced cholesterol metabolism in subjects with elevated VLDL concentrations and of impaired plasma LDL and cholesterol clearance in patients with primary type IIa hyperlipoproteinaemia. The demonstration that HDL-TC concentration is negatively correlated with body cholesterol pool size supports in vitro evidence for a role of HDL IN TISSUE CHOLESTEROL CLEARENCE.


Changing relative proportions of apolipoproteins CII and CIII of very low density lipoproteins in hypertriglyceridaemia. The very low density lipoproteins (VLDL) of fasting serum from subjects with normo- and hypertriglyceridaemia (Type IV and V) were separated into subfractions by density gradient centrifugation. The tetramethylurea (TMU)- soluble apolipoproteins were separated by polyacrylamide gel electrophoresis and the relative proportions of apo CII and CIIIs determined. There were highly significant correlations between the concentration of VLDL triglycerides and apo CII (r---0.92), apo CIII1 (r=+0.88) and the ratio apo CII/apo CIII1 (r= --0.94). It was suggested that the decreasing ratio apo CII/CIII1 with increasing triglyceride levels might cause a resistance to lipoprotein lipase and therefore a defect lipolysis of VLDL based upon a changed ratio apo CII/apo CII1 might be part of the pathogenesis of the hypertriglyceridaemia.


Studies on antistreptolysin O activity generated in serum by microorganisms. An antistreptolysin factor (ASF) was generated in normal human serum by the growth of Staph, aureus and Pseud, aeruginosa. Alpha toxin producing strains of the former were usually positive but activity was not restricted to such strains. Positive strains produce cholesterol esterase which was obtained from DEAE-cellulose column fractions of 18 h broth cultures. Antistreptolysin factor develops slowly in serum, being maximal between the 5th and 10th days and is associated with alterations and disappearance of beta lipoproteins on gel electrophoresis. Activity also appeared in beta lipoproteins precipitated from normal serum with dextran sulphate and redissolved in nutrient broth before inoculation with Staph, aureus. The slow appearance of antistreptolysin activity in serum appears to be due to an esterase inhibitor which is present in high concentrations in some sera. Activity is also modified by the production of a staphylococcal fraction capable of binding to the antistreptolysin factor and reducing its activity. It is suggested that antistreptolysin factor which can be demonstrated in small amounts in normal human serum represents a readily available non-specific defence mechanism capable of binding to certain bacterial products and possible to other foreign protein molecules.


Detection of macrophages in atherosclerotic lesions with cytochrome oxidase. The anaerobic metabolism of the arterial wall allows macrophages to be demonstrated therein by the cytochrome oxidase histochemical method. Monocytes (macrophages) in human fatty streaks (WHO grade I) or fibrofatty (WHO grade II) human atherosclerotic lesions are normally confined to subendothelial regions. Lesions complicated by ulceration, mural thrombosis or intimal haemorrhage (WHO grade III) showed numerous monocytes (macrophages) around newly-formed capillaries in focal areas of organization. By contrast with grades I and II atherosclerotic lesions, macrophages in lipid granulomas induced by subcutaneous injection of cholesterol oleate are more numerous and distributed throughout the lesion. The slow or absent resorption of lipid from atheromatous lesions may in part result from the paucity of macrophages therein.


Further observations on subacute sclerosing encephalitis in adult hamsters: the effects of intranasal infections with Langat virus, measles virus and SSPE-measles virus. Passage by i.c. inoculations of suckling hamsters enhanced the virulence for adult hamsters of Langat virus (TP21), neurotropic strain of measles virus (HNT) and SSPE-measles virus (HBS), not only for i.c. infections but also for intranasal instillations. The various viral strains passaged in hamsters showed a great similarity of behaviour including the ability of producing in a proportion of apparently unaffected survivors a subacute sclerosing encephalitis, leading to atrophy of parts of the brain especially the rhinencephalon. When large groups of animals were used for transmission experiments it became obvious that within one week after intranasal exposure, all the hamsters either died or became clinically affected, or did not show signs of disease but developed acute inflammatory brain lessions. tlater on, between 2-6 weeks following inoculations only 90% of hamsters were affected with either overt signs of disease or subacute brain lesions, suggesting that in about 10% of hamsters the initial infection did not progress further and that in these animals the early brain lesions disappeared. Passage levels, irrespective of the virus used, did not influence the total numbers of infected hamsters but showed a significant effect on the mortality in TP21 and HNT infections where the number of dead and clinically affected increased in the higher passes. In these higher passes the number of survivors with subacute brain lesions decreased. In SSPE-measles virus the number of clinically affected hamsters and those surviving but developing brain lesions remained constant throughout. Vacuolated neurons were present in the brains of hamsters that survived one of the above 3 viral infections. They were seen beginning from 6 weeks after infection only in animals that developed subacute sclerosing lesions and were most commonly found in the amygdaloid nuclei and in the pyriform cortex. There was a dramatic increase in the number of brains with vacuolated neurons in hamsters infected with the high viral passes; however, in the 36th hamster passage of TP21 no vacuolated neurons were present but the total number of survivors was small, the majority had no brain lesions and none had subacute sclerosing changes.


The ineffectiveness of coumarin treatment on thermal oedema of macrophage-free rats. The administration of silica prevents coumarin-stimulated lysis of accumulated abnormal protein. This impairs the resolution of thermal oedema which is normally increased with coumarin administration. Evidence suggests that there is a rapid differentiation and infiltration of monocytes into the  tissues and that these are selectively retained. This is aided by coumarin which increases tissue permeability. Coumarin also injures the vascular endothelium of some vessels, allowing extra protein and fluid into the tissues. Death of recently differentiated macrophages and subsequent release of their lysosomal contents into the extra-cellular spaces may be responsible for the changes in serum enzyme levels. It would seem that macrophages are the only cells in which coumarin stimulates increased phagocytosis, enzyme production and proteolysis.


Experimental infection of monkeys with viruses of the tick-borne encephalitis complex: degenerative cerebellar lesions following inapparent forms of the disease or recovery from clinical encephalitis. Rhesus, patas and vervet monkeys were infected i.c. or i.n. with three viruses of the tick-borne encephalitis complex (TBE) as follows: Turkish tick-borne encephalitis virus (TTE), Louping-ill virus and Central European tick-borne encephalitis virus (CETE). The incidence of overt clinical signs of disease varied according to the virus that was used for the inoculations. TTE proved to be more pathogenic for monkeys than the other two members of the complex, whilst CETE was the least pathogenic. Injections of specific antiserum soon after infection tended to increase both the incidence of clinical signs and the moratlity. A proportion of animals with inapparent infections and a number of monkeys that recovered from the acute phase of the disease developed degenerative lesions in the cerebellum, often superimposed on the involuting inflammatory changes. The changes affected either a few only or many folia of the cerebellum and consisted of neuronal and spongy degeneration of the Purkinje and granular layers, usually accompanied by marked astrocytic proliferation and hypertrophy in the granular and molecular layers.


Enzyme-immunoassays for antibodies in measles, cytomegalovirus infections and after rubella vaccination. Enzyme-immunoassays using an indirect method with alkaline phosphatase conjugated antiglobulins were satisfactory for detection of antibody to Measles and Cytomegalovirus. The antigen was passively adsorbed to polystyrene micro-harmagglutination plates for the assays. IgM antibody to Rubella was also detected by enzyme-immunoassay at 7 and 28 days after vaccination in a person who had negative Rubella serology before vaccination. IgG antibody was detected at those times in another patient who had positive Rubella serology prior to vaccination. The enzyme immunoassays appear to have the potential for routine laboratory use for virological diagnosis.


Lysosomal hydrolases of the epidermis. 5. Variation with depth in the cow snout. The activity of twelve acid hydrolases has been measured in serial horizontal sections of cow snout epidermis. The majority of these enzymes showed a clear peak in the basal and lower spinous cell layers; acid phosphatase, however, was found mainly in the keratinizing zone. These findings suggest that two discrete populations of lysosomes may occur in epidermis. Those present in the lower layers possess a typical pattern of hydrolase activity and probably correspond to the 'orthodox' lysosomes observed by electron microscopy techniques, whereas those found in the keratinizing zone possess an unusual hydrolase profile and may correspond to the bodies identified morphologically as keratinosomes (Odland bodies, membrane-coating granules).


Corticotrophin and tetracosactrin depot-self-administered for the treatment of eczema. The prolonged use of self-administered corticotrophin in the form of 'CMC ACTH' (Crookes) or 'Synacthen Depot' (Ciba) has been considered in twenty-seven patients with severe subactute of chronic eczema. Complications were not troublesome and the therapeutic response was good. Many of the patients were able to control their disease fully without the effort and discomfort of additional local treatment and patients' acceptance of the treatment was high.


Relation between the inhibition of aggregation and the concentration of cAMP in human and rat platelets. Adenosine inhibits the aggregation of human but not of rat platelets whereas both are inhibited by prostaglandin E1 or by the pyrimido-pyrimidine compound RA233. In human platelets all three agents increase adenosine-3'-5'-cyclic monophosphate (cAMP). If the inhibition of aggregation depended on this increase, adenosine might be expected not to increase cAMP in rat platelets. Under conditions in which adenosine inhibited aggregation and increased cAMP in human platelets, adenosine caused a similar increase in cAMP in rat platelets without inhibiting their aggregation. The aggregation of rat platelets was inhibited as effectively as that of human platelets by PGE1 or RA233 at concentrations which caused greater increases in cAMP than did the highest concentrations (2.8 X 10(-4) M) of adenosine it was possible to use. When the increase of cAMP in rat platelets by PGE1 was limited to that produced by adenosine, PGE1 like adenosine failed to inhibit aggregation. Therefore, the difference in the inhibitory effectiveness of adenosine on rat and human platelets was quantitative rather than qualitative and apparently depended on the inability of adenosine to increase cAMP sufficiently in rat platelets. When cAMP had been increased by adenosine, PGE1 or RA233, the addition of ADP caused cAMP to decrease rapidly in both human and rat platelets to between +22 and -18% of control values, except that the decrease in rat platelets was to +40% after RA233 had been present for 0.5 min before ADP. The increase in cAMP produced in rat platelets by adenosine at 5 X 10(-6) to 2.8 X 10(-4) M for 3 min was associated with a small increase in aggregation velocity. It is suggested that the comparative ineffectiveness of adenosine as an inhibitor of platelet aggregation, particularly with rat but less so also with human platelets, is because, unlike PGE1 or RA233, adenosine has two opposing actions on aggregation; one being inhibition by activating adenylate cyclase and increasing cAMP, and the other being potentiation by uptake. This hypothesis accounts for the present results as well as for the earlier observation that dipyridamole which prevents the uptake of adenosine potentiates its inhibitory effect on the aggregation of human platelets.


Surface sulphydryl groups and phagocytosis-associated oxidative metabolic changes in human polymorphonuclear leucocytes. The role of surface sulphydryl (-SH) groups of human polymorphonuclear leucocytes on phagocytosis-associated oxidative metabolic changes was studied. p-Chloromercurybenzene sulphonic acid, a surface -SH group inhibitor, had no effect on phagocytosis, superoxide production during phagocytosis, or killing of S. aureus by the leucocytes, while it inhibited phagocytosis-associated stimulation of hexose monophosphate pathway activity and H2O2 production. In contrast, N-ethylmaleimide, which inhibits both intracellular and surface -SH groups, inhibited phagocytosis-associated oxidative metabolic changes. p-Chloromercurybenzene sulphonic acid also inhibited the stimulation of hexose monophosphate pathway activity by exogenous H2O2, suggesting a regulatory role of plasma membrane on the pathway's activity in human polymorphonuclear leucocytes. The results also suggest that: (I) superoxide production is the primary event of oxidative metabolic changes during phagocytosis, (2) superoxide plays an important role in the killing of S. aureus, and (3) the hexose monophosphate pathway plays a primary role in producing NADPH for H2O2 production from superoxide.


Surface sulphydryl groups and hexose monophosphate pathway activity in resting human polymorphonuclear leucocytes. The role of surface -SH groups on the hexose monophosphate pathway activity in resting human polymorphonuclear leucocytes was studied. Endotoxin, taurocholic acid and concanvalin A stimulated [I-14C]glucose oxidation to a greater degree than [6-14C]glucose oxidation. The stimulation of glucose-I-C-oxidation by endotoxin, taurocholic acid and concanavalin A could be prevented by p-chloromercurybenzene sulphonic acid, an inhibitor of surface -SH groups. In contrast, polymorphonuclear leucocytes from four patients with chronic granulomatous disease failed to show stimulation of glucose-I-C-oxidation by endotoxin, taurocholic acid or concanavalin A.


Adenocarcinoma of the uterine cervix a clinical and pathological appraisal. Twenty-six cases of primary adenocarcinoma of the cervix are analyzed. Their clinical features are described and attention is drawn to the variable histological pattern of these tumors.


Biochemical and pathological changes in tissues of Friesian cattle during the experimental induction of copper deficiency. 1. Copper deficiency was induced in five Friesian cattle offered a semi-synthetic diet containing less than 1 mgCu/kg. Changes in blood and liver Cu contents and in the Cu-containing enzymes, ferroxidase I (caeruloplasmin; EC 1.16.3.1) and monoamine oxidase (EC 1.4.3.4) of plasma and cytochrome oxidase (EC 1.9.3.1) of liver and skeletal muscle were monitored during Cu depletion. 2. Rapid decreases in blood and liver Cu and plasma ferroxidase I activity were found at least 80 d before the first appearance of overt clinical signs of deficiency. Plasma monoamine oxidase and liver cytochrome oxidase activities decreased less rapidly and thus may provide useful indices of chronic Cu depletion.


Differential phosphorylation by GTP and ATP in isolated rod outer segments of the retina. Isolated bovine rod outer segment protein is phosphorylated with GTP-gamma-32P and ATP-gamma 32P and to a much lesser extent by CTP-gamma-32P and UTP-gamma-32P. Phosphorylation with both GTP (GTP-kinase activity) and ATP (ATP-kinase activity) is markedly stimulated by light; phosphorylation with GTP is lower in dark-adapted and higher in light-adapted rod outer segments than is phosphorylation with ATP. Km values of 20 and 200 muM and Vmax values of 2.1 and 5.9 nmol/(mg min(-1)) were calculated using ATP and GTP, respectively, in light-adapted outer segments. When outer segments are incubated with GTP-gamma-32P under the usual conditions employed in these experiments, no formation of ATP-gamma-32P was detected by the techniques of high-pressure liquid chromatography and thin-layer chromatography. In intact, light-bleached outer segments, GTP appears to specifically phosphorylate rhodopsin. Histone and phosvitin are not phosphorylated to any appreciable extent by GTP. Histone appears to block rhodopsin phosphorylation by GTP while histone and, to some extent, phosvitin, both act as substrates for ATP-kinase activity. Cyclic AMP and other adenine derivates have a marked inhibitory effect on GTP-kinase activity. Phosphate also inhibits GTP-kinase activity but stimulates ATP-kinase activity. Such differences in phosphorylation with GTP and ATP indicate that these activities are either due to separate enzyme systems or, if only one enzyme is involved, the activities are under separate physiological control in the photoreceptor unit.


Effects of paramagnetic shift reagents on the 13C nuclear magnetic resonance spectra of egg phosphatidylcholine enriched with 13C in the N-methyl carbons. Effects of paramagnetic shift reagents on the 13C NMR spectra obtained from single-walled vesicle dispersions of egg phosphatidylcholine enriched with 13C in the N-methyl carbons are investigated. Spectra obtained at 25.1 MHz show that, at Yb3+ to phospholipid molar ratios as low as 0.06, complete resolution of the N-methyl carbon resonances is obtained from molecules on the inner and outer faces of the vesicle bilayer. No precipitation of the vesicles is caused by Yb3+ at these concentrations nor is appreciable line broadening observed. Other paramagnetic shift reagents frequently used in proton NMR investigations of phosphatidylcholine vesicles do not give complete separation of the N-methyl 13C signals from the two bilayer surfaces. K3Fe(CN)b,Eu3+, and Pr3+ cause precipitation of the phosphatidylcholine vesicles at concentrations, which give only incomplete resolution of these signals. T1 measurements of the resonances separated by Yb3+ indicate that the choline groups on the inner bilayer surface are less mobile than are the same groups in the outer surface. Gated proton decoupling measurements, which show that the nuclear Overhauser effect is 2.8 +/- 0.1, indicate that the dominant mode of relaxation is dipolar interaction.


Hormonal regulation of pancreatic islet adenyl cyclase. Adenyl cyclase activity of rat pancreatic islet membrane was increased by secretin, pancreozymin, and isoproterenol, while ACTH, glucagon, growth hormone, and insulin had no effect. Both secretin and isoproterenol activations were enhanced by prostaglandin E1 (PGE1) and GTP. Isoproterenol activation was additive with PGE1, as was that of secretin with PGE1, but only in the presence of GTP. Secretin activation in the presence of PGE1 and GTP was equivalent to NaF stimulation. Kinetic analysis indicated that secretin and GTP increased the maximum velocity of the adenyl cyclase and tended to decrease the apparent affinity of the enzyme for ATP. Glucagon activation of islet membrane adenyl cyclase was dependent upon prior treatment of the membrane preparation with EGTA and the use of inhibitors of proteolytic enzymes during the collagenase digestion phase of islet preparation. These results suggest that hormonal regulation of insulin secretion may be affected by PGE1 and guanine nucleotide modulation of the adenyl cyclase activation process.


Determination of molar ratios of vesicular stomatitis virus induced RNA species in BHK21 cells. A modified procedure for analysis of RNA in denaturing formamide-polyacrylamide slab gels containing 6 M urea is described. Using this technique, in conjucntion with fluorographic analysis, we determined molecular weights and molar ratios of the various vesicular stomatitis virus (VSV) induced RNAs in BHK21 cells. A comparison of the molar ratios of virus-specific mRHAs and their putative protein products in these cells suggests that there is little, if any, translational control of viral gene expression during acute VSV infection.


Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate). The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.


Partial purification and characterization of adenosine- and guanosine-3',5'-monophosphate phosphodiesterases from human lung tissue. Crude extracts of human lung tissue were examined for cyclic adenosine- and guanosine-3',5'-monophosphate (cAMP and cGMP) phosphodiesterase activities. Nonlinear reciprocal plots were observed for each substrate. DEAE-Sephadex chromatography of the extracts revealed four main fractions of activity, which were further purified by Sephadex gel filtration. The phosphodiesterase activity of the resulting individual fractions was partially characterized with respect to substrate specificity, kinetic parameters, apparent molecular weight (gel filtration), thermal stability at 30 and 37 degrees C, effect of the cyclic nucleotide not utilized as substrate, and the possible influence of Ca2+-dependent protein activator. The results indicate that the tissue contains phosphodiesterases with strict specificity and a high apparent affinity for each of the two cyclic nucleotides (the Km values determined were approximately 0.3-0.4 muM). The high affinity cAMP phosphodiesterase activity was enriched in two of the purified fractions; both activities probably represent fragments of the native high affinity cAMP specific enzyme. A third purified phosphodiesterase showed mixed substrate specificity. The Km value recorded for hydrolysis of either substrate with this enzyme was approximately 25 muM. A fourth, irregularly occurring, phosphodiesterase activity also showed mixed substrate specificity. The Km value registered for hydrolysis of either substrate with this fraction was approximately 0.4 muM. There was no evidence for a Ca2+-dependent specific activation by a boiled lung tissue supernatant of any of the purified enzymes.


Specific modification of an effector binding site of phosphofructokinase by pyridoxal phasphate. Conditions are described for the covalent modification of rabbit skeletal muscle phosphofructokinase by pyridoxal phosphate plus sodium borohydride to produce an enzyme that appears by a number of criteria to be modified at the citrate binding site. Evidence that modification occurs at this site is as follows. (1) Protection against activity loss due to modification is provided by the combination of MgATP and citrate, whereas neither low concentrations of citrate nor MgATP alone is effective. This is consistent with the increased affinity for citrate that is observed in the presence of MgATP. (2) The extensive changes in activity and equilibrium binding result from the incorporation of only 1 mol of pyridoxal phosphate per mol of protomer. (3) Modification greatly increases sensitivity to MgATP inhibition, an effect consistent with the known synergism between MgATP and citrate. (4) The affinity of the enzyme for both MgATP and MgIPT at the catalytic site is increased by the modification. (5) The sensitivity of the enzyme to citrate inhibition is greatly diminished following covalent modification. (6) Modification abolishes the equilibrium binding of citrate. (7) Enhanced binding of MgATP is observed following modification, a result consistent with the enhancement of MgATP binding by citrate. Phosphofructokinase protected by citrate plus MgATP can also be modified by the incorporation of 1 or more mol of pyridoxal phosphate, but the enzyme so produced is capable of interacting with citrate and shows none of the properties herein described for the enzyme modified in the absence of citrate.


Stimulation of ascites tumor RNA polymerase II by protein kinase. The activity of purified RNA polymerase II from Novikoff ascites tumor cells is stimulated 5-7-fold by a purified protein factor. This protein factor, designated HLF2, has extensive protein kinase activity and catalyzed the incorporation of gamma-32G from ATP into protein under normal RNA polymerase assay conditions. Protein phosphorylation is totally dependent on the presence of HLF2 and is stimulated 2-3-fold by the presence of highly purified RNA polymerase II. The purification procedure developed for the isolation of the polymerase stimulatory factor resulted in a 4000-fold purification of a protein kinase. Chromatography on carboxymethylcellulose, phosphocellulose, and Sephadex G-100 did not resolve polymerase stimulatory activity from protein kinase activity. Adenylimidodiphosphate (AMP-PNP), an inhibitor of protein kinases, inhibited the stimulatory activity of purified factor by 80%. The heat denaturation profile of protein kinase was paralleled by the loss of polymerase stimulatory activity. Concentrations of (NH4)2SO4 which are known to inhibit polymerase stimulation (Lee and Dahmus, 1973) also inhibit protein kinase activity. The protein kinase activity associated with stimulatory factor catalyzes the phosphorylation of basic proteins such as protamine or histone. The protein kinase is not stimulated by cyclic 3', 5'-AMP or -GMP over a concentration range of 10(-6)-10(-4)M. Furthermore, protein kinase activity is not inhibited by either the regulatory subunit of rabbit muscle protein kinase or by the heat-stable inhibitor of cyclic 3', 5'-AMP-dependent protein kinases. Protein kinase activity is stimulated by KCl or NH4Cl and is inhibited by MnCl2. The apparent Km values, determined in the presence of 4 mM Mg2+, are 0.02 mM for ATP, and 4.1 mM for GTP.


Phosphorylation of double-stranded DNAs by T4 polynucleotide kinase. The phosphorylation by T4 polynucleotide kinase of various double-stranded DNAs containing defined 5'-hydroxyl end group structures has been studied. Particular emphasis was placed on finding conditions that allow complete phosphorylation. The DNAs employed were homodeoxyoligonucleotides annealed on the corresponding homopolymers, DNA duplexes corresponding to parts of the genes for alanine yeast tRNA, and a suppressor tyrosine tRNA from Escherichia coli. The rate of phosphoylation of DNAs with 5'-hydroxyl groups in gaps was approximately ten times slower than for the corresponding single-stranded DNA. At low concentrations of ATP, 1 muM, incomplete phosphorylation was obtained, whereas with higher concentrations of ATP, 30 muM, complete phosphorylation was achieved. In the case of DNAs with 5'-hydroxyl groups at nicks  approximately 30% phosphorylation could be detected using 30 muM ATP. A DNA containing protruding 5'-hydroxyl group ends was phosphorylated to completion using the same conditions as for single-stranded DNA, i.e., a ratio between the concentrations of ATP and 5'-hydroxyl groups of 5:1 and a concentration of ATP of approximately 1 muM. For a number of DNAs containing protruding 3'-hydroxyl group ends and one DNA containing even ends incomplete phosphorylation was found under similar conditions. For all these DNAs a plateau level was observed varying from 20 to 45% of complete phosphorylation. At 20 muM and higher ATP concentrations, the phosphorylation was complete also for these DNAs. With low concentrations of ATP a rapid production of inorganic phosphate was noted for all the latter DNAs. The apparent equilibrium constants for the forward and reverse reaction were determined for a number of different DNAs, and these data revealed that the plateau levels of phosphorylation obtained at low concentrations of ATP for DNAs with protruding 3'-hydroxyl group and even ends is not a true equilibrium resulting from the forward and reverse reaction. It is suggested that the plateau levels are due to formation of inactive enzyme-substrate and enzyme-product complexes. For all double-stranded DNAs tested, except DNAs containing protruding 5'-hydroxyl group ends, addition of KCl to the reaction mixture resulted in a drastic decrease in the rate of phosphorylation, as well as in the maximum level phosphorylated. Spermine, on the other hand, had little influence. Both of these agents have previously been shown to activate T4 polynucleotide kinase using single-stranded DNAs as substrates (Lillehaug, J.R., and Kleppe, K. (1975), Biochemistry 14, 1221). The inhibition of phosphorylation of double-stranded DNAs by salt might be the result of stabilization of the 5'-hydroxyl group regions of these DNAs.


Estrogen binding proteins of calf uterus. Inhibition of aggregation and dissociation of receptor by chemical perturbation with NaSCN. Sodium thiocyanate up to 0.5 M is compatible with a stable estradiol-t-receptor complex during sucrose gradient centrifugation; however, the maximum permissible concentration in 0.1 M during Sephadex G-100 and G-200 chromatography. When NaSCN 0.1 M is added to low-salt cytosol (approximately 7 mg of protein/ml); (1) age-dependent aggregation of receptor is inhibited; (2) peaks of estrogen-binding activity in sucrose gradients and on Sephadex chromatography are sharp; (3) instead of the usual larger molecular states ("8S") found in low salt, most of estrogen receptor is under the following form: 4.1S; Stokes radius, 36 A; mol wt 61 000; flfo, 1.25; homogeneous at electrofocusing, with isoelectric point at 6.0. When cytosol containing NaSCN 0.1 M is diluted down to 2-3 mg of protein/ml or, only for sucrose gradients, NaSCN concentration is increased to 0.4-0.5 M, the 61000 dalton species decreases, being substituted, without loss of bound estradiol-t, by the following estrogen-binding entity: 28S; Stokes radius, 28 A; mol wt 32 000; flfo, 1.44. In the presence of NaSCN, KCl up to 0.4 M does not affect in a significant manner the molecular properties of the above forms. When NaSCN is dialyzed out, most receptor reverts to a 8-9S state. When cytosol is preincubated with Ca2+ (4 mM) and KCl (0.4 M) before addition of NaSCN, the above picture is modified only in the following aspects: (1) Sephadex chromatography peaks are broader and slightly but reproducibly shifted toward higher elution volumes; (2) the electrofocusing pattern consists of a two-peak heterogeneous band shifted toward higher pH (isoelectric points 6.4 and 6.6); (3) upon dialysis of NaSCN there is little or no reversion to faster sedimenting states. These modifications appear to depend on limited proteolytic attack of receptor by Ca2+ -activated receptor transforming factor (RTF), not on binding of Ca2+ to receptor. Present data suggest that the 4.1S entity is a dimer resulting from side-by-side pairing of 2.8S subunits. Molecular dimension of larger receptor forms purified from cytosol are consistent with the hypothesis that under native conditions in vivo dimers are coupled end-by-end into tetrameric structures with two stronger (between subunits) and two weaker (between dimers) bonding regions, and that tetramers may further self-associate. While NaSCN reversibly releases native dimers and subunits by direct impairment of intersubunit bonds, Ca2+ activated RTF irreversibly and specifically releases slightly modified, about 60000 mol wt dimers, by preferential proteolytic attack of the weaker bonding regions and indirect destruction of involved bonds. In vivo, this effect of RTF may be instrumental in mobilization and nuclear penetration of receptor-estradiol complex. Heteroassociation of receptor with other proteins of cytosol is not excluded by the above hypothesis.


Characterization of the plasma lipoproteins and apoproteins of the Erythrocebus patas monkey. Patas monkey lipoproteins were fractionated into four distinct classes by a combination of ultracentrifugation and Geon-Pevikon block electrophoresis and characterized with respect to their chemical and physical properties. Very low density lipoproteins (VLDL) were isolated at d is less than 1.006, were triglyceride rich, and were in the size range 300-850 A. They were similar in apoprotein content to the VLDL of man, dog, and swine. The Patas monkey low density lipoprotein referred to as LDL-I had beta mobility and a size which ranged from 190 to 240 A in diameter. Their chemical composition and apoprotein content were similar to those of human LDL. A second low density lipoprotein referred to as LDL-II occurred at a density of 1.05-1.085, ranged in size from 190 to 300 A, and contained the B, arginine-rich, and A-I apoproteins. Differences between LDL-I and LDL-II included a higher sialic acid content for LDL-II and lipid to protein ratios of 3.7 and 3.0 for LDL-I and LDL-II, respectively. In addition, the LDL-II, but not LDL-I, reacted immunochemically with antisera prepared to human Lp(a). The physical, chemical, and immunochemical properties indicated that monkey LDL-II were equivalent to the human Lp(a). Patas monkey HDL, equivalent to human HDL, were protein and phospholipid rich and ranged in size from 70 to 100 A in diameter. The two major HDL apoproteins, A-I and A-II, were isolated from apo-HDL by column chromatography. The amino-terminal sequence of Patas A-I showed striking homology to that reported for human, dog, and swing A-I. The amino acid composition of monkey A-II was very similar to that of human A-II; however, unlike human A-II, the monkey apoprotein was shown to exist as a monomer similar to that reported for Rhesus monkey A-II. The similarities between the plasma lipoproteins of the monkey and of man suggest that the Patas monkey would serve as a suitable model for metabolic studies.


Isolation and characterization of a dog serum lipoprotein having apolipoprotein A-I as its predominant protein constituent. The serum high density lipoproteins (HDL) of normolipemic dogs (beagles) were isolated in the density range of p 1.063 to 1.21 g/ml, and characterized in terms of composition and physical properties (flotation and diffusion coefficients, partial specific volume, molecular weight, electrophoretic mobility, ultraviolet absorption, and circular dichroism). The results indicated that canine HDL is a relatively homogeneous class with a molecular weight of about 230 000 and general properties similar to those reported for human HDL. After delipidation, the resulting apolipoprotein, apo-HDL, was fractionated by Sephadex G-200 column chromatography in urea or guanidine hydrochloride solutions. About 90% of the apo-HDL consisted of a protein with a molecular weight of about 28 000, similar in amino acid composition to human apolipoprotein A-I and having the same NH2 terminus (aspartic acid) and COOH terminus (glutamine) and no carbohydrates. Two other proteins were isolated, one having an apparent mol wt of 55 000 and representing, at least in part, an aggregate of apolipoprotein A-I and the other component with a mol wt of 8000, not yet characterized. The results indicate that canine HDL, as an intact complex, has general physical properties that lie between those reported for human HDL2 and HDL3, and that it differs compositionally from the human products mainly in its predominant content of apo-A-I. These findings together with evidence for the relatively homogeneous nature of the canine HDL provide new prospects for unraveling the relationship between polypeptide composition and HDL structure.


Study of the transverse diffusion of spin labeled phospholipids in biological membranes. I. Human red bloods cells. Spin labeled analogs of phosphatidylcholine were used to study the transverse diffusion (flip-flop) of phospholipids in the erythrocyte membrane. The nitroxide spin label was placed either on the beta acyl chain or on the choline group. These labeled phosphatidylcholine molecules were incorporated into the membrane by incubation of the red cells at 22 degrees C with sonicated spin-labed phosphatidylcholine vesicles from which all traces of free fatty acids and lyso derivatives were carefully removed by bovine serum albumin treatment. This incorporation did not provide any change in the morphology of the cell as indicated by scanning electron microscopy. When spin-labeled phosphatidylcholine, having a nitroxide on the beta chain but near the polar head-group, was incorporated into the erythrocyte membrane, ascorbate treatment at 0 degrees C allows selective reduction of the signal coming from the outer layer of the membrane. When the label was on the polar head-group, the inner content of the erythrocyte rapidly reduced the label facing the cytoplasm, thus creaging a spontaneous anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the erythrocyte membrane was found to be stable at 22 and 37 degrees C for more than 4 h. It is therefore concluded that the rate of outside-inside and inside-outside transition is so slow that the anisotropic distribution of the phospholipids in the erythrocyte membrane can be maintained during cell life.


Study of the transverse diffusion of spin-labeled phospholipids in biological membranes. II. Inner mitochondrial membrane of rat liver: use of phosphatidylcholine exchange protein. Spin-labeled phosphatidylcholine was incorporated into the membrane of isolated "inner membrane+matrix" particles of rat liver mitochondria by incubation with sonicated spin-labeled phosphatidylcholine vesicles at 22 degrees C. When the spin label was on the acyl chain the incorporation of phosphatidylcholine into the membrane was stimulated by the presence of the phosphatidylcholine exchange protein extracted from rat or beef liver. On the other hand no stimulation was observed when the nitroxide was on the polar head-group. When spin-labeled phosphatidycholine was incorporated into the mitochondrial membrane in the absence of phosphatidylcholine exchange protein, ascorbate treatment at 0 degrees C reduced the EPR signal of the spin-labeled membranes by approximately 50%, indicating that fusion incorporates molecules equally on both sides of the membrane. On the other hand when spin-labeled phosphatidylcholine was incorporated in the presence of the exchange protein most of the EPR signal could be destroyed by the ascorbate treatment at 0 degrees C, indicating that the spin-labeled phosphatidylcholine had been selectively incorporated in the outer layer of the membrane. Finally when the label is on the polar head-group the inner content of mitochondria reduces the label facing the matrix, thus creating again an anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the mitochondrial membrane was found to be stable at 25 degrees C for more than 2 h. It is therefore concluded that the rate of outside-inside and inside-outside transitions are extremely slow (half-life greater than 24 h).


Regulation of uracil uptake in Escherichia coli by adenosine 3',5'-monophosphate. Culture of a wild-type strain of Escherichia coli in the presence of cyclic AMP leads to an impairment of uracil uptake. Half maximum inhibition of uracil uptake was observed at 1.5 mM cyclic AMP. The effect seems to be specific since no inhibition was found in cultures supplemented with ATP, ADP or 5'-AMP. Similarly the inhibition was not observed in cultures of a mutant deficient in the cyclic AMP receptor protein. The inhibition in uracil uptake, found in bacteria cultured in the presence of cyclic AMP, is not a consequence of a reduction in the growth rate. On the other hand, this inhibition was observed only in cultures containing glucose or pyruvate as carbon source.


Concanavalin A stimulation modifies the lipid and protein structure or rabbit thymocyte plasma membranes. A laser raman study. (1) We have compared the laser Raman spectra of isolated plasma membranes from resting rabbit thymocytes and cells mitogenically stimulated with concanavalin A. (2) Major alterations in the CH stretching, CH deformation and CC stretching regions indicate a different lipid architecture in the membranes from activated cells. (3) Spectral changes in the Amide I and II regions, by reference to the spectra of model compounds indicate greater protein amidation in the membranes from stimulated cells.


Motion of spin-labeled fatty acids in murine macrophages. Relation to cellular phagocytic activity. Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels. Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings (2 T11) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives. Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 degrees C, or by fixing them with 0.25% glutaraldehyde. No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with cis- or trans-unsaturated fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.


Characterization of the parathyrin receptor in renal plasma membranes by labelled hormone and labelled antibody binding techniques. The parathyrin receptor in renal cortex has been investigated by studying the binding of 125I-labelled parathyrin, or of unlabelled parathyrin detected with 125I-labelled antibodies, to a partially purified plasma membrane fraction. The kinetics of hormone uptake demonstrated a biphasic response in both systems at 22 degrees C but this phenomenon was not detectable at 37 degrees C. Specific displacement of lactoperoxidase labelled 125I-labelled parathyrin occurred with 8 ng unlabelled bovine parathyrin. The apparent affinity constant was 2.3-10(8) M(-1) and the apparent binding capacity of the membranes 1.25 pmol/mg protein. Using the labelled antibody technique the receptor showed maximal binding at pH 7.0-7.5. As little as 80 pg bovine parathyrin produced a significant increase in binding of labelled anti-bovine parathyrin antibody and saturation of binding sites was demonstrated at 2.5 pmol/mg protein. Oxidized hormone showed undetectable binding. Treatment of membranes with phospholipases A or D, or Trypsin greatly reduced subsequent hormone binding. Prior incubation of membranes with 1-34 synthetic parathyrin decreased the binding of intact hormone whereas gastrin, insulin and glucagon had no effect. Growth hormone and calcitonin slightly increased parathyrin binding.


Energy-dependent Mn2+ and Ca2+ uptake by the embryonic chick chorioallantoic membrane. The chick chorioallantoic membrane is an epithelial tissue which actively transports large amounts of Ca2+ during embryonic development. In this paper Mn2+ uptake by the tissue was studied and compared to Ca2+ uptake in parallel experiments. The purpose of these experiments was to determine if Mn2+ could be used to gain more information about the Ca2+ transport system. It was found that Mn2+ uptake was reduced significantly under conditions that reduced Ca2+ uptake and that Mn2+, like Ca2+, was taken up preferentially by the ectodermal side of the tissue. Mn2+ uptake showed saturation kinetics with a Km of 0.33 MM. Mn2+ uptake was also competitively inhibited by Ca2+, and Ca2+ uptake inhibited by Mn2+. Electron microprobe studies showed that Mn2+ was localized in the ectoderm of the tissue in the same way as Ca2+. It was concluded from these studies that significant amounts of Mn2+ were accumulated by the active Ca2+ transport mechanism and that Mn2+ could be useful paramagnetic probe of divalent cation transport in this tissue.


Gramicidin S-synthetase. Electrophoretic characterization of the multienzyme. 1. A method characterizing the fully active gramicidin S-synthetase (EC. 6.3.2.-) multienzyme in protein mixtures by a combination of sedimentation and polyacrylamide gel electrophoretic mobility data has been described. 2. The molecular weight of 280000 has been reevaluated by gradient centrifugation, gel filtration, and polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The size of the multienzyme is not changed by sodium dodecyl sulfate treatment. 3. In polyacrylamide gel electrophoresis dimerisation occurs in Tris, while two bands, which may represent monomer and dimer, are observed in phosphate. 4. Reliability of molecular weight determinations of sodium dodecyl sulfate-protein complexes of sizes up to 300000 daltons has been determined, correlating either mobilities or retardation coefficients.


Gramicidin S-synthetase. A further characterization of phenylalanine racemase, the light enzyme of gramicidin s-synthetase. 1. Chromatography on hydroxyapatite and on aminohexyl-Sepharose as well as isoelectric focusing were introduced as new effective purification procedures for phenylalanine racemase (EC 5.1.1.11). The enzyme preparations obtained were essentially homogeneous, as demonstrated by specific activity measurements and polyacrylamide gel electrophoresis. 2. The enzyme is not dissociable by sodium dodecyl sulfate. 3. Phenylalanine racemase is an acidic protein with an isoelectric point of approx. 4.6 (isoelectric focusing). 4. The Michaelis constants of L-Phe and D-Phe in the aminoacyl adenylate activation are 0.06 and 0.13 mM, respectively. 5. From our studies with structural analogues of phenylalanine we infer that the amino group of this amino acid is essential for its binding to the aminoacyl adenylate reaction center. The carboxyl group is not at all or only weakly bound. The benzene ring of phenylalanine which determines substrate recognition also seems to be of minor importance for substrate binding.


Affinity chromatography of thiamin pyrophosphokinase of rat brain. Affinity column chromatography coupled with thiamin monophosphate absorbs thiamin pyrophosphokinase activity in the crude extract of rat brain, and the enzyme can be eluted from the column by 0.01 mM thiamin with approximately 700-fold purification.


Studies on inosine monophosphate dehydrogenase. Steady state kinetics. The reaction catalyzed by IMP dehydrogenase (IMP: NAD+ oxidoreductase EC 1.2.1.14) from Aerobacter aerogenes has been investigated kinetically at pH 8.1 as a three reactant system by means of steady-state velocity studies in the absence of products, as well as by inhibition studies using products and substrate analogues. The mechanism appears to be a partially random one in which IMP and K+ can bind randomly to the free enzyme while NAD does not react unless K+  or both K+ and IMP are present on the enzyme. While the steady-state velocity data can be analysed adequately on the basis that rapid equilibrium conditions apply, this is only an approximate description of the mechanism since product inhibition studies indicate that there is a significant concentration of an enzyme-XMP (enzyme-K-XMP) complex in the steady-state.


Folates as inhibitors of glutamate dehydrogenase. Folates and tetrahydrofolates inhibit beef liver glutamate dehydrogenase (EC 1.4.1.2). Double reciprocal plats indicate a competitive inhibition for alpha-ketoglutarate-glutamate by folic acid and methotrexate and a complex or mixed type for NAD-NADH site. Pteroic acid is not inhibitory at the concentrations studied. The addition of up to four gamma-linked glutamyl residues to folic and tetrahydrofolic acids increases the inhibition. Further chain elongation of the gamma-peptide had no effect on the inhibitory activity. The p-aminobenzoate poly-gamma-glutamates were less inhibitory than the corresponding folyl polyglutamates.


Protein kinases and their substrates in brown adipose tissue from newborn rats. The 10000 X g supernatant fraction of brown fat from newborn rats catalyzed the cyclic AMP-dependent phosphorylation of both histone and a preparation of proteins from the same subcellular fraction (endogenous proteins). The apparent affinity for ATP was lower for the phosphorylation of the endogenous proteins than for the phosphorylation of histone. In order to discover whether the phosphorylation of histone and the endogenous proteins were catalyzed by different enzymes, the 100000 X g supernatant was fractionated by ion-exchange and adsorption chromatography. Three different cyclic AMP-dependent protein kinases and one cyclic AMP-independent protein kinase were separated and partially purified. Each of these enzymes catalyzed the phosphorylation of both substrates, and the difference in apparent Km for ATP remained. Neither affinity chromatography on histone-Sepharose, nor electrophoresis on polyacrylamide gels resulted in the separation of the phosphorylation of histone from that of the endogenous proteins of any of the partially purified kinases. Moreover, experiments in which the phosphorylated substrates were separated by differential precipitation with trichloroacetic acid showed that the endogenous proteins competitively inhibited the phosphorylation of lysine-rich histone. It is concluded that each of the partially purified kinase preparations contains protein kinase, which catalyzes the phosphorylation of both substrates. The difference in apparent Km for ATP was found to be due to the presence in the endogenous protein preparation of a low molecular weight compound which competes with ATP. This was not ATP nor the modulator protein. The ratio of the phosphorylation of endogenous proteins to that of histone was much higher for the cyclic AMP-independent kinase preparation than for the other enzymes. Electrophoresis of the endogenous substrates in the presence of sodium dodecyl sulphate showed that the enzyme phosphorylated a greater number of proteins than did the cyclic AMP-dependent kinases. The phosphorylation of endogenous proteins relative to that of histone was significantly lower for one of the cyclic AMP-dependent kinases than for the other two. This difference was not reflected in a different pattern of phosphorylation of the individual proteins of the endogenous mixture.


Thiophosphate-activated phosphorylase kinase as a probe in the regulation of phosphorylase phosphatase. Rabbit muscle nonactivated phosphorylase kinase (EC 2.7.1.38) is converted to thiophosphate-activated phosphorylase kinase by cyclic AMP dependent protein kinase, Mg2+ and ATP-gamma-S/adenosine-5'-O-(s-thiotriphosphate)/. The formation of thiophosphate-activated phosphorylase kinase wal also observed in the protein-glycogen complex from skeletal muscle. This new form of kinase is resistant to the action of phosphatase and behaves as a competitive inhibitor in the dephosphorylation of phosphorylase alpha by phosphorylase phosphatase (Ki = 0.04 mg per ml). The fact that the inhibitory effect of thiophosphate-activated phosphorylase kinase is 3 times higher than in the case of nonactivated kinase, may explain the transient inhibition of phosphorylase phosphatase in the protein-glycogen complex. The use of activated (phosphorylated) phosphorylase kinase supports this assumption since it causes a delay in the dephosphorylation of phosphorylase alpha, i.e. the conversion of phosphorylase alpha into beta could start only after the dephosphorylation of activated phosphorylase kinase.


Collagenolytic activity from isolated bone cells. A true collagenase (EC 3.4.24.3) which had been discovered previously in bone culture fluids and extracts of whole bone has now been localized to the cellular component of bone. The cellular enzyme bears the same characteristics as that of bone collagenases described earlier. Moreover, it is directly extractable in relatively large quantities. Attempts to demonstrate the presence of a bone cell procollagease were unsuccessful. It was also observed that the cells secrete significant amounts of collagenase in vitro. With increasing incubation time the extracellular collagenase levels rise and the intracellular collagenase levels drop.


Purification and characterization of phosphodiesterase I from Bothrops atrox. Phosphodiesterase I from the venom of Bothrops atrox has been purified by successive chromatography on phosphocellulose P-11, hydroxyapatite, and DEAE-cellulose DE 52. The final product gave a single band on sodium dodecylsulfate-polyacrylamide gels and was free of endonuclease, 5' -nucleotidase, and unspecific alkaline phosphatase activity. It was concentrated in an Amicon ultrafiltrator without loss of activity and could be stored in 10 mM magnesium acetate and 10% glycerol at 4 degrees C for at least a year. Under optimal conditions, the enzyme reaction required 15 mM Mg2+ and a pH of 9.2. Phosphodiesterase I is relatively thermostable and, in the presence of a macromolecular substrate, was not denatured after 4 h at 55 degrees C. The pure enzyme offers new possibilities for sequence studies on highly structured nucleic acids at elevated temperatures.


Identification of DNA in the core component of avian myeloblastosis virus. Avian myeloblastosis virus (AMV) was found to contain DNA associated with the virion. The viral envelope was removed by treating the virus with a nonionic detergent and the DNA was found in the core fraction. These experiments indicate that the DNA associated with tumor virus is not contaminant associated with the viral envelope and suggest that the DNA is part of the internal core component. The DNA from avian myeloblastosis virus has a density of 1.70 g/cm3.


Electron transfer between protein and DNA in gamma-irradiated deoxyribonucleoprotein. When deoxyribonucleoprotein-proflavine complexes were studied by electron spin-resonance spectroscopy following gamma-irradiation, it was found that stable free radicals were not formed at random on the complex but were preferentially located on proflavine. Since proflavine intercalalated to DNA bases serves as a final acceptor of electrons liberated by ionization, the result of our experiment was regarded as suggesting that the electron transfer from the protein moiety to the DNA moiety occurred in the irradiated deoxyribonucleoprotein.


Repair replication in human cells. Simplified determination utilizing hydroxyurea. A simplified and shortened procedure has been developed for the determination of repair replication of DNA in cultured mammalian cells. The procedure, using the bromodeoxyuridine density label and a radio-isotopic label has been applied to normal diploid human cells (WI38) and to their SV40 transformants (VA13). After incubation with the repair label the cells are lysed and digested for two hours at 50 degrees C with proteinase K. This digest can then be immediately subjected to alkaline cesium chloride density gradient centrifugation with no need for DNA extraction. Hydroxyurea is used to reduce the level of semi-conservative synthesis that a quantitative determination of repair replication can be accomplished by a single centrifugation. The method is not affected by variation in the effectiveness of the inhibitor although a small amount of semi-conservative synthesis normally occurs in the presence of the drug. The time course of repair replication in WI38 cells is unaffected by the drug. The apparent amount of repair synthesis in ultraviolet irradiated cells is increased 25 to 40% in the presence of hydroxyurea when thymidine is used as tracer. Under certain conditions in which the level of semiconservative synthesis is low (e.g., contact inhibited cells, high ultraviolet doses) the use of hydroxyurea is unnecessary.


Possible interaction of cyclic nucleotides with the prolactin stimulation of casein synthesis in mouse mammary gland explants. During a 10-h incubation, cyclic nucleotide phosphodiesterase inhibitors, viz. theophylline and quinine, were found to reduce by 40-50% the rate of [3H] leucine incorporation into casein in mammary gland explants from midpregnant mice. Further, dibutyryl cyclic AMP as well as the phosphodiesterase inhibitors were found to abolish the prolactin stimulation of leucine incorporation into casein. Elevated levels of cyclic AMP therefore appear to impair the functionality of the mammary gland. Although cyclic GMP was previously shown to stimulate RNA synthesis in the mammary gland in a prolactin-like manner, it had no effect on the rate of casein synthesis in mammary gland explants. Preincubation of explants with cyclic GMP did, however, attenuate the time required for the commencement of the prolactin stimulation of the rate of leucine incorporation into casein. A physiological role of cyclic GMP for the regulation of the rate of casein synthesis is thus suggested.


Tryptophan 2,3-dioxygenase: a review of the roles of the heme and copper cofactors in catalysis. L-Tryptophan, 2,3-dioxygenase (EC 1.13.11.11) has been purified to homogenity from L-tryptophan induced Pseudomonas acidovorans (ATCC 11299b) and from L-tryptophan and cortisone induced rat liver. The enzyme from both sources is composed of four subunits and contains two g-atoms copper and two moles heme per mole tetramer. The proteins from the two sources are not identical. Three oxidation states of tryptophan oxygenase have been isolated: (1) fully oxidized, [Cu(II)]2[Ferriheme]2; (2) half reduced, [Cu(i)]2[ferriheme]2; and (3) fully reduced, [Cu(I)]2[ferroheme]2. Catalytic activity is dependent solely on the presence of Cu(I) in the enzyme, the heme may be either ferro or ferri. The presence of Cu(II) in the enzyme results in a requirement for an exogenous reductant, such as ascorbate, in order to elicit enzymic activity. Ligands, such as cyanide and carbon monoxide, can inhibit catalysis by binding to either or to both the copper and heme moieties. Metal complexing agents, such as bathocuproinesulfonate and bathophenanthrolinesulfonate, can inhibit catalysis by binding to Cu(I) resent only in catalytically active enzyme molecules. During catalysis by the fully reduced form of the enzyme, molecular oxygen binds to the heme moieties, while during catalysis by the half reduced form of the enzyme it does not, presumably binding instead to the Cu(I) moieties. Enzymes that catalyze similar reactions have been purified from other sources. Indoleamine 2,3-dioxygenase appears to be a heme protein, but its copper content is unknown. Pyrrolooxygenases appear to be completely different enzymes, although they have not yet been purified to homegeneity.


Analysis of estrogen glucuronides. I--Characterization of estrogen glucuronides by gas chromatography mass spectrometry. The mass spectral characterization of nine kinds of estrogen glucuronides was investigated. The glucuronides were analysed after conversion to n-propyl ester-O-TMS ether derivatives by gas chromatography mass spectrometry. The fragmentation of these derivatives is discussed using deuterium labels and TMS-d9 imidazole. A clear-cut difference was found between aliphatic and aromatic glucuronic acid conjugates. In particular the [M - 362]+- fragment ion was characteristic in the mass spectra of estrogen 3-glucuronides. On the other hand, four isomers of estriol and 16-epiestriol glucuronides containing an aliphatic acetal linkage were also characterized by the fragment ions of m/e value 204, 345, 415, and 431 and their relative intensities. Thus, all nine estrogen glucuronides were discriminated mass spectrometrically.


Characterization of sand as a support for immobilized enzymes. The potential of sand as a support for immobilized enzymes was investigated by preparing alkylamine sand and devising methods to measure the total number of amine groups present and the fraction available for immobilization of enzymes. Alcohol dehydrogenase (alcohol: NAD oxidoreductase, EC 1.1.1.1.) and lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) were immobilized on alkylamine sand, and the stability of the immobilized protein and dehydrogenase activity was measured. Urease (urea amidohydrolase, EC 3.5.1.5) was also immobilized on sand to test the applicability of these methods to larger scale immobilizations. Results suggest that sand shows promise as a support for immobilized enzymes.


[Activity of energy metabolism related enzymes in the myocardium and liver following administration of large doses of noradrenaline]. The activity of the glycolytic enzymes and of the glucose-6-phosphate dehydrogenase (G-6-PDH) were compared with the content of noradrenaline in rat myocardium and the liver after the intraperitoneal injection of high doses of noradrenaline. It was shown that 24 hours after int noradrenaline injection which caused exhaustion of endogenous catecholamine supply, the lactate content and the activities of lactic dehydrogenase were increased in the myocardium; the activity of hexokinase and G-6-PDH in rat myocardium and the liver were also increased, whereas the glucokinase activity was decreased. In these experiments alterations of the enzyme activities were shown to be similar to the alterations in the dystrophic tissues in which the catecholamine content was sharply decreased. The role of the sympathetic nervous system and its mediators in the mechanism of the enzyme regulation of the energy metabolism in the myocardium and the liver is discussed.


[Lipid oxidation in bilayer lipid membranes linked with the reaction of oxidation of NAD.H by atmospheric oxygen]. It is shown that along with NAD.H oxidation with air oxygen peroxide oxidation of lipids forming the membrane takes place in bilayer lipid membranes modified with ubiquinone. During nicotin amide oxidation proton absorption takes place. Peroxide oxidation of lipids results in the liberation of H+ ions, which in its turn brings about the formation of protone-deficient or enriched (against aqueous solution) layers adjacent to the membrane. The potential value on the membrane is shown to depend on nicotine amide and oxygen concentration, on ubiquinone presence and lipid composition of the membrane. It has been also indicated that the transmembrane potential difference is initiated with a sharp change of aqueous solution pH by 0.05--0.4 units.


[Pyruvate kinase activity in the liver of rats with induced tumors]. M-isozyme pyruvate kinase activity proved to be elevated in the liver of rats with hepatoma induced by p-dimethylaminoazobenzene. Sodium fluoride surplus in rat diet prevented the increase of this isozyme activity.


Implicit-shape constancy as a factor in pictorial perception. Schoolboys from the Ivory Coast and from Kenya were tested on a simple task intended to detect the effect of implicit-shape constancy. The presence of such an effect was confirmed. Comparison of two Ivorean subsamples differing in their responses to a request to build a model of a depicted structure showed that those subjects who build flat (2D) models were less influenced by the implicit-shape constancy than those subjects who built three-dimensional structures. This confirms a postulated link between the implicit constancy and perception of pictorial depth.


Enzyme inhibition in colorectal surgery. Anastomotic dehiscence is particularly common after low anterior resection of the rectum. The enzyme collagenase is largely responsible for this high complication rate. Inhibition of this enzyme in an experimental model produced significantly stronger colonic anastomoses. There may be a place for such an inhibitor in clinical surgery.


The treatment of mixed parotid tumours by enucleation and radiotherapy. A series of mixed parotid tumours treated by simple enucleation and radiotherapy between 1962 and 1968 is discussed. There were 73 primary and 8 recurrent cases. Only 2 small recurrences occurred in the 73 primary cases, although 4 other patients were lost to follow-up after 7 years or more. In the 8 recurrent cases, 7 had been treated elsewhere by enucleation alone, thus confirming the opinion that this operation is frequently unsuccessful. Treatment was by further local excision and radiotherapy and no recurrences have occurred. The view is therefore expressed that simple enucleation and radiotherapy is the treatment of choice in all primary mixed parotid tumours.


Ovarian tumours in childhood: a 27-year review. Twenty-five children treated for ovarian neoplasms at the Royal Hospital for Sick Children, Yorkhill,Glasgow, between 1947 and 1974 are reported. Eight of the patients had malignant tumours and 17 benign. The patients with benign tumours were treated by either salpingo-oophorectomy or oophorectomy. The malignant cases were treated with either surgery alone or in combination with deep X-ray therapy and/or chemotherapy. Two patients died of malignant teratoma, but the remaining 6 with malignant teratoma, dysgerminoma or endodermal sinus are well. The management of these patients is discussed.


Plasma cyclic adenosine-3', 5'-monophosphate response to glucagon in patients with liver disease. The change in plasma cyclic adenosine-3', 5'-monophosphate (AMP) was measured after intravenous injection of 1 mg of glucagon in 10 normal subjects and 30 patients with various forms of liver disease. Patients with cirrhosis and those with intrahepatic cholestasis responded normally but in patients with extrahepatic obstruction the plasma cyclic AMP response was considerably increased. Six of the eight patients with cirrhosis and a surgically created portacaval shunt had very reduced responses. This test may prove to be diagnostically important, particularly in differentiating surgical from non-surgical jaundice.


Attachment of gonococci to sperm. Influence of physical and chemical factors. Neisseria gonorrhoeae of pilated T1 and nonpilated T4 colony types attached themselves to human sperm in greatest numbers and most reproducibly when suspensions of the cells were incubated at 35 degrees C in Ringer's solution, pH 6-8. After incubation for 15 or 30 min. in a water bath shaker, 50 per cent. of human sperm had T1 gonococci attached and 25 per cent. T4. Sperm and both types of gonococci were pre-incubated separately with various chemical agents, selected because the agent is found in genital fluids, or has a known effect on bacterial cell walls or sperm membrane. After treatment, sperm were washed or were not washed, and were then tested for attachment by mixture with untreated gonococci. Treated gonococci were handled in the same manner. Change in the percentage of attachment was defined as deviation from the range expected on the basis of a standard curve. Treatment of sperm with the nucleotides, ATP or cAMP, curtailed attachment by T1 gonococci but had no effect on attachment by T4.


Penicillin decreases chloride conductance in crustacean muscle: a model for the epileptic neuron. The effects of penicillin were studied on the neuromuscular preparation of the ghost crab, Ocypoda cursor. Penicillin in doses lower than 2 mM reduced both the amplitude of inhibitory junction potentials and conductance increases induced by external application of GABA. The nature of the latter effect appears to be 2-fold, a weaker competitive inhibition and a more powerful non-competitive effech which may be ionophore blockade. Penicillin in concentrations above 2 mM diminished resting conductance, especially that of chloride. The action of penicillin is, in general, to decrease chloride conductance in this preparation. The crustacean neuromuscular preparation may provide a useful analogue for understanding penicillin evoked epilepsy. The reduced chloride conductance could explain decreased inhibition, increased excitation and depolarization shifts in cortical neurons.


Reduced glucocorticoid binding site concentration in cortical neuronal perikarya from senescent rats. Isolated neuronal perikarya from cerebral cortices of senescent (24-26-month-old) male rats exhibit a 55-65% reduction in the concentration of specific cytosol glucocorticoid binding sites when compared to mature counterparts (12-13 months). Neuronal preparations from the two age groups are essentially identical with respect to yield, protein content, physiochemical integrity and purity as judged by enrichment for free glycine and reduction in carbonic anhydrase specific activity (relative to unfractionated cortical cell suspensions). Adrenalectomy does not alter the number of glucocorticoid binding sites in either group, and age differences do not appear due to differential partitioning of sites between nucleus and cytoplasm. It is suggested that loss of receptors from the cortical neuron population represents a possible mechanism by which glucocorticoid responsiveness may be decreased during aging.


Cytochemical localization of cyclic 3', 5'-nucleotide phosphodiesterase activity in the neuromuscular junction and skeletal muscle of the newt. The localization of cyclic 3', 5'-nucleotide phosphodiesterase activity at the neuromuscular junction and in skeletal muscle of the newt was studied cytochemically. In the axon terminal, activity was localized to a site on the membranes of synaptic vesicles. In skeletal muscle, activity occurred in the sarcoplasmic reticulum but was restricted to the longitudinal tubules. Junctional folds of the motor end plate were unreactive. These findings are suggestive of a role of cyclic AMP in the functions of synaptic vesicles and sarcoplasmic reticulum and indicate its effects are terminated at these sites.


Climbing fiver activation and 3', 5'-cyclic guanosine monophosphate (cGMP) content in cortex and deep nuclei of cerebellum. Harmaline (28 mumoles/kg u.v.), cold exposure (4C) or isoniazid (2.2 mmoles/kg s.c.) increased the cGMP content in rat cerebellar cortex several fold. Isoniazid but not harmaline or cold exposure increased cGMP in the deep cerebellar nuclei (nuclei interpositus, vestibularis and fastigius) and striatum. In rats treated with the nicotinamide antagonist 3-acetylpyridine (3-AP) (0.66 mumoles/kg i.p. 4 days before) the tremorogenic effect of harmaline and the increase of cerebellar cortex cGMP produced by this alkaloid was abated. Similarly the increase of cGMP following exposure to cold was reduced. In contrast isoniazid and glutamate (10 mumoles intraventricularly) increased cGMP to the same extent in control and 3-AP treated rats. Since 3-AP produces in rat a massive degeneration of the inferior olivary nucleus and of the climbing fibers but leaves intact all the other cerebellar elements, these experiments suggest that an increase of cGMP content in postsynaptic cerebellar elements (presumable Purkinje cells) may be an expression of an increased release of an excitatory transmitter from either the climbing fivers or the parallel fibers.


The action of norepinephrine in the rat hippocampus. III. Hippocampal cellular responses to locus coeruleus stimulation in the awake rat. The behavioral and physiological effects of electrical stimulation of the nucleus locus coeruleus (LC) were studied in the awake rat. LC electrodes consistently supported high rates of self-stimulation (SS). LC stimulation also inhibited spontaneous cellular discharges in the hippocampus (HPC). Both the LC-induced inhibition of HPC units and the LC-evoked SS behavior were antagonized by alpha-methyltyrosine and 6-hydroxydopamine. In addition, chlorpromazine and diethyldithiocarbamate antagonized LC-induced inhibition of HPC units. D-Amphetamine facilitated SS behavior and reduced spontaneous HPC unit activity. The reinforcing properties of LC stimulation correlate closely with inhibition of cellular activity in the hippocampus; both actions appear to be mediated by norepinephrine.


The action of norepinephrine in the rat hippocampus. IV. The effects of locus coeruleus stimulation on evoked hippocampal unit activity. Activity of neurons in the hippocampus (HPC) was recorded in awake, freely moving rats. Most cells were inhibited by either a loud auditory stimulus (tone) or by electrical stimulation of the nucleus locus coeruleus (LC). The inhibitory responses to the tone were antagonized by drugs that interfere with central noradrenergic transmission. When LC stimulation was used as the unconditioned stimulus in a classical conditioning paradigm, previously inhibitory responses to the tone were reinstituted. When behaviorally subthreshold LC stimulation preceded a tone which was correlated with food, the existing conditioned response to the tone was potentiated. These data suggest that the generalized inhibitory response of HPC neurons to a tone is modulated by the noradrenergic pathway and that experimental activation of LC can potentiate HPC responses to behaviorally significant conditioned stimuli.


[Subcellular localization of rat adipocyte lipoprotein lipase]. The sub-cellular localisation in rat fat cells of lipoprotein lipase is discussed in this paper. The lipoprotein lipase was found with maximum activity in the microsomal fraction. Some special features of this activity in membrane fraction are pointed out.


[Traumatic deciduoma and delayed nidation in castrated pregnant rat after ACTH injection]. After administration of ACTH it is possible to obtain traumatic deciduomata in rats ovariectomized during early pregnancy and delayed implantation in rats ovariectomized day 4 of pregnancy and injected dayly with oestradiol from day 8 or 9. The substance responsible seems to be Progesterone or Progesterone like corticoid.


[Supppression of paradoxical sleep by chloramphenicol. Absence of effect by thiamphenicol]. The oral administration of 160-250 mg/kg of chloramphenicol selectively suppresses Paradoxical Sleep in the cat for a duration of 10-20 hours. Larger doses (330 mg/kg) suppress Paradoxical Sleep for 27 hours, but aslso decrease Slow Wave Sleep. At the same dose, Thiamphenicol has no effect.


[Corticotrope and melanotrope cells in cat, fox, rat and human fetus adenohypophysis: studies with induced fluorescence, cytoimmunologic technics and lead hematoxyline]. Formaldehyde-induced fluorescence, acid-catalyzed or not, methods, and immunocytology with anti-ACTH (1-24), anti-ACTH(17-39), ANTI-BETA-LPH immunserums were applied on the same preparations of cat, fox, rat and human foetus pituitaries. The superpositions of results showed that the pars intermedia and pars distalis corticomelano-lipotrophic cells of fox and cat pituitary, those of human foetal pituitary, and the purely corticotrophics cells of the rat pars distalis contained a fluorogenic probably granular compound. Moreover, the granules of the same cell types were electively revealed on our lymphilized material by plombic hematoxylin. Only the anti-beta-LPH and/or anti-beta-MSH fixing celpls exhibited hypercyanophilic, PAS-positive and bleu alcian-positive caracteristics.


[Effect of chronic hypoxia (Fi02 = 0.06 - Fi02 = 0.08) on mouse liver cytochrome P450 and b5 content]. During a eight days period of chronic hypoxia (FiO2 : 0,06) the cytochrome P 450 content of mice'liver decreases the 1st day, increases the 2nd and the 3rd day and decreases again and remains at a low levels. During a 13 days period of chronic hypoxia (FiO2 = 0,08) the P 450 level remains inchanged during 5 days. It decreases after until the 13teen day. G6Pase, an endoplasmic reticulum enzyme marker is quite inchanged in these conditions.


Studies on phosphoglyceromutase from chicken breast muscle: number and reactivity of sulfhydryl groups. Phosphoglyceromutase (PGM) from chicken breast muscle was titrated with p-mercuribenzoate (PMB), 5,5'-dithiobisnitrobenzoate (Nbs2), N-ethylmaleimide (NEM), iodoacetate and iodoacetamide. The effect of all of the sulfhydryl reagents, with the exception of NEM was to cause a loss in enzymatic activity. Addition of KCN following reaction with Nbs2 resulted in the recovery of a small amount of enzymatic activity. In the absence of substrate (3-phosphoglyceric acid) or cofactor (2,3-diphosphoglyceric acid) and in the presence or absence of 6 M guanidine hydrochloride, six sulfhydryl groups per mole of enzyme were titrated with PMB.


Biosynthesis of sphingomyelin in rat liver endoplasmic reticulum. Rat liver microsomal sphingomyelin synthetase (CDPcholine: N-acylspingosine choline phosphotransferase (EC 2.7.8.3)) has been shown to be markedly stimulated by ATP and pantothenic acid derivatives such as CoA, pantethine, pantetheine and 4'-phosphopantetheine.


Studies of human papovavirus tumor antigen in experimental and human cerebral neoplasms. Three types of papovaviruses (JC, BK, and SV40) have been isolated from man. All three are oncogenic in hamsters, cause frequent infection of man, and share a common T antigen. Augmentation of the expression of T antigen by in vitro cultivation of SV40-induced tumors of hamsters suggested that growing human brain tumors in vitro might provide an effective screening technique for the SV40 virus. In a series of human brain tumors examined in cryostat sections and in tissue culture, T antigen could not be demonstrated, suggesting that by this immunofluorescent technique SV40 was not implicated in the etiology of these tumors.


Hydatidiform mole metastasizing to the lung. Pulmonary lesions developed in three patients with an invasive hydatidiform mole. At open thoracotomy the modules were found to contain molar tissue. Therapy with twice-weekly methotrexate induced complete clinical and biologic remission of the disease in all three patients.