Plasma, red cell and cerebrospinal fluid concentrations of mannitol and sorbital in patients with severe chronic renal failure. The concentrations of mannitol and sorbitol in plasma, red cells, cerebrospinal fluid and urine were determined in 24 patients with chronic renal failure; 10 of them were on conservative treatment and 14 were haemodialysed three times weekly. The mannitol concentration was significantly increased in the plasma and the cerebrospinal fluid of the uraemic patients compared with the controls. In six out of ten uraemic patients mannitol clearance values exceeded creatinine clearance values. The plasma concentration of sorbitol was undetectable or very low in all patients and control subjects. Red-cell and cerebrospinal fluid concentrations of sorbitol showed a large individual variation in the uraemic patients on conservative treatment and did not correlate with kidney function. During dialysis the mannitol concentration decreased, leading to a small osmotic gradient between the plasma and the red cells. The changes in the concentration of mannitol during dialysis showed no connection with the symptoms of central nervous disturbance which appeared during dialysis treatment. The red cell sorbitol concentration during dialysis increased by about 20%. There was a correlation (p less than 0.05) between t,e increase in sorbital in cereemic patients.


The effect of segmental epidural analgesia on maternal ACTH, cortisol and TSH during labour. Maternal plasma ACTH, cortisol and TSH concentrations were determined during the course of the induced labours of 20 normal parturients. Alternate mothers were given segmental epidural analgesia for pain relief during the first stage of labour. The remaining parturients served as controls. The ACTH level rose in same way in both groups, reaching its peak at the moment of delivery and decreasing rapidly thereafter. Cortisol secretion reached its maximum during the first stage of labour in the moment of delivery. After delivery the cortisol level decreased more rapidly in the epidural group tha. in the control gro,p. Umbilical venous cortisol concentration was the same in both groups. The maternal TSH level did not change significantly during labour in either group.


The regulation of heme biosynthesis.

The regulation of heme biosynthesis. Changes in the levels of the heme biosynthetic enzymes were studied in cells and protoplasts of Saccharomyces cerevisiae during glucose derepression and respiratory adaptation. On aerobic or anaerobic glucose derepression or in the presence of 3', 5' cyclic AMP the levels of beta-aminolevulinic acid dehydratase, protoporphyrinogen oxidase and ferrochelatase increased whereas the levels of the enzymes catalysing the reactions from porphobilinogen to protoporphyrinogen IX remained constant. In contrast, the level of beta-aminolevulinic acid synthetase decreased during aerobic glucose derepression and in the presence of 3', 5' cyclic AMP, but it remained unchanged during anaerobic derepression. The heme content of the cells increased during aerobic glucose derepression but no increase was observed during anaerobic derepression of the cells. It is concluded that, in yeast, the inhibitory effect of anaerobiosis on heme synthesis is due, at least in part, to the requirement for oxygen in the oxidation of protoporphyrinogen IX, and that the effect of glucose on heme biosynthesis is mediated via glucose repression of protoporphyrinogen oxidase and possibly ferrochelatase.


Studies on heme synthesis in the rat adrenal. The activity and regulation of rat adrenal delta-aminolevulinic acid synthetase (EC 2.3.1.37) has been studied. The activity of delta-aminolevulinic acid synthetase in  rat adrenal homogenates is comparable to the activity found in rat hepatic homogenates when untreated animals are employed. Starvation of rats for 24 and 48 hours increased adrenal delta-aminolevulinic acid synthetase activity without altering the hepatic enzyme. The treatment of rats with ACTH gel also increased adrenal delta-aminolevulinic acid synthetase activity. The effect of starvation on rat adrenal delta-aminolevulinic acid synthetase activity appears to be mediated by ACTH and can be prevented by the administration of dexamethasone. Adrenal delta-aminolevulinic acid synthetase appears to be refractory to induction by agents which induce the hepatic enzyme. These results suggest that the adrenal enzyme may be uniquely controlled by neural and endocrine influences which differ from the factors which regulate the activity of the hepatic enzyme.


[Dissociation between the effects of TSH and dibutyryl cAMP on thyroxine secretion by isolated thyroid cells. Similarity of actions on iodide uptake and thyroxine synthesis (author's transl)]. We have compared the effects of TSH and dibutyryl-cAMP (DBC) on three parameters of iodine metabolism in isolated hog thyroid cells: iodide uptake, thyroxine (T4) synthesis and T4 secretion. As WILSON, we observed a similarity between TSH (60 mU/ml) and DBC (3 mM) actions on iodide uptake and T4 synthesis. But DBC did not reproduce the TSH stimulation of T4 secretion. Since TSH increased T4 secretion in the presence of DBC, the lack of effect of DBC was not due to an alteration of the secretory process by DBC. The present observation might suggest that the stimulatory effect of TSH on T4 secretion by isolated thyroid cells is not mediated by the adenyl cyclase-cAMP system, or could indicate that DBC may not act like cAMP on some target enzyme systems.


[Variation of plasmatic adenosine 3'', 5-cyclic monophosphate in irradiated-enterectomised dogs (author's transl)]. In the irradiated dog at different doses 250-400 R., a level plasma AMPc rise has been shown since the fourth hour with a maximum at 24 hours, returning to normal values about the thirtieth day. In irradiated at 250-300 R and secondarily enterectomised animals, nucleotide level rises 24 hours later, however this phenomenon is less intensive and later irradiated at 350 and 400 R.


[Defect of growth hormone receptors in hereditary hypopituitary dwarf mice (author's transl)]. The binding of 125I-growth hormone to its specific receptors was comparatively studied in liver cells isolated from dwarf mice and littermates. The specific binding of growth hormone was about eight-fold lower in cells from dwarf mice than in those from littermates. However, the specific binding of insulin, although lower, was not significantly modified in dwarf as compared to littermates. The defect of growth hormone receptors in hypopituitary dwarf mice is likely to be related to the lack of hormonal effect in this animal when the growth hormone was administered alone.


"Phoenix phenomenon" in the growth of Clostridium perfringens. The "Phoenix phenomenon" was observed with Clostridium perfringens Hobbs' serological type 9 (HT9) in a cooked-meat medium at 81.7 degrees C by a decrease in plate count (phase I), followed by an increase in count to the intiial level (phase II) and a continued increase above the initial count (phase III). The effects of sporulation, age of inoculum, assay medium, anaerobiosis, diluent, and growth inhibitor were studied. This phenomenon was reproduced in experiments with sporulation-negative mutants derived from HT9 inocula of various cell ages, and different assay media (sulfite-iron agar, tryptose-soytone-yeast extract agar, prereduced peptone-yeast extract agar, prereduced veal agar, and veal agar). When strict anaerobic conditions were employed, it was necessary to increase the heating temperature to 52.3 degrees C to observe the phenomenon. The phenomenon was eliminated at 52.3 degrees C when a combination of strict anaerobic conditions, prereduced media, and prereduced veal diluent was employed. The addition of nalidixic acid at the minimum point of the growth curve (end of phase I) had no effect on the appearance of phase II; however, phase III was completely inhibited. This indicated that phases I and II were an injury-recovery process.


Effect of ribavirin on murine cytomegalovirus infection. Ribavirin (1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide), a new synthetic nucleoside, inhibited murine cytomegalovirus in cell culture. This was shown by the inhibition of viral cytopathic effect and plaque formation, as well as reduction in the yield of virus. Despite this in vitro antiviral effect, ribavirin did not protect mice from mortality produced by a high inoculum of cytomegalovirus. When a lower inoculum was used to initiate a chronic infection, the administration of ribavirin for 9 days had no effect on the titer of cytomegalovirus in the salivary gland, kidney, liver, and spleen. Thus, ribavirin was ineffective in the treatment of both acute and chronic murine cytomegalovirus infections.


[Alteration of the catecholamine receptor during hepatic malignancy (author's transl)]. The adenylate cyclase system of a plasma membrane preparation from normal rat liver responds to various catecholamines in the following order: protokylol greater than isoproterenol greater than epinephrine greater than norepinephrine. In the Zadejela ascites hepatoma, the order of sensitivity is modified, indicating a transformation of the receptor from the beta2 to the beta1 type. These observations suggest that the catecholamine receptor might be a single entity, susceptible to secondary, and reversible, modification towards either a beta 1 or a beta 2 type.


[Variations in the estradiol and progesterone receptors in human endometrium during the menstrual cycle]. The concentration of estradiol and progesterone "receptors", both cytoplasmic and nuclear, has been measured in human endometrium during the menstrual cycle using an exchange assay method. A rapid increase of the concentration of the two "receptors" has been observed during the proliferative phase followed by a striking decrease during the secretory phase. New physiological and pharmacological aspects may be derived from these results.


Dapsone-induced peripheral neuropathy.

Dapsone-induced peripheral neuropathy. Peripheral neuropathy occurred in a 32-year-old man who was receiving dapsone (Aviosulfon) for dermatitis herpetiformis. Neurological examination showed polyneuropathy that involved the median and ulnar nerves, with a lesser involvement of other peripheral nerves. After treatment with dapsone was discontinued, the neuropathy reversed itself, with practically complete return of function of all nerves involved.


Cyclic AMP and T-cell differentiation.

Cyclic AMP and T-cell differentiation. We have studied the effects of a circulating thymic factor (TF), cyclic AMP (cAMP) and prostaglandins on T-lymphocyte differentiation in the mouse using three parameters: 1) 0-bearing cell percentage evaluated by a cytotoxicity test with anti-0 serum (A0S) plus complement; 2) 0-antigen presence on rosette forming cells (RFC) assessed by the inhibition of rosette formation with A0S; 3) lymphocyte mediated cytotoxicity (LMC) against allogeneic target cells after in vivo sensitization, using a chromium release assay.


Psychoendocrinological studies in dwarfed children and adolescents. Thirty-two dwarfed children and adolescents were studied clinically, by laboratory assessment, by a battery of psychological tests and a structured interview. Growth hormone deficiency was present in 16 cases, but in the remaining 16 cases there was no endocrine disease. Dwarfed children differed from nornal controls in perception, and a specific personality pattern emerged in the dwarfed children. The effects of age, sex, and socioeconomic status on personality traits were similar for dwarfs as for controls. Intelligence and personality variable were similar in dwarfs with and without endocrine disease. However, symptoms of the psychoendocrine syndrome, namely appetite and thirst disturbances, hypersensitivities, and impulse reduction, were more frequently seen among hypopituitary dwarfs. Social and coping behaviour was impaired in the majority of dwarfs. It is concluded that psychological disturbance occuring inchildren of small stature is a response to being small and is not attributable to any endocrine effect.


Screening for familial hyper-beta-lipoproteinaemia in children in hospital. 1510 plasma cholesterol estimations were made in 1391 children admitted to hospital as part of a biochemical profile. Babies under 1 year and children known to have familial hyperlipoproteinaemia were excluded. The mean concentration was 4-28 mmol/l +/- 1-04 (1 SD) (165-3 mg/100 ml +/- 38-6), and levels exceeded 5-93 mmol/l (229 mg/100 ml) in 68 children. Repeat estimations on 55 of these children showed 34 still to have values greater than 5-93 mmol/l and family studies were performed in 19 of these. In 8 children hypercholesterolaemia was secondary and no familial lipoprotein disorder was present. Familial hyper-beta-lipoproteinaemia (FH) was diagnosed in 3 children and in 2 of the families there was a history of early ischaemic heart disease. In 2 children the diagnosis was in doubt. In the remaining 6 children FH and secondary hyperlipoproteinaemia were excluded so the hypercholesterolaemia was presumably environmentally induced, possibly in association with polygenic inheritance. In the present state of knowledge screening of the childhood population for FH by means of plasma cholesterol determinations cannot be recommended. Studies of lipoproteins should, however, be made in children from families known to have FH or early coronary heart disease.


Stimulation by melanocyte stimulating hormone and dibutyryl adenosine 3'5'-cyclic monophosphate of DNA synthesis in human melanocytes in vitro. The proliferation of human melanocytes in vitro was stimulated by MSH. This stimulation was further intensified by the simultaneous addition of theophylline with MSH. Theophylline alone stimulated proliferation moderately. Dibutyryl cyclic AMP strongly stimulated the proliferation, but sodium butyrate, 5'-AMP and cGMP did not. The stimulation by dibutyryl cyclic AMP was continued up to 4 days so far tested. These findings are directly opposed to those on mouse melanoma cells in culture which responded with retarded growth to MSH and cyclic AMP. It is suggested that the difference of proliferation control may explain the different reaction of the melanocyte and the melanoma cells. The epidermal melanocytes seem to belong to the exceptional group of the cells which responded to cyclic AMP with accelerated proliferation.


Xanthoma secondary to reticulo-histiocyte infiltration. Three cases of xanthomas occurring secondarily are reported: The primary dermatoses were erythroderma, actinic reticuloid and histiocytosis X. Although a slight elevation of pre-beta-lipoprotein was recognized, xanthomas occurring in these particular 3 cases were assumed to be due to the proliferation or hyperplasia of reticulohistiocytes in the skin. The foam cells found in the xanthoma case with erythroderma and actinic reticuloid appeared different from those with histiocytosis X in their fine structures. The lipid constituents of the xanthoma lesion were found to be quite dissimilar from those of serum lipids.


[Behaviour of collagen peptidase in the serum of patients with progressive systemic sclerosis during gestagen therapy (author's transl)]. In 20 patients with progressive systemic sclerosis the activity of collagen peptidase in serum before and after gestagen therapy of 10 resp. 20 days was determined. After 20 days the enzyme activity was significantly decreased. These findings show no correlation to the known gestagen induced alterations of the collagen metabolism.


Tumor vascularity as a prognostic factor for hepatic tumors. The prognostic and therapeutic significance of tumor vascularity was studied in 36 patients with hepatoma or metastatic colon cancer in the liver. All patients had nonresectable tumor and were treated by hepatic artery ligation and hepatic arterial infusion chemotherapy. Chemotherapy consisted of methotrexate, actinomycin-D, 5-fluorouracil and cyclophosphamide. Hepatic tumors were categorized into Grades I to III in the order of increasing vascularity as determined by preoperative hepatic angiography. Tumor vascularity of 15 patients with hepatoma was Grade III in 11 (73%) and Grade II in 4 (27%). No patient with hepatoma had a Grade I tumor. The median survival of patients was 10 and 6 months for Grade III and II hepatomas, respectively, after hepatic artery ligation, and 18 and 8.5 months for Grade III and II, respectively, from the time of diagnosis of hepatoma. Tumor vascularity of 21 patients with metastatic colon cancer was as follows: Grade III in 3 (14%); Grade II in 10 (48%); and Grade I in 8 (38%). The median survival was 11, 10.5 and 4 months for Grades III, II and I, respectively, after hepatic artery ligation, and 17, 14.5 and 7.2 months for Grades III, II and I, respectively, from the time of diagnosis of hepatic metastases of colon cancer. The results indicate that the more vascular the hepatic tumor on angiogram, the better the prognosis following hepatic artery ligation and infusional chemotherapy.


Ectopic ACTH syndrome due to salivary gland adenoid cystic carcinoma. A 68-year-old woman developed Cushing syndrome associated with a submaxillary salivary gland adenoid cystic carcinoma. High levels of immunoreactive adrenocorticotrophic hormone (ACTH) were found in the tumor and peripheral blood. Urinary 17-hydroxycorticosteroid levels decreased following administration of dexamethasone, suggesting possible suppression of ectopic ACTH production. however, there was no specific in vitro binding of dexamethasone by tumor cytosol. A trial of metyrapone therapy resulted in control of hypokalemia and hyperglycemia. Both plasma cortisol and 11-deoxycortisol levels decreased with metyrapone administration, suggesting that this agent may inhibit not only 11-beta-hydroxylation, but also possibly earlier steps in glucocorticoid synthesis.


Liver disease in renal transplant recipients. Significant liver disease developed in 14 patients after renal transplantation. Nine patients had morphologic and functional evidence of chronic active hepatitis. In general, these patients had few symptoms of liver disease, even though the course of chronic active hepatitis was progressive. Despite large doses of prednisone, cirrhosis ultimately developed in five patients. The cause of chronic active hepatitis could not be related to azathioprine or methyldopa therapy because there was no perceptible change in the course of liver disease after treatment with these drugs was stopped. Three patients were persistently positive for hepatitis B surface antigen. Isolated instances of granulomatous hepatitis (Mycobacterium kansasii) and of prolonged intrahepatic cholestasis were encountered in patients with chronic active hepatitis. Two patients had acute cytomegalovirus hepatitis. There was one episode each of fulminant herpes simplex hepatitis and severe fatty metamorphosis.


[Long-term follow-up of 128 children 13-14 years old, with birth weights of less than 2500 grams]. The records of 128 children, whose birth weight was below 2500 g and who were seen again at the ages of 13-14 years, were analyzed. Owing to the knowledge of the data of the last maternal menses in 115 cases, they could be separated in at-term dysmature, premature dysmature and true premature infants. The comparison between the 3 groups led to the following differences: On average, there is little difference between true premature infants and normal neonatal population, on both somatic and psychological points of view. Physical development is less satisfactory in at-term dysmature infants. If their median of I.Q. is close to normal, high intellectual abilities are rare and mental deficiency more frequent. In the physical, auditory, visual and psycho-motor fields, premature dysmature infants are the most underpriviliged.


Murine leukemia induced by viral ultrafiltrates negative for XC plaque activity. Leukemogenic activity has been encountered in 100 nm filtrates of a murine leukemia virus, PLLV-T2, associated with smaller than conventional C type particles. XC plaque assay of these filtrates gave negative results, revealing a lack of correlation between in vivo and in vitro activity. It is postulated that these filtrates contain viral subunits deficient for replication in BALB fibroblasts in vitro but able to transform lymphoid cells in vivo.


ACTH-producing pheochromocytoma.

ACTH-producing pheochromocytoma. A benign adrenal medullary tumor that secreted adrenocorticotropic hormone (ACTH) was associated with bilateral adrenocortical hyperplasia and clinically evident Cushing syndrome. The clinical and chemical features were those usually associated with pituitary Cushing disease, including partial suppression of urinary OH steroids after administration of 8 mg of dexamethasone. The fractionization of the tumor's ACTH revealed 70% little "biologically active" ACTH, which is usually found in this concentration only in pituitary tissue.


[The role of cysteine in cadmium- induced experimental testicular damage (author's transl)]. Simultaneous administration of cysteine and DL-ethionine in food protects, and enhances, respectively, the tissue damage produced by parenteral administration of cadmium (CdCl2) in testicles of rats. The effects are explained as an inactivation of SH-groups by cadmium, and in interference in cysteine metabolism by ethionine, respectively.


Effects of cadmium on carbonic anhydrase activity and hemoglobin content of rat testes. The cadmilm-induced (Cd) damage of mammalian testes is thought to be correlated with an inhibition of carbonic anhydrase (CAH) by Cd. Since Cd causes dose-dependent changes in blood flow of the testes, an inhibition of CAH in the testes could be simulated by a decrease of CAH-rich erythrocytes. Therefore, CAH activities and hemoglobin (Hb) content were determined in blood and testes of untreated and Cd-treated Sprague-Dawley rats as well as in testes perfused via the testicular artery. Cd was intraperitoneally applied as CdCl2 in single doses of 1.5, 3.0, and 5.0 mg Cd2+/kg b.w., respectively. 1. The experiments on perfused testes clearly demonstrated that the CAH activities originate from erythrocytes rather than from a tissue located enzyme. 2. The alterations in blood circulation occurring shortly (0.25-1.0 h) after the Cd administration were characterized by a dose-dependent, transient decrease (1.5 mg Cd2+/kg) as well as an increase (3.0 and 5.0 mg Ck2+, respectively) of the Hb content in the testes. 3. Independent of these minor alterations in a later state (14-24 h after 1.5 mg Ck2+/kg, 7-14 h after 3.0 mg Cd2+/kg, and 1-3 after 5.0 mg Cd2+/kg), Cd induced the well known hemorrhagic alterations of the testes with a high increase of Hb content and CAH activity. 4. By means of the correlations between CAH activities and Hb content in blood and testes an inhibition of the CAH by Cd as the primary cause for the tissue damage of the testes could largely be excluded.


Guanosine 3'-5'-monophosphate in the CSF of neurological patients. Cyclic guanosine monophosphate (cGMP) levels have been measured in the cerebrospinal fluid of patients with various neurological diseases. The subjects with epilepsy or cerebrovascular diseases do not show any difference from the controls. Moreover, in the CSF of patients having cerebral tumors the levels of cGMP are markedly increased. This change is in line with previous in vitro studies on the increase of cGMP during cell growth and cell proliferation showing that the role of the nucleotide is important for the control of the life cycle of the cell.


Pathogenesis of reovirus type 1 hydrocephalus in mice. Significance of aqueductal changes. The pathogenesis of hydrocephalus following reovirus type 1 inoculation of neonatal mice has been examined by light microscopy, radiology, immunofluorescence, and electron microscopy. The reovirus infection causes an acute ependymitis and leptomeningitis, followed by a fibrous arachnoiditis and arachnoid villitis. Hydrocephalus develops in proportion to the degree of inflammatory/fibrotic changes within the cerebrospinal fluid pathways. With the beginning of hydrocephalus there is radiographic evidence of basal cistern blockage. As the hydrocephalic state progresses, axial herniation and compression of the midbrain result in the appearance of aqueduct stenosis. We demonstrate that the stenosis of the aqueduct is a secondary phenomenon, not causally related to the pathogenesis of hydrocephalus, and discuss the significance of this finding to human aqueduct stenosis.


[Hyperlipemias and obliterating peripheral arteriopathies]. 160 patients suffering from radiologically confirmed peripheral obliterating arteriopathy have been studied with respect to frequency of lipid alterations. The resulting data were compared with those observed in a group of clinically healthy subjects. In the arteriopathic series, lipid alterations were encountered in 44% of patients, with a percentage of 12.5% for Fredrikson type II, 7.4% for III and 24% for IV. In women, incidence attained 50% and type classification was 25% for the 2nd, 12.5% for the 3rd and 14.5% for the 4th. An alteration of 18.7% was encountered in the control group. The high frequency of hyperlipidaemia in peripheral vascular diseases reported in the literature and confirmed by the above data, justifies the employement of appropriate dietetic and pharmacological measures for the prevention and treatment of this arteriosclerotic disease risk factor.


Skin punch biopsy in the diagnosis of juvenile neuronal ceroid-lipofuscinosis. A comparison with leukocyte peroxidase assay. Electron microscopical examination of skin punch biopsy specimens was compared with the leukocyte peroxidase assay in establishing the diagnosis of juvenile neuronal ceroid-lipofuscinosis in three families with at least one affected child. Although the leukocyte peroxidase assay failed to distinguish between the patients, heterozygotes, and normal subjects, skin biopsy specimens containing characteristic cytosomes were found only in the children with this disease. In fact, in two families, the three affected members were identified by this method after the biopsy specimens had been randomized. Although only one to four cells containing such cytosomes were identified in each patient, not only cytosomes with curvilinear bodies but also those with rectilinear as well as "fingerprint" profiles were found. Thus, the skin punch biopsy appears to be a useful diagnostic aid the juvenile as well as the late infantile forms of neuronal ceroid-lipofuscinosis.


Acetylcholine receptor protein. Neuromuscular transmission in immunized rabbits. Rabbits injected with purified acetylcholine (ACh) receptor protein produce antibodies against the receptor and develop generalized muscle weakness. The compound muscle action potentials show a decremental fall in amplitude with repetitive nerve stimulation. Both the weakness and the decrement is counteracted by reversible cholinesterase inhibitors. Intracellular recordings from muscle endplates show that the amplitude of the miniature end-plate potentials is considerably reduced. A reduced binding of neurotoxin to muscles from immunized rabbits was observed. Nerve impulses release a normal number of ACh packages (quanta) from the motor nerve terminals. The muscle weakness in immunized rabbits thus has the same features as the muscle weakness in myasthenia gravis and may be a good animal model of myasthenia gravis.


Erythrocyte enzyme activities in diploid and triploid salamanders (Pleurodeles waltlii) of both sexes. Studies of several enzyme activities and of some metabolites in erythrocytes of the newt Pleurodels waltilii are described. The results indicate that differences exist between enzyme activities in males and females and in diploid and triploid animals. Females have higher erythrocyte enzyme activities than males. However, triploid animals have lower enzyme activites in their erythrocytes than diploid animals; this observation is more striking in females than in males.


Kinetic studies on [methionine sulphoxide] cytochrome c. A cytochrome c haem ligand, methionine-80, was photo-oxidized to methionine sulphoxide and the subsequent changes in redox properties and ligand binding were monitored kinetically. Isoelectric focusing of the product showed the presence of a single oxidized species, capable of binding CO when reduced. The binding of CO to the reduced protein was followed in stopped-flow experiments, which revealed the presence of two binding processes, at neutral pH, with rate constants of K+1 = 3.4 X 10(3)M-1-S-1 and k+2 = 5.80 X 10(2)M-1-S-1. When CO was photolytically dissociated from the reduced protein two recombination processes were observed with rates almost identical with those observed in the stopped-flow experiments (k+1 = 3.3 X 10(3)M-1-S-1 and k+2 = 6.0 X 10(2)M-1-S-1). These findings provide evidence of two reduced forms of the protein. The reduction of [methionine sulphoxide]cytochrome c by Cr2+ at neutral pH in stopped-flow experiments showed the presence of a single second-order reduction process (k = 7.2 X 10(3)M-1-S-1, activation energy = 44kJ/mol) and one first-order process. This protein was compared with some other chemically modified cytochromes.


A comparison of bone matrix and tendon with particular reference to glycoprotein content. Bone matrix and tendon are compared in terms of their carbohydrate and non-collagenous protein composition. The collagen content of both tissues was similar (90-91%), but bone matrix had at least three times as much sialic acid (0.28%) as tendon (0.08%). Smaller differences were found in the analysis of hexoses and hexosamines. After digestion with bacterial collagenase, about 9% of the total protein from both tissues was non-diffusible on dialysis, and this contained only 0.15% (bone) and 0.7% (tendon) of the original hydroxyproline; recovery of sialic acid was 86-87%. The collagenase-resistant soluble material amounted to about 9% (bone matrix) and 5% (tendon); the insoluble residues were 1 and 4% respectively. There were clear differences in the carbohydrate contents of the digests, but the amino acid compositions were similar. When the soluble digests were chromatographed on DEAE-cellulose, the elution profiles indicated the presence in each tissue of a variety of glycoproteins and a proteoglycan fraction, and showed clearly that an acidic glycoprotein corresponding to bone sialoprotein was not present in tendon.


The amino acid sequences of the cytochromes c-555 from two green sulphur bacteria of the genus Chlorobium. Amino acid seauences are proposed for the cytochromes c-555 from Chlorobium thiosulphatophilum and from the Chlorobium limicola component of "Chloropseudomonas ethylica 2K". Each is a sincle polypeptide chain, the former of 86, the latter of 99 residues, and, when aligned so as to give the best match, 47 residues are common to the two sequences. The sequences show some resemblance to those of cytochromes c5 and f. The bacteriochlorophyll a-proteins were also isolated and purified, and their amino acid compositions compared (see the Appendix). There are significant differences in the compositions, but not as great as those found for the cytochromes c-555. The significance of these observations for the taxonomy of the Chlorobiaceae and for the further development of the comparative biochemistry of cytochrome c is discussed. Detailed evidence for the sequences of the cytochromes c-555 has been deposited as Supplementary Publication SUP 50073 (36 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1976) 153, 5.


Low-temperature studies on mixed-valence cytochrome oxidase by using magnetic circular dichroism. The spin states of the haem components of mixed-valence cytochrome oxidase were studied at room temperature and at temperature down to 20K by using magnetic circular dichroism. The room-temperature studies show the presence of a low-spin ferrous haem together with a low-spin ferric haem, which we attribute to heams a3 and a respectively. At temperatures below 100K it appears that the CO of the mixed-valence CO complex may be irreversibly photolysed, and that in this case haems a and a3 assume their high-spin states. Thus in this enzyme haem-haem interactions appear possible at temperatures below 100K.


Role of histone kinases as mediators of corticotropin-induced steroidogenesis. In an attempt to determine the role of protein (histone) kinases as mediators of corticotropin-induced corticosterone formation, the ability of homogenates, prepared from adrenals treated with various doses of corticotropin to catalyse the phosphorylation of calf thymus histones was measured. Although corticotropin promoted an increase in histone kinase activity, much more of the hormone was required to induce this response than to stimulate steroidogenesis maximally. In addition, a derivative, nitrophenylsulphenyl-corticotropin, which inhibits the stimulatory effect of corticotropin on cyclic AMP accumulation, stimulated corticosterone synthesis without altering histone kinase activity. Very high doses of nitrophenylsulphenyl-corticotropin were capable of stimulating histone kinase activity. In contrast, when dibutyryl cyclic AMP was used to stimulate steroidogenesis under the same conditions, any dose of the nucleotide which increased adrenal corticosteroid content also increased histone kinase activity. Assuming that histones serve as useful substrates for measurement of total adrenal protein kinase activity, the role of protein kinases as mediators of steroidogenesis is not supported by these studies.


Studies on the control of 4-aminobutyrate metabolism in 'synaptosomal' and free rat brain mitochondria. 1. The specific activities of 4-aminobutyrate aminotransferase (EC 2.6.1.19) and succinate semialdehyde dehydrogenase (EC 1.2.1.16) were significantly higher in brain mitochondria of non-synaptic origin (fraction M) than those derived from the lysis of synaptosomes (fraction SM2). 2. The metabolisms of 4-aminobutyrate in both 'free' (non-synaptic, fraction M) and 'synaptic' (fraction SM2) rat brain mitochondria was studied under various conditions. 3. It is proposed that 4-aminobutyrate enters both types of brain mitochondria by a non-carrier-mediated process. 4. The rate of 4-aminobutyrate metabolism was in all cases higher in the 'free' (fraction M) brain mitochondria than in the synaptic (fraction SM2) mitochondria, paralleling the differences in the specific activities of the 4-aminobutyrate-shunt enzymes. 5. The intramitochondrial concentration of 2-oxoglutarate appears to be an important controlling parameter in the rate of 4-aminobutyrate metabolism, since, although 2-oxoglutarate is required, high concentrations (2.5 mM) of extramitochondrial 2-oxoglutarate inhibit the formation of aspartate via the glutamate-oxaloacetate transaminase. 6. The redox state of the intramitochondrial NAD pool is also important in the control of 4-aminobutyrate metabolism; NADH exhibits competitive inhibition of 4-aminobutyrate metabolism by both mitochondrial populations with an apparent Ki of 102 muM. 7. Increased potassium concentrations stimulate 4-aminobutyrate metabolsim in the synaptic mitochondria but not in 'free' brain mitochondria. This is discussed with respect to the putative transmitter role of 4-aminobutyrate.


Changes in the activities of the enzymes of urea synthesis caused by dexamethasone and dibutyryladenosine 3':5'-cyclic monophosphate in foetal rat liver maintained in organ culture. Liver explants from 19-day foetal rats were maintained in organ culture, in a defined medium, for up to 48h. Both 6-N,2'-O-dibutyryl cyclic AMP, in the presence of theophylline, and dexamethasone caused an increase in the activities of carbamoyl phosphate synthase, argininosuccinate synthetase, argininosuccinate lyase and arginase. These increases could be abolished by simultaneously incubating the explants with cycloheximide. No change in the activity of ornithine transcarbamoylase was found with either hormone. Previous work has shown that injection of corticosteroids into 19.5-day foetal rats in utero did not cause an increase in the arginine synthetase system. Present results suggest that this lack of effect is not due to any incompetence of the foetal rat liver at this stage to respond to this agent. The observations on ornithine transcarbamoylase activity suggest that this enzyme is induced in the liver of the perinatal rat by neither corticosteroids nor hormones acting via cyclic AMP, and it may be that all the enzymes of the urea cycle are induced physiologically by an agent or agents as yet unidentified.


Studies on the phosphorylation of the inhibitory subunit of troponin during modification of contraction in perfused rat heart. 1. Rat hearts were perfused with 32Pi, and contractile force was increased by positive inotropic agents (agents that increase contractility). The inhibitory subunit of troponin (troponin I) was then isolated by affinity chromatography in 8M-urea, and its 32P content measured. Incorporation of phosphate into the subunit was calculated on the basis of the [gamma-32P]ATP specific radioactivity in the hearts. 2. When hearts were perfused with 30 nM-DL-isoprenaline (N-isopropylnoradrenaline), there was an increase in contractile force over 30s which was paralleled by an increase in troponin I phosphorylation. When hearts were perfused for 25s with increasing concentrations of isoprenaline from 1 NM to 0.6 muM, there was again a parallel increase in contractile force and troponin I phosphorylation. The maximum phosphorylation observed was 1.5 mol of phosphate/mol of troponin I, which was reached after 25s with 0.1 muM-isoprenaline. 3. Hearts were stimulated with a 15s pulse perfusion of 30nM-DL-isoprenaline. There was an increase in contractile force which was followed by a return to the control value within 50s. Troponin I phosphorylation increased to a plateau value which was reached within 30s, and remained constant for 60s after the isoprenaline pulse. Phosphorylase a and 3':5'-cyclic AMP concentration showed changes similar to that of the contractile force. There was no change in 3':5'-cyclic GMP concentration. 4. When hearts stimulated with a 15S pulse of isoprenaline were subsequently perfused with 0.6 muM-acetylcholine, the changes in contractile force, phosphorylase a and 3':5'-cyclic AMP were very similar to those seen with the 15s pulse of isoprenaline alone. Troponin I phosphorylation increased to a maximum 30s after the end of the isoprenaline pulse, but then rapidly decreased during the subsequent 30s. This decrease was preceded by a 60% increase in the concentration of 3':5'-cyclic GMP. 5. Hearts were perfused with 0.2 muM-glucagon for periods up to 60s. Contractile force showed little change for the first 30s, but then increased rapidly. This was paralleled by changes in 3':5'-cyclic AMP concentration. Troponin I phosphorylation increased slowly, but the increase in contractile force had reached a maximum before significant phosphorylation had occurred. 6. It is concluded that under certain conditions, e.g. immediately after beta-adrenergic stimulation, there is a good correlation between contractile force and troponin I phosphorylation. However, under other conditions, e.g. when contractile force is decreasing after removal of beta-adrenergic stimulation or in the presence of glucagon, contractile force and troponin I phosphorylation are not well correlated. These results suggest that mechanisms for modifying cardiac contractility, other than troponin I phosphorylation, must be present in rat heart.


[Studies on the Pharmacology of 6alpha,9-difluoro-11beta-hydroxy-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione (diflucortolon valerate) (author's transl)]. The topical and systemic anti-inflammatory action of 6alpha,9-difluoro-11beta-hydroxy-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione (diflucortolone valerate, Nerisona) was studied in the rat in comparison with fluocortolone, diflucortolone and some other corticoids. In addition the effect of the compounds studied on the following parameters of corticoid activity was examined in the rat: body weight and weights of thymus, spleen and adrenals; blood sugar concentration and liver glycogen content; diuresis and Na+ and K+ elimination with the urine; the binding of diflucortolone and some diflucortolone-21-esters to the cytoplasmic corticoid receptor of the rat's thymus was also determined. In all tests diflucortolone was shown to be a corticoid with very potent topical and systemic action. Diflucortolone valerate showed the same potent anti-inflammatory action on topical application as the unesterified compound. After subcutaneous administration, however, the systemic corticoid action of the valerate was considerably inferior to that of diflucortolone on account of the kinetics of the more lipid soluble ester.


[Systemic effect of diflucortolone valerate after dermal application (author's transl)]. The systemic effect of 6alpha,9-difluor-11beta-hydroxy-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione (diflucortolone valerate, Nerisona) 0.1% as an ointment and fatty ointment was investigated in two studies. The parameters used were the plasma 11-OHCS values and the urinary 17-OHCS and 17-KS values, respectively. Suppression of the plasma cortisol values was observed after topical application of both diflucortolone valerate and betamethasone-17-valerate to healthy subjects under extreme conditions of whole-body occlusion. However, it was still possible to stimulate the adrenal cortex with ACTH. No reduction of the 17-OHCS of 17-KS values in the 24-h urine was observed during open, large-surface treatment of patients with skin diseases.


Control of the interaction of cholesterol ester-rich lipoproteins with arterial receptors. Incorporation of 125I-labeled cholesterol ester rich lipoproteins from cholesterol fed rabbits into normal rabbit aorta in vitro was inhibited by heparin, lecithin, and collagenase and by succinylation of the lipoprotein. Aortic uptake of lipoprotein was increased by neuraminidase, proteases, lipase, and beta-glucuronidase. These results suggest that it may be possible to control atherogenesis by controlling the interaction of atherogenic lipoproteins with their arterial receptor.


Binding to plasma lipoproteins of chlorophenoxyisobutyric, tibric and nicotinic acids and their esters: its significance for the mechanism of lipid lowering by clofibrate and related drugs. The binding of chlorophenoxyisobutyric (CPIB), tibric (TA) and nicotinic (NA) acids and CPIB ethyl ester (Clofibrate), TA and NA isopropyl esters (TAPE and NAPE) to human lipoproteins of low density of different classes (LDL2, LDL1 and VLDL) and high density (HDL) were studied by equilibrium dialysis and Sephadex gel filtration. Clofibrate and TAPE bound strongly to lipoproteins, but their acids, CPIB and TA and also NA and NAPE, did not bind. In the same experimental conditions, Clofibrate and TAPE bound only weakly to human serum albumin (HSA) and CPIB bound to HSA with a Ka of 3.3 X 10(5) M(-1) for 1 site of high affinity. The Clofibrate and TAPE bound to lipoproteins did not dissociate either during dialysis or during filtration on Sephadex G 25. The binding percentage remained constant for all drug concentrations studied, and the molar ratio of bound drug rose linearly with increasing concentrations. This suggests that the interaction may be irreversible, and there is some evidence that binding may induce irreversible changes in the lipoprotein molecules. These results, and those already found in experiments made with three other drugs related to Clofibrate, lead to the proposal that in their interaction with lipoproteins, the phenyl groups are necessary and the esterification is contributory. The possible role of this interaction in the lipid-lowering effect of the drugs is discussed with special reference to their possible implication in lipoprotein synthesis within the intestinal and hepatic cells.


Lipoprotein levels in apparently healthy men and women in the west of Scotland. Lipoprotein typing was undertaken in apparently healthy women (419) and men (103) in whom measurements of fasting total cholesterol, triglycerides, and cholesterol content of the major lipoprotein fractions were made. In the female group (30 to 59 years) there was a significant rise in total cholesterol, low density lipoprotein (LDL) cholesterol, and triglycerides with age. Using total cholesterol and triglyceride levels for classification, type II hyperlipoproteinaemia was present in 1 per cent and type IV in 6-7 per cent. When LDL cholesterol levels were also considered, type II occurred in 9-8 per cent ( 8-4% type IIA and 1-4% type IIB). In the male group ( 40 to 59 years) there was no increase in cholesterol or triglyceride levels with age: II per cent had type IV hyperlipoproteinaemia and none had a type II abnormality. When LDL cholesterol levels were also considered, type II occurred in 3 per cent ( 2% type IIA and 1% IIB). The apparent increase in LDL cholesterol frequency of type IIA abnormality in women could be merely the consequence of arbitrary selection of upper limits of normality for LDL cholesterol, but may signify improved detection of a high risk group.


Effect of oestrogens on postexercise electrocardiogram. The effect of three oestrogens (including an oestrogen-progestogen combination) on the postexercise electrocardiogram was studied in 33 men and 18 women who earlier had shown ST segment abnormalities after exercise. When pretreatment exercise tests were compared with tests after two weeks of treatment, the postexercise ST segments which were abnormal before treatment became even more abnormal in 18 (90%) of 20 subjects treated with conjugated oestrogens 10 mg daily, in 16 (89%) of 18 subjects treated with stilboestrol 5 mg daily, and in 12 (92%) of 13 subjects treated with norethynodrel (9-85 mg) and mestranol (0-15 mg)1 daily. The ST segment abnormalities reverted to pretreatment appearance within 6 weeks of stopping oestrogens. When 10 subjects with normal near-maximal exercise tests were treated for 2 weeks with conjugated oestrogens 10 mg daily, the tests remained unchanged in 9. The hypothesis favoured to explain these findings is that of an oestrogen-induced increase in coronary artery smooth muscle tone. An increase in arterial tone would also account for the increased incidence of myocardial (and cerebral) infarction that has been reported among individuals treated with oestrogen, either alone or in combination with progestogen.


Comparison of the serum low density lipoprotein and of its apoprotein in the pig, rhesus monkey and baboon with that in man. The principal form of the serum low density lipoprotein (LDL) in man, baboon, rhesus monkey and pig was isolated by preparative ultracentrifugation in the density interval 1.024-1.045 g/ml. The physicochemical characteristics of pig LDL most closely resembled those of man; thus, electrophoretic studies suggested that both baboon and rhesus LDL have a greater surface charge than that of their human counterpart, and electron-microscopic investigations showed baboon LDL (245 A) to be larger and rhesus LDL (205 A) smaller than those of man (217 A) and pig (228 A). In contrast, the immunological relationship between LDL from the two Old World monkeys and that of man was much closer (80-85% cross-reactivity by micro-immunoprecipitation) than that between pig and man (35% cross-reactivity). The principal difference between pig and human LDL appeared to reside in their protein and carbohydrate moieties. There was a marked resemblance between the protein moieties (apo-LDL) of LDL from the four species. The principal component of each animal apo-LDL was separated by gel-filtration chromatography and amounted to greater than 95% of the total protein; it exhibited a high molecular weight (greater than 250,000) upon SDS-polyacrylamide-gel electrophoresis and was indistinguishable from human apolipoprotein B in amino acid composition. Differences both between the apo-LDL and between the apo-B preparations from the four species, however, were detectable by immunological procedures. Such studies revealed inter-species relationships which were essentially the same as those observed between the respective native LDL preparations. The soluble apolipoproteins, present as minor components (less than 5%) of each apo-LDL, were compared by their electrophoretic mobility in polyacrylamide gel; the pattern seen in baboon and rhesus apo-LDL appeared to be most closely akin to that typical of their human counterpart. It is apparent that many characteristics typical of human serum LDL are found in those of the pig, rhesus monkey and baboon. Moreover, in view of the striking relationship existing between the immunological properties and apo-protein components of the LDL of the two Old World monkeys and that of man, these subhuman primates appear to be highly suitable as animal models for experimental atherosclerosis.


[Hypocalcemic vitamin D-dependent renal rickets]. Two siblings, female 10 years old, and male 15 years old, with the diagnosis of vitamin D-dependent rickets were studied. Another sibling, also with the same diagnosis, died of bronchopnemonia at about 7 months of age. Both patients developed rachitic manifestations since the first year of life, which persisted despite the administration of massive doses of vitamin D intermitently. Severe hypocalcemia, moderate hypophosphatemia and elevated serum alkaline phosphatase were the most characteristic biochemical findings. Both patients showed diminished renal tubular reabsorption of amino acids and phosphates. These alterations were reversible during I.V. calcium gluconate administration. The clinical biochemical and X-ray manifestations disappeared completely after one year of treatment with dihydrotaquisterol. Vitamin D-dependent rickets is an autosomal recessive disease, characterized by a hydroxylation defect of 25 hydroxycholecalciferol at the carbon 1 level, due to abscence of 25 hydroxy-D1-hydroxylase. Thus 1-25 Dihydroxycholecalciferol, the active form of vitamin D3 is not formed, resulting in depression of intestinal calcium absorption and reabsorption from the bones.


Epstein-Barr-virus-specific IgA and IgG serum antibodies in nasopharyngeal carcinoma. The sera of 73 patients with nasopharyngeal carcinoma (NPC), 28 patients with other carcinomas (OC) and 89 healthy subjects (HS) were tested for IgG and IgA antibodies to Epstein-Barr virus (EBV) viral capsid antigen (VCA). The majority of the NPC sera had IgG titres of 160 or above, whereas the majority of the other sera had titres below 160. For IgA reactivity to EBV-VCA, 68 of 73 (93-2%) NPC sera had titres of greater than or equal to 10. In contrast, only 6 of 28 (21-4%) OC sera and none of the HS sera had such titres. The mean serum concentrations of IgG, IgA, IgM and C3' were also determined in 55 NPC and 20 OC patients and 18 HS. They were all significantly higher in the NPC sera than in the HS. Although the concentrations of IgG and C3' were not significantly different in the two carcinoma groups, the concentrations of IgA and IgM were significantly higher in the NPC group than in OC. These findings appear to reflect the intensity of EBV-specific antigenic stimulation in NPC, and the EBV-specific serum IgA reactivity may be a sueful aid to the diagnosis of NPC, especially in cases with an occult primary tumour. It may be also of value as a screening test in people at high risk.


Cytotoxicity of guinea-pig lymphoid cells against guinea-pig hepatoma cells in tissue culture. The cytotoxic effect of guinea-pig lymphoid cells on guinea-pig hepatoma cell lines in tissue culture was investigated, using the microplate technique of Takasugi and Klein (1970). The effect of lymphoid cells from guinea-pigs immunized against tumor cells was compared to that of cells from normal controls. Several ratios of effector to target cells (10 : 1, 50 : 1, 150: 1, 250 : 1) were used. In Hartley guinea-pigs immunized with allogeneic tumour cells, peripheral blood lymphoid cells from 14/16 animals showed significant cytotoxicity against that tumour in culture. In a syngeneic tumour/host system, 7/13 animals showed cytotoxicity. Spleen cells gave less consistent results in both systems. The cytotoxic activity of subpopulations of immune lymphocytes against tumour cells in vitro was investigated. It was found that although both T-cell-enriched and T-cell-depleted cell populations exhibited cytotoxicity against tumour cells, the unfractionated cell population was the most effective. This suggests that some degree of cell cooperation may be involved in the cytotoxicity. Antibody-dependent cellular cytotoxicity was also obtained. A T-cell-depleted population of normal cells was shown to be cytotoxic to tumour cells in the presence of serum from immune animals. This type of cytotoxicity could be obtained concomitantly with cell-mediated cytotoxicity in the same animals.


Localization and activity of tissue bound cyclic nucleotide phosphodiesterase in normal and lack of changes in psoriatic human skin. This study has been undertaken to elucidate the localization and the activity of cyclic nucleotide phosphodiesterase (PDE) in psoriatic epidermis compared to normal. The results showed that the evaluation of cytochemical methods may be difficult because of the various factors which interfere with the reaction and the considerable amount of background staining. Additionally, only the tissue bound particulate enzyme fraction may be demonstrated by cytochemical means. Nevertheless, the method did reveal that the activity of PDE, if any, is localized on the cytoplasmic membranes of the cells, independent of their origin, and not on the cell surface. Moreover, no differences were found between normal and psoriatic skin. It seems, therefore, that the intracellular degradation of cAMP remains unaltered in psoriasis.


Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. A uniform system of classification and nomenclature of the acute leukaemias, at present lacking, should permit more accurate recording of the distribution of cases entered into clinical trials, and could provide a reference standard when newly developed cell-surface markers believed to characterize specific cell types are applied to cases of acute leukaemia. Proposals based on conventional morphological and cytochemical methods are offered following the study of peripheral blood and bone-marrow films from some 200 cases of acute leukaemia by a group of seven French, American and British haematologists. The slides were examined first independently, and then by the group working together. Two groups of acute leukaemia, 'lymphoblastic' and myeloid are further subdivided into three and six groups. Dysmyelopoietic syndromes that may be confused with acute myeloid leukaemia are also considered. Photomicrographs of each of the named conditions are presented.


Relationship of oral contraception to development of trophoblastic tumour after evacuation of a hydatidiform mole. The need for chemotherapy for trophoblastic tumour after evacuation of a hydatidiform mole was found to be significantly increased in patients taking oral contraceptives before normal human chorionic gonadotrophin (HCG) values were obtained. Oral contraception was also found to delay the fall in HDG excretion in patients not requiring treatment with cytotoxic drugs.


Disruption of Escherichia coli outer membranes by EM 49. A new membrane active peptide. A new peptide antibiotic, EM 49, is shown to disrupt the structure of Escherichia coli outer membranes and release outer membrane fragments into the surrounding media. Evidence supporting this conclusion indludes EM 49 stimulated release of outer membrane phospholipids, lipopolysaccharide, and membrane fragments having a phospholipid and polypeptide composition similar to outer membranes. The density of the membrane fragments released by EM 49 was 1.22 g/cm3, which was identical to isolated outer membranes. Approximately 10 to 15% of the E. coli lipopolysaccharide was released upon treatment with EM 49. Both scanning and transmission electron microscopy revealed that the antibiotic caused the formation of numerous protrusions or blebs on the surface of E. coli with apparent release of membrane vesicles from the cells. Direct interaction between EM 49 and outer membranes was demonstrated using outer membranes labeled with the fluorescent dye diphenylhexatriene. Treatment of the fluorescent-labeled outer membranes with EM 49 increased fluorescence intensity and decreased polarization, indicating that the peptide perturbed outer-membrane structure. In addition, strong interactions between EM 49 and purified E. coli phospholipids were detected using the Hummel and Dreyer technique. Association constants between the peptide and phospholipids were approximately 10(5) M-1. A model for the disruptive effect of EM 49 on outer-membrane structure is proposed in which the fatty acid chain of the antibiotic is inserted into the hydrophobic core of the membrane. This orientation would allow the polycationic, peptide portion of the antibiotic to disrupt the antibiotic to disrupt the normal electrostatic interactions between divalent cations and components of the outer membrane. Evidence supporting this conclusion includes specific protection of E. coli from EM 49 by Mg2+ and Ca2+ and inhibition of EM 49 stimulated phospholipid release by these cations. Disruption of the antibiotic to penetrate to the inner membrane, which is probably the primary killing site of EM 49.


Study of abnormal plasma low-density lipoprotein in rhesus monkeys with diet-induced hyperlipidemia. Male rhesus monkeys were divided into three groups: five were fed a regular primate chow diet and were used as controls; four received an "average" American diet; and five a special low-fat primate chow diet supplemented with 25% coconut oil and 2% cholesterol. In all of these animals, the plasma low-density lipoproteins (LDL) were isolated by ultracentrifugal flotation between densities of 1.019 and 1.050 g/ml. The LDL of the five control monkeys had variable molecular weights, with a mean value of 3.12 +/- 0.21 X 10(6) (range: 2.92 X 10(6) to 3.45 X 10(6)), and an average partial specific volume of 0.969 +/- 0.003 ml/g; both were assessed by flotation equilibrium analysis in the analytical ultracentrifuge. In the individual animals, however, the physical properties of LDL were invariant with time. The administration of either an "average" American diet or a coconut oil-cholesterol diet was accompanied by hypercholesterolemia associated with changes in LDL which were characterized by increases in molecular weight to 3.52 +/- 0.21 X 10(6) (average of nine monkeys) and in partial specific volume to 0.973 +/- 0.002 ml/g. These changes were particularly evident when the molecular weight of LDL from monkeys in the normolipidemic state was compared with that obtained from the same monkeys during the hyperlipidemic state. Chemical analyses revealed that the particles from the hyperlipidemic animals had a relatively higher cholesteryl ester content, a slight increase in phospholipids, and a marked decrease to nearly complete absence of triglycerides. The other lipoprotein components, protein, carbohydrate, free cholesterol, and fatty acids, did not vary significantly from those of control LDL. It is concluded that the administration of atherogenic diets causes structural changes in LDL which appear to be accounted for, at least in part, by changes in the composition of the lipid moiety. The changes in physical and chemical properties noted in the LDL of rhesus monkeys with experimentally induced hypercholesterolemia contrast with the apparent structurally normal LDL from rhesus monkeys with spontaneous hypercholesterolemia reported previously.


D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate. Steady-state kinetic studies including initial velocity for mannitol oxidation and fructose reduction and product inhibition for mannitol oxidation using fructose and reduced nicotinamide adenine dinucleotide (NADH) are in accord with a reaction mechanism best described as ordered Bi-Bi with NAD+ and NADH designated as the first substrate, last product, respectively at pH 8.8. All replots of slopes and intercepts from product inhibition studies were linear. Dead-end inhibition studies using mannitol 1-phosphate gave slope-parabolic, intercept-linear noncompetitive inhibition for both NAD+ and mannitol as substrates. The dead-end inhibitor is capable of binding multiply to the E, EA, and EQ forms of the enzyme to an extent that is controlled by the concentration of substrates. The EQ complex is inferred to undergo a conformational change, E'Q equilibrium EQ, since (V1/E1) greater than (KiqV2)/(KqE1), and no evidence for dead-end complex formation with NADH can be adduced. This is interpreted to mean that the release of fructose from the central complex is faster than the isomerization of the E-NADH complex. When mannitol is saturating, the noncompetitive inhibition against NAD+, as the variable substrate, becomes parabolic uncompetitive. A replot of the slopes of the parabola against mannitol 1-phosphate remains concave upward. This situation could arise if the conformational change we infer in the EQ complex opens up additional sites on the protein which can interact with the dead-end inhibitor.


Isolation and characterization of DNA-DNA and DNA-RNA. A simple method for the isolation and characterization of DNA-DNA and DNA-RNA hybrid molecules formed in solution was developed. It was based on the fact that, in appropriate salt concentration, such as 5% Na2HPO4, DNA in either double-stranded (DNA-DNA or DNA-RNA) or single-stranded forms, but not free nucleotides, can bind to diethylaminoethylcellulose disc filters (DE81). Thus tested samples were treated with the single-strand-specific nuclease S1 and then applied to DE81 filters. The free nucleotides, resulting from degrading the single-stranded molecules, were removed by intensive washing with 5% Na2HPO4, leaving only the hybrid molecules on the filters. The usefulness of this method was illustrated in dissociation and reassociation studies of viral (SV40) or cellular (NIH/3T3) DNAs and DNA-RNA hybrid molecules. Using this technique the reassociation of denatured SV40 DNA was found to be a very rapid process. Dissociation studies revealed that the melting curves of tested DNAs were dependent on salt concentration. Thus the melting temperatures (tm) obtained for SV40 DNA were 76 degrees C at 1 X SSC (0.15 M NaCl-0.015 M sodium citrate) and 65 degrees C at 0.1 X SSC, and for NIH/3T3 DNA 82 degrees C at 1 X SSC and 68 degrees C at 0.1 X SSC. MuLV DNA-RNA hybrid molecules were formed by annealing in vitro synthesized MuLV DNA with 70S MuLV RNA at 68 degrees C. The melting temperature of this hybrid in the annealing solution was 87 degrees C. Another important feature of this procedure was that, after being selectively bound to the filters, the hybrid molecules could efficiently be recovered by heating the filters for 5 min at 60 degrees C in 1.5-1.7 M KCl. The recovered molecules were intact hybrids as they were found to be completely resistant to S1 nuclease.


Fidelity of ribosomal ribonucleic acid synthesis by nucleoli and nucleolar chromatin. Isolated nucleoli, nucleolar chromatin, and nucleolar DNA were used as templates for DNA synthesis in appropriately supplemented systems in which RNA polymerases other than RNA polymerase I were blocked by alpha-amanitin. With the aid of nucleotide analysis, DNA-RNA hybridization, and homochromatography fingerprinting, it was found that isolated nucleoli and nucleolar chromatin serve primarily as templates for synthesis of rRNA. However, the products formed with purified nucleolar DNA as a template do not contain the specific rRNA oligonucleotides nor are they appreciably hybridized to the rDNA region on cesium chloride gradients. These results indicate that whole nucleoli and nucleolar chromatin contain control mechanisms that restrict readouts by RNA polymerase I of nucleolar DNA to rDNA.


On the location of the divalent metal binding sites and the light chain subunits of vertebrate myosin. The divalent metal ion binding sites of skeletal myosin were investigated by electron paramagnetic resonance (EPR) spectroscopy using the paramagnetic (Mn(II) ion as a probe. Myosin possesses two high affinity sites (K less than 1 muM) for Mn(II), which are located on the 5,5'-dithiobis(2-nitrobenzoate) (DTNB) light chains. Mn(II) bound to the isolated DTNB light chain gives rise to an EPR spectrum similar to that of Mn(II) bound to myosin and this indicates that the metal binding site comprises ligands from the DTNB light chain alone. Myosin preparations in which the DTNB light chain content is reduced by treatment with 5,5'-dithiobis(2-nitrobenzoate) show a corresponding reduction in the stoichiometry of Mn(II) binding, but the stoichiometry is recovered on reassociation of the DTNB light chain. Chymotryptic digestion of myosin filaments in the presence of ethylenediaminetetraacetic acid yields subfragment 1, but digestion in the presence of divalent metal ions produces heavy meromyosin. Myosin with a depleted DTNB light chain content gives rise to subfragment 1 on proteolysis, even in the presence of divalent metal ions. It is proposed that saturation of the DTNB light chain site with divalent ions protects this subunit against proteolysis, which, in turn, inhibits the cleavage of the subfragment 1-subfragment 2 link. Either the DTNB light chain is located near the region of the link and sterically blocks chymotryptic attack, or it is bound to the subfragment 1 moiety and affects the conformation of the link region. When the product heavy meromyosin was examined by sodium dodecyl sulfate gel electrophoresis, an apparent anomaly arose in that there was no trace of the 19 000-dalton band corresponding to the DTNB light chain. This was resolved by following the time course of chymotryptic digestion of the myosin heavy chain, the DTNB light chain, and the divalent metal binding site. The 19 000-dalton DTNB light chain is rapidly degraded to a 17 000-dalton fragment which comigrates with the alkali 2 light chain. The divalent metal site remains intact, despite this degradation, and the 17 000 fragment continues to protect the subfragment 1-subfragment 2 link. In the absence of divalent metal ions, the 17 000-dalton fragment is further degraded and attack of the subfragment 1 link ensues. Mn(II) bound to cardiac myosin gives an EPR spectrum basically similar to that of skeletal myosin, suggesting that their 19 000-dalton light chains are analogous with respect to their divalent metal binding sites, despite their chemical differences. The potential of EPR spectroscopy for characterizing the metal binding sites of myosin from different sources and of intact muscle fibers is discussed.


Near-neighbor relationships of the subunits of cytochrome c oxidase. Cytochrome c oxidase in detergent dispersion has been cross-linked with two reversible cross-linking agents, dithiobissuccinimidylpropionate (DSP) and dimethyl-3,3'-dithiobispropionimidate (DTBP), and the cross-linked products formed have been analyzed by two-dimensional gel electrophoresis. Under mild reaction conditions, several subunit pairs were seen including II and V, V and VII, IV and VI. With higher levels of DSP, larger aggregates were seen until a cross-linked product with an apparent molecular weight of 140 000 was the predominant band on gels. This is the smallest molecular weight aggregate to contain all seven subunits of the enzyme and most likely represents the "unit" or two heme and two copper containing complex of cytochrome c oxidase.


Topographic distribution of enzymes involved in glycerolipid synthesis in rat small, intestinal epithelium. 1. The enzymes involved in glycerolphosphate and monoacylglycerol acylation of rat small intestine were more active in villi than in crypts. Monoglyceride acyltransferase (EC 2.3.1.22) was found to be absent from crypts. 2. In the villi, the enzymes are mainly localized in microsomes, although low activities of palmitoyl-CoA synthetase (EC 6.2.1.3), glycerolphosphate acyltransferase (EC 2.3.1.15) and cholinephosphotransferase (EC 2.7.8.2) are found in mitochondria. Mitochondria lack monoglyceride acyltransferase and lysolecithin acyltransferase (EC 2.3.1.23), both of which are involved in the reacylation of alimentary partial glycerides. Therefore, this process is confined to microsomes. 3. The monoacylglycerol and lysolecithin acyltransferases, as well as choline-phosphotransferase, are probably localized within the endoplasmic reticulum, since these enzymes are relatively Nagerse resistant (subtilisin; EC 3.4.2.1, compared with palmitoyl-CoA synthetase and glycerolphosphate acyltransferase, which are highly Nagarse-sensitive and therefore probably localized on the outside of the microsomes (and mitochondria). 4. The physical separation of alimentary product reacylation from de novo synthetic processes provides the basis of metabolic compartmentation observed by other workers. 5. The use of sucrose instead of a salt medium for the isolation and homogenization of small intestinal epithelial cells allowed the separation of mitochondria and microsomes by differential centrifugation without mutual contamination. 6. Phospholipids were found to stimulate glycerolphosphate acylation in vitro. 7. The glycerolphosphate and monoacylglycerol acylation pathways are not competitive.


Influence of lysophosphatidylcholine on the metabolism of plasma lipoproteins. Both low density lipoproteins and cellular membranes are known to have a high affinity for lysophosphatidylcholine. In this study lysophosphatidylcholine influenced the retention of lipoproteins by arterial tissue in vitro and the rate of disappearance of low density lipoproteins from the blood in vivo. Pieces of aorta from rabbits or rhesus monkeys were successively incubated for 90 min each in 2 or 3 solutions. After the last incubation the intima plus inner media was dissected from the remainder of the aorta for analysis. The second incubation always contained lipoproteins labeled with [3H]leucine. When lysophosphaticylcholine was included in the first but not in the second incubation fluid, the retention of low, or high density lipoproteins by the intima plus inner media increased. A subsequent incubation of the piece of artery in a fluid with trypsin or lysophosphatidylcholine caused a release of some of the lipoproteins. Lysophosphatidylcholine was bound simultaneously by plasma low density lipoproteins and vascular tissue in vitro and appeared to promote the association of the latter two components. When lysophosphatidylcholine equal to 2--10 times the usual total intravascular content was injected intravenously into control squirrel monkeys or rabbits, it was rapidly cleared from the blood. On the other hand, injected lysophosphatidylcholine persisted in the blood of hyperlipoproteinemic rabbits and was associated with the low density lipoproteins. In control animals, the injection of lysophosphatidylcholine was associated with an increase in the rate of removal of 125I-labelled low density lipoprotein from plasma and of its appearance in liver.


The influence of LP-X and other lipoproteins associated with hepatic dysfunction on the activity of lecithin:cholesterol acyltransferase. A study was made of the in vitro effects of the abnormal serum lipoproteins associated with liver disease on the activity of the enzyme lecithin:cholesterol acyltransferase. At lipoprotein concentrations equivalent to those found in hepatic disease sera, the results indicate that: (1) LP-X levels greater than 2.5 mg/ml produced total inhibition of enzyme activity. (2) LP-X levels remained constant even up to 36 h incubation, despite active cholesterol esterification in the presence of LP-X concentrations less than 2.5 mg/ml. In addition, the specific activity of radiolabelled LP-X, and its electrophoretic properties remained unchanged after incubation showing that the molecule remained intact. (3) Low density lipoproteins other than LP-X stimulated the enzymes activity, but this effect was overcome by LP-X. (4) Additional concentrations of high density lipoproteins also produced enhancement of enzyme activity, but at higher levels inhibition was seen. LP-X prevented the enhancement of lecithin:cholesterol acyltransferase activity. (5) Small samples of pure LP-X, obtained with the minimum of physical manipulation, showed a complete absence of cholesterol ester and triacylglycerol from the molecule. The implications of these results are discussed, particularly in relation to other reports which have presented evidence that LP-X is a substrate for lecithin:cholesterol acyltransferase.


Mössbauer spectroscopic study of compound ES of cytochrome c peroxidase. Mössbauer spectra of Compound ES of cytochrome c peroxidase have been observed over a range of temperature and applied magnetic field. These have been interpreted in terms of a crystal field model of the iron site in which the iron is assumed to be in the Fe(IV) state with unpaired spin S = 1. Detailed least-squares fitting of the spectra fitting of the spectra indicates that both the electric field gradient choice of a single parameter, the axial crystal field, the magnetic properties are well reproduced. The model also provides the observed positive sign for the electric field gradient interaction, but overestimates its magnitude. This apparent discrepnancy may be caused by the presence of significant electronic charge in filled bonding orbitals, a feature which is in keeping with expected covalent charge compensation of the extreme oxidation state. There is no evidence in the Mössbauer spectra of interaction between the iron and the ESR-visible free radical. This suggests they are well separated.


The decreased synthesis of chondroitin sulfate-containing extracellular proteoglycans by SV40 transformed Balb/c 3T3 cells. Balb/c 3T3 cells synthesize 5--10 times more 35SO2/4- -labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular 35SO2/4- -labeled proteoglycans of the Balb/c 3T3 and SV3T3 cells differ markedly in their acid mucopolysaccharide composition. Extracellular Balb/c 3T3 proteoglycans contain about 70--80% chondroitin sulfate, most of which is chondroitin 4-sulfate, and small amounts of heparan sulfate and/or heparin. On the other hand, extracellular SV3T3 proteoglycans contain 65-75% heparan sulfate and/or heparin and less than 15% chondroitin sulfate. Analysis of extracellular 35SO2/4- -labeled proteoglycan by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that Balb/c 3T3 alone synthesizes a class of proteoglycans capable of migrating in a 10% separating gel. This class of proteoglycans, designated as fraction C, accounts for up to 45% of the total extracellular Balb/c 3T3 35 SO2/4- -labeled proteoglycans and contains chondroitin sulfate extracellular SV3T3 proteoglycans. The absence of this and other classes of chondroitin sulfate-containing proteoglycans can account for the 5-10-fold decreased synthesis of 35SO2/4- -labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.


Extracellular metabolism of cyclic AMP.

Extracellular metabolism of cyclic AMP. Intravenously administered cyclic [8-3H]AMP to rats was quickly eliminated from the circulation. After 2 min 93% of the administered radioactivity disappeared from the plasues was recovered mainly in the form of nucleotides, ATP, ADP, AMP and IMP. In vitro contact of cyclic AMP with perfused liver, isolated liver cells and adipose tissue resulted in a rapid breakdown of the nucleotide, presumably on the outer surface of the cells. The degradation products have been identified mainly as adenosine and inosine. Incubation of adipose tissue and isolated liver cells with [3H] AMP also resulted in the breakdown of the nucleotide in themedium. The rate of AMP degradation by these tissues was faster than that for cyclic AMP degradation. The data suggest that cyclic AMP is readily metabolized on the outer surface of cells to products which may be converted within the cells to nucleotides. These findings seem of importance for the quantitative assessments of cellular cyclic AMP outflow during hormonal stimulation.


Cyclic AMP metabolism in mouse parotid glands. Properties of adenylate cyclase, protein kinase and phosphodiesterase. Catecholamines induce unique growth and secretory responses in salivary glands. An analysis of three enzyme activities involved in cyclic AMP metabolism was carried out to identify the specificity of these responses for salivary glands. Although parotid adenylate cyclase has an unusually high specific activity, its kinetic properties and responses to NaF, guanine nucleotides, and isoproterenol are similar to other tissues not stimulated to grow after isoproterenol stimulation. Solubilized adenylate cyclase was separated from other membrane proteins by isoelectric focusing on polyacrylamide gels. There was a single broad peak of activity witha pI of 5.9. Parotid protein kinase has a subcellular distribution and substrate preference similar to hepatic protein kinase. Activation by cyclic AMP is also similar to that reported for other tissues, with a Ka of 1.2 - 10(-7) M. Parotid cyclic AMP and cyclic GMP phosphodiesterases are a heterogeneous group of enzymes with relatively low specific activity as compared with mouse pancreas, liver and brain. Isoelectric focusing of supernatant phosphodiesterases revealed at least sixpeaks of enzyme activity in the pI range of 4-6. Previous reports of a large increase in parotid cyclic  AMP levels after in vivo administration of catecholamines and specific growth and secretion could be the result of a relatively high specific activity adenylate cyclase associated with low specific activity cyclic AMP phosphodiesterases.


The interaction between water and the polar head in inverted phosphatidylcholine micelles. A 2H and 31P relaxation study. 2H and 31P spin-lattice relaxation times (T1) were studied for inverted egg phosphatidylcholine micelles in CCl4 as functions of 2H2O concentration. When the 2h2O/phosphatidylcholine mole ratio changed from 1.0 to 18.0, T1 of 31P increased by about 2.6 fold, whereas T1 of 2H increased by about 50 fold. A quantitative analysis of the deuterium T1 data showed that there is only one water molecule tightly bound to the polar head, and it is in rapid exchange with the rest of the water molecules. The activation  energy for the deuterium T1 was 7.1 +/- 0.8 kcal/mol(30 +/- 3 kJ/mol), and was independent of the 2H2O concentration.


Effects of adenosine and its derivatives on protein kinase activity of beef thyroid. Effects of adenosine and some of its derivatives on beef protein kinase activity were investigated in vitro. Adenosine rapidly inhibited protein kinase activity in a dose-dependent manner. Significant inhibition occurred with 10 muM and half-maximal inhibition at 100 muM adenosine. Inhibition was almost complete with 5 mM adenosine. Inhibition was similar whether protein kinase activity was assayed with or without cyclic AMP. The inhibition  by adenosine was reversed by increasing the concentration of ATP and Lineweaver-Burk analysis indicated that adenosine inhibition was competitive with ATP. Addition of adenosine deaminase to the incubation medium prevented the inhibition induced by adenosine. Intact 1 and N6 positions of adenosine were important for the inhibition since their modification was associated with loss of inhibition. Modification of the 8 position of adenosine decreased, but did not abolish, the inhibition. The 2 and 3 position of ribose did not seem to be critical since 2- and 3-deoxyadenosine produced inhibition similar to that of adenosine.


Subcellular location of adenylate cyclase in rat cardiac muscle. Crude homogenates of rat cardiac muscle were fractionated in order to examine the subcellular location of adenylate cyclase in this tissue. The fractionation procedure employed differential centrifugation of homogenized material followed by collagenase treatment, centrifugation on a discontinuous sucrose density gradient and extraction with 1 M KCl. The particulate fraction obtained by this procedure contained a high specific activity and yield of adenylate cyclase, moderate levels of mitochondria and low levels of sarcoplasmic reticulum and contractile protein as judged by marker enzyme activities. Adenylate cyclase was purified 20-fold with a 33% yield from the crude homogenate, while mitochondrial, sarcoplasmic reticulum and contractile protein yields were 5, 0.4 and 0.7% respectively. The membrane fractions prepared in this manner were examined by sodium dodecyl sulfate - gel electro phoresis. Adenylate cyclase copurfied with ouabain-sensitive (Na+ + K+)-ATPase, a plasma membrane marker enzyme, and not with Ca2+ -accumulating activity, which is associated with the sarcoplasmic reticulum. The distribution of marker enzyme activities indicates that heart adenylate cyclase is not located in the sarcoplasmic reticulum but is localized predominantly, if not exclusively, in the plasma membrane.


Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells. Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E1 also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E1 is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 muM isoproterenol is increased and approaches that produced by5.6 muM prostaglandin E1. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 muM prostaglandin E1 is reduced by as much as 50%, the concentration of prostaglandin E1 required to induce a maximal increase in cyclic AMP is 1/10 that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E1 can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparation from control or choleragen-treated cells. In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblast choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.


Interaction between glucocorticoids and cyclic AMP in the regulation of phosphoenolpyruvate carboxykinase (GTP) in the isolated perfused rat liver. Effects of cordycepin and cycloheximide. The mechanism of the interaction between glucocorticoids and cyclic AMP in the regulation of phosphoenolpyruvate carboxykinase (GTP: oxalocetate carboxylase, transphosphorylating, EC 4.1.1.32) was investigated in the isolated perfused rat liver using inhibitors of transcription or translation. Dibutyryl cyclic AMP produced a rapid increase in P-enolpyruvate carboxykinase activity. The response of the enzyme to the cyclic nucleotide ceased however, at 4 h, but was restored by dexamethasone. The dibutyryl cyclic AMP-induced increase in P-enolpyruvate carboxykinase activity was completely blocked by cycloheximide, but not not by cordycepin. However, cordycepin totalaly suppressed the "permissive" effect of dexamethasone on the response of the enzyme to dibutyryl cyclic AMP. Preperfusion of the livers with dexamethasone and cycloheximide, following by perfusion without the steroid hormone and the inhibitor, resulted in a rapid rise in P-enolpyruvate carbosykinase activity, which was not affect by cordycepin. If livers were preperfused with cordycepin for different time-periods, followed by dibutyryl cyclic AMP stimulation of P-enolpyruvate carboxykinase synthesis, the response of the enzyme to the cyclic nucleotide was progressively reduced, achieving 50% inhibition after 1.5 h of preperfusion. These results suggest that the induction of hepatic P-enolpyruvate carboxykinase to maximum values, brought about by cyclic AMP at the level of translation, depends on the supply of newly synthetized mRNA provided by the transcriptional action of glucocorticoids.


The synthesis of spin-label derivatives of NAD+ and its structural components and their binding to lactate dehydrogenase. Spin-labelled derivatives of NAD+ and its structural components (i.e. adenosine, adenine, AMP, ADP and ADPR) have been synthesized. Their binding to pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been studied and dissociation constants have been determined. The spin-labelled derivatives of ADP and ADPR exhibit a tighter binding than the corresponding NAD+ derivative. This may be attributed to the repulsion of the positively charged nicotinamide ring by an histidine side chain in the active center of the enzyme.


Mössbauer and EPR spectroscopy of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa. Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) from Pseudomonas aeruginosa has been investigated by EPR and Mössbauer spectroscopy. Low temperature Mössbauer data on the native enzyme (Fe3+, S = 5/2) yields a hyperfine field Hsat=-525 kG at the nucleus. This observation is inconsistent with earlier suggestions, based on EPR data of a rubredoxin-like ligand environment around the iron, i.e. a tetrahedral sulfur coordination. Likewise, the dithionite-reduced enzyme has Mössbauer parameters unlike those of reduced rubredoxin. We conclude that the iron atoms are in a previously unrecognized environment. The ternary complex of the enzyme with 3,4-dihydroxyphenylpropionate and O2 yields EPR signals at g = 6.7 and g = 5.3; these signals result from an excited state Kramers doublet. The kinetics of the disappearance of these signals parallels product formation and the decay of the ternary complex as observed in the optical spectrum. The Mössbauer and EPR data on the ternary complex establish the iron atoms to be a high-spin ferric state characterized by a large and negative zero-field splitting, D = approximately -2 cm-1.


Purification and immunochemical characterization of aldehyde dehydrogenase from 2-acetylaminofluorene-induced rat hepatomas. 1. A series of aldehyde dehydrogenase isozymes (aldehyde:NAD (P)+ oxidoreductase, EC 1.2.1.5), has been purified from hepatomas induced in Sprague-Dawley rats by 2-acetylaminofluorene. 2. The functional hepatoma-specific aldehyde dehydrogenase isozymes exist as 105 000-dalton dimers composed to two subunits of 53 000 daltons. Isoelectric points of the purified isozymes are 6.9-7.2. 3. Antiserum to these purified hepatoma-specific aldehyde dehydrogenases has been produced and the immunological relationships of these isozymes to their normal liver counterpart have been studied. Results of Ouchterlony double diffusions, agar-gel immunoelectrophoresis and polyacrylamide gel and agar immunoelectrophoresis indicate that anti-hepatoma aldehyde dehydrogenase antiserum cross-reacts with normal liver aldehyde dehydrogenase.


Deoxythymidine kinase induced in the HELA TK- cells by herpes simplex virus type I and type II. Substrate specificity and kinetic behavior. Deoxythymidine kinases (EC 2.7.1.--) induced in HeLa TK- cells by Herpes simplex Type I and Type II viruses both had a requirement for divalent cations. The enzymes had the highest activities in the presence of Mg2+, followed by Mn2+, Ca2+, Fe2+, and in that order, whereas they were inactive in the presence of Zn2+ and Cu2+. The amount of Mg2+ required for optimal activity was dependent on the amount of ATP present, so that optimal activities were found when the concentration of Mg2+ was equal to that of ATP; an excess of Mg2+ inhibited the reaction. The activities of various nucleoside triphosphates as phosphate donors for Herpes simplex virus Type I deoxythymidine kinase were in the order: ATP = dATP = ara ATP greater than CTP greater than dCTP greater than UTP greater than dUTP greater than GTP greater than dGTP. Those for Herpes simplex virus Type II deoxythymidine kinase were in the order: CTP greater than dCTP = ara CTP greater than dATP greater than ATP greater than UTP greater than GTP greater than dUTP = dGTP. For both deoxythymidine kinases induced by Herpes simplex virus, the nucleoside triphosphates tested exerted cooperative effects. The Km values of ATP and CTP for the Herpes simplex virus Type I enzyme were 30 and 70 muM respectively; whereas those for the Herpes simplex virus Typr II enzyme were 140 and 450 muM. Studies on binding of various thymidine analogs with free 5'-OH to these deoxythymidine kinases indicated that 5-substituted ethyl-, vinyl-, allyl-, propyl-, iodo- and bromo-dUrd as well as iodo5 dCyd and bromo5 dCyd had good affinity to both enzymes. In contrast, vinyl5 Urd, iodo5 Urd and arabinosylthymidine had good affinity only to the Herpes simplex virus Type I enzyme but not to the Herpes simplex virus Type II deoxythymidine kinase. All of these thymidine analogs were competitive inhibitors, with KI values in the range of 0.25 to 1.5 muM. Herpes simplex virus Type I deoxythymidine kinase was less sensitive to either dTTP or iodo dUTP inhibition than Herpes simplex virus Type II. Both dThd and dCyd could serve as substrates and competed with each other for Herpes simplex viruses Type I and Type II induced kinases, but they differed in their Km values for these enzymes. The Km values of dThd and dCyd were 0.59 muM and 25 muM for Herpes simplex virus Type I deoxythymidine kinase; while they were 0.36 muM and 88 muM respectively for the Herpes simplex virus Type II enzyme.


Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes. Determination of specific GTPase (EC 3.6.1.--) activity in turkey erythrocyte membranes was achieved using low concentration of GTP (0.25 muM), inhibition of nonspecific nucleoside triphosphatases by adenosine 5'(beta,gamma-imino-triphosphate (App(NH)p) and suppression of the transfer of gamma-32P from GTP to ADP with an ATP regeneration system. Under these conditions catacholamines caused a 30--70% increase in GTP hydrolysis. The stimulation of GTPase activity by catecholamines required the presence of Mg2+ or Mn2+. DIfferent batches of membranes revealed the following specific activities (pmol 32Pi/mg protein min): basal GTPase (determined in the absence of catecholamine), 6-- 11; catecholamine-stimulated TTPase, 3--7; and residual non-specific NTPase 3--5. The stimulation of GTPase activity by catecholamines fulfilled the stereospecific requirements of the beta-adrenergic receptor, and was inhibited by propranolol. The concentrations of DL-isoproterenol which half-maximally activated the GTPase and adenylate cyclase were 1 and 1.2 muM, respectively. The following findings indicate that the catecholamine-stimulated GTPase is independent of the catalytic production of cyclic AMP by the adenylate cyclase. Addition of cyclic AMP to the GTPase assay did not change the rate of GTP hydrolysis. Furthermore, treatment of the membrane with N-ethylmaleimide (MalNEt) at 0 degrees C which caused 98% inhibition of the adenylate cyclase, had no effect on the catecholamine-stimulated GTPase. The affinity and specificity for GTP in the GTPase reactions are similar to those previously reported for the stimulation of the adenylate cyclase. The apparent Km for GTP in the basal and the catecholamine-stimulated GTPase reaction was 0.1 muM. These GTPase activities were inhibited by ITP but not by CTP and UTP. It is proposed that a catecholamine-stimulated GTPase is a component of the turkey erythrocyte adenylate cyclase system.


Guanosine monophosphate synthetase from Ehrlich ascites cells. Multiple inhibition by pyrophosphate and nucleosides. GMP synthetase (xanthosine-5'-phosphate: ammonia ligase (AMP-forming), EC 6.3.4.1) from Ehrlich ascites cells was found to be subject to multiple inhibition by its reaction product, PPi, and some analogs of adenosine. PPi and the nucleoside (N) inhibitors were also capable of individually inhibiting this enzyme. Under no conditions did the inhibition appear to be irreversible or "pseudoinactivating" in nature. The individual inhibition by PPi was competitive with respect to ATP (KI = 0.42 mM). Conversely, in the absence of PPi, the binding of N was noncompetitive with ATP, but shifted to a competitive pattern when PPi was present. Furthermore, with the inhibitors in concert, there was an apparent lowering of the KI values for both inhibitors. This data was consistent with either PPi functioning to tighten the binding of N at a noncatalytic site (positive cooperativity) or with PPi actually opening a second binding site for N in addition to the non-catalytic site. Although this study did not distinguish which of these events was occurring, it did reveal that the intensity of the effect of PPi appeared to be constant. That is, for various N inhibitors with a range of independently determined KI values from 26 to 1650 muM, the ratio of their KI values determined in the absence of PPi to the values determined in the presence of PPi was always 38 +/- 1.


Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors. Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP  receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors  eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.


Effects of common inhibitors of gastric acid secretion on secretagogue-induced respiration and aminopyrine accumulation in isolated gastric glands. 1. The effects of three inhibitors of gastric acid secretion, atropine, burimamide and thiocyanate, have been studied in isolated glands from the rabbit gastric mucosa. The glands were either resting or stimulated by carbachol, histamine or dibutyryl cyclic AMP. The effects were determined from changes in oxygen consumption and accumulation of the weak base aminopyrine. The latter gives an indirect measurement of the acid production in the glands. 2. Atropine (10 (-6) M) almost totally inhibited the transient response induced by carbachol (10 (-4) M) in both measured parameters. The histamine-induced increase in respiration was inhibited when the atropine concentration was raised to 10 (-4) M. To a lesser extent also, histamine-induced aminopyrine accumulation was reduced. The dibutyryl cyclic AMP stimulated oxygen consumption was not affected by atropine. 3. Burimamide competitively inhibited the histamine responses but was without effect on those of carbachol and dibutyryl cyclic AMP. 4. Thiocyanate (10 (-2) M) inhibited the increase in oxygen consumption induced by all three secretagogues but not down the prestimulatory level, in spite of total abolishment of the aminopyrine accumulation. 5. In unstimulated glands, burimamide (10 (-3) M) or atropine (10 (-4) M) did not alter the normal aminopyrine ratio (aminopyrine in intraglandular water/ aminopyrine in extraglandular water) of approximately 50. This may indicate the existence of preformed acid in resting parietal cells. Thiocyanate, on the other hand, lowered the aminopyrine ratio in unstimulated glands from 46 to 2. Possible mechanisms for the thiocyanate effect are discussed in terms of an inability to separate acid and base in the secreting membrane.


The structure of vesicular stomatitis virus membrane. A phosphorus nuclear magnetic resonance approach. The proton decoupled 40.48 M Hz 31P NMR spectrum of intact and unperturbed membrane-enclosed vesicular stomatitis virus (sterotype Indiana) exhibited two distinct maxima. These can be resolved into a narrow, symmetric line and a broad asymmetric line. The 31P NMR spectrum of a multilamellar (unsonicated) preparation of the extracted viral lipids exhibited a line shape similar to that of the intact virus. A sonicated vesicle preparation of the extracted viral lipids exhibited a narrow symmetric line. The narrow component in the intact virus spectrum may be attributed to small membrane fragments. Phospholipase C digestion of the intact virus resulted in substantial reduction in intensity of both components which suggests that much of the contribution to both peaks is due to phosphate in the phospholipid polar head groups. The phospholipid phosphates in both sonicated and unsonicated preparations of the extracted viral lipids exhibited substantially longer relaxation times than did those in the intact virus. The short relaxation time emanating from the intact virus preparation is caused by immobilization of the phospholipid head groups which could be due to lipid-protein interactions. Trypsin treatment of vesicular stomatitis virions, which results in complete removal of the exterior hydrophilic segment of the membrane glycoprotein, increased the 31P relaxation time to a value similar to that observed in the protein-free total lipid extracts; this finding provides supporting evidence for the role of virus glycoprotein in shortened relaxation times. A reversible temperature-dependent change in apparent line width and absence of an effect of cholesterol on the 31P phospholipid spectrum were also demonstrated.