[Hypoglycaemic coma in Boeck's sarcoidosis (author's transl)].

The diversity of clinical manifestations of Boeck's sarcoidosis may also include endocrine disorders. One year after diagnosis of sarcoidosis in a 29 year-old female patient, endocrinological complications became manifest with amenorrhoea. The course of the disease was additionally complicated by hypoglycaemic episodes. Thorough clinical investigation of the patient revealed sarcoid involvement of the skin, lungs, liver and lymph nodes and an extensive retroperitoneal surgically-verified lymph tumour. After tolbutamide and in reaction to an intravenous glucose tolerance test the blood glucose was found to be very low, whilst the immunoreactive insulin was normal. Further investigation of the endocrine functions of the patient revealed normal functioning of the thyroid gland, subnormal values for the follicle stimulating hormone and extremely low serum ACTH and serum cortisol values, without any diurnal changes in these parameters. The clinical symptoms of the patient and the biochemical findings were regarded as manifestations of secondary adrenal failure due to sarcoid involvement of the hypothalamus and pituitary. Hence, treatment with corticosteroids was started. Hypoglycaemia has not since been observed in this patient and the other clinical features of (secondary) adrenal failure have disappeared slowly.


Smith-Lemli-Opitz syndrome: review and report of two affected siblings.

This paper reports two siblings with the Smith-Lemli-Opitz syndrome and reviews the literature on the subject. SLOS is a syndrome of multiple congenital anomalies with mental and growth retardation, unusual facies, genito-urinary and hand and foot abnormalities inherited as an autosomal recessive trait.


Giant platelets and fibrin-like inclusions in platelets of BALB/c mice after intraperitoneal inoculation of HIPA tumour agent.

2 days after ip inoculation of cell-free extracts of HIPA tumour, giant platelets and platelets with band-like inclusions were found in blood of BALB/c mice. Band-like inclusions with platelets have morphologic structure of fibrin. Their presence in the thrombocytes is probably an accidental finding. Giant platelets in the circulating blood are probably due to a disturbed meagacaryocytopoiesis.


[Oncocytes in a mixed canine mammary gland tumour].

The electronmicroscopic findings of a canine mixed mammary gland tumour containing oncocytes, are described. The tumour consists of small myxoid foci and glandular formations with an organoid structure. The glandular walls are composed of myoepithelial differentiated cells and epithelial cells. Most of the latter are oncocytes and characterized by an extensive amount of pathic mitochondria. The myxoid foci consist of myoepithelial differentiated cells.


Inhibition of prolactin secretion by lergotrile mesylate: mechanism of action.

Lergotrile mesylate (2-chloro-6-methylergoline-8beta-acetonitrile, methane-sulphate salt) was shown to be a potent inhibitor of prolactin secretion in vivo and in vitro. The dopamine receptor blocker, pimozide, was able to reverse the inhibitory effect of lergotrile mesylate (LM) on prolactin release from rat pituitaries in vitro. Alpha-adrenergic or beta-adrenergic receptor blockers were unable to antogonize the action of LM on prolactin release. These findings indicate that ergolines such as LM inhibit prolactin release from pituitaries by activating an adenohypophyseal dopamine receptor. LM is currently undergoing clinical trial as a prolactin inhibitor and a dopamine agonist.


The ontogeny and regulation of corticosteroid secretion by the ovine foetal adrenal.

Adrenal glands of foetal sheep of 40 days gestation to term were incubated with and without ACTH or an increased [K-+]. With ACTH, the 40 day foetal adrenal was capable of producing more cortisol and aldosterone per g body weight than was the term adrenal. ACTH was a potent stimulus to aldosterone and cortisol production in foetuses aged 60-90 days, and this effect declined significantly in the 91-120 day period. An increased [K-+] was stimulatory to aldosterone production only after 120 days gestation. Peripheral blood levels of aldosterone, corticosterone, cortisol, 11-deoxycortisol and 11-deoxycorticosterone were measured in foetuses 60 days to term and the levels of aldosterone and cortisol were significantly lower in 90-120 day foetuses than in the younger or older ones. Direct adrenal vein cannulation proved all five steroids to be secretory products of the foetal adrenal.


The influence of androgens on protein synthesis by cultured rat epididymal tubules.

Rat epididymal tubules maintained in organ culture for 3 days respond to the addition of androgens to the culture media (testosterone and dihydrotestosterone 1 x 10-minus 5 m and 1 x 10-minus 7 m) with an increased incorporation of amino acids into acid-insoluble material. Significant androgenic stimulation is observed only 24 h after addition of hormone, while an inhibitory effect is found at earlier periods. The stimulation seems to be specifically produced by androgens; it is blocked by cyproterone acetate and is not elicited by oestradiol-17beta or corticosterone. The process appears to involve RNA synthesis since actinomycin D suppresses the stimulatory effect of androgen. Evidence suggests that cAMP production is not a primordial step in the response to androgen since dibutyryl cAMP did not mimick the androgenic effect, theophylline did not potentiate the response and alpha,beta-methylene ATP, which competitively inhibits adenyl cyclase, failed to alter the androgenic effect. Radioactive testosterone and dihydrotestosterone added to the culture media showed a preferential intranuclear localization as well as extensive metabolism. DHT was found to be the principal intranuclear steroid.


Growth in monolayer culture of rat pituitary cells which synthetize and release acth.

Cultures of rat pituitary gland cells were developed to study biosynthesis and release of ACTH. ACTH measurement was accomplished by radioimmunoassay. ACTH release was observed following stimulation with theophylline and cAMP in a dose-dependent manner. Biosynthesis was demonstrated by incorporation of 3-H-phenylalanine into the hormone, employing a double antibody technique.


Effect of iodized oil on iodine content, thyroglobulin maturation and on biochemical constituents of endemic goitre in brazil.

The biochemical and morphological changes in thyroid glands were studied in 5 patients with endemic goitre in the region before and one year after the administration of iodized oil. Each patient received a tracer dose of 125-I 3-5 weeks before the biopsy and another tracer of 131-I 12 h before surgery. It was observed that: 1. Iodine deficient goitrous glands had more protein per gram of tissue and that this was corrected by iodination. 2. The goitrous glands exhibited a higher relative proportion of particulate iodoprotein that incorporates more labelled iodine in relation to time of labelling. This abnormal situation is reverted to normal by iodination. 3. A significantly higher proportion of T3 + T4 is synthetized by the glands treated with iodized oil. 4. The increased amount of 127-I per gram of tissue is followed by an increased iodination level of thyroglobulin. 5. The ultracentrifugal pattern of proteins observed for iodine deficient glands differed from the iodinated glands, indicating a failure of maturation of thyroglobulin and possibly a greater than normal degree of heterogeneity. 6. The shift in sedimentation pattern was clearly induced by the increased degree of iodination, as shown by the iodized oil injection. 7. The increased cellularity and the higher rate of protein synthesis in the goitrous glands are reverted to normal after the iodized oil injection.


The excretion of individual adenocartical steroids during normal childhood and adolescence.

The excretion of 7 individual 17-oxosteroids and 7 individual corticosteroids in 24 h urine samples from 62 normal infants, children and adolescents, based on an accurate and specific paper chromatographic method for their separation and quantitation, is reported. The excretion of the 11-deoxy-17-oxosteroids gradually increases from 7 years of age and the increase becomes more rapid 2 or 3 years before the clinical signs of puberty appear. The rise continues throughout puberty and beyond it until the adult level is reached. The increase far exceeds that which would be accounted for by the growth of the individual. The increase in the excretion of the 11-oxy-17-oxosteroids with age is much more gradual. Androgens favour the formation of 5alpha metabolites and the 5alpha:5beta ratio of the total 5alpha 17-oxosteroids and the total 5beta 17-oxosteroids shows a statistically significant increase with age. In addition, a relatively high 5alpha:5beta ratio is noted in male infants, which is likely to be related to their relatively high plasma testosterone levels. The excretion of the 17-hydroxycorticosteroids and the alpha-ketolic metabolites of cortisol gradually rises with age and correlates with body weight. The alpha-ketolic metabolites of corticosterone are relatively high in infancy, but after the age of 4 years their excretion also correlates with body weight. An increase in the 5alpha:5beta ratio of allo-THF to THF is noted at puberty similar to that found with the 5alpha:5beta ratios of the 17-oxosteroids.


Histochemical data on the liver of the marmoset (Callithrix jacchus).

The histochemistry of the hepatic parenchymal cells was studied in four Callithrix jacchus. A large amount of glycogen was noted throughout the lobules while the UDPG-GT and the phosphorylases were found unevenly distributed by the hepatic strands with different degrees of reactivity. Near the central vein one of the livers showed PAS-positive nuclear corpuscles that were more conspicuous in the hepatic cells with a larger amount of cytoplasmic glycogen and weaker UDPG-GT and phosphorylase reactivities. G-6PA (in a larger amount) and LDH (in a moderate amount) were found evenly distributed in the hepatic strands. F-1-6PA was seen sometimes with a stronger reactivity at the peripheral part of the lobules. The enzymes of the pentose shunt (G-6PDH, 6-PGDH and NADPH-2-TR) reacted strongly and as a rule evenly distributed near the hepatic lobules. Occasionally they reacted more intensely in the row of hepatic cells disposed just around the central vein. Cytochrome oxidase showed a very faint reaction. Cis-aconitase and ICDH were weak or moderate. NADH-2-TR more than SDH more than MDH were seen frequently diffused near the hepatic strands. SDH and MDH in some instances showed a stronger reactivity in the row or group of hepatic cells around the central vein. ATPase at pH 6.3 was negative in the marmoset liver; ATPase at pH 7.4 was mainly found in the wall of the portal area vessels; ATPase at pH 8.5 showed a stronger reactivity in the cytoplasm of the hepatic cells and ATPase at pH 9.4 was more abundant in the bile capillaries. The reactivity of the lipid metabolism enzymes was moderate with regard to alpha-GPDH or negligible with regard to beta-OHBDH. Acid phosphatase showed a stronger reaction, but almost limited to the Kupffer cells. The hepatic cells showed only a moderate amount of RNA. Some enzymes of the protein metabolism, such as GDH and leucine aminopeptidase showed a stronger reactivity while some others, such as alanyl aminopeptidase and MAO, were seen diffused near the hepatic lobules in a small amount. Enzymes of the mucopolysaccharide metabolism were not found at all (beta-glucuronidase) or showed only a weak reactivity, such as xylitol dehydrogenase.


Unusual, rod-shaped cytoplasmic inclusions (Hirano bodies) in a cerebellar hemangioblastoma.

The present study reports unusual, long, tubular, rod-shaped cytoplasmic inclusions found in the stromal cells of a cerebellar hemangioblastoma, associated with von Hippel-Lindau syndrome. These inclusions have two layers of circular, laminated filaments, and longitudinally arranged filaments; and appear similar to the so-called "Hirano bodies", which have been previously found in normal and various pathologic conditions of the brain tissue, skeletal muscle cells in myopathy, and interstitial cells of the testis. A review of previous ultrastructural studies of cerebellar hemangioblastoma shows several different types of cytoplasmic inclusions, but none are found to be identical to the present report.


Intra-axonal Corpora amylacea in ventral and lateral horns of the spinal cord.

Deposits similar to corpora amylacea were observed by electron microscopy and light microscopy within thin myelinated axons in the spinal gray matter in various diseases. By electron microscopy the deposits consisted of randomly interlacing short linear structures of various thickness which were different from tangles of neurofilaments. Sometimes they contained dense granules and electron-dense floccules. Though their incidence was relatively high in some cases of degenerative or metabolic disease of the central nervous system, they were not specific to any disease but seemed to be related to aging, indicating a peculiar aspect of chronic degeneration of the axons.


A study on the influence of papovavirus upon encephalitis caused by SSPE agent.

To investigate a possible interaction between the SSPE agent and papovaviruses, histopathological and statistical evaluations were made of encephalitis induced in hamsters by intracerberal inoculation with isolates from SSPE and PML patients, and with BK virus, alone or in combination. With SSPE virus alone, hamster brains showed a reduction of susceptibility to encephalitis with increasing age. A modification of the pathology of SSPE encephalitis was observed after administration of the PML isolate or BK virus: encephalitis was either enhanced or inhibited depending on the time the papovaviruses were administered. A possible mechanism of interaction between SSPE and papovaviruses is briefly discussed.


Studies in cholestasis of pregnancy.

Forty-one pregnant women with pruritus, in whom cholestasis was verified by the presence in their serum of an abnormal lipoprotein, lipoprotein-X (LP-X), were divided into two clinical groups, pruritus gravidarum (PG) (n=20) and hepatosis of pregnancy (HP) (n=21) in relation to serum bilirubin (below and above 1.2 mg/100 ml, respectively) and/or SGOT, SGPT (below and above 50 units/l, respectively). In HP, but not in PG, serum lipids, i.e. cholesterol, phospholipids, triglycerides, pre-beta-lipoproteins (very-low-density lipoproteins), and low-density lipoproteins were increased and high-density lipoproteins decreased when compared with suitable controls. Serum lipids were elevated in proportion to the derangement in the liver function tests, alkaline phosphatase, SGOT, and SGPT. The occurrence of LP-X was inversely related to HDL cholesterol, suggesting a causal relationship between HDL lipid metabolism and the presence of LP-X. Serum TIBC, Simplastin A, and serum iron were elevated in HP in relation to the degree of deterioration of liver function tests. Some of these changes in serum in cholestatic pregnancy may partially (serum triglycerides and pre-beta-lipoproteins) or completely (TIBC and Simplastin A) be explained by an enhanced estrogen influence in promoting increased liver lipid/protein metabolism.


Immunohistochemical demonstration of chorionic gonadotrophin in trophoblastic tumors.

Chorionic gonadotrophin has immunohistochemically been localized to trophoblastic tissue. In normal placenta immunoreactivity was found in syncytiotrophoblastic cells. However, in trophoblastic neoplasms, chorioadenoma destruens and choriocarcinoma, the hormone was also found in cytotrophoblastic cells. The tumor cells showing immunoreactivity appeared to have distinct light microscopic features.


Histological changes in the submandibular glands of rats after intraductal injection of chemical carcinogens.

Carcinogens injected into the excretory canal of submandibular gland of Donryu rats revealed the following histologic changes in salivary glands. 20-Methylcholanthrene induced squamous cell metaplasia, fibrosis in the early stages, and "benign lymphoepithelial lesion"-like pattern after 3 months. Dense hyalinization occurred after 4-5 months with so-called "mixed tumor"-like pattern. In the later stages epidermoid carcinoma and fibrosarcoma were observed. 9, 10-Dimethylbenzanthracene caused degenerative change, metaplasia, fibrosis and cell infiltration, and later carcinoma and sarcoma appeared at a high rate. 4-Nitroquinoline-N-oxide led to dense hyalinization and so-called "mixed tumor"-like pattern was observed in many specimens. N-nitroso-N-methyl urethane and N-methyl-N-nitroso-N'-nitroguanidine revealed metaplastic changes, fibrosis and lymphoid infiltration. Scarlet red induced remarkable infiltration and aggregation of lymphoid cells, showing benign "lymphoepithelial lesion"-like pattern.


Mechanism of the formation of megamitochondria induced by copper-chelating agents. II. Isolation and some properties of megamitochondria from the cuprizone-treated mouse liver.

Megamitochondria have been isolated from the liver of the cuprizone-fed mouse with the aid of bovine serum albumin. Phosphorylating capacities of megamitochondria, specified above, in terms of respiratory control ratios and ADP/O ratios have revealed that they are not uncoupled completely. Biochemical properties of megamitochondria which are related to the metabolism of copper have shown that copper-chelating action of cuprizone may not be directly related to the formation of megamitochondria in vivo. Namely, cytochrome contents, activities of cytochrome oxidase and monoamine oxidase and contents of copper of megamitochondria were unchanged compared with those of the control. However, contents of divalent metals such as Ca-++ and Mg-++, especially that of the former, in megamitochondria decreased significantly. It is suggested that cuprizone may alter Mg-++/Ca-++ ratios when it is administered in vivo, and that changes in the ratio might play a key role in the formation of megamitochondria.


Meniére's disease. Electron microscopy of the vestibular ganglion and end organs after ultrasound.

Material for electron microscopy study was obtained from a patient previously suffering from Meniere's disease and treated by ultrasound. It consisted of parts of the membranous labyrinth as well as the vestibular ganglion. The neuro-epithelium of the macula showed degenerative changes and there was marked vacuolation of the cytoplasm. The endothelial cells lining the semicircular canal showed gross vacuoles contained degenerating mitochondria and some debris which may reach the endolymph. The wall of the semi circular canal was oedematous and the basement membrane lifted off by oedematous fluid. There was obvious change in nerves and neurones. The nerves showed loosening of the myelin sheath and ballooning. The neurones contained lipoid inclusions and multicystic lysosomes. Similar inclusions were also noted in some myelinated nerves.


Immunofluorescent demonstration of plasma protein entry into arterial wall by cholesterol, epinephrine, norepinephrine and angiotensin II.

Thepresence of IgG and beta-lipoprotein in thoracic and abdominal aortas of 33 rhesus monkeys under various conditions was observed by immunofluorescence and interference filter technique. In untreated and placebo control monkeys, a definite fluorescence due to IgG was seen in highly limited areas of the subendothelial space and the innermost layers and a weak but positive fluorescence due to beta-lipoprotein was sometimes show in highly limited spots of the subendothelial space, but not the medial layers. In challenged animals with cholesterol (1 g/kg p.o.) or epinephrine (1 mug/kg i.v.) or norepinephrine (10 mug/kg i.v.) or angiotensin II (1 mug/kg i.v.) or cholesterol (1 g/kg p.o.) plus epinephrine (1 mug/kg i.v.), definite fluorescence showing the entry of beta-lipoprotein and IgG from the vessel lumen into the subendothelial space and then into the medial layers of the arterial wall was clearly shown by serial sacrificing of animals after each challenge. The passage of beta-lipoprotein through the internal elastic lamina into the medial layer, was characteristically delayed as compared with that of IgG. In pretreated animals with pyridinolcarbamate (PDC) (10 mg/kg p.o.), EG467 (1 mg/kg p.o.) and Premarin (5 mg/kg i.v.), the acute entry of those plasma proteins was significantly inhibited.


[Endocrinologic evidence for differential diagnosis and follow-up of expanding intraseller processes].

In expectance of early chromophobic adenomata the transsphenoidal intervention revealed in 5 cases cerebrospinal fluid only, 3 times in closed cysts, 2 times with connection to the endocranial space. In one case the neuro-surgical approach revealed basal arachnoidal cysts. Endocrine studies detected no significant differences between empty sella patients and chromophobic adenomata. Compared with this in suspected acromegalia, the measurement of the different functions of the pituitary gland and especially the repeated control of somatropin proved most effective for early diagnosis, control of the operative effect and long-term success or failure.


Phagocytosis of lymphoblastoid cells and cell destruction of human malignant tumor cells.

On the basis of the previous study, on the cell interaction between malignant tumor cells and other cells, especially with lymphocytes, the present study was carried out by investigating cell to cell interaction of human malignant tumor cells and human lymphoblastoid cells such as T-cell (MOLT-4 cell) and B-cell (Burkitt lymphoma cell). As a result it has been revealed that live lymphoblastoid cells were not adhered on the cell surface of the tumor cells, nor is it ingested by tumor cells, but in thepresence of HVJ (Sendai virus: 2,000 H.A. units) it adheres slightly on the cell surface of tumor cell but no cell fusion of tumor cells and lymphoblastoid cells is observable. On the other hand, the tumor cell as well as T-cell and B-cell all have receptors to concanavalin A (Con. A) on their cell surfaces, and they show a marked cell binding such as tumor cell and T-cell, tumor cell and B-cell, and there can be observed a marked phagocytosis of lymphoblastoid cells by tumor cells. Moreover, the tumor cells that have phagocytized lymphoblastoid cells undergo a marked cell destruction within 4 hours of cell-binding and phagocytosis, which is especially prominent in the case of phagocytosis of E.B cell by tumor cell.


Dynamics of alpha-adrenergic inhibition of the adenyl cyclase-cyclic amp system in human adipose tissue.

In short-term incubations of fat cells isolated from human adipose tissue, noradrenaline (0.2-2x10--5 M), a mixed alpha and beta-adrenergic agonist, stimulated only slightly cyclic AMP accumulation. In the presence of the alpha-adrenergic blocking agent phentolamine (0.05-5mug/ml), noradrenaline markedly augmented the amount of labelled cyclic nucleotide. The stiumlatory effect of noradrenaline and noradrenaline plus phentolamine was abolished by propranolol (0.1-10 mug/ml). Theophylline (10--4-10--3 M) slightly increased the (3-H) cyclic AMP levels per se and magnified the stiumulatory action of other agents, but did not mimic the potentiating effect of phentolamine on noradrenaline action.


Subacute and acute endocarditis due to Pseudomonas cepacia in heroin addicts.

Five heroin addicts were treated for endocarditis caused by Pseudomonas cepacia. Two of these infections occurred in patients with no known heart disease whereas the others occurred at sites of previous endocarditis or valve prostheses. Infection was indolent in four patients but was associated with shock and skin lesions suggestive of ecthyma gangrenosum in the fifth. After failure of chloramphenicol and kanamycin, all patients were treated with a combination of sulfamethoxazole, trimethoprim and polymyxin plus heart valve resection or replacement.


Epibulbar tumors in childhood.

Three hundred two cases of epibulbar tumors in children were retrospectively reviewed histologically and examined by diagnostic categories over a 49-year period. Of the 302 cases, 33% were choristomas; 29% nevi; 11%, epithelial inclusion cysts; 7%, papillomas; 6%, pyogenic granulomas; and 5% granulomas. Three cases of sqamous-cell carcinoma and one of rhabdomyosarcoma occurred as well as a variety of benign lesions.


Proflavine photodynamic viral inactivation in herpes simplex keratitis.

We used the photodynamic inactivation technique with proflavine as the photoactive dye to treat herpetic epithelial keratitis in a preliminary study of patients who had idoxuridine toxicity or resistance. A comparative study with idoxuridine in treating dendritic ulcerations of the cornea showed a good therapeutic effect. But the investigation was suspended when adverse reactions, consisting of a generalized epithelial keratitis and an anterior uveitis, possibly of phototoxic origin, developed in a few patients receiving treatment. The ulcers treated by photodynamic inactivation apparently healed by a process of "debridement" followed by subsequent re-epithelialization.


Immunologic protection of rabbit corneal allografts with heterologous blocking antibody.

We performed exchange penetrating keratoplasties in New Zealand White rabbits by using corneas soaked in heterologous "blocking" antibody (guinea pig antirabbit lymphocyte globulin) under various experimental situations and immunogenic stimuli. Results suggested that the antibody can protect the corneal graft from a weak rejection response possibility by blocking the afferent are of the immunologic rejection reflex, but cannot protect a graft subject to a strong immunologic rejection episode. Based on these results, the use of a blocking antibody in human transplantation would be of potential benefit and offer little risk to a graft recipient.


Experimentally induced malakoplakia.

Malakoplakia was induced experimentally by introducing large amounts of crude endotoxin-antigen comples of 075 Escherichia coli (E. coli 12797 CDC 0 group 75) into the kidneys and testes of rats. First leukocytes, then granulation tissue composed mainly of characteristic macrophages, the so-called Hansemann cells, appeared around the endotoxin-antigen mass. On the eighth day following the injection, deposition of calcium phosphate into the cytosegresomes of macrophages began and acused the formation of the Michaelis-Gutmann bodies necessary for the diagnosis of malakoplakia. The induction of the same process in humans by E coli endotoxin seems to be possible.


Lymphoid cell necrosis, thymic atrophy, and growth retardation in newborn mice inoculated with murine cytomegalovirus.

During studies on the effect of murine cytomegalovirus on the developing retina, virus was inoculated into the eyes of newborn Swiss mice, and the animals were sacrificed at various times thereafter. Controls consisted of mice inoculated with ultraviolet-inactivated murine cytomegalovirus and uninjected mice. Marked lymphoid cell necrosis, thymic atrophy, pronounced growth retardation, bacteremia, and death occurred in the animals inoculated with live virus. this virus-induced injury resulted in a marked depletion of lymphocytes in the subcapsular and cortical areas of the thymus as well as in the spleen, lymph nodes, and Peyer's patches. Areas of necrosis with viral inclusions were present at the site of inoculation and in various other organs including the spleen and bone marrow. Since growth retardation has been associated with thymic atrophy due to other causes, the observed abnormal physical development in the present study was interpreted as a sequel to the thymic injury. An implication of this study is that some human infants with concomitant immune deficiency and viral infection may have a primary viral disease with resultant secondary lymphoid tissue alterations, rather than a thymic disorder with a subsequent viral infection.


Ribosome-lamella complexes in neoplastic hematopoietic cells.

The ultrastructural cellular inclusions referred to as ribosome-lamella complexes were observed in the neoplastic cell population of 4 patients with three types of hematopoietic malignancy, monoblastic leukemia. Waldenstrom's macroglobulinemia, and chronic lymphatic leukemia. The ultrastructural characteristics of the inclusions were similar in the 4 cases. The percentage of cells affected ranged from approximately 90% in 1 patient with monoblastic leukemia to approximately 10% in a patient with Waldenstrom's macroglobulinemia. The complexes appeared to originate from the rough endoplasmic reticulum. Observations suggested a developmental sequence beginning with aggregate strands of rough endoplasmic reticulum, subsequent alignment of the strands of rough endoplasmic reticulum in a concentric configuration, followed by maturation to fully developed ribosome-lamella complexes. Although the ribosome-lamella complex has been found in the neoplastic cells of several patients with leukemic reticuloendotheliosis ("hairy cell leukemia"), its occurrence in these three different hematopoietic disorders indicates a lack of diagnostic specificity of this structure.


Idiopathic acquired cold urticaria in North India.

Forty-three otherwise healthy patients mostly between the ages of 10 and 40 were seen in North India during a two-year period with urticaria which developed on taking a cold water bath, exposure to cold winds, evaporation of rain water or sweat in that order of frequency. There was no familial predisposition to cold urticaria or atopic disorders. The refrigeration test for cryo-proteins was negative. Only one patient developed a wheal on contact with ice but 38 of 43 patients showed an exaggerated erythema response to a cold-pressure test (cryo-stimulation test).


Ultrastructural characteristics of canine transmissible venereal tumor at various stages of growth and regression.

Ultrastructural study of canine transmissible tumors in developing, mature, and regressing stages from 6 dogs revealed the presence of healthy and degenerating tumor cells in all neoplasms. The total number of neoplastic cells seemed to decrease, and the number of degenerating neoplastic cells seemed to increase in mature tumors. Macrophages, lymphocytes, and plasma cells infiltrated mature and regressing tumors. Alteratons in degenerating tumor cells consisted mainly of cytoplasmic changes in early stages and of both nuclear and cytoplasmic changes in cells in which degeneration was more advanced. Amounts of endoplasmic reticulum and ribosomes were decreased. There were swelling and vacuolation of mitochondria. Nuclear chromatin was clumped along the nuclear envelope, and the perinuclear space was widened. Degenerating cells often contained membrane-bound granules and clusters. Lamellar complexes were observed in tumor cells from 2 dogs. Virus particles were not seen.


A case of alveolar cell carcinoma accompanied by fluid and electrolyte depletion through production of voluminous amounts of lung liquids.

A patient is described who showed typical features of alveolar cell carcinoma, including production of a huge amount of clear frothy lung liquid (as much as 4 liter per day), diffuse dissemination of nodular lesions throughout the lung, and tall columnar cell proliferation outlining the alveolar walls uniformly without architectural destruction in the terminal lung unit. From the results of comparative serial analyses of biochemical components in the liquid and the serum, it is suggested that the excessive liquid production in this case should be due to abnormal secretory functioning of diffusely proliferated carcinoma cells.


Alpha1-antitrypsin deficiency and hepatic carcinoma.

A patient with homozygous ZZ alpha1-antitrypsin deficiency and severe pulmonary emphysema was discovered to have a mixed hepatocellular and cholangiolar hepatic carcinoma arising in a noncirrhotic liver. Because of the increasing frequency of hepatomas in patients with alpha1-antitrypsin deficiency, it is suggested that a causal relationship may exist.


Savings from generic prescriptions. A study of 33 pharmacies in Rochester, New York.

Brand name and gereric prescriptions for 12 drugs were surveyed at 33 pharmacies in the Rochester, New York, area to determine how frequently generic prescriptions were filled with a product other than the major brand, and at a savings to the consumer. Generic prescriptions for ampicillin, erythromycin, propoxyphene, and dioctyl sodium sulfosuccinate were often filled at prices lower than comparable brand name prescriptions. Occasional, but often substantial, savings were obtained for papaverine, pentaerythritol tetranitrate, and conjugated estrogens. No savings were obtained for penicillin V, chlorpheniramine, diphenylhydantoin, sulfisoxazole, or methenamine mandelate. The pharmacists included in the survey identified correctly a mean of 18.5 out of 22 drugs as to whether products other than the major brand were available. A sample of physicians identified correctly a mean of 14.1 drugs. Pharmacists who were better informed as to which drugs were available generically were more likely to stock alternative products and more likely to charge lower prices on generic prescriptions than on brand name prescriptions.


Ineffectiveness of subcutaneous cytosine arabinoside in localized herpes zoster.

Cytosine arabinoside (cytarabine) was evaluated in a randomized double-blind controlled study for the treatment of localized herpes zoster. Cytarabine was administered subcutaneously in a dose of 50 mg/m-2 body surface area once daily for 4 days, always within 14 days of onset of the first symptom and usually within 7 days. Thirty patients given cytarabine and 30 patients given placebo were well matched with respect to age, sex, and length and severity of presenting rash and pain as well as underlying disease. There was no difference in the rate of disappearance of pain or rash in either treatment group. More patients given cytarabine than patients given placebo had minimal pain and significantly more cytarabine-treated patients had persistence of neurological symptoms at 6 months' follow-up. Acute side effects, though mild, were significantly increased in the cytarabine-treated patients especially with respect to nausea and vomiting and decrease in platelet count. Cytarabine administered in this dose subcutaneously had no beneficial effect and was associated with mild side effects and persistence of neurological symptoms.


Recent advances in calcium and phosphorus metabolism.

This review considers the most recent developments concerning the metabolism and homeostasis of calcium and phosphorus. The kinetics of the distribution of calcium, theories of calculus formation, hypercalcemia and hypocalcemia are discussed, as well as the role of parathyroid hormone, thyrocalcitonin and 1,25 dihydroxy Vitamin D(3) in maintaining calcium levels and skeletal integrity. In addition, the role of calcium in enzyme activation and inhibition, muscle and nerve function, and intracellular metabolism are considered.


Metabolic and biochemical considerations of bone.

Recognition of the dynamic aspects of bone metabolism can lead to a unified concept involving endocrine and nutritional influences. Although most hormones can influence bone metabolism directly or indirectly, the principal ones involved in skeletal metabolism are parathyroid hormone, calcitonin and 1,25-dihydroxy-vitamin D. The actions of parathyroid hormone and 1,25-dihydroxy-vitamin D result in elevations of circulating extracellular fluid calcium concentration through actions directly on bone, intestine, and kidney. Calcitonin leads to decreases in calcium concentration, primarily by action on bone and kidney. The absorption and retention of calcium by the organism is further influenced by the dietary content of calcium, phosphorus, protein, and fluoride. Chronic dietary deficiencies of calcium and excesses of phosphorus may lead to chronic nutritional secondary hyperparathyroidism with resulting skeletal demineralization. In both experimental animals and in man, the earliest manifestation of this condition may be demineralization of the jaw with resultant paradentosis. Experimental studies in animals and in man have shown that this form of demineralization may be completely reversed by increasing dietary calcium and decreasing dietary phosphrous.


[Determination of penicillinase and acylase by chromatography on a thin layer of sorbent in the case of their joint formation by different strains].

The products of penicillinase and acylase hydrolysis of benzylpenicillin were studied with a method of sorbent thin-layer chromatography. The method provided qualitative determination and differentiation of penicillinase and acylase activity in cultures of E. coli capable of simultaneous production of both enzymes. It was shown that when the cultures of E. coli were grown under conditions optimal for acylase production, the amounts of penicillinase were insignificant.


Varicella-zoster virus and subcutaneous cytarabine: correlation of in vitro sensitivities to blood levels.

The susceptibility of 26 strains of varicella-zoster virus to cytarabine was tested in vitro by measuring reduction in number and size of plaques using human foreskin fibroblast cells. Most strains showed 50% reduction of plaque number by 0.125 mug of cytarabine per ml or less. Cytarabine levels in sera of 12 patients after a single subcutaneous injection of 50 mg/M(2) exceeded this inhibitory level for a period of 75 min. Thus, the subcutaneous route is a potentially useful method of administration of cytarabine for antiviral drug trials.


Phosphonoacetic acid-resistant herpes simplex virus infection in hairless mice.

Phosphonoacetic acid (PAA)-resistant type 1 herpes simplex virus population was isolated by repeated passage of the virus in the presence of this inhibitor. Hairless mice infected percutaneously with the inhibitor-resistant or the parental inhibitor-susceptible virus were treated intraperitoneally with PAA and 9-beta-d-arabinofuranosyl-adenine by using several different dosage schedules. Whereas 9-beta-d-arabinofuranosyl-adenine was effective both in the PAA-susceptible and PAA-resistant herpes simplex virus-induced skin infection, PAA suppressed only the infection induced by the parental PAA-susceptible virus.


Desferrioxamine B, an inhibitor of Escherichia coli motility, reversing the stimulating effect of cyclic adenosine 3',5'-monophosphate.

A substance inhibiting Escherichia coli motility was isolated by "motilometry" assay from the culture broth of Streptomyces sp. strain NR9GG8 and was found to be identical with desferrioxamine B. Its inhibitory effect was reversed by cyclic adenosine 3',5'-monophosphate (cAMP), while the motility stimulation by cAMP was diminished by this inhibitor. Its effects on various enzymes involved in cAMP metabolism of function of cAMP were examined.


Lead. Possible toxicity in urban vs rural rats.

The degree of lead poisoning in wild rats from two environments has been studied. Wild rats captured in an urban area had markedly elevated tissue lead compared with values in rural rats. This elevation may have been caused by differences in factors affecting absorption of ingested lead or an elevated respiratory exposure to airborne lead, or both, and lead in precipitated dust. Changes in several biologic indexes (depression of delta-amino levulinic acid dehydratase in kidney and red blood cells, presence of renal intranuclear inclusion bodies, and increased kidney weight) confirmed lead-poisoning in urban rats.


Radioisotopic detection of osseius metastases. Evaluation of 99mTc polyphosphate and 99mTc pyrophosphate.

A total of 146 patients were investigated for the presence of osseous metastases with 99mTc polyphosphate or 99mTc pyrophosphate bone scans. Results of bone imaging were retrospectively compared to roentgenographic results surveying similar anatomic areas in 128 patients. This comparison revealed that roentgenographic interpretations were in error in 19% of the cases. Thirty-three patients had bone scans and roentgenograms that were in agreement and considered abnormal, but in more than one third of these cases the patients had multiple abnormalities that were shown by the bone scan but were not recognized roentgenographically. In consideration of the low toxicity, ready availability, economy, shortened procedure time, and low radiation dose associated with the use of these new bone-seeking agents, it is concluded that they are superior to roentgenograms and previously utilized radionuclides for early detection of osseous metastases.


The effect of acetylethyleneimine upon a strain of inactivated foot-and-mouth disease virus stored at 4 degrees C.

The presence of either thiosulphate-neutralized or free AEI was shown to degrade inactivated foot-and-mouth disease virus Type O (Hong Kong) antigen during storage at 4 degrees C. Deterioration was evident after 20 weeks of storage and little antigen remained at 36 weeks. Optimum stability was obtained by removing the residual inactivant immediately after inactivation.


Poliovirus-induced infectious double-stranded RNA: Effect of RNA-degrading enzymes.

The infectivity of replicative form RNA (RF-RNA) isolated from poliovirus-infected HeLa cells is completely resistant to the action of T-1 RNase but decreases after exposure to RNase A in the presence of 0.3 M NaCl. Under these conditions neither enzyme produces single-stranded nicks in RF-RNA. Three endonuclease-free exonuleases (RNase II, polynucleotide phosphorylase and spleen phosphodiesterase) rapidly destroy the infectivity of single-stranded RNA, but do not alter the infectivity of RF-RNA. It is concluded that RF-RNA does not contain single-stranded ends essential for infectivity. Indirect evidence suggests that all or most of the poly A region at the 3' end of the plus strand of infectious RF-RNA is base-paired to a poly U region at the 5 end of the minus strand.


Seizure activity and anticonvulsant drug concentration.

Current methods permit frequent, accurate serum anticonvulsant drug concentration measurements and continuous, 24-hour electroencephalographic recording with minimal environmental restriction. These techniques were used to perform longitudinal, 24-hour recordings of electroencephalographic paroxysmal activity and sleep-wake state concurrently with frequent measurements of serum anticonvulsant drug concentrations in two patients with poorly controlled convulsions. Drug administration was designed with the intent of producing high serum concentrations at times of maximum electroencephalographic paroxysmal activity. The suppression of clinical seizures coincided with decreased numbers of paroxysmal bursts in the electroencephalogram and increased serum anticonvulsant drug concentration.


Nonviral intranuclear filamentous inclusions.

Intranuclear filamentous structures have been found in cells of both central and peripheral nervous tissues in eight patients with a variety of neurologic diseases. The structures were curly, fibrillary strands, resembling paramyxoviral filaments morphologically, but larger in diameter (25 to 40nm). Their appearance was granular with indistinct, hazy outlines, and on cross section they were solid rather than tubular. These structures are probably not viral but chromatin fibers. The illustration of sequential development of the fibrillary structures from usual chromatin clumps supports this view. Thus, the finding of intranuclear fibrillary material in the cells of a number of patients with neurologic diseases must be interpreted with caution.


Moebius syndrome in Kallmann syndrome.

A girl born with congenital paresis of cranial nerves III, IV, and VII (Moeblus syndrome) subsequently developed a progressive peripheral neuropathy. There was suggestive evidence of a familial neuropathy with autosomal dominant inheritance in three family members. The patient also had hypogonadotrophic hypogonadism and anosmia (Kallmann syndrome).


Carcinoma metastatic to the eye and orbit II. A clinicopathological study of 26 patients with carcinoma metastatic to the anterior segment of the eye.

In 26 of 227 patients with carcinoma metastatic to the eye or orbit, metastasis to the anterior uveal tract was the predominating feature. There was a definite propensity for the tumor to involve the horizontal meridian of the iris or ciliary body, rather than the upper or lower portions. The site of the primary tumor in the 26 patients was as follows: lung 14; breast, 9; kidney, 2; and rectum, 1. Ocular symptoms and signs produced by the metastatic tumors at onset or during the course of the disease included decreased vision (80%), a visible mass (72%), redness of the eye (56%), pain (56%), glaucoma (56%), iridocylitis (44%), and hyphema (24%). The median survival of the 26 patients with metastasis to the anterior segment of the eye was only 5.4 months from the time of ocular surgery. This is poorer than the median survival (7.2 months) of the patients with metastasis confined to the posterior segment, and much worse than the median survival (15.6 months) of the 28 patients with orbital involvement.


Immunity to chlamydial infections of the eye. IV. Immunity in owl monkeys to reinfection with trachoma.

In a study of trachoma in owl monkeys, it was found that owl monkeys are equally susceptible to low and high doses of trachoma and that resistance to reinfection persisted for six months in the majority of animals. Previous infections with a single trachoma type did not elicit greater resistance than previous infections with two types. Both serum antibodies and eye secretion titers correlated well with resistance to reinfection, but it is not as yet clear if either or both play a substantial role in immunity to trachoma.


Use of refractometry to determine nitrogen accumulation in close d circuits.

We have described a technique by which a portable interference refractometer may be used to determine the accumulation of nitrogen in a totally closed anaesthetic circuit. The inspired mixture contained halothane, oxygen, nitrous oxide and nitrogen. The concentrations of the first two were controlled with u.v. paramagnetic analysers respectively. It was then possible to determine the percentage of nitrous oxide replaced by nitrogen by measuring the refractive power of the mixture.


Totally closed circuit nitrous oxide/oxygen anaesthesia.

Totally closed circuit anaesthesia has been used with nitrous oxide/oxygen gas mixtures, supplemented by halothane for spontaneous breathing, or by opiates for artificial ventilation. Oxygen expenditure averaged 227 ml/min, while nitrous oxide expenditure declined exponentially from an initial value of 462 ml/min to 110 ml/min after 2 hr. These flow rated included gas sampled for analysis. Inspired oxygen concentration was maintained at 30 per cent by monitoring with a paramagnetic analyser. Halothane expenditure average 3.5 ml (liquid) per hr. Halothane concentrations in the operating theatre atmosphere did not increase above 0.03 p.p.m.


DNA polymerase activity of cultured rheumatoid synovial cells.

Cultured synovial cells from 8 patients with rheumatoid arthritis (RA) and 7 subjects without joint disease were assayed and comapred for RNA-directed and DNA-directed DNA polymerase activity. No activity was found in either RA or control specimens using a synthetic RNA template that specificically detects oncornavirus RNA-directed DNA polymerase. The DNA-directed DNA polymerase activity of RA specimens was increased (P less than 0.10) in the high-speed pellet fraction of cell lysates. The possible relationship of these finding to virus infection in RA is disscussed.


[Cystic degeneration of the adrenal cortex associated with hyaline membrane].

Forty-one autopsies in newborns with hyaline membrane were studied. In fourteen cases (34.1%) cystic degeneration of the outermost portion of the adrenal cortex was found. Our results differ from other studies as the lesions were observed in newborns at various gestational ages, with a higher incidence in the group of pre-term babies, small for their gestational age. The constant association of hyaline membrane and adrenal cortex cystic degeneration seem to suggest that both processes may share a common etiopathogenic mechanism, namely hypoxia followed by increased ACTH secretion.


Massive subchorial thrombohaematoma (Breus' mole).

Ten placentae with massive subchorionic thrombohaematomas are reported. This lesion, which is composed of maternal clotted blood, separates the chorionic plate of the placenta from the villous chorion and is traversed by the main cotyledonary branches of the fetal circulation. In some instances it dissects into the layers of the chorionic plate. In anatomical terms it is identical to the lesion which has been described in early pregnancy under the title of Breus' mole. Previous reports suggested that the lesion was confined to early pregnancies and was a consequence of fetal death. In our series all ten placentae were from pregnancies of at least 25 weeks' maturity and six were more than 30 weeks. Seven infants were liveborn and three survived the neonatal period. Differential diagnosis is discussed along with aetiology, which is still obscure although earlier theories implicating fetal death may be discounted.


Lipoprotein lipase. Mechanism of formation of triglyceride-rich remnant particles from very low density lipoproteins and chylomicrons.

The catalytic rate of membrane-supported lipoprotein lipase has been determined for chylomicron and very low density lipoprotein substrates during the formation of triglyceride-depleted ("remnant") particles. Both lipoprotein species and their generated remnant products were competitive substrates for lipase activity. Remnant formation from each species was associated with decreasing kc but an unchanged apparent Km. This finding was confirmed from the rate of plot of total triglyceride catabolism by lipase at low substrate concentrations. When compared with the major very low density lipoprotein fraction (Sf 100-400), a fraction isolated from plasma with a lower flotation rate (Sf 40-100) had a lipid composition and decreased kc compatible with this representing a physiological remnant particle.


Ultraviolet photosensitivity of the estrogen binding protein from rat uterus. Wavelength and ligand dependence. Photocovalent attachment of estrogens to proteins.

Ultraviolet irradiation of the estrogen binding protein in rat uterine cytosol results in a progressive photoinactivation which is rapid at 254 nm and slower at greater than 315 nm. Both unfilled and estradiol-filled sites are inactivated at approimately the same rates at 254 nm (t 1/2 equals 8 min and 11 min, respectively), but at 315 nm, empty sites are consumed much more rapidly (t 1/2 equals 3.4 hr) than filled ones (t 1/2 equals 24 hr). The protective effect of the estrogen ligand at this wavelength appears to depend on its binding to the estrogen-specific binding site, as inactivation rate studies at different concentrations of estrone, estradiol, and estriol show a good correlation between the extent of protection and the fractional saturation of the high affinity estrogen binding sites. Scatchard analysis indicates that inactivation is the result of a loss of binding sites and not a decrease in their affinity, and sedimentation analysis shows that increased heterogeneity and aggregation of the estrogen binding species accompanies the photoinactivation process. Photoinactivation appears to be the result of direct irradiative damage of the animo acid residues, as the inactivation rate is the same under air and nitrogen atmospheres, and is unaffected by nucleophiles, reductants, and radical scavengers. When photoinactivation is measureed by irradiation of cytosol containing [3-H]estradiol, a concurrent photocovalent attachment process is noted; the steroid becomes linked to protein in a solvent-extractable manner (boiling ethanol inextractable). This attachment, however, does not appear to be related to the steroid binding at the estrogen binding site. Its rate is affected by reductants and scavengers. A similar photocovalent attachment reaction occurs when bovine serum albumin or ovalbumin is irradiated in the presence of [3-H]estradiol or [3-H]diethylstilbestrol. The detailed reactions involved in this photocovalent attachment process have not been defined at present.


Effects of cyclic adenosine monophosphate, dexamethasone and insulin on phosphoenolpyruvate carboxykinase synthesis in Reuber H-35 hepatoma cells.

Antiserum prepared against rat liver cytosolic phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) is shown to specifically precipitate the enzyme from Reuber H-35 cells. Synthesis of phosphoenolpyruvate carboxykinase, as measured immunochemically, is increased by dibutyryl cAMP and dexamethasone, the nucleotide maximally producing a sixfold and the glucocorticoid a threefold change in rate. Studies with actinomycin D, cordycepin, and cycloheximide suggest dibutyryl cAMP acts at a translational or post-transcriptional site. Insulin prevents the increase in synthesis of phosphoenolpyruvate carboxykinase produced by either dibutyryl cAMP or dexamethasone. This antagonism is concentration dependent and does not require the simultaneous presence of glucose, pointing to a direct effect of the hormone on liver enzyme induction. It is suggested that hepatic phosphoenolpyruvate carboxykinase activity is regulated predominantly by the antagonistic interaction of cAMP (glucagon) and insulin on enzyme synthesis.


Binding of hormones to receptors. An alternative explanation of nonlinear Scatchard plots.

Binding of 1251-labeled hormones to receptors can usually be inhibited by addition of unlabeled hormone. In analyzing such binding-inhibition data, it is commonly assumed that both labeled and unlabeled hormones are bound with equal affinity. When this assumption is made incorrectly, an artifactually nonlinear Scatchard plot results. Equations to describe these nonlinear Scatchard plots are derived. These results are discussed with regard to previously published observations of nonlinear Scatchard plots for binding of insulin to its receptor.


Mechanism of cytochrome c peroxidase. O-benzoylhydroxylamine as an analog of hydrogen peroxide.

A number of reagents, some of which are electronic analogs of hydrogen peroxide, will replace it in the reactions of cytochrome c peroxidase. These compounds include N-bromosuccinimide, sodium hypochlorite, and the novel oxidizing agent O-benzoylhydroxylamine. If fragments of the oxidant played a functional role in the structure of the oxidized form of the enzyme, it would be expected that the product formed from O-benzoylhydroxylamine would differ from that formed from hydrogen peroxide. The products formed on reaction of the two oxidizing agents with cytochrome c peroxidase are indistinguishable. This results carries implications for the structure of the so-called ES compound. The extension in the range of specific substrates for cytochrome c peroxidase allows identification of the structure which compounds must possess to be oxidizing substrates for the enzyme. A mechanism for the first step of the reaction is suggested. O-Benzoylhydroxylamine is also a reducing agent, and its reaction with the enzyme is analogous to that of hydrogen peroxide with catalase. The final product of the reaction is the inert nitric oxide complex of ferrous cytochrome c peroxidase.


Mercurated polynucleotides: new probes for hybridization and selective polymer fractionation.

Polynucleotides containing covalently bound mercury atoms have been prepared by chemical or enzymatic syntheses and some of their physical and biochemical properties studied. The mercury substituents do not appear to alter significantly normal polynucleotide structure. Mercurated polymers function efficiently as templates for nucleic acid polymerases, they are fully susceptible to degradation by standard nucleases, and their denaturation and reannealing properties resemble those of the corresponding nonmercurated polymers. While the Tm's of DNA duplexes are lowered by extensive mercuration, the Tm's of DNA-RNA hybrids and RNA duplexes are either unaffected or elevated. Mercuration, as would be expected, greatly increases the buoyant density of both DNA and RNA. The introduction of as few as one mercury atom per 200 bases permits the selective and quantitative retention of the mercurated polymer probe (and associated nucleotide sequences) on columns of sulfhydryl-agarose. The use of mercurated nucleotides (as polymerase substrates) and oligonucleotides (as primers) in conjunction with sulfhydryl-agarose chromatography provides a simple and efficient method for the isolation of selected polynucleotide sequences, such as specific in vitro transcription products or terminal fragments of duplex DNA. Products absorbed to the affinity resin are readily recovered for further analysis by eluting with buffers containing mercaptoethanol. Although the mercury-carbon bond is somewhat thermolabile, mercurated polynucleotides are suitable as probes in low temperature hybridization studies.


Enzymes regulating glycogen metabolism in swine subcutaneous adipose tissue. I. Phosphorylase and phosphorylase phosphatase.

Glycogen phosphorylase from swine adipose tissue was purified nearly 700-fold using ethanol precipitation, DEAE-cellulose adsorption, AMP-agarose affinity chromatography, and agarose gel filtration. The purified enzyme migrated as one major and several minor components during polyacrylamide gel electrophoresis. Activity was associated with the major component and at least one of the minor components. The molecular weight of the disaggregated, reduced, and alkylated enzyme, estimated by polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate, was 90,000. Stability of the purified enzyme was considerably increased in the presence of AMP. The isoelectric pH of the enzyme in crude homogenates was 6.3. The sedimentation coefficient of the purified enzyme (7.9 S) and that in crude homogenates (7.3 S) was determined by sucrose density gradient sedimentation. Optimal pH for activity was between pH 6.5 and 7.1. Apparent Km values for glycogen and inorganic phosphate were 0.9 mg/ml and 6.6 mM, respectively. The Ka for AMP was 0.21 mM. Enzyme activity was increased by K2SO4, KF, KCl, and MgCl2 and decreased by NaCl, Na2SO4, D-glucose, and ATP. Inhibition by glucose was noncompetitive with the activator AMP; inhibition by ATP was partially competitive with AMP. The purified enzyme was activated by incubation with skeletal muscle phosphorylase kinase. Enzyme in crude homogenates was activated by the addition of MgCl2 and ATP; activation was not blocked by addition of protein kinase inhibitor, suggesting that phosphorylase kinase in homogenates of swine adipose tissue is present largely in an activated form. Deactivation of phosphorylase a by phosphorylase phosphatase was studied using enzyme purified approximately 200-fold from swine adipose tissue by ethanol precipitation, DEAE-cellulose chromatography, and gel filtration. The Km of the adipose tissue phosphatase for skeletal muscle phosphorylase a was 6 muM. The purified swine adipose tissue phosphorylase, labeled with 32-P, was inactivated and dephosphorylated by the adipose tissue phosphatase. Dephosphorylation of both skeletal muscle and adipose tissue substrates was inhibited by AMP and glucose reversed this inhibition. Several lines of evidence suggest that AMP inhibition was due to an action on the substrate rather than on the enzyme. We have previously reported that the system for phosphorylase activation in rat fat cells differs in some important characteristics from that in skeletal muscle. However, both swine fat phosphorylase and phosphorylase phosphatase have major properties very similar to those described for the enzymes from skeletal muscle.


The catalytic mechanism of amino acid:tRNA ligases. Synergism and formation of the ternary enzyme-amino acid-ATP complex.

Formation of binary and ternary enzyme-ligand complexes was investigated for amino acid:tRNA ligases specific for L-isoleucine, L-leucine, and L-phenylalanine. Each of the enzymes exhibited synergistic binding when a substrate was substituted by a structurally related compound. The strength of coupling between the sites binding the amino acid and ATP was strongly dependent on the structure of ligands. The phenomenon was observed with the L-leucine and L-phenylalanine-specific enzymes only in the presence of magnesium. Spermine was inhibitory for L-phenylalanine:tRNA ligase. From the variation which structure of the strength of the observed synergism a correlation scheme was derived considering the ammonium group, the carboxylate group and the side chain of the amino acid, and the adenosine and triphosphate moieties of ATP. The strength of coupling between the subsites binding various combinations of these moieties was evaluated. We found that binding of the subgroups of the amino acid exerts an intramolecular synergism. The strength intramolecular synergism was similar to the strength of the intermolecular synergism observed for the simultaneous binding of an amino alcohol and ATP (or MgATP-2-). We have derived a molecular mechanism for the formation of the ternary enzyme-amino acid-ATP (or MgATP-2-) complex taking  into account the synergistic phenomena. The complex is considered to involve electrostatic repulsion between the amino acid carboxylate and the ATP triphosphate moieties. When one of the negatively charged groups have been eliminated, the enzymatic rearrangement which facilitates the formation of this complex may be seen as a synergistic coupling.


Phosphorylation of the lysine-rich histones throughout the cell cycle.

The phosphorylating of the lysine-rich histone at various stages in the cell cycle has been studied. In rapidly dividing cell populations the lysine-rich histone is phosphorylated rapidly after synthesis and more slowly once bound to the chromosome. The half-life of hydrolysis of such interphase phosphorylation in 5 hr except during mitosis when the phosphata hydrolysis increases almost three-fold. During mitosis there is extensive phosphorylation at sites different from those phosphorylated during interphase and a smaller measure of sites common to both mitotic and interphase cells. The sites of mitotic phosphorylation are most critically distinguished from those phosphorylated in interphase by the rapidly hydrolysis of M-phase phosphohistone when the cells divide and enter the G1 phase of the cell cycle.


A novel pathway for phosphatidylcholine catabolism in rat brain homogenates.

Rat brain homogenates incubated with exogenous [32-P] phosphatidylcholine liberated: LYSO[32-P] phosphatidylcholine, sn-glycero-3-[32-P] phosphorylcholine, [32-P] phosphorylcholine, sn-gleycero-3-[32-P] phosphate and 32-Pi. Further investigation showed that [32-P] phosphorylcholine was released exclusively from sn-glycero-3-[32-P] phosphorylcholien by a novel diesterase activity. We propose that the enzyme be termed L-3-glycerylphosphinicocholine cholinephosphohydrolase (EC 3.1.4.-). Parallel experiments on rat liver homogenates and a P815Y mouse mastocytoma cell-lysate, revealed no diesterase activity.


Characterization of a membrane-bound phospholipid desaturase system of candida lipolytica.

Several characteristics of the microsomal phospholipid desaturase of Candida lipolytica are described. The phospholipid desaturase reaction required molecular oxygen and reduced pyridine nucleotides as essential cofactors and was inhibited by cyanide but not by carbonmonoxide, indicating that it required cytochrome b5. Desaturation of both 1-acyl-2-[14-C]oleoyl-sn-glycero-3-phosphorylcholine and 1,2-di-[14C] oleoyl-sn-glycero-3-phosphorylcholine appeared to follow Michaelis-Menten kinetics, with apparent Km values of 2.5 10-minus 4 M and 9.5 10-minus 4 M, respectively. Desaturation of the di-[14C] oleoylphosphatidylcholine took place at both position-1 and position-2; the distearoyl or dielaidoyl phosphatidylcholines were not desaturated. Rate of desaturation of the 1=acyl-2-[14-C] oleoyl-glycerophosphorylcholine by microsomes from cold-grown cells was equal to or slightly less than that by microsomes from cells grown at the normal growth temperature of 25 degreesC, measured in the temperature range 10-30 degrees C. However, the rate of desaturation of [14-C]-oleoyl-CoA desaturase was greater with the microsomal preparation from cold-grown cells than with that from 25 degreesC grown cells. These data suggest that the observed increase of diunsaturated fatty acids in cold-grown cells may perhaps be explained by the increased activity of the oleoyl-CoA desaturase acting at the low temperature.


Endogenous steroid production in cellular and subcellular fractions of rat testis after prolonged treatment with gonadotropins.

Steroid production and enzyme activities were examined in preparations of whole testis tissue, isolated interstitial tissue and seminiferous tubules obtained from adult rats with intact pituitaries receiving daily subcutaneous injections of 100 I.U. human chorionic gonadotropin for 5 days and from control animals. After human chorionic gonadotropin administration testosterone concentrations were increased in total homogenates of whole testis tissue, interstitial tissue and seminiferous tubules. The testosterone production from endogenous precursors was enhanced only in total homogenates of whole testis tissue and  interstitial tissue obtained from testes of human chorionic gonadotropin-treated rats. The production of testosterone in the corresponding homogenates of isolated seminiferous tubules was very low. The specific activity of 3 beta-hydroxysteroid dehydrogenase was increased in total homogenates of whole testis tissue, isolated interstitial tissue and seminiferous tubules. No effect was observed on the specific activities of marker enzymes such as cytochrome c oxidase, monoamine oxidase, steroid sulfatase and lactate dehydrogenase, whereas the specific activities of carboxyl esterase were decreased in homogenates of whole testis tissue and interstitial tissue. Total activity of monoamine oxidase was increased in homogenates of interstitial tissue of tests from human chorionic gonadotropin treated rats. After the same prolonged human chorionic gonadotropin treatment the concentration of pregnenolone was increased in mitochondrial fractions of whole testis tissue, interstitial tissue and seminiferous tubules, and the amount of protein isolated in the mitochondrial fraction of interstitial tissue increased by 40%. Steroid production (estimated as pregnenolone) from endogenous precusors by mitochondrial fractions of whole testis tissue and interstitial tissue were increased after human chorionic gonadotropin treatment, for whole testis from 580 pmol/mg mitochondrial protein per h to 1420 pmol/mg per h; and for interstitial tissue from 2665 pmol/mg per h to 7050 pmol/mg per h. The production of pregnenolone in mitochondrial fractions obtaine from isolated seminiferous tubules was very low and contributed hardly at all to the total pregnenolone production in mitochondrial fractions of whole testis tissue from normal rats as well as from human chorionic gonadotropin-treated rats.


Structural differences in ferritins from normal and malignant rat tissues.

Ferritins purified from several normal and malignant rat tissues were examined for amino acid composition, content of tryptic peptides, available sulfhydryl groups and subunit sizes and proportion. Ferritin extracted from adult kidney, neonatal liver and hepatic and renal tumors differed from the ferritin of adult rat liver in migration on electrophoretic gels and in antibody affinity, but did not differ among themselves. Nevertheless, they showed distinctive differences in amino acid composition and tryptic peptide content. All of them and also adult liver ferritin contained two major species of subunits differing in molecular weight. The proportions of subunits, and the available sulfhydryl groups of the intact ferritin molecules, differed among these tissue ferritins. On the basis of amino acid and peptide content, the ferritins of hepatomas and the renal tumor analyzed showec the greatest similarity but not identity. The ferritin of neonatal liver was next most similar. Kidney ferritin differed considerably in composition from tumor and neonatal ferritins, while adult liver ferritin was the most extremely divergent of the series examined. A similar progressive difference was found on examining the proportions of subunits and sulfhydryl groups in these ferritins. However, changes in subunit proportion cannot explain the amino acid and peptide compositional changes.


Radioimmunoassay of human high density lipoprotein apo-protein A-1.

A double antibody radioimmunoassay technique was developed for the measurement of apolipoprotein A-I, the major apoprotein of human high density lipoproteins. Apolipoprotein A-I was prepared from human delipidated high density lipoprotein (d equal to 1.085-1.210) by gel filtration and ion-exchange chromatography. Purified apolipoprotein A-I antibodies were obtained by means of apolipoprotein A-I immunoadsorbent. Apolipoprotein A-I was radiolabeled with 125-I by the iodine monochloride technique. 65-80% of 125 I-labeled apolipoprotein A-I could be bound by the different apolipoprotein A-I antibodies, and more than 95% of the 125-I-labeled apolipoprotein A-I was displaced by unlabeled apolipoprotein A-I. The immunoassay was found to be sensitive for the detection of about 10 ng of apolipoprotein A-I in the incubation mixture, and accurate with a variability of only 3-5% (S.E.M.). This technique enables the quantitation of apolipoprotein A-I in whole plasma or high density lipoprotein without the need of delipidation. The quantitation of apolipoprotein A-I in high density lipoprotein was found similar to that obtained by gel filtration technique. The displacement capacity of the different lipoproteins and apoproteins in comparison to unlabeled apolipoprotein A-I was: very low density lipoprotein, 1.8%; low density lipoprotein, 2.6%; high density lipoprotein, 68%; apolipoprotein B, non-detectable; apolipoprotein C, 0.5%; and apolipoprotein A-II, 4%. The distribution of immunoassayable apolipoprotein A-I among the different plasma lipoproteins was as follows: smaller than 1% in very low density lipoprotein and low density lipoprotein; 50% in high density lipoprotein, and 50% in lipoprotein fraction of density greater than 1.21 g/ml. The amount of apolipoprotein A-I in the latter fraction was found to be related to the number of centrifugations.


The phenoloxidases of the ascomycete Podospora anserina. XI. The state of copper of laccases I, II and III.

1. Laccases I, II and III were (EC 1.14.18.1) prepared from the mycelium of the ascomycete Podospora anserina. The tetrameric laccase I(mol. wt 340 000, 16 copper atoms) and the monomeric laccases II and II (mol. wt 80 000, 4 copper atoms) have been studied by optical absorption-, circular dichroism-(CD)and electron paramagnetic resonance spectroscopy (EPR). 2. The visible and near ultraviolet difference absorption spectrum, which is apparently identical for all three laccases, shows two maxima at 330 and 610 nm and a shoulder at about 725 nm. The molar extinction coefficients of these bands are 4 times larger for the tetrameric laccase I compared to the monomeric laccases II and III which show values similar to other blue copper-containing oxidases. 3. CD spectra between 300 and 730 nm of the tree laccases are similar and contain at least 5-bands in the oxidized enzyme. If the enzyme is reduced, only a band at 307 nm remains. The molar ellipticity values of these bands are 4 times larger for laccase I than the corresponding bands of laccases II and III. It is inferred that the reducible bands are associated with the Type 1 Cu-2+. 4. In all three laccases the EPR-detectable copper accounts for only about 50% of the total copper content. The 9-GHz and 35-GHz spectra, which are identical for all three laccases, consist of two components of equal intensity. One component shows a rather small copper hyperfine coupling and a small deviation from axial symmetry. It is suggested that this copper is associated with the blue chromophore in analogy to Type 1 Cu-2+ in other blue copper proteins. The other component has a broader hyperfine coupling similar to Type 2 Cu-2+ as found in other copper proteins. The assumption that the experimental spectra result from a superposition of the spectra of equal amounts of Type 1 and Type 2 Cu-2+ has been verified by computer simulation. 5. It is suggested that the copper ions which are not detected by EPR are connected to the absorption band at 330 nm and that these ions are also essential for the function of these laccases.


Inhibition of electron transfer from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone by antimycin.

The effect of antimycin on (i) the respiratory activity of the KCN-insensitive pathway of mitochondria of Neurospora grown on chloramphenicol (chloramphenicol-grown) with durohydroquinone and succinate or NADH as substrate, (ii) the electron transfer from the b-type cytochromes to ubiquinone with durohydroquinone as electron donor as well as (iii) the electron transfer from the b-type cytochromes to duroquinone with succinate as electron donor in chloramphenicol-grown Neurospora and beef heart submitochondrial particles was studied. All experiments were performed in the uncoupled state. 1. The respiratory chain of chloramphenicol-grown Neurospora mitochondria branches at ubiquinone into two pathways. Besides the cytochrome oxidase-dependent pathway, a KCN-insensitive branch equiped with a salicylhydroxamate-sensitive oxidase exists. Durohydroquinone, succinate or NADH are oxidized via both pathways. The durohydroquinone oxidation via the KCN-insensitive pathway is inhibited by antimycin, wheras the succinate or NADH oxidation is not. The titer for ful inhibition is one mol antimycin per mol cytochrome b-563 or cytochrome b-557. 2. The electron transfer from durohydroquinone to ubiquinone, which takes place in the KCN-inhibited state, does not occur in the antimycin-inhibited state. 3. The reduction of duroquinone by succinate in the presence of KCN is inhibited by antimycin. The titer for full inhibition is one mol antimycin per mol cytochrome b-566 or cytochrome b-562 for beef heart (or cytochrome b-563 or cytochrome b-557 for Neurospora). 4. When electron transfer from the b-type cytochromes to cytochrome C1, ubiquinone and duroquinone is inhibited by antimycin, the hemes of cytochrome b-566 and cytochrome b-562 (or cytochrome b-563 and cytochrome b-557) are in the reduced state. 5. The experimental results suggest that the two b-type cytochromes form a binary complex the electron transferring activity of which is inhibited by antimycin, the titer for full inhibition being one mol of antimycin per mol of complex. The electron transfer from the b-type cytochromes to ubiquinone is inhibited in a non-linear fashion.


EPR studies on the respiratory chain of wild-type Saccharomyces cerevisiae and mutants with a deficiency in succinate dehydrogenase.

1. Three nuclear mutants of Saccharomyces cerevisiae deficient in succinate dehydrogenase have been isolated. Two of these mutants are allelic. 2. The amount of covalently bound flavin of submitochondrial particles of the two allelic mutants is about 14% and that of the third mutant about 50% of the amount in wild-type particles. The turnover number of succinate dehydrogenase of particles is decreased in all mutants. The turnover number of fumarate reductase is increased in the two allelic mutants, but decreased in the third mutant. 3. EPR spectra, measured at 82 degrees K, show that the amplitude of the g equals 1.93 signal in particles of the two allelic mutants is less than 10% of that in wild-type particles. It is concluded that iron-sulphur centres other than those of succinate dehydrogenase make only a negligible contribution to the line at g equals 1.93 in wild-type particles. 4. EPR measurements below 20 degrees K show that the amplitude of the signal at g equals 2.01 detected in oxidized particles is decreased in particles of the two allelic mutants. 5. A signal with lines at g equals 2.027 and g equals 1.933 is detected at low temperatures in all particle preparations, even in those from a cytoplasmic petite mutant. It is suggested that this signal is derived from a contaminant and not from the inner membrane.


Two mechanisms of near-ultraviolet lethality in Saccharomyces cerevisiae: a respiratory capacity-dependent and an irreversible inactivation.

Near-ultraviolet irradiation of actively growing yeast cells leads to cell death by two distinct mechanisms. The first type of cell death is evident after low doses of near-ultraviolet light (3 times 10-4 ergs times mm- minus 2) and is due to a reversible inactivation of the respiratory capacity of the cell. In studies with yeast mitochondrial membranes the quinones were identified as the site of inactivation by determining the relative levels of the following oxidase activities after irradiation: exogenous NADH, endogenous NADH (via isocitrate dehydrogenase), succinate, and D-lactate oxidases. A second type of cell death is caused after high doses (1.8 times 10-5 ergs times mm- minus 2) and is irreversible. The mechanism of this inactivation is unknown.


Thermodynamic and EPR characterization of iron-sulfur centers in the NADH-ubiquinone segment of the mitochondrial respiratory chain in pigeon heart.

Several iron-sulfur centers in the NADH-ubiquinone segment of the respiratory chain in pigeon heart mitochondria and in submitochondrial particles were analyzed by the combined application of cryogenic EPR (between 30 and 4.2 degrees K) and potentiometric titration. Center N-1 (iron-sulfur centers associated with NADH dehydrogenase are designated with the prefix "N") resolves into two single electron titratins with EM7.2 values of minus 380 plus or minus 20 mV and minus 240 plus or minus 20 mV (Centers N-1a and N-1b, respectively). Center N-1a exhibits an EPR spectrum of nearly axial symmetry with g parellel = 2.03, g = 1.94, while that of Center N-1b shows more apparent rhombic symmetry with gz = 2.03, gy = 1.94 and gx = 1.91. Center N-2 also reveals EPR signals of axial symmetry at g parallel = 2.05 and g = 1.93 and its principal signal overlaps with those of Centers N-1a and N-1b. Center N-2 can be easily resolved from N-1a and N-1b because of its high EM7.2 value (minus 20 plus or minus 20 mV). Resolution of Centers N-3 and N-4 was achieved potentiometrically in submitochondrial particles. The component with EM7.2 = minus 240 plus or minus 20 mV is defined as Center N-3 (gz = 2.10, (gz = 2.10, (gy = 1.93?), GX = 1.87); the minus 405 plus or minus 20 mV component as Center N-4 (gz = 2.11, (gy = 1.93?), gx = 1.88). At temperatures close to 4.2 degrees K, EPR signals at g = 2.11, 2.06, 2.03, 1.93, 1.90 and 1.88 titrate with EM7.2 = minus 260 plus or minus 20 mV. The multiplicity of peaks suggests the presence of at least two different iron-sulfur centers having similar EM7.2 values (minus 260 plus or minus 20 mV); HENCE, tentatively assigned as N-5 and N-6. Consistent with the individual EM7.2 values obtained, addition of succinate results in the partial reduction of Center N-2, but does not reduce any other centers in the NADH-ubiquinone segment of the respiratory chain. Centers N-2, N-1b, N-3, N-5 and N-6 become almost completely reduced in the presence of NADH, while Centers N-1a and N-4 are only slightly reduced in pigeon heart submitochondrial particles. In pigeon heart mitochondria, the EM7.2 of Center N-4 lies much  closer to that of Center N-3, so that resolution of the Center N-3 and N-4 spectra is not feasible in mitochondrial preparations. EM7.2 values and EPR lineshapes for the other iron-sulfur centers of the NADH-ubiquinone segment in the respiratory chain of intact mitochondria are similar to those obtained in submitochondrial particle preparations. Thus, it can be concluded that, in intact pigeon heart mitochondria, at least five iron-sulfur centers show EM7.2 values around minus 250 mV; Center N-2 exhibits a high EM7.2 (minus 20 plus or minus 20 mV), while Center N-1a shows a very low EM7.2 (minus 380 plus or minus 20 mV).


A kinetic completion of the cyclic photosynthetic electron pathway of Rhodopseudomonas sphaeroides: cytochrome b-cytochrome c2 oxidation-reduction.

In Rhodopseudomonas sphaeroides, following a single-turnover flash of light, cytochrome c2 is oxidized by reaction center bacteriochlorophyll, and a cytochrome b is reduced by the primary electron acceptor, probably via ubiquinone. In this report we show that, in the uncoupled state, the rate of re-oxidation of the cytochrome b is identical to the rate of reduction of the cytochrome c2, a kinetic completion of the cyclic photosynthetic electron transport system.


Enzyme profiles of mammalian bile.

The activities of subcellular marker enzymes in bile and liver homogenate from several mammalian species have provided information on the specificity of protein release during bile formation. The presence of significant amounts of the plasma membrane enzymes alkaline phosphodiesterase I and leucyl-beta-naphthylamidase in bile, in addition to alkaline phosphatase and 5'-nucleotidase, and the relative absence of intracellular enzymes lends support to the view that bile salt liberation from the hepatocyte is accompanied by a partial solubilization of the plasma (canalicular) membrane without extensive damage to the whole hepatocyte.


Stimulation of ion transport by ascorbic acid through inhibition of 3':5'-cyclic-AMP phosphodiesterase in the corneal epithelium and other tissues.

Ascorbic acid stimulates active transport of Cl-minus by the isolated intact cornea. The effect is not present in corneas previously stimulated by the theophylline, an inhibitor of 3':5"-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and vice versa, theophylline has no action after stimulation with ascorbic acid. This indicated inhibition of 3':5'-cyclic-AMP phosphodiesterase by ascorbic acid. Assay of phosphodiesterase using 3-H-labeled cyclid AMP of frog and rabbit corneal epithelial homogenates showed an inhibitory effect of ascorbic acid. Concentration of 5 mM produced 16% inhibition with 20 mM producing 46%. This compares with 58% inhibition by theophylline at 5 mM. Phosphodiesterase activity is mostly soluble in frog corneal epithelium but in rabbit 45% is particulate. Soluble and particulate fractions are inhibited by ascorbate, but in rabbits greater inhibition (50%) was observed in the particulate fraction than in the soluble fraction. Other tissues showed inhibition also: frog retina 12%, rat brain (caudate nucleus) 48%, rabbit brain 14%, rabbit liver 16%. It is concluded that ascorbate produces an increase in cyclic AMP content of corneal epithelium and other tissues by inhibition of 3':5'-cyclic-AMP phosphodiesterase. This action may be one of the main functions of the high ascorbic acid content of ocular tissues and explain some of the effects of high dosis of ascorbate in other systems.


Decreased binding of insulin to liver plasma membrane receptors in hereditary diabetic mice.

The interaction of insulin with its receptors was studied in liver plasma membranes of the young non-obese hereditary diabetic mouse (KK strain). Under identical conditions of preparation and incubation, the membranes of the KK mouse bind only 55-70% as much insulin per mg of protein as those of the control mouse (Swiss albino). Scatchard analysis suggests that this decrease in binding is due to a decrease in the number of receptor sites in the membrane of the diabetic mouse. However, the membranes of diabetic and control mice do not exhibit significant differences in hexosamine and sialic acid contents, enzyme activities, and protein and glycoprotein analysis. The decrease in insulin receptors in the KK mouse seems to correlate with the insulin resistance which they exhibit.


Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2.

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 degrees C, but not at 34 degrees C. Its host, Pseudomonas BAL-31, grows at 34 degrees C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 degrees C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place. Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones, prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates amde in its presence contain no PM2-like particles. These experiments, carried out at 25 degrees C, indicate that adamantanone prevents the assembly of stable PM2 virus. Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an "ordered" state at temperatures below about 33 degrees C and undergo a transition to a "disordered" state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 degrees C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the "ordered" or "disordered" state, but that the "ordered" state must be maintanined for PM2 assembly to occur.


Requirement of heat and metabolic energy for the expression of inhibitory action of colicin K.

Escherichia coli B, induced for beta-galactoside permease, can accumulate thio-methyl-beta-galactoside in the cell even at 0 degrees D. At this temperature, cells adsorb colicin K but the adsorbed colicin does not inhibit thiomethyl-beta-galactoside uptake. Inhibition by colicin K is, however, seen at 0 degrees C after exposure of the colicin K-cell complex to a high temperature: a greater degree of inhibition occurs with increasing temperature or duration or exposure. There is a transition point at around 21 degrees C in Arrhenius plots of this colicin K activation reaction. If inhibitors of energy yielding reactions are present during the heat treatment, the inhibitory action of colicin K (as measured by thiomethyl-beta-galactoside uptake after returning the colicin K-cell complex to 0 degrees C and removal of the inhibitors) is prevented. These results indicate that adsorbed colicin K is converted into the active state only in the presence of metabolic energy and that cell surface fluidity appears to be concerned in this process.


Altered cell surface glycoproteins in phytohemagglutinin-resistant mutants of Chinese hamster ovary cells.

Purified membranes from surface-labelled phytohemagglutinin-resistant (Pha(R) and wild-type chinese hamster ovary cells have been analysed by sodium dodecyl sulphate gel electrophoresis. Gel patterns were compared for cells labelled via galactose oxidase and B-3H4 or lactoperoxidase and radioactive iodide. The results suggest that Pha-R cells are altered in the carbohydrate portion of a number of their membrane glycoproteins.


Protection of the active site of mitochondrial malate dehydrogenase from inhibition by potassium tetrachloroplatinate.

Mitochondrial malate dehydrogenase (L-malate : NAD-+ oxido-reductase, EC 1.1.1.37) was inhibited by potassium tetrachloro platinum (II), K-2PtCl-4, in the presence of varying concentrations of NADH, NAD-+ and L-malate and mixtures of NAD-+ and L-malate. It was observed that NADH is an effective protector of the enzyme from inhibition while both NAD-+ and L-malate are poor protectors. Spectral studies have suggested that the protection afforded by the substrates are accomplished by reaction with specific groups on the enzyme rather than by complexation of the substrates with PtCl-4-2-minus. From the above data it has been concluded that the tetrachloroplatinate ion binds only at the active site and that this site which is effectively protected by NADH, and moderately protected by a NAD-+-L-malate complex probably contains one or more sulfur containing amino acid side chains. It is also proposed that when the tetrachloroplatinate complexes with the enzyme there is some effect, possibly a conformational change, which causes the release of NADH at the allosteric site.


A rapid purification method of restriction endonucleases from Haemophilus strains.

A simple and rapid method of purification of restriction endonucleases from different Haemophilus strains is presented. By this method highly purified and stable enzymes can be obtained. Separation of different restriction activities present in the same strain is possible. This method was so far successfully used with Haemophilus influenzae, Haemophilus parainfluenzae and Haemophilus aegyptius strains. The main advantages over previously published procedures reside in the simplication of certain purification steps (for instance the BioGel A 0.5 M filtration is replaced by a hydroxyapatite batch step), elimination of exonuclease activity by fractionation with (NH4) 2SO4, separation of different restriction activities by phosphocellulose chromatography, application of this method to various strains and high purification degree of enzymes.


Assay for adenylate cyclase and cyclic nucleotide phosphodiesterases and the preparation of high specific activity 32-P-labeled substrates.

Simple one step assay methods for adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) and cyclic nucleotide phosphodiesterases (3',5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) have been developed. [alpha-32-P] ATP is used as the substrate for adenylate cyclase. Acid-heat destruction of [32-P] ATP remaining after the cyclase reaction followed by Zn-Ba treatment quantitatively leaves cyclic [32-P] AMP in the supernatant essentially free from other 32-P-containing compounds. This assay method requires no corrections for recovery and routinely yields blank values less than 0.03 per cent. If higher sensitivity is desired, a simple 5 min alumina column step can be introduced into the procedure which quantitatively elutes cyclic [32-P] AMP directly into a liquid scintillation vial and lowers the blank values to less than 0.002 per cent. This method is rapid and easily performed, without sacrificing high reliability, specificity, or sensitivity. One step phosphodiesterase assays are easily accomplished using 32-P-labeled cyclic nucleotides as substrates. Descending paper chromatography of the reaction mixture on individual 2 cm wide paper strips gives a complete and quantitative separation of all possible products including [5'-32-P] AMP and [5'-32-P] GMP from their respective 32-P-labeled 3',5'-cyclic nucleotides in 1-2 h. The paper strips are cut, inserted in scintillation vials without scintillant and the 32-P-products determined by Cerenkov counting. Low blank values of less than 0.5 per cent and the use of high specific activity 32-P-labeled cyclic nucleotide substrates make this method the most reliable and most sensitive phosphodiesterase assay described to date. Because of the simplicity, specificity, and high sensitivity obtainable with these assay methods using 32-P-labeled substrates, we have also devised simple conditions for the preparation and purification of [alpha-32-P] ATP, cyclic [32-P] AMP and cyclic [32-P] GMP with specific activities in excess of 100 Ci/mmol. These high specific activity 32-Plabeled cyclic nucleotides are important for these new assay methods and are also useful to follow purification recovery of endogenous cyclic AMP and cyclic GMP from biological materials before protein binding or radioimmunological isotope displacement assays when performed in the femtomole range.


Stimulation by ATP of alkaline phosphatase in placental plasma membranes.

1. ATP stimulated the p-nitrophenyl phosphatase activity of placental plasma membranes, with an increase in activity of approximately 100% at 5 mM ATP. The stimulation was not dependent on the presence of Mg-2-+. 2. The K-m for p-nitrophenyl phosphate was not changed by the presence of 5 mM ATP. 3. ATP hydrolysis by the plasma membrane preparation under the same assay conditions as for alkaline phosphatase was not influenced by the presence of 5 mM p-nitrophenyl phosphate. 4. Extraction of the plasma membrane preparation with n-butanol abolished the stimulatory effect of ATP, as well as Ca-2-+-activated ATPase activity.


Effect of adenine nucleotides on NAD-dependent isocitrate dehydrogenases in rhizobia and bacteroids of legume root nodules.

ATP, ADP, AMP and cyclic AMP inhibit NAD-dependent isocitrate dehydrogenase (L-s-isocitrate : NAD-+ oxidoreductase, EC 1.1.1.41) from rhizobia but have no effect on the enzyme from corresponding bacteroids. This was observed using three rhizobial strains two of which are effective, and one ineffective, with Lotus pedunculatus. Using partially purified enzyme from each of the three rhizobial strains it was found that the adenine nucleotides inhibit the enzyme by competing with NAD-+, not with isocritrate. The rate of reaction catalysed by the enzyme (expressed as activity per mg protein) in cell-free extracts of each of the effective rhizobial strains was about three times that of the reaction in extracts of the corresponding bacteroids. No correlation was found between effectiveness and NAD-dependent isocitrate dehydrogenase activity in the rhizobial cells.


Comparative structural studies of the active site of ATP: guanidine phosphotransferases. The essential cysteine tryptic peptide of taurocyamine kinase from Arenicola marina.

The active cysteinyl residues of dimeric taurocyamine kinase from Arenicola marina were labelled with N-ethyl-[1-14C]maleimide. The resulting inactivated N-ethyl-[1-14C]succinimido enzyme was then subjected to tryptic hydrolysis. The peptide containing the labelled essential cysteinyl residue was isolated. The amino acid sequence of this peptide is Leu-Gly-Tyr-Leu-Gly-Thr-[14C]-Cys-Pro-Thr-Asn-Ile-Gly-Leu-Arg. This sequence is very similar to that of homologous ATP:guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinase from ox brain and ox muscle, and from rabbit muscle, and lombricine kinase from Lubricus terrestris.


Cyclic AMP binding proteins in early embryos of Drosophila melanogaster.

A variety of effects of cyclic AMP on cellular and subcellular phenomena suggest that there may be other modes of action of cyclic AMP then activation of protein kinase. It is also known that developing embryos contain cyclic AMP and its related enzymes. In order to explore the role of cyclic AMP in embryogenesis, a survey of proteins capable of binding cyclic AMP in the embryonic supernatant of Drosophila melanogaster was carried out. As the result, two cyclic AMP-binding proteins were found and characterized. The one (L) is, as expected, associated with protein kinase and has a dissociation constant of about 10(-9) M. Its molecular weight of 21 000 daltons is extremely small when compared with similar proteins in other organisms. The other (H), whose function is yet to be found, has a molecular weight of about 200 000 daltons and has a dissociation constant of about 10-7 M. Some laxity in binding specificity of the latter protein among adenosine nucleotides was observed, but cyclic AMP is the strongest ligand among them.


Studies on the composition of pig serum lipoproteins. Isolation and characterization of different apoproteins.

1. Different lipoprotein density fractions from pig serum were isolated by phosphotungstate precipitation followed by purification in the preparative ultra-centrifuge. 2. The protein part of very low density lipoproteins was composed of approximately 52 percent lipoprotein B apoprotein and the rest of lipoprotein C II apoprotein and other as yet unidentified peptides. 3. The protein moiety of low density lipoproteins consisted primarily of lipoprotein B apoprotein (over 95 percent); the amino acid compositions of lipoprotein B apoprotein of very low and low density lipoproteins were practically identical. 4. The predominant polypeptide of pig serum high density lipoproteins exhibited an amino acid composition and a molecular weight very similar to human liprotein A I apoprotein. In contrast to human lipoprotein A I apoprotein, the apoprotein from pigs was found to release leucine first followed by alanine, threonine, and lysine upon incubation with carboxypeptidase A. 5. In pig serum the major lipoprotein C apoprotein was found to be a polypeptide similar in amino acid composition to lipoprotein C II apoprotein from human serum. The molecular weight of this polypeptide is approximately 8000. Incubation experiments with carboxypeptidase A indicate serine to be the most likely C-terminal amino acid.


Paramagnetic spin label interactions with the envelope of a group A arbovirus. Lipid organization.

Electron paramagnetic resonance observations were made on nitroxide spin- labeled molecules which were bound to the TC-83 vaccine strain of Venezuelan equine-encephalomyelitis virus. Paramagnetic resonance parameters derived from the observations and their dependence on sample temperature were similar but not identical to those which have been reported for these labels dissolved in lipid bilayer membranes of mammalian and bacterial origin. The data has a mechanical rigidity substantially greater than that of bilayers in cellular membranes. A model is presented which assumes the location of the lipid bilayer outside the nucleoprotein capsid and inside a spherical layer of envelope proteins. The model is in accord with Harrison's X-ray diffraction results for Sindbis virus. The model is discussed in terms of its implications with respects to the role played by lipid in viral maturation and infectivity.


Sulphate transport by rat ileum. Effect of molybdate and other anions.

Kinetic constants for SO4-2- transport by upper and lower rat ileum in vitro have been determined by computer fitting of rate vs concentration data obtained using the everted sac technique. MoO-4-2- inhibition of this transport is competitive, and kinetic constants for the inhibition were similarly determined. Transport is also inhibited by the anions WO4-2-, S2O3-2- and SeO4-2-, in the order S2O3-2- greater than SeO4-2- greater than or equal to MoO4-2- greater than WO4-2-. These anions have no effect on the transport of L-valine. Low SO4-2- transport rates were observed in sacs from animals fed a high-molybdenum diet. The significance of the results with respect to the problem of molybdate toxicity in animals is discussed, and related to the known protective effect of SO4-2-.