Lipids and lipoproteins in normal dogs and in dogs with secondary hyperlipoproteinemia.

Concentrations of serum cholesterol, triglyceride, and free glycerol were neasured, and serum lipoprotein electrophoresis was performed in normal dogs and in dogs with hypothyroidism, diabetes mellitus, and acute pancreatitis. Twelve privately owned dogs and 20 Basset Hounds from a research colony served as normal subjects. Privately owned had higher serum lipid concentrations than did research dogs. On electrophorisis of normal dog serum, lipoprotein bands were detected at the beta, pre-beta, alpha-2, and alpha-1 positions. Hypercholesterolemia was associated with increased intensity of the alpha-2-lipoprotein band, and hypertriglyceridemia occurred with increased lipoprotein at the origin, or the beta or pre-beta positions. Hypothyroid dogs had normal lipid values and lipoprotein electrophoretic patterns, hypercholesterolemia with increased intensity of the alpha-2-lipoprotein band, or hypercholesterolemia and hypertiglyceridemia with prominent beta-, pre-beta, and alpha-2-lipoprotein electrophoretic patterns were changed to near-normal values after levothyroxine administration. Diabetic dogs had increased serum cholesterol and triglyceride content and a predominance of lower density lipoproteins, as detected by electrophoresis. Insulin therapy caused the lipid concentrations to decrease and the electrophoretic pattern to revert to near normal. Dogs with acute pancreatitis had moderately increased serum lipid content and electrophoretic patterns characterized by increased intensity of the beta-lipoprotein band and by altered migration of alpha-1-lipoproteins.


New diagnostic approach to brain lesions in nuclear medicine.  Differential diagnosis of brain lesions with a computed brain scan diagnosis by the likelihood method.

Using 240 true positive brain scans, a computer system for the differential diagnosis of brain lesions has been evaluated.  Eighty-six parameters were extracted from brain scan findings without relationship to neurological signs and symptoms, and the likelihood method was adopted as an example of mathematical logic.  The results of our experiment indicated that the overall accuracy was 77 per cent for the maximum likelihood method.  The digital computer gave satisfactory results, particularly for diseases such as infarct, meningioma, acoustic neurinoma, and subdural hematoma.  In spite of several problems to be solved, this method could provide invaluable help in differential diagnosis of brain lesions.


Survival factors following myocardial infarction recorded by a nurse.

Sixty-five patients were followed closely by a public health nurse following coronary-care unit discharge. A clear-cut pattern was observed in which nonsurvivors had increasing objective signs of physical deterioration and more hospital admissions, but verbalized fewer complaints than survivors. The differences between the eight nonsurvivors and the survivors were significant at the 0.05 level for the observed findings. These findings have implications for organizing health-care delivery.


Adenovirus infection in two calves.

Two 2-week-old calves with pyrexia, diarrhea, and subsequent dehydration were treated symptomatically for 1 week, but without favorable response. At necropsy, foci of necrosis were found in the abomasum and rumen of each calf. The small and large intestineswere dilated by grayish, turbid fluid. Numerous large amphophilic, intranuclear inclusions in endothelial cells of blood vessels in the abomasum and rumen, in endothelial cellsof the adrenal cortical sinusoids and renal glomeruli, and in intestinal epithelial cells were identified as adenovirus particles.


Survey for antibodies to leukemia (C-type) virus in cattle.

Serums from 4,394 dairy cattle in 100 herds and from 2,794 beef cattle in 50 herds were tested for antibody to the bovine (C-type) leukemia virus (BLV), using the agar gel immunodiffusion test. Reactors were found in 66% of the dairy herds (10.2% of the cattle) and in 14% of the beef herds (1.2% of the cattle). The prevalence of reactors was examined with respect to age, herd size, and sex. Few of the reactors were less than 2 years old. There was a high percentage of reactors in small dairy herds (less than 50 cattle). In 22 dairy herds (1,354 cows and 96 bulls), the rate of infection in cows was compared with that in bulls. In those herds, 13.5% of the cows and 10.4% of the bulls were reactors.


Effects of 5-fluorouracil on exocrine glands. III. Fine structure of Brunner's glands of rats.

Effects of a pyrimidine analogue, 5-fluorouracil (Fur), have been studied by electron microscopy and by electron microscopic cytochemical techniques. Previous studies have demonstrated that rats show serious gastrointestinal disturbances 5 days after 3 daily injections of FUR (50 mg/kg). The present investigation demonstrates that Brunner's glands under the same conditions suffer certain cytological changes involving the Golgi apparatus, where a notable reduction in the number of Golgi stacks is observed. The vacuolar components in the Golgi complex appear empty. Cytochemical localizations of uridine diphosphatase and thiamine pyrophosphatase activities, however, are normal. The reaction products are localized in the distal two or three lamellae of the Golgi stack and within the secretory granules nearby. In addition reaction products are present along the apical plasma membrane on the luminal side, suggesting a possible movement of these membranes from the Golgi stack, via secretory granules, to the apical plasma membrane.


Ultrastructure of the submucous nerves of the rat ureter.

The fine structure and distribution of the nerves in the submucosa of the middle part of the ureter were studied in the adult rat. As in the rabbit, the nerves were concentrated on the inner surface of the muscle and beneath the epithelium. The nerves beneath the epithelium were smaller than those on the surface of the muscle, but they were much more numerous. They gave off small branches which ran for only short distances in the subepithelial connective tissue before terminating. Axonal varicosities were common and almost all appeared to represent the terminals of a single type of axon. The varicosities contained clusters of clear vesicles and variable numbers of larger dense-cored vesicles. The dense-cored vesicles were unaffected by short-term treatment with 6-OHDA and in this and other respects compared well with those of the terminals classified by Burnstock (1972) as purinergic. The possibility that the axons are sensory, and are the source of impulses which are recognized as being painful in nature, is discussed.


Observations on the structure of the dorsal muscle in the bottle-nose dolphin (Tursiops truncatus).

The dorsal muscle of five South Australian bottle-nose dolphins has been studied. The ultrastructure and cytochemistry of the red, white and intermediate fibres were essentially similar to those of other mammalian skeletal muscles. Two features of special interest were: (a) the large number of lipid droplets in the red muscle fibres, and (b) the presence of longitudinal tubes linking the transverse tubes of the T-system, both within a sarcomere, and also between adjacent sarcomeres, of the white muscle fibres. The muscle spindles and neuromuscular junctions in the dolphin appeared to be morphologically similar to those of other mammals.


An electron microscopic study of the histogenesis of the parathyroid gland in the sheep.

Definitive chief cells, characterized by the presence of specific secretory granules and typical membrane configurations, were present in the developing parathyroid glands of sheep embryos from 26 days of gestation (term = 150 days). During earlier stages of gestation developing chief cells contained lysosomal systems which appeared to be concerned with the autophagy of retained secretion product. The EM evidence suggests that physiologic release of the hormone does not occur until at least 30 days of gestation, which correlates well with the experimentally established time of onset of functional capacity in the parathyroid gland in the sheep. 'Bark cells' were found in the parathyroid primordia of embryos between 20 and 27 days of gestation. The significance of these "dark cells" is discussed with respect to glutaraldehyde fixation and to their possible relationship to chief cells.


The ultrastructure of the sensory end-organs (baroreceptors) in the atrial endocardium of young mini-pigs.

Light and electron microscopical studies on pig hearts have confirmed the presence in the right and the left atrial endocardium of distinct circumscribed, unencapsulated end-organs (baroreceptors) associated with 4-9 mum thick nerve fibres. The myelin sheath terminates before the entrance of the nerve into the end-organ. The regular presence of thin axon profiles containing clusters of small agranular vesicles, both around the thick nerve fibre and in the periphery of the end-organs, suggests a double innervation. The thick fibre arborizes inside an aggregate of Schwann-like cells to form a large number of terminals which are considered to be the mechano-sensitive receptors of the organ. In addition to the nervous structures and the Schwann-like cells the organs contain a varying number of connective tissue fibrils. The terminals are covered by a basement membrane but are partly or completely devoid of Schwann cell covering. Most of the space inside the terminals is occupied by tightly packed mitochondria. Glycogen granules are regularly present, sometimes abundant. SER occurs widely, frequently forming complexes with glycogen granules. These complexes may be highly organized as parallel or concentric structures, suggesting a close connection between SER and glycogen metabolism in the terminals. The terminals are regularly furnished with small protrusions containing numerous 30-100 nm clear vesicles which resemble pinocytotic vesicles. In addition, a few 80 nm dense-cored vesicles are found. The occurrence of numerous cyto-segresomes and lipofuscin-like bodies suggests a lively turnover of organelles in the terminals.


Ultrastructure of the blastocyst and endometrium of the roe deer (Capreolus capreolus) during delayed implantation.

Transmission electron microscopy of the trophoblast cells during diapause revealed an abundance of electron-dense, membrane-bound granular inclusions and a marked lack of cytoplasmic organelles. The cells also possessed a well-developed covering of branched microvilli, numerous caveolae, micropinocytotic vesicles and a lamina of fine fibrillae. The progressive enlargement of the blastocyst during diapause was correlated with a decline in the height of the trophoblast cells and a reduction in the density of microvilli and caveolae associated with the outer membrane. The granular inclusions also declined in number and electron density during the delay phase, suggesting the progressive utilisation of energy reserves. Embryonic elongation was associated with the disappearance of the granular inclusions, a reduction in the amount of fibrillar material and a dramatic increase in the development of cytoplasmic organelles. During diapause, clear vesicles, apparently derived from the Golgi apparatus, gradually accumulated in the supranuclear region of each gland and non-ciliated duct cell. Embryonic elongation was associated with the sudden release of these vesicles into the glandular lumen and thence into the uterine lumen. Numerous apical protrusions were also observed projecting from the luminal and ductal epithelia at this time, suggesting the formation of an apocrine secretion. Another type of secretion was produced during the early stages of placental attachment by the hypertrophied granular endoplasmic reticulum of the ductal epithelium.


The crystal structure of bonito (katsuo) ferrocytochrome c at 2.3 A resolution. II. Structure and function.

The structure analysis of bonito heart ferrocytochrome c was carried out at 2.3 A resolution by X-ray diffraction, and a Kendrew-type skeletal model was built up. This molecule has an overall egg shape, 35 A in height, 30 A in width and 23 A in thickness; the 5th ligand of the heme iron atom is the N-epsilon atom of the His-18 imidazole ring and the 6th is the Met-80 sulfur atom. Distinct alpha-helix regions are found between the N-terminus and reside 11, between 60 and 69, and between 90 and the C-terminus. The most distinct difference between the conformation of the present molecule and that of the horse oxidized molecule is the location of the Phe-82 phenyl ring. In the present reduced molecule, the phyenyl ring is in closer contact with the iron atom and gives influences on the character of the iron atom. Inside the molecule, at the lower part of the heme pocket, there is an extended hydrogen bond network including the propionic acid residues of the heme group. Both Phe-82 and the hydrogen bond network may play a key role in the function of this molecule.


Dietary and hormonal regulation of serine synthesis in the rat.

The importance of the so-called phosphorylated pathway for the synthesis of serine in rat liver was confirmed by studies on in vitro serine synthesis and also by the existence of a correlation between the serine-synthesizing activity of liver extracts and the activities of the enzymes involved in the pathway under various dietary conditions. Serine-synthesizing activity was found to be distributed in various rat tissues such as kidney, brain, testis, spleen, pancreas, and fat pad. However, only in the liver was the synthesis regulated by dietary protein. In the liver, the three enzymes of the phosphorylated pathway were found to be repressed by high-protein diets or by starvation and induced by low-protein diets. The dietary induction of the enzymes required the presence of insulin and was suppressed by glucocorticoids. A suggestion is made that the effects of diet or hormones may be mediated by changes in the hepatic pool of essential amino acids.


Studies on NADP-+-specific isocitrate dehydrogenase from an extreme thermophile, Thermus flavus AT-62.

1. NADP-+-specific isocitrate dehydrogenase [EC 1.1.1.42] was partially purified by about 440-fold from an extreme thermophile, Thermus flavus AT-62. 2. Remarkable thermostability of the enzyme was confirmed. The enzyme was not inactivated after 60 min at 70 degrees, and the activity was lost only slowly at 80 degrees. Above 90 degrees, however, rapid inactivation was observed. 3. The dehydrogenase was susceptible to concerted inhibition by oxaloacetate plus glyoxylate. In the presence of oxaloacetate plus glyoxylate (each 1 mM), 75 percent inhibition was observed. 4. The degree of inhibition of the enzyme by oxaloacetate plus glyoxylate decreased markedly above 60 degrees. The affinity of the enzyme for isocitrate and NADP-+ was also reduced markedly above 60 degrees. The activation energy calculated from Arrhenius plots below and above 60 degrees were 14,500 and 8,000 cal per mole, respectively. These observations suggest a possible conformation change of the enzyme protein at a transition temperature of 60 degrees, and the physiological significance of this in the adaptation of thermophiles to elevated temperatures is discussed.


Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes.

The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented.


Separation of tetracyclines by high-speed liquid chromatography.

The separation of tetracycline and its four commoninpurities has been studied by high-speed liquid chromatography. A preliminary study of the effectiveness of ion-exchange, adsorption, liquid-liquid partition and reversed-phase ion-pair chromatography indicated that only the last method showed promise. By developing special hydrocarbon-bonded stationary phases a rapid and complete resolution of all five tetracyclines has been obtained within 10 min. Plate heights using a derivatised 18-mum partisil are in the range 0.15-0.3 mm. The method can be used to quantify the impurities in tetracycline at around the 1% level.


Preparation and thin-layer chromatography of bromo-derivatives of unsaturated fatty acid esters. A simple and rapid procedure for fatty acid analysis.

Bromo-addition products of unsaturated long-chain fatty acid esters have been prepared and chromatographed on thin layers of unmodified silica gel. The polarity of these derivatives was found to be directly related to the number of double bonds of the parent fatty acid from which they were derived. This has been made the basis of a simple method for assessing the relative proportions of the main fatty acid classes in a mixture.


Occurrence of big renin in human plasma, amniotic fluid and kidney extracts.

Big renin, a relatively inactive renin which possesses a molecular weight larger than that of normal plasma or renal renin, has been demonstrated by gel filtration in certain human plasma, tumor extracts, and amniotic fluid. Big renin was not present in normal plasma or kidney extracts. Plasma from 3 hypertensive patients with nephropathy contained chiefly big renin. Varying proportions of both big and normal renin activity were present in plasma of other patients with hypertension and proteinuria. The renin present in amniotic fluid, which increased in activity following exposure to acid pH, was shown to be big renin in two patients. Large amounts of circulating big renin apparently can cause hypertension in patients with Wilms' tumors. Furthermore, the relatively inactive big renin may replace normal plasma renin in some patients, resulting in low plasma renin activity.


Failure of somatostatin to inhibit tolbutamide-induced insulin secretion in patients with insulinomas: a possible diagnostic tool.

The effects of somatostatin on tolbutamide-stimulated insulin release were studied in 4 patients with insulin-producing tumors of the pancreas and in 6 normal subjects.  In contrast to its effective inhibition of insulin release in normal subjects, somatostatin, without exception, failed to inhibit tolbutamide-induced insulin release in the patients with pancreatic beta-cell tumors.  This differential effect of somatostatin may prove useful in the diagnosis of insulin-producing tumors of the pancreas.


Inhibition by somatostatin of ACTH secretion in Nelson's syndrome.

Somatostatin (GHIF), when administered as a 1-hr infusion (500 mug/hr) to 5 patients with Nelson's Syndrome;resulted in a sustained, progressive fall in plasma ACTH in each patient to 40% to 71% of basal values with a return toward initail levels after cessation of the infusion.  The meanreduction in plasma ADTH was 48% (p less than 0.005).  These finding suggest that GHIF receptors not fuctional or present in normal pituitary tissue are present in ACTH-producing pituitary tumors.


Effects of sodium depletion on plasma renin activity and on the urinary excretion of cyclic AMP and aldosterone in hypoparathyroid patients.

The effect of sodium depletion on plasma renin activity (PRA), urinary cyclic AMP and urinary aldosterone excretion was studied in hypoparathyroid patients whose basal urinary cylic AMP excretion (urinary cAMP) was less than 50% of that observed in normal subjects. During 7 days of sodium depletion, PRA, urinary aldosterone and urinary cAMP each rose significantly. Administration of the beta-blocker propranolol, 160 mg/day, during 5 further days of sodium depletion produced a fall in PRA and urinary cAMP, but no change in urinary aldosterone excretion. The dissociation in these effects suggests that the increase in aldosterone secretion during sodium depletion may be mediated by pathways other than the renin-angiotensin and adenyl cyclase systems. There was a high degree of correlation between PRA and urinary cAMP (P less than 0.001) during the period of sodium depletion, but not significant relationship between these parameters was found during control and propranolol phases, or in control studies in normal subjects. These findings suggest that beta-adrenergic receptors have a role in mediating the effects of sodium depletion upon renin secretion and adenyl cyclase activity.


Adrenal function in hirsutism I. Diurnal change and response of plasma androstenedione, testosterone, 17-hydroxyprogesterone, cortisol, LH and FSH to dexamethasone and 1/2 unit of ACTH.

ACTH dependency of plasma androstenedione (A) and testosterone (T) was determined in normal and hirsute women by measuring the magnitude of change of A and T between the time of the cortisol (F) peak and F nadir in a diurnal study. There was a significant diurnal rhythm of A synchronous with F in both normal and hirsute women (P less than 0.01). Five of 12 hirsute women had a greater than normal diurnal swing of A (P less than 0.05), but only 2 of the 12 had a greater than normal diurnal swing of T. Responsiveness of A and T to 1/2 unit of intravenous ACTH was determined after dexamethasone 1 mg was given the night before. Plasma A and T were elevated in most of the hirsute women during acute ACTH suppression by dexamethasone, indicating ACTH-independent hypersecretion of androgens. Nine of 17 hirsute women had a greater than normal A response to ACTH (P less than 0.05). Those who had an exaggerated diurnal swing of A also had hyper-responsiveness of A secretion to ACTH. Only 2 hirsute women had an exaggerated T response to ACTH. Some T levels were decreased by ACTH. Seven of the 9 hiruste women who had an exaggerated A response to ACTH had a normal maximum F response, but a greater than normal 17-hydroxy-progesterone (17-OHP) response to ACTH with a high 17-OHP to F ratio, suggesting they have a mild but compensated reduction in 21-hydroxylase or 11beta-hydroxylase activity. Two women with hyper-responsiveness of A secretion had low F and 17-OHP responses to ACTH suggesting reduced C21 but intact C19 3beta-hydroxysteroid dehydrogenase-delta-5,-4 isomerase activity. These apparent reduced enzyme activity may not be congenital, but induced by an altered hormonal milieu such as an abnormal androgen-estrogen ratio. It is concluded that ACTH uniformly stimulated A secretion but not T secretion and that approximately 50% of the hirsute women had ACTH-dependent hypersecretion of A, but most of these also had concurrent ACTH-independent hypersecretion of androgens.


Metabolism of cholesteryl esters of rat very low density lipoproteins.

Rat very low density lipoproteins (d smaller than 1.006), biologically labeled in esterified and free cholesterol, were obtained form serum 6 h after intravenous injection of particulate (3-H) cholesterol. When injected into recipient animals, the esterified cholesterol was cleared form plasma with a half-life of 5 min. After 15 min, 71% of the injected esterified (3-H) cholesterol had been taken up by the liver, where it was rapidly hydrolyzed. After 60 min only 3.3% of the amount injected had been transferred, via lipoproteins of intermediate density, to the low density lipoproteins of plasma (d 1.019-1.063). Both uptake in the liver and transfer to low density lipoproteins occurred without change of distribution of 3-H in the various cholesteryl esters. 3-H appearing in esterified cholesterol of high density lipoproteins (d greater than 1.063) was derived from esterification, presumably by lecithin: cholesterol acyltransferase, of simultaneously injected free (3-H) cholesterol. Content of free (3-H) cholesterol in the very low density lipoproteins used for injection could be reduced substantially by incubation with erythrocytes. This procedure, however, increased the rate of clearance of the lipoproteins after injection into recipient rats. These studies show that hepatic removal is the major catabolic pathway for cholesteryl esters of rat very low density lipoproteins and that transfer to low density lipoproteins occurs to only a minor extent.


Metabolism of bovine parathyroid hormone. Immunological and biological characteristics of fragments generated by liver perfusion.

The metabolism of bovine parathyroid hormone (PTH) by the perfused rat liver was studied. Labeled hormone, with or without cold hormone, was infused into the circulating perfusion medium containing various calcium concentrations. Pefusate samples at various time periods after the introduction of PTH into the system were chromatographed on Bio-gel P-10; radioactivity and/or immunoreactivity were measured in eluted fractions. Before the perfusion, all immuno- and radioactivity eluted in a single peak, with an apparent mol wt of 9,500 (peak I). After perfusion for 15 min, two other peaks with approximate mol wt of 7,000 (peak II) and 3,500 (peak III) were discernible. Peak I contained both NH2-terminal and COOH-terminal immunoreactivity and was biologically active at all time periods tested. The relative contribution of NH2-terminal and COOH-terminal immunoreactivity to the total immunoreactivity remained constant in this peak throughout the perfusion. In every respect, peak I had the characteristics of intact hormone. At all times, peak II consisted of only COOH-terminal immunoreactivity and was biologically inactive. At early time periods, peak III contained predominantly NH2-terminal immunoreactivity and was biologically active. With time, the relative contribution of NH2-terminal immunoreactivity decreased strikingly while that of COOH-terminal immunoreactivity increased. The three peaks identified in these experiments were analogous in  size, biological activity, and immunological characteristics to those we have previously described for fractionated human hyperparathyroid serum. The rate of metabolism of PTH appeared to be regulated by the calcium concentration in the medium. At a high concentration of calcium (greater than 11 mg/100 ml), PTH metabolism was greatly retarded. At a low concentration of calcium (smaller than 5 mg/100 ml), the rate of metabolism was greatly increased. The physiological significance of our observations on the metabolism of PTH by isolated perfused rat liver is not known. However, since such metabolism results in a biologically active fragment, it is suggested that metabolism of intact hormone may be required before full biological expression is possible.


The role of superoxide anion generation in phagocytic bactericidal activity. Studies with normal and chronic granulomatous disease leukocytes.

The capacity of human phagocytes to generate superoxide anion (O2-), a free radical of oxygen, and a possible role for this radical or its derivatives in the killing of phagocytized bacteria were explored using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Superoxide dismutase, which removes O2-, consistently inhibited phagocytosis-associated nitroblue tetrazolium (NBT) reduction indicating the involvement of O2- in this process. Similarly, superoxide dismutase inhibited the luminescence that occurs with phagocytosis, implicating O2- in this phenomenon, perhaps through its spontaneous dismutation into singlet oxygen. Subcellular fractions from homogenates of both normal and CGD leukocytes generated O2- effectively in the presence of NADH as substrate. However, O2- generation by intact cells during phagocytosis was markedly diminished in nine patients with CGD. Leukocytes from mothers determined to be carriers of X-linked recessive CGD by intermediate phagocytic reduction of NBT elaborated O2- to an intermediate extent, further demonstrating the interrelationship between NBT reduction and O2- generation in phagocytizing cells. Activity of superoxide dismutase, the enzyme responsible for protecting the cell from the damaging effects of O2-, was approximately equal in homogenates of normal and CGD granulocytes. Polyacrylamide electrophoresis separated this activity into a minor band that appeared to be the manganese-containing superoxide dismutase associated with mitochondria and a more concentrated, cyanide-sensitive, cytosol form of the enzyme with electrophoretic mobility that corresponded to that of erythrocyte cuprozinc superoxide dismutase. Superoxide dismutase inhibited the phagocytic killing of Escherichia coli, Staphylococcus aureus, and Streptococcus viridans. A similar inhibitory effect was noted with catalase which removes hydrogen peroxide. Neither enzyme inhibited the ingestion of bacteria. Peroxide and O2- are believed to interact to generate the potent oxidant, hydroxyl radical (.OH). A requirement for .OH in the phagocytic bactericidal event might explain the apparent requirement for both O2- and H2O2 for such activity. In agreement with this possibility, benzoate and mannitol, scavengers of .OH, inhibited phagocytic bactericidal activity. Generation of singlet oxygen from O2- and .OH also might explain these findings. It would seem clear from these and other studies that the granulo cyte elaborates O2- as a concomitant of the respiratory burst that occurs with phagocytosis. To what extent the energy inherent in O2- is translated into microbialdeath through O2- itself, hydrogen peroxide, .OH, singlet oxygen, or some other agent remains to be clearly defined.


Inorganic pyrophosphate pool size and turnover rate in arthritic joints.

Recent studies have shown elevated inorganic pyrophosphate (PPi) levels in most knee joint fluid supernates from patients with pseudogout (PG) or osteoarthritis (OA) and more modestly elevated levels in some supernates from patients with gout or rheumatoid arthritis (RA) relative to PPi levels found in the venous blood plasma of normal or arthritic subjects. We measured the intraarticular PPi pool and its rate of turnover to better understand the significance of the joint fluid-plasma PPi gradient. Preliminary studies in rabbits showed that (32-P)PPi passed from joint space to blood and vice versa without detectable hydrolysis. Incubation of natural or synthetic calcium pyrophosphate dihydrate (CPPD) microcrystals with synovial fluid in vitro in the presence of (32P)PPi tracer showed no change in PPi specific activity in the supernate over a 19-h period so that exchange of PPi in solution with that in CPPD microcrystals could be ignored. Clearance rates of (32P)PPi and of (33P)Pi, as determined by serially sampling the catheterized knee joints of volunteers with various types of arthritis over a 3-h period, were nearly identical. The (32P)PPi/(32P)Pi was determined in each sample. A mixture of a large excess of cold PPi did not influence the clearance rate of either nuclide. The quantity of PPi turned over per hous was calculated from the pool size as determined by isotope dilution and the turnover rate. The residual joint fluid nuclide was shown to be (32P)PPi. The PPi pool was generally smaller and the rate of turnover was greater in clinically inflamed joints. The mean plus or minus SEM pool size (mu-moles) and turnover rate (percent/hour) in PG knees was 0.23 plus or minus 0.07 and 117 plus or minus 11.9, hydrolysis rate (%/h) to Pi was 27.7 plus or minus 13.2; in OA knees: 0.45 plus or minus 0.26 and 72 plus or minus 9.2, hydrolysis 6.9 plus or minus 0.9; in gouty knees: 0.8 plus or minus 0.41 and 50 plus or minus 11.6, hydrolysis 9.8 plus or minus 2.8; and in RA knees: 0.14 plus or minus 0.14 and 114 plus or minus 35.8, hydrolysis 236 plus or minus 116. PPi turnover (mumoles/hour) correlated with the degree of OA change present in the joint as graded by radiologic criteria irrespective of the clinical diagnosis. Mean PPi turnover in joints with advanced OA was greater than in those with mild or moderate changes (P smaller than 0.001), but the mild and moderate groups showed no significant difference. We conclude that synovial PPi turnover and elevated PPi fluid concentrations are not specific for PG patients, and that these factors alone cannot be the only determinants of CPPD crystal deposition.


Plaque accumulation on composite surfaces after various finising procedures.

A clinical study was undertaken to determine the rate at which plaque accumulates on the surfaces of composite restorations on which four finishing techniques were used. Plaque on these surfaces was compared with that on control surfaces with polished metallic or glazed porcelain restorations, and unrestored enamel surfaces. Composite surfaces were covered with plaque after 24 hours regardless of the finishing technique used. Plaque did not collect on adjacent control teeth to this degree until the third day, although the diffence remained statistically significant. Surface discrepancies on the polished composites were shown in scanning electron micrographs and with laboratory measurements.


Effect of Bordetella pertussis extract and vasoactive amines on vascular permeability.

Vasxular permeability to Evans blue dye and 131-I-labeled human serum albumin was studied in normal mice and in mice treated with alkaline saline extracts (SE) from Bordetella pertussis cells. Skin sites inoculated intracutaneously with small doses of histamine, serotonin, or a combination of these 2 substances were more permeable in SE-treated mice than in normal animals. Intravenously administered catecholamines were able to reduce in varying degrees the vascular permeability induced by serotonin or by histamine in normal mice; in SE-treated mice the catecholamines were less effective. The relative effectiveness of intravenously administered catecholamines to reduce vascular permeability in normal or SE-treated mice was ranked as follows: isoproterenol greater than epinephrine greater than norepinephrine. When catecholamines were given concomitantly with histamine and serotonin in the skin test site, the permeability in both normal and SE-treated mice was again reduced or blocked, but isoproterenol was only weakly effective in this instance. Their relative effectiveness was epinephrine greater than norepinephrine greater than isoproterenol. The possible explanations for these results are discussed.


Evidence for the complex nature of the ganglioside receptor for cholera toxin.

Choleragenoid binds more slowly and less strongly than cholera toxin to intestinal mucosal cells, and even less strongly to free ganglioside in solution. However, binding to ganglioside is greatly enhanced when the ganglioside is in the form of an insoluble complex with cerebroside. These findings suggest that both the binding and the active components of the toxin molecule may be necessary for optimal binding of the toxin to the intact cell, and that the ganglioside in the cell receptor is in a complex form. Choleragenoid only partially blocks the action of the toxin on ruptured cells. This observation indicates that, while binding to a membrane receptor is necessary for the action of the toxin on the whole cell, it is possible to activate adenyl cyclase in a perforated cell by a process apparently independent of membrane binding; however, this activation may be possible only if the toxin preparation contains the active component dissociated from choleragenoid.


Antiviral activity of an adenosine deaminase inhibitor: decreased replication of herpes simplex virus.

A unique seven-membered heterocyclic-ring inhibitor of adenosine deaminase was studied. One preparation of the compound inhibited replication of herpes simplex virus in the absence of adenine arabinoside. In this capacity, the minimal inhibitory concentration of deaminase inhibitor for herpes simplex virus type 1 (HSV-1), with 50 percent reduction of plaque-forming units as the end point, was 37.7 mug/ml. This activity compared favorably with the inhibitory activity of ara-hypoxanthine (34.1 mug/ml). Another preparation of deaminase inhibitor lacked antiviral activity. On the other hand, the adenosine deaminase inhibitor was active at a concentration of 0.009 mug/ml as a potentiator of the inhibition of HSV-1 by adenine arabinoside. The potentiation of adenine arabinoside by deaminase inhibitor is about 4,000 times more potent than the activity of the direct inhibitory effect on HSV-1. The nature of the possible contaminant of the preparation in question is unknown. Coformycin, another inhibitor of adenosine deaminase, had no antiviral activity in the absence of adenine arabinoside.


The effect of dexamethasone on the control of plasma aldosterone concentration in normal recumbent man.

In order to evaluate the control of the circadian rhythm of plasma aldosterone concentration in man, plasma aldosterone, cortisol, sodium and potassium concentrations, and plasma renin activity (PRA) were measured in samples obtained at 20-minute intervals from 0200 to 0800 in sodium repleted (180 mEq. per day of diet) and sodium depleted (22 mEq. per day of diet) normal subjects. During sodium replete studies, plasma aldosterone and cortisol concentrations were significantly correlated (p greater than 0.01) in all 4 subjects. Plasma aldosterone also correlated with PRA (p greather than 0.01) in 2 subjects and with potassium (p greater than 0.01) in one. Episodic increases in plasma aldosterone concentration were observed despite suppression of ACTH by dexamethasone treatment. Plasma aldosterone concentrations were significantly correlated with PRA (p greater than 0.05 and p greater than 0.01) in only 2 of 4 subjects under these conditions. Following sodium restriction, plasma aldosterone concentrations were not significantly correlated with plasma cortisol and only infrequently with either PRA or potassium. When dexamethasone was administered during the low sodium diet, correlations with PRA (p greater than 0.001) were seen in 2 of 3 subjects and with potassium (p greater than 0.01) in 1 of 3 subjects. There was no significant correlation between plasma aldosterone and sodium concentration during any of the studies. These results are compatible with the concept that the relative importance of PRA, ACTH, and potassium in inducing changes in aldosterone production during the early morning hours is partially dependent upon dietary sodium, but also varies between individuals studied during similar sodium diets. The episodic alterations of plasma aldosterone concentration continued after ACTH was suppressed by dexamethasone pretreatment. These changes, in the absence of a consistent significant correlation with PRA and sodium and potassium concentrations, further suggest that another factor(s) may be important in controlling aldosterone production in recumbent normal subjects.


Conservative surgical treatment of mixed tumours of the parotid gland.

The author presents 44 cases of mixed tumours of the parotid gland, operated on by simple enucleation and not radical removal of the gland, which most authors maintain must always be carried out. There were 6 recurrences (13.6 percent), and the last appeared in 1965. He maintains that simple enucleation is justified. It is reasonable to hope for a further decrease in the number of recurrences with future improvement in surgical techniques.


Gel filtration of human urinary immunoreactive luteinizing hormone.

In human urinary concentrates and also in a urinary gonadotrophin standard (2nd IRP-HMG), gel-filtration analysis revealed three main peaks of immunoassayable luteinizing hormone (LH). A similar analysis of LH extracted from human pituitaries showed most of the activity in a peak of larger molecular weight, and only minor fractions in the positions of the urinary peaks. In an extract of normal human serum, analyss showed only one similar peak of large molecular weight, which also emerged before the urinary peaks. During an i.v. infusion of pituitary LH into normal men, the urinary LH activity increased but was still found only in the same three peaks on gel filtration, and all were of a molecular weight smaller than that of the infused material; but a higher proportion of the urinary LH was found in the earliest of these peaks compared with that found before infusion. Conversely, 20-35 h after the i.v. infusion, there was a slightly higher proportion of LH activity in the third peak of smallest molecular weight. These findings suggest that the urinary immunoassayable LH, which is found in three peaks of different molecular weights, is derived from the pituitary or serum LH of higher molecular weight. The changes in the proportions of larger or smaller molecular weight fractions in the urine during and after LH infusion suggest that the earliest peak may be disaggregated serum LH, while the last or smallest molecular weight peak may comprise metabolites of LH.


Physiological variation in abundance of oestrogen specific high-affinity binding sites in hypothalamus, pituitary and uterus of the rat.

High-affinity binding of [2,4,6,7-3-H]oestradiol-17 beta has been studied in cytosols prepared from hypothalami, pituitaries and uteri of female rats killed at different stages of the oestrous cycle, and in cytosols prepared from the hypothalami and pituitaries of male rats. In all cases the equilibrium dissociation constant of reaction was of the order of 10- minus 10 mol/1. The number of available high affinity sites per tissue (n) varied with physiological state. In females, n fluctuated with the oestrus cycle. In hypothalamus and pituitary, n fell by about 60 and 40% respectively in pro-oestrus, replenishment occurred during oestrus but could be delayed by phenobarbitone administration during the afternoon of pro-oestrus. In the uterus, n varied biphasically, there being peaks during dioestrus and oestrus, and troughs at pro-oestrus and metoestrus. The numbers of available sites at metoestrus were 12-5 times 10-9, 10-6 times 10-10 and 24-4 times times 10-10 for hypothalamus, pituitary and uterus respectively. In male rats, values for n were similar to those obtained for females at pro-oestrus (n/hypothalamus equals 6-8 times 10-9, n/pituitary equals 4-2 times 10-10). Binding was oestrogen specific in all the tissues studied.


A comparison of the effects of the calcitonins, steroid hormones and thyroid hormones on the response of bone to parathyroid hormone in tissue culture.

A bone culture system was used to compare the effects of several hormones on the response of 5-day-old mouse calvaria to parathyroid hormone (PTH). The results showed that salmon calcitonin was almost 10-5 times more active than any other hormone in preventing the PTH-induced release of calcium and caused a dose-related inhibition of calcium release over a range of 0-2-200 milli MRC units/culture. A high dose of calcitonin (200 milli MRC units) caused a net accretion of calcium in the absence of PTH. Progesterone and testosterone were more active than the naturally occurring oestrogens although a synthetic oestrogen (stillboestrol diphosphate) had approximately the same potency. High concentrations of these hormones caused a net accretion of calcium whether or not PTH was present. Cortisol was only effective at high doses, as was the steroid precursor cholesterol. In the present culture system the thyroid hormones (triiodothyronine and thyroxine) inhibited the action of PTH. It was concluded that these agents acted in a similar fashion to the oestrogens. That is, they prevented the accumulation of citric acid induced by PTH by reducing the rate of glycolysis. None of the hormones affected the inhibition of citrate oxidation caused by PTH. The results also showed that, whilst these hormones inhibited PTH-mediated bone resorption, they had an action on bone independent of PTH. Experiments with clomiphene citrate failed to demonstrate an oestrogen receptor in bone.


Development of parthenogenetic membranes in double-yolked and injected chicken eggs.

Development of parthenogenetic eggs is expressed in a Dark Cornish stock by either membrane or embryo formation. Double-yolked eggs have membranes developing in zero, one, or two yolks. The incidence of double-yolked eggs with two membranes was higher than expected when compared with the incidence of single-yolked eggs from the same hens. Membrane formation was increased over that of the controls by injecting saline, Ringer's solution, or ground crude membranes. Yolk and membranes filtered through a 3 mu filter or autoclaved did not increase membrane formation over that of the controls. Fertile membranes behaved like parthenogenetic membranes.


The physiological function of nitrate reduction in Clostridium perfringens.

Fermentation-balance studies have been carried out on Clostridium perfringens grown in the presence and absence of nitrate in the medium. Nitrate is able to serve as an electron acceptor for these bacteria, permitting increased growth yields over those obtained in its absence. This increase is due to an increase in the proportion of metabolite molecules which can participate in substrate-level phosphorylation reactions when an inorganic acceptor is available. The nitrate reduction can be regarded as a primitive form of anaerobic respiration in these bacteria, since it is clearly coupled to their energy metabolism and is not assimilative in function. We believe that the existence of this kind of energy metabolism in these bacteria has significant evolutionary implications.


The induction of intractranial neoplasms by the inoculation of avian sarcoma virus in perinatal and adult rats.

The induction of intracranial neoplasms following the intracerebral inoculation of avian sarcoma virus (ASV) in neonatal mammals is well established. The present study demonstrates the susceptibility of adult rats and compares the incidence and morphology of tumors induced by a uniform inoculum of the Bratislava-77 strain of ASV in adult, neonatal, and fetal Fischer 344 rats. Post-inoculation mortality varied significantly between groups inoculated at 1, 10 and 100 days and was most precipitous in perinatally inoculated rats. Percentage of tumor induction declined from 100% among rats inoculated at 1 day of age to 50% among rats inoculated at 100 days of age. The mean number of tumors/animal was inversely proportional to the logarithm of the age at inoculation. A large majority of tumors in each group were glial; the remainder were mesenchymal and mixed glial and mesenchymal. Neuroglial tumors included: mixed gliomas with oligodendroglial and astrocytic elements; and gemistocytic, pilocytic, fibrillary, anaplastic and protoplasmic astrocytomas. Tumors induced in perinates were more heterogeneous in histological pattern while tumors induced in perinates were more heterogeneous in histological pattern while tumors induced in older animals tended to be purely astrocytic and of uniform cell type. Mesenchymal tumors occurred primarily in the meninges and were common among animals inoculated perinatally but were rare among animals inoculated as adults. No neuronal tumors were encountered even among rats inoculated as early as 16 days of gestation.


Herpes simplex virus encephalitis in suckling mice: ultrastructural studies of virus replication.

Ultrastructural studies concerning the replication and release of a neuro-adapted strain of herpes simplex virus were carried out on acutely encephalitic mice. It was found that envelopment of nucleocapsid particles at the nuclear membrane of brain cells was inefficient, resulting in accumulation of unenveloped nucleocapsids in the cell cytoplasm. These nucleocapsids were associated with arrays of microtubules within cytoplasmic processes. Enveloped particles were rarely present in tissur spaces. Those that were seen appeared to be in a state of disintegration. This replication process of herpes simplex virus in the murine encephalitis model differs from that observed in tissue culture cells. The implications of these differences are discussed.


Ultrastructure of the terminals of an identified dopamine-containing neurone markedby intracellular injection of radioactive dopamine.

The terminal processes of an identified dopamine-containing neurone in the central nervous system of the water snail were located for study with the electron microscope. This was achieved by intracellular injection of 3-H-dopamine followed by light and electron microscope autoradiography. The neurone showed extensive branching in ganglia whereprevious electrophysiological work had shown the presence of postsynaptic neurones. The terminal processes in these ganglia contained dense-cored vesicles of 50-250 nm diametersimilar to those in the perikaryon. Vesicles were found in large numbers in certain lacalized regions which may be synaptic terminals. The vesicles, which appear to take updopamine, tended to be smaller in the presumed terminal regions than in the preterminals and the perikaryon. No membrane specializations associated with typical synapses were observed.


Possible intervention of insulin, cyclic AMP, and glucocorticoids in protein-sparing action of dietary carbohydrate in rats.

The present study was carried out to examine the possible intervention of some hormones in the "protein-sparing/actions of dietary carbohydrate and fat. Administration of either a carbohydrate meal or a fat meal to fasted rats caused a reduction in the urinary output of urea and total nitrogen with a concomitant decrease in rate of urogenesis in liver. as well as the activities of some amino acid-catabolizing enzymes in liver. The sparing action of carbohydrate but not that of fat was abolished in alloxan-diabetic rats. Feeding rats a carbohydrate meal caused a marked reduction in the amount of cyclic AMP in liver. The change was coincident with a reduction in the level of plasma urea and the urinary output of urea and total nitorgen. Administration of dibutyryl cyclic AMP abolished the carbohydrate-induced depression of urinary output of urea and total nitrogen as well as partially the activity of serine dehydratase in liver. Feeding a carbohydrate meal resulted in a significant reduction in the level of corticosterone in plasma. However, the possible intervention of glucocorticoids in the protein-sparing action of carbohydrate was ruled out inasmuch as the action of carbohydrate was also observed in the adrenalectomized rats. The overall results suggest that the protein-sparing action of dietary carbohydrate may be exerted in a different fashion from that of fat, that is, through depression of cyclic AMP in liver and thus the reduction of the degradation of amino acids in liver


Cyclic AMP metabolism and nucleic acid content in the lymphocytes of the thymus, spleen, and lymph nodes of protein-deficient rats.

The concentration of cyclic AMP (cAMP) and its metabolites (5'-AMP and adenosine) as well as the adenyl cyclase, cAMP phosphodiesterase, and 5'-nucleotidase activities were determined in lymphocytes of thymus, spleen, and lymph nodes of control and protein-deficient rats. The values of these parameters, when expressed as per milligran DNA and as per 10-8 cells, but not always when expressed as per milligran protein, were much lower in the thymus as compared with the spleen and the lymph nodes in the control rats. The protein-deficient diet increased the nucleotide concentrations in the thymus and spleen lymphocytes on a per milligram DNA basis except those of thymic cAMP, which did not change. The same diet also increased the activities of the enzymes involved in the cAMP metabolism in thymic, splenic, and lymph node lymphocytes. Such a peculiarity could be related to the reduction of the mitotic activity of lymphocytes caused by protein deficiency since an inverse relationship has been reported between this activity and the synthesis of cAMP. On the other hand, it was noted that purified lymphocyte suspensions contained paradoxically higher amounts per cell of DNA, RNA, and protein in the thymus, spleen, and lymph nodes of protein-deficient rats as compared with those of the control rats. However, when the cell preparations were not purified, only the lymph node cells displayed a strong increase in their DNA content. Prolongation of the S phase of the cell cycle in these lymphocytes is suggested.


Timed-release depot for anticancer agents.

The timed-release of anticancer agents from composities with poly (lactic acid) was studied in rats. In the case of cyclophosphamide-poly (lactic acid) composites, 67% of the administered dose was released within 34 days. With cis-dichlorodiammineplatinum (II), the amount of drug released was only 9.3% within the same period. This difference might be attributed to the different solubilities of these two drugs in the polymer. Electron spectroscopy chemical analysis, a new tool, was used to investigate the extent of diffusion of drugs in polymer films.


Quantitative GLC determination of conjugated estrogens in raw materials and finished dosage forms.

A GLC method is described for the quantitative analysis of conjugated estrogens. The procedure was used for the analysis of various estrogen mixtures obtained from formulations containing estrogen sodium sulfate salts. The method involves slurrying or dissolving a portion of the formulation pH 5.2 buffer, hydrolysis with sulfatase enzyme, extraction with ethylene dichloride, anf finally, GLC separation and quantitation of the corresponding trimethylsilyl ethers on a 2.5% diethylene glycol succinate column.


Release of norepinephrine and dopamine-beta-hydroxylase by nerve stimulation. IV. An evaluation of a role for cyclic adenosine monophosphate.

The effects of several cyclic nucleotide analogs and of phosphodiesterase inhibitors on the release of norepinephrine (NE) and dopamine-beta-hydroxylase activity (DBH) by electrical stimulation were studied in the isolated, perfused cat spleen. N-6-butyryl-3',5'-adenosine monophosphate (mbcAMP), 8-methylthio-3',5'-adenosine monophosphate, 8-bromo-3',5'-guanosine monophosphate (8-Br-cGMP) and two potent phosphodiesterase inhibitors: 1-methyl-3-isobutylxanthine and 4-(3-butoxy-4-methoxy-benzyl)-2-imidazolidinone (Ro 20-1724) enhanced the overflow of NE and total H and reduced pressure responses elicited by nerve stimulation. A concomitant outflow of DBH activity was observed in the presence of mbcAMP, 8-Br-cGMP or Ro 20-1724. Synergistic effects on the nerve stimulation-mediated overflow of NE and DBH were obtained with low concentratons of Ro 20-1724 and mbcAMP (5 muM). Adenosine 5'-monophosphate produced a very slight increase in nerve stimulated release of NE and DBH activity in concentrations which inhibited pressor responses considerably. cAMP produced slight inhibition of pressure responses but failed to influence the release of either NE or DBH activity during nerve stimulation. In contrast to the enhanced overflow of NE and DBH activity induced by nerve stimulation, with the exception of Ro 20-1724, the spontaneous release of these substances was not modified by any of the cyclic nucleotide analogs or phosphodiesterase inhibitors examined. This effect of Ro 20-1724 can probably be explained by the ability of this compound to inhibit the activity of monoamine oxidase and therefore reduce the formation of deaminated metabolites. The present results suggest that cyclic nucleotides are not directly responsible for the release of the adrenergic neurotransmitter, but may facilitate the normal process of release by nerve stimulation. Phentolamine, a blocker of the alpha adrenergic receptors, produced a marked increase in the nerve stimulation-mediated overflow of NE, total H and DBH activity and inhibited pressure responses. This effect was several times greater than that produced by either cyclic nucleotide analogs or phosphodiesterase inhibitors. In addition, the effect of phentolamine was not modified by prior treatment with 1-methyl-3-isobutylxanthine or Ro 20-1724, suggesting that the effect of phentolamine is not related to its ability to inhibit phosphodiesterase and is probably not mediated via an increase in cAMP.


Increased sensitivity to dopaminergic agents after chronic neuroleptic treatment.

Male CF-1 mice were treated for 14 days with diets containing haloperidol, thioridazine HCl and 4'-fluoro-4[[4-(p-fluorophenyl)-4-methoxycyclohexyl]-amino]-butyrophenone HCl (U35,777A). At various times during and after neuroleptic treatment, spontaneous and d-amphetamine-stimulated motor activity were measured. Two days after cessation of treatment, the mice displayed enhanced spontaneous and d-amphetamine-stimulated motor activity. This effect was no longer apparent 9 days after neuroleptic intake was terminated. With a quantal test based on the climbing activity induced by apomorphine, it was determined that mice were also supersensitive to apomorphine at 2 days but not 9 days after withdrawal from chronic haloperidol. In an attempt to correlate this supersensitivity to a biochemical parameter related to receptor function, dopamine-stimulated adenyl cyclase activity was assayed in striatal homogenates of mice 2 days after haloperidol withdrawal. No alteration in this parameter was observed. Likewise, the ability of apomorphine to elevate striatal cyclic adenosine monophosphate concentrations in vivo was unaltered by withdrawal from chronic haloperidol. Chronic treatment with neuroleptics results in a brief supersensitivity to dopaminergic agents. This effect does not appear to be accompanied by increases in dopamine-stimulated adenyl cyclase activity in the corpus striatum.


Increased extrajunctional acetylcholine sensitivity produced by chronic acetylcholine sensitivity produced by chronic post-synaptic neuromuscular blockade.

1. Anaesthetized rats were paralysed for periods of up to 3 days by chronic administration of D-tubocurarine (DTC), succinylcholine or alpha-bungarotoxin. 2. After 3 days of treatment with DTC, the phrenic nerve remained active. Neuromuscular transmission and spontaneous miniature end-plate potentials (m.e.p.p.s) were restored after removal of the DTC. Resting potentials and input resistances of muscle fibres that had been paralysed for 3 days were similar to those in denervated fibers. 3. Chronic neuromuscular blockade increased the binding of [125-I]-alpha-bungarotoxin by extrajunctional regions of muscle. The time course of the increase was similar to that seen after denervation. Binding to muscles from animals that were anaesthetized and respirated, but not paralysed, was not increased. 4. Three days of paralysis increased the sensitivity of the extrajunctional muscle membrane to acetylcholine (ACh) applied by iontophoresis. 5. Approximately the same proportion of muscle fibres from muscles paralysed for 3 days gave overshooting action potentials in the presence of tetrodotoxin 10-minus 6 g/ml. as did fibres form muscles denervated for 3 days. 6. Chronic paralysis did not change the accumulation of acetylcholinesterase above a ligation in the sciatic nerve. 7. These results are consistent with the idea that extrajunctional ACh sensitivity is normally controlled by muscle activity.


The distribution of acetylcholine sensitivity at the post-synaptic membrane  of vertebrate skeletal twitch muscles: iontophoretic mapping in the micron range.

1. The distribution of acetylcholine (ACh) sensitivity was mapped in skeletal twitch muscles of the snake, frog and mudpuppy with iontophoretic methods that provide a resolution in the mum range. 2. The preparations were thin sheets of muscle fibres that were viewed with Nomarski optics, giving sharp definition of cellular detail. The muscles in the snake were especially suitable. Their motor nerves terminate in a compact cluster of synaptic boutons that rest in distinct craters on the muscle surface. After treatment with collagenase the motor nerve and its terminal boutons can be removed, exposing the subsynaptic membrane in the craters. 3. The slopes of dose-response curves obtained by iontophoretic application of ACh were expressed in mV/nC and used as an index of ACh sensitivity. The areas of highest sensitivity, tested either with the terminals in place or removed, were those immediately under the presynaptic terminals. The greatest subsynaptic sensitivities were about 5000 mV/nC, and the time course of the potentials caused by ACh released iontophoretically closely matched that of synaptic potentials set up by ACh released by the nerve. 4. The sensitivity of the extrasynaptic surface less than 2 mum away was at least 50 times lower than that of the subsynaptic membrane. The low extrasynaptic sensitivity declined still further at greater distances. 5. Acetylcholinesterase was shown physiologically to be confined to subsynaptic  areas. No activity of the enzyme was detected in extrasynaptic areas beyond about 2 mum from the edge of the synapse. 6. The confinement of high densities of receptors and of acetylcholinesterase to the subsynaptic membrane in muscles is also a feature in parasympathetic neurones. It is suggested that similar specialization may be a widespread property of neurones with chemical synapses.


Active chloride secretion by in vitro guinea-pig seminal vesicle and its possible relation to vesicular function in vivo.

1. The guinea-pig seminal vesicle in vivo is characterized by a transmural electrical potential difference of approximately 11 mV with the lumen electrically negative with respect to the interstitial fluid. The concentrations of Na, Cl and K in the vesicular fluid are 13, 15, and 0-4 mM, respectively. 2. When mounted as a flat sheet in a short-circuit apparatus, guinea-pig seminal vesicles initially undergo a decline in the transmural electrical potential difference and short-circuit current ('low phase') followed by a spontaneous increase in these parameters ('high phase'). 3. During the low phase, net C1 movements across the tissue do not differ significantly from zero, and there is a small 'residual' current that is unaccounted for. 4. During the high phase, there is a significant active C1 secretion into the mucosal solution, not detectable net movement of Na and an unaccounted for or 'residual' current that is equal to that found in the low phase. 5. Theophylline, dibutyryl-3'-5' cyclic adenosinemonophosphate,prostaglandin E1 and prostaglandin F2alpha markedly stimulate the transmural electrical potential difference and short-circuit current during the low phase, but have no effect when added to the bathing solution during the high phase. 6. Diffusion potentials determined across in vitro seminal vesicles suggest that the spontaneous transmural electrical potential difference in vivo may be attributable to the large ionic asymmetries between the vesicular fluid and the plasma. 7. It is postulated that two phases are involved in the elaboration of seminal vesicular fluid. The initial phase, following emptying of the vesicle, is characterized by the secretion of electrolytes, organic molecules and water. Active C1 secretion presumably regulated by intracellular cyclic adenosinemonophosphate and/or prostaglandins may be the driving force for this initial secretion of electrolytes. Following this secretory phase, electrolytes and water are reabsorbed, thereby concentrating the organic components in the vesicular reservoir.


On the association between transmitter secretion and the release of adenine nucleotides from mammalian motor nerve terminals.

1. Conventional electrophysiological techniques were used to record from isolated rat phrenic nerve-hemidiaphragm preparations. After periods of rest (20 min) or nerve stimulation (7/sec for 20 min) the bathing medium of the preparation was removed and assayed for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) using a sensitive modification of the firefly luciferase method (Silinsky, 1974). 2. In the presence of tubocurarine and normal (2 mM) calcium, fourteen periods of nerve stimulation (eight preparations) caused the appearance of ATP and/or ADP in amounts ranging from 28 to 641 p-mole. Experiments using carbachol (30 muM or 1 mM) suggested that this nucleotide efflux was not produced by a secondary action of released acetylcholine (ACh). 3. Stimulation of isolate phrenic nerve trunks at 7/sec for 20 min did not cause the efflux of ATP or ADP. 4. In solutions of normal osmotic pressure and reduced calcium concentrations (0-1 mM or 'calcium-free'), stimulation failed to release adenine nucleotide from non-contracting preparations. 5. Diaphragms were bathed in normal calcium and indirectly stimulated at 11/sec for 80-90 min in the presence of 5 times 10-minus 5 M hemicholinium-3. After all detectable signs of ACh release were eliminated, nerve stimulation failed to release ATP or ADP. 6. These results in conjunction with experiments on the hydrolysis of exogenous ATP suggest that ATP is released from the motor nerve ending and is subsequently degraded by enzymatic activity. It is also suggested that the released nucleotide may be derived from the cholinergic vesicle.


A note of the mechanism by which inhibitors of the sodium pump accelerate spontaneous release of transmitter from motor nerve terminals.

1. The actions of 0-1 mM ouabain and of K-free Ringer have been examined at the frog neuromuscular junction. 2. After a delay of more than 30 min, ouabain produces an increase in the miniature end-plate potential (m.e.p.p.) frequency. This increase occurs unchanged in Ca-free Ringer containing 1 mM-EGTA and is therefore unlikely to be due to an entry of Ca into the motor nerve terminals. 3. If the nerve to the preparation is stimulated repetitively in Ca-free Ringer containing 0-1 mM ouabain and 1 mM-EGTA the response of the m.e.p.p. frequency depends on the timing of the tetanus relative to the beginning of the ouabain treatment. 4. During the first 30 min of exposure to ouabain, the tetanus produces a small, transient increase in the m.e.p.p. frequency similar to that which occurs before ouabain is present. After about 30 min the same tetanus produces large, irreversible increases in the m.e.p.p. frequency. 5. Superfusion of an end-plate with K-free Ringer causes an immediate exponential rise in the m.e.p.p. frequency that is unaffected by the presence of external Ca ions. On replacing the normal K of the Ringer (2 mM) the m.e.p.p. frequency recovers quickly to its original value. 6. Late in an exposure to 0-1 mM ouabain the m.e.p.p. frequency becomes extremely sensitive to changes in the external Na concentration, [Na]o. Reducing [Na]o increases the m.e.p.p. frequency. The sensitivity to [Na]o is independent of external Ca ions or whether the isotonic substitute for NaCl is LiCl or sucrose. 7. It is suggested that the spontaneous release of transmitter is facilitated, in some way, by the changes in the monovalent cation content of the nerve terminals that result from blocking the Na-K exchange pump. The Na sensitivity of the m.e.p.p. frequency that develops simultaneously can be explained if a Na-dependent Ca efflux system is present in the membrane of the presynaptic terminals.


The relationship of adenosine triphosphatase activity to tension and power output of insect flight muscle.

1. On a simple model of actomyosin interaction, the tension cost (ATP hydrolysed/unit tension) and the frequency of low amplitude oscillation optimum for work production are both determined by the rate of detachment from the actin filament of the myosin crossbridge. To test this model, the two parameters were measured under different conditions using glycerol-extracted Lethocerus cordofanus dorsal longitudinal flight muscle fibres. 2. The ATPase activity of the static muscle rose by an amount approximately proportional to the rise in tension as the muscle was stretched. 3. When the muscle fibres were sinusoidally oscillated at 5-10 Hz by 2% of their resting length they produced a large amount of mechanical power and hydrolysed approximately twice as much ATP per unit mean tension as they did when static. The ATPase activity was linearly related to the mean tension during oscillation. 4. The experiments were repeated at temperatures between 12 and 30 degrees C and the tension cost and the optimal frequency of oscillation of the fibres were found to rise with temperature. 5. Removal of phosphate from the incubating medium reduced both the tension cost and the optimal working frequency. Addition of pyrophosphate or sulphate reduced both parameters still further. 6. From these results the tension cost of static muscle was shown to be proportional to its optimal working frequency. 7. ATPase activity rose monotonically with power production at work-producing frequencies and at moderate degrees of stretch. A high absolute efficiency was found under a wide range of conditions. 8. The proportionality between tension cost and optimal frequency is evidence for the proposed model of actomyosin interaction.


Behavior modification approach in a partial day treatment center.

Since the beginning of the program in September 1970, we have found that a combined behavioral program of individual therapy, home intervention, and group therapy to be the most effective for maintaining a person in his community. In addition, we have not seen a need for a full eight-hour day in the clinic. Rather, we operate on the principle that it is more adaptive to keep a patient functioning in the community (natural environment) with support from us. For the extremely or severely disorganized person, we have found the application of behavior modification principles to be the most useful in the terms of establishing and maintaining adaptive behaviors, of interruptin and decreasing maladaptive behaviors and of offering support and guidance (generalization) to patients and families. We have also increased the amount of individual therapy and intensified the home intervention program. These changes have been based upon subjective data as well as objective data (change in patient behavior  while receiving and following a particular mode of therapy). The program has undergone many changes since its inception. We have found the behavior modification modality to be effective for establishing a program that patients can generalize to their community setting. Our constant thrust is to utilize already existing agencies in the community and to optimally assist the patient so that he may remain in his community. We have gone from an eclectic program to a systematic behavior modification program. The present behavioral program has been demonstrated to be an effective modality for the training of personnel. We are grateful to our staff for their cooperation and suggestions in making the program successful. In addition, we are indebted to our late Director, Curtis W; Gifford--a loyal and dedicated friend. We particularly appreciate his administrative skills and encouragement in developing an autonomous program.


Accountability: a right of the rape victim.

Accountability by the professional is especially important in a crisis situation.  Rape is experienced by victims as a life threatening situation and it thus triggers an acute stress reaction which requires them to go through a long-term reorganization process.  As a crisis situation, it makes them hypersensitive to the attitudes of those people to whom they turn for help and assistance.  Clients--in this case rape victims--benefit when professionals are careful to be accountable to them.  The victim counselors defined and redefined their role to the victims through their work with the victim as well as explaninig the services they could provide.  In reviewing the reactions of 146 victims to treatment by police, hospital, and counselors, one theme emerged again and again: clients want explanation from professionals about role expectations and about procedures that are to be done.


Tactics for counteracting staff apathy and hopelessness on a psychiatric unit.

In summary, then, the five following tactics are minimally demanding, and can provide high rewards in the battle against apathy and hopelessness on a psychiatric ward.  1. Identifying social system mechanisms.  2. Validating perceptions of ward happenings with a trusted other.  3. Giving (and hopefully receiving) "informational-support."  4. Using physical presence or being highly visible.  5. Chatting informally with other staff members.


Nutritional concerns of addicts in treatment.

Some of the subcultural phenomena affecting diet patterns of methadone patients have been utilized to derive principles of treatment intervention.  Attitudes and values related to time, treatment personnel, and group identification are important cultural determinants of beliefs and behaviors affecting nutrition.  The proposed program of intervention is based on ethnographic observations.  This suggested program emphasized the inclusion of cultural identity, sense of uniqueness and group information sharing and reinforcement.  These characteristics are valued highly in the informants' subcultural group, and are important components of acceptable proprosals for change within this group.


Chemotherapeutic nitroheterocycles. 18. 2-(5-Nitro-2-imidazolylmethylene)-1-indanones, -1-tetralones, and -acetophenones substituted by aminoalkoxy groups.

2-(5-Nitro-2-imidazolylmethylene)-1-indanones, -1-tetralones, and -acetophenones substituted by aminoalkoxy groups and related compounds (41-69, table ii) were synthesized and their antimicrobial activities were evaluated (table iii). Some of these compounds (e.g. 47, 52, and 59) suprisingly exhibited a broad antibacterial spectrum including Proteus species and Pseudomonas aeruginosa. Extraordinary antitrichomonal activities could also be observed in vitro (MIC of compound 59, 0.0004 pg/ml) and six of the title compounds (48, 49, 52, 58, 64, 66) displayed in vivo activity in mice against Trichomonas vaginalis comparable to that of metronidazole (70).


Immunologic, virologic, and genetic aspects of mammary tumor virus-induced cell-surface antigens: presence of these antigens and the Thy 1.2 antigen on murine mammary gland and tumor cells.

The distribution of the normal differentiation antigen Thy 1 and the mammary tumor virus (MTV)-induced antigens or antigen complexes MLm and MLr were studied in mouse mammary gland cells, mammary tumor cells, and other cell types, by use of ascites leukemia cells of the GR mouse strain as target cells in the cytotoxicity test. The Thy 1.2 antigen was detected by an AKR antiserum to C3Hf thymocytes. MLm was shown by a homologous C57BL antiserum to GRSL2 leukemia (absorbed in vivo in GR mice); MLr was detected by a rabbit heterologous antiserum (absorbed in vivo in C57BL or GR mice and in vitro with BALB/c milk) prepared against Tween 80- and ether-treated purified B particles. Sera from Sprague-Dawley rats bearing murine leukemia virus (MuLV)-producing syngeneic tumors were not cytotoxic or only slightly cytotoxic for GR leukemias transplanted in vivo, which indicated that MuLV-induced antigens were absent or present in very low quantity in such leukemias. The MLr and MLn antigens or antigen complexes were possibly identical to the mammary leukemia (ML) antigen, since they could be detected not only on GR but also on DBA/2 leukemia cells and since their distribution was exactly the same as that of MTV. Both the MLr and MLm antigens were present in purified B particles, and antigenic activity were present in purified B particles, and antigenic activity was enhanced by destruction of the purified virus particles. The antigens were about eightfold enriched in a preparation of B-particle envelopes, as shown by quantitative cytotoxicity absorption (CYTA) tests. Purified nucleoid fractions of B particles were only lightly positive for the antigen, probably due to envelope contamination. One dominant gene was responsible for the expression of MLr, as shown by CYTA tests with mammary glands of individual animals of segregating crosses between the GR strain with high mammary cancer incidence and strains with low incidence. This gene was closely linked with or was possibly identical to 1) the gene for cytoplasmic MTV gs antigen expression as seen by fixed cell immunofluorescence, and 2) the gene causing mammary tumors in the GR mouse strain.


Distribution and antibody-induced redistribution of a mammary tumor virus-induced and a normal antigen on the surface of mouse leukemia cells.

By means of the indirect membrane immunofluorescence test, the distribution and antibody-induced redistribution (patching and capping) of a mammary tumor virus-induced (MLr) and a normal (Thy 1.2) cell-surface antigen were compared on mouse thymocytes and leukemia cells (GRSL2). At 0 degrees C Thy 1.2 fluorescence was ringlike and more intense on GRSL2 cells than on thymocytes, whereas MLr fluorescence on GRLS2 cells at this temperature was patchlike and brighter than Thy 1.2 fluorescence. At 20 or 37 degrees C, capping of Thy 1.2 on both cell types was readily achieved but MLr capping occurred only in a few GRSL2 cells and was less pronounced. However, after addition of the secondary antibodies, MLr capping was markedly increased by gradual cooling of cells to about 17 degrees C. Conversely, after addition of antibodies at 0 degrees C, gradual warming of cells under the fluorescence microscope resulted in extensive capping both of MLr and Thy 1.2 at approximately 13-14 degrees C. Rapid cooling or rapid warming led to almost instantaneous capping. These results may be explained by the occurrence of phase transitions or phase separations in the particular temperature range. Another difference between capping of Thy 1.2 and MLr was that the former caps were small and eventually were endocytosed, whereas the MLr caps were large and were exfoliated from the cells.


Protective tumor immunity induced by potassium chloride extracts of guinea pig hepatomas.

Tumor-specific antigens of cells of the diethylnitrosamine-induced hepatomas in strain-2 guinea pigs were extracted with 3 M KCl. Immunization of normal animals with the extracted tumor antigens in adjuvant protected them against a subsequent challenge with viable tumor cells. Extracted tumor-specific antigens were less effective immunogens than viable tumor cells for both of two antigenically distinct lines.


Isolation and characterization of viruses from natural outbreaks of reticuloendotheliosis in turkeys.

We have described the in vitro isolation of type-C RNA viruses from two outbreaks of a fatal neoplastic disease in turkeys we diagnosed as reticuloendotheliosis. The virus had a density of 1.16 g/ml in sucrose gradients, had a DNA polymerase capable of using both endogenous and exogenous (synthetic) templates, and was infectious in vitro for turkey and chick cells. The culture-propagated virus was oncogenic for turkeys. The virus isolates were unrelated to avian leukosis virus and antigenically identical to reticuloendotheliosis virus (REV) strain T. Thus our studies suggested that REV is a causative agent of naturally occurring, fatal leukosis in turkeys.


Hormonally induced renal neoplasia in the male Syrian hamster and the inhibitory effect of 2-bromo-alpha-ergocryptine methanesulfonate.

One group of male Syrian hamsters received diethylstilbestrol (DES) over a period of 9 months. All developed widespread and severe renal tumors. Another group of male Syrian hamsters was given DES plus 2-bromo-alpha-ergocryptine methanesulfonate (CB154) over the same period. These hamsters either did not develop renal tumors or had renal tumors that were minimal in severity. DES treatment induced hyperplastic and neoplastic changes in the intermediate lobe of the pituitary glands of treated animals, increased the number of prolactin-secreting cells, and decreased the number of somatotrophin-secreting cells. Adding CB154 significantly inhibited those changes in the pituitary gland. Histopathologic and ultrastructural examinations indicated that the renal tumors were carcinomas originating from proximal convoluted tubules.


Clinicopathologic characterization of Herpesvirus saimiri malignant lymphoma in New Zealand white rabbits.

Twenty-two of 39 rabbits inoculated with Herpesvirus saimiri developed malignant lymphoma and either died or were killed between 17 and 165 days after inoculation. No clinical signs were present in animals developing the disease before 46 days, but all other rabbits had a severe conjunctivitis, nasal discharge, and dyspnea resulting from a lymphocytic invasion of the ocular and nasal tissues. Four rabbits developed terminal leukemia. Pathologically, the disease resembled H. saimiri malignant lymphoma in nonhuman primates; there was extensive diffuse infiltration of most organs and tissues with either a lymphocytic or lymphoblastic infiltrate. Tumor nodules or masses seen in some forms of malignant lymphoma were not present. In contrast to nonhuman primates, all affected rabbits showed invasion of the skin of the nose and eyelids, conjunctiva, iris, ciliary body, and choroid. In 3 rabbits there was slight infiltration into the brain, not noted in nonhuman primates. The susceptibility of rabbits extended the host range of H. saimiri beyond the order Primates.


Nuclear magnetic resonance studies of cancer. VI. Relationship among spin-lattice relaxation times, growth rate, and water content of Morris hepatomas.

Relaxation time(s) (T1), growth rates, and the water content of six Morris hepatomas, several murine tumors, and a selection of normal tissues indicated that malignant tumors did not always exhibit longer T1 values than any normal tissue, as previously suspected. This overlap raised the possibility of confusion between normal and malignant tissues studied by this method. Tissue T1 values depended primarily on the hydration of the tissue and correlated well with water content determinations. A rough correlation of T1 and water content with tumor growth rate was also observed.


Maturation and differentiation of B16 melanoma cells induced by theophylline treatment.

Recent studies suggested that 3',5'-cyclic AMP (CAMP) may be involved in the regulation of cell proliferation and differentiation. Theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, elevated intracellular cAMP. A melanotic clone of the B16 melanoma was treated with theophylline and studied in vitro and in vivo. With 12 hours after 1.0 mM theophylline was added to growing cultures, the number of cells incorporating tritiated thymidine (3-H-TDR) and the rate of uptake of 3-H-TDR into DNA were significantly reduced. After 7 days, the number of cells in the control cultures increased twenty-four times, whereas theophylline-treated cells increased only sixfold. Compared to the controls, the theophylline-treated cells contained ten times the melanin and an elevated cAMP content. Stimulation of melanogenesis and inhibition of proliferation increased progressively with duration of exposure to theophylline. After 5 days of culture with theophylline, cells were assayed for plating efficiency in theophylline-free medium. Although the number of colony-forming cells was unaffected by previous exposure to theophylline, the colonies were composed of fewer cells inoculated into syngeneic hosts were less tumorigenic than untreated cells. However, theophylline treatment of hosts bearing B16 tumors failed to reduce the tumor growth rate, and theophylline did not potentiate the growth inhibition resulting from treatment with the synthetic polyribonucleotide, polyinosinic-polycytidylic acid.


R-type virus-like particles in avian sarcoma virus-induced rat central nervous system tumors.

Morphologically distinct virus-like particles (VLP), similar to R-type VLP, were observed by electron microscopy in experimental rat central nervous system tumors induced with the B-77-C strain of avian sarcoma virus (ASV). R-type VLP have a characteristic internal radial structure and were observed previously only in hamster cells and in an established bovine cell line. They were not observed in the B-77 ASV inoculum used to induce the rat tumors or in the B-77 induced hamster glioma cells from which the B-77 was rescued. Nevertheless, the genome of an endogenous hamster R-type particle also might have been rescued and carried in the B-77 inoculum. Alternatively, R-type VLP may exist in a number of animal species, including the rat, and may be expressed in certain conditions such as neoplastic transformation.


Polyoma virus strain with enhanced synthesis of capsid protein.

A study of the immunochemical characteristics and the synthesis of the capsid proteins of two polyoma virus strains (3049 and 1pS) was carried out to determine the mechanism responsible for the unique accumulation of those structural polypeptides in the cytoplasm of cells infected with the 3049 strain. Antisera prepared against disaggregated virus peptides and whole virus were used to measure the quantity of virus-specific antigens in cells infected by the two strains by using an indirect radioimmunoassay technique. The 3049-infected mouse embryo cells were found to contain several-fold more antibody-binding material than those infected with the 1pS strain. Furthermore, the cytoplasmic fraction of 3049-infected cells also contained more antibody-binding activity, supporting the hypothesis that the phenotype of the 3049 virus (cytoplasmic capsid protein) was a reflection of the increased synthesis of the capsid polypeptides.


Comparative studies on the structural phosphoproteins of mammalian type C viruses.

The major phosphoprotein common to woolly monkey sarcoma virus, gibbon ape lymphosarcoma virus, and type C viruses of the lower mammalian species (mouse, rat, cat), with the exception of the endogenous cat virus (RD-114), is the polypeptide of about 12,000 molecular weight. The protein-phosphate bond in this polypeptide of several viruses is of the phosphoserine variety excepting gibbon ape virus, which contains both phosphoserine and phosphothreonine. The primary phosphoprotein of RD-114 virus and the endogenous baboon type C virus, on the other hand, is the polypeptide of about 15,000 molecular weight which contains phosphothreonine as its phosphoamino acid. A second major phosphoprotein of molecular weight of 10,000 is detected only in viruses genetically related to rat species including those derived from the RPL cell line, from Sprague-Dawley rat embryo cells, and the Kirsten mouse sarcoma virus which was recovered from a mouse erythroblastosis virus after in vivo propagation through rat. These phosphorylated polypeptides of molecular weight 15,000, 12,000, or 10,000 are present in the virion structure in several different but nonrandom phosphorylated states.


Iodination of herpesvirus nucleic acids.

A simple method is described for the iodination of herpes simplex virus (HSV) DNA. The procedure involved synthesis of 125-I-labeled 5-iodo-dCTP which was subsequently used as a precursor for the in vitro repair synthesis of HSV DNA. Synthesis of 5-iodo-dCTP and purification from oxidation and reduction reagents, buffer salts, unreacted dCTP and Na125-I was accomplished in a single chromatographic step. It was possible to prepare 125-I-labeled HSV DNA in vitro with specific activities exceeding 10-8 counts/min/mu-g. The DNA prepared by this method reassociated with DNA extracted from HSV-infected HEp-2 cells but not with HEp-2 cell DNA. Iodinated HSV DNA was susceptible to S-1-endonuclease digestion once denatured but was resistant to digestion in the native form. This method was used to synthesize 125-I-labeled ribo-CTP (5-iodo-CTP) which was used to prepare cytomegalovirus-specific complementary RNA. The method should be of value in the preparation of viral probes and for use in autoradiography of viral nucleic acids.


Anatomy of herpes simplex virus DNA. III. Characterization of defective DNA molecules and biological properties of virus populations containing them.

We have characterized the virus progeny and its DNA from plaque-purified and undiluted passages of herpes simplex virus 1 in HEp-2 cells. Secifically, (i) infectious virus yields declined progressively in passages 1 through 10 and gradually increased at passages 11 through 14. The yields correlated with PFU/particle ratios. (ii) In cells infected with virus from passages 6 through 10, there was an overproduction of an early viral polypeptide (no. 4) and a delay in the synthesis of late viral proteins. In addition, the virus in these passages interfered with the replication of a nondefective marker virus. Cells infected with passage 14 virus produced normal amounts of polypeptide 4 and, moreover, this virus showed minimal interfering capacity. (iii) In addition to DNA of density 1.726 g/cm-3, which was the sole component present in viral progeny of passage 0, passages 6 through 14 contained one additional species (p 1.732) and in some instances (passages 6 and 10) also DNA of an intermediate buoyant density. The ratio of p 1.732 to p 1.726 DNA increased to a maximum of 4 in passages 6 through 9 and gradually decreased to 1 in passages 10 through 14. (iv) p 1.732 DNA cannot be differentiated from p 1.726 DNA with respect to size; however, it has no Hin III restriction enzyme cleavage sites and yields only predominantly two kinds of fragments with molecular weights of 5.1 x 10-6 and 5.4 x 10-6 upon digestion with EcoRI enzyme. (v) Partial denaturation profiles of purified p 1.732 DNA from passage 14 revealed the presence of two types of tandemly repeated units corresponding roughly in size to the EcoRI fragments and situated in different molecules. (vi) In addition to the two kinds of p 1.732 molecules consisting of tandem repaeat units of different sizes, other evidence for the diversity of defective DNA molecules emerged from comparisons of specific infectivity and interfering capacity of the progeny from various passages. The data suggest that some of the particles with DNA of normal buoyant density (1.726) must also be defective since the capacity to interfere and to produce an excess of polypeptide 4 did not appear to be proportional to the amount of high-buoyant-density defective DNA. The data suggest that defective interfering particles are replaced by defective particles with diminished capacity to interfere and that more than one species of defective DNA molecules evolves on serial preparation of HSV.


Regulation of tumor antigen synthesis by simain virus 40 gene A.

Simian virus 40 gene A has previously been shown to promote the replication of viral DNA and the transcription of late viral RNA in productive infection and to maintain the growth characteristics of some transformed cells. The present study examines the effect of the A function on proteins synthesized during productive and transforming infections. Under restrictive conditions, temperature-sensitive A mutants induce the overproduction of a 100,000-dalton protein both in productively infected monkey cells and in transformed rabbit cells. Immunoprecipitation of the induced protein with antisera, prepared against simian virus 40-induced tumors in hamsters, was used to identify the induced protein as tumor antigen. The same protein can be precipitated from extracts of cells infected by wild-type virus but not from uninfected cells. Furthermore, the mutant-induced protein is more rapidly degraded in vivo and is less tightly bound to intranuclear components than the protein induced by wild-type virus. The presence of the same virus-induced protein in infected cells from different species and the altered behavior of that protein in mutant infection strongly suggest that the protein is virus coded. Because the protein is large enough to account for the entire coding capacity in the early region of the simian virus 40 genome, the 100,000-dalton protein may well be the primary product of the only early gene identified by complementation studies, the A gene. If the 100,000-dalton protein that is overproduced in mutant infection is the A protein and the only early protein, then functional wild-type A protein must regulate its own synthesis in both productive and transforming infections.


In vitro transformation of rat and mouse cells by DNA from simian virus 40.

Primary rat kidney cells and mouse 3T3 cells can be transformed by DNA of simian virus 40 when use is made of the calcium technique (Graham and van der Eb, 1973). The transformation assay in primary rat cells is reproducible, but the dose response is not linear.


UV-induced reversion of a temperature-sensitive late mutant of simian virus 40 to a wild-type phenotype.

Irradiation of a simian virus 40 temperature-sensitive late mutant before infection of CV1 cells produces viruses that are phenotypically wild-type revertants. This probably occurs through suppression rather than true reversion because the revertants are produced at relatively high frequency and still have some properties of the original temperature-sensitive virus.


Simian virus 40 DNA replication, transcription, and antigen induction during infection with two adenovirus 2-SV40 hybrids that contain the entire SV40 genome.

The Ad2++hey hybrid virus population produces simian virus 40 (SV40) efficiently during lytic infection, whereas Ad2++ley does not, although both hybrids contain a complete SV40 genome. In this report, we demonstrate the synthesis of nonhydrid SV40 DNA in Ad2++HEY-infected Vero cells, but only early SV40 RNA is transcribed efficiently in Ad2++LEY-infected cells. Ad2++HEY induces SV40 U, T, and V antigens during lytic infection of African green monkey kidney cells, whereas Ad2++LEY induces only SV40 U and T antigens. These variations in the behavior of Ad2++HEY and Ad2++LEY regarding expression of SV40 functions probably reflect differences in the rate of SV40 excision from the hybrid genomes.


Role of gene 59 of bacteriophage T4 in repair of UV-irradiated and alkylated DNA in vivo.

Nonsense mutants in gene 59 (amC5, amHL628) were used to study the role of this gene in the repair of UV-damaged and alkylated DNA of bacteriophage T4 in vivo. The higher sensitivity to UV irradiation and alkylation of gene 59 mutants after exposure to these agents was established by a comparison of the survival fractions with wild type. Zonal centrifugal analysis of both parental and nascent mutant intracellular DNA molecules after UV irradiation showed that immediately after exposure the size of single-stranded DNA fragments was the same as the wild-type intracellular DNA. However, the capability of rejoining fragmented intracellular DNA was greatly reduced in the mutant. In contrast, the wild-type-infected cells under the same condition resumed DNA replication and repaired its DNA to normal size. Methyl methanesulfonate induced more randomly fragmented intracellular DNA, when compared to UV irradiation. The rate of rejoining under these conditions as judged from their sedimentation profiles was also greatly reduced in mutant-infected cells. Further evidence is presented that UV repair is not a simple consequence of arrested DNA replication, which is a phenotype of the mutant when infected in a nonpermissive host, Escherichia coli B (su minus), but rather that the DNA repair function of gene 59 is independent of the replication function. These and other data presented indicate that a product(s) of gene 59 is essential for both repair of UV lesions and repair of alkylation damage of DNA in vivo. It is suggested that gene 59 may have two functions during viral development: DNA replication and replication repair of DNA molecules.


Chromatographic separation and antigenic analysis of proteins of the oncornaviruses. V. Identification of a new murine viral protein, p15(E).

Profiling of murine leukemia virus (MuLV) proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) has revealed a low-molecular-weight protein which does not appear in the corresponding region of viral protein profiles obtained by gel filtration in 6 M guanidine hydrochloride. This protein species, termed p15(E), is easily demonstrable in MuLV isolates for which the viral p15 and p12 proteins have almost identical electrophoretic mobilities; this leaves a protein slightly larger than these two in the PAGE system unaccounted for in the gel filtration system. However, antiserum against the void volume fraction of the gel filtration eluate precipitated the p15(E) component from solubilized, radiolabeled virions, as shown by SDS-PAGE analysis of such immunoprecipitates. Comparative radioprecipitation analyses of this type revealed that for various MuLV isolates p15(E) was distinguishable from p15 in terms of serological reactivities, relative mobilities in gel electrophoresis, and relative efficiencies of labeling with individual amino acids. Thus it appears that, as is the case for avian oncornaviruses, MuLVs contain seven major structural proteins.


Fate of polyoma form IDNA during replication.

The fate of polyoma form IDNA generated during replication was investigated in resting BALB-3T3 cells. The experiments showed that there was extensive re-entry of such molecules into replication. This process took place over a period of several hours and appeared to be random. Progeny form I molecules must, therefore, spend some time in a nonreplicating pool before reinitiating replication. We propose that two factors affect the fate of progeny form IDNA. (i) The rate of reinitiation of progeny molecules is determined by the capacity of the initiation machinery. (ii) The extent of re-entry is determined by the availability of maturation proteins which divert form I from replication.