Studies on a defective variant of simian virus 40 that is substituted with DNA sequences derived from monkey. II. Structure of DNA. The structure of a substituted, reiterated defective variant of the simian virus 40 genome has been analyzed. The DNA of the defective variant is a closed circular duplex resistant to restriction endonuclease R-EcoRI and slightly shorter than the genome of wild type simian virus 40. Analysis of the double-stranded DNA segments produced by the action of a variety of restriction endonucleases on the defective genome allow description of the molecule as follows. (a) The full DNA molecule contains four tandem repeats of a DNA segment containing both SV40 and monkey DNA sequences. (b) Three out of the four segments are identical and are about 23% of the wild type genome in length: one segment of the four is larger and contains an additional peice of DNA about 4.3% of a wild type genome in length, but is otherwise identical with the other three segments. (c) Portions of the defective variant that contain monkey DNA can be isolated as discrete segments by restriction endonuclease digestion. (d) Some of the sequences originating from monkey DNA are derived from the highly reiterated class of monkey DNA sequences; others may represent sequences derived from infrequently reiterated or single copy monkey sequences.


Adenosine 3':5'-monophosphate-regulated phosphoprotein system of neuronal membranes. I. Solubilization, purification, and some properties of an endogenous phosphoprotein. An endogenous substrate for adenosine 3':5'-monophosphate-dependent protein kinase has been solubilized, and purified about 5,000-fold to apparent homogeneity, from a particulate fraction of bovine cerebral cortex enriched in synaptic membranes. This endogenous substrate, referred to as Protein I, is apparently specific to nervous tissue, and is composed of two types of polypeptides, present in a proportion of 1 (Protein Ia, 86,000 daltons) to 2 (Protein Ib, 80,000 daltons). In the presence of cAMP-dependent Protein I kinase purified from the same membrane fractions, Proteins Ia and Ib incorporated 0.83 and 0.81 mol of phosphate into serine/mol of peptide, respectively. Proteins Ia and Ib have similar amino acid compositions and have isoelectric points of 10.3 and 10.2, respectively. Both types of polypeptide have a relatively high content of glycine and proline, and both are degraded to a peptide of 48,000 daltons by highly purified collagenase, suggesting that Proteins Ia and Ib contain some sequences similar to those observed in collagen. The sedimentation coefficient of Protein Ia and Protein Ib was determined to be 2.9 S. The data suggest that both Protein Ia and Protein Ib have an elongated shape.


On the mechanism of ATP-induced shape changes in human erythrocyte membranes. II. The role of ATP. In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.


Metabolism of cationized lipoproteins by human fibroblasts. Biochemical and morphologic correlations. Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 mug/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.


The structure of postsynaptic densities isolated from dog cerebral cortex. I. Overall morphology and protein composition. A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.


Studies on the mechanism for entry of vesicular stomatitis virus glycoprotein G mRNA into membrane-bound polyribosome complexes. Glycoprotein mRNA (G mRNA) of vesicular stomatitis virus is synthesized in the cytosol fraction of infected HeLa cells. Shortly after synthesis, this mRNA associates with 40S ribosomal subunits and subsequently forms 80S monosomes in the cytosol fraction. The bulk of labeled G mRNA is then found in polysomes associated with the membrane, without first appearing in the subunit or monomer pool of the membrane-bound fraction. Inhibition of the initiation of protein synthesis by pactamycin or muconomycin A blocks entry of newly synthesized G m RNA into membrane-bound polysomes. Under these circumstances, labeled G mRNA accumulates into the cytosol. Inhibition of the elongation of protein synthesis by cucloheximide, however, allows entry of 60 percent of newly synthesized G mRNA into membrane-bound polysomes. Furthermore, prelabeled G mRNA associated with membrane-bound polysomes is released from the membrane fraction in vivo by pactamycin or mucomycon A and in vitro by 1mM puromycin - 0.5 M KCI. This release is not due to nonspecific effects of the drugs. These results demonstrate that association of G mRNA with membrane-bound polysomes is dependent upon polysome formation and initiation of protein synthesis. Therefore, direct association of the 3' end of G mRNA with the membrane does not appear to be the initial event in the formation of membrane-bound polysomes.


Acute actions of 1,25-dihydroxy-vitamin D3 in normal man: effect on calcium and parathyroid status. The present study was undertaken to evaluate the acute effect of 1,25-dihydroxy-vitamin D3 (1,25 (OH)2D3) on serum Ca, P and immunoreactive parathyroid (iPTH) and urinary Ca, P. and cyclic AMP. In 8 normal subjects, samples were collected over intervals of 30 to 60 min during a control day and on a treatment day following oral ingestion of 1,25(OH)2D3, 2.7 microgram. For the entire group there were no significant changes in serum Ca. P, iPTH or urinary P. Urinary Ca increased significantly 7 h after administration of 1,25(OH)2D3, and urinary cAMP decreased at 12 h. In 4 patients (group A). showing an increase in serum Ca by 0;2 to 0.4 mg/dl, serum iPTH decreased in 3, and the decrease in urinary cAMP appeared sooner. Among 4 patients showing no change in serum Ca after 1,25(HO)2D3 (group B), 3 showed an increase in iPTH. These data document the early onset of action of 1,25(OH)2D3 following its administration to normal man; increments in urinary Ca provide the most sensitive index of its action. The data provide no support for the view that 1,25(OH)2D3 exerts any direct inhibitory effect on the secretion of parathyroid hormone.


The effect of conjugated estrogens on the renin-angiotensin system. In a survey of 3 California communities by the Stanford Heart Disease Prevention Program, we obtained data on blood pressure, medications, age, height and weight, blood for measurement of plasma renin activity (PRA), plasma renin concentration (PRC), plasma renin reactivity (RR), and plasma renin substate concentration (PRS), and urine for measurement of urinary sodium and creatinine. No effect of conjugated estrogens (Premarin) on blood pressure could be discerned when the blood pressure, corrected for age and relative weight, of 575 women on no medication was compared to that of 82 women taking only Premarin. Premarin increased PRA, PRS, and RR, but had no effect on PRC. We also found in both Premarin-treated woman and controls 1) that RR was positively correlated with PRS, and 2) that PRA is dependent on PRC and PRS. These data indicate that the reninrenin substrate reaction of plasma, even at normal substrate concentration, is strongly deprendent on PRS.


Studies on phenolic steriods in human subjects. XXI. renal metabolism, conjugation and excretion of four androgens: a comparison with estriol. The present study was conducted in order to ascertain whether the human kidney can conjugate androgens to an extent similar to that of estriol (E3). Differently labeled androgens (testosterone, DHT and androstenedione) were injected simultaneously into a peripheral vein and the renal artery. The excretion of the radioactivity in the early urine collections served as an index of the ability of the kidney to conjugate and/or metabolize the various steroids administered. It was shown that the human kidney can conjugate testosterone to some extent as the 17-glucuronide of DHT, but to a much lesser degree that E3. Androstenedione was not conjugated by the kidney and the excretion DHT was paradoxically lower following its renal artery administration than following its peripheral injection. We interpret the latter to indicate that some kidney cells may contain receptors with very high affinity for DHT, thus leading to the lower excretion observed. The administration of androstenediol (into the renal artery) and E3 (peripherally) indicated that the diol was not conjugated as readily as E3. The results point to the ability of the kidney to conjugate testosterone to some extent; however, in no case was it able to conjugate an androgen with the same facility as it does E3.


Insulin receptors in patients with insulinomas: changes in receptor affinity and concentration. We have measured the specific binding of 125I-insulin to insulin receptors on circulating monocytes from 5 patients with insulinomas. In all patients the concentration of insulin receptors was inversely related to the basal, circulating level of insulin (corrected for proinsulin content). In the four patients with chronically elevated concentrations of plasma insulin, receptor concentrations were decreased. In three of these patients there were also marked alterations in receptor affinity. With insulinomas, correlations with the clinical state appear to be clearer when receptor concentration and especially receptor affinity are considered in addition to circulating insulin and proinsulin levels.


Results of intermittent treatment of growth hormone deficiency with human grwoth hormone. The growth in stature of two groups of growth hormone deficient children has been compared. The frist group received intermittent treatment with human Growth Hormone (hGH), 1 year on, 1 year off, subsequent years on; the second group received continous treatment. This latter group had a significantly better mean growth response. The reduction in treatment velocity in years subsequent to the first was not prevented by the year off treatment, the mean velocity of the intermittent group in their 3rd year being the same as that of the continous group in their second. We conclude that intermittent hGH therapy is of less values than continous treatment and should be avoided, at least pending further evidence.


Serodiagnosis of viral hepatitis A: detection of acute-phase immunoglobulin M anti-hepatitis A virus by radioimmunoassay. A modified micro solid-phase radioimmunoassay (RIA) for antibody to hepatitis A virus (anti-HAV) was developed. This double antibody procedure was performed by coating the surface of a polyvinyl microtiter plate "well" with 200 microliter of a 1:1,000 dilution of a patient's test serum. Purified HAV and 125I-labeled immunoglobulin G (IgG) anti-HAV were then sequentially added to form an antibody sandwich. The specificity and sensitivity of the RIA procedure for anti-HAV were verified by examination of coded human and chimpanzee serum specimens. Radioimmunoassay of early-acute-phase serum specimens from human cases of hepatitis A revealed the presence of anti-HAV activity. Differential examination by RIA of IgG and IgM fractions of acute-phase sera from experimentally infected chimpanzees demonstrated that IgM contained the bulk of the anti-HAV activity. A modification of the RIA procedure for anti-HAV (RIA-IgM blocking), incorporating an incubation step with anti-IgM (Mu chain specific), was further shown to differentiate acute- from convalescent-phase hepatitis A sera. This adapted RIA-IgM blocking procedure required less than 1 microliter of a single acute-phase serum specimen for the diagnosis of viral hepatitis A.


Rapid biochemical tests for characterization of the Mycoplasmatales. Methods are described for the rapid detection of beta-D-glucosidase and phosphatase in mycoplasma cultures using fluorogenic 4-methylumbelliferone substrates. These methods were applied to a selection of mycoplasma cultures and were compared with the conventionally used tests for these enzymes. Results were similar by both methods, but the fluorogenic tests could be read after 1 h, whereas the conventional tests took several days.


Variables affecting viral plaque formation in microculture plaque assays using homologous antibody in a liquid overlay. A liquid antibody microculture plaque assay and the variables that govern its effectiveness are described. The assay is based on the principle that low concentrations of homologous antibody can inhibit secondary plaque formation without inhibiting formation of primary plaques. Thus, clear plaques that followed a linear dose response were produced. The assay was found to be more rapid, less cumbersome, and less expensive than assays using agar overlays and larger tissue culture plates. It was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique in measuring infectious coxsackievirus type B-3. It was more sensitive in assaying adenovirus type 3 and Western equine encephalomyelitis, vesicular stomatitis, Semliki forest, Sendai, Sindbis, and Newcastle disease viruses than were liquid, carboxymethylcellulose, and methylcellulose microculture plaque assays. The variables influencing sensitivity and accuracy, as determined by using coxsackievirus type B-3, were: (i) the inoculum volume of virus; (ii) the incubation period of virus; and (iii) the incubation temperature.


Radioimmunoassay of human apolipoprotein CII. A study in normal and hypertriglyceridemic subjects. A specific, precise, and sensitive double-antibody radioimmunoassay for the measurement of human apolipoprotein CII (apoCII) was developed. ApoCII was labeled with (125)I (chloramine-T) and monospecific antibody was raised in rabbits. No appreciable cross-reactivity with apolipoproteins CI, CIII, AI, AII, low density lipoproteins, and lipoprotein-free plasma was observed. Lipoproteins containing apoCII displaced the standard curve in parallel. ApoCII measurement was not affected by pretreatment of plasma with tetramethylurea, ethanol-diethyl ether, or heating. Mean (+/-SE) plasma-immunoreactive apoCII in 47 normotriglyceridemic subjects was 51.8+/-3.2 mug/ml, generally comparable with previous estimates of its concentration by other methods. ApoCII levels in 9 subjects with type IIB lipoprotein pattern, 14 with the type IV lipoprotein pattern, and 5 with type V lipoprotein pattern were respectively, 89.9+/-4.6, 85.4+/-6.9, 132.8+/-21.0 mug/ml, all higher than normals (P < 0.001). Plasma apoCII and triglyceride concentrations correlated in normo- and hypertriglyceridemics (r = 0.36 and 0.58, P < 0.05). Plasma triglycerides correlated inversely with the fraction of total apoCII in very low density lipoprotein (VLDL)-free plasma (r = -0.75, P < 0.01). There was no correlation between plasma apoCII and high density lipoprotein cholesterol. In normotriglyceridemics, VLDL apoCII levels correlated with in vitro lipoprotein lipase (LPL) activator activities (r = 0.89, P < 0.01). In hypertriglyceridemic subjects the mean concentrations of apoCII per milligrams VLDL protein, LPL activator activity per milligrams VLDL protein, and LPL activator activity per micrograms VLDL apoCII were all lower than in normotriglyceridemics, P < 0.05. As plasma triglycerides and apoCII increase, apoCII is redistributed from high density lipoprotein to VLDL. However, the amount of apoCII per milligram VLDL protein and its LPL activator potency per milligram VLDL protein are reduced. These factors may contribute to impaired VLDL catabolism.


Isolation and characterization of an abnormal high density lipoprotein in Tangier Diesase. The nature of the high density lipoproteins has been investigated in five patients homozygous for Tangier disease (familial high density lipoprotein deficiency). It has been established that Tangier high density lipoproteins, as isolated by ultracentrifugation, are morphologically heterogenous and contain several proteins (Apo B, albumin, and Apo A-II). An abnormal lipoprotein has been isolated from the d = 1.063-1.21 g/ml ultracentrifugal fraction by agarose-column chromatography which contains apoprotein A-II as the sole protein constituent. In negative-stain electron microscopy, these lipoproteins appeared as spherical particles 55-75 A in diameter. By a variety of criteria (immunochemical, polyacrylamide electrophoresis, amino acid composition, and fluorescence measurements), apoprotein A-I the major apoprotein of normal high density lipoproteins and the C apoproteins were absent from this lipoprotein. As demonstrated by (125)I very low density lipoprotein incubation experiments with Tangier plasma, C apoproteins did not associate with lipoproteins of d = 1.063-1.21 g/ml. Tangier apoprotein A-II, isolated to homogeneity by delipidation of the apoprotein A-II-containing lipoprotein or Sephadex G-200 guanidine-HCl chromatography of the d = 1.063-1.21 g/ml fraction, was indistinguishable from control apoprotein A-II with respect to amino acid composition and migration of tryptic peptides in urea-polyacrylamide electrophoresis. The ability of Tangier apoprotein A-II to bind phospholipid was demonstrated by in vitro reconstitution experiments and morphological and chemical analysis of lipid-protein complexes. It is concluded that normal high density lipoproteins, as defined by polypeptide composition and morphological appearance, are absent from Tangier plasma and that as a consequence, the impairment of C apoprotein metabolism contributes to the hypertriglyceridemia and fasting chylomicronemia observed in these patients.


The measurement of apolipoprotein A-I and A-II levels in men and women by immunoassay. To study apolipoprotein A-II, a simple, precise, and accurate immunodiffusion assay was developed and applied in a population sample of industrial employees. Apolipoprotein A-II (A-II) did not increase with age in men (r = -0.20, n = 172), but showed a slight increase with age in women (0.1 mg/dl per yr, r = 0.20, n = 188). A-II correlated significantly with apolipoprotein A-I (A-I) (r = 0.71) and high density lipoprotein (HDL) cholesterol (men, r = 0.64; women, r = 0.49). The A-I/A-II ratio was significantly related to HDL cholesterol (men, r = 0.29; women, r = 0.44). Women on no medication (n = 92) had A-II levels similar to men (34+/-5 and 33+/-5 mg/dl, mean+/-SD, respectively), whereas women on oral contraceptives or estrogens had significantly higher levels (39+/-6 mg/dl, n = 75, P < 0.01). The plasma A-I/A-II weight ratio was 3.6+/-0.4 for men and 3.8+/-0.5 for women. In the d = 1.10-1.21 subfraction, both males and females had similar A-I, A-II, and HDL cholesterol levels (men: mean, 97, 27, and 32 mg/dl, respectively; women: mean, 104, 28, and 36 mg/dl, respectively). Women had approximately twice the amount of A-I, A-II, and HDL cholesterol than men in the d = 1.063-1.10 fraction (men: mean, 10, 2, and 10 mg/dl, respectively; women: mean, 24, 4, and 19 mg/dl, respectively). The A-I/A-II weight ratio in the d = 1.063-1.10 fraction (men, 5.1+/-0.7; women, 6.1+/-1.3) was significantly greater (P < 0.01) than that in the d = 1.10-1.21 fraction (men, 3.7+/-0.2; women, 3.8+/-0.2). Furthermore, the weight ratio of cholesterol to total apoprotein A in the d = 1.063-1.10 fraction (men, 0.75+/-0.09; women, 0.67+/-0.05) was significantly higher (P < 0.01) than that found in the d = 1.10-1.21 fraction (men, 0.26+/-0.04, women, 0.28+/-0.05). Thus, the compositions of HDL hydrated density subclasses are significantly different from each other. These results suggest that the differences in HDL between men and women are due primarily to differences in the relative proportions of HDL subclasses rather than to the intrinsic differences in HDL structure.


Stimulation of gastrin secretion and synthesis in antral organ culture. The purpose of the present study was to examine stimulation of gastrin release and the synthesis of gastrin directly by measurement of incorporation of [(3)H]tryptophan into gastrin in rat antral mucosal explants maintained in organ culture. Gastrin synthesis and secretion were assessed simultaneously at intervals over the 24-h duration of explant culture. Antral mucosal explants from fed female Wistar rats (4-5 wk, 100-150 g) were cultured at 37 degrees C (95% O(2)/5% CO(2)) in medium containing 70% Trowell-T8 and 10% NCTC-135 without unlabeled tryptophan, 10% dialyzed fetal calf serum and [(3)H]tryptophan (100 muCi/ml). Antral tissue was harvested at regular intervals during 24-h culture periods. Incorporation of [(3)H]tryptophan into immunoreactive gastrin was determined by techniques utilizing double-antibody immunoprecipitation. Antral tissue protein synthesis was assessed by measurements of incorporation of [(3)H]tryptophan into tissue protein of cultured antral explants. In paired experiments, gastrin synthesis and secretion in the presence of dibutyryl cAMP (DBCAMP) were compared to those observed under control conditions. Gastrin and protein specific activity progressively increased with time. Gastrin specific activity at 30 min increased from 3.3+/-0.5 (SEM) to 55.2+/-10.6 fmol [(3)H]tryptophan/pmol gastrin (or from 1.57+/-0.48 to 26.28+/-5.05 pmol [(3)H]tryptophan/mug gastrin) at 24 h: specific activity of antral tissue protein at 30 min increased from 33.6+/-8.4 to 1,660+/-236 fmol [(3)H]tryptophan/mug protein at 16 h. Culturing of explants for 4 h in the presence of cycloheximide (100 mug/ml) inhibited both gastrin synthesis and protein synthesis by greater than 90 and 95%, respectively. DBCAMP (10 mM) significantly increased both the synthesis and secretion of antral gastrin when compared with control cultured explants. Results of these experiments provide direct demonstration of gastrin synthesis by rat antral mucosal explants in organ culture, indicate that both gastrin and total antral protein synthesis are inhibited by cycloheximide, and demonstrate DBCAMP-induced stimulation of both gastrin synthesis and secretion, suggesting the potentially important role of cyclic AMP in gastrin cell function.


Interaction between high density and low density lipoproteins uptake and degradation by cultured human fibroblasts. High density lipoprotein (HDL) inhibited the binding (trypsin-releasable radioactivity), internalization (cell-associated radioactivity after trypsinization), and degradation (TCA-soluble non-iodide radioactivity) of (125)I-low density lipoprotein ((125)I-LDL) by cultured normal human fibroblasts. At HDL:LDL molar ratios of 25:1 (protein ratios about 5:1), these parameters were reduced by about 25%. Unlabeled LDL was about 25 times more effective in reducing (125)I-LDL binding, implying that if HDL and LDL bind at common sites the affinity of HDL for these sites is very low or that the interaction is on some other basis. The fractional reduction in (125)I-LDL binding at a given HDL: (125)I-LDL ratio was independent of (125)I-LDL concentration and occurred equally with fibroblasts from a subject with homozygous familial hypercholesterolemia. Reciprocally, the binding, internalization, and degradation of (125)I-HDL were reduced by LDL. Preincubation of fibroblasts with HDL (or LDL) reduced the subsequent binding of (125)I-LDL (or (125)I-HDL) during a second incubation. In other studies HDL reduced the net increase in cell cholesterol content induced by incubation with LDL. HDL alone had no net effect on cell cholesterol content. These findings suggest that HDL reduces both the high affinity and the low affinity binding of LDL to human fibroblasts and that this in turn reduces the internalization and degradation of LDL. The effect of HDL on the LDL-induced changes in cell cholesterol content could be in part on this basis and in part on the basis of an HDL-stimulated release of cholesterol from the cells. These effects of HDL in vitro may be relevant to the negative correlations reported from in vivo studies between plasma HDL concentration and both body cholesterol pool size and the prevalence of clinically manifest atherosclerosis but further studies will be needed to establish this.


Stimulation by alcohols of cyclic AMP metabolism in human leukocytes. Possible role of cyclic AMP in the anti-inflammatory effects of ethanol. In this study ethanol and certain other short-chain aryl (benzyl and phenethyl) and aliphatic (methyl, propyl, butyl, and amyl) alcohols produced up to 10-fold increases in cyclic AMP (cAMP) concentrations in purified human peripheral blood lymphocytes. Ethanol concentrations as low as 80 mg/dl produced significant elevations in lymphocyte cAMP. Significant but less marked augmentation of cAMP in response to alcohols was observed in human platelets, human granulocytes, and rabbit alveolar macrophages. The mechanism of the alcohol-induced cAMP accumulation is probably secondary to membrane perturbation and consequent activation of adenylate cyclase, because ethanol directly stimulated this enzyme in lymphocyte membrane preparations but had no effect on lymphocyte phosphodiesterase activity. Lysosomal enzyme release, by phagocytosing human leukocytes, and aminoisobutyric acid transport in mitogen-stimulated human lymphocytes were shown to be inhibited by ethanol and other alcohols at concentrations which also elevate cAMP. In general, the magnitude of the inhibition of these inflammatory processes correlated with the ability of the alcohol to elevate cAMP concentrations. Lectin-and anti-thymocyte globulin-induced lymphocyte mitogenesis was inhibited or unaffected depending upon both the concentration and type of mitogenic stimulus and the concentration and type of alcohol utilized. Inflammatory mediator release from rat mast cells also was inhibited by ethanol and certain other alcohols, but whole cell cAMP was not increased. Ethanol may alter these inflammatory responses and other biologic processes at least in part by modulating cellular levels of cAMP.


Hormonal control of adrenocortical cell proliferation. Desensitization to ACTH and interaction between ACTH and fibroblast growth factor in bovine adrenocortical cell cultures. A primary bovine adrenocortical cell culture system responsive to physiological concentrations of ACTH has been established. When added to cultures, ACTH inhibited DNA synthesis and cell division over the same concentration range required for stimulation of fluorogenic steroid production (0.01-10 nM). With chronic exposure to ACTH, cells became desensitized to the growth inhibitory effects of ACTH. Though cell growth was initially completely inhibited by ACTH, cells ultimately began to grow in its continued presence. The lag time to initiation of cell growth, the rate of growth, and the final density achieved depended on the ACTH concentration. Desensitization to ACTH(1-39) was not induced by monobutyryl cyclic AMP nor by ACTH(11-24). Specificity of desensitization was apparent because cells which had become desensitized to ACTH(1-39) remained fully responsive to monobutyryl cyclic AMP, prostaglandin E(1), and cholera toxin. Though the effects of ACTH on cell growth were readily reversible upon hormone removal, the desensitized response to readdition of ACTH persisted for at least 8 h. Fibroblast growth factor (FGF) stimulated both the growth rate and saturation density achieved. FGF did not alter the growth inhibitory effects of ACTH nor the reduced growth rate observed in desensitized cells maintained in ACTH. However, FGF greatly increased the saturation density achieved by cultures maintained with ACTH. Through the process of desensitization, adrenocortical cells are able to grow in the presence of high concentrations of ACTH and to respond to the effects of a growth factor by increasing the cell density achieved. This pattern of response may be a general one for growth control under the combined effects of antimitotic and mitotic factors.


Evidence for hydroxyl radical generation by human Monocytes. A number of highly reactive oxygen species have been implicated in the oxygen-dependent mechanisms involved in bactericidal activity of phagocytic leukocytes. Hydrogen peroxide and superoxide, two agents known to occur during phagocytosis, are thought to interact to generate hydroxyl radical, singlet oxygen, and other potentially reactive molecules. Using an assay system of ethylene generation from methional, cell preparations of human monocytes were demonstrated to generate hydroxyl radical or a similar agent during phagocytosis of zymosan particles. The generation of ethylene was impaired by agents which reduce superoxide or hydrogen peroxide concentrations as well as by agents reported to be hydroxyl radical scavengers. The ethylene generation did not appear to be dependent on myeloperoxidase in that azide enhanced ethylene generation. Monocytes from a patient with chronic granulomatous disease failed to generate ethylene during phagocytosis. This assay technique may be useful in exploring the metabolic events integral to the bactericidal and inflammatory activity of phagocytic leukocytes.


Aetiology of cirrhosis, hepatic fibrosis, and hepatocellular carcinoma. Histological study of 69 cases of cirrhosis, 9 of severe generalised hepatic fibrosis, and 19 of hepatocellular carcinoma showed an association with alcohol, hepatitis B surface antigen (HBsAg) or a1-antitrypsin bodies in, respectively, 41 (cirrhosis), 5 (fibrosis), and 9 (carcinoma). Eight of the cirrhotic cases and two of the carcinoma cases had double associations, HBsAg being present in all. Torcein and aldehyde fuchsin staining gave both false positive and false negative results when compared with immunofluorescence and immunoperoxidase methods for HBsAg. Large amounts of copper were found in four cirrhotic livers, and moderate amounts in 13: the diagnostic value of copper staining is questioned.


Pancreatic scanning using retrograde injection of technetium-99m-labeled compounds. With increased clinical availability of endoscopic retrograde pancreatography, a method for retrograde injection of radionuclides into the pancreas became available. A dog model was developed to evaluate resolution and toxicity of retrograde imaging prior to human use. Two groups of technetium-99m-labeled compounds were used. The first included ionic carriers--pertechnetate and pyrophosphate--and the second included particulate carriers--sulfur colloid and albumin microspheres. Parenchymal visualization, absorption patterns, and toxicity in dogs were studied. The results suggest that pyrophosphate, sulfur colloid, and albumin microspheres would all be feasible technetium carriers for use in humans.


Myocardial imaging with Cc-99m pyrophosphate in patients on adriamycin treatment for neoplasia. Technetium-99m pyrophosphate was utilized for myocardial imaging in 15 patients on adriamycin treatment for neoplasia. We have noted abnormal accumulation of the pyrophosphate in several patients, particularly in those in whom the so-called poor-risk factors were operative, namely prior radiation, cyclophosphamide therapy, and ischemic heart disease.


Diagnosis of acute myocardial infarction in patients undergoing open heart surgery: a comparison of serial myocardial imaging with cardiac enzymes, electrocardiography, and vectorcardiography. In 44 consecutive patients undergoing elective open heart surgery (OHS), serial electrocardiograms (ECG), vectorcardiograms (VCG), serum CPK, cardiac isoenzymes (CPKMB), and myocardial images using Tc-99m pyrophosphate were obtained, before and after the operation, for the detection of acute myocardial infarction (AMI). Twenty-nine patients developed one or more positive tests postoperatively. Two patients had positive myocardial scintiscans; both had other evidence of infarction. Conversely, the appearance of CPKMB, or new ECG and VCG changes, occurred frequently without evidence of infarction, and were not associated with the development of a positive scintiscan. The results show that false-negative results are infrequent in patients imaged early after OHS, and that cardiac surgical procedures do not cause a high incidence of false-positive scintigrams. Consequently, radionuclide imaging for AMI offers an important adjunct for excluding acute infarction following open heart surgery.


The significance of calcific valvular heart disease in Tc-99m pyrophosphate myocardial infarction scanning: radiographic, scintigraphic, and pathological correlation. Technetium-99m pyrophosphate (PPi) is currently considered the best scanning agent for the diagnosis of acute myocardial infarction. False-positive scans have been reported in association with unstable angina, alcoholic cardiomyopathy, and ventricular aneurysms. In this study, 86% of patients (12/14) with either calcific aortic or mitral valvular heart disease had positive PPi cardiac scintiscans and the location of the PPi uptake was limited to the calcific valve in all (9/9) of the patients who underwent valve replacement surgery. Six patients with valvular disease without radiologic evidence of calcium had negative PPi heart images. Three of these patients had surgical valve replacement, and in none was there increased uptake in the resected valve. Seventy-five percent of the patients with calcified aortic valves had localization of the PPi activity to the area of the aortic valve, whereas 50% of the patients with calcified mitral valves showed a diffuse pattern of uptake on the cardiac image. In vitro demonstration of increased radioactivity in surgically removed cardiac valves warrants the conclusion that Tc-99m PPi is taken up by calcified heart valves. We conclude that while PPi heart scanning is a sensitive indicator of acute myocardial infarction, false-positive scans can occur in the presence of calcific valvular disease, due to localization of PPi in the calcified portion of the valve.


Hemostasis by premedication with estrogen in hair-transplant surgery. Premedication with water-soluble conjugated estrogens administered the previous day and a half-hour before operation diminished bleeding and allows for a drier field, better vision and quicker work. Post-operative bleeding is negligible. Their short-term use has not led to feminization nor presented cardiovascular problems.


Comparison of Tc-99m methylene diphosphonate with Tc-99m pyrophosphate in the detection of acute myocardial infarction: concise communication. Myocardial scans were obtained in ten patients with proven myocardial infarction. Scintigraphy on consecutive days was performed with technetium-99m methylene diphosphonate and technetium-99m pyrophosphate. Images obtained with the two tracers were generally equal in quality. Those with PPi exhibited denser uptake, while background activity was lower with MDP.


A comparative study on lysosomal accumulation of gallium-67 and indium-111 in Morris hepatoma 7316A. Intracellular localization of Ga-67 and In-111 was investigated in Morris hepatoma 7316A and in normal Buffalo rat liver cells by a cell fractionation method at 48 hr after an intraperitoneal injection of the nuclides. Lysosomal fractions of the tumor and normal liver cells had the highest relative specific radioactivities of the nuclides (p less than 0.001). In a gradual solubilization experiment, the release of the nuclides at the same time as the acid phosphatase from the lysosomal fractions (p less than 0.001) was thought to indicate that lysosomes are the site of accumulation for both nuclides whether in tumor or normal liver cells. Fragility of the tumor lysosomes might be inferred from the significantly greater regression coefficient in relation to the lysosomal fraction of tumor cells than that of normal liver cells when labeled with Ga-67 (p less than 0.001). The poorer confidence limit for the regression coefficient in relation to the lysosomal fraction of tumor cells labeled with Ga-67 seemed to indicate that Ga-67 determines lysosomal functions of tumor cells more precisely than In-111.


Absence of the effect of ephedrine on levels of cyclic adenosine monophosphate in human lymphocytes. Concentrations of 10(-6) to 10(-3) M ephedrine did not increase cyclic adenosine monophosphate (cAMP) levels of normal human lymphocytes even in the presence of theophylline or cortisol, which enhance the response to isoproterenol or prostaglandin E1. Preincubation for 2 hr with ephedrine did not reduce the subsequent effect of isoproterenol, although preincubation with isoproterenol itself did. Therefore, ephedrine is not a direct beta-adrenergic agonist on human peripheral blood lymphocytes.


The ocular manifestations of congenital infections produced by toxoplasma and cytomegalovirus. Chorioretinitis is the most common ocular manifestation of congenital toxoplasmosis occurring in both symptomatic and the more freqeunt subclinical form of the disease. Chorioretinitis also occurs in symptomatic congenital cytomegalovirus (CMV) but not in the subclinical form of this congenital congenital infection. Diagnosis cannot be made from ocular signs alone. Knowledge of the epidemiology of both congenital infections along with information about the postnatal history will assist in the differentiation.


Dietary control of hyperlipidaemia. Dietitians in the EEC: 2. Republic of Ireland. One hundred and ten adult out-patients entered a prospective study of the effect of dietary control of hyperlipidaemia. Seventy-nine were male and 31 female. Eighty-four patients had primary type II and 26 had primary type IV hyperlipidaemia. Cases of secondary hyperlipidaemia were excluded. Patients with type II hyperlipidaemia were treated with a diet low in saturated fat and cholesterol, with substitution of polyunsaturated fats. Additional carbohydrate and alcohol restriction was applied to patients with type IV hyperlipidaemia. Calorie restriction was prescribed for overweight patients. In type II patients, mean serum cholesterol fell from 322 to 286 mg per 100 ml, and weight from 159 to 152 lb over the first six months of treatment. In type IV patients cholesterol fell from 328 to 281 mg per 100 ml, triglycerides from 389 to 241 mg per 100 ml and weight from 166 to 156 lb over the same period. All reductions were statistically significant at the 1 per cent level. No statistical correlation was observed between reduction of weight and reduction in lipid levels. Over a three-year follow-up period no tendancy for lipid levels or weight to 'rebound' was noted. It is concluded that diet can produce a significant reduction in plasma lipid levels over six months in some hyperlipidaemic patients. Further follow-up is continuing to determine whether this effect can be maintained over the longterm.


Increased binding and killing of neuraminidase-galactose oxidase-treated tumor cells by normal macrophages. The binding of Line 10 hepatoma cells to normal syngeneic guinea pig macrophages is increased when the tumor cells are treated with neuraminidase and galactose oxidase (NAGO) before they are added to the macrophage monolayers. The effect is abolished by exposure of the NAGO-treated tumor cells to sodium borohydride. Line 1 hepatoma cells treated with NAGO or with sodium periodate are killed to a greater extent than untreated tumor cells. This effect can also be reversed by sodium borohydride. Further, periodate-treated macrophages become cytotoxic for unmodified tumor cells. These results demonstrate that increased tumor cell killing occurs when artificial contacts (presumably via Schiff bases) are established between normal macrophages and tumor cells. They are consistent with the hypothesis that close cell to cell contact is necessary for macrophage-mediated cytotoxicity.


Migration inhibition by an agarose microdroplet assay: monitoring of tumor-associated antigens on a simian virus 40-induced sarcoma. Macrophage migration inhibition assays, with a direct agarose microdroplet method, were used to monitor TAA activity of preparations of SV-40-induced mKSA cells. These preparations included cell-free crude membranes, papain-solubilized and NP40 detergent-solubilized membrane extracts from mKSA tumor cells. The assay was extremely sensitive and could detect migration inhibition reactivity with all three types of antigenic preparations with concentrations as low at 250 ng protein/ml. The reactivities were quite reproducible from experiment to experiment using the same or different lots of these antigen preparations, and the reactivities were specific in that peritoneal exudate cells from BALB/c mice, immunized with antigenically unrelated but syngeneic plasmacytomas, were not inhibited by these antigens. The results demonstrated the usefulness of this assay in rapidly detecting small concentrations of partially purified TAA preparations by using small number of immune cells.


Reactivity to SV40 T antigen in athymic (nude), anti-thymocyte serum-treated, and normal mice. Antisera prepared in syngeneic mice by hyperimmunization with intact SV40-transformed mouse cells or with somatic cell hybrids between SV40-transformed human and normal mouse cells exhibit anti-SV40 tumor (T) antigen reactivity. Athymic mice bearing tumors formed by SV40-transformed mouse, human or mouse-human hybrids were not reactive with SV40 T antigen. Anti-thymocyte serum (ATS)-treated mice also lacked T antigen reactivity during suppressive treatment but developed antibody to T antigen after discontinuing ATS treatment and tumor regression. We conclude that that presence of growing tumors in the mouse is not necessary for the production of anti-SV40 T antigen antibodies but that helper thymus-derived cells are essential for the humoral response.


Cytotoxic interactions of virus specific effector cells with virus infected targets of different cell type. The action of Sendai virus specific cytolytic T lumphocytes (CTL) against Sendai virus infected macrophages was found to be H-2 restricted while Sendai virus infected cell lines, including fibroblasts and tumour cells, were lysed across the H-2 barrier to some extent. The properties of the Meth-A tumour cell, which was resistant to lysis by allogenic killer cells was investigated. Sendai virus specific CTL failed to kill Sendai virus infected Meth-A cells but after vaccinia virus infection these target cells were susceptible to lysis by vaccinia virus specific CTL.


A linkage study of rabbit serum high density lipoprotein (HDL) allotypes. Close linkage between the two allotypic systems of rabbit serum high density lipoprotein (HDL), Hl 1 and R 67, has been confidently excluded, the recombination fraction being at least 0-20. No suggestion of linkage between either HDL allotype and the immunoglobulin allotypes a1a2a3/b4b9, or the red blood cell system (RBC) ADF was obtained. Close linkage was confidently excluded for the Hl 1-ADF, Hl 1-a1a2a3, R 67-ADF, R 67-a1a2a3, R 67-b4b9, a1a2a3-b4b9, and b4b9-ADF relationships.


Controlling influences of the autonomic nervous system. Recent information about the localization of sympathetic nerves and catecholamine-containing cells suggests sites of action not usually described in the neuroscience textbooks. In this study, we focused on the autonomic controls that affect ganglia, heart, gut, and chemoreceptors. As a result of some speculation derived mainly from histochemical observations and partially from physiologic data, we concluded that at the organ level the interplay between a nerve terminal-receptor serves as a local control. Additional controls may function at the ganglion level where catecholamine-containing chromaffin cells may serve as interneurons. We suggest that all peripheral catecholamine-containing elements which function in a modulatory role are not vital to the survival of the individual but rather serve as "fine tune" adjustment that do not involve the central nervous system.


Disorders of peripheral cutaneous nerves. The histopathology of leprosy is described with particular reference to its effects on peripheral cutaneous nerves. Mycobacterium leprae invade the Schwann and perineurial cells of peripheral cutaneous nerves preferentially. The organisms are eventually destroyed with their host cells by a cell-mediated immune response. The effect is a dying-back phenomenon without the formation of neuromata. The sensory effects are gradually increasing anesthesia and localized nerve trunk pain but seldom any peripheral sensory reference or paresthesiae. Peripheral nerves are shown to be zones where there is some degree of immunologic privilege for Myco. leprae.


C-peptide suppression test for insulinoma. During hypoglycemia induced by an infusion of porcine insulin, impaired suppression of endogenous insulin secretion as measured by C-peptide was demonstrated in 11 of 12 patients with insulinoma. During hypoglycemia (plasma glucose less than or equal to 40 mg/dl) the mean C-peptide immunoreactivity (CPR) of normal subjects was less than or equal to 1.2 ng/ml, whereas 11 of 12 insulinoma patients had a mean CPR of larger than or equal to 1.9 ng/ml. One patient showed normal CPR suppression by these criteria but may have shown impaired CPR suppression for glucose less than or equal to 30 mg/dl. Impaired CPR suppression during insulin-induced hypoglycemia may prove to be a useful test for insulinoma.


99mTc-pyrophosphate bone scans in patients with metastatic carcinoid tumors. Three patients with foregut (bronchial), hindgut (rectal) or (ovarian) carcinoid tumors had symptomatic bone metastasis with abnormal 99m Tc pyrophosphate bone scans and bone roentgenograms. Six patients with midgut (small intestine or caecal) carcinoid) carcinoid tumors who had no symptoms of bone metastasis had no evidence of bone metastasis on bone scan or bone roentgenographic examination. This study supports the clinical impression that patients with midget carcinoid tumors have a low incidence of bone metastasis.


Dynamics and mechanics of corticosteroid feedback at the hypothalamus and anterior pituitary gland. Corticosteroid feedback mechanisms were investigated at the hypothalamic level using the rat hypothalamus in vitro and the pituitary level using basal hypthalamic-lesioned rats. Both fast and delayed corticosteroid feedback effects were demonstrated at the level of the hypothalamus and pituitary gland with doses of corticosteroids within or near the physiological range. These two phases of feedback were separated temporally by a 'silent period' during which no feedback was apparent. Studies on the mechanism of action of corticosteroids at the hypothalamic level showed that the fast feedback mechanism acts by inhibition of release whilst the delayed feedback mechanism acts by inhibition of both synthesis and release. The fast feedback action of corticosterone does not appear to act by excitation of neuroinhibitory pathways since neither picrotoxin nor phentolamine prevented the feedback action of corticosteroids in vitro. Corticosterone inhibition of corticotrophin releasing factor release was overcome by depolarization of the membrane with K+ suggesting that the mechanism of action of the fast feedback of corticosteroids is by membrane stabilization.


Temporal changes in ovarian ornithine decarboxylase and cyclic AMP in immature rats stimulated by exogenous or endogenous gonadotrophins. Pregnant mare serum gonadotrophin given intravenously to immature rats caused a maximal (x 70) increase in ornithine decarboxylase activity (ODC) at 3 h; enzyme activity declined to about ten times the control levels by 9 h and a second rise began after about 20 h. Anti-PMSG given 30 min after PMSG reduced the peak response by 70%. Actinomycin D, or cycloheximide, completely prevented an increase in ODC when given with PMSG, but only cycloheximide lowered the enzyme activity when given 18 h later. Ovine FSH plus LH also produced a peak in ODC at 3 h but the activity decreased quickly and by 9 h it was at the control level. Secretion of endogenous FSH and LH, induced by hourly injections of LH releasing hormone (LH-RH) increased ODC to the same extent as did the exogenous hormones; ODC was still higher than in the controls 4 h after the last dose of LH-RH. Increased endogenous levels of FSH and LH did not consistently raise ovarian cyclic AMP content and the increases found were much less than those obtained after injection of PMSG or FSH+LH. The results indicate that increased ODC is induced and maintained by the continual presence of gonadotrophin. The dependence of increased ODC upon increased cyclic AMP cannot be unequivocally determined because of important differences in the timing of the responses and the difficulty in determining biologically significant changes in cyclic AMP.


Binding of human chorionic gonadotrophin to rat ovary during development. Ovarian tissue of prenatal, newborn, and 5-day-old rats does not specifically bind 125I-labelled HCG. Specific binding of HCG was first observed in ovaries of 10-day-old animals and binding increased with age. These results indicate that, contrary to rat testis, the HCG receptor in the rat ovary is not present during foetal and early postnatal development. Thus, the insensitivity of the ovary to endogenous and exogenous LH or HCG during this developmental period is due to the lack of specific receptors.


Renal uptake and metabolism of adrenocorticotrophin analogues in the rat: an autoradiographic study. Renal resorption of tritiated adrenocorticotrophin analogues was studied in the rat using light microscopic and quantitative electron microscopic autoradiography. The synthetic corticotrophins used were Synacthen (corticotrophin-(1-24)-tetracosapeptide) and C41795-Ba ([D-Ser1,Lys17,Lys18]-corticotrophin-(1-18)-octadecapeptide amide), the tetracosapeptide being tritiated in either the tyrosine residue of position 2 or 23 or the phenylalanine of position 7 and the octadecapeptide in the tyrosine of position 2. Inspection of autoradiographs showed that peptides injected intravenously were resorbed into proximal tubules by endocytosis to produce vesicles whose radiolabel later appeared in lysosomes, a route previously elucidated for other peptides and proteins. The use of two techniques for analysis of electron microscopic autoradiographs, however, suggested that apical tubules also acquire label and are in some way involved in the transfer of resorbed labelled material from endocytotic vesicles to lysosomes. In addition, the autoradiographic analyses revealed that the duration of lysosomal labelling depends upon the position of tritium in the chain. Thus, when the CO2H-terminus of Synacthen was labelled, silver grains were more transiently associated with lysosomes than was the case when the NH2-terminal or core regions were tritiated, indicating a greater resistance of these portions of the peptide to attack by intracellular peptidase. The label from the chemically protected C 41795-Ba was also less readily expelled from the lysosomes of the proximal tubules.


Pituitary regulation of Leydig cell function in the adult male rat. The effects of hypophysectomy on serum testosterone, 125I-labelled hCG binding to testicular membranes and on testicular responsiveness were studied in adult rats. Serum testosterone decreased rapidly over the first 6 h after hypophysectomy. LH receptors were determined (pmol/testis) by measuring the specific binding of 125I-labelled hCG in membrane preparations of testes of rats hypophysectomized 1, 2, 3, 6, 9, or 15 days earlier. Hypophysectomy did not result in a decrease in 125I-labelled hCG binding on day 1 but this had decreased to 40% of that in intact controls by day 2. A gradual decline was found between days 2 and 6 at which time hCG binding had decreased to 15%. No further decrease occurred between days 6 and 15. Scatchard analysis indicated that the decline in hCG binding was due to a decreaffinity. FSH, testosterone, dihydrotestosterone, and oestradiol were unable to prevent the decline in hCG binding. Although serum testosterone, testicular testosterone content, and 125I-labelled hCG binding decreased rapidly after hypophysectomy, testicular responsiveness to LH was biphasic. The intraperitoneal administration of 25 microgram LH 2 h before decapitation increased testosterone in the circulation to a greater extent extent in animals hypophysectomized for 1 day than in intact controls while hCG binding affinities and capacities had not changed. Two or three days after hypophysectomy testicular responsiveness to LH was similar to that of intact controls even though hCG binding in hypophysectomized animals had decreased to 40 and 28% of intact controls respectively. It is concluded that (1) the testis is dependent on anterior pituitary hormones for maintenance of testicular LH receptors and testosterone secretion, (2) FSH, testosterone, dihydrotestosterone, or oestradiol cannot prevent the decline in testicular LH receptors resulting from hypophysectomy, and (3) steroidogenic capacity of the testis persists significantly longer than the hCG binding capacity of the testis.


Differentiation of memory T cells to virus plaque-forming cells and cytotoxic T lymphocytes. The aims of this study were to define the T-cell subpopulation(s) detected by the virus plaque assay, and particularly to determine whether the virus plaque assay could be used to enumerate cytotoxic T lymphocytes. In addition, studies were undertaken to ascertain whether cell proliferation was required for development of cytotoxic effector function and virus plaque formation by these subpopulations. The results of experiments with a secondary mouse mixed lymphocyte culture (MLC) model indicated that 70 percent of virus plaque-forming cells bore the Ly 1 phenotype and 30 percent the Ly 2,3 phenotype. Three lines of evidence suggested that cytotoxic T lymphocytes (CTL) can be detected by this assay: the fact that some virus plaque-forming cells (V-PFC) bear the same Ly phenotype as CTL; the use of an inhibitor of DNA synthesis indicated that proliferating cells could be eliminated with no effect on V-PFC production and cytotoxic activity of the Ly 2,3 cell population; and that infection of primed lymphocyteswith vesicular stomatitis virus before (MLC) stimulation eliminated cytotoxic activity. In primary MLC, development of V-PFC and CTL was completely abolished by cytosine arabinoside. In contrast, in secondary MLC, some CTL and V- PFC were generated by antigenic stimulation in the absence of proliferation. However, the development of both functions became progressively more susceptible to cytosine arabinoside as the time between primary immunization and in vitro boosting is increased. It is suggested that there may be a considerable disparity between the number of existing effector cells at any given time and the cytotoxic potential, i.e. the number of cells capable of being generated by antigenic stimulation.


Dengue viruses and mononuclear phagocytes. II. Identity of blood and tissue leukocytes supporting in vitro infection. Studies were made on the identity of human and monkey mononuclear leukocytes permissive to antibody-enhanced dengue 2 virus (D2V) infection. In cultures of peripheral blood leukocytes (PBL) inoculated immediately after separation, it was concluded that only mononuclear phagocytes support dengue infection. This is based upon observations that D2V-permissive cells were resistant to 1,200 rads, were both plastic adherent and nonadherent, were removed when passed through nylon wool columns in 10 percent fetal bovine serum or 100 percent autologous serum, and were destroyed by incubation with 100 mug/ml particulate silica. On direct immunofluorescence staining, perinuclear dengue antigen was visualized at 24 h, becoming maximal at 60 h. Antigen-containing cells had ample cytoplasm, ruffled cytoplasmic membrane, and 73 percent were actively phagocytic. As further evidence of the infection of mononuclear phagocytes, antibody-enhanced D2V replication was observed in bone marrow cultures from five of five rhesus monkeys, but not in cell cultures of spleen, thymus, or lymph nodes prepared from the same animals. It is hypothesized that dengue virus complexed with non-neutralizing antibody is internalized by immune phagocytosis in a mononuclear phagocyte with a defective virus-destroying mechanism. Dengue permissiveness may depend upon cellular immaturity since bone marrow leukocytes could be infected even when held for 4 days before infection while PBL held for this time decreased in permissiveness. In vitro antibody-dependent infection of mononuclear phagocytes should prove useful as a model for study of immunopathologic mechanisms in human dengue.


Preleukemic expression of TL antigens in x-irradiated C57BL/6 mice. Anomalous appearance of TL (thymus-leukemia) antigens is a characteristic feature of radiation-induced leukemias of C57bl/6 mice. We now report that thymocytes of irradiated C57BL/6 mice express TL antigens long before the development of overt leukemia. Thus, TL is a marker for preleukemic changes occurring during radiation leukemogenesis. Low levels of murine leukemia virus (MuLV)-related antigens are also detected on preleukemic thymocytes. Comparative tests on individual mice show no direct correlation between TL and MuLV antigen expression.


Expression of AKR murine leukemia virus gp71-like and BALB(X) gp-71-like antigens in normal mouse tissues in the absence of overt virus expression. By competition radioimmune assays with antisera against AKR murine leukemia virus (MuLV) gp 71 or antisera against xenotropic virus, and iodinated AKR MuLV gp71 or BALB(X) gp71, antigens serologically indistinguishable from the viral antigens can be detected in tissues of normal mice in the absence of overt virus expression. An antigen serologically indistinguishable from AKR MuLV gp71 can be readily detected in normal bone marrow cells of the common strains of mice including NIH Swiss, 129/J, and SWR/J, as well as in Mus cervicolor and Mus musculus casteneus. In contrast, this antigen is not detected in normal spleen, thymus, lymph nodes, or serum. Similarly, an antigen serologically indistinguishable from BALB(X) gp71 was found in all normal mouse sera examined. This antigen was not present in fetal liver, perfused adult liver, thymus, spleen, lymph nodes, or bone marrow of the mice examined. An equivalent antigen was detected in sera from Mus musculus casteneus but not in sera from Mus cervicolor.


Influence of local anesthetics upon human polymorphonuclear leukocyte function in vitro. Reduction of lysosomal enzyme release and superoxide anion production. Cationic local anesthetics have been reported to influence cellular responses to surface stimuli by interfering with the function of microtubules and microfilaments. Since unimpaired microtubule and microfilament functions are required by human polymorphonuclear leukocytes in order to respond normally to surface stimulation, we have studied effects of the local anesthetic, tetracaine on the function and morphology of these cells in vitro. Tetracaine (0.25--1.0 mM) significantly reduced extracellular release of the lysosomal enzymes, beta-glucuronidase and lysozyme from polymorphonuclear leukocytes exposed to serum-treated zymosan (a particulate stimulus), zymosan-treated serum (a soluble stimulus), and to the surface-active lectin, concanavalin A. Tetracaine also significantly reduced superoixde anion production (superoxide dismutase-inhibitable cytochrome c reduction) by these cells. Tetrancaine was not cytotoxic and its effects could be reversed completely by washing cells once with buffer. Electron microscope examination of tetracaine-treated cells revealed marked alterations of surface membranes. Microtubules and microfilaments appeared normal in "resting" polymorphonuclear leukocytes, but the increase in microtubules normally observed in stimulated cells was not seen after tetracaine treatment. These results suggest that tetracaine interferes with those interactions between immune reactants and the polymorphonuclear leukocyte cell surface which provoke exocytosis and increased oxidative metabolism.


Suppression of in vitro Epstein-Barr virus infection. A new role for adult human T lymphocytes. Studies have been performed on in vitro infection by Epstein-Barr virus (EBV) of subpopulations of human lymphocytes. B cells of adult peripheral or fetal cord blood transform with equal efficiency, whether assayed by DNA synthesis induction or by outgrowth of transformed lymphocytes. In contrast, unfractionated adult lymphocytes transform much less efficiently than those from fetal cord. Reconstitution experiments of different cell preparations indicated that this difference was due to a suppression of B-cell proliferation by adult Ig-negative lymphocytes which fetal Ig-negative lymphocytes were unable to perform. Separation of Ig-negative lymphocytes into various subpopulations revealed that the suppression was performed by T cells. Macrophages and null cells play little or no role in suppression. The relevance of this phenomenon to infection and recovery from EBV infection during and after infectious mononucleosis is discussed.


The developmental cycle of Chlamydia trachomatis in McCoy cells treated with cytochalasin B. The growth of a genital trachoma-inclusion conjunctivitis agent strain of Chlamydia trachomatis in McCoy cells treated with cytochalasin B was studied by quantitative infectivity estimations and by light and electron microscopy. Provided that infection of the monolayer was initiated by centrifuging the infectious particles on to the cells before incubation, this chlamydial strain grew as fast and to as high a titre [approximately 10(7) inclusion-forming units (i.f.u.) per culture] as those chlamydiae which infect cell cultures in vitro without centrifugation. Each i.f.u. inoculated yielded approximately 600 i.f.u., and extracellular infectivity was detected soon after intracellular infectivity appeared. Inclusions were recognized by fluorescent antibody staining techniques early in the developmental cycle when cultures were not infectious and when only reticulate bodies were seen by electron microscopy. Inclusions were recognized in Giemsa-stained preparations examined by dark ground microscopy only when elementary bodies appeared in the inclusions. Iodine staining was not a reliable indicator either of the number of inclusions present or of their infectivity.


The uncoupled relationship between the temperature-sensitivity and neurovirulence in mice of mutants of vesicular stomatitis virus. Inoculation of wild-type (wt) VSV intracerebrally (i.c.) in Swiss weanling mice results in a rapidly fatal illness with death in two to three days. In contrast, i.c. inoculation of temperature-sensitive (ts) VSV mutants G3I and G22, but not ts GII or ts G4I, results in a more slowly progressive central nervous system (CNS) disease with distinct neurological signs. Studies undertaken to evaluate the neurovirulence of ts VSV mutants indicated that the ability of ts mutants to produce pathological changes in the CNS of mice appeared related to their ability to replicate to high titre in brain and spinal cord. However, replication of ts VSV mutants in brain alone was not sufficient to produce clinical illness. More importantly, the ability of ts VSV mutants to replicate at non-permissive temperatures in vitro did not appear to correlate with neurovirulence. VSV harvests from brains and spinal cords of mice infected with each of the ts mutants were temperature-insensitive. In spite of their temperature-insensitivity, the biological behaviour of viruses recovered from CNS tissues was, surprisingly, not that which was characteristic of revertant clones. Virus isolates recovered from infected CNS tissues, despite their temperature-insensitivity, behaved biologically like the orignal stocks of ts mutant virus. These data suggest that temperature-sensitivity is not directly correlated with the unique pathogenesis elicited by infection with ts VSV mutants.


Studies of L cells persistently infected with VSV: factors involved in the regulation of persistent infection. Infection of interferon-treated L cells with VSV led frequently to the establishment of L cells persistently infected with VSV (LVSV cells). These cells were characterized by the following properties; (I) no supplement of antiviral factors such as anti-VSV antiserum, interferon, was required for their maintenance; (2) virus antigens were detected in about 5 to 30% of the cells by immunofluorescence staining; (3) the cells were not only resistant to superinfection by homologous virus, but also resistant to challenge by heterologous viruses such as Mengo virus; (4) the cells were destroyed by co-cultivation with heterologous cells susceptible to VSV infection; (5) the cells could be cured by serial cultivation in medium containing antiviral antibody, and the cured cells were as susceptible to VSV as normal L cells. It was shown that at least three factors (interferon, defective interfering [DI] particles and a selection of small-plaque temperature-sensitive [ts] mutants) took part in the maintenance of LVSV cells although it was difficult to evaluate exactly the relative importance of these factors. The effect of antiviral antibody, interferon and incubation temperature upon the maintenance of LVSV cells are discussed further.


Biochemical mapping of the foot-and-mouth disease virus genome. Four primary cleavage products, mol. wt. 10(3) X 100, 88, 56 and 52 (P100, P85, P56 and P52 respectively) are present in BHK 2I cells infected with foot-and-mouth disease virus (FMDV). However, no precursor polyprotein equal to the sum of their mol. wt. was detected, even when amino acid analogues and proteolytic enzyme inhibitors were used. Three of the primary products were shown to cleave to smaller polypeptides, including the capsid polypeptides of the virus. Polypeptide P88, which was shown to be the precursor of the capsid polypeptides, is translated from the gene located at the 5'-end of the genome. The order of the structural polypeptides, determined by the use of emetine, is VP4, VP2, VP3, VP1. The order of the remaining primary cleavage products is P52, P56 and P100. P56 is a stable product, identical with the virus infection associated (VIA) antigen found in virus harvests. The function of the other two products P52 and P100 is not known. EMDV thus differs from other picornaviruses in that there is an extra primary cleavage product, apparently resulting from translation of more of the virus genome.


Effects of interferon on vesicular stomatitis virus transcription and translation. The effect of interferon on the synthesis of the RNA species and proteins of vesicular stomatitis virus has been studied in two cell types. Virus protein synthesis is inhibited by interferon despite the apparent presence of near normal amounts of virus RNA with sedimentation values characteristic of virus messenger RNA. The synthesis of those virus RNA species which are completely dependent on virus protein synthesis is preferentially inhibited in interferon-treated cells. These results are most consistent with a model of interferon action postulating a primary effect on translation of virus messenger RNA.


Multiplicity-dependent replication of varicella-zoster virus in interferon-treated cells. The inhibitory effects of interferon (IF) on the replication of varicella-zoster (VZ) virus in human foreskin fibroblast (HFF) cultures inoculated with infected cells or with cell-free virus were assayed by measuring (1) yields of infected cells, (2) plaques, (3) microfoci and (4) cytopathic effects. More IF was needed to reduce yields of infected cells at high input multiplicities of challenge than at low input multiplicites, and still more IF was needed to prevent cytopathology due to VZ virus. A cell-free virus inoculum was more sensitive to the inhibitory effects of IF than an inoculum of infected cells. With the latter, but not with cell-free virus, the continuous presence of IF in the medium was necessary for it to express its maximum antiviral activity. To explain these results, it is suggested that some herpesviruses may establish "reservoirs' of infectivity and thus provide a prolonged challenge to IF-treated cells which are not uniformly resistant to infection.


Diurnal rhythms in cyclic nucleotide metabolism in Helix nervous system. Concentrations of cAMP (cyclic adenosine 3',5'-monophosphate) and cGMP (cyclic guanosine 3',5'-monophosphate), in ganglia from the garden snail Helix pomatia, vary considerably over the course of the day. There is a maximum in the concentration of both cyclic nucleotides between 08:00 and 12:00 (lights on 06:00 to 18:00), with the cAMP maximum occurring slightly later than that in cGMP. In addition there can be several smaller maxima in cAMP and cGMP levels; the timing of these can be  markedly different from experiment to experiment, with cAMP and cGMP sometimes in and sometimes out of phase with each other. This pattern is observed in Helix which had been activated from the dormant state 4-6 days earlier, but is not present in dormant or in long-active animals. The cyclic nucleotide rhythm can be seen in ganglia maintained in organ culture, and persists for at least 24 hours after removal of the tissue from the animal. There appears to be little change in the level of basal or NaF-stimulated adenylate cyclase activity in Helix ganglia over the course of the day. On the other hand, both cAMP and cGMP phosphodiesterase activities exhibit rhythms which are consistent with the rhythms in cAMP and cGMP concentrations.


Inking in Aplysia californica. III. Two different synaptic conductance mechanisms for triggering central program for inking. 1. In the previous two papers we described the motor elements which mediate all-or-none inking behavior in Aplysia (4) and the contribution of specific membrane properties of the ink-gland motor cells to the central program for inking (5). In the present paper we show that the central program for inking (a characteristic accelerating burst of action potentials in the motor cells) can be triggered in two different ways: 1) by means of a suprathreshold stimulus delivered to a single site on the animal; and 2) by means of two subthreshold stimuli closely spaced in time, delivered either to the same or to different sites on the animal. 2. The single suprathreshold stimulus triggers the central program primarily by means of a complex increased-conductance EPSP. The two subthreshold stimuli trigger the program by means of a complex decreased-conductance EPSP which is capable of augmenting subsequent afferent synaptic input. 3. The synaptic augmentation produced by the decreased-conductance EPSP is attributable to two properties of the EPSP: 1) it increases the input resistance of the ink-gland motor cells so that the synaptic current from afferent input onto the cells produces larger, more effective EPSP's; and 2) it increases the electrotonic coupling among the motor cells, thereby producing increased positive feedback among them. Thus, the decreased-conductance EPSP provides a novel mechanism for spatial and temporal summation.


Peptidyl transferase activity in rat skeletal muscle ribosomes after protein restriction. Rats were fed for 6 consecutive days a diet containing casein supplemented with 3 g/kg methionine. The protein level was either 20% or 3%. Skeletal muscle ribosomes were isolated and the activity of the ribosomal enzyme peptidyl transferase was determined. [3H]puromycin acted as acceptor for tRNA bound peptide provided it was located at the ribosomal peptidyl site. The results showed that the rate of peptidyl transferase activity was 13% lower after protein restriction. The differences amounted to 6% when diphtheria toxin and NAD were added as an inhibitor of elongation factor2. This factor catalyzes translocation of peptidyl-tRNA from ribosomal acceptor to peptidyl site. Between 0.11 and 0.15 pmoles peptidyl-tRNA per pmole ribosome were originally accessable as a substrate for peptidyl transferase. The results indicate that peptidyl transferase contributes to a decrease in protein  synthesis after protein restriction; but that other components also contribute to the decrease observed.


Characterization of sterol carrier protein binding with 7-dehydrocholesterol and vitamin D3. The interaction of rat liver sterol carrier protein (SCP) and 7-dehydrocholesterol or vitamin D3 was analyzed by the method of Scatchard plots and gel filtration on Sephadex G-100. Scatchard plots of binding data revealed that the binding behavior for 7-dehydrocholesterol was monophasic, while that for vitamin D3 was biphasic. In 7-dehydrocholesterol, the apparent number of binding sites and the apparent assocation constant K were 0.72 nmoles/mg protein and 8.75 X 10-7M-1, respectively. On the other hand, vitamin D3 showed two types of binding sites differing in affinity. The apparent number of binding sites and the K for high affinity binding were 0.65 nmoles/mg protein and 7.08 X 10-7M-1, those for low affinity binding were 1.51 nmoles/mg protein and 0.36 X 10-7M-1, respectively. Gel filtration of SCP on Sephadex G-100 column gave three protein peaks (peaks I, II and III protein according to the elution orders; Ve/Vo=1.0, 1.56 and 2.29, respectively). 7-Dehydrocholesterol was able to bind with peak III protein, while vitamin D3 was bound peaks II and III protein, respectively. The molecular weight of peak II protein was estimated to be 44,000 and that of peak III protein was 16,000. From these results, it was clearly demonstrated that SCP was involved in the transformation of 7-dehydrocholesterol to cholesterol and could also bind vitamin D3.


The identification of particulate matter in biological tissues and fluids. Sections, 1-2 micron thick, of Araldite embedded tissue were prepared to provide a link between optical and electron microscopy for the identification of crystals. This technique permits examination of individual particles by means of polarising light microscopy, electron microscopy, X-ray energy spectroscopy and electron diffraction. It provides a reliable method for routine and research studies. It has led to the positive identification of individual crystals of calcium pyrophosphate dihydrate and hydroxyapatite in synovial fluids. The method can be applied to the identification of other particulate matter in a variety of biological specimens.


Increased purine nucleotides in adenosine deaminase-deficient lymphocytes. It was found that ATP and cyclic AMP were greatly increased in human blood lymphocytes which were deficient in ADA. Certain other purine and pyrimidine nucleotides were elevated but to a lesser degree. Energy production in these cells may be inhibited by the increase in nucleotides since the ATP:ADP ratio was significantly below normal. Thus it appears that the immunologic deficiency in human ADA deficiency is related to increased nucleotide concentrations in the lymphocytes.


Vitamin D-resistant rickets associated with epidermal nevus syndrome: demonstration of a phosphaturic substance in the dermal lesions. A 5-year-old boy was found to have severe rickets in association with hyperpigmented, linear, verrucous, epidermal tumors, typical of the epidermal nevus syndrome. Normocalcemia (9.6 mg/dl), hypophosphatemia (2.0 mg/dl), elevated serum alkaline phosphatase concentration (313 IU), decreased renal tubular reabsorption of phosphorus (35%), radiologic evidence of rickets, and lack of response to usual therapeutic doses of vitamin D suggested hypophosphatemic vitamin D-resistant rickets. Therapy with vitamin D in doses to 750,000 IU and oral phosphate, 2.0 gm/day, failed to induce healing of the rickets. A subtotal parathyroidectomy performed when the patient was 9 years old was also without effect. When he was 12 years old several fibroangiomas on the face and left lower limb were excised. Within three months all biochemical abnormalities resolved and radiologic evidence of healing was observed. A portion of excised tissue was homogenized and injection of the supernate into a 6-week-old puppy induced excessive phosphaturia. The data suggest that the rickets was induced by a phosphaturic substance extractable from the tumors.


Enteropathogens associated with pediatric diarrhea in Mexico City. Enteropathogens were investigated as possible agents in pediatric diarrhea occurring in Mexico City during the summer of 1975. Pathogens were identified in 47 (76%) of 62 cases. Rotavirus particles were detected in 16 cases. Enterotoxigenic Escherichia coli was detected in 29 cases; 11 were positive for heat-labile enterotoxin and 18 were positive for only the heat-stable form of enterotoxin. Multiple pathogens were found simultaneously in 15 (24%) of the study population. This study indicates that the etiology of pediatric summertime diarrhea in Mexico City is diverse. ETEC and RV were the most frequently encountered pathogens, yet they frequently occurred together and with other pathogens. ST-only strains of toxigenic E. coli were as frequently recovered as LT-E. coli suggesting that both forms of ETEC must be sought in future field studies.


Clinical features of acute gastroenteritis associated with human reovirus-like agent in infants and young children. Between January, 1974, and June, 1975, infection with a human reovirus-like agent was detected in 47% of 152 infants and children hospitalized with acute gastroenteritis. Certain epidemiologic, clinical, and laboratory findings appear to be helpful in distinguishing gastroenteritis due to HRVLA from other causes in those children sick enough to require hospitalization. Age: 76% of infants and children seven through 12 months of age and 76% of those 13 through 24 months of age had infection with the HRVLA, whereas such infection was found in only 21% of infants under six months of age and 23% of children 25 through 60 months of age. Time of Year: 61% of patients studied during the cooler months had HRVLA infection and such infection was not found from June to October. Frequency of vomiting and dehydration: Twice as many patients infected with HRVLA as those who were not had vomiting (92%) and significant dehydration (83%).


Parainfluenza viral infections in children: correlation of shedding with clinical manifestations. Children presenting with acute respiratory disease to a private group practice in the fall of 1975 were studied to: (1) evaluate the efficacy in a pediatric office of a simple technics of obtaining nasal washes for the diagnosis of parainfluenza virus infections and (2) to determine the quantities of virus shed in relation to clinical characteristics. The nasal wash technic proved feasible for an office or clinic. Parainfluenza virus type 1 was recovered from 26 (74%) of 35 children with croup and from 40 (56%) of the total 72 children presenting with any form of respiratory illness. Virus was recovered significantly more often from children with croup and from those of younger age. The mean quantity of virus in 26 nasal washes was 2.97 log10 TCID50/ml. The shedding of greater quantities was correlated with younger age and the more frequent occurrence of laryngitis, pharyngitis, and fever.


Stannous-ion quantitation in pyrophosphate and polyphosphate radiopharmaceutical kits using differential pulse polarography. Stannous salts are used as reducing agents in many radiopharmaceutical kits. Differential pulse polarography, with 1 M sulfuric acid as the supporting electrolyte, is a relatively simple and effective technique for stannous quantitation and can be used in the quality assurance testing of pyrophosphate and polyphosphate radiopharmaceutical kits.


The pharmacokinetics of d-tubocurarine in man with and without renal failure. Plasma and urine concentrations of d-tubocurarine (d-TC) were determined and twitch tension was monitored before and after the intravenous administration of d-TC, 0.5 mg/kg in patients anethetized with halothane and nitrous oxide with (N=5) and without (N=5) renal failure. Terminal half lives of d-TC of 231 and 330 minutes predicted from other studies for patients with and without renal function were consistent with plasma concentrations measured. In patients with normal renal function, 38% of unchanged d-TC was eliminated in urine in 25 hours as opposed to 13% in those patients with a newly transplanted kidney. However, we still conclude that a newly transplanted kidney can eliminate d-TC effectively since the kidney was inserted 3.5 hours after administration of d-TC. Absence of renal function significantly prolonged the duration of neuromuscular blockade but did not alter the plasma concentrations of d-TC required for neuromuscular blockade. Thus, prolonged neuromuscular blockade from d-TC in patients with renal failure is due to decreased elimination rate of the drug as a consequence of lack of renal function rather than increased sensitivity.


Extraocular muscle surgery in the presence of complete paralysis of the fifth, sixth and seventh cranial nerves. A nine-year-old boy developed paralytic left esotropia secondary to an intracranial lesion. He also exhibited total loss of function of the left fifth and seventh cranial nerves. Extraocular muscle surgery in this situation was felt to be dangerous to the operated eye. The degree of risk however could not be determined from published reports. Surgery was performed and was complicated by severe corneal ulceration. Use of a therapeutic contact lens presumedly helped in finally achieving a satisfactory result.


Mechanism of cardiovascular effects of clonidine in conscious and anesthetized rabbits. The actions of intravenous clonidine [2-(2,6 dichlorophenylamino)-2-imidazoline hydrochloride] on blood pressure and heart rate were examined in conscious rabbits. Complete transection of thecervical spinal cord increased the intensity and duration of the hypertensive effect of 30 microgram/kg of clonidine and completely abolished the fall in blood pressure. Heart rate slowing by clonidine was reduced. Result were similar 1 hour, 24 hours and 7 days after cord transection. Bilateral aortic sinus nerve section (baroreceptor deafferentation) increased both the hypertensive and hypotensive action of clonidine but reduced the bradycardia. When cervical cord transection was combined with batensive action, were abolished. We conclude that whereas the hypertensive action results from a direct effect on peripheral adrenoceptors, the fall in blood pressure is related to a reduction in sympathetic tone mediated at the level of the brain stem or more rostrally. The heart rate slowing results from both a reduction in sympathetic tone and also an enhanced vagal outflow. The increase in vagal tone seems to be dependent on the intergrity of baroreceptor afferent pathways.


Inhibition of canine adrenergic transmission by an analog of dopamine: GJH-166. Experiments were conducted to investigate the effect of GJH-166 [Trans-4-methyl-7,8-dihydroxy-1,2,3,4,4a,5,6,10b-octahydrobenzo (f) quinoline HBr], a potential dopamine agonist, on responses to adrenergic vasoconstrictor stimuli in dogs. The hindlimb, gracilis muscle and spleen were isolated and perfused with arterial blood at constant flows. Responses of the nictitating membrane to adrenergic stimuli were aso studied. Intravenous injections of GJH-166 in doses of 0.25 to 4.00 micron/kg impaired responses to sympathetic nerve stimulation of the perfused hindlimb, gacilis muscle and spleen, and also inhibited responses of the nictitating membrane. GJH-166 had a preferential inhibitory effect on responses induced by low frequency stimulation of nerves. Haloperidol antagonized the inhibitory effect of GJH-166 on sympathetic transmission. The changes induced by norepinephrine at the perfused sites were not affected by the compound. Pressor responses to norepinephrine and tyramine were not inhibited by GJH-166. As a result of this study, the suggestion has been made that GJH-166 impairs sympathetic transmission by acting on certain inhibitory dopaminergic receptors located on the adrenergic nerve terminals.


Termination of transmitter release by stimulation of sodium-potassium activated ATPase. 1. The release of acetylcholine (ACh) from Auerbach's plexus of guinea-pig ileum has been measured in eserinized Krebs solution using longitudinal muscle strip preparations. 2. Removal of the external K ions enhanced both the resting and stimulated release of ACh from the plexus. This effect was not affected by tetrodotoxin. 3. On readmission of K+ to tissues which had been suspended in K-free Krebs solution the release of ACh was promptly reduced in both stimulated and unstimulated tissues. The extent of the reduction of ACh release depended on the exposure time to K-free solution, the recovery being delayed by longer exposure. 4. The ACh releasing effect of (1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) was completely inhibited by the readmission of K ions to tissue which had been kept in K-free Krebs solution. 5. Rb+ substitution for K+ produced no change in ACh release and addition of 5-9 mM-Rb after K removal reduced the release of ACh as K did readmission. When the K ions were substituted by Cs+, both the resting and stimulated release were enhanced. The amount of ACh released by a stimulus was enhanced both at low and high frequency of sustained stimulation. 6. Removal of the external K ions increased the release of tritiated noradrenaline (NA), from isolated rat iris; however, when K+ (5-9 mM) was readmitted the release was reduced even below the control value. 7. It is concluded that the stimulation of (Na+-K+)-activated ATP-ase in the membrane inhibits the release of transmitter, and under physiological condition Ca-fluxes and the subsequent inhibition of membrane ATP-ase may be involved in triggering the release of transmitter.


The effects of strontium and barium ions at synapses in sympathetic ganglia. 1. A study has been made of the effects of Sr2+ and Ba2+ ions at synapses in isolated superior cervical ganglia of guinea-pigs. Intracellular recordings of membrane potential were made from ganglion cells in the presence of different concentrations of Ca2+, Sr2+ and Ba2+ ions. 2. The addition of Sr2+ (2-5 mM) caused little change in resting membrane potential; in contrast, Ba2+ (1-6 mM) always depolarized the cells and prolonged the duration of action potentials. 3. The resting frequency of spontaneous miniature excitatory post-synaptic potentials (min. e.p.s.p.s) was briefly accelerated by the addition of either Sr2+ or Ba2+, but subsequently returned to about control levels. 4. Following replacement of Ca2+ by Sr2+, e.p.s.p.s could always be evoked during repetitive stimulation of preganglionic axons at a fixed latency after the nerve impulses ('phasic' transmitter release). Replacement of Ca2+ by Ba2+ produced many asynchronous e.p.s.p.s during trains of impulses ('residual' transmitter release). 5. By analysis of the interaction between Sr2+ and Ca2+, Sr2+ was shown to have a partial agonist action on 'phasic' transmitter release. The same analysis applied to Ba2+ failed to demonstrate either a partial agonist or antagonist action. 6. Both Sr2+ and Ba2+ prolonged e.p.s.p.s. Changes in Sr2+ could mainly be attributed to its effect on cell input resistance; Ba2+ may also prolong the time course of transmitter release. 7. The increased frequency of min. e.p.s.p.s which occurs during repetitive stimulation was potentiated by both Sr2+ and Ba2+, Ba2+ being about twice as potent as Sr2+. This activation of 'residual' transmitter release is independent of the action of these ions on 'phasic' release. 8. It is concluded that the reported maintenance by Ba2+ of acetyl-choline output from perfused ganglia results from the asynchronous release of large numbers of quanta during trains of impulses.


Muscarinic, alpha-adrenergic and peptide receptors regulate the same calcium influx sites in the parotid gland. 1. Carbachol, phenylephrine and substance P were each capable of producing a transient release of K (86 Rb) from rat parotid slices in the absence of extracellular Ca. 2. Each of the agonists was also capable of producing "cross-receptor inactivation"; that is, if a transient response was elicited by any one of the agonists then no transient response could be obtained by either of the other two. 3. Removal of carbachol from muscarinic receptors with atropine did not reverse the cross-receptor inactivation of the substance P response unless Ca was added with substance P or unless Ca was present when atropine was added. 4. It is concluded that the K release response in the parotid gland is mediated by receptor-controlled Ca influx sites. These influx sites also bind Ca at a location inaccessible to EGTA and release the bound Ca upon receptor activation. 5. It is also conlcuded that all three receptors (muscarinic, alpha-adrenergic, and peptide) appear to regulate the same Ca influx sites.


Changes in attitudes about mental illness among nursing students following a psychiatric affiliation. The purpose of this study was to determine changes in nursing students' attitudes about mental illness following a twelve-week psychiatric affiliation program in a state mental hospital. Data were collected by means of two combined opinions about mental illness scales -- Cohen and Struening's factor analytically-derived Opinions About Mental Illness (OMI) questionnaire and Ellsworth's empirically-derived Opinions About Mental Illness Scale. The sample consisted of 95 student nurses from three diploma schools of nursing. The subjects were administered the instrument at the beginning and end of their psychiatric affiliation. Following the affiliation, student nurses' attitudes about mental illness changed significantly in a favorable direction on the following attitudinal dimensions: Authoritarianism, Mental Hygiene Ideology, Social Restrictiveness, Interpersonal Etiology, Nontraditionalism, and Restrictive Control. Although significant changes were not found on Benevolence and Protective Benevolence, there was a trend toward favorable changes on both attitudinal dimensions.


Porcine respiratory disease in the western Cape Province. The aetiology and pathogenesis of respiratory syndromes in pigs are reviewed with emphasis on the role of environmental factors and diagnosis. Therapeutic and control measures are suggested and the cost of various regimens is dealt with in detail.


Antiviral activity of some beta-diketones. 1. Aryl alkyl diketones. In vitro activity against both RNA and DNA viruses. The discovery that 4-[3-ethyl-6-[(3,4-methylenedioxy)phenyl]-3-hexenyl]-3,5-heptanedione (40) exhibited an in vitro inhibitory effect against equine rhinovirus led to a structure--activity study to establish the criteria for optimum activity. Modification of the bridge included removal of the ethyl group and reduction of the double bond. The heptanedione was replaced with hexanedione and pentanedione with a minimal effect. The effect of replacing the heptanedione with beta-keto esters and monoketones was also investigated. Maintaining the hexamethylene bridge and heptanedione, the methylenedioxy group was replaced with various substitutents. In general, most substituents did not adversely affect activity particularly against equine rhinovirus although there was some variation in activity against herpesvirus. Strongly hydrophilic groups significantly reduced activity. Finally, the effect of varying the length of the alkyl bridge was examined in the 4-hydroxyphenyl series, where peak activity was attained with n = 8.


Antiviral activity of some beta-diketones. 2. Aryloxy alkyl diketones. In vitro activity against both RNA and DNA viruses. A series of aryloxy alkyl diketones II was synthesized and screened in vitro for antiviral activity. The effect of various substituents on the phenyl ring, as well as the length of the alkyl bridge, was examined to establish the requirements for optimum activity. One of the most active members of the series, 4-[6-(2-chloro-4-methoxy)phenoxy]hexyl-3,5-heptanedione (56), exhibited a high level of activity against both DNA and RNA viruses in both the tissue culture and organ culture screens and was particularly effective against herpesvirus type 1 and 2.