Identification of liver plasma membrane glycoproteins which bind to 125I-labelled concanavalin A following electrophoresis in sodium dodecyl sulfate. Following electrophoresis of ovalbumin in sodium dodecyl sulfate (SDS) this glycoprotein bound 125I-labelled concanavalin A (Con A). The reaction was specific and proportional to the amount of glycoprotein present on the gel. This technique was used to study the Con-A-binding glycoproteins of liver cell surfaces. Mouse liver plasma membranes were purified and subfractionated to yield two fractions corresponding to the bile canalicular surface and the surface between adjacent hepatocytes (Evans, W.H. (1970) Biochem. J. 116, 833-842). Both fractions bound 125I-labelled Con A, the former binding two to three times more lectin than the latter. Following SDS gel electrophoresis individual membrane glycoproteins reacted with 125I-labelled Con A. Both membrane subfractions yielded qualitatively similar Con A binding profiles, seven binding proteins being present in each. The results are consistent with a generally uniform distribution of glycoproteins over the hepatocyte surface. The reaction of lectins with glycoproteins following SDS gel electrophoresis should find general application in the study of membrane composition.


Lipogenesis in muscle, liver, and adipose tissue of normal and dystrophic chickens. The in vitro rate of oxidation and incorporation of [U-14C]glucose into lipids of muscle, liver, and adipose tissue of dystrophic and control chickens at 1 week, 1, 6, and 12 months of age were determined. The muscle and liver from dystrophic birds showed a high rate of [U-14C]glucose oxidation and incorporation into lipids during 1 week, 6 and 12 months. Insulin did not have any consistent stimulatory effect in all the tissues on the rate of glucose oxidation and incorporation into lipids. The contributory role played by the liver in the accumulation of lipids in dystrophic muscle is discussed.


The synthesis and some pharmacological properties of (2-L-DOPA)-oxytocin. The first reported synthetic analogue of a naturally occurring peptide with a residue of L-3,4-dihydroxyphenylalanine (L-DOPA) was prepared by coupling N-carbobenzoxy-S-benzylcysteinyl-L-DOPA azide with isoleucylglutaminylasparaginyl-S-benzylcysteinylprolylleuclglycinamide. The protecting groups were removed from the resultant nonapeptide derivative by sodium in liquid ammonia and the peptide analogue was formed by short term oxidation of the dithiol-containing compound. It was isolated by sequential partition chromatography and exclusion chromatography on Sephadex G-25. It was unstable at neutral or alkaline pH. [2-L-DOPA]-oxytocin was found to possess a minimum milk-ejection-like activity of 54 +/- 9 U/mg and uterotonic activity of 26 +/- 4 U/mg. These potencies are approximately 12% and 5% of the corresponding potencies of oxytocin.


Synthesis and some pharmacological properties of (2-tryptophan)-oxytocin. Previous studies have suggested that an aromatic amino acid residue at position 2 in oxytocin facilitates the expression of the hormone's biolgocial activities. [2-Tryptophan]-oxytocin, in which a residue of tryptophan has replaced that of tyrosine in oxytocin, has been synthesized by the method of azide coupling of the N-terminal dipeptide and C-terminal heptapeptide amide. It was found to have approximately 0.1% of the potency of oxytocin in milk ejection and uterotonic biological activities.


Sulfhydryl analogues of adenosine diphosphate: chemical synthesis and activity as platelet-aggregating agents. 2-Thioadenosine 5'-diphosphate (2-SH ADP), 2,2'-dithiobisadenosine 5'-diphosphate (2,2'-(S-ADP)2), 8-thioadenosine 5' diphosphate (8-SH ADP), and 6-mercaptopurine riboside 5'-disphosphate (6-MPRDP) were synthesized as potential affinity labels for ADP receptors on the blood-platelet membrane. The mean relative activities of these compounds in aggregating human platelets suspended in homologous plasma were 155% (2,2'-(S-ADP)2), 74% (2-SH ADP), 0.65% (8-SH ADP), and 0.08% (6-MPRDP). The mean relative activities against washed platelets were 249% (2,2"-(S-ADP)2) and 115% (2-SH ADP), whereas no aggregation occurred with 8-SH ADP or 6-MPRDP. The last two compounds were found to be weak inhibitors of ADP-induced aggregation. Therefore, thio-substitution at postition 2 followed by oxidation to a disulfide appears to be the most promising approach to further studies of affinity labelling of membrane ADP-receptors.


Pyruvate dehydrogenase activity in liver and brown fat of the developing rat. The total activity of pyruvate dehydrogenase (EC 1.2.4.1) and the fraction of the enzyme in the active form were assayed in brown fat and liver throughout the development of the rat. In brown adipose tissue, the total activity increased until the late suckling period. After weaning, a decrease was noted. The fraction of the enzyme in the active form did not increase until after 10 days of age, reached its highest level in the late suckling period and remained at this level after weaning. Pyruvate dehydrogenase in liver decreased in both total activity and percentage activity in the early neonatal period. Both parameters increased after this period, reaching their highest levels in the late suckling period. In both fetal liver and fetal brown fat, the total activity of pyruvate dehydrogenase was increased by in vitro incubation with insulin.


The isolation and characterization of the tropomyosin binding component (TN-T) of bovine cardiac troponin. The tropomyosin binding component (TN-T) of troponin was purified from bovine cardiac muscle using a combination of ion exchange chromatographies in the presence of urea. Sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-T of 36 300 +/- 2 000, consistent with a value of 37 000 +/- 1 000 determining by polyacrylamide gel electrophoresis. Calculations based upon circular dichroism spectra indicate an apparent alpha-helical content of 43 +/- 3% for TN-T. Polyacrylamide gel electrophoresis and the effects of the calcium binding component (TN-C) upon the solubility of TN-T suggest that the two cardiac troponin components can interact with each other. Cosedimentation analysis of solutions containing cardiac tropomyosin and TN-T provide evidence for complex formation involving these two proteins. The data presented on the physical and chemical properties of TN-T, as well as the interaction studies indicate that the cardiac muscle regulatory system operates in a manner similar to that proposed for skeletal muscle.


Properties of the citrate transporter in rat heart: implications for regulation of glycolysis by cytosolic citrate. The efflux of [14C]citrate from rat heart mitochondria was significantly greater with L-malate as the extramitochondrial substrate as compared with [12C]citrate, isocitrate or phosphoenolpyruvate. The concentration of L-malate required for half-maximal rate of efflux of citrate was 0.45 mM and the maximum velocity was 0.36 nmol min-1 mg-1 mitochondrial protein at 23 degrees C. This citrate transporter was inhibited by 1,2,3-benzenetricarboxylate and palmitoyl-CoA but not to the same extent as these compounds inhibit the tricarboxylate carrier in rat liver mitochondria. The apparent inability of these mitochondria to transport citrate in the inward direction necessitates the presence of a cytosolic citrate removal pathway. We propose that the enzymes of this pathway in rat heart could be ATP citrate (pro-3S)-lyase (EC 4.1.3.a) and carnitine acetyltransferase (EC 2.3.1.7), both of which we demonstrate to have adequate activity in both the fed and fasted state. An hypothesis has been put forward to account for the inhibition of rat heart phosphofructokinase by citrate in the fasted state incorporating these properties of the citrate transporter and ATP citrate (pro-3S)-lyase.


In vitro biosynthesis of gamma-lipotropic hormone. Sheep gamma-lipotropic hormone (gamma-LPH) is a pituitary polypeptide made of 58 amino acids and is formed of the first 58 residues of beta-lipotropic hormone (beta-LPH). The C-terminal portion (41-58) of gamma-LPH is identical with the structure of beta-melanophore-stimulating hormone (beta-MSH). We hypothetized in 1967 that beta-LPH could be the biological precursor of beta-MSH and that gamma-LPH could be an intermediate compound. We demonstrated in 1974 that beta-LPH is actively synthesized in the bovine pituitaries. We now studied the biosynthesis of gamma-LPH by monitoring the incorporation of radioactive amino acids in beef pituitary slices. We separated gamma-LPH from the other radioactive proteins with a method previously described. We characterized the radioactive proteins by ion-exchange chromatography, gel filtration and polyacrylamide gel electrophoresis. Our results show that radioactive gamma-LPH was actively synthesized. This gamma-LPH has all the chemical characteristics of nonradioactive gamma-LPH. However, in the conditions used, we were unable to demonstrate biosynthesis of beta-MSH. These results suggest that gamma-LPH is biosynthesized more slowly than beta-LPH and that the conversion into beta-MSH, if it exists, is a slow or subactive process in the species studied.


The absence of cell-specific histone in erythroid cell from rabbit marrow. The histone complements of erythroid cells from anemic rabbit marrow and cell fractions enriched in orthochromatic erythroblasts were examined using a variety of techniques. None of these methods revealed the presence of a histone comparable to H5, the erythrocyte-specific histone of birds and fish. Purified histones H1, H2A, H2B, H3 and H4 from rabbit marrow nuclei showed no unusual amino acid compositions.


Lipid composition of developing Xenopus laevis embryos. The total lipid content, amount of phospholipid, proportions of major polar and neutral lipid classes, and the overall fatty acid composition were examined in Xenopus laevis embryos. No obvious differences were observed in any of the parameters between fertilization and hatching or between eggs produced by different females. The average lipid content per egg was 113 mug, 31.6 mug of which was phospholipid. The major phospholipids were phosphatidylcholine and sphingomyelin. The major fatty acids were palmitic and oleic acids, but polyunsaturated fatty acids were also present in substantial amounts. The results suggest that significant de novo synthesis of lipids does not occur until after hatching.


Observations on the effect of salicylate in fever and the regulation of body temperature against cold. Prostaglandins appear to be mediators, within the hypothalamus, of heat production and conservation during fever. We have investigated a possible role of prostaglandins in the nonfebrile rabbit during thermoregulation in the cold. Shorn rabbits were placed in an environment of 20 degrees C, and rectal and ear skin temperatures, shivering and respiratory rates were measured. A continuous intravenous infusion of leucocyte pyrogen was given to establish a constant fever of approximately 1 degree C, and after observation of a stable febrile temperature for 90 min, a single injection of 300 mg of sodium salicylate, followed by a 1.5 mg/min infusion was then given. After the salicylate infusion was begun, rectal temperature began to fall, and reached nonfebrile levels within 90 min. Shivering activity ceased, respiratory rates increased, and in two animals, ear skin temperature increased. When these same rabbits were placed in an environment of 10 degrees C, at a time they were not febrile, and an identical amount of salicylate was given, rectal and ear skin temperatures, shivering and respiratory rates did not change. These results indicate that prostagladins do not appear to be involved in heat production and conservation in the nonfebrile rabbit.


Longitudinal extension of the pulmonary trunk during breathing in young rabbits: a possible factor in elastin fragmentation. Corrosion casts of the pulmonary trunk and major branches of the pulmonary arteries of 39 rabbits were made from a silicone polymer. In half of the rabbits the casts were made with the lungs expanded and half with the lungs collapsed. The length of various segments of the casts were measured from magnified photographs. It was found that in rabbits less than 23 days old the pulmonary trunk is significantly longer when the lungs are expanded than when they are collapsed. This suggests that a repeated longitudinal extension  of the vessel occurs during breathing. This effect disappears after 30 days of age, possibly because of elastin fragmentation. Between the ages of 23 and 30 days the growth  in length and diameter of the pulmonary trunk undergoes a rapid acceleration. This may also be the result of elastin fragmentation. Whereas it may be that repeated longitudinal stress in the pulmonary trunk during breathing causes elastin in its media to fragment, one cannot exclude the possibility that other factors such as growth are responsible.


Dexclamol, a candidate for neuroleptanalgesia. Dexclamol, a potent neuroleptic, was found to potentiate the anesthetic actions of halothane when given at relatively small dose to albino rats. This effect was further enhanced by the addition of phenoperidine, a potent analgesic, at doses that per se did not influence the effects of halothane. Droperidol was used as a standard. In potentiating the effects of halothane, dexclamol behaved both qualitatively and quantitatively in a manner similar to droperidol. Dexclamol, however, was approximately 37 times less potent than droperidol in antagonizing the vasopressor effects of epinephrine.


Effective vascular compliance and venous diameter in dogs. Total effective vascular compliance was measured repeatedly in open-chest dogs without circulatory arrest, utilizing a closed-circuit venous bypass system with a constant cardiac output. Mutual inductance coils were used to measure the diameter of the inferior vena cava above the diaphragm at the position where the pressure change was recorded during a volume load (lambde V). In all experiments, there was a relationship which tended to be curvilinear between the diameter of the inferior vena cava and the venous pressure before lambde V. No relationship was demonstrated between the initial diameter or pressure and the calculated effective vascular compliance. During aortic constriction or infusion of noradrenaline, the effective compliance was reduced in value at any given initial venous diameter and pressure. An unaltered venous diameter and plasma volume excluded the possibility of a large change in initial venous volume as a cause of the observed changes in compliance during aortic constriction or during infusion of noradrenaline. A relationship was observed between compliance and calculated venous wall tension so that as the wall tension, developed during a fixed volume load, increased, there was an associated reduction in compliance. These results demonstrate that the measurement of effective compliance provides an assessment of combined active and passive venous wall tension and venous tone.


Unmasking of sympathetic vasoconstruction of the coronary vessels after acute administration of reserpine to dogs. Acute intravenous administration of reserpine or pretreatment of dogs with Segontin selectively abolished coronary vasodilation and unmasked a constrictor response to stimulation of the cardiac sympathetics. In view of earlier findings of separate coronary vasomotor and cardiostimulatory sympathetic innervation, the results are interpreted to indicate the existence of reserpine-resistant short (vasomotor) and reserpine-sensitive long (cardiostimulatory) sympathetic postganglionic neurons.


Effect of 6-hydroxydopamine on diet-induced hyperlipidemia and atherosclerosis in the rat. The effect of 6-hydroxydopamine (6-OH-dopamine) and of a cholesterol rich diet on the plasma and aortic cholesterol of female rats were studied. Both the diet and the 6-OH-dopamine produced an important increase in plasmatic and aortic cholesterol. A synergistic effect of these two treatments was observed on the plasma but not on the aortic cholesterol. The mechanism by which 6-OH-dopamine produces hypercholesterolemia and a increase in aortic cholesterol remains to be explained.


Species differences in the reactivation of organophosphate-inhibited plasma esterases by diacetylmonoxime. A study was conducted to assess whether the protection afforded to organophosphatepoisoned animals by diacetylmonoxime (DAM) was correlated with the reactivation of non-essential aliesterases (AliE). In vitro, the DAM-catalyzed reactivation of plasma AliE and cholinesterases (psi ChE) of rat, rabbit and guinea pig inhibited by 10-5 M diisopropylphosphorofluoridate (DFP) and O,O-dimethyl-2,2-dichlorovinyl phosphate (DDVP) was investigated. Marked reactivation of the rat plasma enzymes was achieved with 10mM DAM. Higher concentrations (30 mM) were necessary for the slow reactivation of rabbit and guinea pig plasma AliE. Reactivation of the psiChE of these species was comparatively slow. Reactivation of DDVP-inhibited esterases proceeded in all species at a more rapid rate than those inhibited by DFP. The dependence of psiChE reactivation upon concomitant more rapid reactivation of AliE by DAM was demonstrated using Sephadex fractionated AliE and psiChE but only a marked effect was observed with the rat, suggesting that the plasma AliE of this species is functionally different. The in vitro observations were confirmed by in vivo studies in rats and rabbits. DAM (50 or 150 mg/kg), administered to atropinized rats 15 min before a lethal dose of DFP, protected the animals. Few severe toxic signs were observed and reactivation of both plasma AliE and psiChE occurred. In contrast, DAM protected the rabbit against a lethal dose of DFP but only reactivation of the erythrocyte acetylcholinesterase was observed.


Characteristics of accumulation of ephedrine in rabbit atria. The characteristics of uptake of (not equal to)-(beta-14C)ephedrine were studied in isolated rabbit atria. Ephedrine was rapidly accumulated against the concentration gradient. From 5 X 10-7 to 10-2 M, uptake occurred at a uniform initial rate. Uptake was slightly inhibited by high concentrations of ouabain, cocaine, desipramine, lidocaine and phenethylamines, and by a reduction in the external Na+ concentration. Uptake was not, however, reduced by omission of K+ from the medium, by metabolic inhibitors or by a variety of drugs known to inhibit the extraneuronal uptake and binding of noradrenaline. Pretreatment of animals with 6-hydroxydopamine very significantly reduced the uptake of (not equal to)-(3H)metaraminol, but did not alter the uptake of ephedrine. It was concluded that the uptake of ephedrine in rabbit atria occurred predominantly in extraneuronal tissues possibly as a result of passive diffusion followed by binding.


The growth of nitrogen-fixing Azotobacter chroococcum in continuous culture under intense aeration. Azotobacter chroococcum (ATCC 7493) was grown in continuous culture with intense vortex aeration (stirring rate 1750 rpm) with up to 50% O2 in the gas phase. Under these conditions the dissolved O2 generally remained at zero while the cell growth rose to about twice the normally accepted value. The meaning of the term "O2-limitation" in N2-fixing A. chroococcum cultures is critically examined.


[The Palmier date palm and Fusarium infection. II. Report on the stereochemistry of monosaccharides and their various derivatives and the growth of Fusarium oxysporum f.sp. albedinis (Killian et Maire) Gordon]. Fusarium oxysporum albedinis was grown in the presence of 20 different sugars or their derivatives. The results obtained were correlated with the stereochemical structure of the main oses. Osmotic phenomena as well as active and selective transport were used to explain differences in utilization of oses of very near structure. Based on the affinity of the fungus for mannose, a hypothesis is formulated concerning the fungus action in the host.


Effect of chemicals used as nitrification inhibitors on the denitrification process. Several chemicals used as nitrification inhibitors were tested to determine their effect on dentrification by a Pseudomonas sp. and in soil. Denitrification by the bacterium was suppressed by 2-chloro-6(-trichloromethyl)-pyridine (N-Serve) at a concentration of 50 ppm, while 2,5-dichloroaniline caused the accumulation of nitrite in the culture medium. The nitrification inhibitors had little effect on the denitrifying activity in soil under anaerobic conditions. 2-Sulfanilamidothiazole inhibited denitrification to some extent and samples supplied with potassium azide produced N2O rather than N2 as the predominant gas.


Urease-negative strains of Clostridium sordellii. Twenty-seven of 37 non-toxigenic, urease-negative strains originally identified as Clostridium bifermentans that were isolated in the Antarctic are reidentified as C. sordellii by the tests for DNA-DNA homology, by the absence of mannose in the cell wall, and by growth inhibition of mannose. The test for cell wall sugar components of urease-negative and -positive strains of C. sordellii revealed that glucose, mannose, and rhamnose could not be detected in any of eight urease-negative strains used by galactose was detectable in seven of the eight strains and that glucose or galactose or both of the two sugars were present in the urease-positive strains tested.


Preparation and chemical properties of the outer membrane of a moderately halophilic gram-negative bacterium. Outer membranes, almost free from peptidoglycan components, were prepared from a moderately halophilic gram-negative bacterium grown in a medium containing 2 M NaCl. The outer membrane was easily released, leaving mureinoplasts, by mild desalting in a 20% sucrose solution containing 50 mM tris(hydroxymethyl)aminomethane-HCl buffer, pH 7.8. The membrane was recovered by treatment with DNase I and CsCl buoyant density centrifugation. Chemical analyses revealed that the outer membrane was mainly composed of 31% protein, about 20% extractable lipids (mainly phospholipids), and lipopolysaccharides. The proteins had about 18 mol % excess of acidic over basic amino acids. The phospholipids comprised phosphatidyl ethanolamine, phosphatidyl glycerol, cardiolipin, and an unidentified phospholipid containing glucose, which seemed mainly associated with the outer membrane. The content of lipopolysaccharides in the outer membrane was calculated arbitrarily as 30% from the heptose content. A unique feature of these lipopolysaccharides seemed to be higher lipid content than found in lipopolysaccharides of other gram-negative bacteria. The major fatty acids of bound lipids of the outer membrane resembled those of the lipopolysaccharides obtained from cell envelope preparation and contained high concentrations of 3-hydroxy lauric acid.


Two toxic indole alkaloids from Penicillium islandicum. Two new isomeric metabolites, toxins A and B, were isolated from culture extracts of Penicillium islandicum. Both toxins had a molecular formula of C16H15O2N2Cl, and physical and chemical analyses indicated that they were indole alkaloids. LD50 of toxin A was about 75-125 mg/kg in day-old cockerels. Insufficient toxin B was available to determine accurate LD50 values. Toxin A moderately inhibited growth of wheat coleoptiles but did not inhibit growth of week-old intact corn and bean plants. Toxin B showed no plant-growth inhibition.


Nematophagous fungi: three new species of Myzocytium. Three species of Myzocytium parasitic on nematodes are described as new. In M. papillatum the zoospores encyst directly on the host cuticle before penetration. This species produces smooth, spherical oospores. In M. glutinosporum the biflagellate zoospores do not attack the host directly; after encystment they produce a spherical adhesive bud which allows the spores to adhere to the cuticle of passing nematodes. This species produces echinulate, spherical oospores. In M. anomalum the primary spores are aplanospores. After a dormant phase, and when suitably stimulated, these aplanospores change into biflagellate zoospores and the latter encyst on the host cuticle. No sexual state is known in this species. Persistence is by means of thick-walled, spherical chlamydospores.


Release of bacterial alkaline phosphatase in the rumen of cattle fed a feedlot bloat-provoking diet or a hay diet. Alkaline phosphatase (APase) was present in the bovine rumen in both cell-free and cell-associated states and levels of the enzyme varied with dietary regime. Reaction product deposition showed that the enzyme was associated with the mixed bacterial population. No enzyme was observed to be associated with protozoa. Trace activity of APase was also detected in the saliva. The presence of large amounts of APase in cell-free rumen fluid of cattle fed fine concentrate feed is believed to be due, in part, to the breakage of bacterial cells that occurs in the rumen.


Comparative zone electrophoresis of esterases of Staphylococcus species isolated from mammalian skin. The electrophoretic mobilities of non-specific esterases in vertical polyacrylamide slab gels were determined for 184 strains of staphylococci, representing a total of 18 proposed species and subspecies. Markedly uniform esterase patterns were seen within species demonstrating a high degree of human host specificity, while those species demonstrating a wide host range were polytypic and often showed considerable polymorphism. The unique banding patterns found in several species indicate that this technique may serve as a valuable aid to existing taxonomic schemes. Starch gel electrophoresis of representative strains usually produced sharper esterase bands than were found with polyacrylamide electrophoresis. However, the additional molecular-sieving effect produced by the polyacrylamide gels differentiated esterases to a greater extent.


Some properties of an unidentified halophile: growth characteristics, internal salt concentration, and morphology. An unidentified halophile isolated from plates of a complex agar medium containing 4.25 M NaCl showed optimum growth in broths containing 0.5-1.0 M NaCl but exhibited a wide range of growth from 0.045-4.5 M. The organism can be classified as a facultative halophile with wide salt tolerance. Logarithmic phase cells grown in media containing 0.5 M NaCl were rod-shaped in long chains which changed to smaller, single, or paired cells in stationary growth. The internal Na+ and K+ concentrations were 0.05 M and 0.34 M for logarithmic phase cells and 0.29 and 0.32 M for stationary phase cells. In 4.3 M NaCl media the cells were rod-shaped throughout the growth cycle, occurring primarily in pairs. The internal Na+ K" concentrations in cells in logarithmic phase growth were 0.62 M and 0.58 M while in stationary phase growth these values were 1.01 M and 0.66 M respectively. In contrast, logarithmic phase cells of the extreme halophile Halobacterium cutirubrum had internal Na+ and K+ concentrations of 0.80 M and 5.32 M when grown in 3.3 M NaCl. The internal Na+ and K+ concentrations, therefore, in the unidentified halophile do not resemble those found in H. cutirubrum but are much closer to those present in Escherichia coli.


The effect of ribonuclease on the penetration of R17 phage A-protein and RNA. In an attempt to throw further light on the relationship of R17 phage RNA and A-protein during the early stages of infection, studies were carried out to determine the effect of ribonuclease (ribonuclease I, EC 3.1.4.22) on the ability of these two phage components to penetrate into host bacteria. It was found that the penetration of phage RNA is affected by ribonuclease concentrations as low as 0.1 mug/ml, while the penetration of phage A-protein was unaffected by ribonuclease concentrations as high as 20 mug/ml. In addition, it was found that a significant fraction of the phage RNA is resistant to the ribonuclease effect. This RNase-resistant portion of the phage population increased with increasing phage concentrations, and gave rise to the penetration of intact, 28S RNA molecules that produced the expected number of infectious centers. These findings are discussed in terms of a model for phage RNA injection in which the A-protein functions both as an attachment organelle and a pilot protein that guides the RNA from the capsid to the exterior surface of the F pilus, and thence into the host bacterium.


Effects of temperature on the growth of psychrophilic bacteria from glaciers. Growth of five strains of psychrophilic bacteria (four Arthrobacter and one Pseudomonas) isolated from glacial deposits was studied at different temperatures. Three strains were facultative psychrophiles, having an optimum temperature for growth at about 25-28 degrees C and a maximum at about 32-34 degrees C. The two Arthrobacter glacialis strains were found to be obligate psychrophiles with an optimum at 13-15 degrees C and a maximum at 18 degrees C. Arrhenius plots showed that A. glacialis could compete with the facultative psychrophilic bacteria only at 0 degrees C, that is, the temperature of its natural environment. The psychrophilic Arthrobacter species studied here are more resistant to thermal stress than are marine psychrophilic bacteria. For Arthrobacter, in contrast to Pseudomonas, temperatures above the optimum induced formation of filaments and abnormal cells. The culture turbidity increased 10 to 30 times, whereas viable count tended to decrease. The thermal block seems to prevent cell wall synthesis and septation, but at a different step for each species.


The fine structure of conidial development in the genus Torula. III. T. graminis Desm. Torula graminis produced blastoconidia in acropetalous chains after the evagination of a characteristic conidiogenous cell. Conidia consisted of up to 15 cells and their cell wall was differentiated into an outer melanized zone and an inner hyaline zone. A consistent cytoplasmic feature of conidial cells was the presence of dictyosomal-like membranous stacks often closely associated with the nucleus. Vesicles that developed from the dictyosomal-like cisternae were probably involved in conidial wall synthesis.


Repression of the acid phosphatase of Saccharomyces bisporus in relation to the polyphosphate content of the cells. The relationship between the level of stored polyphosphate in growing cells of Saccharomyces bisporus and the repressing or derepression of the synthesis of the enzyme acid phosphatase (EC 3.1.3.2) was investigated. Time-course studies showed that there is no correlation between the cellular concentrations of either polyphosphate or orthophosphate and the ability of the cells to form this enzyme. The only compound investigated that was capable of repressing acid phosphatase synthesis was orthophosphate in the growth medium (i.e. orthophosphate outside the cell).


[Antibacterial effect of various mycotoxins and fungal metabolites against Bacillus thuringiensis (Berliner) strains sensitive or resistant to aflatoxin B1]. Antimicrobial activity of pure preparations of mycotoxins and fungal metabolites was studied against strains of Bacillus thuringiensis (Berliner). Two resistant strains, called stable-variant, were isolated after treatment with high concentrations of aflatoxin B1. These strains were then resistant also towards compounds with a double furan system (aflatoxins B1, B2, G1, G2, and sterigmatocystin).


Long-chain n-alkanes occurring during microbial degradation of petroleum. Five axenic cultures and a mixed culture were examined for ability to degrade South Louisiana, Brass River Nigerian, Anaco Venezuelan, and Altamont crude oils. A wax was observed during microbial degradation of Altamont crude oil, but not during weathering of the oil. The high-boiling n-alkanes in the wax were associated with microbial degradation of the oil and appeared to be similar to components of tarballs found in the open ocean.


Electron microscopy of antibody-labelled cells of Streptococcus mutans. Examination of immune complexes between cells of Streptococcus mutans and homologous antiserum by the techniques of thin-sectioning and freeze-etching revealed that the cells were embedded within and extensive matrix 80-90 nm thick with defined boundaries.


Distension arthrography of the shoulder joint. Pain and stiffness resulting from degenerative disease of the rotator cuff of the glenohumeral joint is not always self-limiting. In a few patients, the symptoms persist despite medication and physiotherapy. Distension arthrography, a method of hydrostatic manipulation, has been used in six such patients; five recovered full range of abduction and rotation and the sixth was improved. It is concluded that distension arthrography should be part of the treatment of intractable pain stiffness of the shoulder joint.


Knee arthrodesis: long-term results.

Knee arthrodesis: long-term results. A review of 41 patients in whom knee fusion was performed mainly for degenerative arthritis and sepsis disclosed that only 15% of patients were without complications. Complications were numerous and appeared both early and late - persistent knee and back pain were the most common. Many patients were unable to return to work or to participate in social activities. The optimal position for fusion is 15 to 20 degrees of flexion, which results in a smoother gait and less difficulty for the patient in driving a car. Because of the continued refinement in total knee joint replacement, the indications for knee arthrodesis should be confined to the treatment of chronic infection and failed total joint replacement.


Value of intraoperative arteriography in arterial embolectomy surgery. Clinical assessment of the adequacy of arterial embolectomy and reconstruction is often unsatisfactory and inadequate. Objective information on the state of the distal circulation can be readily obtained at the time of surgery by arteriography. Illustrative cases demonstrating the usefulness of control arteriography obtained during the surgical procedure are presented.


Fatal cerebral embolus - a complication of left ventricular venting. A 3-year-old child died following a routine tetralogy of Fallot repair. Death was caused by a cerebral embolus. Injury to the left ventricle during left ventricular venting resulted in an apical myocardial infarct. The embolic source was a mural thrombus covering the area of infarction. A technique is described whereby left ventricular venting can be avoided during open-heart surgery in small patients.


Surgical rates in the Canadian provinces, 1968 to 1972. Canadian and provincial rates for eight elective and seven nonelective operations were determined from 1968 through 1972. Of the elective procedures considered, the Canadian tonsillectomy rate decreased substantially and rates for hysterectomy and cholecystectomy showed large increases. The provinces reflected national trends for these three operations. Except for coletomy, which increased 36%, rates for the nonelective procedures showed little change. Correlations between the numbers of surgical personnel and the elective surgery rates in the provinces were demonstrated in 3 of the 5 years; these are best shown at the extremes. Newfoundland had the fewest surgeons, a bed: population ratio below the national average and the lowest combined elective surgical rate; Alberta ranked high in all three categories. In the seven other provinces, there was less variation in the number of surgeons, hospital beds and elective surgical rates. Provinces with extensive insurance coverage prior to universal medical insurance reduced their combined elective surgery rate, while provinces where fewer people were insured showed increases. In the absence of different methods of payment for surgical services and marginal changes in the number of hospital beds and the number of surgeons, more insured persons produced more elective surgery.


Technique for cardiopulmonary bypass in the goat. The goat is an ideal animal for intracardiac surgical investigation. The animal is docile and easy to care for; it has an ideal heart size, a high cardiac output and a long life expectancy. The animal tolerates cardiopulmonary bypass well and does not require blood transfusion. Anoxic cardiac arrest is tolerated for up to 35 minutes. Initial experience in 36 animals with operations on the right side of the heart resulted in a 72% long-term survival.


Sudden hemolysis indicating prosthetic valve dysfunction. Ten months after aortic valve replacement (Björk-Shiley prosthesis) a 57-year-old man had thrombotic encapsulation of his prosthetic valve. The only clinical manifestation of this serious complication was gross hemoglobinuria. The management of the patient is described. The slightest change in the condition of a patient after prosthetic valve replacement should be viewed as a possible sign of thrombosis.


Gallium-67-citrate scintiscanning in the search for occult primary malignant tumours. Fifty consecutive patients in whom occult primary malignant disease was suspected underwent scintiscanning of the head, neck and trunk with gallium-67-citrate. In 17 patients a carcinoma was correctly identified as to presence and approximate location. In another seven patients the site of the primary tumour was identified from the scan and confirmed by radiologic study but no histopathological diagnosis, except for metastatic anaplastic carcinoma, was made. In another five patients the many abnormalities on the scan did not permit identification of a possible site of the primary tumour. Eleven patients had normal scintiscans and no evidence of malignant disease on follow-up. In seven patients with proven carcinoma false-negative results were obtained: no uptake of the radioisotope was detected, even at the sites of known disease. In three patients false-positive uptake of the radiotracer was observed; two had benign disease and one had a malignant tumour remote from the scan abnormality.


Evaluation of the normal range of values for uptake of radioactive iodine by the thyroid gland. Uptake of radioactive iodine by the thyroid gland after oral administration of 75 muCi was determined in 1971-72 in 60 euthyroid female volunteers from the North Shore of Vancouver. Values were as low as 3% at 4 hours and 7% at 24 hours in subjects otherwise proven to be euthyroid. The highest values found were 13 and 26% at 4 and 24 hours. The effective thyroxine ratios were all within the accepted normal range. However, more than half of the volunteers showed some enlargement of the thyroid gland. These results suggest an increased iodine pool in the subjects, the likley source being iodine in mild, dairy products and erythrosine, a colouring agent in foods and pharmaceutical products. Potassium iodate, used sometimes in bread baking, contributed little to this pool in our sample. Subjects were, on the average, 9 to 25% heavier than their peers 20 years ago.


Application of special diagnostic techniques in the management of nodular goitre. The primary challenge in the management of nodular goitre is to establish which thyroid nodules are malignant. Since selection of patients for operation on the basis of palpation of nodules alone gives a low yield of malignant disease, physicians have sought criteria for selection that combine the information obtained from special laboratory procedures with thoughtful clinical appraisal. Such special procedures, which include radioisotope scintiscanning, echography by B-mode ultrasonography, and either large- or fine-needle aspiration and cytologic examination of the aspirate, are considered valuable in a proposed clinical approach to the management of thyroid nodules.


A functioning pleomorphic carcinoma of the thyroid. A metastasizing functioning pleomorphic carcinoma of the thyroid displayed a morphologic piture ranging from follicular to sarcomatous and carcinoid-like patterns. Spindle-cell elements were believed to be the result of metaplasia of the follicular epithelium. Development of carcinoid-like lesions in the secondary deposits of tumour supported the contention that so-called parafollicular or c-cells may be a phenomenon of metaplasia and not a histologic entity. The tumour was associated with a chromophobe adenoma of the pituitary and a benign schwannoma of the stomach. Clinically the patient did not show any other endocrinologic abnormalities and lived for 4 years from the time of diagnosis of the thyroid tumour.


False left ventricular aneurysm as a complication of open heart surgery. A false aneurysm developed at the site of left ventricular venting in an 8-year-old boy who had undergone aortic valve commissurotomy under cardiopulmonary bypass. The diagnosis was made by clinical examination and chest radiography and confirmed by left ventricular cineangiography. The aneurysm was successfully resected.


Yellow-nail syndrome: report of three cases. The yellow nail syndrome, a combination of yellow discolouration of and dystrophic changes in the nails, pleural effusions and lymphedema, is thought to be relatively rare; to date 44 cases have been reported. Of a further three patients with this syndrome, one had all three features, one had the yellow nails alone and the other had pleural effusions and lymphedema without classic nail changes. Each had recurrent lower respiratory tract infections; and of all 47, chronic pulmonary infections occurred in approximately one quarter and were frequently associated with chronic sinus infections. The underlying abnormality is presumed to be a congenital defect of the lymphatics, but so far this has not been demonstrated to be the cause of the nail changes, the pathogenesis of which remains obscure.


Forty years of stress research: principal remaining problems and misconceptions. An overview of the main problems and misconceptions in the clinical application and theoretic evaluation of the stress concept reveals that the same 10 problems appear to cause the greatest difficulties in its application, irrespective of the specialty in which it is used: (1) the correct definition of stress, stressors and the general adaptation syndrome; (2) the concept of nonspecificity in biology and medicine; (3) the conditioning of stress responses by diverse endogenous (mainly genetically determined) and exogenous (environmental) factors; (4) the relation between the genral and the local adaptation syndromes; (5) the difference between direct and indirect pathogens; (6) the definition of the morbid lesions in whose pathogenesis stress plays a particularly prominent role--the so-called diseases of adaptation; (7) the role of genetics versus that of factors under voluntary self-control in mastering biologic stress; (8) the mode of action of syntoxic and catatoxic hormones, drugs and behavioural attitudes; (9) the so-called first mediator of the stress response, which carries the message that a state of stress exists from the directly affected area to the neurohormonal regulatory centres; and (10) the prophylaxis and treatment of stress-induced damage by pharmacologic and behavioural techniques.


Care of the terminally ill at home.

Care of the terminally ill at home. Care of the terminally ill at home demands the attention of the medical and paramedical community. Patients who choose to remain at home while death approaches must be given full physical, emotional and psychological support by the attending physician and home care services personnel. In 1974 the Vancouver early hospital discharge home care service provided such care to 47 patients. Generally both patient and family were happy with the program. A few families were unable to cope for more than a few days but most continued the care almost to the end, a large number (14) keeping the patient at home until death occurred. Added benefits are the lower costs and the freeing of hospital beds.


A numerical method of evaluating and categorizing hospital emergency departments. The capabilities of individual hospital emergency departments participating in an emergency medical services system must be evaluated as the first step im improving the services in the region. By means of a weighted point-scoring method, personnel, facilities, equipment and organization can be assessed and the department categorized. This not only provides an evaluation of the present status of the department but also sets standards for improvement. Categorization provides the basis for regionalization of services, whereby each department has a defined responsibility to provide treatment of specified sophistication.


Normal-pressure hydrocephalus due to tentorial meningioma. Normal-pressure hydrocephalus and the associated triad of dementia, apraxic gait and urinary incontinence may be casued by various, sometimes unsuspected, lesions, usually those that block the flow of cerebrospinal fluid (CSF) around the tentorium. A 58-year-old woman with insidious onset of behaviour and gait problems had occult, normal-pressure hydrocephalus and a tentorial meningioma, resection of which produced complete recovery. This case demonstrates that a distinction must be made between the syndrome of normal-pressure hydrocephalus resulting from disturbance in CSF dynamics and the pathophysiologic features of the underlying lesion.


Diagnosis and management of microinvasive (stage IA) carcinoma of the uterine cervix. One hundred and sixty-two cases of Stage IA microinvasive carcinoma of the cervix are presented. These patients represent the combined experience at the University of Miami School of Medicine, Miami, Florida, and Downstate Medical Center, Brooklyn, New York. The criteria used in both institutions are 1) penetration of invasive carcinoma beneath the basement membrane of less than 1 mm and 2) absence of invasion of blood vessel or lymphatic spaces. All tissue specimens have been measured accurately by use of calibrated optics. The literature has been reviewed for criteria of diagnosis of microinvasive cancer, as well as methods of management. Our method of accurately determining depth of penetration is described and the evolution of microinvasive cancer is presented in a series of photomicrographs in which measurements are accurate to 0.1 mm. In the literature, when depth of penetration of up to 5 mm is used as criterion for microinvasive carcinoma, the incidence of nodal metastasis may be as high as 3.5%. Since, in our combined institutions, the mortality rate with radical hysterectomy is less than 1% and the incidence of ureterovaginal fistulas is 1.2%, we conclude that simple hysterectomy is not adequate therapy for lesions with stromal invasion to a depth of 5 mm.


The stilbestrol-adenosis-carcinoma syndrome. This disease complex is one of the few entirely new and previously unsuspected discoveries of the recent past. The first case, seen in 1966, ushered in an era of suspicion. A study and report on the first seven cases published in 1970 inaugurated the era of hot pursuit which culminated in 1971 in an epidemiologically structured and controlled investigation of possible etiologic factors. With the establishment of the stilbestrol association a Registry of Cases was initiated. , heralding the era of verification. This Registry, while accelerating and embossing confirmation of the suspected relationship, served an even more useful purpose by collecting under one roof and in front of one cluster of observers all the necessary and relevant data on a sufficiently large number of cases to enable rapid (1973-1974) wide dissemination of knowledge about the occurrence and behavior of the disease and its response to treatment. The behavior of these tumors is comparable to that of other cancers at this location. When they are still localized, cure is possible by either surgery or radiation therapy. Since all the individuals at risk can be identified by their clinical history, screening examination for presymptomatic cancer is entirely feasible. At the same time we can and should note the presence and nature of any morphologic anomalies of the kind, which are seen in a very high proportion of the women exposed before the 18th week of fetal life.


Colposcopy.

Colposcopy. The main clinical value of colposcopy in modern gynecology is in the clinical diagnosis of patients with abnormal cytology. In colposcopically directed biopsies it is possible for an experienced colposcopist to sample with a high degree of accuracy the most advanced histopathologic changes. This significantly decreases the frequency of diagnostic conization. Colposcopy can efficiently select patients for outpatient treatment of cervical intraepithelial neoplasia, since it precisely defines the size and localization of epithelial changes. A new application of colposcopy is in the evaluation of young girls who have been exposed to stilbestrol in utero. In these patients colposcopy can improve the diagnosis of changes in cervical-vaginal epithelium.


Screening techniques in endometrial cancer. Carcinoma of the uterus is the most frequently seen malignancy of female genital tract. Screening techniques have lowered the incidence and morbidity of cervical neoplasia. Attempts to identify premalignant and silent cancers of the uterus have been made. A single diagnostic procedure has not been uniformly successful. It would appear that multiple techniques, using both cytologic and histologic material, would increase the possibility of achieving the goal of early diagnosis in uterine disease.


Use of diagnostic ultrasound in trophoblastic neoplasms and ovarian tumors. Analyses of the data on the ultrasonic diagnostic accuracies are presented for the cases of hydatidiform mole and ovarian tumors. These data are derived from two sources: the State University of New York Downstate Medical Center (SUNY-DMC) and the Chiba University School of Medicine (Chiba U.), Japan. Diagnosis of hydatidiform mole was correct in 47 (92.2%) of 51 (SUNY-DMC) and 66 (91.6%) of 72 cases (Chiba U.). The incidences of lutein cysts were 29.4% (SUNY-DMC) and 18.1%. (Chiba U.). Ultrasound plays an important role in the postmolar follow-up: increased intrauterine echoes in the subinvoluted uterus may signal a developing invasive mole or choriocarcinoma. Ultrasonic diagnoses of 223 (SUNY-DMC) and 183 (Chiba U.) ovarian tumors have been analyzed and the rates of diagnostic errors for the individual tumors are presented. Solid ovarian tumors appear to be more difficult to diagnose than cystic ovarian tumors.


Pretreatment staging.

Pretreatment staging. Renewed emphasis on pretreatment staging of gynecologic malignancies has resulted from a number of reports on surgical staging of malignant cervical, endometrial, and ovarian lesions. These reports have shown that by pretreatment staging, in significant numbers of patients, more advanced disease was detectable than by the usual clinical methods. From 32% to 46% of patients with stage III cervical carcicarcicarcinoma have been found to have metastatic disease in the para-aortic nodes. Diaphragmatic and para-aortic node metastases have been reported in patients with ovarian carcinoma recently in significantly higher incidences than as previously detected. The pelvic lymph nodes have been shown to be involved in 11% to 13.5% of patients with stage I endometrial carcinoma. Twelve and one-half percent had metastases to the para-aortic nodes. These data suggest the need for more accurate definition of extent of disease prior to treatment. Improvement in survival rates will not necessarily follow, but modifications in treatment must be tried.


Cryosurgery. Its use for the abnormal Pap smear. During a two-and-one-half year period, 317 women were seen in the Colposcopy Clinic for evaluation of an abnormal Pap smear. Seventy-four (23%) desired to maintain their childbearing potential and were treated conservatively. Ten (14%) had inadequate colposcopy and underwent cold knife conization of the cervx. The remaining 64 were treated by adequate colposcopy, combined with cryosurgery; they are discussed in detail. All were managed economically as outpatients without anesthesia and no serious complications occurred. To date, only four have recurrent abnormal cervical cytology and several have delivered children. Adequate colposcopic examination combined with biopsies must proceed all cryosurgical procedures performed for abnormal cervical cytology.


Pelvic exenterative therapy for gynecologic malignancy: an analysis of 70 cases. During the 10-year period from June 1, 1965 to June 1, 1975 70 patients with gynecologic malignancy were treated with some type of pelvic exenteration at the University of Michigan Medical Center. Total pelvic exenteration was performed in over 75% of the cases. The most recent figures suggest a 3-year and 5-year survival rate of over 60%. The surgical mortality is 1.4%: the hospital mortality is 1.4%. Those patients with squamous cell disease, such as recurrent carcinoma of the cervix, vagina or vulva, have a better prognosis than do those patients with gland cell disease. Regional lymph node involvement gives a bad prognosis. Advances in therapeutic techniques during this 10-year period, including: 1) use of prophylactic antibiotics: 2) transverse abdominal incision; 3) so-called "ski-position" on the operating table; 4) prophylactic compartmentalization of the inferior vena cava; 5) uretero-sigmoid conduit for urinary diversion; 6) peritoneal graft as a "lid" over the pelvic vault' and 7) split-thickness skin graft vaginoplasty as part of the rehabilitation program, are discussed.


Current status of the treatment of epidermoid cancer of the vulva. Epidermoid cancer accounts for 81% of the malignancies of the vulva. Although the etiology has not been delineated, chronic vulvitis is associated with cancer in almost one-third of the patients. The staging system should utilize the most precise and accurate parameters delineated for improved treatment. A new staging system is utilized. Proven treatment is primarily surgical.


Clinical studies with adjunctive surgery and irradiation therapy in the treatment of carcinoma of the cervix. The majority of patients with cancer of the cervix are treated adequately by irradiation alone, and surgery is not necessary. A role for operation developsin certain patients with Stage I-II cancer of the cervix, who are prone to develop central recurrence if treated by irradiation alone. In these patients the primary lesion is massive. The vasculature to the center of these bulky or barrel-shaped cancer is insufficient, and a population of the cancer cells, perhaps related to anoxia, is more resistant to irradiation. The postirradiation conservative hysterectomy was incorporated into the treatment plan for 212 selected patients and the number of local recurrences was reduced. Incomplete cure of the peripheral component of the disease is another mechanism of treatment failure. This may occur, even though the primary lesion is cured. Patients with cervical cancers greater than 5 cm diameter have a high incidence of node metastasis, regardless of clinical stage. In those patients with positive nodes, the standard portals for external irradiation were expanded to include nodes at higher levels. The site of metastases was established by pretreatment laparotomy and the irradiation portals were then extended to include the involved nodes. Survival rates were not improved, because recurrences were not prevented, and death from treatment complications were high.


The evolution of modern treatment of corpus cancer. The treatment of endometrial cancer has been surgical, traditionally, with preoperative radiation gaining favor in the past three decades. This surgical treatment has been confined to simple hysterectomy and bilateral salpingo-oophorectomy in most clinics because of the pattern of spread of corpus cancer and the alleged benignity of this disease. Even now, the role of radical surgery, the timing of radiation therapy and its mode, and the place of hormonal treatment for primary disease are still the subject of discussion and clinical investigation. The definition of virulence factors and an appropriate staging dependent upon these factors has helped to clarify some of these problems and will help the analysis of cure rates when we can understand the selection of the material. With such a prospect for quality control and establishment of a protocol of individualization of treatment, we have a right to expect that appropriate surgical and radiotherapeutic measures will gain their proper priority and adjuvant hormonal therapy and chemotherapy may be incorporated.


Current status of the treatment of gynecologic cancer by site: ovary. Cancer of the ovary is the leading cause of death from gynecologic cancer. The constant challenge presented by ovarian cancer is that about 11,000 women die from ovarian cancer each year and the results in 1974 are no better than have been achieved in the previous two decades. Standard practice of treatment for truly invasive common epithelial ovarian cancer includes total hysterectomy, bilateral salpingo-oophorectomy, appendectomy, omentectomy, and post-surgical insertion of tubes and administration of P32 (if the disease is of limited extent). Although it is occasionally necessary to resect isolated segments of bowel, exenterative or ultraradical surgery in the management of ovarian cancer is not usually chosen because of the natural history of the disease. However, aggressive surgery is indicated not so much because it is curative, but because it potentiates other forms of treatment. All stages I through IV are treated surgically, to remove as much tumor as possible without running a risk of a gastrointestinal or genitourinary fistula. Radiation therapy has been utilized in addition to the surgical therapy in stage IV to control supraclavicular and/or inguinal node involvement. Single agent alkylating chemotherapy is chosen for the treatment of common epithelial ovarian cancers. Combination chemotherapy does not produce better results at this time, except in the treatment of embryonal tumors. The treatment of the common epithelial tumors by stage is outlined. The treatment of germ cell tumors, gonadal stromal tumors, ovarian tumors in childhood, ovarian tumors in pregnancy, as well as tumors not specific for the ovary, will also be discussed.


Monitoring central venous pressure: principles, procedures and problems. When caring for patients with CVP lines, nursing assessments are aided by knowledge of the interdependence of physiological factors such as cardiovascular volume and flow, cardiac efficiency and central vein capacity. Comprehension of the underlying principles facilitates adaptation of the procedure to patient needs and to the available resources.


Coping with the aggressive patient: an alternative to punishment. Extremely aggressive patients in mental institutions constitute a threat to staff and other patients that usually necessitates the use of physical restraints. With chronically aggressive patients this cycle of behaviors is defeating for both the staff and the patient. In this case history the authors describe how positive and negative reinforcement and shaping were used to alter the previously unmanageable aggression of a 36-year-old male psychiatric patient.


Understanding the patient in emergency.

Understanding the patient in emergency. Psychological care should be integrated with physical care even when one works in a busy 'acute care' environment, be it an emergency department, an intensive care unit, or at the roadside as a member of the ambulance team. The quality of care is enhanced by awareness of psychological needs. Initially a conscious effort is needed to remember to have cues verified and to be open to noting subtle changes in behavior. If a continuous effort is made by each health team member, this verification process will become automatic without being time-consuming. All members of the health team must be aware that they are individuals with a multiplicity of factors affecting their thinking, feeling and behavior. They care for individuals who are also unique. A form of support proving successful with one patient may not be effective with another. Each patient must be assessed as an individual before appropriate psychological care can be effectively given. By being open to cues given by the patient and his relatives, and by having perceptions of these cues verified, an important step is taken in deciding on appropriate psychological care. With an increased awareness of the total psychological impact of the 'emergency' situation on the patient, his relatives--in fact, on the entire health team--individualized patient care will be administered in a more thorough and understanding way.


Effect of combination treatment with cyclophosphamide and nonspecific passive immunization on a transplantable tumor in WKA rats. Combination of cyclophosphamide (CY) and passive immunization with lymphoid cells sensitized to allogeneic tumor was studied in the treatment of a methylcholanthrene-induced transplantable fibrosarcoma in WKA rats. For determination of the most effective timing of combination treatment, rats given an injection of CY on Day 3 received passive transfer of the sensitized lymphoid cells on Day 0, 1, 2, 3, 4, or 6. A remarkable therapeutic effect was observed only when the passive transfer was combined on Day 4 with CY on Day 3. Rats inoculated with tumor succumbed in all cases without any treatment. After i.v. injection of CY on Day 3, 2 of 28 rats were cured (7.1%). Passive immunization with the sensitized lymphoid cells on Day 4 resulted in no therapeutic effect. After combination of CY on Day 3 and transfer of nonsensitized normal lymphoid cells on Day 4, 2 of 15 rats survived (13.3%). However, combination of CY and passive transfer of the nonspecifically sensitized lymphoid cells resulted in 23 survivors of 29 rats (79.3%).


Comparison of in vitro methods to determine drug-induced cell lethality. In proliferating cell populations, the inability to reproduce indefinitely is the only relevant criterion to assess cell lethality. The in vitro colony formation technique (CF) used to determine reproductive death is, however, too slow and has several technical limitations. For finding suitable, more rapid techniques that assessed drug-induced cell killing, a human lymphoma cell line was exposed in vitro to increasing concentrations of adriamycin, bleomycin, and 1,3-bis(2-chloroethyl)-1-nitrosourea for 1 hr. Survival was assayed immediately after treatment and at regular intervals thereafter. Data from CF were compared to those resulting from the following tests: doubling time, labeling index, dye exclusion, 51Cr release, and rate of [3H]thymidine uptake (scintillation index). Dye exclusion and 51Cr release failed to demonstrate any killing effect for the 3 drugs. The percentage of killing calculated from doubling time determinations, although dose dependent, failed to correlate with CF. Scintillation and labeling index values displayed similar temporal fluctuations but were not clearly dose dependent and did not correlate with CF. Thus, CF appears as the most reliable, dose-dependent index of cell lethality. Tests that measure metabolic death grossly overestimate or underestimate killing activity induced by 3 of the most effective antitumor drugs.


Interaction of the carcinogen 3,3-dimethyl-1-phenyltriazene with nucleic acids of various rat tissues and the effect of a protein-free diet. The methylation by 3,3-[14C]dimethyl-1-phenyltriazene of nucleic acids in various rat tissues was investigated. Following a single s.c. injection of 3,3-[14C]dimethyl-1-phenyltriazene (50 mg/kg), approximately 40% of the radioactivity was subsequently exhaled as 14CO2. Expiration of 14CO2, metabolic 14C labeling of liver proteins, and formation of 7-[14C]methylguanine, the major reaction product with nucleic acid bases, were completed within about 15 hr. Minor alkylation products detectable in DNA were O6-methylguanine and 3-methyladenine. In cytoplasmic RNA, 1-methyladenosine, 3-methylcytidine, and O6-methylguanosine were present in addition to 7-methylguanosine. Concentrations of 7-methylguanine were highest in nucleic acids of kidney and liver. Among the other organs investigated (brain, lung, spleen, small intestine), 7-methylguanine levels showed little variation but were 4 to 7 times lower than those in liver and kidney. Feeding of a protein-free diet prior to 3,3-[14C]dimethyl-1-phenyltriazene administration reduced the formation of 7-methylguanine in liver and kidney RNA, whereas in the remaining organs the extent of methylation was markedly increased. The results are discussed with respect to the significance of methylation at specific sites in nucleic acids for the initiation of malignant transformation and the possible role of 3-methyl-1-phenyltriazene as the systemically distributed proximate carcinogen of 3,3-dimethyl-1-phenyltriazene.


Fine structural changes in hepatocytes after simultaneous treatment with single doses of 3'-methyl-4-dimethylaminoazobenzene and cycloheximide. Male rats were given single doses p.o. of 3'-methyl-4-dimethylaminoazobenzne (3'-MeDAB), either alone or at the same time as single s.c. injections of cycloheximide. They were killed either 24 or 48 hr after treatment or at intervals up to 21 months, and their hepatic tissues were examined by electron density, combined treatment with both chemicals failed to produce this acute toxic effect. However, the fine structure of the hepatic cells of rats given this combined treatment with cycloheximide and 3'-MeDAB closely resembled that obtained by chronic exposure to carcinogenic azo dyes. Of the changes thus produced, the granular endoplasmic reticulum in particular became permanently altered, both quantitatively and morphologically. Other persistent changes included mitochondrial abnormalities and glycogen depletion. Cycloheximide appears to protect the liver cell against the nonspecific acute toxic action of 3'-MeDAB, while facilitating the expression of effects that may possibly be associated with the carcinogenic action of this azo dye. Although this experimental model does not result in the appearance of tumors, it demonstrates that a single exposure to a carcinogen may induce permanent changes that are similar to those observed during carcinogenesis.


Relationship between nucleolar microspherule formation by urethan and inhibition of RNA and protein synthesis. Liver tissues of suckling C3Hf mice treated with urethan, a carcinogen and inhibitor of RNA synthesis, and with cycloheximide, an inhibitor of protein synthesis, were studied by electron microscopy and by light microscope radioautography. At 4 and 12 hr after treatment with a single dose of urethan (1 mg/g body weight). [3H]uridine incorporation in hepatocyte nuclei was 38 and 9% lower than in controls and became 32% higher at 24 hr. Nucleolar microspherules were found 1.5 and 4 hr after the treatment, increased considerably in number at 10 and 12 hr, and decreased at 24 hr. These results suggest an inverse relationship between RNA synthesis and formation of microspherules. Cycloheximide alone (3 mg/kg body weight) produced microspherules, whereas in combination with a dose of 1 or 2 mg of urethan per g body weight it partially prevented or potentiated, respectively, the microspherule production.


Two forms of thymidine kinase in normal and tumor tissues of animals. The thymidine kinases extracted from the spleen of mice infected with Friend virus and from Yoshida sarcoma in rats were separated into two active peaks by diethylaminoethyl cellulose column chromatography, while those of normal tissues have been found to consist of only the first peak (P-1). The second peak (P-II) was also found in the enzyme from the extract of the spleen when the animals were treated by i.p. injections of 1-acetyl 2-phenylhydrazine. The two P-II peaks from tumor tissue and from spleen enlarged by anemia-inducing agents were indistinguishable on the chromatographic profile. On the other hand, the thymidine kinase extracted with Triton X-100 from a mitochondrial fraction of normal liver was found to consist of only one peak in the same position as the above P-II's on the chromatogram, but its faculty for deoxythymidine triphosphate inhibition was not identical to that of tumor tissue. This treatment with the detergent might cause dissociation of a certain component from the enzyme complex to make the extra peak (P-IIb), but it eventually shifts to P-II on the chromatogram.


Karyotypes of vasoformative sarcomas arising from BALB/3T3 cells attached to polycarbonate plates. Four vasoformative sarcoma in vivo tumor lines arising from the s.c. implantation of 3 X 10(4) BALB/3T3 cells attached to a 1- X 5- X 10-mm polycarbonate platelet were shown to be quite similar in chromosome constitution to the parental 3T3 line. All tumors contained the same marker chromosomes that were present in the parent BALB/3T3 cells. The findings conclusively showed that the tumors had derived from BALB/3T3 cells and not from host cells or from the in vivo fusion of the inoculated 3T3 cells with host cells. Each tumor line had a unique karyotype that was markedly more homogeneous than that of the parental BALB/3T3 cells, strongly suggesting that each tumor represented a separate clone. An M1 marker chromosome was consistently present in each of the four tumor lines but was present in only about 50% of the parent BALB/3T3 cells; it therefore appeared to be a distinct and stable feature of the tumors.


Correlation of antitumor chemoimmunotherapy with bone marrow macrophage precursor cell stimulation and macrophage cytotoxicity. The present investigations have assessed the effects of prolonged cyclophosphamide (CY) and Corynebacterium (CP) treatment on the production of bone marrow macrophage precursors [colony-forming cells (CFC)] and on the cytotoxicity of macrophages comprising colonies produced by the CFC. The findings have been correlated with tumor growth in animals receiving the immunochemotherapy. In addition, studies have been directed toward ascertaining whether the administration of CP with CY might lessen the myelosuppressive effects of the latter. Following each consecutive weekly dose of CY (even after as many as 11), there was a significant depression in the number of bone marrow cells (BMC's) but, by the next injection, marrow cellularity had returned to normal. When the number of BMC's was reduced, the proportion of the remaining cells, which consisted of CFC, was increased. Upon reconstitution of the marrow, the proportion of CFC returned to the level of the controls. The total number of CFC in marrow was at no time following CY therapy significantly less than the number in marrow of untreated mice. The addition of CP to the treatment regimen with CY resulted in an absolute as well as relative increase in CFC at all times during administration of the combined therapy, i.e., when there was a depression in total numbers of marrow cells, as well as when marrow restoration had occurred. Although CP stimulated the number of cells entering into differentiation, it failed to affect the total numbers of marrow cells, as well as when marrow restoration had occurred. Although CP stimulated the number of cells entering into differentiation, it failed to affect the total BMC's had been neither increased nor prevented from decreasing, by CP administration, indicating that the use of total cellularity as an index of the CP marrow-sparing effect is without merit. The present results relative to cytotoxicity of macrophages derived from the CFC concur with and extend our previous findings indicating that the cytotoxic property of macrophages originates in its ancestral stem cell or CFC and that factors responsible for increasing the CFC population do not selectively stimulate precursor cells responsible for production of the cytotoxic macrophage. Although the proportion of cytotoxic macrophages was not altered by CP when administered with CY, the absolute number of such cells was increased. Since the increase in macrophage colony production and, consequently, in cytotoxic macrophages correlates with increased inhibition of tumor growth when CP was used with CY, it is suggested that macrophage precursors are the cells of primacy in CP immunopotentiation. Their stimulation, resulting in enhanced cytotoxic macrophage formation, could be responsible for the inhibition of tumor growth observed in our model system. The findings also suggest that when myelosuppression is a limiting factor in the use of a chemotherapeutic agent, the concomitant use of CP may be advantageous.


Heterotopic urinary bladder with a communicating reservoir. A heterotopic bladder attached to an Ommaya reservoir was transplanted to the back of a syngeneic rat. Following transplantation, the mucosa underwent ischemic necrosis. However, it was rapidly reepithelialized in 2 weeks as a result of spreading of the epithelium originating from the ureterotrigonal region. By light microscopy the regenerated epithelium was indistinguishable from the normal transitional epithelium, and the cannula connecting the reservoir to the bladder remained patent throughout the observation period of 22 weeks. One bladder, when examined at 22 weeks, contained a transitional cell papilloma with squamous metaplasia. Although further study is needed, this model is potentially useful in studies related to bladder carcinogenesis.


Developmental phase-specific alkaline phosphatase isoenzymes of human placenta and their occurrence in human cancer. Alkaline phosphatase electrophoretic patterns characteristic of three phases in early human trophoblast development are described in this preliminary communication. Phase 1 (6 to 10 weeks) consists entirely of two heat-sensitive, L-homoarginine-inhibited bands, the slower one of which possesses antigenic determinants of live-bone-type alkaline phosphatase, whereas the fast band lacks any of the known alkaline phosphatase antigenic determinants. Phase 2 pattern (11 to 13 weeks) is that of a mixture of Phase 1 and Phase 3 isozyme components, the latter exhibiting two isozyme bands with the characteristics of term placental alkaline phosphatases correspond in order to non-Regan isoenzyme, a mixture of Regan and non-Regan isoenzymes and Regan isoenzyme in a variety of human cancer tissues. The biochemical profile characteristic of trophoblast developmental Phase 1 alkaline phosphatase is expressed as 78.5% heat-sensitive inhibition (5 min at 65 degrees), 66.3% L-homoarginine inhibition, and 17.3% L-phenylalanine inhibition where n = 12. It is hypothesized that the alkaline phosphatase of human tumor tissues reflects the expression of placental genes corresponding to one or more phases of trophoblastic development.


Alterations in the transcriptional capacity of hepatic DNA-dependent RNA polymerase I and II by N-hydroxy-2-acetylaminofluorene. Using hepatic RNA polymerases I and II, acetylaminofluorene modification of DNA in vitro results in reduction of RNA elongation but not in an altered frequency of incorporation of [alpha32P]ribonucleotides. In contrast, in vivo modification of RNA polymerases by a single dose of N-2-hydroxy-2-acetylaminofluorene results in an alteration of their capacity for transcribing a normal RNA product but does not affect the product size transcribed from normal or carcinogen-modified templates.


The effect of a growing tumor and its removal on the cytotoxicity of macrophages from cultured bone marrow cells. Previous investigations by us have demonstrated that there is a significant but transient (4 to 14 days) increase of colony-forming cells (CFC) in bone marrow following implanation of a syngeneic mammary tumor in C3H mice. Those CFC gave rise to enhanced macrophage colony production when cultured in semisolid medium. The present studies have for the first time demonstrated that macrophages from the colonies were cytotoxic to cells from the immunizing tumor, and they continued to possess that characteristic for as long as a tumor was present in the animal from which bone marrow was derived. By 21 days after tumor removal cytotoxicity was no longer evident. The findings provide evidence to suggest that the cytotoxicity displayed by the macrophage originates in its ancestral CFC or in the antecedent stem cell. They also suggest that receptor sites of the CFC (or stem cell) that respond to a stimulus for self-replication are probably different from those sites that, when activated, result in cytotoxic properties of their progeny. The present findings also indicate not only that quantitation of macrophage production is of relevance in determining the efficacy of a therapeutic regimen but also that knowledge concerning the specific properties, i.e., ctytotoxicity, of such cells is of equal or greater importance.


Comparison of the products of the reaction of 7-methylbenz(a)anthracene 5,6-oxide and RNA, with those formed in 7-methylbenz(a)anthracene-treated cells. RNA was isolated by a phenol extraction method from mouse embryo cells treated in culture with either [G-3H]-7-methylbenz(a)anthracene or [G-3H]-7-methylbenz(a)anthracene 5,6-oxide (the K-region epoxide). The RNA was degraded to ribonucleosides, mixed with ultraviolet-absorbing quantities of the epoxide ribonucleoside products isolated from RNA that had reacted with 7-methylbenz(a)anthracene 5,6-oxide in aqueous ethanol solution, and chromatographed on a column of Sephadex LH-20 eluted with a methanol:water gradient. The 7-methyl-benz(a)anthracene 5,6-oxide ribonucleoside products formed in cells were identical to those formed in aqueous solution, although the relative amounts of the products varied. The majority of these epoxide-ribonucleoside products were not identical to the products formed in cells treated with the parent hydrocarbon. These results suggest that the major reactive form of 7-methylbenz(a)anthracene that binds to RNA in mouse embryo cells is not the K-region epoxide of this hydrocarbon.


Growth of HeLa cells in diffusion chamber cultures in vivo. In order to investigate some characteristics of Millipore diffusion chamber cultures (DCC) and their potential for the quantitation of tumor cell growth kinetics, HeLa S-3-cells were grown in diffusion chambers implanted i.p. in mice. HeLa cells grew pseudologarithmically in DCC with a population-doubling time of approximately 2.8 days when 9.2 to 12.0 X 10(5) cells were placed in the chambers initially. The population-doubling time varied as a function of the initial innocula size but was always longer than the in vitro population-doubling time of 1.2 days. Comparable harvests of cells from experiment to experiment varied quantitatively by no more than a factor 1.8. This variaton somewhat limits the use of DCC for the quantitation of subtle effects. The fate and morphology of HeLa cells in DCC were determined from scanning electron microscopy. DCC might be exploited in clinical studies of the growth of patients' tumors and the effects of various therapies in a host-mediated system.


Guanine-specific DNA repair after treatment of mouse skin cells with N-methyl-N'-nitro-N-nitrosoguanidine. The incorporation of [3H]thymidine and [3H]deoxyguanosine into DNA was studied during DNA repair in mouse skin cells treated with the skin tumor initiator N-methyl-N'-nitro-N-nitrosoganidine. At high, toxic levels of N-methyl-N'-nitro-N-nitrosoguanidine, repair (incorporation of the precursor into DNA which had not replicated) was demonstrated with both precursors. At lower, less toxic doses of the carcinogen, repair could not be demonstrated with [3H]-thymidine, but it was clearly demonstrable with [3H]deoxyguanosine. Thus, we are apparently observing two kinds of DNA repair, one in which a single base (in this case, guanine) replaces a base lost by chemical or enzymatic depurination and the second in which more than one base is replaced, indicating synthesis of longer stretches DNA after extensive enzymatic excision. The guanine-specific repair shown at relatively nontoxic dose levels of N-methyl-N'-nitro-N-nitrosouanidine may be more relevant to the survival of cells than the repair demonstrated with [3H]thymidine at higher doses.


Identification of singlet oxygen as the cytotoxic agent in photoinactivation of a murine tumor. Singlet oxygen, a metastable state of normal triplet oxygen, has been identified as the cytotoxic agent that is probably responsible for in vitro inactivation of TA-3 mouse mammary carcinoma cells following incorporation of hematoporphyrin and exposure to red light. This photodynamic inactivation can be completely inhibited by intracellular 1,3-diphenylisobenzofuran. This very efficient singlet oxygen trap is not toxic to the cells nor does it absorb the light responsible for hematoporphyrin activation. We have found that the singlet oxygen-trapping product, o-dibenzoylbenzene, is formed nearly quantitatively intracellularly when both the furan and hematoporphyrin are present during illumination but not when only the furan is present during illumination. The protective effect against photodynamic inactivation of the TA-3 cells afforded by 1,3-diphenylisobenzofuran coupled with the nearly quantitative formation of the singlet oxygen-trapping product indicates that singlet oxygen is the probable agent responsible for toxicity in this system.


Energetics and efficiency of photoinactivation of murine tumor cells containing hematoporphyrin. Hematoporphryrin derivative at an intracellular concentration in TA-3 mouse mammary carcinoma cells of 0.6 or 0.9 mM required input of 3.0 X 3.6 X 10(9) quanta/cell of red light (620 nm) to achieve a 90% kill. At an intracellular concentration of 1.2 mM, this light requirement drops to 1.5 X 10(9)quanta/cell. The energy for this photodynamic process is about 100 times higher than that required for ionizing radiation to achieve the same level of kill for these cells. The quantum yield for singlet oxygen formation (the cytotoxic agent in most photodynamic processes) from hematoporphyrin derivative is 0.75 +/- 0.07 in ethanol but only 0.16 +/- 0.07 within TA-3 cells, indicating possible intracellular complexing and quenching.


Effect of avitaminosis A and hypervitaminosis A on urinary bladder carcinogenicity of N-(4-(5-Nitro-2-furyl)-2-thiazolyl)formamide. The effect of vitamin A deficiency and hypervitaminosis A on the urothelial carcinogenicity of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formanmide (FANFT) was determined in female weanling Sprague-Dawley rats. Vitamin A deficiency resulted in squamous metaplasia of the urinary bladder and high incidences of cystitis, ureteritis, and pyelonephritis. Administration of FANFT to vitamin A-deficient rats appeared to accelerate the carcinogenic process, with earlier appearance of urinary bladder tumors and the development of ureteral and renal pelvic carcinomas. Most of these tumors were squamous cell, occasionally with transitional cell foci. Hypervitaminosis A prevented the appearance of squamous metaplasia and squamous cell neoplasia in rats fed FANFT, but it did not inhibit the formation of transitional cell hyperplasia or neoplasia in comparison to rats receiving normal levels of vitamin A and FANFT.


Distribution and disposition of platinum following intravenous administration of cis-diamminedichloroplatinum(II) (NSC 119875) to dogs. cis-Diamminedichloroplatinum(II) is an antineoplastic drug that is undergoing a renewed clinical interest as a drug for use in combination regimens. In order to increase the understanding of the pharmacokinetics of this drug, the plasma clearance and organ distribution of platinum were followed in female beagle dogs treated with a single i.v. dose of cis-diamminedichloroplatinum(II). Plasma levels of platinum were determined by flameless atomic absorption spectrometry and showed a distinctly biphasic clearance pattern with a rapid-phase half-time of considerably less than 1 hr and a slow-phase half-time of nearly 5 days. During the first 4 hr after treatment, plasma levels fell by 90% while 60 to 70% of the applied dose was recovered in the urine. Sixteen tissues plus plasma, bile, and urine were routinely analyzed for platinum content. An easily measurable plasma concentration of platinum was still detectable 12 days after treatment, with no significant change in plasma concentration between Days 4 and 12. Initial concentrations of platinum were highest in organs of excretion, gonads, spleen, and adrenals but remained significantly elevated only in kidney, liver, ovary, and uterus, where a tissue: plasma ratio of 3 to 4 was maintained for as long as 6 days posttreatment. The apparent in vitro binding of platinum to dog plasma and to bovine serum albumin was studied by ultrafiltration and increased progressively during 48 hr of incubation at 37 degrees.


Comparative adduct formation of 4-aminobiphenyl and 2-aminofluorene derivatives with macromolecules of isolated liver parenchymal cells. Isolated parenchymal cells of rat liver have been used in a study of the metabolic activation of derivatives of the carcinogens 4-aminobiphenyl and 2-aminofluorene. The formation of adducts of these compounds with cellular RNA and protein has been taken as evidence of their transformation to metabolites that are capable of spontaneous reaction with tissue macromolecules. The hydroxamic acid N-hydroxy-N-4-acetylaminobiphenyl was bound to RNA to a greater extent than were the amino-, hydroxylamino-, nitroso-, nitro-, acetylamino-, or azoxybiphenyl derivatives. RNA adducts of the hydroxamic acid retained little of the acetyl group. The structural requirements for binding and the nature of the bound derivatives are consistent with the activation of N-hydroxy-N-4-acetylaminobiphenyl by N leads to O acyltransfer. Approximately equal quantities of 4-nitrosobiphenyl and the hydroxamic acid were bound to protein, but far less of the nitroso derivative was incorporated into RNA. Adduct formation of N-hydroxy-N-2-acetylaminofluorene with RNA occurred with retention of the acetyl group and was dependent on the concentration of sulfate in the media. Consequently, reaction of the fluorenyl derivative with RNA probably resulted from conjugation of the hydroxamic acid with sulfate.


Effect of 6-hydroxydopamine on tumor growth. The growth of C-1300 neuroblastoma was markedly slowed in 6-hydroxydopamine-treated mice. The growth of the A-10 breast adenocarcinoma was also significantly retarded in 6-hydroxydopamine-treated mice but the growth of B-16 melanoma was not affected. In mice axotomized by pretreatment with 6-hydroxydopamine, the growth of C-1300 neuroblastoma was slowed but the growth of the A-10 tumor was not affected. It is suggested that an intact functional sympathetic nervous system may be a factor that determines the rate of growth of certain tumors in vivo.


Alteration of mammalian cells by interaction with artificial lipid vesicles. Artificial lipid vesicles interact with a variety of mammalian cells, including blood cells, spleen cells, and tumor cells, and during this interaction components can be transferred from the vesicle to the cell and from the cell to the vesicle. Transfer of intravesicular material is observed when artificial lipid vesicles carrying an intravesicular marker, 99mTc (as TcO4- ion), are incubated with mammalian cells. When vesicles prepared with [14C] phospholipid are incubated with mammalian cells, the labeled lipid is also transferable to mammalian cells. Conversely, if the mammalian cell surface is radiolabeled (with 125I), the cell marker is in part transferable to the vesicles. Thus, interaction of mammalian cells with vesicles alters the characteristics of the cell in several ways. There is a loss of some cell surface components, a gain of vesicle lipid components, and an acquisition of intravesicular contents. Such alteration may affect the biological behavior of the cell in vivo. Thus, the in vivo distribution in the mouse of isologous red blood cells is altered after interaction with vesicles; the liver and spleen remove large proportions of such cells in comparison to control erythrocytes labeled with 51Cr. The behavior of vesicle survivors from a cell interaction is also altered. Upon reexposure of such vesicles to a fresh population of cells, the intravesicular marker is no longer transferable to cells; and upon injection of such vesicles into mice, the liver accumulation of vesicular label is reduced as compared with that of nonincubated vesicles.


Chemically induced smooth muscle tumors of the mouse urinary bladder. Occasional submucosal tumors of indeterminate origin and without definite connection to the overlying mucosa have been reported in mice given various carcinogenic agents. Two Swiss mice fed niridazole developed such tumors. Electron microscopy of 1 of the tumors revealed thin myofilaments with oval dense bodies, marginal dense plaques, and gap junctions, identifying the tumor as originating from smooth muscle cells. No desmosomes, tonofilaments, secretory granules, striated muscle, or collagen were present in the tumor cells. Since the biology of these lesions is unknown, classification as benign or malignant cannot be made at this time. Their recognition is important in evaluating possible bladder carcinogens in mice.


The effects of prolonged feeding with aflatoxin B1 on adult rat liver. The effects of feeding adult rats for 6 weeks with a carcinogenic regimen of aflatoxin-contaminated diet are described. Effects on the histological appearance of liver sections are related to changes observed in nuclear separations carried out using zonal centrifugation. Changes in the levels of nuclear RNA and DNA synthesis have been studied in the populations of hepatic nuclei separated in the zonal rotor. The first 3 weeks of the feeding period was accompanied by continuing inhibitions of nucleic acid synthesis, terminating in a loss of the majority of the tetraploid hepatocyte nuclear population. The subsequent 3 weeks of feeding was predominantly a period of proliferation, restoration of the lobular architecture, and recovery of nucleic acid-synthetic activity. The possible bases of these two opposite effects, inhibition followed by stimulation, which occurred sequentially during the continued feeding of the toxic diet, are discussed.


The metabolism of rhodium(II) acetate in tumor-bearing mice. Rhodium(II) acetate has been shown to have carcinostatic activity in Swiss mice bearing Ehrlich ascites tumors. For metabolic studies, single therapeutic doses of rhodium(II) [1-14C]acetate that had been given i.p. implantations 3 days previously of 50-fold 10(6) Ehrlich ascites tumor cells. The tissue distribution and excretion of the rhodium (measured by atomic absorption spectrometry) and the acetate (measured by 14C label) were followed at designated time intervals up to 24 hr after injection. Rhodium(II) acetate, a neutral cage complex, breaks down to rhodium and acetate ionic species within 2 hr after i.p. injection, as measured by the rapid exhalation of 14CO2. Both the rhodium and 14C label disappear rapidly from the ascites fluid, with a small but variable amount of each species being incorporated into the tumor cells. Both species were detected mainly in the blood plasma, and the primary organ of deposition was the liver. No measurable quantity of rhodium was found in the brain tissue. During the first 24 hr following drug administration, only 5% rhodium was eliminated in the urine.


Estimation of tumor growth fraction in murine tumors by the primer-available DNA-dependent DNA polymerase assay. The tumor growth fraction measured by the percentage labeled mitoses method has been determined in transplantable solid and ascites murine tumors, the latter being measured at different times after transplantation. These values were compared to an in vitro, autoradiographic assay that determines the fraction of cells in a given population (primer-available DNA-dependent DNA polymerase index) that have both nuclear DNA-dependent DNA polymerase and DNA capable of acting as primer-template. It appears that almost all cells with a short G1 phase duration (less than 19 hr) that are within the proliferative cycle are primer-available DNA-dependent DNA polymerase positive. The results of the comparison indicate that the primer-available DNA-dependent DNA polymerase index estimation of growth fraction is very nearly identical to the growth fraction measured by the percentage labeled mitoses method.


Covalent interaction of metabolites of the carcinogen trichloroethylene in rat hepatic microsomes. Trichloroethylene (TCE), a structural analog of vinyl chloride, is known to induce hepatocellular carcinoma and other tumors in C57BL/6 X C3H/He F1 (hereafter known as B6C3F1) hybrid mice. TCE epoxide, a possible metabolite, is expected to be highly reactive toward cellular nucleophiles, e.g., proteins and nucleic acids. Hence, the microsomal metabolism of TCE and its covalent binding to microsomal protein were examined. Rat liver microsomes were incubated in vitro with [14C]TCE. The results showed that TCE binds covalently to microsomal protein since extensive organic extractions and Pronase digestion do not dissociate the TCE-protein complex. The binding was decreased by 7,8-benzoflavone, blocked by SKF-525A, and enhanced by i.p. administration of phenobarbital. The possibility that TCE epoxide, once formed, could be converted to water-soluble products through enzymatic hydrolysis by epoxide hydrase was also investigated. Addition of 3,3,3-trichloropropene oxide, a potent inhibitor of epoxide hydrase, to the incubation system markedly enhanced the binding of TCE. These observations support the view that, in order to bind to protein, it is necessary for TCE to be metabolized to its epoxide, a reactive intermediate that is most likely involved in TCE carcinogenesis and toxicity.


Pulmonary carcinogenesis by derivatives of polynuclear aromatic alkylating agents. Simple alkylating derivatives of polycyclic aromatic hydrocarbons have been found to be much more carcinogenic in the Strain A mouse than are the parent hydrocarbons. It has also been shown that the carcinogenicity of these halomethyl hydrocarbons is not a function of the first-order solvolysis rate. The acridine antitumor agent and mutagen ICR 170, 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride, has been shown to be a potent carcinogen in the same system when administered i.v., superseding data in the literature indicating inactivity when the drug is administered i.p. Stimulation of the immune system has been shown to have a marked inhibitory effect on the carcinogenic activity of this compound.


Molecular structures of the chemical carcinogens 7-chloromethylbenz(a)anthracene and 7-chloromethyl-12-methylbenz(a)anthracene. The three-dimensional structures of two carcinogens, 7-chloromethyl-12-methylbenz[a]anthracene and 7-chloromethylbenz[ai1anthracene, have been determined by X-ray crystallographic techniques. Both compounds are carcinogenic and are believed to act by alkylating DNA. However, the first has a nonplanar ring system, whereas the second has a planar ring system. The nonplanarity of 7-chloromethyl-12-methylbenz[a]anthracene results from steric hindrance between a hydrogen atom of the 12-methyl group and a hydrogen atom on the [a] ring. This molecule cannot be made planar unless the 12-methyl group or the [a] ring is removed. It is concluded that the carcinogenic activity of these compounds does not correlate with planarity of the ring system. This implies that, if DNA is the critical target of attack by these carcinogens, complete intercalation of the aromatic ring system of the carcinogen between the bases of DNA is not a likely mechanism of carcinogenic action in this system of compounds. The results presented here and those of others are more consistent with a model for a common interaction of the carcinogens 7-chloromethyl-12-methylbenz[a]anthracene and 7-chloromethylbenz[a]anthracene with DNA, in which they alkylate the bases of DNA and then lie with their long axes approximately parallel to the helix axis, probably in the major groove.


Elevation of dihydrofolate reductase, thymidylate synthetase, and thymidine kinase in cultured mammalian cells after exposure to folate antagonists. Roswell Park Memorial Institute 4265 human lymphoblasts were grown with three dihydrofolate reductase inhibitors: a 2,4-diaminopteridine, methotrexate; a 2,4-diaminoquinazoline, chlorasquin; and, a 2,4-diaminotriazine, triazinate. In the absence of inhibitor, dihydrofolate reductase activity increased to a peak at mid-log growth and then declined during the later growth stages. When cells were grown with 10(-8) M antifolate, cell growth was not affected, but dihydrofolate reductase activity (assayed at pH 7.0) remained at approximately initial levels throughout the growth cycle. This represented 60 to 70% less activity at the mid-log stage of growth, as compared to control cells. Dihydrofolate reductase activity in cells grown with 10(-8) M methotrexate, when assayed at pH 8.5, reached levels twice those in control cells. Enzyme activity in cells grown with 10(-8) M chlorasquin, when assayed at pH 8.5, was also higher than at pH 7.0, but it was not as high as that observed in methotrexate-treated cells. Activity in cells grown with 10(-8) M triazinate was approximately the same when assayed at either pH 7.0 or 8.5. At 10(-8) M, the three antifolates had no effect on the activities of thymidylate synthetase, thymidine kinase, serine trans-hydroxymethylase, 5,10-methylenetetrahydrofolate dehydrogenase, 10-formyltetrahydrofolate synthetase, and thymidylate kinase. However, when concentrations were used which completely inhibited growth (10(-7) to 10(-5) M methotrexate or chlorasuin; 10(-6) to 10(-5) M triazinate), dihydrofolate reductase was progressively inhibited, and there was a two- and a threefold elevation of thymidylate synthetase and thymidine kinase activity, respectively. Quantitatively, the elevation of either enzyme was similar over the range of growth-inhibitory concentrations studied. The activities of the other enzymes were unaffected. Methotrexate and chlorasquin inhibited thymidylate synthetase in a noncompetitive manner (with respect to 5,10-methylenetetrahydrofolate) with approximate Ki values of 4.5 X 10(-5) M and 4.9 X 10(-6) M, respectively. Triazinate, at 10(-3) M, had no significant effect on thymidylate synthetase activity. At 10(-3) M, the antifolates produced a negligible inhibition of thymidine kinase. Deoxyuridine 5'-monophosphate (10(-5) M) effectively protected thymidylate synthetase from heat inactivation in vitro. Dihydrofolate or 5,10-methylenetetrahydrofolate, at 10(-3) M, only partially protected thymidylate synthetase. Concentrations of methotrexate (10(-7) to 10(-6) M), chlorasquin (10(-7) M), and triazinate (10(-6) to 10(-5) M), which produced thymidylate synthetase elevation in vivo, did not protect the enzyme from heat inactivation in vitro. Methotrexate at 10(-5) M and chlorasquin at 10(-6) M gave slight protection. Thymidine kinase was stabilized only by thymidine.