Alterations in uptake and metabolism of aspartate and glutamate in brain of thiamine deficient animals. The high affinity uptake systems of aspartate, glutamate, glycine and taurine were studied in synaptosomal preparations isolated from the cerebellum, medulla-pons and the telencephalon of rats made thiamine deficient (TD) by diet or pyrithiamine (PT). There was a significant enhancement in the uptake of asparate/glutamate (probably transported by the same carrier) by the synaptosomal preparations of the cerebellum only, in both groups of thiamine-deficient animals as compared to controls. This was due to an increase in the number of uptake sites and not to an alteration of the binding affinity. Aspartate levels decreased significantly in all three brain areas of PT-treated animals and this change was greatest in the medulla-pons and the cerebellum and least in the cortex. Glutamate and serine levels were significantly decreased only in the medulla-pons whilst the concentration of glutamine was significantly increased in the three brain regions studied. The changes in both uptake and levels of amino acids in TD rats were reversed by thiamine therapy. Though the uptake studies do not discriminate between an altered aspartergic or glutamergic system, the changes in the levels of aspartate in the cerebellum suggest that the aspartergic system is involved. Since earlier studies showed a selective impairment in the high affinity uptake of serotonin by cerebellar synaptosomes, thiamine deficiency could impair cerebellar function by inducing an imbalance in its neurotransmitter systems.


The effect of phenytoin on the action potential of a vertebrate spinal neuron. The effect of phenytoin (PTN) 20 microgram/ml was tested on the passive membrane properties and the action potential of the dorsal cells in the spinal cord of the river lamprey. Dorsal cells are primary sensory neurons with no synaptic input, and thus allow examination of membrane properties in the absence of contaminating synaptic currents. PTN did not affect the resting membrane potential and slightly raised the input resistance. However, it greatly raised the threshold voltage and current for activation of action potentials by intracellularly injected current. It also reduced the maximum rate of rise of the action potential, the spike overshoot and the spike undershoot, while increasing the spike duration. In contrast to findings in other vertebrate sensory neurons, dorsal cell action potentials were blocked in zero sodium or tetrodotoxin, and not affected by zero calcium or 1 mM manganese. Thus they PTN on dorsal cells is that it partially blocks the activated sodium conductance increase of the action potential. Because a long delay was observed for maximal effect of PTN and for washout, it is postulated that the drug may require partitioning into the lipid membrane, or entry into the cell for its pharmacological action.


Origin of descending afferents to the rostral part of dorsal cap of inferior olive which transfers contralateral optic activities to the flocculus. A horseradish peroxidase study. Neurons in the ventromedial tegmental area that project to the rostral part of the dorsal cap of the ipsilateral inferior olive were labeled by the technique of retrograde transport of horseradish peroxidase (HRP). Following iontophoresis of HRP into the rostral part of the dorsal cap a cluster of labeled cells are found ipsilaterally in the area ventromedial to the red nucleus as well as in the area between the substantia nigra and the medial lemniscus. Following HRP injections into the medial accessory olive just beneath the dorsal cap, labeled cells are found exclusively contralaterally in the deep layers of the superior colliculus and in the adjacent reticular formation. These findings clearly show that neuronal groups in the ventromedial tegmental area are involved in the pathway which from the contralateral eye via the rostral half of the dorsal cap conveys impulses to the flocculus, recorded as climbing fiber activation.


A comparison of the effect of mid-thoracic spinal hemisection in the neonatal or weanling rat on the distribution and density of dorsal root axons in the lumbosacral spinal cord of the adult. Transecting the thoracic spinal cord of the rat has markedly different effects on behavioral responses of the hindlimbs if the lesion is made at the neonatal or weanling stage of development. The present investigation tested the possibility that the behavioral differences were related to a difference in the distribution or density of dorsal root connections in the lumbosacral spinal cord. In order to use each animal as its own control the distribution and density of dorsal root axons was compared on the two sides of the L5-S1 segments of the lumbosacral spinal cord in adult rats given a mid-thoracic spinal hemisection at the neonatal or weanling stage of development. Comparing the experimental (initially hemisected side) and control sides of the cord, we found no evidence for a change in the distribution of dorsal root axons. The distribution of Fink-Heimer stained degeneration 4--6 days after bilateral spinal root section was virtually identical on the two sides of the cord from animals hemisected at either stage. However, in rats spinally hemisected at the neonatal stage (n = 8), a significantly greater density of dorsal root degeneration was found within the intermediate nucleus of Cajal (INC) on the experimental side using coded material and a blind analysis. No difference in the density of dorsal root degeneration was detected in the group of rats spinally hemisected at the weanling stage (n = 6). Controls indicated that the increased density of degeneration was not due to compression resulting from shrinkage of the INC or to degeneration remaining from the initial hemisection. We conclude that the increased amount of argyrophilia within the INC of neonatally hemisected rats is due to an increased density of dorsal root axons in this zone. This result supports the hypothesis that the behavioral differences found when comparing animals transected at the neonatal or weanling stages of development are related to an increased number of dorsal root connections within the lumbosacral spinal cord.


Nicotinic cholinergic receptors in brain synaptosomes. In order to elucidate pharmacological characteristics of nicotinic receptors in the brain, competitive binding of nicotine analogues or cholinergic agents to brain particles was studied utilizing [3H]nicotine. The binding of [3H]nicotine to brain crude nitochondrial or synaptosomal fraction was progressively inhibited by the addition of increasing amounts of native nicotine or nornicotine, but cotinine had little effect. Of the myelin, synaptosomal and mitochondrial subfractions of the crude mitochondrial fraction, [3H]nicotine binding was almost exclusively confined to synaptosomes. In this binding, the affinity constant was 1.97 X 10(9)/M and the maximum binding capacity was 7.30 X 40(-8) M/g of protein. This binding was reduced by 50% in the presence of 4 X 10(-7) M D-tubocurarine and 10(-6) M carbamylcholine, while atropine was the least effective of the drugs tested. These findings suggest the possible existence of nicotinic cholinergic receptors in the synaptic region. A study of regional differences in nicotinic cholinergic receptors showed that higher specific bindings of nicotine to synaptosomes occurred in the hypothalamus, hippocampus and thalamus. These findings suggest that the nicotinic cholinergic mechanism plays an important role in these regions of the central nervous system.


Dissociation of active from immobility components of sexual behavior in female rats by central 6-hydroxydopamine: implications for CA involvement in sexual behavior and sensorimotor responsiveness. Ovariectomized female rats were given a hormone treatment (2 X 8 micrograms/kg estradiol benzoate) that normally supports only low levels of lordosis responding and no soliciting behavior in tests with sexually active males. When subjected to an intraventricular 6-hydroxydopamine (6-OHDA) procedure (with pargyline pretreatment) that produced 85% and 95% depletions of caudate dopamine and cortical norepinephrine respectively, these females exhibited a dramatic increase in the intensity and frequency of lordotic responding but no soliciting behavior over 3 weekly tests. The increase in lordosis was not due to a drug- or stress-induced release of adrenal progesterone, since dexamethasone suppressed the progesterone levels, as documented by radioimmunoassay, but not the higher receptivity of 6-OHDA treated females. In other ovariectomized females given a hormone regimen (2 X 50 micrograms/kg estradiol benzoate plus 500 micrograms progesterone) that supported maximal levels of lordosis and soliciting, the same 6-OHDA treatment prolonged the average duration of lordosis while actually decreasing the incidence and duration of soliciting. The hypothesis is put forward that the differential effects of interfering with catecholamine, and more likely dopamine function on the soliciting and lordosis components of female sexual behavior might best be understood as a dissociation between mutually antagonistic behavior patterns such that responsiveness involving active orientation and forward locomotion is suppressed, whereas responses requiring immobility are augmented.


Incorporation of [3H]leucine into hypothalamic nerve and glial cells. A comparison by EM autoradiography. Incorporation of L-[3H]leucine into the arcuate and supraoptic nuclei of rat and mice hypothalamus was studied by EM autoradiography at 30 min after intracerebral and intravenous application, respectively. The analysis applied provides information about (1) the distribution and (2) the concentration of radioactivity in the various tissue compartments, in particular nerve and glial cells. In the supraoptic nucleus, the proportion of radioactivity present in neuronal perikarya markedly exceeds that in the neuropil. Similar amounts of radioactivity in neuronal perikarya and neuropil were found for the arcuate nucleus. In both hypothalamic nuclei, only a small percentage of radioactivity is localized in glial cell bodies. However, the concentration of radioactivity in glial cells is nearly as high as in nerve cells and is lowest in the neuropil. Furthermore analysis of the subcompartments of the neuropil reveals that a considerable proportion of its radioactivity can be attributed to astrocytic processes. Conditions are discussed under which grain densities can be taken as measurements of relative synthesis rates of proteins. The results which are at variance with earlier autoradiographic studies are largely consistent with those of more recent biochemical studies on brain tissue separated into neuronal and glial fractions.


Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells. The effects of various agents on the binding and degradation of 125I-labelled insulin by isolated rat hepatocytes and cultured H4 hepatoma cells were studied. Various lysosomotropic agents, including chloroquine, ammonium chloride, and the topical anesthetics, lidocaine and procaine inhibited insulin degradation by H4 hepatoma cells but had little effect on the binding of the hormone. Similarly, tosyl-L-lysyl chloromethyl ketone selectively inhibited the degradation of 125I-labelled insulin by isolated hepatocytes, as did the sulfhydryl reagents, p-hydroxy- and p-chloromercuriphenyl sulfonic acid. Inhibitors of energy production, including sodium fluoride, sodium azide, and dinitrophenol, also selectively inhibited the degradation of insulin by hepatocytes, although cyanide had no effect under the conditions used. Lectins and antimicrotubular agents, which are known to affect the mobility of plasma membrane proteins or of intracytoplasmic vesicles, selectively inhibited insulin degradation by hepatocytes to varying degrees, whereas agents which inhibit the function of microfilaments had no effect. At temperatures below 20 degrees C, insulin degradation was negligible but rose rapidly between 20 and 37 degrees C, suggesting that a membrane-related step is rate limiting in the overall degradative process. These results are all consistent with a model of insulin uptake by target tissue involving pinocytosis of receptor-bound hormone followed by intralysosomal degradation.


Structural relationships in the two-zinc insulin hexamer. The refinement of the crystal structure of two-Zn pig insulin using 1.5-A (1A = 0.1 nm) resolution data by Fourier and fast Fourier least-squares methods allows us to make detailed comparisons between the two independent molecules present in the two-Zn insulin dimer and to describe their interactions in the monomer, dimer, and hexamer. The main chain structures for the two molecules agree well except at the N terminus of the A chain and the C terminus of the B chain. The residues along the line of the local two-fold axes, apart from the B25 side chain, conform extremely closely to the two-fold symmetry, although the discrepancies are much more apparent away from this axis. The ability of the insulin molecule to adopt different conformations may be an important factor in the expression of its biological activity.


Effect of thyrotropin on the phosphorylation of thyroid chromosomal proteins. Thyrotropin (TSH) stimulated the phosphorylation of histone H1 in calf thyroid slices but had no effect on other classes of histones. Phosphorylation of total phenol-soluble nonhistone chromosomal proteins was not affected by incubation with TSH. However, when these phenol-soluble nonhistone chromosomal proteins were analysed by two-dimensional gels involving isoelectrofocusing and dodecyl sulfate-polyacrylamide gel electrophoresis, TSH was shown to stimulate the phosphorylation of two specific groups of phosphoproteins with molecular weights between 35,000 and 45,000 and isoelectric points at pH values of 5.4-6.0. This increase in phosphorylation with TSH stimulation was confirmed by quantitative analysis of one-dimensional isoelectrofocusing gels.


Immunological and biological activities of fragments and analogs of bradykinin. Using a number of analogs and fragments of a short-chain peptide bradykinin, a series of experiments have been carried out to assess the effect of modifications to the basic structure of the parent molecule on its myotropic and immunoreactive properties. Binding kinetics of both an antibody raised against the authentic nonapeptide and its specific biological receptor found in the guinea pig ileum were used to study these alteration effects. Peptide derivatives of bradykinin with an extension at the N-terminal (Lys- and Met-Lys-bradykinin) cross-react with the antibody raised to bradykinin 59 and 70% respectively. On the other hand, internal fragments with intact C-termini (2-9 and 3-9 bradykinin) react with this same antibody to an extent of 250 and 875% respectively, indicating that they are more potent antigens than the vasopressor molecule itself. Other internal fragments, as well as 9-substituted analogs effectively and not interact. These results indicated that the C terminal arginine of bradykinin is indeed essential in the binding mechanism with its antibody. This in turn illustrates the role of the carrier ovalbumin in the development of antiserum to the ovalbumin-toluene-diisocyanate-bradykinin complex. The physiological experiments with the guinea pig bioassay preparations lead to similar conclusions. Most internal fragments of bradykinin are devoid of activity, whereas N-terminal fragments (2-9, 3-9, and 5-9 bradykinin) have retained some activity again indicating a need for an intact arginine residue at the C-terminus of the molecule. Any modification in position 9 results in severe impairment of biological activity. Thus, the C-terminal residue of bradykinin must be conserved in order that the molecule may retain its immunological and physiological activities. Any extensions, deletions, or modifications of this site will severely retard these functions.


Effect of nucleotides on UDP-N-acetylglucosamine pyrophosphatase and N-acetylglucosaminyltransferase activities in microsomal membranes. Rat liver microsomes solubilized by incubating with lysolecithin or Triton X-100 showed very active UDP-N-acetylglucosamine pyrophosphatase activity leading to the hydrolysis of the substrate into N-acetylglucosamine-P and N-acetylglucosamine. ATP, GTP, CDPcholine, and CDPglucose exerted a considerable inhibitory effect on the solubilized membrane pyrophosphatase activity. CDPcholine and CDPglucose, in addition, appeared to stimulate the transfer of N-acetylglucosamine into endogenous and exogenous acceptor proteins. Evidence is also presented of an inhibitory effect of ATP (and to some extent GTP) on N-acetylglucosaminyltransferase activity. This inhibitory effect of ATP and GTP became clearly evident when the pyrophosphatase activity in the membranes was virtually eliminated in the presence of CDP-choline and CDPglucose. The effect of ATP and GTP on the solubilized membrane enzymes indicated that the inhibition of pyrophosphatase activity alone did not determine the rate of transfer of sugar to protein. The results also suggested that the UDP-N-acetylglucosamine pyrophosphatase and N-acetylglucosaminyltransferase activities were controlled independently and the effect of each nucleotide on these enzymes should, therefore, be carefully evaluated to understood its role in glycopolymer biosynthesis. Also, a possible role of choline and its derivatives in glycoprotein synthesis is discussed.


Chemical synthesis of sn-3-phosphatidyl sulfocholine, a sulfonium analog of lecithin. A sulfonium analog of lecithin has been reported to replace the ubiquitous phosphatidyl choline in a non-photosynthetic diatom, Nitzschia alba. The structure of this sulfonium analog has now been established by chemical synthesis using the following methods: (i) condensation of sn-3-phosphatidic acid (dimyristoyl-, dipalmitoyl-, distearoyl-, distearoyl-, and dioleoyl-) with sulfocholine chloride in the presence of triisopropylbenzenesulfonylchloride in chloroform-pyridine (9:1); and (ii) phosphorylation of 1,2-dipalmitoyl-sn-glycerol with monophenylphosphoryl-dichloridate followed by a reaction with sulfocholine in the presence of pyridine and finally removal of the blocking phenyl group by catalytic hydrogenolysis. The desired synthetic products were obtained in overall yields of 50-70% and 11% for methods (i) and (ii), respectively, and were characterized by elemental analyses; infrared spectroscopy, nuclear magnetic resonance spectrometry, and mass spectrometry; optical rotation; and thin-layer chromatography mobilities. Comparison of the synthetic analogs with the natural sulfolecithin showed them to be identical, except for the nature of the fatty acid chains, thus establishing the natural product as sn-3-phosphatidyl sulfocholine.


On hepatic mitochondrial monoamine oxidase activity in lipid deficiency. A marked decrease in liver mitochondrial monoamine oxidase activity was noticed in rats fed a fat-free diet as compared with that of their controls. In lipid-deprived rats, the specific activity of this enzyme was very low towards different substrates studied. The activity of kynurenine 3-monooxygenase, which like monoamine oxidase is localized on the mitochondrial outer membrane, was similarly depressed under conditions of lipid deprivation. On the other hand no major changes were observed in the activity of the inner membrane enzyme, kynurenine amino-transferase. Mitochondria from fat-free diet-fed rats were deficient in essential fatty acids whereas no appreciable variations were found in the relative proportions of phospholipids in comparison with those of control mitochondria. Mitochondrial monoamine oxidase activity of the deficient rats retained its sensitivities to inhibitor drugs like clorgyline and deprenyl. No changes were noticeable in the substrate specificity of monoamine oxidase in these rats. When we switched the fat-free diet-fed rats to a diet supplemented with a source of essential fatty acids, there was an elevation in the activities of both monoamine oxidase and kynurenine 3-monooxygenase, their levels approaching those of the control rats.


Lipid requirements for the aggregation of CTP:phosphocholine cytidylyltransferase in rat liver cytosol. Two forms of CTP:phosphocholine cytidylyltransferase were identified in rat liver cytosol by gel filtration chromatography. The low molecular weight form (L form) is the major form in fresh cytosol. The enzyme associates into a high molecular weight form (H form) upon storage of the cytosol at 4 degrees C. Aggregation of the purified L form of cytidylyltransferase is caused by total rat liver lipids, neutral lipids, diacylglycerol, or phosphatidylglycerol. Diacylglycerol was the only lipid isolated from the rat liver that caused aggregation of the purified enzyme. Although the addition of diacylglycerol to the cytosol did not change the amount of aggregation of the enzyme, a 2.5-fold increase in H form was observed in cytosol pretreated with phospholipase C, or in cytosol from rats fed a high cholesterol diet. In both of these cytosolic preparations, the concentration of diacylglycerol was elevated twofold. Phosphatidylglycerol did not seem to affect the association of the enzyme in cytosol since it is present in very low concentrations in the rat liver cytosol, and its degradation in cytosol by a specific phospholipase did not affect the rate of aggregation. The results suggest that diacylglycerol in an appropriate form is required for association of cytidylyltransferase in rat liver cytosol.


Suitability of L-[35S]methionine for studying the biosynthesis of the polypeptides of mouse liver endoplasmic reticulum membrane fractions in vivo. The use of L-[35S]methionine (500-700 Ci/mmol (1 Ci = 37 GBq) for labelling the polypeptides of liver rough (R) and smooth (S)endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 muCi of the isotope and killed at various times (2'min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 microgram protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.


Resolution of saturated and unsaturated 5 beta-cholanoic acids by gas-liquid and thin-layer chromatography. The thin-layer and gas-liquid chromatographic properties of the methyl ester acetates were determined for a series of 20 monounsaturated 5 beta-cholanoic acids representing the simple chemical and enzymic dehydration products of the common bile acids. The unsaturated acids were generally indistinguishable from their saturated analogues by thin-layer chromatography on plain silica gel, but resolution was achieved on silica gel impregnated with silver nitrate for compounds having sterically exposed double bonds. The gas-liquid chromatographic behaviour of the unsaturated bile acids on the OV-225, SE-30, and Poly-S-179 liquid phases was closely similar to that observed for the saturated bile acids. The 5 beta-cholenoic acids obeyed the general rules of chromatographic mobility based on the overall shape of the molecule and the number and configuration of functional groups, with a constant retention factor attributable to the olefinic bond. The structural information provided by the chromatographic behaviour of the standard unsaturated bile acids allows a distinction to be made among most of the isomeric 5 beta-cholenoates. A complete identification of all isomeric olefins is possible when chromatographic and mass spectrometric data are combined.


Effects of dietary carbohydrate on cholesterol metabolism in rats and rabbits. Semipurified diets produce a marked hypercholesterolemia in rabbits and tend to elevate the level of plasma cholesterol in rats. They also decrease rates of oxidation of [26-14C]cholesterol to respiratory 14CO2 and excretion of label in fecal lipid, compared with commercial feed. In both species, the hypercholesterolemia was prevented and the rate of oxidation of [26-14C]cholesterol increased by using unmodified potato starch as the carbohydrate component of the semipurified diets. Potato starch was poorly digested by rats but appeared to be well utilized by rabbits. A semipurifed diet containing cooked potato starch gave results in rats comparable to those obtained with diets containing other types of carbohydrate. Cooked potato starch produced diarrhea in rabbits, thus complicating interpretation of the results. When the diarrhea was treated with antibiotics, the results were similar to those obtained with other carbohydrates. Rats fed commercial diet which had been heated in an oven or autoclaved had higher plasma levels than those fed untreated commercial diet but no significant differences in rates of oxidation or excretion of cholesterol were observed.


Binding of mitochondrial malate dehydrogenase to mitoplasts. The binding of 14C-labelled bovine and porcine malate dehydrogenase (EC 1.1.1.37) to rat liver mitochondria and mitoplasts was examined. The bovine enzyme was found to associate nonspecifically with isolated mitochondria and sonicated mitoplasts. Scatchard plot analysis suggested a specific binding to mitoplasts of the order of 5 pmol malate dehydrogenase per milligram of mitoplast protein. Porcine malate dehydrogenase dimer but not monomer exhibited a similar binding. The results are discussed in relation to the mechanism of uptake of the enzyme by mitochondria after synthesis on cytosolic ribosomes.


Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1. Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.


Chick pineal serotonin acetyltransferase: a diurnal cycle maintained in vitro and its regulation by light. We have reproduced in vitro the diurnal cycles in levels of serotonin acetyltransferase activity found in the chick pineal gland in vivo. The more closely the lighting conditions of culture matched those under which the birds were raised, the closer was the similarity between cycles in levels of enzyme activity in vitro and in vivo. Repetitive cycles in levels of acetyltransferase activity persisted in culture for at least 4 days under a diurnal cycle of illumination, and at least 2 days in continuous darkness. When glands were explanted into culture in the light phase of a cycle, short periods of further exposure to light markedly stimulated subsequent increase of acetyltransferase in the dark (after a short lag). Prolonged exposure to light in culture markedly inhibited increase of enzyme activity. Cycles in the levels of enzyme activity in glands cultured under altered light cycles were regulated primarily by changes in illumination. However, the endogenous biological 'clock' remained at least partly entrained to the original light cycle. Increase of acetyltransferase activity in vitro was markedly stimulated by theophylline plus compound Ro. 20.1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone) under all lighting conditions. Kinetics (to the time of attaining maximum levels in situ) of the increase under diurnal lighting and in constant darkness were indistinguishable from those in vivo. A high concentration of dl-propranolol markedly stimulated an increase in acetyltransferase activity in glands cultured in constant darkness but had little effect on glands under diurnal lighting or continuous illumination.


Structural homologies in alanine-rich acidic ribosomal proteins from procaryotes and eucaryotes. The amino acid composition and amino-terminal sequence have been determined for the alanine-rich, acidic ribosomal 'A' protein (equivalent to Escherichia coli L7/L12) from three procaryotic cell types that live under extreme environmental conditions (Arthrobacter glacialis, Clostridium pasteurianum, and Bacillus stearothermophilus) as well as from wheat germ, a eucaryote source. These data are compared with previously published 'A' protein sequences from other procaryotes and eucaryotes. All the procaryotic 'A' proteins, with the exception of the very acidic 'A' protein from Halobacterium cutirubrum, show similar charge, size, and amino acid composition, as well as an extensive sequence homology in the N-terminal region. Some differences are observed between gram-negative and gram-positive bacteria. The 'A' proteins from eucaryotes contain two tyrosine molecules, an amino acid absent in procaryotic 'A' proteins, as well as a reduced number of valine residues and an increased amount of aspartic acid. The N-terminal sequence of wheat germ 'A' protein shows considerable homology with other eucaryotic 'A' proteins and also with H. cutirubrum. It also shows some sequence homology with E. coli 'A' proteins.


Electron microscopy of serum amyloid protein in the presence of calcium; alternative forms of assembly of pentagonal molecules in two-dimensional lattices. The P component is a protein believed to be of serum origin commonly found in the highly ordered proteinaceous tissue deposits called amyloid. Both the protein recovered from the tissue and its serum counterpart have an identical characteristic appearance in the electron microscope, consisting of pentagonal plates which are often assembled as columns of stacked discs. These images, seen in the absence of calcium, are replaced by three more complex assemblies when low concentrations of calcium are present. One of the structures is crystalline (III), the other two appear to be planar lattices, one having a distinguishable structure with threefold symmetry (1). The structural elements of the other lattice (II) which takes the form of a cylinder are less distinct. It is suggested that the regular arrays which P component forms in the presence of calcium may be the basic framework on which amyloid deposits form.


The amino acid sequence of porcine intestinal calcium-binding protein. The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometric analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.


The interaction between beef cardiac troponin T and troponin I as demonstrated by ultraviolet absorption difference spectroscopy, circular dichroism, and gel filtration. The specific interaction of bovine cardiac troponin T with troponin I has been demonstrated at a 1:1 molar ratio by absorption difference spectroscopy, near and far ultraviolet circular dichroism, and gel filtration chromatography. The maintenance of the sulfhydryl groups of both proteins in the reduced state was essential in order to demonstrate interaction between cardiac troponin I and troponin T using the aforementioned methodology. Carboxamido-methylated troponin I and troponin T samples were prepared by reaction with iodoacetamide. Spectrophotometric titration of the two proteins with 2-chloromercurinitrophenol and amino acid analysis of their carboxamidomethylated derivatives revealed that cardiac troponin I possesses two cysteine residues while cardiac troponin T has one. The modified troponin T possesses properties identical to those of the native molecule. The modification of troponin I is accompanied by an increase in secondary structure and a loss in ability to interact with troponin T at 0.5 M NaCl ionic strength. However, at 0.3 M NaCl the modified troponin I was shown by gel filtration chromoatography to interact very weakly with troponin T. On the other hand, the modified troponin I interacts with troponin C in a manner identical to the native protein, indicating that the troponin T interaction domain of the molecule is distinct from that region which interacts with troponin C.


Lysyl dipeptide and tripeptide model systems for racemization studies in amino acid and peptide chemistry. Four series of diastereomeric peptide pairs (X-Lys, Lys-Y, Gly-X-Lys, and Gly-Lys-Y; where X and Y = Ala, Leu, Val, Ile, Phe, and Pro) have been synthesized by conventional procedures using benzyl-based protecting groups. Chromatographic separation of each pair, except Gly-Lys-Pro, has been achieved by elution from a 15-cm Aminex A-5 resin column using pH 5.5 buffer for tripeptides, pH 6.5 buffer for dipeptides, and pH 7.5 buffer for phenylalanyl peptides. The isomers have been identified by chromatography of the L-L isomers. Examples of the use of the model peptides for optical purity determinations and as tests for racemization are presented. The series Gly-X-Lys allows comparison of the tendencies to racemize of residues X during couplings. The separations of other peptides where Y = MeVal and X = N- and O-methylhydroxyamino acids are also described.


Lysozyme catalysis: kinetics of the hydrolysis of cell wall oligosaccharides. The cleavage of cell wall tetrasaccharide, the beta(1 leads to 4)-linked dimer of the basic repeating disaccharide N-acetyl-D-glucosamine-beta(1 leads to 4)-N-acetyl-D-muramic acid, by lysozyme has been studied at various concentrations of lysozyme and over long time ranges. A theoretical analysis of the kinetic results indicates that direct hydrolysis of the tetrasaccharide by binding in subsities CDEF of the active site of lysozyme is significant at long times relative to the transglycosylation pathway. The binding constant for tetrasaccharide in CDEF is shown to be 10(3) times larger than that determined on the basis of an analysis of kinetic data over a more restricted range of times and concentrations.


Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase from porcine liver. Location of the activities in two domains of the multifunctional polypeptide. Chymotryptic cleavage of the trifunctional protein methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase from pig liver yields a fragment of two-thirds the original polypeptide that retains only synthetase activity. A smaller polypeptide corresponding to about one-third of the original polypeptide was shown earlier to retain dehydrogenase-cyclohydrolase activity. On immunodiffusion, the synthetase fragment cross-reacts and shows partial identity with antibodies raised against the uncleaved enzyme but shows nonidentity with the dehydrogenase-cyclohydrolase fragment, suggesting that the two fragments are derived from different regions of the polypeptide. Amino-terminal analysis of the peptides and uncleaved enzyme indicate that the dehydrogenase-cyclohydrolase activities are located at the amino-terminal region and the synthetase near the carboxyl-terminal portion of the polypeptide.


Relationships among purine nucleoside metabolism, adenosine triphosphate catabolism, and glycolysis in human erythrocytes. In human erythrocytes incubated with both naturally occurring purine nucleosides and with a variety of purine nucleoside analogs, ATP catabolism was accelerated and lactate accumulation was increased. Tubercidin was a particularly potent inducer of ATP catabolism. In cells incubated with tubercidin, the major route of adenylate metabolism was deamination, whereas in cells incubated with deoxyglucose, the major pathway was dephosphorylation.


The ribosomal RNA of the trypanosomatid protozoan Crithidia fasciculata: physical characteristics and methylated sequences. When extracted and analyzed under conditions which maintain noncovalently associated RNA-RNA complexes, the bulk cellular RNA of Crithidia fasciculata contains species of apparent molecular weights 1.3, 0.825, 0.08, 0.065, and 0.045 x 10(6) in addition to 5S rRNA and tRNA. Heat denaturation results in the disappearance of the 1.3 x 10(6) dalton RNA and the appearance of three new species having molecular weights of 0.67, 0.575, and 0.059 x 10(6). In addition, the apparent molecular weight of the 0.825 x 10(6) dalton component is reproducibly lowered to 0.81 x 10(6) after heat treatment. With the exception of tRNA, all of the RNA species are present in close to equimolar amounts in either undenatured or heat-denatured C. fasciculata bulk cellular RNA. On the basis of previous observations on the ribosomal RNA of the closely related organism, Crithidia oncopelti (Spencer, R. & Cross, G.A.M. (1976) J. Gen. Microbiol. 93, 82-88), the 1.3 and 0.825 x 10(6) dalton RNA's are considered to be components of the large and small subunits, respectively, of C. fasciculata ribosomes, but the subunit localization of the other RNA's described here has not yet been determined. O2'-Methylnucleosides account for about 1.4 mol% of the total nucleoside constituents of unfractionated C. fasciculata rRNA. Quantitative analysis suggests that the rRNA molecules in a C. fasciculata ribosome contain a total of 95-100 O2'-methyl groups, distributed in 80-85 Nm-Np sequences (including four 'hypermodified' Nm-Np, each containing a modification of a base or base-sugar linkage in addition to sugar methylation), six different Nm-Nm-Np sequences, and one Nm-Nm-Nm-Np sequence. While the specific pattern of O2'-methylation in the rRNA of C. fasciculata is distinct, both qualitatively and quantitatively, from the pattern observed in other organisms, Crithidia rRNA does contain certain 'universal' O2'-methylated sequences which appear to have been extensively conserved in evolution. The base-methylated nucleoside, N6,N6-dimethyladenosine (m26A), has been isolated from both C. fasciculata and wheat embryo rRNA in the form of the alkali-resistant dinucleotide, m26A-m26Ap. This dinucleotide and its enzymatic degradation products have been characterized by examination of their ultraviolet absorption spectra and electrophoretic and chromatographic properties.


Protamine messenger RNA: partial purification and characterization of a heterogeneous family of polyadenylated messenger components. Poly(A)+ protamine mRNA (pmRNA) components were isolated after separation on denaturing preparative polyacrylamide gels. The four size classes of protamine mRNA described previously were found to contain poly(A) tracts of different lengths. The pmRNA1 was found to be associated with (A)110, pmRNA2 with (A)90, pmRNA3 with (A)85, and pmRNA4 with (A)69. Following deadenylation with RNase H after duplex formation with oligo-dT, the isolated mRNAs were found to be still heterogeneous, although highly enriched in certain of the deadenylated components. DNA complementary to the isolated mRNAs (cDNA) was synthesized in vitro. Following depurination, the oligopyrimidine maps indicated that C7T4, corresponding to an Arg-Arg-Gly-Gly sequence in protamine and originally thought to be characteristic of all mRNA components, is present in only one or possibly tow of the components. Cross-hybridizations between the cDNAs and the four poly(A)+ pmRNAs indicated that a basic polynucleotide unit of substantial length is common to all four mRNAs and that the existing nucleotide sequence variations probably originate from one or both of the non-coding portions of the mRNA molecules.


Conservation of nucleolus organizer regions during evolution in sheep, goat, cattle and aoudad. There are ten nucleolus organizer regions (NORs) in domestic sheep (Ovis aries L.). cattle (Bos taurus L.), goat (Capra hircus L.) and aoudad (Ammotragus lervia Blyth) and these are located terminally on chromosomes with homologous (G-banding patterns. The similarity in number of nucleolus organizer regions in these species may indicate that their ribosomal DNA regions are infrequently involved in exchange events which could lead to different numbers of active nucleolus organizer regions. Other possible explanations of the conservation of number of nucleolus organizer regions in these species are discussed. The homology of NOR location in these species supports the idea that the Bovidae karyotype tends to be fairly stable apart from changes due to centric fusion events.


The use of the Salmonella/microsomal assay to determine mutagenicity in paired chemical mixtures. The mutagenicities of two sets of chemicals acting singly and in pairwise combinations were determined by use of the Salmonella/microsomal assay. The first set consisted of the promutagens of benzo(a)pyrene and benzo(rst)pentaphene. The second set contained the direct-acting mutagens methyl-nitro-nitroso-guanidine and ethyl methane sulfonate. In the tests with the promutagens, the quantities of S-9 mix were varied over the range of 0.05 ml to 1.0 ml with increasing quantities of each chemical. The mutagenic responses or production of revertant colonies of the promutagens, acting singly and in pairwise combinations failed to show an additive effect. Excess quantities of S-9 mix appeared to inhibit partially or totally the mutagenic activity of each chemical, although for each particular dose there was an optimal quantity of S-9 mix to induce maximum activity. However, the direct-acting mutagens produced, individually, almost linear dose responses with increasing concentrations. In pairwise combinations, these chemicals also showed linear responses that closely approximated the theoretical additivity indicating that the mutagenicity of the mixtures was the sum of the activities of each component.


Frequencies of centromeric heteromorphisms of human chromosomes 3 and 4 as detected by QFQ technique: can they be identified by RFA technique? One hundred normal Caucasians were studied by sequential QFQ and RFA banding techniques in order to estimate the type and frequency of heteromorphisms in the centromeric regions of chromosome 3 and 4. Intensity variants were classified into 1 of 5 levels of QFQ banding. QFQ intensity heteromorphisms (greater than or equal to level 3) for chromosomes 3 and 4 were 62 and 15 percent respectively. The interrelationship between QFQ and RFA variants were also examined. When the centromere was brilliant by QFQ, it was found that it was deep red by RFA; when it was pale by QFQ, it was light red by RFA. Neverthless, a blind coded study could not pick up these color variants by RFA. QFQ banding showed variations of the centromeric regions of chromosomes 3 and 4 while RFA banding failed to demonstrate it. It was concluded that QFQ  is the most useful technique in detecting the different intensity levels in the centromeric regions of chromsomes 3 and 4.


Sperm size abnormalities in homozygous and heterozygous In(5)9Rk mice. Sperm abnormalities were scored in In(5)9Rk homozygotes, heterozygotes and in the inbred strains, C57BL/6J, C3H/HeJ, DBA/2J and JUFaCt, used to produce the geterozygotes. The sperm abnormalities of the inversion heterozygotes were remarkable, about 40% were either double-headed or abnormally large in size. The double-headed sperm had head dimensions similar to those of normal-sized sperm, but the large sperm heads were 1.2 times longer and 1.3 times wider and presumably had twice the usual nuclear volume. This observation suggests that the anaphase bridge formed in inversion heterozygotes resulting from the paracentric inversion may constitute an impediment to cytokinesis and may lead to two genomes being packaged into a single sperm. If this is a general phenomenon, sperm morphology may provide a useful screen for paracentric inversions.


Considerations of a method of analyzing diallel crosses of atlantic salmon. Inferences derived from a proposed mixed model analysis of a diallel cross involving four stocks of Atlantic salmon (Salmo salar) are illustrated with growth data on weight and length. Variation between stocks was more apparent when samples represented dams as opposed to sires, thus stressing the relative importance of maternal effects. However, the ranking of stocks was not altered when either the means of sire sources or the means of dam sources were considered. No heterotic effects were found for the growth traits studied.


The chromosomes of the Canadian Beaver Castor canadensis. A chromosome analysis of 24 Canadian beavers, Castor canadensis Kuhl (12 males and 12 females), captured in Laurentides Park, Qébec, has been performed from preparations of blood lymphocyte and skin cultures. The chromosome number was found to be 2n = 40. Measurements were made to determine relative lengths and arm ratios of chromosomes, which are metacentric or submetacentric. Results are in agreement with those already published regarding the chromosome number, but differ in the identification of the X chromosome, and in the morphology of the Y and some autosomes. C- ad G-banding techniques allowed the precise identification of individual chromosome pairs. A detailed idiogram of G-bands is presented.


Direct genetic and postnatal maternal genetic effects on body composition in mice selected for body weight. Line crossfostering techniques were used to study differences among selected and control lines of mice in direct genetic and postnatal maternal genetic influences on preweaning (day 12) body weight and composition. The lines were selected for high (H6) and low (L6) 6-week body weight and the control line (C2) was maintained by random selection. There were positive correlated responses to selection in both direct genetic and postnatal maternal genetic effects on body weight and weights of all body components (P less than 0.01) except for water and ash weight in H6. The correlated responses in postnatal maternal genetic effects were of the same order of magnitude as those in direct genetic effects. Correlated responses were greater in L6 than in H6. Correlated responses in direct genetic effects were positive (P less than 0.01) for water percent in H6 and ether extract percent in L6, and negative (P less than 0.01) for water percent and lean percent in L6. Correlated responses in postnatal maternal genetic effects were positive for ether extract percent and negative for water percent (P less than 0.01). Correlated responses were far greater in L6 than in H6 and were greater for postnatal maternal genetic effects than for direct genetic effects. Analyses of covariance results indicated line differences in the relative growth rates of the body components.


Electrophoretic variation and differentiation in four strains of domesticated rainbow trout (Salmo gairdneri). A total of 1462 rainbow trout (Salmo gairdneri Richardson) representing four California domestic strains, were examined electrophoretically at 24 gene loci to investigate the amount of genetic variability within strains and genetic differentiation between strains, relative to wild populations. Estimated heterozygosities of 0.071 to 0.134 were similar to those reported for wild populations and thus indicated that the domestic strains were at least as variable as wild populations. Genetic distances between strains ranged from 0.011 to 0.067, values typical of the level of differentiation observed between local conspecific populations in a variety of organisms. It was suggested that this high level of heterozygosity is due to mixing of populations in the strains' histories and perhaps also to balancing selection.


Can mutation theories of carcinogenesis set priorities for carcinogen testing programs? The recent activity in designing, validating and implementing short-term tests for carcinogens has been spurred by the fairly convincing correlation between the carcinogenicity and mutagenicity of chemicals and by the assumption that mutations are somehow involved in neoplastic transformation. Moreover, it has been tacitly assumed that the mutagenic capacity alone of compounds would induce regulatory agencies to pass rules for their removal from man's environment, and would lead the public to avoid them. The actual response, however, is quite different. Government departments shy away from making any decisions on the basis of in vitro test systems, the public at large is becoming irritated by daily announcements that many of their cherished habits could adversely affect their health, and industries feel threatened and may reduce their search for new beneficial chemicals. The reluctance to accept wholeheartedly the mutagenicity tests for the detection of carcinogens is partly due to the uncertainty about the involvement of mutations in the formation of benign and malignant tumors. Following the initial rapid advances in the detection of environmental chemicals with carcinogenic and mutagenic properties, we seem to have arrived at the cross roads: we must now set new priorities for future research, and must make an unbiased assessment of the actual hazard of a compound to man and the human population.


Structure of nitrogen-fixing nodules formed by Rhizobium on roots of Parasponia andersonii Planch. The structure of nitrogen-fixing nodules produced by Rhizobium infection of the non-legume Parasponia andersonii was examined by light and electron (both SEM and TEM) microscopy. Comparisons were made with the nodules previously described on P. rugosa. Like the nodules on different non-legumes formed by other types of endophytes, the Rhizobium nodules on Parasponia resembled modified roots by having a central vascular bundle surrounded by an endophyte-infected zone. The intimate association between the Rhizobium and the host nodule cell was compared with the Rhizobium association found in legumes. The rhizobia were not released from the infection thread as happens in the legume. The infection thread, which propagates the Rhizobium infection to new cells, was transformed within a nodule cell from a darkly stained (light microscopy) or very electron-dense (TEM) structure to a number of thread types. The walls of the threads varied greatly in thickness and often the thread structures were without rigid walls and were only enclosed by a plasma membrane. If the rhizobia are transformed into bacteroids, as in the legumes, it would have to occur when the threads had reached their mature size, when bacterial division had ceased. Nitrogen fixation was considered to occur in all thread types.


Deoxyribonucleic acid relationships among members of the genus Aeromonas. Polynucleotide sequences among 24 motile and 11 non-motile aeromonads were studied by analysis of deoxyribonucleic acid - deoxyribonucleic acid (DNA-DNA) duplexes with endonuclease S1. In addition, DNA base composition (mole % guanine and cytosine (G + C)) and relative genome sizes were determined for selected strains. Large variations in genome size were found and % GC ranged from 57.1 to 62.9%. On the basis of the strains examined, the Genus Aeromonas consists of two genotypically legitimate groups: a diverse group of motile aeromonads, and the genetically more homogeneous non-motile aeromonads, comprising the species Aeromonas salmonicida. Internal homology groups could not be demonstrated within the motile aeromonads, and significant divergence in related sequences was indicated. This diverse motile group forms the single species Aeromonas hydrophila.


Effect of glycerol on viability and other properties of starved Mycobacterium fortuitum. Cells of Mycobacterium fortuitum kept in 0.85% saline solution containing 0.1% Tween 80 (without glycerol) survive for a long time. In glycerol-enriched medium, they continue to lose their viability at a high rate; after 71 days of exposure the percentage of survival as indicated by colony formation and respiratory and dehydrogenase activities is lower than 1%. Surviving cells starved in medium without glycerol revealed unchanged sensitivity to streptomycin, p-aminosalicylic acid, and isoniazid.


Mannose transport in Amphora coffeaeformis var. perpusilla (Bacillariophyceae). Amphora coffeaeformis var. perpusilla is able to concentrate but not metabolize mannose. The sugar, which can be accumulated against a concentration gradient, is taken up only after a short lag. Preincubation with glucose allows immediate mannose uptake. Substances that reduce the ATP content of the diatom cells also inhibit mannose uptake as do sugars of similar stereochemical configuration. Mannose appears to act as a non-metabolizable analogue of glucose in these respects.


Effect of thiols on macroconidia of Fusarium sulphureum. Treatment of Fusarium sulphureum macroconidial cells with five thiols alters their morphology. Macroconidial cells incubated in dithiothreitol (DTT), dithioerythritol (DTE), or thiourea differentiate into thick-walled, chlamydospore-like cells (thiol-induced spores). These cells appear similar in size and shape to chlamydospores in the light microscope, but differ markedly in cell wall structure when viewed in the electron microscope (EM). Incubation of macroconidia with both DTT and DTE also leads to the formation of large swollen cells (giant cells) which have a parietal cytoplasm and electron-tranparent cell walls; most of these giant cells lyse within 3 to 7 days of incubation. Thiourea-induced spores are characterized by the deposition of a thick, electron-dense, extracellular layer and an accumulation of mitochondria. DTT and DTE, at the concentrations used, inhibit macroconidial germination while thiourea, mercaptoethanol, and cysteine do not. With the latter three thiols, the newly formed hyphal cells become elongated with either one or both ends swollen. Mercaptoethanol-treated cells contain an abundance of mitochondria. The DTT-induced spore differs from both macroconidia and chlamydospores with respect to cellular lipid and cell wall composition. While the thiols have different effects on the macroconidia, the fact that they all induce cell expansion suggests that they react at some common sites.


[Effect of the saccharide chain length of bacterial lipopolysacchrides on the stimulation of immune system]. Three lipopolysaccharides (LPS) of different chain length were used as adjuvant. The one of shortest chain length is the most effective in enhancing antibody titer in rats against sheep red blood cells, suggesting that lipid A and KDO were responsible for the stimulatory effect of LPS.


A dominant temperature-sensitive assembly mutant of adenovirus type 2. An assembly negative temperature-sensitive mutant of Ad2, ts48 was shown to exert dominance over other ts mutants and wild-type virus during coinfection, by inhibiting virion assembly. Dominance was only expressed at the nonpermissive temperature.


Immunologic evidence that staphylococcal alpha toxin is oriented on membranes. Antibodies to staphylococcal alpha toxin were separated into two distinct populations. One population prevented binding of alpha toxin onto erythrocyte membranes. The other population neutralized after the toxin was bound onto erythrocytes and thereby brought about an indirect hemagglutination reaction. The data suggest that alpha toxin has a membrane-binding region.


Isolation of Azospirullum from diverse geographic regions. We have isolated Azospirillum (Spirullum lipoferum) from roots of grasses of several genera collected from a number of tropical and subtropical-temperate locations. Pure cultures were obtained from a small percentage of samples; no higher percentage was secured from tropical than from other grasses. Acetylene reduction and distinctive growth in N-free soft agar deeps were inadequate to identify this genus, although helpful in initial screening. Fluorescent antibody tests with antiserum against characterized strains were helpful. There is some evidence that this genus of bacteria may be favored in the rhizoplane.


[Study of a new group of Enterobacteriacea (group H1) related to Enterobacter cloacae strain]. A DNA-DNA hybridization study was carried out on a new group of enterobacteria (group H1) previously studied by numerical taxonomy work on the genus Enterobacter. This group showed a very high genetic homogeneity since the average relative binding ratio of nine analysed strains is equal to 91%. The taxonomic position of this group into the family of enterobacteria is discussed with the species E. cloacae (37 to 61%), K. pneumoniae (44 to 60%), K. oxytoca (57-58%), L. malonatica (syn. Citrobacter intermedius, a:46 to 54%), L. amalonatica (syn. C. intermedius, b: 51%), and the group H3 (52-61%). The group H1 is defined on phenotypic and genetic data.


Thiosulfate formation and associated isotope effects during sulfite reduction by Clostridium pasteurianum. During growth of Clostridium pasteurianum on sulfite, approximately half the sulfite was reduced to sulfide and half to thiosulfate. Sulfide was enriched in 32S or 34S at different stages of growth and thiosulfate was enriched in 32S, particularly in the sulfane atom. It is suggested that thiosulfate in these bacterial cultures arose from a secondary chemical reaction. The chemical formation of thiosulfate from sulfide and sulfite was also accompanied by sulfur isotope fractionation. The implications of these results with respect to 'inverse' isotopic effects are discussed.


Minute tubular forms in soil.

Minute tubular forms in soil. Large numbers of minute, flat, long, straight, but often broken structures were observed in aqueous extracts of soil. We have applied the purely descriptive term 'tubules' to these structure, because they apparently became flattened during preparation for electron-microscopic viewing. The tubules appeared to be composed of parallel fibers held together by protein. Most of the tubules fell within a ranged of 10 to 50 nm in width. Lengths (of broken pieces) ranged from 0.2 to 1 micron or more. A few unbroken ends were found. They were rounded. Tubular structures of similar dimensions were found surrounding lysed cells in sectioned preparations of bacteria that had been separated from soil. The tubules were present in surface soils but not a subsurface sample. Their numbers decreased during bacterial multiplication in soil or broth containing soil. No evidence was found of cleared areas or increase in number of tubules when the tubules were plated with a heterogeneous microbial flora from soil.


Effect of oxygen on batch and continuous cultures of a nitrogen-fixing Arthrobacter sp. Growth, acetylene reduction, and respiration rate were studied in batch and continuous cultures of Arthrobacter fluorescents at different oxygen partial pressures. The optimum pO2 values for growth and acetylene reduction were 0.05 and 0.025 atm, respectively, but microorganisms can tolerate higher pO2 values. The growth of cultures provided with combined nitrogen was dependent on oxygen availability, and strict anaerobic conditions did not support growth. Acetylene reduction of a population grown in continuous culture and adapted to low pO2 (0.02 atm) was much more sensitive to oxygenation than that of a population adapted to high pO2 (0.4 atm). Their maximum nitrogenase activity, at their optimal pO2 values, were quite different. The respiratory activity of nitrogen-fixing cultures increased with increasing oxygen tensions until a pO2 of 0.2 atm. At higher pO2 values, the respiration rate began to decrease.


Investigation of antibodies against Fasciola hepatica in moose Alces americana. A serological investigation was carried out to check the presence of antibodies against Fasciola hepatica in a collection of 125 sera moose Alces americana killed in the Province of Quebec. As revealed by hemagglutination tests, two moose had titers suggesting contact with this parasite.


Chlortetracycline and sulfonamide resistance of fecal bacteria in swine receiving medicated feed. The fecal bacterial flora of swine receiving a ration supplemented with chlortetracycline, sulfamethazine, and penicillin was tested for resistance to chlortetracycline and sulfamethazine using anaerobic techniques and medium M-10. Approximately 15.5 and 1.4% of the flora grew in the presence of 25 and 100 microgram of tetracycline/mL, respectively. Higher numbers of bacteria grew in the presence of similar concentrations of sulfamethazine. Thirty-five chlortetracycline-resistant isolates were tentatively identified by genera. Nine different genera were identified, four of these were Gram-positive and five were Gram-negative. The most common genera isolated were Streptococcus and Eubacterium. This demonstrates that in the fecal flora of swine fed rations supplemented with chlortetra-cycline, a wide variety of bacterial genera can be resistant to this antibiotic.


Fingerprinting bacterial chromosomal DNA with restriction endonuclease EcoRI: comparison of Rhizobium spp. and identification of mutants. Total cellular DNA from Rhizobium trifolii, R. melitoti, and R. japonicum strains 110 and 117 were prepared. DNA fragments generated with restriction endonuclease EcoRI from these DNA samples were compared in agarose gels after electrophoresis. DNA cleavage patterns generated from R. japonicum strain 110, R. trifolii, and R. meliloti were clearly distinguishable from each other. Restriction endonuclease cleavage patterns of DNA from R. japonicum strain 110 and presumptive R. trifolii mutant strains that nodulate soybean were found to be similar. Rhizobium trifolii mutant strains were also lysed by a phage specific for R. japonicum strain 110. These results show that "R. trifolii mutant strains" are indeed derivatives of R. japonicum strain 110 and not R. trifolii.


The fine structure of mature and germinating chlamydospores of Fusarium oxysporum. A number of features not described previously has been revealed in electron-microscope studies of mature chlamydospores of Fusarium oxysporum. On the maturation of one isolate, many spores formed a thick matrix-like layer containing electron-dense particles on the exterior surface of the spore wall. In thin sections of mature chlamydospores of the same isolate, cisternae of endoplasmic reticulum surrounding, and in close apposition to, the limiting boundary of the lipid bodies were revealed. The germination of chlamydospores was accompanied by (a) the rapid appearance of polysaccharide deposits and changes in the configuration of some subcellular organelles, (b) the formation of a new wall layer between the plasma membrane and the innermost layer of the spore wall, (c) the rupture of the outermost coats of the spore wall, and (d) the emergence of the germ tube as an extension of the new wall layer.


Bacterial utilization of a hydrazine derivative as nitrogen source for growth. The ability of microbes to metabolize the N--N bond seems rare. Pseudomonas sp. from soil can utilize 1,4,5,6-tetrahydro-6-oxo-3-pyridazinecarboxylic acid as C and N source. This appears to be the first report that a microbe can cleave a nitrogen--nitrogen bond in an organic compound and use the products for growth.


The ultrastructure of the major species of an enriched methanogenic culture utilizing acetic acid. The ultrastructure of the cells of the major component of an enriched culture of a presumed methanogen which utilized acetic acid was studied by transmission and scanning electron microscopy. The filaments were composed of Gram-positive, rod-shaped cells, 1--2 micrometer in length and about 0.5 micrometer in breadth, attached end to end. Septa between cells were complex, with a central, electron-dense sheet which had a spherical enlargement in the center separating the cell walls. The cells walls themselves were of variable thickness with a light, fluffy, thin portion on the outside and a denser, thicker portion within. They contain a series of rings stacked side by side which are composed of material that stains strongly and positively with phosphotungstate ion. The cytoplasmic membrane of these cells had an outer leaflet which stains more densely with uranium and lead ions than the inner leaflet. There were no recognizable organelles in the cytoplasm other than ribosomes. It is shown in these observations that the presumed methanogen may likely be a new species.


[Study of the structural polypeptides of Chilo suppressalis iridescent virus (Iridovirus type 6)]. We report a procedure for the purification of Chilo iridescent virus (Iridovirus type 6), an evaluation of the purification procedure, and the results of analyses of the virion proteins by acrylamide gel electrophoresis. Purity was evaluated in three ways, i.e., by analysis of purified virions from artificial mixtures of infected and labeled uninfected larvae, electrophoresis at neutral pH, and electron-microscopic examination. Analysis of the polypeptides of purified CIV gave the following results: (i) after solubilization with SDS-B-mercaptoethanol, 16 polypeptides could be resolved in Coomassie brillant blue-stained electrophoretograms with molecular weights ranging from 18,000 to 115,000; (ii) after solubilization with SDS-urea, 26 polypeptides could be resolved with molecular weights ranging from 10,000 to 230,000 daltons.


Evidence of a competitive hierarchy among coprophilous fungal populations. Evidence is presented that interference competition may be important in later states of fungal colonization of cattle feces from a semiarid grassland in Colorado. Cultural antagonism was examined among fungal isolates representing early sporulating colonists (Ascobolus furfuraceus and Saccobolus truncatus), later sporulating colonists (Iodophanus carneus, Coniochaeta discospora, Hypocopra merdaria, and Poronia punctata), and one early successional species that is able to persist (sporulate) through later stages (Podospora decipiens). Poronia punctata, a comparatively slower-growing and later-appearing colonist (18- to 54-month-old fecal pats), is uniformly antagonistic to all of seven earlier-appearing and co-occurring fungal species. Antibiosis is believed to account for the observed antagonism. The authors suggest that the evolutionary product of interference competition among coprophilous fungal populations may be a pattern of competitive hierarchy in which certain slower-growing, later-successional species can limit the reproductive potential of other fungal colonists on fecal substrates.


The effect of 5-fluorouracil and adriamycin on heterotransplantation of Noble rat prostatic tumors in congenitally athymic (nude) mice. The Nb rat prostatic adenocarcinoma is a well-characterized, hormonally induced family of tumors that are all readily transplantable into congenitally athymic (nude) mice. Because of this versatile heterotransplantation model, multiple replicate copies of individual tumors can be studied "in rodent." We have extended this by studying the chemotherapeutic response of such tumors and believe that this provides a useful avenue for evaluation of cytotoxic agents. Indeed, this combination of both animal model systems may provide a useful experimental tool to evaluate tumor growth, histopathologic changes and responsiveness to appropriate therapy. We report herein that two Nb rat prostatic carcinomas (2 Pr-129-D-11A and Pr-90) and thie responsiveness to Adriamycin and 5-fluorouracil are objective by studying both growth rates and tumor histology.


Remission of giant lymph node hyperplasia with anemia after radiotherapy. We have described a 51-year-old patient with unresectable mesenteric giant lymph node hyperplasia of the plasma cell type, severe systemic manifestations, and profound anemia. Supression of erythropoiesis may have been related to the presence of a circulating erythropoietic inhibitor produced by the lymphoid tumor. Markedly elevated titers to Epstein-Barr virus capsid antigen suggest that this virus may be important in the etiology of the abnormal lymphoid proliferation. The marked clinical response and decrease in the size of the tumor following irradiation suggests that radiation therapy may be an alternative form of treatment for similar patients with unresectable lesions.


Slipped capital femoral epiphysis (SCFE) following pelvic irradiation for rhabdomyosarcoma. A 7-year-old child developed slipped capital femoral epiphysis (SCFE) 3 years following pelvic irradiation for rhabdomyosarcoma. The reasons for this occurrence are discussed. This is the second report of radiation-induced SCFE and it is anticipated that more such cases are likely to occur.


Gallium67 scanning compared with physical examination in the preoperative staging of malignant melanoma. Sixty patients with Stage II malignant melanoma were evaluated in order to compare preoperative gallium67 total body scan, physical examination findings, and ultimate histologic findings in regard to the site of the primary lesion and status of the regional lymph nodes. Physical examination was a more efficient and a more sensitive method of evaluation compared to gallium67 scanning in these patients when assessing nodal involvement.


Inhibition of spontaneous AKR leukemia by multiple inoculations of Corynebacterium parvum. In previous studies we have shown that AKR mice could be protected from spontaneous leukemia by specific immunotherapy in combination with splenectomy. In this experiment we investigated the effects of nonspecific immunotherapy with C. parvum in a similar regimen. It was found that bi-weekly, ip inoculations of 0.7 mg of C. parvum could significantly protect AKR mice from spontaneous tumors, and that splenectomy could not modify this effect. Thus, the spleen does not appear to play a determinant role in mediating the protective effects of C. parvum, as had been suggested in other systems.


Plasma IgA, IgG and IgM and their relationship to breast cancer in British, Japanese and Hawaiian-Japanese women. The plasma levels of immunoglobulins IgA, IgG and IgM have been measured in 35 British, 44 Hawaiian-Japanese and 37 Japanese healthy adult women. Previous investigations showed that the mean levels of all three immunoglobulins were higher in Japanese than in British normal women. The present study finds that Hawaiian-Japanese women have "Japanese" levels of IgA, "British" levels of IgM and are intermediate for IgG. Thus, plasma IgM concentrations correlate with breast cancer incidence rates in the three racial groups and the reduced amounts of plasma IgM found in Japanese patients with breast cancer support this association.


Intralesional immunotherapy of malignant melanoma with mycobacterium smegmatis cell wall skeleton combined with trehalose dimycolate (P3). The clinical efficacy of intralesional immunotherapy utilizing Mycobacterium smegmatis cell wall skeleton (CWS) and trehalose dimycolate attached to oil droplets was investigated in 15 patients with advanced malignant melanoma. Patients received 300 microgram to 1050 microgram of the CWS combined with one-half that amount of trehalose dimycolate every 1 to 2 weeks for a total of 8 treatments. Therapy was continued if regression of injected lesions only occurred. Therapy was discontinued if regression of noninjected disease also occurred. Six of the 15 patients had regression of at least one injected lesion. Four of these 6 patients also had regression of noninjected disease lasting 4+, 6, 16 and 18+ months. Response was highly related to immune status. Six (83%) of 7 patients who reacted to one of a battery of skin tests responded. All 8 patients who did not react to skin tests failed to respond to therapy. There was no correlation of response with sex, prior therapy, disease-free interval or presence of visceral disease. Mycobacterial CWS and trehalose dimycolate is an effective immunotherapeutic agent. Additional studies of purified immunoadjuvants are warranted.


Electron microscopy of Hodgkin's disease tissue cultures. Cells from 9 monolayer tissue cultures prepared from Hodgkin's disease tumors in the spleen were examined in the electron microscope. Three established culture lines (carried in vitro for greater than 3 years and passaged greater than 200 times) that contained aneuploid karyotypes were composed of oval cells with numerous interdigitating surface microvilli. The nuclei were complex and convoluted with multiple large nucleoli and dispersed chromatin. The cytoplasm contained lysosomes, microfilaments, a complex Golgi apparatus, nondilated rough endoplasmic reticulum, polyribosomes, fat, and glycogen. One Hodgkin's disease monolayer with aneuploid chromosomes examined from the 4th to 48th passage in culture was composed of larger cells with fewer microvilli and numerous multinuclear giant cells. Two monolayers derived from transplanted tumors in nude mice inoculated with Hodgkin's disease cultured cells were similar to the original cell lines. The ultrastructural features of these 6 cultures with aneuploid karyotypes differed from those of 3 monolayers which, although prepared from Hodgkin's disease splenic tumors, were composed of fibroblastic cells with diploid chromosomes. The aneuploid Hodgkin's disease cultures did not resemble 6 normal spleen, thymus, or lung monolayers, Raji lymphoblastoid suspension cultures, or Hela cells. Our electron microscopic studies indicate that adherent cells which replicate in some monolayer tissue cultures derived from Hodgkin's disease tumors are related to and possibly derived from neoplastic macrophages.


Necrolytic migratory erythema, presenting as candidiasis, due to a pancreatic glucagonoma. A 54-year-old male with diabetes, weight loss, glossitis and Candidiasis presented with the typical cutaneous eruption of necrolytic migratory erythema. The suspicion of pancreatic glucagonoma was confirmed by an elevated plasma glucagon level. Surgical removal of the pancreatic alpha cell tumor resulted in a complete disappearance of all symptoms. The importance of the recognition of the skin eruption of necrolytic migratory erythema as a clue to the presence of pancreatic glucagonoma is emphasized.


Significance of squamous components in endometrioid carcinoma of the ovary. Forty-nine cases of ovarian endometrioid carcinoma with squamous foci were reviewed. Of particular interest was the biologic behavior of the neoplasms in relation to the appearance of the epithelium. As previously noted for the analogous uterine endometrial tumors, the ovarian adenosquamous lesions occurred later in life, were more advanced, were associated with less differentiated adenocarcinomatous components, and had a poorer prognosis. The five-year survival rate for patients with adenosquamous endometrioid ovarian carcinoma was 21% in comparison with the 90% survival found in patients with ovarian endometrioid adenocanthoma.


Ultrastructure of a primary fibrosarcoma of the human thyroid gland. A primary fibrosarcoma of the thyroid occurring in a patient with a nodular goiter is described. Light microscopy showed interwoven bundles of spindle cells admixed with plump ovoid cells, and foci of multinucleated giant cells. Remnants of distorted thyroid follicles were found only at the periphery of the tumor. "Transitional" epithelial elements were not discernible but the tumor resembled an anaplastic giant cell carcinoma. Multiple blocks studied by electron microscopy, however, revealed that the tumor cells, including the giant cells, have the ultrastructure features of a fibroblast.


Clear-cell chondrosarcoma: a report of five cases including ultrastructural study. Five cases of clear-cell variant of chondrosarcoma (Unni et al.) are reported. The tumors occurred in the epiphyseal region of long bones; three in the femoral head. Roentgenographically, the lesion was usually a well-defined and benign appearing one, either purely lytic (3 cases) or with central radiodensity (2 cases). Histologically, all five cases had areas of conventional chondrosarcoma; however, the greater portion of the tumor was made up of sheets of clear-cells intermixed with nonneoplastic bone trabeculae but devoid of chondroid matrix. Electron microscopic studies showed that these clear-cells possess cytoplasmic microvilli, abundant glycogen particles and prominent golgi complexes, like normal or tumorous chondroid cells usually have. In our experience, the best treatment seemed to be en bloc resection with joint replacement; indeed, despite the fact that they are true chondrosarcomas, these tumors usually have a very slow rate of growth.


Biochemical studies of the matrix of craniovertebral chordoma and a metastasis. Glycosaminoglycans of human chordoma and a metastasis were studied. The glycosaminoglycans were derived from the extracellular matrix and consisted of chondroitin 4- and 6-sulphate, keratan sulphate and hyaluronate. The ratio of chondroitin sulphate to keratan sulphate was much lower in the metastasis than in the primary. Proteoglycan extracted with 4M guanidinium chloride and purified by associative density-gradient centrifugation was assessed on Sepharose 2B before and after reduction and alkylation. The result of this experiment suggested that only a small proportion of the proteoglycans were aggregated.


Family study of serum carcinoembryonic antigen in inherited medullary carcinoma of the thyroid. Serum carcinoembryonic antigen (CEA) and calcitonin were assayed in 8 patients with medullary carcinoma of the thyroid (MCT) and 14 unaffected family members, from 4 pedigrees of Sipple's syndrome and one pedigree with inherited MCT. Serum CEA ranged from 5.4 to 44.5 ng/ml in the patients, but less than 2.5 ng/ml in the unaffected. This is in contrast with retinoblastoma and cancer family syndrome, where not only the patients but also unaffected family members show high serum CEA, and cytoplasmic or selective maternal inheritance of serum CEA level is presumed. In inheritable MCT, serum CEA increases in association with the development of MCT, and serum CEA level per se is not inherited.


Relationship between proliferative activity and estrogen receptors in breast cancer. The proliferative activity of breast cancer has been analyzed in relation to the hormonal characteristics of the host and of the tumor for 199 patients. The analyses of labeling index frequency distributions of estrogen receptor positive (ER+) and negative (ER-) cancers from premenopausal and postmenopausal patients have allowed us to identify three different kinetic groups. A first group, with a very low proliferative activity, includes ER+ cancers from postmenopausal patients; a second group, with an intermediate proliferative activity, includes ER+ cancers from premenopausal and ER- cancers from postmenopausal patients; and a third group, with a very high proliferative activity, includes ER- cancers from premenopausal patients. Generally, the amount of estrogen receptors in ER+ cancers is inversely correlated with the proliferative activity. Lower levels of ER in premenopausal in comparison to postmenopausal patients were found in low proliferative activity tumors.


Prognostic value of estrogen receptors in primary breast cancer. The estrogen receptor status in 335 primary breast carcinomas was correlated with disease-free interval, survival and site of recurrent disease. Estrogen receptor positive carcinomas had a longer disease-free interval, a longer survival (mastectomy-death) and a longer time interval between recurrence and death. These parameters were also influenced by the lymph node status at mastectomy. Estrogen receptor positive cancers had a significantly better chance of survival independent of lymph node status. Estrogen receptors also delayed recurrence in node-positive carcinomas, but this advantage gradually disappeared with increasing interval after mastectomy. Estrogen receptor positive or estrogen receptor negative primary carcinomas did not show any predilection for spread to any particular site.


Calcitonin as a marker for bronchogenic cancer: a prospective study. A prospective study was done of serum calcitonin (HCT) levels in 61 patients with bronchogenic cancer. Initially, 52% of patients had hypercalcitonemia. Hypercalcitonemia was not confined to patients with any particular histologic type. Seventy-eight percent of those with high calcitonin remained normocalcemic. There was no correlation between high calcitonin levels and osseous metastases. Selective thyroid venous sampling delineated two types of hypercalcitonemia: thyroidal and ectopic. To date, the ectopic type has been associated with the small cell bronchogenic carcinoma. High initial calcitonin levels decreased significantly in 75% of patients on antitumor therapy. In 13 evaluable patients calcitonin levels mirrored clinical status changes 67% of the time. Calcitonin may be a useful marker to assess the results of therapy in patients with bronchogenic cancer.


Multicystic, peritoneal mesothelioma: a report with electron microscopy of a case mimicking intra-abdominal cystic hygroma (lymphangioma). A 27-year-old Caucasian female complained of persistent, poorly localized abdominal pain of several months' duration, found to be due to a multicystic diffuse lesion involving omentum, peritoneum and pelvic viscera. Three resections were required within a ten-month period for symptomatic control. The tumor showed many of the clinical, operative, gross and light microscopic features of intra-abdominal lymphagioma. Tissues removed at the third operation, however, were examined by electron microscopy, and found to consist of a myriad of small cysts and channels lined by mesothelial cells. In view of these findings, the lesion was regarded as a cystic peritoneal mesothelioma. Similarities between lymphangioma and peritoneal mesothelioma are discussed. It is suggested that electron microscopic examination of lining cells of cystic lesions which are considered grossly consistent with lymphagiomas may yield additional similar cases.


Giant cell tumor in Paget's disease of bone: familial and geographic clustering. Four patients with benign giant cell tumor and one patient with probable benign giant cell tumor associated with Paget's disease of bone are reported. The familial and geographic clustering of these cases is unique in that three patients were related and all patients traced ancestral roots to the same area of southern Italy. Tumors arose from the cranial or facial bones in three patients and from vertebral bodies in two patients. All caused symptoms by local compression, and treatment by curettage or radiotherapy was successful in all patients. Three separate tumors in one patient shrank dramatically in response to treatment with high doses of dexamethasone, and one patient whose tumor caused spinal cord compression showed marked improvement in neurologic function on therapy with dexamathasone.


Male breast cancer: a natural history study. Eighty-seven cases of male breast cancer seen over a 30-year period were reviewed. The overall observed and corrected five-year survival rates of 42% and 53% correspond well with the results in other series. Factors predicting disease-free interval and survival included size of primary lesion, ipsilateral axillary status, and presence of ulceration, which appear to be similar to those observed for female breast cancer. Eighteen patients (21%) had a second primary tumor (seven with cutaneous malignancies) and were of an older age group with a higher incidence of positive family history of breast cancer as well as other tumors. The overall prognosis of breast cancer in males does not appear to be less favorable than that in females.


Blindness as initial manifestation of meningeal carcinomatosis in breast cancer. The sudden onset of blindness in adults, with or without a history of malignancy should raise the possibility of meningeal carcinomatosis. The diagnosis is best confirmed with examination of the spinal fluid. The actual mechanism by which blindness occurs is probably a combination of tumor cuffing around the optic nerve, direct infiltration and chronic papilledema. We are reporting two patients with disseminated breast cancer who presented with blindness as the first manifestation of meningeal involvement. Treatment with whole brain radiation and intrathecal Cytosine Arabinoside produced temporary remissions, but both died a few months later of their cancer.


Prognostic factors in metastatic and hormonally unresponsive carcinoma of the prostate. Eighty-eight patients with hormone-resistant Stage IV prostate cancer were treated with a five-drug chemotherapy program. Patient demographic data, prior therapy, symptoms, extent of disease, and laboratory studies were analyzed statistically to evaluate the association of these parameters with survival from the onset of chemotherapy. Factors associated with short survival included age greater than 65, severe bone pain, poor performance status, presence of soft tissue metastases, anemia, elevation of serum LDH, SGOT, alkaline and acid phosphatases, and prolactin, and hypoalbuminemia. Race, stage at initial diagnosis, prior radiation therapy, prior orchiectomy, and elevation of CEA had no prognostic association. We suggest that clinical trials of new therapies of hormone-resistant prostate cancer take into account the presence of these prognostic factors in the analysis of the results of therapeutic programs.


The thorax as the initial site for systemic relapse in malignant melanoma: a prospective survey of 324 patients. Thirteen of 324 patients with malignant melanoma followed during a 24 month period experienced dissemination. The thorax was the initial site for relapse in 12, all of whom were asymptomatic. Ten gave no evidence of extrathoracic disease. Retrospective analysis of previous x-rays originally interpreted as negative revealed metastases in 33%. Life table analysis demonstrated a significantly longer survival for the subset with isolated intrathoracic metastases treated surgically than for their counterparts with metastases no longer amenable to surgery and treated by other modalities. We conclude that the thorax is the site of predilection for initial systemic relapse in malignant melanoma, that detection of early, surgically resectable metastases correlates with longer patient survival, and that routine chest roentgenography is inadequate in reliably uncovering such early disease. These data suggest the potential value of more vigorous radiographic surveillance (with either computed tomography or conventional full lung tomography) in patients at high risk for relapse.


Leukoerythroblastosis and cancer frequency, prognosis, and physiopathologic significance. This investigation was carried out on 100 bone marrow biopsies with metastases and 56 autopsies on patients with evidence of cancer. Leukoerythroblastosis was found in 44% of the patients with bone marrow mestastases and was more frequent in prostatic and gastric carcinoma. Moreover, the postmortem study of patients who died with cancer showed that leukoerythroblastosis was always the sign of bone marrow metastasis. A significant correlation was found between these blood changes and bone marrow fibrosis around the metastasis. Furthermore, leukoerythroblastosis seems caused by hepatosplenic extra medullary hematopoiesis.


Swallowing disorders in three types of head and neck surgical patients. This study examined swallowing transit times and motility problems in three groups of patients following ablative surgery for oropharyngeal carcinoma and in a control group of 10 normal subjects. A total of 30 patients was studied: 10 after anterior floor of mouth resection, 12 after tonsil/base of tongue resection, and 8 after supraglottic laryngectomy. Videofluoroscopic studies of liquid, thin paste, thick paste, and thick paste plus liquid swallows were completed 1 week post-initiation of oral feeding following surgery. From the videotapes, oral and pharyngeal transit times were measured, and motility disturbances were defined during each stage of the swallow. All three types of patients in this study showed severe problems with swallowing. The anterior floor of mouth resection patients had problems with preparation for the swallow and oral transit. Tonsil/base of tongue resection patients had slowing in the preparation for the swallow and in the oral and pharyngeal stages. After supraglottic laryngectomy, patients showed only slight slowing in oral transit and pharyngeal transit as compared to other types of surgical patients.


Asbestos, dental X-rays, tobacco, and alcohol in the epidemiology of laryngeal cancer. A case-control study of 47 laryngeal cancers in males of three counties of Washington State was conducted. Personal interview was used to obtain information on smoking, alcohol use, exposure to asbestos, and other substances, and x-rays of the head and neck area. Smoking and alcohol consumption were found to increase risk of laryngeal cancer independently, with a clear dose-response relationship. Neither asbestos exposure nor exposure to other substances was found to significantly increase the risk of laryngeal cancer, although the relative risk with asbestos exposure was 1.75. Lifetime history of exposure to dental x-rays on five or more occasions was associated with significantly increased risk of laryngeal cancer among heavy smokers but not among light smokers. The importance of tobacco and alcohol in the epidemiology of laryngeal cancer was re-affirmed, the importance of asbestos exposure was brought into question, and a possible relationship of laryngeal cancer with exposure to dental x-rays among heavy smokers was demonstrated.


Surgical repair of esophagobronchial fistula following successful treatment of Hodgkin's disease. A 33-year-old man with stage III-BS nodular sclerosing Hodgkin's disease received combination chemotherapy and subsequently developed a right middle lobe infiltrate in the lung unresponsive to antibiotic therapy. Thoracotomy revealed an esophagobronchial fistula with no evidence of malignancy. Surgical correction of the fistula was technically difficulty but proved lifesaving for this patient.


Evaluation of response criteria in advanced lung cancer. Because we found it illogical to attempt measurement of nonmeasurable but visible tumors in patients with advanced lung cancer, we devised and used a separate set of response criteria for patients with evaluable, nonmeasurable tumors. Patients with evaluable disease who had obtained a tumor regression after therapy were compared to patients with measurable disease who had obtained a tumor regression according to standard criteria. Among 191 cases evaluated (54% with measurable and 46% with evaluable disease), 59 tumor regressions were found (35 in patients with measurable disease and 24 in patients with evaluable disease). Evaluating regressions in patients with both types of disease, we could not detect statistical differences in regression rates, times to regression, durations of regression or survival. We believe the response criteria used for patients with evaluable, nonmeasurable lung cancer to be valid in assessing response to therapy.


Gastric remnant carcinoma.

Gastric remnant carcinoma. One hundred cases of gastric carcinoma at The New York Hospital-Cornell Medical Center included 7 cases of gastric remnant carcinomas, which were analyzed and compared with prior reports in the literature. Gastric remnant carcinoma is a distinct entity, noted in greatest incidence beginning approximately 15 years after surgery for benign gastric disease. Although the etiology is unknown, the high incidence of bile reflux and concomitant existence of histologic change may account for a premalignant environment in which the stomach assumes some absorptive function. Such new activity may result in the influx of potentially carcinogenic compounds and subsequent tumor development. Early diagnosis of remnant carcinoma following gastric surgery is important to improved prognosis, and endoscopic followup is recommended for all such patients beginning 10-15 years postoperatively.


Hemangioendothelial sarcoma of penis.

Hemangioendothelial sarcoma of penis. Hemangioendothelial sarcoma of the penis in a 44-year-old man was treated by preoperative radiation, penectomy, and chemotherapy. The patient was free of evident disease 2 years later. Electronmicroscopy showed differentiated vascular structures at the periphery of the lesion and anaplastic cells throughout the remainder of the tumor.


Ureteral obstruction secondary to metastatic breast carcinoma. An uncommon manifestation of breast cancer is ureteral obstruction secondary to metastatic disease. Five patients who recently developed this complication from two to 20 years after the diagnosis of breast cancer are described. Only two of the five patients had urinary symptoms. All of the patients were older, postmenopausal females who had bone metastases and all had responded to previous hormonal manipulation. Bone scanning was useful in detecting unsuspected hydronephrosis in two patients. Retroperitoneal disease appears to be a complication of long standing breast cancer which is usually hormonally dependent. Routine examination of the bone scan for renal asymmetry may aid in the diagnosis, especially in asymptomatic patients.


Adjuvant chemotherapy of breast cancer.

Adjuvant chemotherapy of breast cancer. One hundred women with primary breast cancer with 4 or more metastatic axillary nodes were treated for 9 months postoperatively with vincristine, prednisone, cyclophosphamide, methotrexate, and fluorouracil (VPCMF). Sixty-five women have been observed for a minimum of 5 years or until failure and the rest for 3 years or more. For 73 women who received adjuvant chemotherapy only, observed for 5 1/2 years median, disease-free status by life table analysis is 68% at 8 years. No significance difference was found between response of pre- and postmenopausal women in disease-free interval or survival. Mortality compared to expectation was sharply reduced; only 9 of 73 have died. These findings demonstrate the long term effectiveness of relatively short-term surgical adjuvant combination chemotherapy in pre- and post-menopausal patients with breast cancer at high risk.


Analysis of proliferative compartments in human tumors. I. Renal adenocarcinoma. Vascular perfusion of 16 renal adenocarcinomas with radioactive DNA precursors provides a possibility of characterize proliferative compartments of this tumor type. Immediately after resection of the tumor-bearing kidney, the organ is perfused via renal artery with dextran-diluted, heparinized oxygenated blood at physiological temperature, pH, flow, and pressure in a recirculation system. DNA synthetizing cells are labeled by addition of 3H- or 14C-thymidine or both isotopes at different intervals. Beta camera scans and whole-tumor autoradiograms disclose a striking proliferative heterogeneity of the tumor. Cell proliferation depends on intratumoral localization, cellular differentiation, histological structure and vascular supply. Subpopulations of high proliferative activity are found at the invasive borderline near normal kidney, focally in subcapsular areas and in intrarenal metastases, but also immediately adjacent to necrotic areas in the tumor center. Quantitative evaluation of autoradiograms yields, at the cellular level, a significantly higher labeling index in granular cells (3.21%) than in clear cells (0.65%), with a large variability dependent on the histological structure. The highest number of DNA synthetizing cells is seen in papillary and mixed solid-tubular zones and at peripheral parts of solid areas, whereas in central parts of solid tumor cords and in highly differentiated tubular areas lower labeling indices are observed. The labeling index decreases exponentially as a function of the distance from the supporting blood vessel. In solid cords, no labeled cells are seen at a distance of more than 200 micron from the capillary. The ts determined by 3H/14C-thymidine double labeling is between 9.9 and 16.8 hr for granular cells and about 9.2 hr for clear cells. Potential population doubling time calculated for various subpopulations yields values between 4 and 50 days. It is concluded that cell loss is high, for granular cells in particular. Besides cell loss, a large nonproliferating compartment contributes to a delay of the tumor volume doubling time. Proliferative heterogeneity of advanced human tumors, as exemplified by the renal adenocarcinoma, bears important implications for therapy and prognosis.