Prolactin and the development and progression of early neoplastic mammary gland lesions. Chronic suppression of prolactin by 2-bromo-alpha-ergocryptine (CB-154) in young nulliparous or mature multiparous C3H mice sharply suppressed the development of preneoplastic mammary gland lesions (hyperplastic alveolar nodules) and markedly inhibited the progression of these preneoplasias to carcinomas. This effect was also observed in C3H mice treated with either 17beta-estradiol or the oral contraceptive Enovid. Chronic CB-154 induced prolactin suppression was more effective than ovariectomy in the suppression of hyperplastic alveolar nodule development and comparable to ovariectomy in the suppression of mammary carcinoma development. Evidence is also provided indicating that human placental lactogen, a peptide chemically and physiologically similar to prolactin, promotes growth both in vitro (organ culture) and in vivo (athymic "nude" mouse) of the epithelium contained in benign human breast tumors. Whether or not human pituitary prolactin is capable of mimicking the mammotrophic action of human placental lactogen and whether a dysplastic, prolactin-sensitive lesion comparable to hyperplastic alveolar nodules exists in the human breast remains to be determined.


Effect of parity on recovery of inapparent nodule-transformed mammary gland cells in vivo. Inapparent nodule-transformed cells were recovered from five late-pregnant, first-pregnancy BALB/cfC3H females 4 months of age and from five late-pregnant multiparous females 6 to 7 months of age. Mammary tissues were removed from each donor and dissociated by means of the enzyme collagenase (0.1%), hyaluronidase (0.1%), and pronase (1.25%). Aliquots of 100,000 viable cells in 0.01 ml of media were injected into the gland-free mammary fat pads of 3-week-old syngeneic host mice. Ten weeks after the injection the outgrowths were classified as ductal, nodule, tumor, or combinations of these types of outgrowths. The recovery of nodule outgrowths indicated the presence of nodule-transformed cells in the cell suspension that was injected. All donors yielded nodule outgrowths, and the percentage of outgrowths was significantly greater than was the percentage recovered from virgin BALB/cfC3H females of the same age groups. The latent period for the emergence of nodules and tumors was reduced from 8 to 9 months in virgin females to 4 months in parous females. The incidence of both nodules and tumors was greatly increased. The data suggest that parity significantly increases the numbers of nodule-transformed cells in donor tissue, decreases the time required for the emergence of nodules and tumors, and increases the number of overt nodules and tumors.


Role of prostaglandins in the production of hypercalcemia by tumors. This report summarizes the data from two animal and cell culture systems that serve as models that show how certain malignant tumors produce hypercalcemia by means of a humoral mechanism. Studies with the HSDM1 murine fibrosarcoma and the VX2 carcinoma in the rabbit have led to the conclusion that these two tumors produce hypercalcemia in the host by means of a mechanism that utilizes prostaglandin E2 as the mediator between the neoplasm and bone. Analogous or identical mechanisms may operate in a small number of human tumors.


Structure of chick progesterone receptors. Oviduct progesterone receptors have been purified from both immature chicks and laying hens. The subunits, progestophilins A and B, have different molecular weights, but each is the same from either tissue source. Immunological data show that the subunits are cross-reactive. The hen B protein as isolated to homogeneity contains nonradioactive progesterone in stoichiometric amounts as shown by gas chromatography-mass spectrometry. These studies further confirm our earlier determinations on these proteins.


Receptor and centriole pathways of steroid action in normal and neoplastic cells. An experimental system designed for tracing the estradiol kinetics in target cells by specific antibodies has been applied to human breast cancer. Several major defects of the estradiol receptor mechanism have been demonstrated. The detected changes (lack of cytoplasmic receptors, impaired nuclear transfer of estradiol:receptor complexes, and abnormal nuclear retention of these complexes) have been demonstrated in most human breast cancers that appear to be composed of hormone-dependent and autonomous mixed-cell populations. These abnormalities could be the biological background for the overall or partial unresponsiveness of breast cancer to endocrine management. The participation of steroids in the regulation of centriole activities is taken into account since immunoreactive steroids are traceable by UV and electron microscopy at the level of this cell organelle by steroid antibodies. Moreover, the presence of steroids in the pericentriolar material correlates well with the modulating appearance and activity of the centriole throughout the cell cycle. A new centriole pathway is suggested by which steroid hormones can regulate cell proliferation.


Comparison between different forms of estrogen cytosol receptor and the nuclear receptor extracted by micrococcal nuclease. As an approach to the mechanism of the nuclear translocation of estrogen receptor, the estradiol nuclear receptor (RN) of lamb endometrium was extracted with micrococcal nuclease at 2--4 degrees and compared to the "native" 8S and to the Ca2+-transformed cytosol receptors. After extensive digestion of chromatin, giving up to 10% perchloric acid-soluble DNA and a majority of nucleosome monomers, up to 80% of the RN was extracted and under low ionic strength. This RN was found to be completely different from the partially proteolyzed Ca2+-transformed cytosol receptor. It migrated with a sedimentation constant of 4 and 6 S. The Stokes radius of the predominant form as determined by ACA 34 chromatography was 5.3 nm. The calculated apparent molecular weights were 130,000 and 90,000, respectively. The RN was able to bind DNA and was eluted from a diethylaminoethyl cellulose column at 0.23 and 0.30 M KCl. We conclude that the mechanism proposed by Puca et al., according to which the Ca2+-transformed cytosol receptor is split by a Ca2+ receptor-transforming factor into a smaller form able to cross the nuclear membrane, is very unlikely.


Effects of ethidium bromide on the extractability of nuclear receptor-estrogen complex. In previous reports we challenged the concept that uterine nuclei of rats contain two forms of estrogen receptors, one salt extractable and the other salt resistant. Although it is likely that a certain fraction of the nuclear bound receptor-estrogen complex exists as a ternary high-affinity acceptor-receptor-estrogen complex, current salt extraction procedures do not allow discrimination between receptor-17beta-estradiol complexes associated with high-affinity and low-affinity nuclear binding sites. Recent reports suggested that the DNA-intercalating agent ethidium bromide selectively extracted those sites that appeared to be salt resistant. In view of contradictory reports to this effect, we have attempted to clarify this issue. The data presented indicate that ethidium bromide is not a useful tool for the identification of a specific class ("salt-resistant") of nuclear binding sites for receptor-17beta-estradiol complexes. This conclusion is based on measurement of nuclear bound receptor-17-beta-estradiol following KCl and/or ethidium bromide extraction by using both the direct assay and the nuclear exchange assay.


Restoration by adrenalectomy of weight and binding capacity of glucorcorticoid receptors of the thymus in AH 130 tumor-bearing rats. The binding capacity of specific receptors for dexamethasone (11beta,17alpha,21-trihydroxy-9alpha-fluoro-16alpha-methyl-1,4-pregnadiene-3,20-dione) in the cytoplasm of the thymus of rats bearing AH 130 (solid-type) tumors decreased significantly during tumor growth. Adrenalectomy restored the binding capacity of the glucocorticoid receptors and the weight of the thymus to nearly the normal values in rats bearing tumors. The affinity of these receptors for dexamethasone was very low in rats bearing tumors, but it was also restored to nearly the normal value by adrenalectomy.


Specific binding of estrogens in different fetal tissues of guinea pig during fetal development. Cytosol and nuclear specific estradiol binding was evaluated in the fetal uterus, kidney, lung, and brain of guinea pig during fetal development and after birth. The quantity of specific binding sites increases during fetal development and decreases after birth, particularly that of the nuclei. An exception is the binding in the cytosol fraction of lung in which the number of available binding sites continues to increase in newborn animals. In the fetal uterus the amount of specific binding of estradiol and estrone is similar. The selective uptake of radioactivity and its localization by autoradiography in cell nuclei are in accordance with the high levels of estrogen receptors, particularly at the end of gestation.


Glucorticoid resistance in murine lymphoma and thymoma lines. The thymoma line W7 contains 30,000 dexamethasone-binding sites per cell and gives rise to glucocorticoid-resistant variants at a frequency less than 1.6 X 10(-9); the lymphoma S49 contains one-half that amount of receptor, is resistant to low concentrations of dexamethasone, and gives rise to fully resistant variants at high frequency. These results suggest that S49 is functionally haploid (r+/-) for a gene coding for the receptor while W7 appears diploid for that locus (r+/+). Derivatives of the W7 (r+/+) line, selected for resistance to low concentrations of dexamethasone, have the same properties as S49. These putative W7's (r+/-) give rise to the same types of receptor variants as S49 and in the same proportion; 80 to 90% are "receptorless" (r-) while 10 to 20% are "nuclear transfer defective" (nt-). A total of 127 variants resistant to high concentrations of dexamethasone were derived from the W7 (r+/+) line after mutagenesis. All are receptor variants but N-methyl-N'-nitro-N-nitrosoguanidine and ethyl methanesulfonate induce only 60 to 70% r- variants, confirming the presence of two r+ alleles in the parental line. Ultraviolet light induces a higher proportion (87%) of r+ variants, as expected from the introduction of breaks and deletions in DNA.


Steroid receptors in human breast cancer. The measurement of cytoplasmic estrogen receptor in tumors from patients with breast cancer is now well established. Potential uses include prognosis of early recurrence following mastectomy, stratifying patients for adjuvant therapies, and selecting or rejecting endocrine therapy in advanced breast cancer. The use of progesterone receptor measurements to improve our selection process has a good theoretical basis, and early reports now emerging indicate that the presence of both estrogen and progesterone receptor in a breast tumor predicts a high response rate to endocrine therapy. Further work in this area is required, however, since patients with estrogen receptor but not progesterone receptor still have an appreciable response rate.


Estrogen receptor assay in primary breast cancer and early recurrence of the disease. Estrogen receptor assays of primary breast tumors have been related to early recurrence of the disease. A significantly longer disease-free interval was found in women whose primary tumor was estrogen receptor positive. Although there was no relationship of receptor content to stage of disease at mastectomy, the greatest difference between recurrence rates was found when the tumor had spread to the lymph nodes, especially to those in the apex of the axilla or in the internal mammary chain. Presence of estrogen receptor is closely related to histologically well-differentiated tumors, but it was found that poorly differentiated estrogen receptor-negative tumors recurred earlier than poorly differentiated receptor-positive tumors and had a very unfavorable prognosis.


Prognostic value of estrogen receptor determinations in patients with breast cancer. The estrogen receptor content of human breast cancer specimens is related to the degree of differentiation (grade) of the tumor. In addition, patients with estrogen receptor-positive tumors experience fewer recurrences and remain disease free for a longer priod of time than do patients with receptor-negative tumors. The presence of estrogen receptor is not correlated with lymph node infiltration.


Some comments on the necessity of receptor determination in human breast cancer. Estrogen receptors (ER's) were determined by agar gel electrophoresis in 105 human mammary tumor biopsies. Fifty-three tumor specimens revealed measurable amounts of receptor, and 64% of these patients showed an objective remission after endocrine treatment. If we also include cases with partial remission, previous response, and arrested growth but no actual remission, the benefit rate increases to 87%. The corresponding percentage in the ER-negative groups is 13%. In a retrospective study of 42 patients with human breast cancer, a correlation between ER finding and remission rate after chemotherapy was found. Of the ER-negative patients, 71% showed an objective remission after polychemotherapy, whereas only 43% of the ER-positive cases revealed such remission. The presented data are discussed with special regard to their clinical relevance.


Relation between steroid receptor content and the response to hormone addition in isolated human breast cancer cells in short-term culture. The hormone dependence of human breast tumors has been investigated in two ways: (a) estradiol and progesterone receptor determination in tumor tissue; and (b) measurement of the growth-stimulating effect of hormones on cells in short-term culture. Preliminary results from 15 tumors suggest that such a model may be used for human breast tumors. The behavior of the cultures under hormonal stimulation depended on their receptor content. In this series of patients, the tumors containing both estradiol and progesterone receptors were stimulated by addition of insulin and estradiol while the tumors containing either estradiol receptor alone or no steroid receptor were not modified by hormonal addition.


Correlation between clinical response to hormone therapy and steroid receptor content in prostatic cancer. Hormonal therapy is the dominating form of treatment for prostatic carcinoma. The majority of cases (80%) are well controlled for varying times with this regimen. However, thus far there have been no adequate methods to predict in which cases hormonal therapy is of less benefit. Measurement of cancer tissue content of intracellular hormone receptors constitutes progress toward a more individualized therapy in prostatic carcinoma. In this study biopsies from 16 cancer patients were taken before therapy was given, and the specimens were analyzed with regard to content of specific methyltrienolone-binding sites. A correlation has been made between receptor content and clinical response to hormonal therapy in each case. Twelve specimens contained measurable amounts of steroid receptors. Of these, one patient died during irradiation therapy before onset of hormonal treatment. However, of the remaining 11 patients, 9 responded well to hormones (9/11 approximately 82%). The two receptor-positive nonresponders had the lowest measurable receptor levels in the series. Four specimens contained no detectable amounts of receptors. Three of these patients showed no response to therapy (3/4 = 75%) but one was "false negative." Our data indicate that steroid receptor analysis may become a valuable diagnostic tool in individualizing the therapy for prostatic cancer.


Animal models of the hormone-sensitive and -insensitive prostatic adenocarcinomas, Dunning R-3327-H, R-3327-HI, and R-3327-AT. The Dunning R-3327-H is a well-differentiated transplantable rat prostatic adenocarcinoma that contains both hormone-sensitive and -insensitive cells. The component composed of hormone-insensitive cells has been permitted to grow in a castrated male, and a new slow-growing, well-differentiated hormone-insensitive subline of the tumor has been established and designated R-3327-HI. In addition, a rapidly growing hormone-insensitive anaplastic tumor has been developed, R-3327-AT. These three tumor lines have been characterized, and their histological and biochemical profiles are compared.


Chemical modification of neamine.

Chemical modification of neamine. The aminocyclitol antibiotic neamine has been modified by changing the configuration of one or two hydroxyl groups of the aminocyclitol moiety to elucidate the relationship between configuration and antimicrobial activity. 5-Epi-, 6-epi-, and 5,6-diepineamine have been prepared and their antimicrobial activity has been determined against several micro-organisms.


Isolation and characterization of glycoconjugates from B16 mouse melanoma tumors. The major glycosaminoglycans isolated from B16 mouse melanoma tumors after Pronase digestion were shown to be a family of chondroitin 4-sulfates with different degrees of sulfation and a wide molecular-weight range. Ultracentrifugation data gave molecular weight values as high as 88 000, in contrast to that of costal cartilage chondroitin 4-sulfate which is about 14 000. A mucin-type sialoglycopeptide, isolated from the tumors by cetylpyridinium chloride precipitation of the Pronase digest, was shown to contain O-glycosylically linked tetra- and tri-saccharides consisting of sialic acid, galactose, and N-acetylgalactosamine. The sialoglycoprotein, which on Pronase digestion gave rise to the glycopeptide, was isolated from the tumor by extraction with lithium diiodosalicylate and affinity chromatography on a wheat-germ agglutinin-Sepharose 4B column. It was homogeneous on the basis of gel filtration on Sepharose 4B and Sephadex G-200 columns, lectin affinity, and ion-exchange chromatography. The compounds isolated from the B16 mouse melanoma tumors are similar to those produced by the cultured melanoma cells, which suggests that the latter compounds are not artifacts of the culture system.


beta-D-xylosidase from Bacillus pumilus PRL B12: hydrolysis of aryl beta-D-xylopyranosides. The influence of substituents on the binding and hydrolysis of several substituted beta-D-xylopyranosides by beta-D-xylosidase from Bacillus pumilus PRL B12 has been investigated. From a comparison of the inhibition constants of 1-thio-beta-D-xylopyranosides with the apparent Michaelis-Menten constants of the substrates, it followed that the latter constants are good approximations of the true equilibrium constants. The influence of the substituent on the rate and activation parameters is small. The results are in agreement with, but do not prove, a one-step mechanism without the formation of a glycosyl-enzyme intermediate.


Immunochemical studies on dextran-specific and levan-specific myeloma proteins from NZB mice. Two dextran-specific (PC 3858 and PC 3936) and one levan-specific (PC 3660) NZB myeloma proteins were studied by quantitative precipitin and precipitin-inhibition assays. Both myeloma antidextrans were alphaD-(1 leads to 6) specific and precipitated strongly with a synthetic, linear dextran, molecular weight 35,500, and with other dextrans. The two myeloma antidextrans differed with respect to their relative reactivities with dextrans containing various proportions of alpha-D-(1 leads to 6), alpha-D-(1 leads to 4)-like, and alpha-D-(1 leads to 3)-like linkages. In inhibition assays, the two antidextran myeloma proteins behaved differently from each other, from alpha-D-(1 leads to 6)-specific BALB/c myeloma antidextrans, and from the human antidextrans previously studied. Isomalto-oligosaccharides IM3, IM4, and IM5 were all equal in inhibitory power but were only about 60% as potent as IM6 and IM7, which also inhibited equally on a molar basis. Although precipitation with linear dextran suggests that both may have groove-type sites, as previously inferred for QUPC 52, the size of their combining sites is uncertain. It is not clear whether the sites are only as big as three glucose residues with the increased inhibition by six and seven glucose residues being attributable to partial bivalence and to their ability to combine in several ways along the chain, or whether the site is as big as six glucose residues with the increment in binding by the fourth and fifth glucose residues being minimal and the sixth contributing considerable additional binding-energy. The fructan-specific myeloma protein did not react with inulin, but reacted with many levans and with perennial rye-grass levan containing only beta-D-(2 leads to 6) links. The levan-antilevan reaction was not inhibited by beta-D-(2 leads to 1)- linked oligosaccharides. The findings suggest that PC 3660 has a specificity for (2 leads to 6)-linked chains.


The chemistry and immunochemistry of carrageenans from Eucheuma and related algal species. Carrageenans from several species of Eucheuma have been fractionated into KC1-soluble and KC1-insoluble fractions and analyzed by the usual chemical procedures. An anti-kappa-carrageenan, the reactivity of which is directed to kappa-structures (i.e., 3-linked galactose 4-sulphate, and 4-linked 3,6-anhydrogalactose) was used to analyze these carrageenans immunochemically. The antibody preparation shows only a small amount of cross-reactivity with iota-type carrageenans and thus could be used to distinguish kappa- and iota-type carrageenans, the latter having an index of homology of less than 0.2. A comparison of chemical and immunochemical data yielded further information as to the nature of the carrageenan-anti-carrageenan interaction, as well as elucidating the finer structure of carrageenans.


Prolapse of the mitral valve: clinical, hemodynamic, angiographic and echocardiographic correlations. Among 1,519 patients undergoing diagnostic cardiac catheterization over a 2-year period, angiographically unequivocal mitral valve prolapse was identified in 79 cases (5.2%). Mitral valve prolapse was clinically unsuspected in 30 of these 79 patients. Echocardiographic studies were available in 44 cases, but only 55% of the patients with prolapse of the mitral valve documented by angiography had positive echocardiographic findings. One third of the patients had associated coronary artery disease, aortic valve disease or atrial septal defects. 14 patients (18%) had moderate or severe mitral regurgitation. There was a wide spectrum of hemodynamic and ventriculographic abnormalities that could not entirely be explained by the degree of prolapse, by the severity of mitral regurgitation or by the presence of associated diseases.


A method for demonstrating the efficacy of interventions designed to limit infarct size following coronary occlusion: beneficial effect of hyaluronidase. In order to measure the protective effect of interventions following coronary artery occlusions in dogs, the creatine kinase activity of myocardial tissue was assayed after 24 h and related to the myocardial blood flow of that tissue measured with 85Sr labelled microspheres injected 15 min after occlusion. This assay showed normal levels when flow exceeded 50 cm3.min-1.100 g-1. In myocardium with flow reduced to 0 to 15 cm3. min-1.100g-1, creatine kinase activity was 7.6 +/- 0.6 IU.mg-1 protein in control dogs and 13.1 +/- 1.8 IU.mg-1 protein (P less than 0.01) in dogs given 500 NF units.kg-1 of hyaluronidase 20 min after occlusion. Where myocardial blood flow was reduced to 16 to 50 cm3. min-1. 100g-1, creatine kinase activity was increased from 14.1 +/- 1.1 to 20.5 +/- 1.4 IU.mg-1 protein by hyaluronidase. This method therefore assesses ischaemic damage independent of electrophysiological measurements and confirms myocardial preservation by hyaluronidase.


Effects of oxyfedrin: a beta-adrenoreceptor stimulant, on infarct size following acute coronary artery ligation. Regional left ventricular blood flow and the extent of myocardial ischaemia were studied after acute coronary artery occlusion in open-chest dogs before and after infusion of oxyfedrin, a beta-adrenergic stimulant. Regional blood flow was measured with radioactive tracer microspheres and local tissue injury was estimated by the S-T segment elevation in epicardial electrocardiograms. Animals receiving oxyfedrin were divided into two groups: 1 and 2. Oxyfedrin was infused intravenously in a dose of 0.80 to 0.94 mg.kg-1 in dogs of group 1 and 1.45 to 1.60 mg.kg-1 in dogs of group 2. The rate of infusion in the animals of both groups was 0.61 mg.min-1. Oxyfedrin caused further S-T segment elevation over ischaemic myocardium and increased the extent of ischaemic injury in group 1 dogs. Conversely, in this same group of dogs, the blood flow was unchanged in low flow regions ( less than 0.3 cm3.g-1.min-1) and increased in higher flow areas, inside the ischaemic region. In the animals of group 2, oxyfedrin caused further S-T segment elevation over ischaemic myocardium and increased the extent of ischaemic injury. Concomitantly, blood flow was significantly reduced both inside and outside the ischaemic region. These observations in dogs of group 1 (ie increased blood flow inside the ischaemic region by infusion of oxyfedrin, in flow zones higher than 0.3 cm3.g-1.min-1, with a further S-T segment elevation over ischaemic myocardium, and an increase in the extent of ischaemic injury) may be explained by a primary effect of oxyfedrin on oxygen demands with secondary changes in blood flow.


Haemodynamic effects on the myocardial blood flow supply/oxygen demand ratio in pacing induced angina pectoris. Angina pectoris results from an imbalance between oxygen supply and demand in the subendocardium. The haemodynamic effects contributing to this imbalance have been studied in 10 patients with coronary artery disease. Myocardial oxygen demand was estimated from the tension time index (TTI), potential subendocardial flow from a diastolic pressure time index (DPTI), and the oxygen supply/demand ratio from (DPTI/TTI. With progressively increasing pacing rates up until induction of angina, no significant change in TTI was found whereas a significant fall in DPTI and DPTI/TTI occurred (P less than 0.001). During pacing runs with induction of angina DPTI/TTI reached a minimum value 5 s before,and at the onset of angina. No such relationship was seen for TTI or DPTI alone. A significant rise in LVEDP (P less than 0.05) and fall in dP/dt min (P less than 0.01) occurred at angina both contributing to a further reduction in DPTI and DPTI/TTI. Changes in DPTI/TTI may then reflect changes in the myocardial blood flow supply/oxygen demand ratio in the presence of coronary artery disease and haemodynamic changes before and at the induction of angina lead to a further reduction of this ratio.


Static mechanical properties and chemical composition of the aorta of spontaneously hypertensive rats: a comparison with the effects of induced hypertension. The static elastic properties and medial scleroprotein content of the aorta have been examined in spontaneously hypertensive rats (AS strain) aged 6 and 20 weeks (group SH). The results are compared with data from two previous studies on normal (group N) and induced hypertensive animals (group H). Spontaneous hypertension is associated with a relative increase in elastin and decrease in collagen when compared with the normal aorta. These changes are similar to, although smaller in magnitude than, those associated with induced hypertension. Elasticity measurements show that the functional stiffness (incremental strain) of the aorta in group SH is greater than normal. However, this difference diminishes with age, suggesting an adaptive response which tends to maintain the functional stiffness near to normal levels.


Significance of epicardial Q waves as an acute marker of myocardial necrosis in dogs. This study examines the significance of epicardial Q waves as a marker of myocardial cell necrosis. Ischaemia was produced in dogs by two methods: coronary artery occlusion sustained for 24 h (Group 1) and occlusion for 1 h followed by reperfusion (Group 2). Q waves did not appear until after 3 h of sustained occlusion, but were present within 40 min of reperfusion. In both groups, Q waves were not transient but persisted for at least 24 h. CPK levels were determined at 24 h in specimens from each lead site. In Group 1, Q sites had 66.6 +/- 5.9% (mean +/- SEM) less CPK than R wave sites (P less than 0.005). In Group 2, Q sites had only 28.2 +/- 4.5% less CPK than R sites. These results suggest that the extent of necrosis was greater at Q sites with sustained occlusion than with reperfusion. A similar relationship existed for the levels of ATP and CP determined at Q and R sites at 24 h. Histological examination by light and electron microscopy confirmed that in both groups, Q sites corresponded to areas of necrosis, while R sites indicated normal myocardium. However, the type of necrosis depended on the pathogenesis. Our results demonstrated that epicardial Q waves were a reliable marker of cell death, but that the morphological picture and extent of cell death depended on the mechanism and manner of injury. These conclusions were tested in a final series (Group 3) in which propranolol was given before and with release of the occlusion (0.5 mg.kg-1 at each time). In 47 sites at risk, in five dogs only two Q waves appeared. In each of these two, cell death was confirmed by evidence of CPK depletion and morphological alteration. In the remaining sites, no CPK depletion occurred. Histological examination revealed only infrequent small islands of subendocardial necrosis. The results confirm the validity of the epicardial electrocardiographic findings and illustrate the role of propranolol in preventing reperfusion necrosis.


Use of chronically-implanted ultrasonic crystals in the intact heart of the cat. Methodology has been developed for utilising ultrasonic crystals in closed chest cats to measure alterations in regional left ventricular function. The technique is a modification of one previously utilised to monitor regional left ventricular length and thickness changes in closed chest dogs. The ultrasonic crystals we have utilised in the cat heart can monitor regional left ventricular epicardial length changes chronically and are able to identify changes in inotropic state produced by the administration of either isoprenaline or propranolol. These ultrasonic crystals should be useful in the acute and chronic monitoring of left ventricular function in closed chest and relatively small experimental animals that might be studied in a variety of different protocols.


Alterations in brain serotonin during congestive heart failure in the cardiomyopathic Syrian hamster. Serotonin and its major metabolite 5-hydroxyindoleacetic acid were measured in the pons-medulla, midbrain, posterior hypothalamus, anterior hypothalamus, thalamus, and cerebellum of cardiomyopathic hamsters and their age-matched controls during the early and terminal stages of the cardiomyopathy. During the stage of cardiac decompensation, significant increases were observed in the concentration of serotonin in pons-medulla (2.30 +/- 0.07 vs 1.74 +/- 0.04; P less than 0.001) and posterior hypothalamus (3.49 +/- 0.04 vs. 3.04 +/- 0.11; P less than 0.005) and in 5-hydroxyindoleacetic acid in pons-medulla (1.42 +/- 0.05 vs 0.97 +/- 0.05; P less than 0.001) and posterior hypothalamus (1.65 +/- 0.02 vs 1.32 +/- 0.07; P less than 0.001). No changes were noted in any of the brain regions of control animals. We conclude that congestive heart failure may be associated with alterations in activity of bulbohypothalamic serotonergic nerves.


Early development of noradrenaline-containing sympathetic nerves in the choroid plexus system of the rabbit. The development of the adrenergic sympathetic innervation of the rabbit choroid plexus system was studied prenatally and up to two months after birth by a combination of fluorescence histochemistry (formaldehyde and glyoxylic acid methods) and quantitative enzymatic determinations of noradrenaline. The first signs of adrenergic nerves are found in the plexus of the third ventricle within the first day after birth. Fluorescent fibres subsequently appear in the choroid plexuses of the lateral ventricles (five days post partum) and the fourth ventricle (two weeks post partum). During the following development nerve fibres grow along blood vessels to form a plexus located between small vessels and the overlying epithelium. The nerve plexus, with varicose axon terminals, is fully developed at three weeks post partum, and maturation is then established by an increase in the number of terminals within the network of axons. There is a good agreement between (a) the development of the fluorescent nerves and histochemically visible adrenergic innervation, and (b) the tissue level of noradrenaline in the various choroid plexuses. Against the background of available information on the development of the secretory functions in choroid plexus, it is concluded that possibilities for a sympathetic neurogenic influence on the formation of cerebrospinal fluid exist already a few weeks after birth.


The T-tubule system in the myocardia of the sand rat and mouse as demonstrated by horseradish peroxidase. The transverse tubule (T-tubule) system in papillary muscles of the sand rat and the mouse were studied with the aid of a diffusion tracer (horseradish peroxidase). The T-tubule system in the sand rat showed a typical mammalian pattern with sarcolemmal tubules invaginating at the Z-band level of the sarcomere. These tubules follow a transverse direction in the cell with frequent longitudinal side-branches which connect tubules at different Z-band levels. In the mouse myocardium, the T-tubules also start as lateral invaginations from the sarcolemma at the Z-band levels. In the cell interior, however, the tubules ramify and brake up into a complicated system of spirally running tubules. These spirals, of relative small diameter (400A--700A), frequently expand and form lobulated cisternae at the Z-band levels.


Types of endocrine cells in the human colon and rectum. At least four types of endocrine-like cells have been detected histochemically in the mucosa of the human colon and rectum, i.e. argentaffin cells storing 5-hydroxytryptamine (5HT) and non-argentaffin cells reacting with glucagon, somatostatin and bovine pancreatic peptide (BPP) antibodies. Ultrastructurally, four main types and three rare types of endocrine-like cells have been identified. Among the former cells were: (1) argentaffin EC1 cells, known to store 5HT and substance P, (2) poorly argyrophil L cells, corresponding to the glucagon-immunoreactive cells storing enteroglucagon or glucagon-like immunoreactivity (GLl), (3) inconstantly argyrophil F-like cells, possibly corresponding to BPP-immunoreactive cells, and (4) fairly argyrophil H cells of unknown function. Rare D cells, corresponding to somatostatin cells, N cells, corresponding to neurotensin cells, and P cells, of unknown function, have been also found.


Sequential differentiation of intestinal endocrine cells in the fetal mouse. The duodenum and ileum of 15 to 18-day old Swiss ICR mouse embryos were studied with the electron microscope to follow the differentiation of endocrine cells. Endocrine cells were classified on purely morphological characteristics. EC cells appeared at 16 days of gestation in both segments. At the same stage G cells were seen in the duodenum and K cells in the ileum. ECL, and S cells were identified in the duodenum at 17 days. Finally, D cells were seen at 18 days of gestation in the duodenum and ileum. With the electron microscope, endocrine cells were not identified in the small intestine of the mouse before the formation of villi.


Catharanthine: a novel stimulator of pancreatic enzyme release. The plant alkaloid, catharanthine, was shown to stimulate release of amylase from pancreatic fragments and to cause extensive degranulation of pancreatic acinar cells with accumulation of membrane material in the Golgi region. The extent and time course of maximal catharanthine stimulation was comparable to that induced by the cholinergic analog bethanechol. Antimycin inhibited the action of catharanthine while atropine did not. Removal of Ca2+ from the incubation medium inhibited amylase release induced by catharanthine but did not affect release induced by bethanechol. Catharanthine induced a delayed release of 45Ca2+ from prelabeled pancreatic fragments as compared to bethanechol. It is suggested therefore that catharanthine activates the physiological pathway controlling amylase release by causing a rise in cytoplasmic Ca2+ but the mechanism by which this occurs is different from that caused by physiological secretagogues.


Influence of autonomic denervation upon the kinetics of the ileal epithelium of the rat. The effect of autonomic denervation upon the kinetics of the ileal epithelium of rats was studied by tracing 3H-thymidine labelled nuclei and counting mitotic figures and goblet cells. Counts of labelled nuclei and goblet cells provided information about cell migration along the intestinal epithelium. The mitotic index and turnover time of this population were calculated from the mitotic figures counts. Comparing denervated animals with sham operated controls, it was possible to conclude that autonomic denervation, either sympathectomy or parasympathectomy results in a decrease in mitotic activity. As a result of lower mitotic activity in the crypts, the turnover time increases and there is a delay in the migration of cells from the crypts towards the villi. Sympathectomy causes an early short-lived effect upon the kinetics of the crypt cell population, since after 27h there is a tendency to normality. Parasympathectomy produces a slower but continuous decline in mitotic activity.


An autoradiographic study on the biosynthesis of the capillary basal lamina in the chick embryo telencephalon. The elaboration of the basal lamina of telencephalic capillaries has been studied by autoradiography in chick embryos from day 16 to 19 of incubation. The cerebral vascular basal lamina is actively synthesized during the period of these stages of development. The animals were sacrificed 2, 15, 40 and 180 min after intravenous injection of 3H-proline. The hydroxylation of proline to hydroxyproline in the "secretory" cells permits their identification. Our observations suggest that endothelial cells, pericytes and perhaps glial cells participate in this elaboration. The various stages of the synthesis are performed sequentially in the ergastoplasm and then in the Golgi apparatus before the secretory material is released by exocytosis.


Contractile vacuoles in cells of a fresh water sponge, Spongilla lacustris. Forty or more independently functioning contractile vacuoles (CVs) occupy the central region of fresh water sponge pinacocytes. Eash CV undergoes a cycle of enlargement by fusion, movement, shape change, rounding up, and emptying over the course of 5--30 min. Diameter at discharge varies between 1 and 13 micron. CVs in all cell types are associated with submicroscopic coated vesicles. Filled CVs are bounded by an unmodified trilaminar membrane, but vacuoles with excess membrane frequently show coated evaginations. These evaginations are thought to pinch off as coated vesicles, providing an avenue for membrane recycling in the CV system.


An ultrastructural study of periderm granules in the regenerating feather of the jungle fowl. Periderm granules in the support cells of regenerating feathers of mature male Jungle Fowls were studied ultrastructurally and histochemically. Histochemical results showed the absence of carbohydrate and lipid, and the presence of protein in the periderm granules. The periderm granules were measured at successive levels of feather regeneration. The mean size of the periderm granules increased significantly as the regenerating feather matured, and this observation was suggestive of a storage function, perhaps of surplus of waste protein. The cells in which the periderm granules are found also contain glycogen. There are numerous desmosomal junctions on their interdigitating plasma membranes. These transient cells may collect waste, provide nutrition, and serve as a protective barrier for the definitive cells of the regenerating feather.


A further type of transient cytoplasmic organelle in human spermatids. Osmiophilic granules with surrounding vesicles resembling flower-like structures occur transiently during the differentiation of human spermatids. These organelles are incorporated into the residual bodies when mature spermatids are released from the germinal epithelium.


Evidence for the zonation of interrenal tissue in the adrenal gland of the duck (Anas platyrhynchos). Although interrenal tissue from the intact duck does not show a clear zonation when examined by light microscopy, the tissue does develop a well defined zonation following exposure to high and low levels of corticotropic stimulation. Under these conditions clear ultrastructural differences are seen between cells of the subcapsular  zone (SCZ) and the inner zone (IZ). Based on these observations, the ultrastructure of tissue from intact birds was examined retrospectively and in addition, cell sizes and the relative volumes and areas of intracellular components were measured morphometrically. These analyses reveal morphological and quantitative differences between cells from the IZ and the SCZ. Cells of the IZ have small rounded nuclei, extensive smooth endoplasmic reticulum (SER) and mitochondria with tubular cristae, whereas cells from the SCZ contain pleomorphic nuclei, less SER and mitochondria with shelf-like cristae. The mean cell volume in the IZ is significantly less than that in the SCZ. In the SCZ cells the volume densities (volume per unit volume cytoplasm) of mitochondria and lipid droplets and the surface densities (area per unit volume cytoplasm) of the outer mitochondrial membranes are significantly greater than those in IZ cells. Conversely, in the cells of the IZ the volume densities of the nuclei and dense bodies and the surface density of the SER are greater than the corresponding values estimated for the cells of the SCZ. Although the mitochondria comprise a smaller fraction of the mean volume of IZ cells than SCZ cells, the total surface area of the cristae is approximately the same in the cells of both zones.


Light and electron microscopy of the ducts and their subepithelial tissue in the rat ventral prostate. The ducts of the rat ventral prostate have been studied by light and electron microscopy for elucidation of their role in prostatic function. The epithelium of the main duct consists of simple columnar cells and polymorphic basal cells. The columnar cells show no indication of secretory activity. The basal cells contain bundles of filaments of 5--6 nm thickness and numerous pinocytotic vesicles. The ducts are surrounded by layers of circular smooth muscle cells interspersed with nerve axons. On ultrastructural grounds the ducts do not appear to secrete material into the seminal fluid, but apparently the muscular coat actively helps drain the gland during ejaculation.


Studies on the ultrastructure and permeability of the hemotrichorial placenta. I. Intercellular junctions of layer I and tracer administration into the maternal compartment. The distribution of lanthanum chloride and horseradish peroxidase within the full-term chorioallantoic placenta of the rat was investigated 1 to 20 min after administration of these tracers into the maternal blood circulation. Both tracers rapidly penetrate trophoblastic layer I and diffuse into the interspace between layers I and II. They are localized in extensive infoldings and caveolae of the outer surface of layer II. The syncytial character of layer II is confirmed at this development stage of the placenta. There is no vesicular uptake or penetration beyond layer II until 20 min after tracer administration. Our results indicate that trophoblastic layer II is the main barrier in the chorioallantoic rat placenta preventing the permeation of macromolecules from maternal to fetal compartments. With freeze-fracturing, particulate strutures of variable arrangement and size are found between adjacent cells of layer I. With goniometric analysis small gaps between the appositions of the membrane are observed. These structures are interpreted as tight and/or gap junctions during stages of assembly or disassembly.


Studies on the ultrastructure and permeability of the hemotrichorial placenta. II. Fetal capillaries and tracer administration into the fetal blood circulation. The distribution of horseradish peroxidase and lanthanum chloride within the full term chorioallantoic placenta of the rat was examined after administration of these tracers into the umbilical artery. Both tracers rapidly traverse the capillary endothelium. Transendothelial channels, fenestrations and micropinocytotic vesicles provide the main pathways. Intercellular clefts which are either patent or interrupted by leaky intercellular junctions, also contribute to a rapid passage of low and high molecular weight substances. Deep channel-like invaginations, effecting an increase of the exchange area of layer III, are freely accessible to the tracers from the interspace between the capillary endothelium and trophoblastic layer III. The invaginations, however, are not in continuity with the interspace between layers II and III, verifying the syncytial character of layer III. Neither an uptake of the tracers nor a passage across layer III is observed. The main permeability barrier to feto-maternal transfer within the chorioallantoic placenta is localized in the syncytiotrophoblastic layer III. This layer controls the passage of low molecular weight substances and restricts the penetration of high molecular weight substances.


Intra- and extrahypothalamic vasopressin and oxytocin pathways in the rat. Pathways to the limbic system, medulla oblongata and spinal cord. Vasopressin and oxytocin pathways were specifically localized in glutaraldehyde-paraformaldehyde fixed rat brains, with the use of the unlabelled antibody enzyme method and purification of the first antiserum. Vasopressin and oxytocin containing pathways were traced from the paraventricular nucleus towards the dorsal and ventral hippocampus, the nuclei of the amygdala, substantia nigra and substantia grisea, nucleus tractus solitarius, nucleus ambiguus and to the substantia gelatinosa of the spinal cord. In addition, a vasopressin containing pathway between the suprachiasmatic nucleus and the lateral habenular nucleus was demonstrated. The possible nature (axons or dendrites) and role of these extrahypothalamic fibres is discussed in relation to water balance, milk ejection and avoidance behaviour.


Studies on bone marrow histogenesis: morphometric and autoradiographic studies of regeneration marrow stroma in extramedullary autoimplants and after evacuation of marrow cavity. The marrow cavity of the rat tibia was mechanically evacuated and autoimplanted to the subcutaneous tissue. The regenerative process which restored the integrity of marrow stroma and hemopoiesis, was morphometrically evaluated in whole mount of tibia. Following evacuation, the clot filled the cavity. The granulation tissue then appeared and expanded, penetrating and replacing the clot. The fibroblasts of the granulation tissue differentiated into osteoblasts forming osteoid bone. Within its interstices, the primordial marrow consisting of loose connective tissue and vascular sinuses appeared and hemopoiesis resumed. Expansion of hemopoiesis resulted in the resorption of bone and within three weeks the tibial cavity was restored to the pre-evacuation state. Autoradiography indicated that the labeling index was initially high in fibroblasts and osteoblasts but was subsequently reduced while it increased in osteocytes, cells of Haversian canals, stromal and hemopoietic cells of marrow. The finding is in disagreement with the view that the regenerative process originates from the Haversian canal. When the label was introduced on day 4 post-operatively, it subsequently appeared in osteocytes, cells of Haversian canal, stromal elements of the marrow, but not in the hemopoietic cells. This indicates complete dissociation of marrow stroma and hemopoietic stem cell.


Ultrastructure of venules in the cat brain. Intracerebral venules of the cat were examined to establish criteria for a distinct separation between the venous and arterial system, and to characterize, in greater detail, the mural construction of individual venules. The intracerebral venules compared with those of other organs. Venules do not have a vascular wall composed clearly of endothelium, media, and adventitia, as is characteristic of arteries and arterioles. The venous endothlium has a similar structure to that of capillaries. The periendothelial cells of the venule differ in shape depending on the vascular diameter. The number of periendothelial cell processes in postcapillary venules increases progressively. Segments in which the basal lamina of the endothelium merges with that of the glia cover a smaller portion of the circumference than in venous capillary loops. In collecting venules, the endothelium is almost completely enveloped by periendothelial cells which have a larger number of filaments. There are no typical smooth muscle cells in the intracerebral venules. The perivascular space becomes wider in collecting venules, contains adventitial cells, phagocytes and a great number of collagen fibers.


Ependyma and ependymal protrusions of the lateral ventricles of the rabbit brain. The ependymal lining of the lateral ventricles of the rabbit brain was studied by means of scanning (SEM) and transmission electron microscopy (TEM). There exist cells devoid of cilia in the anterior horn over the region of the caudate nucleus, in the inferior horn over the hippocampus and on the opposite side over cortical regions. On the surface of some of these ependymal cells, accumulations of cytoplasmic folds and globules can be found. They bulge at different height over the ependymal cells. Clots of these cell particles are tied off from the cell, coming to lie as globules either on or between the cilia of the ependyma. TEM reveals that these protrusions are cell debris consisting of different sized vesicles, cell organelles, tubuli and cell filaments. They originate from the ependymal layer but may reach down to subependymal cells. Multivesicular protrusions into the ventricular lumen are also observed. Possible causes of these protrusions are discussed; they are likely to be related to the age of the animals. On the ependyma of the caudate nucleus cilia, microvilli, microblebs and supraependymal neuronal cell processes are distributed unevenly over the surface. Within regions where cilia predominate there are cells which are tightly covered with microvilli. A certain direction of the course of the supraependymal neuronal fibers could not be found.


The effect of sex hormones on the proximal tubules in the rat kidney. The three segments (S1, S2, S3) of the proximal tubule of the rat kidney were investigated, with special reference to lysosomes, after castration, estradiol application, and at the end of pregnancy. Especially in S1 and S2 castration induces an increase of cellular autophagy. The nuclei become smaller; endoplasmic reticulum (ER), ribosomes, and Golgi apparatus are reduced; catabolism predominates. In S1 more giant lysosomes occur; the total number of lysosomes increases whereas acid phosphatase activity decreases at the same time. Sex differences which exist in untreated animals disappear. Substitution with estradiol causes an activation of the proximal tubule cells: Heterophagy predominates, and cellular autophagy is reduced. Nuclear size is unchanged; ER, ribosomes and Golgi apparatus show a clear increase. Giant lysosomes are absent in S1. On the whole lysosomes are larger, but less numerous than after castration. Acid phosphatase is highly active. All changes are most evident in S3. At the end of pregnancy the proximal tubule cells are "stressed" considerably: Pinocytotic activity increases, and large numbers of cell organelles and many lipid vacuoles can be observed. The basal lamina in S1 and S2 becomes thicker. Lysosomes enlarge and increase in number in all segments; giant lysosomes are absent in S1; acid phosphatase is extremely high. The results indicate that sex hormones directly influence the regulation of the proximal tubule cell; moreover, they are indirectly important for the functioning of the kidney via changes in the whole organism.


Nucleotide sequence of turnip yellow mosaic virus coat protein mRNA. The primary structure of the coat protein messenger RNA of turnip yellow mosaic virus is presented. This sequence is the first complete nucleotide sequence of the coat protein messenger of a plant virus to be reported. The coding region, consisting of 567 nucleotides, is flanked by a 5' noncoding region of 19 nucleotides (not including the initiation codon and the cap structure) and by a 3' noncoding region of 109 nucleotides (including the termination signal). The coat protein mRNA has a base composition identical to that of the genome RNA with, in particular, the same high content in cytosine (38%). The codons that govern the incorporation of amino acids into the coat protein are nonrandomly utilized: is greater than 50% of the time the third base of the codons used is a cytosine. This pattern of codon preference is particularly marked for Leu, lle Val, Thr and Cys.


The incorporation of uracil into animal cell DNA in vitro. In the presence of dUTP, net DNA synthesis in vitro is substantially reduced. Small DNA fragments that arise during in vitro DNA synthesis in the presence of dUTP are produced as a result of dUMP incorporation and subsequent post-replication excision repair process initiated by uracil-DNA-glycosylase. The size of the fragments is dependent upon the amount of dUMP incorporated, but unlike the normal 4S intermediates of DNA synthesis, these repair products are not precursors to high molecular weight DNA but are further degraded. The high levels of dUTPase as well as the presence of RNA primers on most nascent DNA pieces (Tseng and Goulian, 1977) suggest that repair of uracil-containing DNA does not contribute to the generation of the small, nascent DNA pieces found during DNA synthesis in this in vitro system.


Organization of human delta--and beta-globin genes in cellular DNA and the presence of intragenic inserts. We have analyzed human cellular DNA for its delta--and beta-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with 32P--beta-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3' end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the delta and beta genes and those containing sequences flanking either 5' to the delta gene or 3' to the beta gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, delta--and beta-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.


Sea urchin nuclei use RNA polymerase II to transcribe discrete histone RNAs larger than messengers. RNA transcribed in isolated sea urchin nuclei and assayed by hybridization to histone genes cloned in E. coli contains sequences homologous to each of the five histone genes. Histone RNA is synthesized exclusively from the same DNA strand which is the template in vivo. Synthesis of the histone gene transcripts is sensitive to alpha-amanitin concentrations which inhibit RNA polymerase II activity. The fraction of histone RNA synthesized in vitro is comparable at two developmental stages to the fraction synthesized in vivo. The nuclear histone transcripts contain sequences homologous to spacer DNA regions present between the coding regions of the 6500 base pair (bp) histone gene repeat unit. The transcription of spacer sequences was demonstrated by hybridization of the nuclear transcripts to subcloned spacer DNA. Although the bulk of the RNA transcripts are greater than 2000 bases long, the histone-specific transcripts are of discrete sizes ranging from 100 bases to about 1100 bases long. Each histone gene hybridizes with at least one of the larger transcripts and with a different subset of smaller RNAs. We do not detect any giant polycistronic transcript spanning the entire histone repeat unit.


Synthesis of H1 histones by BHK cells in G1. The synthesis of histones and DNA was examined in BHK cells arrested in G1 by isoleucine starvation and in cells progressing into the S phase upon isoleucine refeeding. Approximately 2-3% of the cells were not arrested in G1 and synthesized DNA. The rate of synthesis of DNA and nucleosomal histones observed in cells starved for isoleucine could be accounted for by the presence of these asynchronous cells. Synthesis of H1 histones by cells in G1, however, was 3 times that of the nucleosomal histones and approximately 15% of the rate of H1 histone synthesis in mid-S. Upon entry into S, the histones were synthesized in the same molar ratio in which they are present in chromatin. The possible biological significance of H1 histone synthesis in G1 cells and its implications for the regulatory mechanisms controlling histome synthesis are discussed.


Specific representation of cloned repetitive DNA sequences in sea urchin RNAs. Nine cloned repetitive sequences were labeled, strands-separated and individually hybridized with RNA extracted from the nuclei of gastrula stage sea urchin embryos and of adult sea urchin intestine cells. The concentration of transcripts complementary to each cloned sequence was measured by RNA excess hybridization kinetics and by a DNA excess titration method. Transcripts of certain of the repeat families are present at over 100 times the concentration of transcripts of other families in each RNA. The set of repetitive sequence families highly represented in intestine nuclear RNA is different from that highly represented in gastrula nuclear RNA. Together with the results obtained with mature oocyte RNA and presented in the accompanying paper by Costantini et al. (1978), these findings show that quantitative patterns of repetitive sequence representation in RNA are specific to each cell type. Both strands of all of the nine cloned repeats are represented at some level in all the RNAs studied. Usually, though not always, the concentration of transcripts complementary to the two strands of each repeat do not differ by more than a factor of two. The cloned tracers do not react with polysomal messenger RNA, and the nuclear RNA molecules with which they hybridize are many times larger than the repetitive sequences themselves.


Low molecular weight RNAs hydrogen-bonded to nuclear and cytoplasmic poly(A)-terminated RNA from cultured Chinese hamster ovary cells. A group of RNAs 90--100 nucleotides long were isolated by melting them from poly(A)-terminated nuclear or cytoplasmic RNA from cultured Chinese hamster ovary cells. Conditions that favor hydrogen bond formation allowed the reassociation of these low molecular weight RNAs with poly(A)-terminated RNA. The nuclear poly(A)-terminated molecules contained 1.3 moles of the low molecular weight RNAs per mole of poly(A), while the cytoplasmic poly(A)-terminated RNA contained only one seventh as much. These low molecular weight RNAs were also isolated from the total 4S RNA of either the nucleus or cytoplasm by polyacrylamide gel electrophoresis. They formed a prominantly labeled band of RNA in the gels after cells had been labeled with H(3)32PO4 for 4 hr. The low molecular weight RNAs melted from the nuclear poly(A)-terminated RNA were slightly different (although not necessarily in primary nucleotide sequence) from those melted from the cytoplasmic poly(A)-terminated RNA.


Regulation of gene expression by site-specific inversion. A site-specific inversion event is responsible for phase transition in Salmonella, as indicated by heteroduplex analysis of recombinant molecules carrying the gene coding for H2 flagellin in Salmonella. The inversion region corresponds to approximately 800 base pairs in length, and the inversion process does not appear to be dependent upon the E. coli RecA recombination pathway. Specific deletion derivatives of the cloned fragments no longer produce H2-specific flagella, effectively mapping the H2 gene within about 300 bp of the inversion region. Recombinant products of the hybrid molecules arose spontaneously, and they were used in the mapping of restriction sites within the inversion region. The restriction maps further demonstrate the extent and nature of the inversion.


One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Erythrocyte ghosts containing a known number of molecules of purified fragment A of diphtheria toxin with a constant amount of FITC-BSA as a fluorescence marker were prepared by dialyzing a mixture of erythrocytes and these substances against hypotonic solution. These substances were then introduced into diphtheria toxin-resistant mouse L cells by virus-mediated cell fusion of the cells with the ghosts, and mononuclear recipients that has fused with only one erythrocyte ghost were separated in a flourescence-activated cell sorter (FACS) on the basis of their cell size and fluorescence intensity. After separation, the viability of cells containing known numbers of fragment A was examined by measuring colony-forming ability. The results demonstrated that a single molecule of fragment A was sufficient to kill a cell. This fact was confirmed by introduction into cells of fragment A from an immunologically related mutant toxin, CRM 176 (fragment A176); this has a completely functional fragment B region, but in cell extracts, the enzymic activity of its fragment A is about 10 fold less than that of wild toxin. The cytotoxicity of CRM 176 is about two hundredths of that of the wild-type (Uchida, Pappenheimer and Greany, 1973). As expected, about 100-200 fold excess of fragment A-176 was needed to kill the cells.


Analysis of the beta-delta-globin gene loci in normal and Hb Lepore DNA: direct determination of gene linkage and intergene distance. Total human DNA was cleaved with a variety of restriction enzymes, and the fragments were fractionated by gel electrophoresis and transferred to nitrocellulose filter strips. The restricted DNA was then hybridized to nick-translated radioactive recombinant plasmid DNA containing sequences derived from human beta-globin messenger RNA. Under suitable conditions, this probe hybridizes with both the beta--and delta-globin genes. Using this probe, a restriction map of the human beta--and delta-globin genes and the surrounding genomic DNA regions has been constructed. The beta-globin gene contains a nonglobin DNA insert approximately 899-1000 base pairs in length, present within the sequence coding for amino acids 101-120 of the 146 amino acid long globin polypeptide. A similar sequence may be present within the same sequence of the delta-globin gene. The distance between the beta--and delta-globin genes is approximately 7000 nucleotide pairs, and the delta-globin gene is to the 5' side of the beta-globin gene, as predicted by genetic evidence. Both genes are transcribed from the same DNA strand. The structure of the Hb Lepore gene is shown to be a fused delta--and beta-globin gene, and to be completely consistent with the derived map of normal beta--and delta-globin genes. [Restriction enzyme nomenclature follows that of Smith and Nathans (1973) and Roberts (1976). A genomic DNA restriction fragment containing part or all of one globin gene will be designated by that globin chain--for instance, the Pst I fragment containing the beta-globin gene sequence will be designated Pst I beta. A similar convention will be used for double digests. Throughout this paper, when reference is made to the 5' or 3' side or fragment of a gene, this refers to the 5' or 3' side of the mRNA coded by that sequence. Thus the 5' side (N terminal) of the beta-globulin gene is the sequence to the 5' side of the anti-sense strand.].


Recessive male-determining genes.

Recessive male-determining genes. The autosomal dominant gene polled (P) causes hornlessness in goats. Chromosomal females (XX) that are P/P homozygotes develop testes or ovotestes. Thus with respect to its testis-determining properties, P or a closely linked gene acts as an autosomal recessive. Polled intersex goats are H-Y+. This finding is consistent with the view that there may be a cluster of testis-determining H-Y genes on the Y chromosome, and that translocation of a subcritical portion of these genes may generate a recessive mode of sex determination.


A possible mammary stem cell line.

A possible mammary stem cell line. The cell line Rama 25 is derived from a mammary tumor induced in a rat by dimethylbenzanthracene. During rapid proliferation, Rama 25 cells appear as a single undifferentiated epithelial type; at high cell densities, however, small numbers of two other cell types are formed, which respectively resemble secretory and myoepithelial cells of the mammary gland, as judged by light and electron microscopy and immunofluorescent staining for casein (milk proteins). These additional cell types cannot be explained as contaminating cell populations since the cell line has been cloned several times; furthermore, the proportion of either can be increased by dimethylsulphoxide under different conditions. Specific epithelial features are seen by histological and ultrastructural examination of tumors formed by Rama 25 cells in immunodeficient mice. A line of the myoepithelial-like cells, Rama 29, isolated from a Rama 25 culture by cloning, is also described. We propose that the undifferentiated cell type is a form of mammary stem cell which can differentiate in culture.


The primary sequence of rabbit alpha-globin mRNA. The rabbit alpha-globin DNA insertion in the chimeric plasmid pHb 72 (Liu et al., 1977) has been sequenced by the method of Maxam and Gilbert (1977). This has enabled us to determine the messenger RNA(mRNA) sequence beginning in the 5' untranslated region 9 nucleotides before the initiation codon and extending through the first 361 nucleotides of the translated region. The data reported here overlap and are in complete agreement with sequences determined by Baralle (1977) for the 5' end of the mRNA and by Proudfoot et al. (1977) for the 3' end. Our sequence is also in agreement with the partial complementary RNA (cRNA) sequencing data which we reported previously (Paddock et al., 1977), this work marks the completion of the primary sequence of the rabbit alpha-globin mRNA. These observations reaffirm the high fidelity with which gene copies can be synthesized in vitro, cloned in a bacterial plasmid and maintained in the host. The general features of the mRNA nucleotide sequence are duscussed with particular attention given to the base composition and codon preferences observed and to comparison of this sequence with other completed mRNA gene sequences. A new computer program has been used to search for the most stable base-pairing arrangement of the completed mRNA.


Post-meiotic gene activity in spermatogenesis of the mouse. Mouse male germ cells at middle-late pachytene and early spermatid stages obtained by velocity sedimentation at unit gravity in albumin gradients were labelled in culture with [3H]uridine. The newly synthesized RNAs extracted from polysomes of the 2 cell types were studied by sucrose gradient fractionation and poly(U) Sepharose chromatography. The results showed that round spermatids, as well as pachytene spermatocytes, synthesize both ribosomal and polyadenylated RNA molecules. Since these latter are engaged in polysomes they are presumably active messenger RNA molecules.


Cell surface modifications in neuronal maturation. Changes in carbohydrate composition of the cell surface related to neuronal maturation have been studied on neuroblastoma and embryonic dorsal root ganglia (DRG) cultures by using fluorescein conjugated lectins. In neuroblastoma cells, it has been found that the surface of the fibers differs from that of the cell body as shown by concanavalin A (Con A) and WGA binding. In primary cultures of embryonic DRG, lectin binding has also shown that the neuron surface undergoes changes during maturation. In fact, lectin binding which is absent at early stages (5--6 day old embryos) becomes first detectable at the 7th day and then increases progressively. At day 7, the Con A binding pattern resembles that observed in neuroblastoma cells. The possibility of correlating these surface changes with cell adhesive properties and cell differentiation is discussed.


DNA-dependent DNA polymerase species in male germ cells of the mouse. Quasi-homogeneous fractions of male mouse germ cells at definite stages of meiosis and spermiogenesis were obtained by using a separation method based on sedimentation velocity in an albumin gradient. In the various cell types, the total DNA-dependent DNA polymerase activity was determined, and the major enzymatic forms were characterized. The DNA polymerase species present in premeiotic, meiotic and post-meiotic cells were analyzed by glycerol gradient sedimentation. Two types of DNA polymerase were identified in fractions enriched in spermatogonia and preleptotene spermatocytes. One showed a sedimentation coefficient of about 7.5 S and was sensitive to N-ethylmaleimide (NEM); the other exhibited a sedimentation coefficient between 3 and 4 S and was resistant to NEM. On the basis of their sedimentation coefficients, their sensitivity to NEM and their template specificities, these 2 enzymes were identified respectively as alpha and beta DNA polymerases as reported in mammals. The gradient analysis performed on fractions enriched in meiotic and post-meiotic cells revealed the presence of DNA polymerase beta only. A quantitative analysis showed that the activity of the DNA polymerase beta reaches a maximum at middle-late pachytene stage and then drops gradually during spermiogenesis. Although any conclusion as to the biological role of this high level of DNA polymerase activity in pachytene spermatocytes is premature, it is tempting to suggest that this enzyme is involved in meiotic recombination.


Effects of estradiol on early messenger RNA in male Xenopus laevis liver. Cytoplasmic RNA isolated from male Xenopus liver between 1 and 6 h of hormone treatment was separated into distinct classes by Sepharose 4B chromatography. Assay of the mRNA activity of these RNA species in the wheat germ cell-free system demonstrated 2 populations of mRNA activity (peak 2 and peak 3). The activity of 1 population of mRNA (peak 2) was inducible by estradiol while the other (peak 3) remained unaltered for at least 6 h. mRNAs obtained by Sepharose 4B chromatography were also translated in the Xenopus oocyte. The translation products were analyzed by Sephadex chromatography and SDS-gel electrophoresis.


The lack of an inhibitory effect of hyaluronate on chondrogenesis in chick limb-bud mesoderm cells grown in culture. The effect of hyaluronate on chondrogenesis in cultures of chick limb-bud mesoderm cells, derived from stage 20--21, 23--24 and 26 embryos grown at different cell densities and in 3 different culture media, was studied. The results show that hyaluronate at a concentration of 500 microgram/ml, does not consistently produce an inhibition of chondrogenesis in cultures of stage 20--21, 23--24 or 26 limb-bud mesoderm cells in contrast to what has been reported by Toole et al. (1972). It was demonstrated that under optimal conditions, stage 26 cells grown in the absence of hyaluronate do not form as many cartilage colonies in culture as do cells from stage 20--21 or 23--24 embryos. It was determined that culture medium composed of Eagle's MEM supplemented with 7% horse serum, 3% fetal calf serum and 5% 10-day chick embryo extract supported chondrogenesis significantly better than Ham's F-12 supplemented with 10% fetal calf serum. Our results suggest that the inhibition of chondrogenesis by hyaluronate reported earlier is most likely due to the sub-optimal conditions of growth medium, cell density and embryonic stage than to the hyaluronate treatment.


[Carcinoma of the thyroid gland with foamy cells in the stroma]. Foamy cells were found in the stroma of 23 out of a total of 1,073 differentiated carcinomas of the thyroid gland investigated during the 1946--1977 period, i. e. in 2.1% of the cases concerned. Of these, 15 carcinomas with foamy cells showed purely papillary differentiation, while the remaining 8 showed mixed papillofollicular structures. Apart from the papillary structure, the development of foamy cells may have been associated with hypercholesterolaemia due to hypothyroidism.


[Acute pulmonary histoplasomis. 1st findings in Czechoslovakia]. A 57-year-old man, who for many years was treated for a pleomorphic lymphoma with cytotoxic drugs and x-ray therapy, died from the generalized tumor process. At autopsy, an extraordinary finding was found in form of acute pulmonary mycosts, which histologically appears to be histoplasmosis. Culture was not attempted, but the diagnosis is based on morphology, both of the organism proper and its intracellular location, and on results obtained by immunofluorescence studies. In numerous pulmonary vessels, endothelial pillows of histiocytes with yeast cells are found, typical for histoplasmosis, but no generalization was found in other organs. The source of the infection is not clear.


[Morphological changes in liver tissue in experimental porphyria]. Miscroscopic changes in the liver tissue of mice became detectable 42 hours after the administration of gricine through the appearance of porphyrins in the plasma of hepatocytes marked by the enlargement of their volume, nuclei, and nucleoli. 72 hours after gricine, porphyrins were also found in the lumina of capilary bile ducts and hepatic ducts. This was accompanied by dispersion extinction of hepatocytes and pigment accumulation in the plasma of Kupffer's cells. There was histochemically proved increased activity of alkaline and acidic phosphatase, G6PDH, NADH and NADPH tetrazolium reductase. Under electron microscopy, the porphyrins showed crystalline structure, rotated the plane of polarized light, and were marked by red fluorescence.


[Histological, electron-optical and histochemical findings in mycosis fungoides]. Skin excisions were investigated in 5 patients with verified mycosis fungoides. Findings yielded by light and electron microscopy helped to confirm the findings anticipated, and were correlated with histochemical observations. One of the cases involved a substantially higher activity of lysosomal enzymes, particularly KF and beta-glucuronidase in the skin infiltrate mycotic cells. In the other cases, this sort of activity was low. The significance of high ATPase activity in the peripheral cell membrane remains unclear. In one case, involvement in the T lymphocyte series was confirmed by the formation of rosettes.


[Lymphoepitheloid malignant lymphoma (Lennert)]. A group of 22 lymfoepitheloid malignant lymphomas was singled out from a wider group by means of demonstating nuclear atypias in epitheloid cells. There were three roughly equal subgroups: the first corresponding to malignant lymphogranuloma, the second to different malignant lymphomas of the ono-Hodgkin type. At first, the third group could not be recognized as a malignant lymphoma other than according to different sorts of atypia of each of the epitheloid cells in granulomas.--The general behaviour of malignant lymphomas of the first two subgroups was in keeping with the properties of the respective basic tumour. The third subgroup was more independent. At each subsequent check-up, the epitheloid granulomatous component of the tumour was seen gradually disappearing. It appeared to a secondary feature in most cases. The cause of atypia in epitheloid cells remained unexplained. Nonetheless, these atypias are an important diagnostic facilitating the identification of a more independent form of lymphoepitheloid malignant lymphoma, the subsequent development of which mostly shows signs of a moderately pleiomorphnous malignant lymphoma, sometimes obviously belonging in the T series.


[Arthrotic manifestations on the patella in the x-ray and microscopy pictures]. 40 patellae of different age groups were examined by X-ray and histology. X-ray changes found in the cartilage, corticalis and cancellous bone were in full agreement with histological findings. However, more than a quarter of the patellae were found to be affected by microscopic fibrillation of the surface of the cartilage without any demonstrable changes in the cartilage proper. In all those cases except one, however, roentgenograms helped to detect changes in the corticalis or also cancellous boneof the lamellae such as are associated with bone reconstruction, mostly in the form of subchondral bone lamella dilatation and of the thickening of adjacent cancellous bone trabeculae.


trans-Stilbene oxide: an inducer of rat hepatic microsomal and nuclear epoxide hydrase and mixed-function oxidase activities. The administration of trans-stilbene oxide to rats resulted in increased hepatic microsomal and nuclear epoxide hydrase (with styrene oxide (SO), benzo[a]pyrene 4,5-oxide (4,5-BP) as substrates) and aryl hydrocarbon hydroxylase (AHH) activities. Hepatic microsomal aminopyrine N-demethylase, benzphetamine N-demethylase, and ethylmorphine N-demethylase activities were also increased. These increases in microsomal enzyme activity were dose- and time-dependent (about 100% at 200 mg/kg body weight, administered for 2 consecutive days). However, only marginal increases in hepatic microsomal NADPH-cytochrome c reductase activity and cytochrome P-450 content were observed. No apparent proliferation of hepatic endoplasmic reticulum occurred in trans-stilbene oxide pretreated rats. The administration of trans-stilbene oxide has no effect on hepatic glutathione S-transferase activities (with SO or 4,5-BPO as substrates). None of the parameters were affected in pulmonary microsomes from treated rats. The in vitro addition of trans-stilbene oxide (10(-6)--10(-2) M) did not affect hepatic epoxide hydrase or glutathione S-transferase activities.


Excision of hydrocarbon-DNA adducts and consequent cell survival in normal and repair defective human cells. Survival curves for normal human cells and xeroderma pigmentosum variant cells (XP4BE) after ultraviolet radiation were indistinguishable. In comparison, cells from xeroderma pigmentosum complementation group A (XP12BE) were very sensitive to ultraviolet radiation. Complementation group C (XP2BE) cells were almost as sensitive as group A cells. These survival phenomena parallel to known unscheduled DNA synthesis responses of these cells to ultraviolet radiation, which, compared with normal cells, are: XP4BE, 100%; XP2BE, 20%; XP12BE, 2%. The relative capacities of these cells to excise 7-bromomethylbenz[a]anthracene-DNA adducts and to survive treatment with the carcinogen were similar to the responses to ultraviolet irradiation, except that the XP2BE cell line both excised and survived this damage far better than anticipated from its response to ultraviolet irradiation. Moreover, whilst in the normal cells and variant cells the ratio of hydrocarbon-adenine adduct to hydrocarbon-guanine adduct remaining in DNA decreased notably with excision, this ratio did not change significantly with excision in the XP2BE cell line. The relationship between greater excision capacity and increased cell survival in the experiments with the chemical carcinogen indicates that the unexcised damage is responsible for the cell-killing action of this agent. The different relative repair and survival responses of these cell lines to ultraviolet irradiation on the one hand, and to 7-bromomethylbenz[a]anthracene chemical carcinogen treatment on the other, indicate that in at least one of these cell lines (XP2BE), and possibly in all the lines, different cellular mechanisms are involved in the repair of DNA damage resulting from ultraviolet irradiation and that resulting from the chemical carcinogen treatment.


Epoxide metabolizing enzyme activities in rat testes: postnatal development and relative activity in interstitial and spermatogenic cell compartments. Microsomal epoxide hydrase (EH) and 176 000 g supernatant fraction glutathione-S-transferase (GSH-S-T) activities were determined with styrene oxide as substrate in rat testes during postnatal development. The development of these enzymes was also followed in liver for comparison. Testes of 6-day-old rats had high GSH-S-T activities (66 nmol/min/mg protein), which were about 50% of the adult levels. Transferase activity then developed slowly and reached a maximum by 165 days of age. Specific testicular GSH-S-T activities of 6-day-old rats were 3--4 times those of hepatic GSH-S-T activities in the same animals. In contrast, EH activities of both liver and testes were very low in prepubertal rats, but they increased dramatically at the onset of puberty and reached maximum activities by 45 days of age. Microsomal and microsomal supernatant fractions prepared from adult rat spermatogenic cells had about twice the EH and GSH-S-T specific activities (with styrene oxide or benzo[a]pyrene 4,5-oxide as substrates) of similar fractions prepared from interstitial cells. On the other hand, benzo[a]pyrene hydroxylase (AHH) activity and cytochrome P-450 content were at least 2-fold greater in microsomes from interstitial cells than in those from spermatogenic cells.


Effects of halogenated antibacterials on the erythrocyte membrane. The influence of halogenated antibacterials on membrane structure and function was investigated using the human erythrocyte membrane as a model. Measurements of hemolysis in isotonic solution, altered membrane permeability, and stabilization against hypotonic hemolysis resulting from exposure of erythrocytes to halogenated antibacterials served as criteria of membrane-related effects. The hemolytic potency of the compounds studied differed widely, decreasing in the order hexachlorophene (HCP) greater than 2,2'-methylenebis(3,5-dichlorophenol) (3,5-TCP) greater than 2,2'-methylenebis(3,4-dichlorophenol) (3,4-TCP) approximately equal to 2,2'-methylenebis(4,6-dichlorophenol) (4,6-TCP) greater than 2,2'-methylenebis(4-chlorophenol) (DCP) greater than 3,4'-tribromosalicylanilide (TBS) approximately equal to 3,3',4',5-tetrachlorosalicylanilide (TCSA). Each of the antibacterials tested stabilized the erythrocyte against hypotonic hemolysis, although there were marked differences in the concentrations required to afford maximum stabilization as well as in the extent of protection. The observed order of protective effectiveness was HCP greater than 3,4-TCP greater than 4,6-TCP greater than DCP approximately equal to TCS greater than TBS. As shown by measurements of the first-order rate constant for K+ efflux, the permeability of the erythrocyte membrane to K+ was increased upon exposure to the antibacterials, with the effect of HCP greater than 3,4-TCP greater than 4,6-TCP approximately equal to 3,4-TCP greater than DCP approximately equal to TCS greater than TBS. These results indicate that halogenated antibacterials are capable of perturbing mammalian membranes, a feature which may account in part for their mammalian toxicity.


Interactions of vinyl chloride with rat-liver DNA in vivo. 9beta-D-2'-Deoxyribofuranosyl-imidazo-[2,1-i]purine ("etheno-deoxyadenosine") and 1beta-D-2'-deoxyribofuranosyl-1,2-dihydro-2-oxo-imidazo-[1,2-c]pyrimidine ("etheno-deoxycytidine") are identified in the enzyme hydrolysates obtained (i) from calf-thymus DNA which had been modified by chemical reaction with chloroacetaldehyde and (ii) from liver DNA prepared from rats which had been exposed orally to vinyl chloride in their drinking water (250 ppm) for approx. 2 years. Thus, vinyl chloride-derived chloroethylene oxide and/or chloroacetaldehyde behaves as a bifunctional alkylating agent towards deoxyadenosine and deoxycytidine residues of DNA. The separation of deoxyribonucleosides and the two etheno-deoxyribosyl-nucleosides by liquid chromatography, and the mass spectra of etheno-deoxyadenosine and etheno-deoxycytidine and of their O-bis-(trimethylsilyl) derivatives are described. In the animal experiment (ii), the resulting proportion of etheno-deoxyadenosine is small compared with that of etheno-deoxycytidine. Imidazo-[2,1-i]purine (etheno-adenine) is identified: (a) in the supernatant after sedimentation of the modified DNA in the model experiment (i), and (b) in the product resulting from the reaction between chloroacetaldehyde and deoxyadenosine. The effect on the structure of DNA of the imidazo-cyclization of deoxyadenosine and deoxycytidine residues and of the depurination of etheno-deoxyadenosine residues is discussed in relation to vinyl chloride oncogenicity.


The ontogeny of nuclear aryl hydrocarbon hydroxylase. The ontogeny of rat liver nuclear aryl hydrocarbon hydroxylase (AHH) ws studied. AHH which was barely detectable in 18--20 day fetal rat liver nuclei increased rapidly post-partum reaching a value which was over 200 times greater than the fetal liver specific activity. Nuclear AHH was induced upon administration of 3-methylcholanthrene (3-MC) to rats. The level of induction was dependent upon the age of the rat. Thus, the highest induced nuclear AHH specific activity was observed in the 3--4-week-old rat, i.e., 50--70 g in weight. The greatest fold increase in nuclear AHH after 3-MC administration was observed in the 6--8 g, or 1--2-day-old rat. Fetal rat liver nuclear AHH was also induced after 3-MC administration to the dam. The basal and induced levels of AHH were compared in nuclei and microsomes from 1--2 and 10--12-day-old rats. In the former, the ratio of the microsomal to nuclear AHH was 1.4 and 1.8 under basal and induced conditions, respectively.


Antitumor action of rhodium (I) and iridium (I) complexes. Several rhodium(I) and iridium(I) complexes displayed different degrees of antitumour activity when tested in mice bearing Ehrlich ascites carcinoma. Rhodium (I) and iridium (I) acetylacetonate derivatives caused a high percentage of cures. The rhodium (I) dimers were particularly interesting, since (bis(cycloocta-1,5-diene)micromicron' dichlorodirhodium(I) [RhCODCl]2) was highly effective, whereas its analogues, bis(bicyclo[2,2,1]hepta-2,5-diene)micromicron'-dichlorodirhodium(I) [RhNBDCl]2) and bis(1,5-hexadiene)micromicron' dichlorodirhodium(I) [RhEDCl]2) were virtually inactive. The absence of significant inhibition of DNA, RNA and protein syntheses in tumour cells found for [RhCODCl]2 at therapeutically active dosages, indicates that this substance has a different mechanism of action from that of cis-dichlorodiammine Pt(II) (cis-PDD). The amount of rhodium found in tumour cells after administration of active [RhCODCl]2 was higher than that for [RhEDCl]2, while the rhodium concentration in the ascitic fluid was much higher for [RhEDCl]2. A mechanism based on the chemical properties of the complexes is tentatively proposed for explaining these findings and selective toxicity for tumour cells.


The major metabolite of aflatoxin B1 in the rat is a glutathione conjugate. [14C]aflatoxin B1 (AFB1) was injected i.p. into female Wistar rats. Half of the dose was eliminated into the bile mostly as polar non-extractable metabolites. Among these a glutathione conjugate was the main component. The same conjugate was formed when rat liver postmitochondrial supernatant was incubated with AFB1 and [3H]glutathione. The conjugate was purified by ion exchange chromatography, gel-filtration and thin layer chromatography (TLC). It was tentatively identified as 2,3-dihydro-2-(S-glutathionyl)-3-hydroxy aflatoxin B1 (AFB1-GSH-conjugate). This structure was derived mainly from amino acid analysis, ultraviolet spectra and the enzymatic requirements for its formation in in vitro experiments. In the rat this detoxification product of the potentially ultimate reactive AFB1-epoxide constitutes about 10% of the administered dose and thus underlines the quantitative importance of this activating pathway.


Interactions between cadmium and zinc in cytoplasm of duck liver and kidney. Ducks were collected from a marine environment known to be polluted with heavy metals. Gel elution profiles were determined for both liver and kidney tissue using Sephadex G-75 gel. Zn increased linearly on the high molecular weight protein pool until this pool was apparently Zn saturated; saturation levels were 0.06 mumole cytoplasmic Zn/g kidney (wet wt.) and 0.14 mumole cytoplasmic Zn/g liver (wet wt.). Seventy-five percent of cytoplasmic Cd was found on the high molecular weight protein pool unless it was Zn saturated. If this pool was Zn saturated, then excesses of Zn and up to 75% of cytoplasmic Cd appeared on metallothionein. Results are discussed in terms of a competition of Cd and Zn for Zn binding sites on metalloenzymes. Cu increased linearly on both the high molecular weight protein pool and metallothionein, with increases of Cu in tissue.


Comparative actions of 1,10-phenanthroline nickel(II) chelates and their constituent metal ion and ligands on the guinea-pig isolated atrium. The actions of fully co-ordinated, inert nickel(II) chelates of 1,10-phenanthroline and 3,4,7,8-tetramethyl-1,10-phenanthroline have been compared on the guinea-pig isolated atrium with those of their constituent metal ion and ligands. In general, each test substance showed distinctive actions suggesting that the effects of the metal chelates are mediated by the intact chelate cation and not by liberated metal ion or ligand. Differences in the actions of the unsubstituted and the highly methylated compounds may result from the greater capacity of the methylated chelate and ligand to induce more profound and sustained conformational changes in the responsive atrial pre- and postsynaptic membranes thereby promoting more rapid and prolonged influx of Ca2+.


Mutagenicity of dichlorvos and other structurally related pesticides in Salmonella and Streptomyces. The following pesticides: azinphosmethyl, diallate, dichlorvos, EPTC, fenchlorphos, mevinphos, monocrotophos, noruron, parathionmethyl, triallate, trichlorphon and vegadex were tested for the ability to induce his+ revertants in four histidines requiring strains of Salmonella typhimurium--TAI 535(missense), TAI 536, TAI 537 and TAI 538 (frame-shift)- and resistance to low levels of streptomycin in Streptomyces coelicolor. Dichlorvos, which is a phosphoric ester with a dichlorovinyl group as side chain, and trichlorphon, which is known for its spontaneous conversion in dichlorvos, are both mutagenic in Salmonella (strain TAI535) and Streptomyces. Five organophosphorus pesticides similar to dichlorvos but devoid of the vinyl group are not mutagenic. Three carbamates, diallate, triallate and vegadex, which contain a chloroallyl group similar to the vinyl group of dichlorvos are mutagenic in Streptomyes; triallate and vegadex are powerful mutagens also in Salmonella (strain TAI535); two other carbamates devoid of the chlorinated group are not mutagenic. The results suggest that the presence of a vinyl chloride or allyl chloride group in the molecule of these pesticides is responsible for the ability to induce point mutations in Salmonella and Streptomyces.


Comparative effect of a family of substituted thiopseudoureas on protein synthesis by rat liver and Walker carcinoma ribosomes. In order to assess the role played respectively by the pseudothiourea group and the alkylic chain in the inhibition of protein synthesis and tumour growth caused by compound AHR-1911, a series of eight related substances were studied. The blockade of protein synthesis on liver and Walker carcinoma ribosomes and on suspensions of Walker carcinoma cells, depended essentially on the length of the alkylic chain and the substitution in C-1. The minimum chain was 9 carbons and a plateau in the activity was reached at 11 carbons. Replacement of the thiourea group in C-1 by an NH2 group did not change the pattern. A double bond in the distal section of the chain (AHR-1911) increased inhibition on intact cells with a parallel decrease in cytotoxicity, and reduced the aggregation of ribosomes, protein synthesis factors and other proteins. The antitumor effect depends on the pseudothiourea group and is not caused primarily by interference with protein synthesis. Aminoacyl tRNA binding and transfer appeared to be targets of AH4-1911, but this did not affect significantly tRNA change or nascent peptide release. Drug binding to ribosomes and their subsequent aggregation can be regulated by K+ concentration and temperature. It is assumed that the inhibition of protein synthesis is caused by AHR-1911 effects on elongation factors, impairing their interaction with ribosomes.


Computer-generated graphic models of the N2-substituted deoxyguanosine adducts of 2-acetylaminofluorene and benzo[a]pyrene and the O6-substituted deoxyguanosine adduct of 1-naphthylamine in the DNA double helix. Computer models of three deoxyguanosine-carcinogen adducts in double-helical DNA are presented. The carcinogen moiety is rotated and the best fit within the double helix is evaluated. The 2-acetylaminofluorene (AAF) derivative, 3-(deoxyguanosin-N2-yl)-AAF, is found to be situated within the minor groove, has very little freedom of rotation and causes little helical distortion. The (+)-anti-benzo[a]-pyrene (BP)-diol epoxide-N2 adduct, 10beta-(deoxyguanosin-N2-yl)-7beta, 8alpha,9alpha-trihydroxy-7,8,9,10-tetrahydro-BP, has a similar fit with a greater degree of steric interaction, suggesting that this adduct could cause some local destabilization. The 1-naphthylamine (NA) derivative, N1-(deoxyguanosine-O6-yl)-1-NA, resides within the major groove, does not perturb the helix and has considerable freedom of movement.


Covalent binding of ethinylestradiol and estrone to rat liver DNA in vivo. The covalent binding of [6,7-3H] ethinylestradiol (EE) and [6,7-3H] estrone (E) to liver DNA of 200 g female rats was measured 8 h after the administration of 80 microgram (9.2 mCi) estrogen by gavage. The binding is 1.5 for EE and 1.1 for E, expressed as binding to DNA/dose, in units of mumol hormone/mol DNA phosphate/mmole hormone/kg body wt. It is the same order of magnitude as for benzene and about 10 000 times below the binding of typical liver carcinogens, such as aflatoxin B1 or N,N-dimethylnitrosamine.


Sulfate conjugation of benzo[alpha]pyrene metabolites and derivates. Sulfate conjugation of benzo[alpha]pyrene(BP) metabolites and derivatives was studied. The reaction sequence consisted of two steps; activation of sulfate ion to 3'-phosphoadenosine-5'-phosphosulfate and transfer of the activated sulfate to the BP-derivatives. Both reactions were carried out by enzymes located in the rat liver 105 000 g supernatant. The reactions required MgCl2. Phenol and quinone derivatives were generally good substrates for sulfate conjugation and different reactivities were observed with the dihydrodiol derivatives. Sulfate conjugates were more polar than their parent BP-derivatives and except for quinone conjugates were easily extracted with ethyl acetate. The role of sulfate conjugation in BP carcinogenesis is discussed.


DL-2-Oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine. A sulfhydryl metabolite of levamisole that interacts with microtubules. DL-2-Oxo-3-(2-mercaptoethyl)-5-phenylimidazolidine (OMPI) a sulfhydryl metabolite of levamisole, unlike the parent compound, is shown to interfere with the morphological and functional integrity of microtubules in cultured cells at high concentrations (1.6-10(-4) M). Lower concentrations do not affect the cell morphology, viability or growth rate in any appreciable way. Both levamisole and OMPI, at low concentrations (10(-5)-10(-6) m), markedly enhance the antimicrotubular effect of mercaptoethanol. High concentrations of OMPI (+/- 10(-4) M) inhibit the self-assembly of microtubules in a cell free system. Low concentrations (+/- 10(-6) M) markedly enhance the polymerization rate of tubulin. Levamisole has no effect on tubulin polymerization. The effects of OMPI on microtubules in cells and in the polymerization system can be reversed by reduced glutathione, cysteine and dithiothreitol. The data indicate that OMPI interacts in a biphasic manner with microtubule formation probably through interaction with critical SH-groups on the tubulin molecule. It seems of interest to further investigate the hypothesis that the immunomodulating properties of levamisole are at least partially due to the formation of its metabolite (OMPI) which could enhance microtubule integrity and function in leukocytes.


The toxicity of thallium(I) to cardiac and skeletal muscle. The effects of (a) partial or complete replacement of K+ by Tl+ in saline perfusing isolated rat heart and diaphragm preparations and (b) pulse injections of high concentrations of Tl+ or K+, have been studied. The immediate effect of Tl+ resembles that of higher concentrations of K+ and may reflect its more rapid penetration into the tissue. Tl+ appears to replace K+ on a 1:1 basis to an extent dependent upon the relative abundance of the two cations in the perfusion solution. However, analysis of diaphragm preparations after perfusion with salines containing increasing Tl+ but constant [K+ + Tl+] showed a related and progressive increase in total cation content. This effect, which was not seen in the presence of constant high (normal) K+ concentrations, may reflect an increase of the intracellular space brought about by the thallium. Functional effects of Tl+ were (a) preferential block of the phrenic nerve or neuromuscular junction over the muscle fibre and (b) transient but marked acceleration of cardiac frequency following pulse injections, which may be of value in analysing the pacemaker mechanism of the heart. In both tissues Tl+ is eventually toxic and probably irreversibly so.