Frequency of the gene for cystic fibrosis with a view of replacement and recognition effects and reproduction by homozygotes. The apparent frequency of the recessive autosomal allele for cystic fibrosis (cf) is too high to be satisfactorily explained by mutation equilibrium. However, higher reproductive fitness by heterozygotes (expressed by t greater than 0) or higher gametic viability of the cf allele (expressed by a greater than 0.5) can provide reasonable explanations of the present frequency. Furthermore, birth replacement in families with CF children (RPE) can account for the estimated historical values of t. Today, however, t is probably at or approaching zero as a result of discontinuation of RPE. In the future we can look for an imperceptibly slow decrease in the incidence of CF. Reproduction by CF females will not cause an increase in incidence; it will only slightly slow the rate of decrease.


Renal tubular abnormalities in a patient with the phenotypic appearance of C-trisomy mosaicism. In 1971, SINHA et al. described a patient with isochromosomes arising from a C-group autosome. This patient had the phenotypic appearance of C-trisomy mosaicism. Six years later she presented with complaints of polydipsia and polyuria and was documented to have nephrogenic diabetes insipidus and renal tubular acidosis. The abnormal cell line of the patient had disappeared at the time of this observation.


Esterase D phenotypes in psoriasis vulgaris, atopic diseases and healthy controls. Esterase D phenotypes have been determined in 162 patients with psoriasis vulgaris, 181 patients with atopic diseases and 110 healthy controls. All these samples are from the area of Rheinland-Pfalz (West Germany). The two patient samples show somewhat higher frequencies of Es D 1 and lower of Es D 2-1 phenotypes as compared to controls. These differences are, however, statistically not significant. The Es D1 frequency in controls (0.9045) is in accordance with those reported from other European populations.


The acetylator polymorphism in four populations of Afghanistan. Studies of the acetylator polymorphism in Pushtoons, Tajiks, Hazaras and Usbeks living in Afghanistan revealed a lower frequency of the allele ACS in the last two populations. The results were compared with those of other populations. The importance of this polymorphism for therapy and a possible relation to the use of alkaloids in form of spices and drugs is discussed.


Gene differentiation in three tribes of American Indians. The amount of gene differentiation in the subdivided populations of three American Indian tribes, the Papago, Makiritare and Yanomama were studied and found to vary from 2 to 7%. These results indicate that only small fractions of the total gene differences are attributable to between subpopulations and the remaining 93-98% to within subpopulations.


Development of an attached strain from a continuous insect cell line. A continuous attached cell strain has been developed from the IPRI-CF-124 line of the spruce budworm, Choristoneura fumiferana. This was done by discarding suspended cells at each passage, rinsing attached cells with 0.05% trypsin and using only the strongly attached cells for subculturing. The method is very effective in that the proportion of attached cells increased from 6% in the parent cell line to 97% in the new cell strain after 20 passages. The attachment and growth properties are stable after storage of cells in liquid nitrogen. The new cell strain is designated IPRI-CF-124T and has a population doubling time comparable to that of the parent cell line.


Comparative studies on human skin fibroblasts: life span and lipid metabolism in medium containing fetal bovine or human serum. Three strains of human skin fibroblasts were cultivated in nutrient medium supplemented either with human serum or fetal bovine serum, and growth and lipid synthesis were compared. Rates of cellular growth were similar in both kinds of medium, but the replicative life spans of all three strains were curtailed significantly in human-serum medium. Incorporation of label into the major classes of neutral lipids from [14C]acetate and 3H2O was increased also in human-serum medium. Since human serum contained higher concentrations of cholesterol known to reduce endogenous cholesterol synthesis, these results were unexpected. Nonlipid factors in human serum may account for the shortened cellular life spans and increased lipogenesis and perhaps for the potential to develop atherosclerosis.


Effects of perinatal estrogen on mouse mammary response to corticoids in vitro. Effects of three adrenal corticoids on in vitro mammary differentiation were compared in neonatally estrogenized (E) and uninjected control (N) BALB/c Crgl female mice. E mice were injected with 10 microgram of 17 beta-estradiol on each of the first 5 days of life. At 4 weeks of age, all mice were pretreated with 1 microgram  17beta-estradiol and 1 mg progesterone for 9 consecutive days. Groups of 10 or more entire mammary glands then were organ-cultured for 5 days at 37 degree C on chemically defined medium in 95% O2/5% CO2 with various combinations of hormones: mammotropin (M), somatotropin (S), insulin (I), thyroxine (T); and corticosterone (B), aldosterone (A), or cortisol (F). Differentiation followed a quantitative pattern of MSIT less than BMSIT less than AMSIT less than FMSIT for both E and N tissues. E tissues formed more alveoli and more lobules in vitro than N tissues with each of the corticoids tested. These findings may have pathologic significance.


In vitro development of plants from microspores of rice. Rice (Oryza sativa L., 2n=24) anthers containing microspores in the early-uninucleate to first-mitosis stages were induced successfully to develop into plants in vitro through an intermediary step of callus formation. Callus initiation occurred with highest frequency in anthers containing mid-uninucleate imcrospores. The callus derived from different stages of microspore development differed in the potential to differentiate into plants. The plants regenerated from pollen callus were predominantly haploid or diploid; polyploid and aneuploid plants were relatively infrequent. The first division of the uninucleate microspores was asymmetrical, resulting in the formation of large vegetative and small generative nuclei. The vegetative nucleus divided repeatedly and assumed the major role in the formation of callus, whereas the generative nucleus degenerated rapidly. Simultaneous division of the two nuclei was observed in a few pollen grains. Nuclear fusion during the very initial stages of pollen development was postulated to account for the occurrence of the diploid and polyploid plants.


Intestinal absorption of botulinum toxins of different molecular sizes in rats. During a period of 10 to 12 h after injection of type B 16S (L) toxin into the ligated duodenum of rats, 0.01 to 0.1% of the total toxicity administered was found in the lymph drawn by cannulation of the thoracic duct. The recovery was 50 to 100 times higher than that of the rat given type B 12S (M) or 7S (S) toxin. During the same period, 0.6 to 1.5% of the specific antigens were recovered, regardless of the molecular size of the toxin that had been administered. In lymph of the B-L or B-M toxin recipient, the toxic and nontoxic components were detected in comparable quantities, indicating that the undissociated progenitor toxin molecule is absorbed through the intestinal wall. Although the toxic component had lost its toxic activity, the two components of B-M toxin appearing in lymph reassembled to reconstruct the 12S molecule, whereas those of B-L toxin did not, although the toxic component was still active. Type B-L, B-M, and B-S toxins showed similar stabilities to in vitro exposure to rat lymph (pH 8.2), but B-L toxin showed a considerably higher stability to intestinal juice (pH 7.0) than did B-M toxin. Thus, the toxicity of lymph of rats administered botulinum toxin intraduodenally depends not upon the rate of absorption, but largely upon the stability in the intestine.


Effect of warfarin on the induction and course of experimental Staphylococcus epidermidis endocarditis. The effect of warfarin treatment on an experimental Staphylococcus epidermidis endocarditis was studied. Warfarin was found to affect both the induction and course of the infection of catheter-induced endocardial vegetations. In warfarin-treated rabbits, larger bacterial inocula were needed to induce an infection, and the degree of infection of the vegetations was also significantly lower, eventually resulting in the total elimination of the bacteria from the vegetations. Thus, warfarin treatment seems to have an inhibitory effect on the induction and development of an S. epidermidis infection of the endocardium. The results differ from previous findings in studies done with Streptococcus anguis, where warfarin was found to have no effect on the induction or course of the infection of endocardial vegetations, which suggests that different mechanisms are involved in the pathogenesis of endocarditis caused by these two species of bacteria.


Regulation of invertase of Actinomyces viscosus. The regulation of diethylaminoethyl-partially purified invertase (EC 3.2.1.26; beta-D-fructofuranoside fructohydrolase) from the 37,000 X g-soluble intracellular fluid of Actinomyces viscosus serotype 2 strain M-100 was studied. Glycolytic intermediates, mono-, di-, and triphosphate nucleotides, inorganic phosphate, and various divalent cations were tested for regulatory effects. Fructose-6-phosphate (F6P) and fructose-1,6-diphosphate (FDP) were found to act as noncompetitive inhibitors of invertase. The Ki values for F6P and FDP were found to be 3.4 and 5.1 mM, respectively. The Hill coefficient for sucrose was 1.03 and remained unchanged in the presence of varying amounts of F6P or FDP.


Chemiluminescence response of human leukocytes: influence of medium components on light production. Light-producing reactions have been reported to occur after phagocytosis of opsonized particles by human polymorphonuclear neutrophils, eosinophils, and monocytes. Such chemiluminescence appears to be related to the generation of singlet oxygen, superoxide, and hydroxyl radicals, which have also been implicated as microbicidal agents. In examining the influences of various medium components on leukocyte chemiluminescence, we have observed that the amount of light measured is increased by addition of soluble protein, the amino acids tyrosine and tryptophane, or excess zymosan to the reaction medium. These agents appear to produce their effect not by increasing the rate of phagocytosis, but by providing substrate for secondary light-producing reactions. Polystyrene particles do not provide a suitable substrate for such secondary light-producing reactions. This is evidenced by the failure of ingested latex to stimulate high levels of chemiluminescence in the cellular system and their failure to augment light production in two noncellular chemiluminescent reactions. Some of the light generated in the cellular chemiluminescence response may derive from secondary reactions, which occur outside of the phagocyte. Support for this phenomenon is provided by two experiments. In one, addition of supplementary, nonopsonized zymosan to the reaction, after phagocytosis of opsonized zymosan is complete, resulted in an increased level of chemiluminescence. In another, addition of nonopsonized zymosan, together with latex particles, resulted in a significant increase in chemiluminescence. The results of the latter experiment also support our hypothesis that latex does not provide an appropriate substrate for secondary light-producing reactions. These observations suggest that leukocytes activated by phagocytosis generate electronically activated radicals which act intra- and extracellularly and that the amino acids tyrosine and trytophane may provide one substrate through which these agents act.


Recovery of Streptococcus mutans after amino acid deprivation. The recovery of Streptococcus mutans FA-1 in a complete, chemically defined medium was examined after 1, 3, and 6 h of essential amino acid deprivation. Amino acids could be divided into two groups based on their effect on the relative rates of recovery: those amino acids (leucine and cystine) that are precursors of protein only, and amino acids (glutamate/glutamine or lysine) that are incorporated into both protein and cell wall peptidoglycan. Culture turbidity, deoxyribonucleic acid, ribonucleic acid, protein and cell wall peptidoglycan measurements indicated rapid recovery after leucine/cystine starvation periods. However, a 6-h leucine/cystine deprivation resulted in a slower exponential rate of growth (180-min doubling time compared to the normal doubling time of 85 to 90 min) after recovery. Glutamate/glutamine starvation, on the contrary, resulted in greatly extended recovery periods, especially after 3- and 6-h amino acid deprivations. Macromolecular synthesis was most severely affected by 6-h glutamate/glutamine starvation and required 6 to 10 h for recovery of an exponential rate. A delay in the recovery of deoxyribonucleic acid and cell wall peptidoglycan synthesis beyond that of the other macromolecules was observed after 1 and 3 h of deprivation with either leucine/cystine or glutamate/glutamine. However, after a 6-h amino acid deprivation, deoxyribonucleic acid synthesis recovered more rapidly than that of the other macromolecules studied. The results are discussed in terms of the nutritional environment of the oral cavity and its effect on the growth and survival of S. mutans.


Effective immunity to dental caries: passive transfer to rats to antibodies to Streptococcus mutans elicits protection. Rat dams, given intravenous injections of heat-killed Streptococcus mutans 6715, mutant C211 demonstrated significant agglutinin activity to the homologous S. mutans in colostrum, milk, and serum. This antibody activity was associated with the immunoglobulin G (IgG) class. High titers of anti-S. mutans antibody associated with the IgG class were also exhibited in the sera and saliva of the offspring that suckled these dams. After challenge with the homologous, live S. mutans, these offspring developed significantly fewer caries on all molar surfaces than did nonimmunized infected controls. A secretory immune response (manifested by the presence of specific IgA antibody to S. mutans in colostrum and milk) was elicited (i) in rat dams locally injected, in the region of the mammary gland, with heat-killed S. mutans antigen, and (ii) in other rat dams that were provided formalin-killed S. mutans in their drinking water. Offspring suckling these dams were challenged with virulent S. mutans before weaning and developed significantly fewer caries than did their infected controls. These findings clearly suggest that passively derived IgG or IgA antibodies to S. mutans are protective against dental caries.


Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Fibronectin, a plasma protein immunologically identical with a major surface protein of normal fibroblasts, was found to bind to collagen and gelatin. A solid phase enzyme immunoassay was used for the binding tests. Collagen, gelatin or various control proteins were adsorbed to a plastic surface. Binding of fibronectin was detected using purified fibronectin antibodies conjugated to alkaline phosphatase. Circulating fibronectin and fibronectin obtained from fibroblast cultures both showed specific binding to collagen and gelatin. Preparative affinity chromatography of plasma on gelatin coupled to Sepharose gave electrophoretically and immunologically pure fibronectin in high yields. Malignantly transformed fibroblasts lack surface fibronectin. Our findings suggest the possibility that this results in a lack of anchorage to the surrounding intercellular matrix, which could contribute to the malignant growth behavior.


The effect of azathioprine on host cell infiltration and growth of a murine fibrosarcoma. Azathioprine (AZ) was injected into C57 Black mice before or after IM implantation of the syngeneic fibrosarcoma FS6, and observations were made on tumour growth and on the cellular composition of the fibrosarcomas. When multiple injections were given to the tumour-bearing host the tumours regressed. The percentage of tumour-associated macrophages remained essentially stable as regression occurred whereas the percentage of theta-antigen-positive cells increased. In the case of single IP injections of AZ, changes in the cellular composition of tumours were seen only when AZ was given 1-72 h before implantation but not when it was given after implantation. Compared with controls there was a 2- to 3-week delay in the appearance of large numbers of tumour-associated macrophages. The percentage of theta-antigen-positive cells within the tumours mass was elevated 7 days post AZ treatment but the level declined to control values between days 10 and 13. The significance of the results is discussed in relation to the dependency of the macrophage content of this tumour upon the development of an immune response towards the tumour.


A proliferation gradient in three-dimensional colonies of cultured human glioma cells. The growth of colonies of human glioma cells cultured in agarose has been studied. The mitotic activity and the fraction of cells which incorporated tritiated thymidine decreased nearly exponentially with the depth in the colonies, the shape and inclination of the proliferation gradient being independent of the colony diameter. This explains why the volume doubling times were rather similar for colonies of different sizes. It is also shown that the stability of the gradient during growth gave rise to nearly exponential growth, although a large part of the centrally situated cells were out of the cell cycle or had a reduced proliferative rate. The incorporation of tritiated thymidine into labelled cells was similar in the peripheral and the central regions as evaluated from grain counting. This indicated that the length of the S-phase was constant.


Inhibition of the growth of lymphoid tumours in syngeneic athymic (nude) mice. A comparison of the growth of a number of BALB/c tumours in BALB/c, BALB/c.nu/ + (heterozygous nude) and BALB/c.nu (homozygous) nude mice has shown that a majority of BALB/c lymphoid tumours grow at a significantly reduced rate in BALB/c.nu mice. The three non-lymphoid BALB/c tumours (carcinomas and sarcomas) tested and a minority of lymphoid tumours, however, grew as well, or better, in the BALB/c.nu mice. Furthermore a correlation, without exception to date, has emerged from a comparison between the susceptibility of a tumour cell line to in vitro lysis by spleen cells from syngeneic nude mice and its growth in vivo in syngeneic nude mice. Only those lymphoid tumour cell lines lysed in vitro show the reduced growth rate in syngeneic nude as compared to syngeneic normal mice. Tumour cell lines, both lymphoid and non-lymphoid, which are resistant to in vitro lysis do not show the reduced in vivo growth rate in syngeneic nude mice. Both the in vivo and in vitro effect can be partially abolished by irradiating the nude mice prior to use,  suggesting that a radiation-sensitive non-T-cell surveillance of lymphoid tumours is operating in nude mice. While the identity of the antigens is still unknown, the specificity of the phenomena suggest that C-type RNA oncogenic viral determined antigens are involved.


Sequential evaluation of cutaneous delayed hypersensitivity responses to recall and to lymphoid cell line antigens in Burkitt's lymphoma. Delayed cutaneous hypersensitivity reactions to standard recall antigens (candidin, mumps and PPD), to crude membrane extracts of a cell line derived from Burkitt's lymphoma (Raji) and to cell line derived from normal lymphocytes (F265) were sequentially evaluated in 44 patients with Burkitt's lymphoma. Sixteen patients (36%) manifested delayed hypersensitivity responses to the standard antigens and seven (16%) to the Raji membrane extract at presentation. Following successful chemotherapy, there was prompt and significant improvement of reactivity to both the standard and Raji antigens (p greater than 0.001), suggesting that the initial impairment of delayed hypersensitivity was most likely related to tumor burden. By 9 months after treatment, all patients in sustained remission expressed reactivity to Raji and 21 of 22 to the standard antigens. None of the patients skin-tested with the F265 extract at presentation gave a positive response and only one subsequently expressed reactivity after remission was induced. On relapse, reactivity to the standard antigens was more readily lost (4 of 11) then reactivity to the Raji extract (1 of 7). Pretreatment delayed hypersensitivity to the standard antigens also correlated better with long-term survival than to pretreatment responses to Raji. It remains to be determined whether the antigens expressed in the Raji extract are indeed tumor-specific or related to Epstein-Barr virus.


Tumor necrosis and cell detachment.

Tumor necrosis and cell detachment. Cell detachment from and around Walker 256 tumors grown in rats is quantitated by a standardized in vitro shaking procedure. The volume of cells detached from cancers of similar size is virtually the same in tumors growing in the liver, the spleen, in intramuscular and in subcutaneous sites. In cystic tumors, greater volumes are detached from the innermost regions of the walls adjacent to necrotic material than from the outermost parts. More liver perenchymal cells are shaken free of liver adjacent to a tumor interface than from regions 0.5 and 1.0 cm distant from it. Detachment of tumor and liver cells is also enhanced by prior incubation of tissue samples with necrotic extracts. It is suggested that the necrotic regions of tumors, and products derived from them, facilitate the detachment of tumor cells and cells composing the normal tissues surrounding them, thereby potentially promoting metastasis and invasion.


Asbestos and lung cancer: an analysis of the epidemiological evidence on the asbestos-smoking interaction. Three simple models for the asbestos-smoking interaction on human lung cancer production are considered. In the first model the excess incidence of lung cancer independently due to asbestos and to smoking adds together when both agents are present (additive model). In the second the addition of each one of the two agents produces an effect (increase in lung cancer incidence) which is proportional to the effect of the other (multiplicative model). In the third, asbestos can only increase lung cancer incidence in the presence of smoking. As previously found by other investigators, the additive model appears the least plausible in the light of the data from two published epidemiological studies. A discrimination between the other two models is attempted through a detailed analysis of the five published epidemiological studies today available which provide information on occupational asbestos exposure, smoking habits and lung cancer risk. Although the data do not allow a definitive discrimination, the multiplicative model appears to be more plausible, being also consistent with a multi-stage carcinogenic mechanism and with evidence from animal (rat) experiments. It is relevant both for biology and for public health that in this model asbestos and smoking are regarded as independently capable of producing lung cancer in humans and that they act synergistically when exposure to both occurs.


Increased ferritin synthesis and release by Hodgkin's disease peripheral blood lymphocytes. Peripheral blood lymphocytes from patients with all stages of untreated Hodgkin's disease and from normal healthy adults were shown to synthesize and release ferritin in vitro. Ferritin synthesis was confirmed by immunoelectrophoresis, double immunodiffusion and autoradiography. Hodgkin's disease lymphocytes synthesized ferritin 4.2 times faster and released it 2.4 times faster than did normal lymphocytes, whereas total protein synthesis was faster in normal lymphocytes. Patients with nodular sclerosis and perhaps those with absence of fever had the highest synthetic rates; however no relationship was observed between relative rates of lymphocyte ferritin synthesis and sex, age, anatomical stage and presence of splenic or hepatic involvement by tumor. Addition of iron to normal human lymphocytes produced little or no change in ferritin synthesis. These data indicate that part of the intracellular ferritin detected in peripheral blood lymphocytes from patients with Hodgkin's disease and from normal individuals resulted from de novo synthesis rather than from uptake and storage of serum ferritin, and suggests that elevated ferritin levels detected in the serum and tumor tissue of Hodgkin's disease patients originate from lymphocytes.


Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. II. Is the complement receptor necessarily present on the killer cells? The effect of various lymphocyte depletion techniques on SLMC effector cell activity against the cell line K562 could be attributed to the effects that these procedures had on the proportions of Fc/C3 receptor-bearing cells in the preparations. depletion of cytotoxic activity by removal of Fc receptor-bearing cells could be augmented by the presence of complement on the 7S-EA indicator cells used for rosette depletion. Two methods of assessing the presence of surface receptors on cytotoxic cells are presented: (1) linear regression analysis of spontaneous cytotoxicity plotted as a function of the proportions of receptor-bearing cells remaining after various depletion techniques; and (2) the use of a mathmatical formula to estimate the proportion of cytotoxic cells with any particular receptor, based on the effects of receptor-bearing cell depletion. Linear regression analysis of cytotoxicity vs the proportions of SRBC, Fc or C3 receptor-bearing cells demonstrated that all cytotoxic cells had the Fc receptor, but that cytotoxicity could still occur in the absence of cells with the C3 receptor. This indicated that the C3 receptor is not necessary for the demonstration of SLMC activity. The mathematical formula to predict cytotoxicity after depletion of C3 receptor-bearing lymphocytes showed, however, that a significant proportion of the Fc receptor-positive killer cells were also C3 receptor-positive. No direct evidence was found to support a role for T cells in SLMC, although E rosette depletion resulted in lower than expected cytotoxicity in view of the number of Fc receptor-bearing cells remaining in the preparation.


Natural cell-mediated cytotoxicity in mice against non-lymphoid tumor cells and some normal cells. Lymphocytes from normal mice were found to have cell-mediated cytotoxicity, in a short-term 51Cr release assay, against a variety of non-lymphoid tumor cells as well as against lymphomas. Some of the non-lymphoid tumors were as susceptible to natural cytotoxicity as the standardly used lymphoid lines. Some tissue culture cell lines and in vivo passaged tumor lines were susceptible to lysis, as were some primary virus-induced lymphomas. Tumor which arose in nude mice, which have high levels of natural cytotoxic activity, were all resistant to lysis. In addition to the susceptibility of transformed cells to natural cell-mediated cytotoxicity, some untransformed cultured cells and cells from normal tissues were targets for this mechanism. Low levels of cytotoxicity were seen with normal thymus cells, bone-marrow cells, and short term cultures of macrophages, whereas normal spleen and lymph-node cells were completely resistant to lysis. These results indicate a broader spectrum for mouse natural cell-mediated cytotoxicity reactivity than has been previously recognized.


Soluble tumor antigen-induced lymphocyte proliferation: effects of serum from normal and tumor-bearing mice. The effect of serum from mice bearing methylcholanthrene-induced fibrosarcomas on the lympho-proliferative responses of their spleen and lymph-node cells to 3 M KCl-solubilized tumor antigens has been investigated. Low concentrations of serum (less than or equal to 2.5%) collected throughout the course of tumor growth were inhibitory. Normal sera collected from age-matched control mice were often slightly inhibitory, but to a significantly lesser extent at identical serum concentrations. The inhibitory effect of tumor-bearing serum was not limited to homologous tumors, but was also observed when cross-reacting syngeneic tumor antigens or serum from animals bearing other syngeneic tumors were added to the assay mixture. At the low concentrations used, tumor-bearing sera inhibited only tumor-antigen-induced proliferation, and not proliferation induced by T cell mitogens. The inhibitory effects of tumor-bearing sera on lympho-proliferative responses to soluble tumor antigens appear to be similar to the inhibition seen as "serum blocking" in other assays of tumor immunity.


The binding of benzo(alpha)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine to subnuclear fractions of AKR mouse embryo cells in culture. The marked localization of a carcinogenic polycyclic aromatic hydrocarbon, benzo(alpha)pyrene, and its metabolites and a carcinogenic alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, to a specific subnuclear fraction (fraction I) from AKR-2B mouse embryo cells in culture is described. Fraction I is isolated by sucrose gradient centrifugation of sheared nuclei from cells exposed to the carcinogens. The association of tritiated benzo(alpha)-pyrene to fraction I consisted of loosely associated radioactivity which is extractable by organic solvents, and of tightly bound (termed "covalently" bound) radioactivity which is not extractable by organic solvents. Increases in the extent of metabolism of benzo(alpha)pyrene and in the amount of "covalently" bound radioactivity occur with increasing periods of incubation of the cells with the labelled carcinogen. This observation, together with the fact that these increases are dramatically reduced by inhibiting polycyclic aromatic hydrocarbon metabolism (using the inhibitor 7,8-benzo-flavone), suggests that a time-dependent metabolism of benzo(alpha)pyrene is required for "covalent" binding to muclear material. Data are presented suggesting that a two-step reaction may be involved in the binding of benzo(alpha)pyrene to subnuclear macromolecules. The fraction I localization of such structurally diverse chemical carcinogens as benzo(alpha)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine suggests that this fraction may localize all species of chemical carcinogens and that this localization may be involved in the chemically induced malignant transformation of cells.


Morphology and pathogenesis of tumors of the thymus and stomach in Sprague-Dawley rats following intragastric administration of methyl nitrosourea (MNU). Intragastric intubation of MNU (20 mg/kg twice weekly for 9 weeks) into young Srague-Dawley rats resulted in a 100% incidence of thymic lymphomas and gastric carcinomas in animals surviving more than 14 weeks. Progressive thymic atrophy followed the successive MNU administration culminating in marked lymphoid depletion by the 5th week of MNU treatment. At 6 weeks, repopulation of the thymus with lymphoblastic cells was observed, which progressed to thymic neoplasia. The first thymic lymphoma appeared at the 6th week. By the 15th week all rats were either moribund or had succumbed to anoxia caused by pleural effusions associated with thymic neoplasia. Twenty-six of 31 thymic tumors were classified as lymphocytic lymphomas, 2 as histiocytic, 1 as mixed lymphocytic-histiocytic, 1 as bimorphic and 1 as an epithelial thymoma. All but 2 lymphomas were restricted to the thymus. Of these 2 (both histiocytic lymphomas), 1 involved the spleen, liver and abdominal lymph nodes and the other infiltrated locally into cervical and thoracic regions. Significant thymic enlargement occurred only after MNU treatment was stopped (MNU acting as an anti-cancer drug). The gastric carcinomas were restricted to the non-glandular portion of the stomach and developed as a final step following ulceration, hyperplasia and neoplastic transformation. The short latency period and the high incidence of thymic neoplasms represent valuable criteria for future use of this model for pathomorphogenetic as well as immuno- and chemotherapeutic investigations.


ADCC effector cells in a murine adenocarcinoma. I. Evidence for blood-borne bone-marrow-derived monocytes. The origin and identity of effector cells for anti-body-dependent cell-mediated cytotoxicity and/or growth inhibition (ADCC) found in the murine mammary adenocarcinoma T1699 were investigated. Non-phagocytic, non-adherent ADCC effector cells were identified in bone marrow, peripheral blood and the host fraction of the tumor but not from lymphoid organs. These cells were shown to be distinct from the specific growth-inhibitory "armed monocytes" also identified in situ. The ADCC effector cells possess low levels of EA Fc receptors and appear to express a macrophage-associated antigen. Both the bone-marrow and the in situ effector cells became phagocytic after overnight incubation, providing further evidence for the monocyte-macrophage lineage of these cells. The results suggest that bone-marrow-derived blood-borne monocytes are the principal ADCC effector cells localizing in the mammary adenocarcinoma T1699. The parallels between tumor-induced immunity and cellular resistance to infectious agents involving hypersensitivity reactions and blood-borne monocytes are discussed.


An analysis of the G-banded chromosomes of the golden hamster. Chromosomes from 20 G-banded karyotypes of golden hamster cheek pouch cells were precisely measured to determine total lengths and arm ratios. Mean values were used to establish an idiogram which may serve as the basis for a standard hamster karyotype.


Inferior vena cava ligation versus the Mobin-Uddin filter for prevention of recurrent pulmonary embolism. Sixty-nine patients (37.5%) presented recurrent pulmonary embolic phenomena. Of these, 62 patients underwent interruption of the inferior vena cava by total surgical ligature (11 patients) or partial interruption by means of a transvenous Mobin-Uddin umbrella filter (51 patients). The mortality was 0.9% in the ligature group and zero in the Mobin-Uddin filter group. Eight patients developed mild to moderate late sequelae in the inferior extremities following ligature. No sequelae were observed following the filter method. None of the patients treated by either form of inferior vena caval interruption developed evidence of recurrent pulmonary embolism. In our opinion, pulmonary perfusion isotopic scanning is the most accessible and reliable method for the diagnosis and follow-up of recurrent pulmonary embolic disease. The Mobin-Uddin inferior vena cava filter is an effective device for the control of recurrent pulmonary embolism, relatively easy to apply and highly satisfactory for the management of the seriously ill patient.


Amebic peritonitis.

Amebic peritonitis. Fifteen cases with generalized amebic peritonitis have been described. Early surgical intervention combined with intensive resuscitation and specific chemotherapy has given good results. Mortality associated with colonic perforation may be reduced by performing staged procedures. Chances of survival may be improved through early diagnosis by suspecting an amebic cause in cases of generalized peritonitis in endemic areas.


Intraperitoneal rupture of amebic liver abscess. The traditional management of amebic liver abscess which has ruptured into the peritoneal cavity has been conservative and 70% to 90% fatal. Laparotomy with complete toilet of the abscess and the peritoneal cavity has shown encouraging results. A comparative study of the management of 36 cases of intraperitoneal rupture of amebic liver abscess was carried out. Twenty-one cases were treated by laparotomy with 85.5% survival. Mortality for 15 cases treated conservatively was 80%. We believe that intraperitoneal rupture of amebic liver abscess can now be treated by laparotomy with much better results than were possible before the advent of drugs such as metronidazole.


Beneficial effect of omental wrapping of unsafe intestinal anastomoses. An experimental study in dogs. The effect of omental wrapping on the safety of intestinal anastomosis was studied experimentally in 12 dogs. In six animals serving as controls, complete division of the small intestine and immediate restoration of continuity was carried out, after rendering the intestinal segment ischemic by ligating the mesenteric vessels for a distance of 10 cm. In a second group of animals, the ischemic anastomosis was wrapped with a pedicled strip of omentum. Gangrene and peritonitis developed in all animals of the control group, whereas the omental graft afforded complete protection against leakage from the devascularized anastomosis and peritonitis in the test animals.


Renal toxicity. Tobramycin and gentamicin. The new aminoglycoside, tobramycin sulfate, has been shown to be effective against the same microorganisms for which gentamicin is currently employed. This study compares the effects of these two antibiotics on renal function and ultrastructure in guinea pigs. Significant differences in the blood urea nitrogen of treated and nontreated groups, as well as between the gentamicin- and tobramycin-treated groups were observed. Gentamicin produced greater, more rapid elevations of the blood urea nitrogen, although all elevations were reversible. The ultrastructural pathology produced by tobramycin was similar to that previously described for gentamicin.


Ureterocolostomy. An antireflux technique. A new antireflux ureterocolonic urinary conduit was developed and evaluated in sheep. It combined the principles of the Wallace conjoined ureterointestinal conduit with the Lich extravesical ureteroneocystostomy. The procedure accomplished the following with a single anastomotic technique: (i) joined both ureters to the intestinal stump; (ii) provided constant ureteral orifice position in the antimesenteric butt of the intestinal segment; (iii) eliminated the blind intestinal stump; (iv) prevented intestinoureteral reflux; and (v) allowed an average of 12 times the normal urinary flow rate before exceeding mean human midureteral peristaltic pressure. The projected long term animal experiments and the sound urologic surgery principles involved indicate that the procedure may soon have clinical application.


The effect of caffeine on uretheral motility. An in vivo ureteral perfusion technique combined with a recently described methodology was used to study the effects of caffeine on ureteral motility patterns. Effects of the drug on the disused ureteric stump were also noted. The results showed a dose-related inhibition to complete cessation of peristalsis. Proposed mechanisms of caffeine effect on the ureter are discussed.


Studies on the neurophysiology of the vas deferens. In order to investigate the mechanism of sperm transport along the genital ducts, intraluminal pressure of isolated segments of the vas deferens was recorded in vivo. Responses to filling and mechanical as well as pharmacologic and electric stimulation of the autonomic nervous system were monitored. Contraction waves were initated in response to the stretch of filling and from mechanical stimulation. Pharmacologic response was variable. Low doses of alpha-adrenergic stimulant produced an increase in frequency of the contraction wave. Large doses of the same drug induced stroking contraction of the entire vas. alpha-Blocking drugs did not alter the rhythmic activity of the vas. beta-Adrenergic stimulation blocked peristaltic activity while administration of parasympathomimetic drugs increased the force of contraction. Electric stimulation of the hypogastric nerve produced strong sustained contractions. These data suggest that, whenever stretched, the vas deferens responds by regular peristaltic waves of low amplitude. These peristaltic waves can be enhanced by sympathomimetics or electric stimulation of the sympathetic system. The contents of the vas are propulsed into the urethra through strong rhythmic contractions of the entire vas.


Changes in canine bladder perfusion with distension. Using a radioisotope-labeled microsphere technique, canine bladder perfusion was investigated. Blood flow was measured in in flaccid and distended conditions. A slight decrease in total bladder blood flow was recorded. There was a relative decrease in bladder mucosal flow with distension. There was no difference in perfusion of dome versus trigone.


Prostatic tissue and secretion concentrations of rosamicin and erythromycin. Experimental studies in the dog. In dogs with normal and infected prostate glands, a new antibacterial substance, rosamicin, chemically a lipid soluble basic macrolide similar to erythromycin but with a better activity against Gram-negative bacteria, was tested, and compared to erythromycin in normal dogs. The two antibiotics were administered by constant intravenous infusion for 4 hr and their concentrations in plasma, urine, prostatic tissue, and secretion were determined by bioassay technique. Rosamicin was highly concentrated in the secretion of both the normal and the infected prostate gland, although in the latter the antibiotic levels were significantly lower. Erythromycin was significantly less concentrated than rosamicin in the prostatic secretion from normal prostates and was also less concentrated in normal prostatic tissue. Inasmuch as rosamicin has an improved antibacterial spectrum, which is well suited for the treatment of bacterial prostatitis, it should be investigated in this respect.


Abnormalities in pelvic visceral nerves. A basis for neurogenic bladder in the diabetic Chinese hamster. In order to determine whether bladder dysfunction and hydronephrosis in diabetic Chinese hamsters are associated with nerve pathology, the pelvic visceral nerves of diabetic and normal hamsters were examined with histochemical and electron microscopic techniques. Acetylcholinesterase activity was reduced in the nerves and on smooth muscle fibers in the urinary bladder of diabetic hamsters when compared to controls. Depression of enzyme staining was most marked in those hamsters with the most severe hydronephrosis. Frequent examples of aberrant myelination were found in the pelvic plexus and urinary bladder of diabetics. Many of these myelinated fibers exhibited wide periaxonal spaces lined by unusual processes of Schwann cells. An increase in the number of microtubules in axons and circular profiles of Schwann cells, which failed to enclose axons, gave evidence of axonal degeneration or Schwann cell injury in diabetic nerves. These findings suggest that pathologic changes in pelvic visceral nerves may underlie urinary bladder dysfunction in the diabetic Chinese hamster.


Evaluation of methods for perfusing rat testes. Organ perfusion methods offer a number of advantages in biologic studies but require full characterization before application. Two new methods for perfusing rat testes were characterized and compared with recirculating hemicorpus system. These preparations, selective and isolated testicular perfusion, are nonrecirculating and consequently, allow direct measurement of testosterone secretion. In both systems, testosterone production was a fuction of the dose of human chorionic gonadotropin in the perfusion medium up to 1000 mIU per ml which appeared to be inhibitory. The isolated testis method, in comparison with the selective, is more sensitive to human chorionic gonadotropin, requires less perfusion medium, maintains normal blood flow rates and water content, and is associated with no ischemia at commencement of perfusion. However, this system does not retain normal levels of ATP and GTP after 3 hr of perfusion. Whereas both procedures may be used for studies of testosterone secretion and androgen receptors, the inability to maintain testicular ATP and GTP levels indicates that present methods are not suitable for study of processes dependent upon high energy phosphate metabolism.


Inhibition of urease by miscellaneous ions and compounds. Implications for the therapy of infection-induced urolithiasis. One hundred forty-eight drugs and other organic and inorganic substances were screened for their ability to inhibit the enzyme urease in an in  vitro system modeled on infected urine. The reported urease-inhibiting properties of ascorbic acid, tetracyclines, and sulfanilamide were not confirmed. At least 50 per cent inhibition was observed in the presence of kanamvcin, hydroxguanidine, benzoquinone, 1,2-naphthaquinone-4-sulfonate, chloramine-T, N-bromoacetamide, copper, mercury, and fluoride. It is, however, unlikely that therapeutically effective concentrations can be attained in urine without giving dosages likely to result in toxic effects. Hydroxyurea, at the dose level used in cytotoxic therapy, may be expected to produce effective inhibition of bacterial urease in the urinary tract, providing renal function is unimpaired and providing urinary volume does not exceed 1 liter per 24 hr. Acetohydroxamic acid is potentially the most useful drug for the treatment of infection-induced urinary stone disease available at present.


Inhibitory effects of urinary macromolecules on the crystallization of calcium oxalate. Protective (inhibitory) effects of human urinary macromolecules on the crystallization of calcium oxalate were found when these urinary macromolecules were added to water or to salt solutions, which mimic human urine with respect to the concentrations of the most abundant small molecular weight species. The aspects of calcium oxalate crystallization affected by urinary macromolecules were: (i) an inhibition of nucleation (increased metastable limit), (ii) an inhibition of the crystallization rate, (iii) a decrease in the supersaturation level, and (iv) a decrease in adhesion of crystals to container surfaces.


[Conjugal psoriasis and its relation to histocompatibility antigens. A family study]. The higher risk of the occurrence of psoriasis vulgaris in the progeny of conjugal psoriasis marriages was repeatedly reported. In this paper a family, where the parents and both the children are suffering from psoriasis is described. The disease occurs also in some of the blood relatives of the father and mother. The occurrence of the disease and the segregation of the HLA antigens was followed in the kinship. It seems that a genetic factor linked to the HLA system influences the developement of psoriasis vulgaris.


[Tinea nigra plantaris].

[Tinea nigra plantaris]. A second case of tinea nigra occuring in Germany is recorded. The patient was a 33 year old women, who supposedly acquired the infection in Israel, which was not diagnosed until two years after the onset. Scrapings from a dark macule on her sole yielded Cladosporium werneckii. The lesion cleared with topical treatment of a keratinolytic ointment and a haloprogin-cream.


[Fibrous papules of the nose--a clinical and histologic entity?]. Twelve patients with 19 fibrous papules of the nose were studied. Usually the papules appear domeshaped and are flesh colored or reddish-brown. The majority of the lesions are asymptomatic and firm, a few of them are soft. The microscopic changes of the fibrous papules of the nose were usually those of fibromas or angiofibromas with increased cellularity of the upper parts of the cutis. The cell shape varied greatly and included dendritic, stellated and strap-shaped forms as well as multinucleated giant cells, similar to those of juvenile melanomatas. It is considered that the fibrous papule of the nose is a special form of regressive nevus cells.


[A case of circumcision tuberculosis].

[A case of circumcision tuberculosis]. A case of primary tuberculous complex following ritual circumcision is described. Long-lasting ulceration of lymph nodes required tuberculostatic therapy.


Histochemical studies of noradrenergic innervation of the liver in untreated and daunomycin-pretreated guinea-pigs. The pattern of noradrenergic innervation of guinea-pig hepatic tissue was examined by using the fluorescence method of Falk et al. (1962). The distribution of monoaminergic nerve fibers in untreated animals was clearly evident at the level of the portal vein, the hepatic artery and the bile duct. The hepatic artery was the most densely innervated. Mast cells and Kupffer cells had no connection with the adrenergic fibres. In daunomycin-pretreated animals, the hepatic cells appeared to be deep red in colour owing to the presence of the antibiotic. Some noradrenergic nervous fibres, never previously described, were unexpectedly distinguishable in the liver parenchyma against the background red colour of daunomycin-pretreated tissue. They appeared to be contiguous to the hepatocytes and were of different shapes: comma-like, V-like, or like a string of pearls. The nature and the functional role of these elements is briefly discussed.


Atypical staphylococcal mastitis in a dairy herd. A herd of cattle with a history of increased prevalence of clinical and nonclinical mastitis was investigated. Bacteriologic analysis of milk samples indicated approximately 50% of the herd was producing milk containing coagulase-positive staphylococci. Of these staphylococcal isolates, 55% had characteristics consistent with those of human strains of staphylococci, based on hemolysin production and phage patterns. Human beings in contact with the herd were nasal carriers of these staphylococci, which produced a granulartype coagulase reaction in bovine plasma, rather than the usually expected clot-type reaction. In the herd, the staphylococci caused mainly nonclinical mastitis, which was largely unresponsive to antibiotic therapy.


Effect of three anthelmintics on weight gain of feedlot cattle. Three groups of 50 heavy feedlot steers were treated with 1 of 3 anthelmintics (haloxon, tramisol, or thiabendazole). One group of 50 steers was not treated and served as a control. The initial degree of infection with gastrointestinal nematodes was light and decreased during the course of the study. There were no significant differences in weight gains among any of the groups at the end of a 113-day feeding period.


Suspected infectious necrotic hepatitis (black disease) in Oregon cattle. Suspected infectious necrotic hepatitis (black disease) in a herd of 436 cattle in Douglas County, Oregon, resulted in 79 deaths during a 2-week period. Although Clostridium novyi could not be isolated from hepatic lesions, the clinical course of the disease, gross and histopathologic findings, and fluorescent antibody identification of C novyi in various tissues were suggestive of the disease. The epizootic was preceded by a long drought, during which grazing conditions were sparse. A few days before the 1st dead animal was found, the drought was relieved by about 10 cm (4 in) of rainfall, resulting in the growth of young succulent grass. The cattle, attempting to eat this new grass lying close to the ground, consumed large quantities of soil. It was speculated that the soil contained C novyi and that the proliferation of these ingested organisms in necrotic tissue cuased by Fasciola hepatica resulted in fatal toxemia.


Phycomycosis associated with encephalitis caused by Haemophilus somnus in a heifer. Numerous fungal hyphae resembling those of a phycomycete were found in thrombi, vessel walls, and areas of inflammatory cell infiltration within a large focus of necrosis in the brainstem of a 2-year-old heifer. Haemophilus somnus and Pasteurella multocida were isolated from the same lesion. Antemortem hyperglycemia was also demonstrated.


Myodegeneration associated with selenium-vitamin E deficiency in a pregnant heifer. One of group of pregnant Holstein-Friesian heifers became recumbent the day after it was transported approximately 100 km. The heifer died after 10 days of recumbency, in spite of symptomatic treatment, which included an injection of a selenium-vitamin E (Se-E) preparation on day 5. Serum creatine phosphokinase activity was 21,000 IU/L on day 3 and 6,290 IU/L on day 9. Gross and histopathologic findins were characteristic of Se-E deficiency. Hepatic selenium content on day 10 was low (0.13 ppm) in spite of the Se-E injection given earlier.


Bromide intoxication of horses, goats, and cattle. During the summer and fall of 1973, a few horses, goats, and cattle in the Napa Valley of California became intoxicated by bromide via the ingestion of volunteer oat hay that had been cut from a field treated with methyl bromide, a soil fumigant. The bromide content of the hay ranged from 6,00 to 8,400 ppm. Signs of intoxication were lethargy, weakness, and ataxia. Animals experimentally fed the contaminated hay developed signs of intoxication between the 7th and 9th days.


Disseminated septic meningitis in a mare. Physical, neurologic, and clinicopathologic findings in a 3-year-old female Morgan horse with anorexia and nasal discharge suggested suppurative meningitis. Necropsy findings substantiated the tentative diagnosis. Actinomyces sp was cultured from a meningeal abscess surrounding the pituitary gland and from resolving lung abscesses.


Airborne mold survey--Edmonton.

Airborne mold survey--Edmonton. An outdoor airborne mold survey was recently carried out in the Edmonton area revealing some previously unrecognized molds, especially Rhodotorula. Hormodendrum was by far the commonest mold identified and was present all year round. It was also revealing that outdoor molds were quite prevalent in the Edmonton area throughout all seasons of the year but certainly more prevalent in the spring and early summer months. It would seem wise to have outdoor mold surveys done periodically in one's own area of practice when one is using immunotherapy with them.


Effect of triiodothyronine on bronchial asthma. II. Triiodothyronine (T3) administration to patients suffering from chronic bronchial asthma or patients suffering from nasopharyngeal allergy was reported to ameliorate the attacks. Twelve patients aged less than 35 years suffering from chronic bronchial asthma for more than 5 years, who were maintained on usual anti-asthmatic drugs with the exception of steroids and chromoglycate and who were not the subject of repeated attacks of bronchitis were chosen for the study. Each patient was given T3 orally as 40 micron/day divided into two doses, for a period of 60 days. The drug increased the peak flow in all cases. The increase averaged 24.6% +/- 8.48 (P less than 0.001). Three cases reported marked improvement and four were able to reduce the dose of anti-asthmatic drugs. There was no significant change in the pulse rate, ECG, body weight, blood pressure, or appetite. One patient complained of insomnia and another of increased anxiety. It is possible that T3 exerts its beneficial effect through correcting the level of C-AMP, which is known to be low in asthmatics, through improvement of body mechanism for antibody formation, or through other unidentified mechanisms.


Isolation of reconstitutively active succinate dehydrogenase in highly purified state. Existing procedures for the isolation of mammalian succinate dehydrogenase yield preparations of high purity or retain reconstitution activity, but not both. A new procedure is described for the isolation in good yield of virtually homogeneous preparations with full reconstitution activity, and retaining iron-sulfur center 3 and the "low Km" reaction site of ferricyanide. On reincorporation of the soluble enzyme into alkali-treated membranes, the same high turnover number (approximately 21,000/min at 38 degrees) is obtained in catalytic assays as with intact inner membrane preparations.


Resolution of some components of adenylate cyclase necessary for catalytic activity. Adenylate cyclase activity in a Lubrol 12A9 extract of wild type S49 lymphoma plasma membranes is completely inactivated by incubation at 37 degrees for 20 min. Activity is restored by mixing this heated extract of wild type membranes with an unheated detergent extract of membranes from a variant clone that lacks measureable adenylate cyclase activity (AC-). The factor(s) donated by the AC- extract is labile to heating at 30 degrees (t1/2 = 3 min) or to treatment with N-ethylmaleimide or trypsin. The factor(s) donated by the heated wild type extract is also sensitive to proteases or N-ethylmaleimide. This extract displays more complex inactivation kinetics at 50 degrees, consistent with the existence of separate factors necessary for the stimulatory effects of NaF and guanyl-5'-yl-imidodiphosphate. We suggest that at least two proteins are necessary for adenylate cyclase activity and that one of these is retained in the phenotypically adenylate cyclase-deficient variant.


Glycosyl moiety of the lima bean lectin. Pronase digestion of the lima bean lectin (Phaseolus lunatus (alpha-D-GalNAc greater than alpha-D-Gal)) afforded a glycopeptide of molecular weight 1380. Amino acid and carbohydrate analysis showed the glycopeptide to contain fucose, mannose, glucosamine, and aspartic acid in the molar ratios, 0.8:4.4:1.9:1.0. Methylation analysis, Smith degradation, and sequential degradation by specific glycosidases permit the formulation of a tentative structure:.


Lectins as probes of chromatin structure. Binding of concanavalin A to purified rat liver chromatin. Concanavalin A (Con A) binds specifically to rat liver chromatin. The extent of binding is directly proportional to both chromatin and concanavalin A concentration. It is reversible and inhibited by specific sugars for which concanavalin A has a binding site. Scatchard analysis reveals the presence of one type of Con A-binding site, with an apparent dissociation constant of 3 X 10(-7) M. A maximum of 10 pmol of Con A binds to 10 microgram of chromatin, indicating an average of one binding site/1400 base pairs of DNA. To identify the polypeptide chains which contain Con A-binding sites, chromosomal proteins were separated by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. Con A receptors were localized by incubating the gel in 125I-Con A and subsequent autoradiography. Three major polypeptide bands which bind Con A were identified among the nonhistone chromosomal proteins. The apparent molecular weights of these glycoproteins are 135,000, 125,000, and 69,000. We suggest that lectins may serve as probes for the study of the organization of specific components in chromatin.


In vivo metabolism of ceramides in rat brain. Fatty acid replacement and esterification of ceramide. Three double-labeled ceramides, [1-14C]lignoceroyl D-erythro-[1-3H]sphingosine, [1-14C]palmitoyl D-erythro-[1-3H]sphingosine and D-[1-14C]cerebronoyl D-erythro-[3-3H]-sphingosine, were prepared and injected separately into the brains of 18-day-old rats. The animals were killed after 2 h and various sphingolipids were isolated and purified. These lipids were further fractionated into subgroups depending on their fatty acid content (nonhydroxy or alpha-hydroxy, shorter chain or longer chain), 3H/14C ratios obtained in ceramides, cerebrosides, and sphingomyelins containing different fatty acids from the injected material were much higher than the initial ratio. This observation indicates that a replacement of the fatty acid occurred in the injected ceramide. Comparison of the 3H/14C ratios of the cerebrosides and sphingomyelin in a specific fatty acid group with the ratios of the corresponding ceramide indicated that most of these lipids were derived directly from the ceramide. The 3H/14C ratio of the hydroxycerebroside obtained from cerebronyl sphingosine-injected brain was nearly twice the ratio of the injected material. In addition to these lipids, considerable radioactivity was incorporated into an unknown nonpolar material in the brains which had been injected with [1-14C]lignoceroyl or [1-14C]palmitoyl [1-3H]sphingosine. This material was tentatively identified as ceramide fatty acid ester. The 3H/14C ratio of the ester was identical to that of the injected material.


Effect of glucagon on pinocytosis by the yolk sac of the rat. The uptake of macromolecular markers by fluid pinocytosis in the rat yolk sac was inhibited by glucagon, with half-maximal effect at a hormone concentration of approximately 3 X 10(-8) M. Glucagon had no effect on the cellular distribution of the marker subsequent to its uptake. Rates of uptake promptly returned to normal when the yolk sacs were transferred from a glucagon-containing to a glucagon-free medium. Epinephrine also inhibited, but only at much higher concentrations. The effect of the latter was augmented by theophylline. Insulin (10(-6) M) had no effect when added alone or with an inhibitory level of glucagon (10(-7) M). The presumption that the hormone effect was mediated by cyclic AMP was supported by the findings that the cellular levels of cyclic AMP were elevated in the presence of glucagon and that dibutyryl cyclic AMP could replace glucagon as an effective inhibitor. The conclusion that the hormone effect was on uptake rather than on subsequent regurgitation was based on the linearity of accumulation in both the presence and absence of glucagon and the inability of glucagon to stimulate loss of invertase from preloaded cells. Colchicine and vinblastine also inhibited uptake. This finding and those of others which are discussed suggest the possibility that effects of cyclic nucleotides on certain cell functions may involve their regulation of microtubular status.


Purification and characterization of a thiol:protein disulfide oxidoreductase from bovine liver. A thiol:protein disulfide oxidoreductase which degrades insulin into its A and B chains has been purified to homogeneity from bovine liver. The initial extraction procedure used 1% Triton X-100, which increased the soluble insulin degrading activity 100 to 800% as a result of solubilizing a membrane-associated enzyme. The criteria for homogeneity of the isolated protein include sodium dodecyl sulfate and disc gel electrophoresis. Characterization of the homogeneous protein by sedimentation equilibrium centrifugation indicates that a single protein is present which has a mass of 60,000 daltons. A single amino-terminal end group and molecular weight estimation by sodium dodecyl sulfate gel electrophoresis indicated that the thiol:protein disulfide oxidoreductase consists of a single polypeptide chain. The pure enzyme was also examined by gel filtration chromatography and has an apparent molecular weight of 92,000. The amino acid composition and carbohydrate content were also determined. The carbohydrate analysis demonstrated the presence of D-mannose, D-galactose, 2-acetamido-2-deoxy-D-glucose, and N-acetylneuraminic acid which comprise a total of 12% by weight of the isolated protein.


Cystine requirement of continuous human lymphoid cell lines of normal and leukemic origin. A number of continuous, human lymphoid cell lines from healthy and leukemic donors have been studied. Those from healthy individuals were found to be cystine prototrophs (cys+), while those from leukemic donors were observed to be cystine auxotrophs (cys-). The former can grow in either cystine or cystathionine-containing media. The latter grow only in cystine-containing medium. The former contain readily measurable levels of cystathionase, the last enzyme in the mammalian cysteine biosynthetic pathway, while the latter contain much less of this activity. The apparent molecular weight, overall charge, pH versus activity, Km, and [pyridoxal phosphate] versus activity properties of the enzyme from both cell types were indistinguishable. No soluble cystathionase inhibitor could be detected in cys- cell lines. Thus, it is reasonable to hypothesize that those leukemic cell lines studied synthesize less active cystathionase protein than their cys+ counterparts and that this difference in enzyme production underlies the differential growth properties in cystathionine-containing medium.


Studies on bovine adrenal estrogen sulfotransferase. Inhibition and possible involvement of adenine-estrogen stacking. The inhibition of bovine adrenal estrogen sulfotransferase has been studied utilizing representative androgens and estrogens with structural changes in rings A, B, and D. These investigations have shown oxygen functions in positions 3, 16, or 17 to be required for the binding of estrogens or androgens to the enzyme. 5alpha-Androstanes (all transfused rings) are weak noncompetitive inhibitors and bind more tightly than the 5beta isomers (cis-fused A and B rings). The competitive inhibition observed with the estrogens does not require a free 3-phenolic hydroxyl, however, groups larger than 3-methoxy limit binding. While the product, estrone sulfate, is not inhibitory, phosphate esters on positions 3- or 17beta or a sulfate moiety on the 17beta-hydroxyl group of estrogens slightly inhibit the enzyme. The stacking of adenine (in adenosine 3'-phosphate-5'-phosphosulfate) with the A ring of estrogens is postulated for the enzyme-bound transition state. This structure, which facilitates the hydrogen bonding between the 6-amino group of adenine and the 4-nitro, 4-bromo, or 6-keto substituents on estrogens, readily explains the unusual substrate and inhibitory properties of these compounds. Certain of these estrogen derivatives, which possess little or no hormonal activity, are efficient inhibitors of estrogen sulotransferase.


Hydrogen isotope effects in the cyclization of D-glucose 6-phosphate by myo-inositol-1-phosphate synthase. myo-Inositol-1-phosphate synthase (EC 5.5.1.4) from rat testis, Acer pseudoplatanus L. cell culture and Oryza sativa L. cell culture, converted D-[5-3H]glucose 6-phosphate to myo-[2-3H]inositol 1-phosphate at rates ranging from 0.21 to 0.48 that of unlabeled substrate. D-[3-3H]- and D-[4-3H]glucose 6-phosphate were converted at approximately the same rate as that of unlabeled substrate. In the case of testis enzyme, storage as a frozen solution further lowered the rate with D-[5-3H]glucose 6-phosphate as substrate. When the reaction was run in [3H]water, no 3H appeared in myo-inositol 1-phosphate but a small amount was recovered in substrate isolated from the final reaction mixture. These data support the involvement of carbon 5 of D-glucose 6-phosphate in the mechanism proposed for this conversion.


Formation of alpha-1,2-mannosyl-mannose by an enzyme preparation from rabbit liver. Rabbit liver microsomes catalyzed the formation of 2-O-alpha-D-mannosyl-D-mannose when incubated with GDP-mannose and free D-mannose. Formation of the disaccharide required the addition of divalent metal ions and was inhibited by EDTA and guanosine nucleotides. An enzyme present in the microsomal preparation transferred mannose from GDP-mannose to an endogenous lipid and to exogenous dolichol-P resulting in the formation of a product tentatively identified as dolichol-P-mannose. Formation of the mannosyl-lipid was also metal ion-dependent and was inhibited by EDTA and guanosine nucleotides. A second enzyme in the preparation catalyzed mannosyl transfer from dolichol-P-mannose to free mannose resulting in the formation of 2-O-alpha-D-mannosyl-D-mannose. Transfer from the mannosyl-lipid required the addition of Triton X-100 but was metal ion-independent. The time course of the transfer of mannose from GDP-mannose to the endogenous lipid and to the exogenous saccharide acceptor suggested that the mannosyl-lipid was an intermediate in the formation of the mannobiose. Other evidence, such as similar requirements for divalent metals, inhibition by guanosine nucleotides, and acceptor specificity, also supported the involvement of a mannosyl-lipid as an intermediate in the formation of alpha-1,2-mannosyl-mannose linkages by rabbit liver microsomes.


Determination of the primary structure of cholera toxin B subunit. The primary structure of cholera toxin B subunit, responsible for the binding of the toxin to cell surface ganglioside Gm1, has been determined as: Thr-Pro-Gln-Asn-Ile-Thr-Asp-Leu-Cys-Ala-Glu-Tyr-His-Asn-Thr-Gln-Ile-His-Thr-Leu-Asn-Asn-Lys-Ile-Phe-Ser-Tyr-Thr-Glu-Ser-Leu-Ala-Gly-Lys-Arg-Glu-Met-Ala-Ile-Ile-Thr-Phe-Lys-Asn-Gly-Ala-Thr-Phe-Glu-Val-Glu-Val-Pro-Gly-Ser-Gln-His-Ile-Asp-Ser-Gln-Lys-Lys-Ala-Ile-Glu-Arg-Met-Lys-Asn-Thr-Leu-Arg-Ile-Ala-Tyr-Leu-Thr-Glu-Ala-Lys-Val-Glu-Lys-Leu-Cys-Val-Trp-Asn-Asn-Lys-Thr-Pro-His-Ala-Ile-Ala-Ala-Ile-Ser-Met-Ala-Asn The number and the sequence of amino acid residues has been established by separation and analyses of the peptides obtained through enzymatic and chemical cleavage of the B subunit. The chymotryptic peptides from the citraconylated B subunit proved to be useful in the alignment of the tryptic peptide.


Covalent structure of the beta chain of cholera enterotoxin. The complete amino acid sequence of the beta chain of cholera enterotoxin was determined: NH2-Thr-Pro-Gln-Asn-Ile-Thr-Asp-Leu-Cys-Ala-Glu-Tyr-His-Asn-Thr-Gln-Ile-His-Thr-Leu-Asn-Asn-Lys-Ile-Phe-Ser-Tyr-Thr-Glu-Ser-Leu-Ala-Gly-Lys-Arg-Glu-Met-Ala-Ile-Ile-Thr-Phe-Lys-Asn-Gly-Ala-Thr-Phe-Gln-Val-Glu-Val-Pro-Gly-Ser-Gln-His-Ile-Asp-Ser-Gln-Lys-Lys-Ala-Ile-Glu-Arg-Met-Lys-Asn-Thr-Leu-Arg-Ile-Ala-Tyr-Leu-Thr-Glu-Ala-Lys-Val-Glu-Lys-Leu-Cys-Val-Trp-Asn-Asn-Lys-Thr-Pro-His-Ala-Ile-Ala-Ala-Ile-Ser-Met- Ala-Asn-COOH. The sequence was obtained from automated sequence analysis of intact beta chain, cyanogen bromide fragments, and a fragment generated by cleavage at a single tryptophan with 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine as well as from manual sequence analysis of tryptic peptides using 5-dimethylaminonaphthalene-1-sulfonyl-monitored Edman methodology. The tryptic peptides accounted for all 103 residues established for the complete primary structure.


An electron microscope study of the interaction between fructose diphosphate aldolase and actin-containing filaments. The interaction of fructose diphosphate aldolase with F-actin, F-actin-tropomyosin, and F-actin-tropomyosin-troponin has been studied by using negative staining. In the absence of troponin, minor aggregates of aldolase and the F-actin filaments are formed. A well-ordered lattice structure is only formed in the case of the fully reconstituted filament when the filament-to-filament spacing is 18nm, and the cross-bridge spacing is 38.7 nm. Evidence is presented that the lattice is due to an interaction between troponin and aldolase. The minimum subunit structure of troponin, still capable of giving rise to a lattice, is the troponin-IT complex, which indicates that troponin-C is not involved in aldolase binding.


A mode of formation of tubular myelin from lamellar bodies in the lung. A mechanism is suggested by which the membranes of lamellar bodies are converted to tubular myelin (TM) in the lung. It is argued that a simple corrugation of the membranous sheets can produce the TM formation. Such corrugation would occur in response to simple stresses acting on the lamellar body membranes. The intersections of the tubular figures are formed by fusion of adjacent corners in the corrugations. This results in a more stable hydrophobic bonding of phospholipid molecules. Strong supportive evidence for the mechanism is given by electron micrographs of TM formations.


The proliferative response of hepatic peroxidomes of neonatal rats to treatment with SU-13 437 (nafenopin). The repeated administration of the hypolipidemic agent Su-13 437 (nafenopin) to neonatal rats roughly doubled the number of peroxisomes in the liver tissue and caused a sixfold volumetric expansion of the peroxisomal compartment. During the proliferative response, the size-distribution of the peroxisomes was reversibly altered, enlarged particles appearing in numbers varying according to the dose given. By means of a new method for quantitative autoradiography, it was shown that (a) the concentration of silver grains over peroxisomes was comparable to that found over the endoplasmic reticulum; (b) the peak incorporation of [3H]arginine into the peroxisomes was dealyed in comparison with that into the endoplasmic reticulum; (c) the label, once incorporated into the expanding peroxisomal compartment, displayed the same shift to large particles as did the whole population. These results are compatible with the biosynthetic pathway for peroxisomal catalase proposed earlier (cf. reference 12), and with the notion that the drug-induced size-shift might have resulted from progressive growth of a particular class of peroxisomes formed in the presence of the agent. Evidence is presented to show that during the recovery period the larger peroxisomes are removed preferentially.


The probable role of phosphatidyl cholines in the tannic acid enhancement of cytomembrane electron contrast. Unsaturated natural and synthetic phosphatidyl cholines (PCs), when treated with tannic acid and OsO4, demonstrated a substantial increase in contrast as compared to PC treated only with OsO4. This was not observed when phosphatidyl ethanolamine (PEA) was similarly exposed to tannic acid. The increased electron density observed in the lamellar organization of the PC phospholipids was limited to the hydrophilic layers corresponding to the polar regions of the phospholipid molecules. The repeating periods of lamellae were identical in PC, treated with both tannic acid and OsO4, and when treated only with OsO4. In each case, this approximated 45 A. The enhancement of membrane contrast by tannic acid in the presence of OsO4 is interpreted as being at least in part due to its multivalent capacity, binding to reactive sites on choline, as well as with OsO4.


Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly. Two structures on the distal ends of Chlamydomonas flagellar microtubules are described. One of these, the central microbutule cap, attaches the distal ends of the central pair microtubules to the tip of the flagellar membrane. In addition, filaments, called distal filaments, are observed attached to the ends of the A-tubules of the outer doublet microtubules. Inasmuch as earlier studies suggested that flagellar elongation in vivo occurs principally by the distal addition of sublnits and because it has been shown that brain tubulin assembles in vitro primarily onto the distal ends of both central and outer doublet microtubules, the presence of the cap and distal filaments was quantitated during flagellar resorption and elongation. The results showed that the cap remains attached to the central microtubules throughout flagellar resorption and elongation. The cap was also found to block the in vitro assembly of neurotubules onto the distal ends of the central microtubules. Conversely, the distal filaments apparently do not block the assembly of neurotubules onto the ends of the outer doublets. During flagellar elongation, the distal ends of the outer doublets are often found to form sheets of protofilaments similar to those observed on the elongating ends of neurotubules being assembled in vitro. These results suggest that the outer doublet microtubules elongate by the distal addition of subunits, whereas the two central microtubules assemble by the addition of subunits to the proximal ends.


Fibroblast-collagen interactions in the formation of the secondary stroma of the chick cornea. Fibroblasts invade the primary corneal stroma of the 6-day-old chick embryo eye. The way in which these cells build the secondary stroma has been studied by microscope examination of the stroma during the subsequent 8 Days. Eyes were embedded in low viscosity nitrocellulose, and 30-micrometer tangential sections of cornea were cut and stained with azan (giving blue collagen and red cells). These sections were sufficiently thick to include enough cells and collagen for stromal organization to be visible under Nomarski optics. Three days after invasion, the fibroblasts extend along collagen bundles in the posterior region of the stroma; surprisingly, fibroblasts near the epithelium are more rounded. The collagen itself is organized in orthogonal bundles rather than in sheets. Measurements show that posterior bundles increase in size with time while anterior stroma si similar in diameter to primary stroma. These observations confirm that the epithelium continues to deposit primary stroma up to at least the 14th day. They show, moreover, that fibroblasts deposit collagen fibrils on extant stroma and that the farther a bundle is from the epithelium, and hence the longer the period since it was first laid down, the wider it is likely to be. Analysis of the results and existing data on hyaluronic acid levels in the stroma suggests that Bowman's membrane, the region of anterior stroma that remains uncolonized by cells, is, during this period at least, primary stroma laid down but as yet unswollen.


Adult rat hepatocytes in primary monolayer culture. Ultrastructural characteristics of intercellular contacts and cell membrane differentiations. Primary monolayer cultures were obtained in 60-mm petri dishes by incubating 3 X 10(6) isolated hepatocytes at 37 degrees C in Dulbecco's medium supplemented with 17% fetal calf serum. The ultrastructure of monolayer cells was examined after various incubation periods. Within 4 h of plating, the isolated spherical cells adhere to the plastic surface, establish their first contacts by numerous intertwined microvilli, and form new hemidesmosomes. After 12 h of culture, wide branched trabeculae of flattened polyhedral cells extend in all directions. Finally, after 24 h of culture, bile canaliculi are reconstituted, and a biliary polarity is recovered: the Golgi elements, which are scattered throughout the cytoplasm in the isolated cells, are reassembled in front of the newly formed bile canalculi, symmetrically in the adjacent cells; lysosomes are concentrated in that region, and microtubules reappear. Concomitantly, plasma membrane differentiations, namely desmosomes and tight junctions, develop. Tight junctions sealing the bile ducts constitute a barrier to the passage of ruthenium red and horseradish peroxidase. De novo formation of these junctions was studied by the freeze-etching technique: 10-nm particles compose a network of anastomosed linear arrays in the vicinity of the bile canaliculi; in the next step of differentiation, the particles fuse, form short ridge segments and finally continuous branched smooth strands, characteristic of the mature tight junction.


Lectin-binding sites on the plasma membranes of rabbit spermatozoa. Changes in surface receptors during epididymal Maturation and after ejaculation. MODIFICATIONS IN RABBIT SPERM PLASMA MEMBRANES DURING EPIDIDYMAL PASSAGE AND AFTER EJACULATION WERE INVESTIGATED BY USED OF THREE LECTINS: concanavalin A (Con A); Ricinus communis I (RCA(I)); and wheat germ agglutinin (WGA). During sperm passage from caput to cauda epididymis, agglutination by WGA drastically decreased, and agglutination by RCA(I) slightly decreased, although agglutination by Con A remained approximately unchanged. After ejaculation, spermatozoa were agglutinated to a similar degree or slightly less by Con A, WGA, and RCA(I), compared to cauda epididymal spermatozoa. Ultrastructural examination of sperm lectin-binding sites with ferritin- lectin conjugates revealed differences in the densities of lectin receptors in various sperm regions, and changes in the same regions during epididymal passage and after ejaculation. Ferritin-RCA(I) showed abrupt changes in lectin site densities between acrosomal and postacrosomal regions of sperm heads. The relative amounts of ferritin-RCA(I) bound to heads of caput epididymal or ejaculated spermatozoa. Tail regions were labeled by ferritin RCA(I) almost equally on caput and cauda epididymal spermatozoa, but the middle-piece region of ejaculated spermatozoa was slightly more densely labeled than the principal-piece region, and these two regions on ejaculated spermatozoa were labeled less than on caput and cuada epididymal spermatozoa. Ferritin-WGA densely labeled the acrosomal region of caput epididymal spermatozoa, although labeling of cauda epidiymal spermatozoa was relatively sparse except in the apical area of the acrosomal region. Ejaculated spermatozoa bound only a few molecules of ferritin-WGA, even at the highest conjugate concentrations used. Caput epididymal, but not cauda epididymal or ejaculated spermatozoa, bound ferritin-WGA in the tail regions. Dramatic differences in labeling densities during epididymal passage and after ejaculation were not found with ferritin-Con A.


A retarded rate of DNA replication and normal level of DNA repair in Werner's syndrome fibroblasts in culture. We investigated the cloning efficiency, DNA repair, and the rate of DNA replication in the skin fibroblasts from patients with Werner's syndrome (WS) of an autosomal recessive premature aging disease. Five WS strains exhibited normal levels of sensitivity toward X-ray and UV killings and repair of X-ray induced single strand breaks of DNA (rejoining) and UV damage to DNA (unscheduled DNA synthesis). The sedimentation of newly synthesizing DNA in alkaline sucrose gradients demonstrated a characteristic feature that only the elongation rate of DNA chains, estimated by the molecular weight increase, was significantly slower during early passages in WS cells than in normal Hayflick Phase II fibroblasts. In addition, plating efficiencies as well as the replicative potentials of five WS strains were more limited than those of normal cells under the identical culture conditions. It seems therefore that at least in the WS cells tested, the slow rate of DNA replication may be more related to the shortened lifespan and enhanced cell death, as manifestation of premature senescence at the cellular level, than be the DNA repair ability.


Increased messenger RNA from protein synthesis inhibited human fibroblasts. Numerous reports have demonstrated that specific protein synthesis in response to specific inducers is markedly stimulated by a simultaneous brief exposure to protein synthesis inhibitors such as cycloheximide. This phenomenon is known as "superinduction" and is most often attributed to the accumulation of cytoplasmic messenger RNA during the inhibition period. Messenger RNA, as defined by rapid labeling, oligo (dt)-cellulose binding, and cell free protein synthesis stimulation was measured in cycloheximide treated human fibroblasts. In spite of a consistent 40% decrease in total polysomal 3H-uridine labeled RNA, a 1.5- to 2-fold increase in extractable mRNA was observed. These data provide direct evidence that protein synthesis inhibition stimulates the appearance of cytoplasmic mRNA and/or completely blocks its degradation and, are consistent with the hypothesis that mRNA accumulation partly underlies the superinduction phenomena.


Selective lethal effect of thymidine on human and mouse tumor cells. Human cell lines derived from a melanoma and a colon carcinoma, and cultures of human melanocytes and intestinal epithelial cells, as well as a mouse mesenchymal non-neoplastic cell line and a malignant subline of the same have been quantitatively studied in tissue culture for their sensitivity to thymidine. All three tumor lines produced solid tumors when injected into nude thymus-deficient mice. No tumors were obtained by injecting cells of the human normal long-term cultures or the non-neoplastic mouse line. The tumor-producing lines showed a greater sensitivity to the lethal effects of high concentrations of thymidine than their non-tumor-producing counterparts. Less than 23% of the tumor cells survived 72 hours in the presence of 1 mg/ml of thymidine, in contrast to 60% or more of the non-tumor cells. Colony formation was much more inhibited by thymidine and the differential between normal and tumor cells was even more pronounced. Tumor cells which also were treated for 72 hours with 1 mg/ml of thymidine and then plated in fresh medium formed very few colonies. If the plating efficiency of the untreated controls is considered as 100%, 4.3% or less of the treated tumor cells formed colonies, in contrast to 33% or more of the non-tumor cells.


Responses of protein synthesis and degradation in growth control of WI-38 cells. The overall rates of protein synthesis and degradation in perfusion-grown WI-38 cells were followed in the three days after a stepdown in the serum concentration of the culture medium, from 10% to 0.3%. Within three hours after the stepdown, the rate of protein synthesis had decreased and the rate of protein degradation had increased, the combined result being the cessation of protein accumulation. The degradation rate returned over the next three days to its original value, but a zero rate of accumulation was retained because the synthesis rate continued to decline. The rate of DNA synthesis remained constant for six hours after the stepdown. It then declined steadily until reaching a minimum about eight hours later. The results show that extracellular control of protein accumulation depends on adjustments in both protein synthesis and protein degradation, and that the adjustments take place rapidly. This behavior suggests that the cell cycle is arrested after a stepdown because post-mitotic cells are unable to accumulate additional protein. However, an alternative interpretation of the data is that at least part of the changed accumulation is the result, rather than the cause, of the cycle arrest, and that the arrest is caused by other, more specific, reactions than those of general protein metabolism.


Cell surface glycosaminoglycans: identification and organization in cultured human embryo fibroblasts. A morphologically detectable cell coat, composed of glycoprotein, glycolipid, and glycosaminoglycan, is present on the external surface of most vertebrate cells. We have investigated the composition and organization of glycosaminoglycans in the cell coat of cultured human embryo fibroblasts by labeling cells with 3H-glucosamine and Na235SO4 and subsequently treating cultures with specific enzymes. Components released were identified by chromatography and specific enzymatic digestion. In situ incubation with leech hyaluronidase (4 microgram/ml) removed only hyaluronic acid from the cell surface whereas testicular hyaluronidase (0.5 mg/ml) removed both hyaluronic acid and chondroitin sulfate. Trypsin (0.1 mg/ml) released a large mass of glycopeptides in addition to hyaluronic acid, chondroitin sulfate, and heparan sulfate. The affinity of the cell coat for the cationic dye, ruthenium red, was reduced by leech hyaluronidase treatment. Sequential enzyme digestions of the cell surface showed that hyaluronic acid could be removed without the concomitant or subsequent release of sulfated glycosaminoglycans, suggesting that the hyaluronic acid is not a structural backbone for glycosaminoglycan complexes of the external cell surface.


Proliferative response of human diploid fibroblasts to intermittent light exposure. Three- to four-hour exposure to fluorescent light, one to three times weekly, reproducibly enhanced the proliferation rate of human diploid fibroblasts. This enhancement was observed in WI-38 and a line from whole embryo mince at late population doubling level (PDL) as well as in a line from adult skin at early PDL. Single or multiple exposures of short duration stimulated proliferation, whereas exposures of long duration were cytotoxic. This proliferative response is reversible, and is mediated through the culture medium, Dulbecco Vogt's supplemented with 10% fetal bovine serum. Apparently light produces some mitogenic substance(s) in the culture medium that accumulates in the cells and is toxic or growth-stimulatory depending on its concentration per cell. Another possibility is that light produces in the medium both cytotoxic and growth-stimulatory substances.


Parathyroid hormone and the hypercalcemia of immobilization. Serial measurements of serum calcium and immunoreactive parathyroid hormone (PTH) were performed in two young patients with hypercalcemia of immobilization. Serum PTH was elevated in both patients. With mobilization, both serum PTH and serum calcium returned to normal levels and remained so during six months of follow-up. The hyperparathyroidism of immobilization is an unexplained, reversible disorder that should be treated by medical measures and aggresive attempts at early mobilization.


Absence of hCG-like activity in the blood of women fitted with intra-uterine contraceptive devices. Blood was obtained at various stages of the menstrual cycle from 201 women fitted with intra-uterine contraceptive devices (IUDs), and plasma levels of hCG were determined by radioimmunoassay. Urine samples were obtained from 117 of these women and tested, before and after sephadex gel filtration, in a haemagglutination inhibition test for pregnancy (Pregnosticon). Plasma hCG was undetectable (less than 25 MLU/ml) in all but one of the 201 women and, in this instance, the hCG-assay appeared to be measuring a midcycle peak of LH, as evidenced by high plasma FSH levels. All unextracted urines gave negative results in the Pregnosticon test but, after extraction, 18 of the 117 urines gave positive reactions, most of these being from women at midcycle or in the luteal phase. We conclude that the IUD does not permit the development of the embryo to a point where it is capable of secreting measurable amounts of hCG. Recent claims to the contrary are probably due to  cross-reaction of LH or non-specific interference in the assays used for measuring hCG.


Failure of triiodothyronine to inhibit TSH-mediated thyroid hormone release in man. We studied the effect of short-term triiodothyronine administration on thyroid gland responsivity to exogenous thyrotropin in four euthyroid human subjects. Thyroidal iodine release and serum thyroxine during daily im injections of bovine TSH were not significantly inhibited, despite a four-fold elevation in serum T3 concentrations. This negative finding contrasts with earlier positive reports of a regulatory "short-loop" effect of elevated circulating T3 on the thyroid gland. This difference may be due either to the use in previous murine or in vitro studies of non-physiologic, high doses of exogenous T3, or failure to control the withdrawal of the trophic effect of endogenous TSH in man on the subsequent glandular response.


Potentiation of insulin action: a probable mechanism for the anti-diabetic action of sulfonylurea drugs. Insulin action was assessed by measuring insulin-mediated glucose disposal (KI) in 13 non-ketotic diabetic patients both before treatment and after normalization of plasma glucose by diet (5 patients) or sulfonylurea (glipizide) therapy (8 patients). While the KI of the diet-treated group was unchanged by therapy, that of the glipizide-treated group was significantly increased. The data show that chronic glipizide therapy is associated with a potentiation of insulin action, which may account for the major anti-diabetic effect of this drug.


A methodological study of factors affecting the judgment of misarticulated /s/. Two studies involving judgments of /s/ misarticulation are described. The first involved simultaneous audio and video recordings of three clinically different /s/ misarticulating children. Their recorded /s/ productions, embedded in 54 sentences with varying phonetic contexts, were judged on an acceptable/unacceptable/questionable basis. There was a large proportion of judgments in the questionable category, relatively low interjudge reliability, and few clear enhancing contexts. To better define the problems inherent in using recorded /s/ misarticulations, a second study was undertaken in which six specially chosen /s/ defective and eight normally speaking subjects spoke a set of 12 sentences containing a total of 24 /s/ target words. These productions were judged dichotomously as correct/incorrect under live conditions and from tape recorded conditions. Both normal and /s/ defectives were easily and reliably detected under live speaking conditions; judgments from tape recordings showed a reduction in reliability of judgment and reduced ability to detect the /s/ defective speakers' errors. Examination of the recorded /s/ defective productions revealed that certain phonetic contexts, especially adjacent apical stops, appeared to encourage judgments of correct /s/ productions. Thd discussion includes suggestion concerning techniques for sampling and judging /s/ misarticulations.


The effects of examiner race on the articulation responses of black school-aged children. Ninety 6-, 7-, and 8-year-old black children were administered the Watts Articulation Test for Screening by a white examiner and by a black examiner. The subjects' obtained significantly better articulation scores when the test was administered by the white examiner than when it was administered by the black examiner. Age of subjects and the interactions of age and sex, and of examiner age and sex were found to be significant.


Generalization of an operant remediation program for syntax with language delayed children. Generalization of the Monterey Behavioral Sciences Institute operant language program was assessed. Six male and three female children (4 years, 4 months to 6 years, 3 months) receiving language remediation were randomly assigned to (I) the Monterey program for the syntactic structure "is interrogative" (including the home carryover phase) plus an extended transfer program devised by the investigators (II) the Monterey "is interrogative" program alone (including the home carryover), or (III) a control activity (articulation training). Language samples evoked by a variety of language tasks were collected outside the treatment settling preceding and following treatment. Standard within-clinic measures indicated that groups I and II improved a significant, equal amount. However, the extraclinic language measures showed that group I demonstrated significantly greater improvement than group II; neither group II nor group III showed significant extraclinic improvement. Extraclinic generalization occurred, therefore, only for the group receiving the special extended transfer training.


Diagnosis and intervention of adult articulation errors based on distinctive feature theory and the Mysak feedback model of therapy. This article explores the applicability of distinctive feature theory to the phonological problems of high school and college students and to a feedback model of therapy. Results suggest that high school and college students with numerous substitutions have phonological errors that can be accounted for on a distinctive feature basis. Therefore, diagnosis of such students should include a distinctive feature analysis. Pilot data also indicate that a client can be taught to differentiate between correct and incorrect features rather than correct and error phonemes, and that distinctive feature theory is applicable to a feedback model of therapy.


The effect of task variables on speech during oral anesthesia. A normal, adult female performed a variety of speech tasks before and after a series of nerve-block injections that anesthetized the oral cavity. The tasks included diadokokinesis, imitation of unfamiliar Swedish phonemes, production of one-, two-, three-, and four-syllable words, and of two prose passages. Four experienced judges scored misarticulations. Intelligibility was determined in a series of listening studies involving college students. The highest percentage of misarticulations occurred on the single-syllable word list and the most complex prose passage. There were virtually no misarticulations on the two- or three-syllable words. Intelligibility tended to covary with articulation, but the specific nature of the relationship depended on the particular speech task. A misarticulation was especially likely to result in unintelligibility on the most difficult passage, although not all unintelligible words on that passage were attributable to phonemic errors.


Simultaneous communication with autistic and other severely dysfunctional nonverbal children. Despite its widespread application, the teaching of speech does not appear to be effective with all autistic and other severely dysfunctional, nonverbal children. A body of recent evidence points to the peculiarities in the information processing of these children and to the importance of gestures as aids or alternatives to speech. The present study reports on the use of simultaneous communication (gestures plus speech) with five such children. The fact that four of the five children showed marked gains in their ability to communicate over a 5-week period raises some theoretical and methodological issues pertinent to the treatment of autistic and autistic-like children and to our general understanding of the processes involved in nonverbal communication.


The distribution of 3H-leucine labeled protein in the retinula cells of the crayfish retina. The synthesis and distribution of 3H-leucine labeled protein was studied under conditions of diurnal lighting in the retinula cells of the crayfish retina with both light and electron microscopic autoradiography. Times ranging from two minutes to seven days after an intracardiac injection were analyzed. Quantification of the electron microscopic autoradiograms revealed that labeling of the cytoplasm was greater than the rhabdome at 2, 5, and 30 minutes and reached a peak at 12 hours. The rhabdome showed increasing activity after 5, 30, and 60 minutes, also reaching a peak at 12 hours. Radioactive label in cytoplasmic multivesicular bodies was higher than activity measured in either the total cytoplasm or rhabdome at all times except two minutes. Two temporally different microvillar labeling patterns were seen under diurnal lighting conditions. (1) Microvilli forming the slightly enlarged distal tip of the rhabdome retained their radioactivity at 1, 3, and 7 days, when labeling of the rest of the rhabdome microvilli was decreasing. (2) In the remainder of the microvilli, labeling at 1 and 12 hours appeared as a gradient which declined toward the proximal end of the rhabdome. This gradient subsequently reversed itself, showing heavier proximal labeling at three days. In a second experiment, labeling patterns in light and dark adapted rhabdomes were compared. In the dark, a distinct gradient of activity was observed with radioactivity concentrated distally and declining toward the proximal end of the rhabdome. A more even distribution of label was present in the light adapted eye, but a slight distal-proximal gradient was still present. The dark adapted rhabdomes had more radioactivity per unit area than those exposed to light.