Influence of Candida albicans glycoprotein on 3-methylcholanthrene induced malignancy in newborn rodents

In the experimental conditions reportedCandida albicans glycoprotein has a stimulating effect on the course of 3-methylcholanthrene carcinogenesis in newborn rodents. Stimulation of 3-methylcholanthrene carcinogenesis in newborn rats was found following injections of 9.5 µg/g and 18.5 µg/g ofCandida albicans glycoprotein. Subcutaneous tumors occurred in the experimental animals earlier, with higher frequency and attained larger dimensions than in the control animals. Similar effects were observed in mice. In the experimental mice there was also a significantly higher number of thymomas. From the evidence presentedCandida albicans glycoprotein appears to act as a cocarcinogenic substance to 3-methylcholanthrene.This work was supported partially byDora Kaplan, Joan Sloan, Cathy Cooper Memorial Funds and the Leo Roon Foundation.     

Electron microscope autoradiographic studies of RNA metabolism in Trillium erectum microspores

3H-Uridine has been used to investigate the sites of RNA synthesis in the post-meiotic G₁ phase of Trillium microspores using electron microscope autoradiography. The dilute, non-condensed component of the nucleus has been found to be the site of synthesis. When the labelled cells were further incubated in non-radioactive medium the label was found to shift towards the condensed chromatin regions within the nucleus. Two hypotheses to explain the observations are considered, one involving migration of the RNA from the relaxed to condensed regions, the other involving a change in state of the nuclear regions involved in the synthesis. The data are interpreted as favoring the latter possibility.This study supported by grants from the Jane Coffin Childs Memorial Fund for Medical Research and from the Swiss National Fund, Grant Nr. 3202.Fellow of the Jane Coffin Childs Fund for Medical Research.     

Seizure induction by pentylenetetrazol in rodents receivingCandida albicans glycoprotein orally

Oral administration of extracellularCandida albicaus glycoprotein produces increased proneness to seizures in mice and rats when tested with pentylenetetrazol in the experimental conditions reported.Dosages of 0.25 ug/g of body weight in mice and rats applied for a period of five weeks were enough to produce increased proneness to seizures. It appears that the substance is absorbed from the stomach or intestine and also that its detoxication or excretion is at such a slow rate that it accumulates. Introducing to the stomach much larger dosages could cause increased proneness to seizures to be obtained within six hours. Due to the frequent presence ofCandida albicans in humans and the high probability ofCandida albicans glycoprotein production in vivo, it is possible that the level of this substance may accumulate to produce increased proneness to seizures. The states of tenseness, increased sensitivity and hyperactivity could possibly occur in the same pathological process.A renewal of interest in the possible role of intestinal intoxication in human pathology appears to be indicated. Fungi which are harbored in the digestive tract could be the important source of such toxic substances.This work was supported partially byDora Kaplan, Joan Sloan, Cathy Cooper. Memorial Funds and the Roon Foundation.     

Blood changes and fertility derangements following the injection of Candida albicans into the spleen of the experimental animals (occurrence of hypersplenism)

Summary The injection ofCandida albicans (under experimental conditions used here) into the spleens of Wistar rats and Swiss mice produces profound hematological changes which are similar in rats and mice. After injection of the organisms there was steady lowering of the white cells and of the platelets in mice and in rats. Following splenectomy there was a sharp rise of both platelets and white cells in mice and rats. Mild normocytic, normochromic anemia was observed during the experiment. Following the splenectomy the level of hemoglobin returned to normal in fourteen weeks in the rats and in thirteen weeks in the mice. An increased level of reticulocytes and siderocytes was observed during the experiment. The mean value of the spleen weights in the experimental animals was higher than in the controls. Hyperplasia was the constant histological feature of these spleens. Bone marrow was either normal or hyperplastic. plastic. These changes correspond to the picture known in human pathology as hypersplenism. The injected organisms could still be cultured from the spleen about 4–6 months after operation. Attempts to isolate the injected organisms after 6 months were unsuccessful. The experimental mice showed derangement of fertility. Of the forty females mated with normal males only seven became pregnant. The litters were normal, 7–12 babies. Thirty percent of the young died before one month. Many of them showed growth retardation.This work was supported by a Damon Runyon Memorial Fund Grant No. 720.     

Glutamicin CBII,a bacteriocin-like substance produced by

Corynebacterium glutamicum CBII, in the stationary phase of growth, was found to produce spontaneously a substance resembling bacteriocins by its bactericidal properties. This substance designated glutamicin CBII was observed to exhibit bactericidal activity against coryneform bacteria (12 species tested) but not against unrelated gram-positive (3) and gram-negative (3) bacteria, while its action on bacteria with no quite known relatedness to the coryneform group (14) was found to be variable. Glutamicin CBII was partially purified by precipitation with ammonium sulphate (70% saturation), selective heat precipitation and gel chromatography on Sepadex G-50. The antibacterial substance diffused through cellophane membrane with an approximate cut-off of 10000 dalton and its sedimentation coefficient was determined to be 1.1. S by ultracentrifugation. Heating at 100°C for 30 min had no effect on its activity. Glutamicin CBII was proved to be resistant to chloroform, trypsin, chymotrypsin, pronase, and subtilisin. According to its staining behaviour and ¹H NMR spectra it probably represents a glycoprotein containing only a minor protein component.

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Action of light and gibberellic acid on the growth of excised embryos from Phacelia tanacetifolia seeds

Summary Germination of Phacelia tanacetifolia seeds is inhibited by light. Embryos freed of endosperm grow irrespective of light. However, if they are held in 0.3 M mannitol plus 2% sucrose, light sensitivity is reinstated: growth (i. e., germination) occurs in darkness but not in light. Gibberellic acid (GA₃) releases the inhibition due to light. These results suggest that (a) the photoreceptor site of the seed is in the embryo; (b) GA₃ acts directly on the embryo; and (c) darkimbibed embryos appear to have a higher water-uptake potential.This work was supported in part by the Jane Coffin Childs Memorial Fund for Medical Research, and by the U.S. Atomic Energy Commission under Contract No. AT(11-1)-1338.     

Seizure induction by pentylenetetrazol in animals experimentally infected with Candida albicans

Summary The prolonged presence ofCandida albicans in the spleen of experimental rats and mice predisposes them to pentylenetetrazol induced seizures. The seizure threshold of the experimental animals is lowered. This becomes statistically significant at ten months after the operation. In the experimental animals the latent period was sometimes reduced. The convulsions in experimental rats and mice were prolonged, sometimes repeated, and on three occasions (2 rats and 1 mouse) ended in death. Electroencephalographic studies confirmed the increased proneness to seizures in the animals to whichCandida albicans was introduced to the spleen.This work was supported by the Damon Runyon Grant no 720, and Joan Sloan Memorial Fund.     

Nutritional Requirement for Production of Antifungal Substance by Enterobacter aerogenes and Bacillus subtilis Antagonists of Phytophthora cactorum1

Anker's medium with glucose and Thornton's medium were most suitable for growing Enterobacter aerogenes and Bacillus subtilis respectively, antagonists of P. cactorum, the causal agent of apple crown rot. Calcium nitrate was thebest source of nitrogen for growing cultures of E. aerogenes and B. subtilis. E. aerogenes produced the maximum amount of antifungal substance at 200 and 400 mg/l of nitrogen in the medium. Phosphate supplied either in the potassium or calcium form did not change the growth of either antagonist. An addition of 200 mg/l of N and 400 mg/l of P significantly enhanced the production of antifungal substance by E. aerogenes on Anker's medium with glucose. Thornton's medium supplemented with 200 mg/l of N and 100 mg/l of P produced the maximum amount of antifungal substance from B. subtilis. Generally, soil extracts without enrichment did not support the growth of either antagonist; E. aerogenes required at least 400 mg/lof, both N and P while B. subtilis required 200 mg/l of N and 800 mg/l of P for the maximum production of antifungal substance. When ammonium phosphate was added to soil extracts, only a small amount of antifungal substance was produced by E. aerogenes and none by B. subtilis. These results indicate that E. aerogenes and B. subtilis need N and P fertilization of the sterilized soil for the maximum production of the antifungal substance that inhibits the growth of P. cactorum.     

Re‐evaluation of the role of a transmitting tract‐specific glycoprotein on pollen tube growth

It has been proposed that a stylar glycoprotein, the transmitting tissue-specific (TTS) protein isolated from Nicotiana tabacum, serves both as a growth stimulant, by providing a source of nutrients, and as an attractant for pollen tubes during their growth through the style. Working with a galactose-rich style glycoprotein (GaRSGP) that is the N. alata homologue (97% homology) of the TTS protein, a series of experiments, similar to those done with the TTS protein, was performed. Evidence was found for inhibition of pollen tube growth at high concentrations, but no evidence was found for stimulation of growth at concentrations up to 2 mg ml^–1. No effect as a pollen tube attractant was detected. The discrepancies in the features and functionality between these homologous glycoproteins in the closely related Nicotiana species warrants further investigation before a general function is assigned to these molecules.     

Metabolic properties of erythrocytes of normal and genetically anemic mice

The murine hemolytic anemias microcytosis (gene symbol mk), normoblastosis (nb), spherocytosis (sph), and hemolytic (ha) are inherited as autosomal recessive diseases and resemble the human hereditary hemolytic anemias caused by defective enzyme activities in erythrocytes. The activities of 14 different enzymes of the glycolytic and pentose phosphate pathways were compared in erythrocytes from normal and anemic mice, but no quantitative differences suggesting enzyme deficiency were found. There were no major changes in reduced glutathione, NAD, NADP, or methemoglobin content. The rate of entry of glucose into the glycolytic and hexose monophosphate shunt pathways of intact erythrocytes was higher in mk/mk erythrocytes than predicted. Interpretation of studies of erythrocytes from anemic mice is generally complicated by the extremely high reticulocyte and nucleated cell counts in ha/ha, sph/sph, and nb/nb mice.Investigations in Kentucky (Dr. Hutton) were supported by Research Career Development Award 1-K4-AM-70, 186-01 and NIH Research Grant AM 16013-01 from the National Institute of Arthritis and Metabolic Diseases, and those at The Jackson Laboratory (Dr. Bernstein) by NIH Research Grant HD-00254 from the National Institute of Child Health and Human Development, by U.S. Atomic Energy Commission Contract AT(30-1)-1800, and in part by the George W. Perkins Memorial Fund and by income from the Endowment Funds of The Jackson Laboratory. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

The effects of syntheticdl-dormin (Abscisin II) on the growth of the oat mesocotyl

Summary The growth-inhibitory properties of syntheticdl-dormin (Abscisin II) and itstrans, trans isomer were assayed using the oat mesocotyl section. Growth of this tissue was promoted by IAA and GA; dormin inhibited the elongation caused by both compounds and also the small amount of growth that occurred in blank buffer. A given concentration of dormin inhibited growth to about the same proportion in all these cases. IAA and GA mixtures stimulated growth in the presence of 2.0 mg/l dormin slightly more than the sum of growth with IAA+2.0 mg/l and GA+2.0 mg/l dormin. At lower concentrations of dormin (0.02 mg/l and below) the relationship was reversed. Dormin inhibited growth slightly at 0.02 mg/l but below that concentration it had no observable effect.Kinetin did not affect mesocotyl elongation, nor did it interact with the inhibition of IAA- or GA-stimulated growth.Thetrans, trans isomer had 1/30th the activity of the natural 2-cis-4-trans compount; when they were assayed together in mixtures of different proportions the inhibitions were additive.Most experiments were carried out with GA₃ but dormin also inhibited the action of gibberellins 1, 4, 5, 6, 7 and 9. The action of dormin was not competitive with that of IAA or GA.The effect of dormin as an inhibitor of plant growth is discussed.     

Isolation,and biochemical and biological characterization of an a-mating-type-specific glycoprotein responsible for sexual agglutination from the cytoplasm of a-cells,in the yeast Saccharomyces cerevisiae

An a-mating-type-specific substance responsible for sexual agglutination was purified to 397-times in specific activity (units/mg protein) from the cytoplasm of a-mating type cells. The purified substance gave a single band stained with PAS reagent but not with both Coomassie brilliant blue and silver staining reagent by polyacrylamide gel electrophoresis in the presence of 8 M urea. However, incorporation of [³⁵S]methionine and Lowry reaction clearly indicate that the substance is a glycoprotein. The substance specifically masked sexual agglutinability of cells of the opposite mating type , indicating univalent action. The substance is a glycoprotein with a carbohydrate content of 90%, a pI of 4.5, and a molecular weight of 130,000. The substance was inactivated by 2-mercaptoethanol and proteolytic enzymes but not by glycolytic enzymes. The substance formed a complementary complex having no biological activity when mixed with -agglutination substance from the wall or cytoplasm of -cells in vitro.Non-common abbreviations PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

Transformation-specific cell killing by a cancer-associated galactosyltransferase acceptor and cellular binding

Cancer-associated galactosyltransferase acceptor (CAGA glycoprotein), a small glycoprotein purified from human malignant effusion that selectively kills transformed cells, was tritiated by reductive methylation in the presence of NaB³H₄. CAGA-glycoprotein-sensitive cells (baby-hamster kidney cells transformed by polyoma virus and chick-embryo fibroblasts infected with Ts68 temperature-sensitive mutant of Rous sarcoma virus grown at 37°C, the permissive temperature) bound 3–5-fold more ³H-labelled CAGA glycoprotein than did their CAGA-glycoprotein-resistant non-transformed counterparts. The Rous-sarcoma-virus-infected chick-embryo fibroblasts grown at non-permissive temperature (41°C) bound an intermediate amount of ³H-labelled CAGA glycoprotein; however, this intermediate amount appeared to be sufficient to induce inhibition of cell growth when the infected chick-embryo fibroblasts treated at 41°C were switched to 37°C. Binding of ³H-labelled CAGA glycoprotein was time- and temperature-dependent and was not inhibited by monosaccharide. Binding was completely inhibited by the oligosaccharide liberated by endoglucosaminidase H treatment or by exhaustive Pronase digestion of intact CAGA glycoprotein. However, the isolated oligosaccharide failed to demonstrate the growth-inhibition characteristics of the intact glycopeptide. Binding of ³H-labelled CAGA glycoprotein was unaffected by co-incubation with the peptide core released by endoglucosaminidase H treatment. ³H-labelled CAGA glycoprotein bound to intact cells could be removed by trypsin treatment up to 4h after addition of the glycoprotein but not thereafter. This time course paralleled the decreasing reversibility of growth inhibition. However, all ³H-labelled CAGA glycoprotein was found in the supernatant when cells were first disrupted by sonication followed by trypsin treatment for up to 12h. ³H-labelled CAGA glycoprotein linked to Sepharose 4B failed to cause growth inhibition in CAGA-glycoprotein-sensitive cells. These findings suggest that binding of CAGA glycoprotein occurs via its oligosaccharide moiety. Binding appears to be a necessary but not sufficient condition to induce cell killing. Growth inhibition appears to depend on internalization of the glycoprotein and the presence of a transformation-specific cell process.     

Growth Stimulating Substance for Microorganisms Produced by Escherichia coli Causing the Reduction of the Lag Phase in Microbial Growth and Identity of the Substance with Pyrroloquinoline Quinone

The finding that most strains of microbes produce a growth stimulating substance for microorganisms was demonstrated and confirmed with the culture broth of Escherichia coli grown on a glucose-mineral medium. Addition of culture broth of E. coli to the culture media of the others markedly reduced the lag phase in microbial growth but not growth rate in the subsequent exponential phase nor the total cell yield in the stationary phase. The growth stimulation causing reduction of the lag phase was dependent on the amount of culture broth added. Occurrence of cell growth was essential for the excretion of the growth stimulating substance by E. coli. Under identical inoculum size, even with a heavy inoculum, a further reduction of the lag phase was observed by the addition of culture broth of E. coli. The substance was only effective at the initial growth phase but inert when the substance was added to a growing culture at the exponential phase. Finally, the substance was identified as pyrroloquinoline quinone, a newly established coenzyme, through chromatographic, spectroscopic and enzymatic criteria.     

A physicomathematical model of sodium balance

A model is introduced in which the reabsorption of sodium is governed by an enzymatic process. This process is in turn assumed to be influenced by the extracellular volume which depends on the amount of sodium in the body at a given time. The model allows for damped oscillations when the sodium intake lies within range of values and thus can account for observed oscillations. This investigation was supported by the U.S. Public Health Service (grants 5-K6-GM-18, 420 and A-4668), and by a grant from the Dr. Wallace C. and Clara A. Abbott Memorial Fund of the University of Chicago.     

In vitro drug screening system using membrane alteration in macrophages byMycobacterium leprae

The observation that liveMycobacterium leprae on entry into macrophages from lepromatous leprosy patients reduced the number of EA rosetting macrophages, was extended to macrophages from Swiss white mice also. Further, the fact that deadMycobacterium leprae do not bring about such a change in macrophages from mice, allowed us to develop this into a bacterial viability testing system. Thus drug treated macrophages in the presence ofMycobacterium leprae showed normal rosetting ability ifMycobacterium leprae are inactivated by the drug, but showed reduced level of rosetting when bacteria were not susceptible to the drug. It was shown that a drug like dapsone, does act onMycobacterium leprae based on its permeability, quantity available inside the macrophages and inhibition of its action by Para amino benzoic acid. The inactivation ofMycobacterium leprae by sulphone and rifampicin was also proved by the flourescence diacetate method, which showed poorly viable bacteria after exposure to drugs. Thus it has been possible to develop a rapid drug screening method for testing the activity of unknown compound againstMycobacterium leprae.     

Structure:function studies of receptors for thyrotropin and tetanus toxin: lipid modulation of effector binding to the glycoprotein receptor component.

¹²⁵I-labeled tetanus toxin interacts with the glycoprotein component of the thyroid thyrotropin receptor when this component is in solution or when it is incorporated into a liposome. Binding can be inhibited by both unlabeled thyrotropin and tetanus toxin but not by unlabeled prolactin, glucagon, insulin, ACTH, or growth hormone; binding can also be inhibited by a purified fragment of the glycoprotein component of the receptor. Changing the phospholipid of the liposome matrix from dipalmitoyl phosphatidylcholine to dioleoyl phosphatidylcholine significantly increases the binding of ¹²⁵I-TSH to the glycoprotein component of the receptor but does not affect ¹²⁵I-tetanus toxin binding.     

Bioavailability of tobramycin after oral delivery in FVB mice using CRL-1605 copolymer, an inhibitor of P-glycoprotein

Tobramycin is an aminoglycoside used in the treatment of infection against gram-negative bacteria. Tobramycin cannot be delivered orally probably due to efflux of drug by a P-glycoprotein pump in the brush border of the small intestine. In this report we demonstrate oral delivery of tobramycin in FVB mice using CRL-1605 copolymer as a vehicle. This copolymer is known to inhibit P-glycoprotein. Two different doses of tobramycin (25 mg/kg and 200 mg/kg) were used. The concentration of CRL-1605 copolymer was 132 mg/kg. The liquid formulation was fed to mice by gavage and serum tobramycin concentrations were measured after one and two hours using the fluorescence polarization immunoassay. We observed significant increases in serum tobramycin concentrations when the drug was delivered orally with the copolymer compared to when the drug was delivered alone. We also performed a bioassay using Bacillus subtilis to confirm antibacterial effect of tobramycin in mice sera. This was to ensure that tobramycin did not undergo structural change during oral absorption when delivered in the copolymer vehicle. We observed minimal inhibition in growth of Bacillus subtilis in sera obtained from mice fed with tobramycin alone. In contrast, we observed almost complete inhibition of growth (most specimens) in sera obtained from mice fed with tobramycin in the presence of CRL-1605 copolymer. We conclude that tobramycin delivered orally in mice using copolymer 1605 is also bioactive.     

Effect of a coat color locus on kidney lysosomal glycosidases in the house mouse

Activities of three lysosomal glycosidases, -galactosidase, -glucuronidase, and N-acetyl--hexosaminidase, have been shown to differ in bf/bf and bf/+ mice. Thus bf/bf mice usually have much higher activities of these enzymes in their kidney cells than bf/+ animals. There seem, however, to be some exceptions to this general pattern, especially for galactosidase of females from the C57BL/6J strain. A likely interpretation of the difference is that the bf locus has pleiotropic effects. An alternative explanation, less likely, is that a gene closely linked to bf is involved. There is also a differential response to dihydrotestosterone in different groups of mice reflected in activity changes of the three enzymes.This work was supported by the Hierta Memorial Fund and the Swedish Natural Science Research Council.     

Role of phospholipids in the structure and function of the thyrotropin receptor.

Phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine interact with 125I-thyrotropin and inhibit its binding to thyroid plasma membranes; phosphatidylcholine is not similarly effective. The interaction has been monitored by column chromatography on Sephadex G-100 which shows, for example, that 125I-labeled thyrotropin forms an adduct with phosphatidylinositol but not with phosphatidylcholine. Formation of the 125I-labeled thyrotropin-phosphatidylinositol adduct is dependent on the phosphatidylinositol concentration but can be reversed by both unlabeled thyrotropin and excess membranes. The efficacy of the phospholipid interaction and the phospholipid inhibition of thyrotropin binding to thyroid membranes is paralleled by changes in fluorescence and fluorescence polarization imposed on the 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of thyrotropin. These changes are reversed by unlabeled thyrotropin but not by prolactin, placental lactogen, or growth hormone; similar changes are not observed when phospholipids are incubated with dansylated growth hormone, prolactin, and placental lactogen. Monovalent potassium, sodium, and lithium salts neither prevent nor reverse the formation of the phospholipid-dansyl-thyrotropin adduct; these results contrast with the effects of the same salts on the formation of ganglioside adducts with dansyl-thyrotropin. Despite their ability to interact witw 125I-thyrotropin in solution, neither phosphatidylinositol, phosphatidylserine, nor phosphatidylethanolamine, when incorporated in a liposome, binds the 125I-labeled ligand. These same phospholipids have no effect on ganglioside binding of 125I-labeled thyrotropin when gangliosides are incorporated in a liposome. These phospholipids do, however, modulate the expression of the glycoprotein component of the thyrotropin receptor when it is imbedded in a liposome. The phosphatidylinositol in this case serves as a negative modulator, both by decreasing the incorporation of the glycoprotein component of the receptor into the liposome and by inhibiting the binding activity of the glycoprotein component which is incorporated. Speculation is offered as to a possible role of the phospholipids in the message transmission process which would be consistent with current studies demonstrating a direct interaction of acidic phospholipids with thyrotropin. The effect of phospholipids on liposomes containing the glycoprotein component of the thyrotropin receptor raises the possibility that phospholipids and, in particular, phosphatidylinositol, may also play a role in regulating the insertion and expression of this receptor component in thyroid plasma membranes.