Zinc binding properties of human prostatic tissue, prostatic secretion and seminal fluid

When studied by gel filtration, zinc in prostatic cytosol (10(5) g, 1 h) was associated with high (greater than 80000), medium (3000-80000) and low molecular weight (less than 3000) molecules in approximately equal proportions. The molecules of high and medium Mr were not secreted into the prostatic fluid where all the zinc was associated with molecules of low Mr (probably citric acid). After ejaculation much of the zinc is redistributed and becomes bound to molecules of high and medium Mr of vesicular origin.     

Detection and characterization of quorum sensing signal molecules in Acinetobacter strains

Quorum sensing is a widespread regulatory mechanism among Gram-negative bacteria. In this study, Acinetobacter strains were assayed for the presence of quorum sensing signal molecules capable of activating N-acylhomoserine lactone biosensors. By using an Agrobacterium tumefaciens reporter strain it was shown that all the cultures produced two to four detectable signal molecules with different chromatographic patterns. In A. calcoaceticus BD413 supernatants four compounds were detected in a time-dependent manner, and maximal activity was reached at stationary phase. The number of signal molecules was dependent on medium composition; typically, cultures in minimal medium displayed one or two more signals, as compared to complex medium. None of the Acinetobacter supematants showed autoinduction activity with an Chromobacterium violaceum reporter strain, neither in direct or competition assays.     

Stable, resealable pores formed in sea urchin eggs by electric discharge (electroporation) permit substrate loading for assay of enzymes in vivo.

We describe a simple electroporation procedure for loading suspensions of unfertilized sea urchin eggs with impermeant small molecules under conditions that allow close to 90% successful fertilization and development. Poration is carried out in a low-Ca2+ medium that mimicks the intracellular milieu. The induced pores remain open for several minutes in this medium, allowing loading of the cells; resealing is achieved by adding back millimolar calcium ions to the medium. While the pores are open, an influx of exogenous molecules and efflux of endogenous metabolites takes place, and the eggs can lose up to 40% of their ATP content and still survive. Introduced metabolites are utilized by the cells, e.g., introduced 3H-thymidine is incorporated into DNA. This procedure will be useful for loading impermeant substrates into eggs, permitting in vivo assessment of metabolism, and also for introducing other interesting impermeant molecules, such as inhibitors, fluorescent indicators, etc. Though the details may differ, the principle of electroporation in an intracellular-like medium may prove to be useful for loading other cell types with minimal loss of viability.     

The susceptibility of membrane vesicles to interaction with ethanol

The susceptibility of membranes to interaction with ethanol is an important consideration in the further understanding of the ethanol-membrane interaction. Interaction of membrane vesicles, including passive diffusion of ethanol across membranes, leakage of internal molecules out of membranes and membrane-membrane interaction, were examined systematically using two populations of fluorescent probe-encapsulated phospholipid bilayer vesicles, each prepared with 1,2-dimyristoyl phosphatidylcholine, cholesterol and a fluorescent probe. Fluorescence quenching experiments with these vesicles were performed in a medium containing a wide range of ethanol concentrations (0.30-3.5 M). In the presence of a lower concentration of ethanol in the external medium, passive diffusion of ethanol across membrane vesicles occurred. This was demonstrated by an interaction of ethanol with the encapsulated fluorescence probe molecules inside the vesicles, resulting in an increase in the fluorescence intensity and a shift of the fluorescence emission spectrum to a shorter wavelength. While, in the presence of a higher concentration of ethanol in the external medium, a strong perturbation of lipid bilayers by ethanol was found, leading to an over expansion of membranes and consequently causing the membrane leakage. As a result of this, the initially encapsulated probe molecules leaked out of the vesicles so as to interact with the other probe molecules in the external medium. Consequently, fluorescence quenching was observed. Moreover, studies of the mixture of two populations of fluorescence probe-encapsulated membrane vesicles revealed that ethanol acted on individual membranes and did not promote membrane-membrane interactions. The implication of the present results to the alcohol-mediated expansion of membranes is discussed.     

非洲爪蟾孵化酶分子对卵黄膜作用方式的研究

非洲爪蟾的孵化液对卵黄膜和二甲基酷蛋白具有降妥活性。用非洲爪蟾孵化酶的特异性抗ＧＳＴ－ＵＶ．２抗体进行Ｗｅｓｔｅｒｎ杂交的结果表明,孵化液中出现一种分子量为６０ｋＤ的大组分,有时也会出现一种分子量为４０ｋＤ的小组分。     

Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC3H1 cell line

We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC3H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, 2H, 13C, 15N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme (s = 7.40 +/- 0.04 S, n = 36). Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme (i.e. Km for ATP and EC50 for Mn2+ or Mg2+) did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control (cells in light medium). After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value indicating that the heavy isotope-labeled enzyme replaced the pre-existing light form of the molecule. These observations show that the rapid decrease in adenylate cyclase activity and the synthesis of heavy adenylate cyclase molecules are two separate events. The relative amounts of heavy and light components of forskolin-stimulated adenylate cyclase obtained in sucrose gradient differential sedimentation were determined as a function of time beginning 24 h after the transfer into the heavy medium. The decrease of the pre-existing light form could be represented by simple first order kinetics with a half-time of 40 h. This result suggests that the metabolic renewal of forskolin-stimulated adenylate cyclase is comparable to that of most plasma membrane proteins.     

Thermodynamic and spectroscopic features of the behavior of amphotericin B in aqueous medium

The interaction between amphotericin B molecules in aqueous medium solution was studied using absorption and circular dichroism approaches. The results showed that at concentrations below 1 microM of amphotericin B, an equilibrium between the monomer and aggregate occurred with a constant of approximately 0.6x10(6) M(-1). The aggregate formation constant was dependent on the experimental conditions of the medium: its value increased at acidic pH values, while alkaline medium induced the equilibrium displacement to the monomer formation. Either neutral salts or chaotropic agents such as urea prevented the formation of the aggregate. The presence of net electrical charge on the amine and carboxyl groups plays a role in the thermodynamic stability of the aggregate. A hydrophobic effect was also found between the monomer form and the water molecules of neighbours. In the aggregate formation water molecules were released contributing to an increase in the entropic change.     

Resonant interactions between biological molecules

Forces between localized, oscillating vibrations on molecules show strongly resonant dependence on the relative frequencies of the two oscillators; i.e. there is a force between the vibrating parts of two molecules if the frequencies are the same but none if the frequencies are different. A vibrating site on a large molecule can attract another specific type of molecule out of a medium, one which has a vibrational mode at the same frequency, without influencing other types of molecules in the medium. These resonant forces are thus highly selective. Other pairs of molecules could similarly interact using vibrations at a different frequency. In a complex medium such as a cell, many different types of interactions could be assisted by these resonant forces wtihout interfering with each other. These forces could be found to be useful in assisting some metabolic reactions. It is interesting that they are vulnerable to selective suppression or enhancement by externally applied electromagnetic radiation of correspondingly specific frequencies. Supported by NIH Grant #GM 24443-01 and Amer. Cancer Society Grant #IN17Q.     

Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules

When culture medium, conditioned by any of several cell types, is applied to a polycationic substratum, a substance is adsorbed that causes neurons cultured on that substratum to extend processes (neurites) rapidly and profusely. We have purified the factor responsible for this effect from medium conditioned by bovine corneal endothelial cells, and have shown that it is composed of the glycoprotein laminin and two associated laminin-binding molecules: a sulfated protein known as entactin, and a large heparan sulfate proteoglycan. Of these molecules, only laminin was found to be present throughout the purification in all fractions possessing neurite outgrowth-promoting activity and absent from all fractions lacking activity. Laminin, purified from other sources, has been shown previously to promote extensive outgrowth by cultured neurons. These and other data presented here support the conclusion that laminin is responsible for the neurite outgrowth-promoting activity of the conditioned medium factor. Evidence is also presented that the association of a proteoglycan with laminin promotes efficient attachment of laminin to polycationic substrata, particularly in the presence of competing molecules.     

Cell swelling induced by the permeant molecules urea or glycerol induces immediate high amplitude thyrotropin and prolactin secretion by perifused adenohypophyseal cells

The permeant molecules, urea and glycerol, evoked a prompt secretory burst of TSH and PRL when added to the extracellular medium of acutely dispersed anterior pituitary cells. Secretion of both hormones was proportional to the concentration of urea or glycerol between 26 and 104 mM (r greater than 0.89, P less than 0.001). Equivalent concentrations of the impermeant molecule, mannitol, did not induce secretion. The acute TSH and PRL secretory responses to TRH, hyposmolarity, and permeant molecules were qualitatively indistinguishable. These data support our hypothesis that cell swelling and resultant plasmalemma expansion is a potent inducer of hormone secretion. Since the secretory response to permeant molecules was not reduced in a Ca2+-free medium containing 0.1 mM EGTA, an increase in Ca2+ transport across the plasmalemma to raise cytosol Ca2+ concentration does not appear involved.     

Hormonal induction of tyrosine aminotransferase and RNA synthesis in the cells of Morris hepatoma strain 7777

The transfer of Morris hepatoma cells induced by the hormone within 10-60 min in to a hormone-free medium is associated with the augmentation of tyrosine aminotransferase synthesis. The kinetics of this process does not differ from that of the hormone-induced enzyme. The return of tyrosine aminotransferase synthesis to the basal level occurs 15-20 hours after the hormone withdrawal from the medium, although the concentration of the intranuclear hormone sharply decreases already after 3 hours. It was demonstrated that the presence in the hepatoma cell nuclei of 20-25% of the initially bound hormone for at least 20 hours after the cell transfer to the hormone-free medium is not sufficient for maintaining a high level of tyrosine aminotransferase gene expression. Using two-dimensional electrophoresis of 3H-labeled hepatoma cell proteins, it was demonstrated that the observed high activity of tyrosine aminotransferase is due to the de novo synthesis of enzyme molecules rather than to the existence of preformed long-living tyrosine aminotransferase molecules inside the cell. Study of [14C]uridine incorporation into non-ribosomal nuclear RNA of hepatoma cells showed a long-term presence of the label in the RNA throughout the chase experiment. It was assumed that the high activity of the enzyme for 10-15 hours after the hormone release from the hepatoma cell nuclei is due to the accumulation in the nuclei of long-living pre-mRNA molecules synthesized after the hormone addition to the cells and during the first hours after the cell transfer to the hormone-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiochemical studies on interactions between DNA and RNA polymerase. Unwinding of the DNA helix by Escherichia coli RNA polymerase.

In a medium containing 10mM Tris, pH 8, 10 mM MG++, 50 mM K+ and 10 mM NH4, the binding of an E. coli RNA polymerase holoenzyme unwinds the DNA helix by about 240 degrees at 37 degrees C. In this medium the total unwinding of the DNA increases linearly with the molar ratio of polymerase to DNA. The number of binding sites at which unwinding can occur is very large. If the K+ concentration is increased at 200 mM, the enzyme binds to only a limited number of sites, and the bound and free enzyme molecules do not exchange at an appreciable rate. The unwinding angle of the DNA per bound enzyme in this high salt medium is measured to be 140 degrees at 37 degrees C. The total unwinding angle for a fixed number of bound polymerase molecules per DNA is strongly temperature dependent, and decreases with decreasing temperature.     

Quantitative Effects of 2,4-Dichlorophenoxyacetic Acid on Growth of Suspension-cultured Acer pseudoplatanus Cells: II. Influence of 2,4-D Metabolism and Intracellular pH on the Control of Cell Division by Intracellular 2,4-D Concentration

The utilization of 2,4-dichlorophenoxyacetic acid (2,4-D) molecules by Acer pseudoplatanus cells is governed mainly by a glucosylation process. Evidence that 2,4-D glucoside molecules are biologically inactive is presented. 2,3,5-Triiodobenzoic acid (TIBA), by inhibiting 2,4-D glucosylation, has a sparing effect on 2,4-D molecules; thus TIBA treatments increase growth yield (expressed as the ratio of the maximum number of cells produced to the initial concentration of 2,4-D in the culture medium).     

The effect of metal ions on the synthesis of extracellular enzymes by sporulating bacteria]

The action of metal ions which are present in nutritious medium on the synthesis of extracellular enzymes by sporulating bacteria is analysed. An important role of these ions in post-secretory modification of protein molecules and formation of functionally active molecules of enzyme is shown. The effect of metal ions on some cell envelope properties and extracellular enzyme secretion is under discussion.     

Growth inhibitory factor diffusing from chick embryo fibroblasts

Density-dependent inhibition (DDI) of growth is assumed to be the result of diffusion in the medium of growth inhibitory molecules. In this work, we demonstrate the presence of inhibitory molecules (IDFc: chicken inhibitory diffusible factor) in the medium of chick embryo fibroblasts (CEF) cultures. IDFc partially purified by Bio-Gel P150 chromatography followed by reverse phase FPLC. The dose-response curve showed that 250 ng/ml IDFc inhibited 50% DNA synthesis. IDFc was also able to inhibit the growth of sparse cultures of CEF; this inhibition was reversible. IDFc was unable to prevent the DNA synthesis in cells transformed by v-src gene expression. These results suggest that IDFc is involved in the DDI of CEF growth.     

Relationships between Escherichia coli cells and the surrounding medium during survival processes

In Escherichia coli, during survival under adverse conditions, namely starvation and luminous radiation, two things occur. On the one hand organic substances are released into the surrounding medium and on the other there is a transition from the culturable state to viable but non-culturable (VBNC). An analysis of organic molecules released into the surrounding medium showed the presence of proteins, dissolved free amino acids, and dissolved monomeric carbohydrates. The concentration of these substances in the medium changed with exposure time, type of stress and type of molecule. The proteins accumulated in the medium and in some cases their identification revealed the presence of components of the outer membrane. Variations in the concentration of amino acids and carbohydrates point to a twofold process of excretion and uptake. Indeed, cell free supernatants supported the growth of several generations of a population of 10(4) cells ml(-1). The survival of E. coli in supernatants previously colonized by cells in the VBNC state was greater than that observed in the control experiments, with a short delay in the loss of culturability. It was thus clear that organic molecules released into the medium play a role in the transition from culturable to VBNC state.     

"Phantom" structure of solvents and packing of dual-chain nucleic acid molecules in liquid crystal dispersion particles

The properties of mesomorphic dispersions of double-stranded nucleic acids were studied. A comparison of these properties indicates that their diversity cannot be explained unambiguously in terms of the conception of Van-der-Waals interactions in particles of mesomorphic dispersions without regard for the specific properties of the solvent, water, in the vicinity of adjacent nucleic acid molecules. It was assumed that, with small distances between the molecules of nucleic acids, a specific "phantom" structure of the solvent appears in their vicinity, which acts as an elastic medium that modifies the interactions between nucleic acid molecules and as a medium in which a collective tunneling of protons can occur. The combination of the two effects determines the "recognition" of nucliec acid molecules and the stabilization of the cholesteric structure of mesomorphic dispersions of nucleic acids.     

Electron microscopy of ribosomal RNA mounted by adsorption in the absence of protein.

A method is described for visualizing ribosomal RNA in the electron microscope by an adsorption technique. The visual configuration of the molecules is sensitive to the composition of the medium from which they are adsorbed, and is reproducible under a given set of conditions. Regular structural features occur in the larger rRNA species similar to those observed with protein monolayer techniques. The dimensions of adsorbed molecules, however, are more consistent with current models of ribosomal RNA as it exists in solution.