The diffusion of gibberellins into agar and water during early germination of Pharbitis nil Choisy

Agar diffusion of imbibed seeds yielded significant amounts of diffusible Gibberellin-like substances. An analysis of the extractable and diffusible gibberellin-like substance, including an analysis of the remaining imbibition water of the seeds, indicated that a significant part of these gibberellin-like substances could be attributed to a net biosynthesis of these substances in the imbibing seeds. At the same time it was found that water diffusion yielded considerably more gibberellin-like activities than comparable agar diffusions i.e. 10 to 12 fold in general.Agar as well as water diffusion showed a temperature effect with regard to the yield of gibberellin-like substances particularly during the first 6 h of diffusion. The yield of these substances is lower at 10°C, and remains lower as shown with consecutive diffusions, in comparison with the yields at 20°C or 30°C.With both agar and water diffusion the sum of activities obtained with consecutive diffusions is always higher, often considerably higher, than equal periods of continuous diffusion which is probably due to inactivation and/or interference of inhibitory substances with the bioassay responses. Finally, water diffusates of both seeds and seedlings of the normal growing cv. Violet of Japanese morning glory contained considerably more gibberellin-like activities than those of the dwarf cv. Kidachi which indicated that normals synthesize more gibberellins than dwarfs.     

FURTHER CHEMICAL STUDIES ON BLOOD-AQUEOUS HUMOR DYNAMICS

The proportion of SCN, Br, PO₄, urea, levulose, and Fe which remains freely diffusible when added to plasma has been determined by ultrafiltration and "differential" dialysis through a cellophane membrane. After injecting each of these substances as well as Mg and Li into rabbits a continuous record of their concentration in the plasma was obtained for each animal and the concentration in the aqueous humor was also determined and related to the maximum diffusible concentration in the plasma.     

Determination of Extracellular Space in Amphibian Muscle

The volumes of distribution of inulin and dextran in the sartorius, stomach, and cardiac muscle of the frog agree rather closely. That these spaces represent the volume of extracellular water is supported by the observation that efflux of sucrose can be divided into a fast and a slow phase and that the fast-moving fraction corresponds closely with inulin space determined in the same muscle. These and other findings confirm that sugars and related substances penetrate slowly into part of the fiber water and that, therefore, their volume of distribution does not accurately represent the volume of extracellular water. The kinetics of efflux of sucrose is consistent with the assumption that the movement of sugars is determined by the resistance of the cell surface as well as by internal diffusion. In connective tissue, sucrose and inulin are excluded only from a small part of the total water.     

Molecular and cellular aspects of axon-glia interaction in CNS regeneration

The relationships of neurons and non-neuronal cells are vital for the maintenance and function of neurons. Trauma alters these relationships causing proliferation of non-neuronal cells and, in adult mammalian CNS, presumably disturbs the environmental support needed for regeneration. A supportive environment can be restored by introducing a regenerating nerve to injured mammalian CNS. This response is probably due, at least in part, to diffusible substances secreted by the non-neuronal cells. We have obtained diffusible substances from either regenerating fish optic nerves or neonatal rabbit optic nerves and applied them around crushed adult rabbit optic nerves. This manipulation caused the adult nerve to show regenerative changes: a general increase of protein synthesis in the retinas; selective increase in synthesis of a few polypeptides in the retinas; sprouting from the retinas in vitro; increased viability of nerve fibers as shown by HRP staining; and the appearance of growth cones adjacent to glial limitans in the injured nerves. We termed these diffusible, active substances "Growth Associated Triggering Factors" (GATFs). In addition to the phenomena described above, the active substances (obtained in the form of media conditioned by regenerating fish optic nerve or neonatal rabbit optic nerve) caused various other changes in the injured nerve itself: acceleration of non-neuronal cell proliferation; changes in the protein pattern, e.g. an increase in a 12 kDa polypeptide which might be a second mediator in the cascade of events leading to regeneration; increased laminin immunoreactive sites in the nerve; and the acquisition of growth supportive activity in media conditioned by the implanted injured nerves.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxyribonueleate solutions: Sedimentation in a density gradient,partial specific volumes,density and refractive index increments,and preferential interactions

Density increments (?ρ/?c₂)°_μ in solutions of NaDNA in NaCl and CsDNA in CsCl were determined over a wide range of salt concentrations; calf thymus DNA, fragmented by sonic irradiation to a molecular weight of 4–6 × 10⁵ was used. The partial specific volume v?₂° of NaDNA at 25°C was found to ho 0.500 ml/g in water, and that of CsDNA 0.440 ml/g. Both values increase with increasing NaCl and CsCl concentration. Refractive index increments under various experimental conditions were also determined. The relevance of the density increments (at constant, chemical potential of diffusible solutes) to equilibrium sedimentation in a density gradient and the evaluation of molecular weights is discussed. Distribution coefficients of diffusible components, sometimes referred to as preferential solvation or net hydration, were derived from the density increments and partial volumes and compared with direct experimental results, whenever available, from membrane distribution and isopiestic distillation. The thermo-dynamic significance of the distribution coefficients as well as possible interpretations in terms of specific molecular mechanisms are considered.     

Residence time distribution of diazepam in the isolated perfused rat liver. Analysis with the axial dispersion model.

The application of the axial dispersion model to diazepam hepatic elimination was evaluated using data obtained for impulse-response experiments with diazepam in the single-pass isolated perfused rat liver preparation. The transient form of the two-compartment dispersion model was applied to the output concentration versus time profile of diazepam after bolus input of a radiolabelled tracer into the hepatic portal vein (n = 4), providing DN and CLint estimates of 0.251 +/- 0.093 and 135 +/- 59 ml min-1, respectively. In contrast, the one-compartment form of the axial dispersion model, which assumes instantaneous transversal distribution of substance to the accessible spaces within the liver, could not adequately describe the residence time distribution (RTD) of diazepam. Furthermore, the magnitude of DN, a stochastic parameter which characterizes the axial spreading of solutes during transit through the liver, is similar to that determined for non-eliminated substances such as erythrocytes, albumin, sucrose and water. These findings suggest that the dispersion of diazepam in the perfused rat liver is determined primarily by the architecture of the hepatic microvasculature.     

Concentrations of glycolytic enzymes and other cytosolic proteins in the diffusible fraction of a vertebrate muscle proteome

We used a novel microvolumetric technique based on protein diffusion to characterize the subproteome of muscle that consists of diffusible proteins, including those involved in cell metabolism. Muscle fiber segments were mechanically demembranated under mineral oil and transferred into drops of relaxing solution. After the fiber segment was depleted of diffusible proteins, the content of each drop and residual segment was analyzed by one-dimensional polyacrylamide gel electrophoresis. Proteins were identified through peptide mass fingerprinting and quantified using purified protein standards. Ten of the most abundant cytosolic proteins, distinguished by their ability to readily diffuse out of the skinned fiber, were glycolytic enzymes whose concentrations ranged from 2.6+/-1.0 g liter-1 (phosphoglucose isomerase) to 12.8+/-1.1 g liter-1 fiber volume (pyruvate kinase). The concentrations of the other five most abundant cytosolic proteins were as follows: glycogen phosphorylase, 6.0+/-2.3 g liter-1; phosphoglucose mutase, 2.2+/-0.2 g liter-1; adenylate kinase, 1.6+/-1.3 g liter-1; phosphocreatine kinase, 6.6+/-2.6 g liter-1; and parvalbumin, 0.7+/-0.4 g liter-1. Given the molecular weight and subunit number of each enzyme, the combined concentration of the 15 most abundant cytosolic proteins was 82.3 g liter-1; the volume fraction was 0.093. The large volume fraction of diffusible proteins favors nonspecific interactions and associations, particularly if the glycolytic enzymes and diffusible phosphocreatine kinase are restricted to the I-band as previous studies suggest. The relative molar concentration of glycolytic enzymes is roughly consistent with a stoichiometry of 1:2 for enzymes catalyzing the hexose and triose sugar reactions, respectively, a stoichiometry that may favor metabolic channeling of intermediates during glycolysis. Our results indicate that subcellular fractionation of muscle proteins, in which cytosolic constituents are distinguished by their ability to diffuse readily from demembranated cells, is a promising microvolumetric technique that allows conclusions to be drawn about native protein-protein interactions based on concentration and stoichiometry.     

An Electron Microscope Study of Basophile Substances of Frozen-Dried Rat Liver

Small pieces of liver from rats subjected to different dietary regimes were fixed by freeze-drying, and postfixed by in vacuo heating and denaturation with alcohol. Specimens were digested with ribo- or deoxyribonuclease, and stained with gallocyanin-chromalum, azure II, the Feulgen procedure or alcoholic platinic tetrabromide. Some specimens were reserved as controls of the effects of enzyme treatment. Stained and unstained specimens were embedded in methacrylate and examined by light and electron microscopy. Basophilic and Feulgen-positive substances, after contact with watery reagents, were found by electron microscopy to exist as small dense granules embedded in a less dense homogeneous matrix, forming the walls of submicroscopic vacuoles. These granules were absent after digestion with nucleodepolymerases. In specimens (unstained, or stained with platinic tetrabromide) which had not passed through water, the dense (basophile) substances in nuclei and cytoplasm were found to exist, not as granules, but as ill defined submicroscopic concentrates which blended imperceptibly into the homogeneous matrix of the vacuolar walls. Objections to the use of stains for improving contrast conditions in electron microscopy of tissues are discussed, and it is concluded that the reagents do not necessarily produce the observed increases in contrast by selectively stabilizing certain structures. The concept of microsomes as pre-existing distinct morphological entities in intact (unhomogenized) cells is thought to be inconsistent with the distribution of basophile substances in frozen-dried liver.     

An electron microscope study of basophile substances of frozen-dried rat liver

Small pieces of liver from rats subjected to different dietary regimes were fixed by freeze-drying, and postfixed by in vacuo heating and denaturation with alcohol. Specimens were digested with ribo- or deoxyribonuclease, and stained with gallocyanin-chromalum, azure II, the Feulgen procedure or alcoholic platinic tetrabromide. Some specimens were reserved as controls of the effects of enzyme treatment. Stained and unstained specimens were embedded in methacrylate and examined by light and electron microscopy. Basophilic and Feulgen-positive substances, after contact with watery reagents, were found by electron microscopy to exist as small dense granules embedded in a less dense homogeneous matrix, forming the walls of submicroscopic vacuoles. These granules were absent after digestion with nucleodepolymerases. In specimens (unstained, or stained with platinic tetrabromide) which had not passed through water, the dense (basophile) substances in nuclei and cytoplasm were found to exist, not as granules, but as ill defined submicroscopic concentrates which blended imperceptibly into the homogeneous matrix of the vacuolar walls. Objections to the use of stains for improving contrast conditions in electron microscopy of tissues are discussed, and it is concluded that the reagents do not necessarily produce the observed increases in contrast by selectively stabilizing certain structures. The concept of microsomes as pre-existing distinct morphological entities in intact (unhomogenized) cells is thought to be inconsistent with the distribution of basophile substances in frozen-dried liver.     

Temperate phages and bacteriocins of the gliding bacterium Cytophaga johnsonae

A collection of 30 independently isolated strains of Cytophaga johnsonae was screened for the presence of temperate bacteriophages. Two strains were found to harbour phages. The newly isolated phages differ in several respects from the 43 previously isolated phages for C. johnsonae. Both phages are polyhedral, approximately 60 nm in diameter, and have no apparent tail structure. They are chloroform sensitive, and plaque formation is inhibited by agar. Both are capable of establishing a stable association with host cells. Twenty-nine of the 30 strains produced diffusible substances that specifically inhibited the growth of other C. johnsonae strains or closely related species and that could not be propagated. These substances appear to be bacteriocins, some of which, like bacteriophages, are active only against motile cells, while other inhibit nonmotile as well as motile cells. One of each of these two types of bacteriocins was partially characterized and both were found to be proteinaceous in nature and bactericidal in effect.     

A new biochemical marker for foot-specific cell differentiation in hydra

Summary Mucous cells in the basal disk of hydra contain a peroxidase-like enzyme allowing specific staining of these cells with substrates for peroxidases. The peroxidase activity provides an excellent marker for foot mucous cell, differentiation and was used to follow the reappearance of footspecific cells during foot regeneration after amputation. By choosing the appropriate either soluble or precipitable substrate the peroxidase reaction was used both for a qualitative and for a quantitative evaluation of foot-specific differentiation in hydra. For histological studies diaminobenzidien was found to be a suitable substrate which forms a dark brown precipitate within the cells containing the peroxidase activity. For a quantitative evaluation of foot regeneration the soluble substrate 2,2-azino-di(3-ethyl-benzthiazoline-sulfonic acid-6) ammonium salt was used which after reaction with the enzyme gives rise to a diffusible green reaction product the concentration of which can be measured by its specific absorption at 415 nm. Based on the diffusible enzyme product a new quantitative assay for foot regenration was developed and applied to confirm the effect and specificity of morphogenetic substances which either inhibit or activate foot or head regeneration in hydra.     

Toward an understanding of the epithelial requirement for osteogenesis in scleral mesenchyme of the embryonic chick

Explants of scleral tissue from chick embryos of H.H. stage 29-36 (6-10 days of incubation) were used to determine if the epithelial-mesenchymal interaction which initiates scleral bone formation is cell contact, extracellular matrix, or diffusion mediated. Transfilter tissue recombinations, in which explanted interacting tissues are associated across interposing Nuclepore filters of various pore sizes and thicknesses, were performed with scleral mesenchyme and epithelium. When filters with pore sizes which would allow the passage of cell processes and diffusible substances were used, osteogenesis was initiated in the scleral mesenchyme. When cell processes were blocked with thicker filters or smaller pore sizes, bone formation still occurred, indicating that a diffusible substance mediates this tissue interaction. Further support for a diffusion-mediated interaction came from transfilter experiments using dialysis membranes to discriminate the size of the molecule(s), and Millipore filters to determine the distance over which these molecules travel. These experiments revealed that the scleral epithelial diffusible factor has a molecular weight of between 3500 and 6000 daltons, and acts over distances between 150 and 300 microns.     

Preservation of the diffusible cations for SIMS microscopy. I. A problem related to the state of water in the cell.

The notion of diffusible ions is reviewed in the light of recent knowledge on the stage of water in biological matrices. It appears that ion distributions would be little affected as long as water-macromolecular equilibrium is maintained, but they risk to be significantly modified during dehydration, because the transformation of bound water into highly solvating free water can produce ion displacements. In addition, differently hydrated areas may undergo unequal volume variations. The principal modes of preparing material for SIMS (secondary ion mass spectrometry) microscopy are envisaged from this viewpoint.     

Introduction of 2,4-dichlorophenoxyacetic acid into soil with solvents and resulting implications for bioavailability to microorganisms

Slow equilibration of introduced chemicals through tortuous pore space limits uniform substrate distribution in soil biodegradation studies. The necessity of introducing poorly soluble xenobiotics via organic solvents, the volume of which is minimized to limit toxicity, likely also affects xenobiotic distribution. Our objective was to investigate relative effects of carrier solvent choice and volume on xenobiotic distribution, apparent solvent toxicity, and soil degradation of 2,4-dichlorophenoxy acetic acid. Incubations using four carrier solvents ranging in properties showed that the fraction of 2,4-D mineralized was a hyperbolic function of solvent volume used (0.02–10 μl g⁻¹), attributed to compensating effects of herbicide bioavailability and solvent toxicity. Substrate concentration influenced mineralization of herbicide introduced with organic carriers, but not water. Fraction of material readily desorbed increased when water was the carrier. Results suggest that solvent toxicity should be balanced with uniformity of substrate distribution when using organic carriers in soils. Substrate bioavailability is a ubiquitous issue in terrestrial microbiology research, thus limitations observed herein broadly apply to microbiology questions about introduced substances in soil. We advocate the development of tools to characterize variable conditions among soil compartments, estimates of substrate bioavailability, and linkage of this information to microbial data.     

Effects of diffusible products of peroxidation of rat liver microsomal lipids

The effects on cellular structures of products of peroxidation of rat liver microsomal lipids were investigated. A system containing actively peroxidizing liver microsomal fraction was separated from a revealing or target system by a dialysis membrane. The target system, contained in the dialysis tube, consisted of either intact cells (erythrocytes) or subcellular fractions (liver microsomal fraction). When liver microsomal fractions were incubated with NADPH (or an NADPH-generating system), lipid peroxidation, as measured by the amount of malonaldehyde formed, occurred very rapidly. The malon-aldehyde concentration tended to equilibrate across the dialysis membrane. When the target system consisted of erythrocytes, haemolysis occurred abruptly after a lag phase. The lysis was greatly accelerated when erythrocytes from vitamin E-deficient rats were used, but no haemolysis was observed when erythrocytes from vitamin E-treated rats were used. When, in the same system, freshly prepared liver microsomal fractions were exposed to diffusible factors produced by lipid peroxidation, the glucose 6-phosphatase activity markedly decreased. A similar decrease in glucose 6-phosphatase activity, as well as a smaller but significant decrease in cytochrome P-450, was observed when the target microsomal fractions were exposed to diffusible factors derived from the peroxidation of liver microsomal lipids in a separate preincubation step. These and additional experiments indicated that the toxicological activity is relatively stable. Experiments in which the hepatic microsomal fractions destined for lipid peroxidation contained radioactively labelled arachidonic acid, previously incorporated into the membranes, showed that part of the radioactivity released from the microsomal fraction into the incubation medium entered the dialysis tube and was recovered bound to the constituents of the microsomal fractions of the target system. These results indicate that during the course of the peroxidation of liver microsomal lipids toxic products are formed that are able to induce pathological effects at distant loci.     

Sexual Differences in Cell Proliferation in the Ventricular Zone,Cell Migration and Differentiation in the HVC of Juvenile Bengalese Finch

Song control nuclei have distinct sexual differences and thus are an ideal model to address how brain areas are sexually differentiated. Through a combination of histological analysis and electrical lesions, we first identified the ventricle site for HVC progenitor cells. We then found that there were significant sex differences in the cellular proliferation activity in the ventricular zone of the HVC, the number of migrating cells along the radial cells (positive immunoreactions to vimentin) and differentiation towards neurons. Through co-culturing of male and female slices containing the developing HVC in the same well, we found that the male slices could produce diffusible substances to masculinize the female HVC. By adding estrogen, an estrogen antagonist, brain-derived neurotrophic factor (BDNF) or its antibody into the culture medium, separately or in combination, we found that these diffusible substances may include estrogen and BDNF. Finally, we found that 1) estrogen-induced BDNF upregulation could be detected 48 hr after estrogen treatment and could not be blocked by a vascular endothelial growth factor (VEGF) receptor inhibitor and 2) the amount of VEGF mRNA expressed in the developing HVC and its adjacent area did not display any significant sex differences, as did the distribution of VEGF and laminin-expressing endothelial cells in the developing HVC. Because these findings are largely different from previous reports on the adult female HVC, it is suggested that our estrogen-induced BDNF up-regulation and the resultant sexual differentiation might not be mediated by VEGF and endothelial cells, but instead, may result from the direct effects of estrogen on BDNF.     

Mating Reaction in Saccharomyces cerevisiae. IV. Retardation of Deoxyribonucleic Acid Synthesis

When a and a type haploid cells of Saccharomyces cere-visiae were mixed and cultured, deoxyribonucleic acid synthesis was retarded but ribonucleic acid and protein syntheses were not. It was found that culture filtrate of a type cells inhibited deoxyribonucleic acid synthesis of a type cells and that of a type cells inhibited that of a type cells. Thus, sex-specific diffusible substances secreted by opposite mating type cells are thought, at least partly, to be responsible for the retardation of deoxyribonucleic acid synthesis.     

Water- and solute-accessible spaces of purified peroxisomes. Evidence that peroxisomes are permeable to NAD+.

Peroxisomes were purified from liver homogenates from rats, treated with the peroxisome proliferator clofibrate, by a combination of differential centrifugation and isopycnic centrifugation in iso-osmotic self-generating Percoll gradients. Structural integrity of the peroxisomes appeared to be preserved as evidenced by a high degree of catalase latency, the absence of catalase release during purification and the exclusion of inulin (mol.wt. +/- 5000). Spaces for water and solutes were measured after incubation of the peroxisomes in iso-osmotic sucrose with radioactive water or solutes and separation of the organelles from their media by centrifugation through an organic layer. Extraperoxisomal water was corrected for by the use of radioactive dextran or inulin. The sucrose, glucose, urea, methanol and acetate-accessible spaces were identical, suggesting that these spaces represent the volume in which molecules that can cross the membrane distribute. This volume equalled 50-65% of the water space. Urate and NAD+, a cofactor of peroxisomal beta-oxidation of fatty acids, also distributed in this volume, but were also partly bound. Urate and NAD+ binding was not abolished by sonication, which released the bulk of matrix catalase activity, but NAD+ binding was seriously diminished. The peroxisomal water and sucrose spaces were estimated to be 107 microliters and 55 microliters per g of liver tissue from a clofibrate-treated rat. From quantitative morphometric data [Anthony, Schmucker, Mooney & Jones (1978) J. Lipid Res. 19, 154-165] and our marker enzyme analyses, as well as from our experimentally determined water spaces of mitochondrial and microsomal fractions, it could be calculated that the volume contamination by lysosomes, mitochondria and microsomes did not exceed 1, 8 and 6% respectively. Our data indicate that apparently intact peroxisomes are permeable to a number of small molecules, including NAD+. Whether the NAD+-binding sites in sonicated peroxisomes mirror the likely existence of a membrane carrier requires further investigation.     

Control of mitochondrial volume by mitochondrial metabolic water

It is well-known that mitochondrial volume largely controls mitochondrial functioning. We investigate whether metabolic water produced by oxidative phosphorylation could be involved in mitochondrial volume regulation. We modulated the generation of this water in liver mitochondria and assess their volume by two independent techniques.In liver mitochondria, the mitochondrial volume was specifically decreased when no water was produced independently of energetic parameters and uncoupling activity. In all other conditions associated with water generation, there was no significant change in mitochondrial metabolic volume.Altogether these data demonstrate that mitochondrial volume is regulated, independently of energetic status, by the mitochondrial metabolic water that acts as a signal.     

Distribution of opiate-like substances in rat tissues

Rat tissues were tested for their ability to inhibit the binding of [³H]dihydromorphine or [³H]naloxone to membrane-bound opiate receptors. By this criterion, morphine-like substances were found in lung, heart, liver, and kidney as well as in brain. The relative activity of the extracts, based on initial tissue weight, differed with the radioactive ligand employed. With dihydromorphine, the order was as above. With naloxone, lung was most active, followed by heart, brain, liver, and kidney. The ability of all tissue extracts to inhibit opiate binding was reduced by 100 mM NaCl and slightly reduced by 1 mM MnCl₂. Gel filtration using Sephadex G-25 indicated that the inhibitory Substances were heterogeneous in molecular weight. Only with brain and kidney extracts was there significant activity at the elution volume where enkephalins would be expected. Fraction tion using Amberlite XAD-2, a resin which selectively absorbs hydrophobic materials, again indicated that the major portion of activit in all tissue extracts was due to substances other than enkephalins.