Patterns of junctional communication in skin: Studies on cultured keratinocytes

Junctional communication has long been suggested to play a role in coordinating the development of multicellular tissues. A better understanding of the patterns of communication between cells in such tissues is important for the identification of areas where this process may have a role. We have investigated the patterns of communication in cultures of human epidermal keratinocytes by iontophoretic injection of Lucifer Yellow CH, using involucrin expression as a marker of cells undergoing terminal differentiation. Cells that lack involucrin (i.e., the basal, proliferating cells) transfer dye preferentially to other involucrin-negative cells, whereas involucrin-positive cells either are not coupled or transfer dye with similar frequency to involucrin-positive and involucrin-negative neighbors. This decrease in communication associated with terminal differentiation was observed in both the presence and the absence of assembled desmosomes. Our observations lead us to speculate that loss of junctional communication may influence the commitment of basal keratinocytes to terminal differentiation.     

Sugar transport by the bacterial phosphotransferase system. Reconstitution of inducer exclusion in Salmonella typhimurium membrane vesicles

The accompanying articles (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253; Mitchell, W.J., Saffen, D. W., and Roseman, S. (1987) J. Biol. Chem. 262, 16254-16260) show that "inducer exclusion" in intact cells of Escherichia coli is regulated by IIIGlc, a protein encoded by the crr gene of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The present studies attempt to show a direct effect of IIIGlc on non-PTS transport systems. Inner membrane vesicles prepared from a wild type strain of Salmonella typhimurium (pts+), carrying the E. coli lactose operon on an episome, showed respiration-dependent accumulation of methyl-beta-D-thiogalactopyranoside (TMG) via the lactose permease. In the presence of methyl-alpha-D-glucopyranoside or other PTS sugars, TMG uptake was reduced by an amount which was dependent on the relative concentrations of IIIGlc and lactose permease in the vesicles. The endogenous IIIGlc concentration in these vesicles was in the range 5-10 microM, similar to that found in whole cells. Methyl-alpha-glucoside had no effect on lactose permease activity in vesicles prepared from a deletion mutant strain lacking the soluble PTS proteins Enzyme I, HPr, and IIIGlc. One or more of the pure proteins could be inserted into the mutant vesicles; when one of the two electrophoretically distinguishable forms of the phosphocarrier protein, IIIGlc Slow, was inserted, both the initial rate and steady state level of TMG accumulation were reduced by up to 40%. The second electrophoretic form, IIIGlc Fast, had much less effect. A direct relationship was observed between the intravesicular concentration of IIIGlc Slow and the extent of inhibition of the lactose permease. No inhibition was observed when IIIGlc Slow was added to the outside of the vesicles, indicating that the site of interaction with the lactose permease is accessible only from the inner face of the membrane. In addition to the lactose permease, IIIGlc Slow was found to inhibit both the galactose and the melibiose permeases. Uptake of proline, on the other hand, was unaffected. The results are therefore consistent with an hypothesis that dephosphorylated IIIGlc Slow is an inhibitor of certain non-PTS permeases.     

Sugar transport by the bacterial phosphotransferase system. The intrinsic fluorescence of enzyme I

Enzyme I of the bacterial phosphoenolpyruvate: glycose phosphotransferase system has 2 tryptophan residues/monomer, as determined spectrophotometrically. The tryptophan fluorescence has been investigated with the aid of nanosecond time-resolved techniques. The decay of the fluorescence intensity was analyzed in terms of a biexponential function. The contribution of the emission associated with the shorter decay constant increases from 17-19% at 1 degree C to 43-44% at room temperature. Decay-associated spectra obtained with Enzyme I indicate different spectral distributions associated with the two decay constants. The measurement of tumbling of Enzyme I as a function of temperature revealed a transition of rotational rates between 5 and 15.5 degrees C. Global analysis allowed decomposition of the anisotropy decay into a formulation consistent with monomer and dimer rotational contributions.     

The sugar-specific adhesion/deadhesion apparatus of the marine bacterium Vibrio furnissii is a sensorium that continuously monitors nutrient levels in the environment

Our earlier studies on cell adhesion to immobilized carbohydrates are extended here to a marine bacterium, Vibrio furnissii. Apparently one lectin mediates the binding of these cells to glycosides of N-acetylglucosamine, mannose, and glucose covalently linked to Agarose beads. Kinetic studies show that protein synthesis is required for initiating and for maintaining adhesion to the glycosides. Furthermore, a pro- mutant binds to GlcNAc-beads at Pro concentrations insufficient to support cell growth. Expression of the functional lectin therefore predominates under conditions of limiting protein synthesis. Thus, cells adhere to the sugars in an environment compatible with protein synthesis, and deadhere when depleted of any required nutrient, presumably to migrate to a more favorable locale. The adhesion-deadhesion apparatus thereby permits constant monitoring of the surrounding environment, comprising a "nutrient sensorium".     

Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells

Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using¹²⁵I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS phosphate-buffered saline - STS sucrose-Tris-serum solution - NGF nerve growth factor - C collagen - PL polylysine - BBG bovine brain ganglioside mixture - GM₁ gafactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1a [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1a [N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosyl ceramide - GD_1b galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide - GT_1b [N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide - NANA N-acetylneuraminic acid     

Acute Uteroplacental Ischemic Embryo: Lactic Acid Accumulation and Prostaglandin Production in the Fetal Rat Brain

A new experimental model for studying the effects of acute ischemia on brain development in the near-term fetal rat has been devised. Ischemic conditions are achieved by complete clamping of blood vessels branching from the uterine vasculature into each individual fetus for designated times followed by removal of the clamps to permit reperfusion. Accumulation of lactic acid in the fetal brain depends on the length of the restriction period, reaching a plateau level of 29 mumol/g tissue at about 30 min. It also depends on the reperfusion time. Thus after a period of 15 min of restriction lactate levels show an increase over the next 30-min reperfusion to a value of 25.5 mumol/g followed by a rapid decrease to normal values by 3 h of reperfusion. Restriction of 15 min followed by reperfusion of 45 min causes an elevation of prostaglandin E2 (PGE2) level from 12.4 +/- 0.86 ng/g to 21.1 +/- 0.6 ng/g (p less than 0.001). This elevation in PGE2 level is less apparent after 20 min of restriction. No effects are seen on the level of PGF2 alpha. Both PGE2 and PGF2 alpha accumulate in vitro in a time-dependent manner by brain particulate fraction. In vitro synthesis of both PGE2 and PGF2 alpha is inhibited by indomethacin (100% and 68%, respectively) and AA861 (94% and 76%, respectively). BW755c and nordihydroguaiaretic acid do not affect PGE2 formation but enhance PGF2 alpha production by 112% and 152%, respectively. Particulate fractions from restricted brain produce less PGF2 alpha than control brains (6.38 +/- 1.62 versus 11.43 +/- 2.2, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrophobicity of the peptide C=O...H-N hydrogen-bonded group

The hydrophobicity of the peptide C=O ... H-N hydrogen-bonded group is an important parameter that determines the structure of proteins in water and in biological membranes, and therefore the free energy of transferring this group from water to non-polar solvents should be determined accurately. The essential work on this problem was carried out by Klotz and co-workers, and has been summarized elsewhere. Using N-methylacetamide as a model peptide, the free energies of the following processes were determined; (1) formation of the C=O ... H-N bond in water, (2) formation of the C=O ... N-N bond in CCl4, and (3) transfer of N-methylacetamide from water to CCl4. (4) From (3), the free energy of transferring the non-hydrogen bonded (C=O, H-N) group from water to CCl4 was calculated. When the free energies of (1), (2) and (4) are combined, one finds that the free energy of transferring the C=O ... H-N group from water to CCl4 is a surprising -1.4 kcal/mol (1 cal = 4.184 J). This number does not seem reasonable, since it implies that the C=O ... H-N group is about as hydrophobic as an isopropyl group, i.e. the side-chain of valine. In the present report, it is shown that this apparent hydrophobicity results from an underestimation of the free energy contribution that the methyl groups make to the transfer of N-methylacetamide from water to CCl4. When appropriate methyl group transfer free energies are used, one finds that the free energy of transferring the C=O ... H-N group from water to CCl4 is +0.62 kcal/mol. Therefore, this group is relatively insensitive to solvent polarity. A similar calculation shows that the free energy of transferring the C=O ... H-O hydrogen-bonded group from water to benzene is +0.55 kcal/mol.     

Bulb mite,Rhizoglyphus robini (Astigmata: Acaridae) as an experimental animal

Rhizoglyphus robini Claparède (Acari: Astigmata: Acaridae) is proposed as a model laboratory animal for biological, ecological, physiological and toxicological studies. The mite is easy and inexpensive to rear, quite fecund, convenient to manipulate, and may rapidly be raised to gram quantities. Examples are presented of its use in soil pest ecology and control studies, and in physiological, biochemical and toxicological investigations. Efforts to explore the induction of detoxification systems by various chemicals, and a demonstration of its control by solarization, are also described.     

Enzymes Associated with Defence against Toxic Oxygen Species in PCNB-Tolerant and Sensitive Soil Fungi

The specific activities of enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX) and glutathione reductase (GR), which are involved in protection against toxic species of oxygen, were determined in mycelia extracts of pentachloronitrobenzene (PCNB)-tolerant and susceptible soil fungi. The organisms assayed were the highly PCNB-sensitive Rhizoctonia solani and Rhizopus arrhizus; Sclerotium rolfsii and Trichoderma harzianum, which are moderately susceptible to PCNB, and the fungicide-tolerant Fusarium oxysporum f. sp. melonis and Pythium aphanidermatum. No GPOX activity was detected in the six examined fungi. Significant differences in the specific activities of the other enzyme systems among the fungi were evident. Remarkably low levels of CAT activities were measured in R. solani. Except for T. harzianum, no meaningful differences regarding SOD, CAT and GR activities with age of the fungi cultures were observed. The electrophoretic patterns of SOD and CAT displayed dissimilarities among the fungi under study. P. aphanidermatum is more polymorphic with respect to both SOD and CAT enzyme systems as compared to the other fungi. The SOD of F. oxysporum f. sp. melonis, R. arrhizus and T. harzianum is a cuprozinc enzyme, while the mangano-SOD species was detected in S. rolfsii, R. solani and T. harzianum.