COLLAGEN SYNTHESIS AND CELL GROWTH IN CHICK EMBRYO FIBROBLASTS: INFLUENCE OF COLCHICINE,CYTOCHALASIN B AND CONCANAVALIN A

Culturing of chick embryo fibroblasts in the presence of colchicine or cytochalasin B with and without concanavalin A (Con A) demonstrated that colchicine induces greater neosynthesis of endocellular type I collagen, whereas cytochalasin B boosts secretion. The effects are modified by the addition of Con A, which increases α₂more than a1 chain production.³H-thymidine incorporation is unaffected by cytochalasin B, but stimulated by colchicine. Con A neutralizes the stimulatory action of colchicine. It would therefore seem that Con A exerts transmembrane control of effects induced by colchicine and cytochalasin B by binding to cell surface receptors and so triggering rearrangement of the cytoskeleton.     

Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking

These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C₁-BODIPY-C₁₂ in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis–Menten kinetics; the apparent efficiency (k_cat/K_T) of this process increases over 2-fold (2.1 × 10⁶–4.5 × 10⁶ s⁻¹ M⁻¹) upon adipocyte differentiation. The V_max values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 × 10⁶ s⁻¹ M⁻¹ versus 1.5 × 10⁶ s⁻¹ M⁻¹). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V_max values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The same cells had reduced efficiency for fatty acid transport (ranging from 0.82 × 10⁶ s⁻¹ M⁻¹ to 1.35 × 10⁶ s⁻¹ M⁻¹).     

α-Factor inhibition of the rate of cell passage through the “start” step of cell division in Saccharomyces cerevisiae yeast: Estimation of the division delay per α-factor·receptor complex

A highly sensitive, kinetically unambiguous assay for α-factor-induced delay of cell passage through the “start” step of cell division in yeast is presented. The assay employs perfusion with periodic microscopy to monitor the bud emergence kinetics on the 20% of cells within an exponentially growing population which exist prior to the α-factor execution point of start. The t_1/2 for cell passage through start by this population of cells is 31 min in the absence of α-factor. The inhibition constant, K_I, represents the α-factor concentration which produces a 50% inhibition of this rate and is equal to 2×10⁻¹⁰M. A second assay for maximal cell division arrest by α-factor on whole populations of cells is presented. This assay shows a maximum cell division arrest time of 125±5 h at saturating α-factor, and a K₅₀ (that is, an α-factor concentration which produces a half-maximal response) of 2.5×10⁻⁸M. Both assays were performed in the effective absence of α-factor inactivation. Values of the dissociation constant K_D and total number of receptors per cell which specifically mediate cell division arrest or delay were estimated to be 2.5×10⁻⁸M and 10⁴, respectively. These estimates, along with the quantitative dose-response data for division arrest which are presented here, are consistent with each receptor·α-factor complex which is present on the cell at equilibrium producing a 43±10 s delay of cell passage through start. Surprisingly, this number is constant within twofold over the entire range of cellular division arrest responses to α-factor, that is, from a 1.9-fold inhibition of the rate of cell passage through start at 0.17 nM α-factor to a 125±5 h maximum arrest at saturating α-factor concentrations of >170 nM. The possible significance of this observation toward the mechanism of α-factor-induced cell division arrest is discussed.     

Studies on the mechanism of action of isopropyl N-phenyl carbamate

The binding of [¹⁴C]isopropyl N-phenyl carbamate (IPC) to microtubular protein isolated from chick brains, and the effect of isopropyl N-phenyl carbamate (IPC) on the in vitro reassembly of microtubules was investigated. While [¹⁴C]colchicine binds to microtubular protein, [¹⁴C]IPC does not. Concentrations from 1 × 10⁻⁴ M to 1 × 10⁻³ M IPC do not prevent in vitro repolymerization of microtubular protein. IPC (1 × 10⁻⁴ M) does not affect the rate of reassembly of microtubules. We conclude that IPC does not exert its effect through an interaction with microtubular protein; we suggest that IPC probably interacts with microtubule organizing centers.     

Characterization of galactomannans isolated from legume endosperms of Caesalpinioideae and Faboideae subfamilies by multidetection aqueous SEC

Galactomannans isolated from legume seed endosperms, including those of commercial interest, have been characterized by multidetection aqueous SEC. Galactomannans derived from seeds of the Faboideae subfamily had substantially higher M_w than those from Caesalpinioideae seeds (M_w,Fab = 2.4–3.1 × 10⁶ g/mol, M_w,Caes. = 0.86–2.1 × 10⁶ g/mol) and within the latter botanical subfamily, an apparent correlation between M_w and the degree of galactose substitution DG was found. The molar mass distributions were unimodal and differed primarily by a scale factor, with distributional widths narrower than a true Flory ‘most-probable distribution’; good fits to Schulz–Zimm model were obtained. Across subfamilies no differences were found in the exponents of [η]–M and R_v–M relationships (0.61 ± 0.02, 0.54 ± 0.01, respectively), the Flory chain stiffness ratio (C_∞ = 20 ± 1 (BSF analysis)), or the persistence length (L_p = 5.5 ± 0.2 nm) obtained from SEC fraction data. However, it was found that prefactors in the [η]–M and R_v–M relationships as well as the unperturbed parameter K_Θ decrease in proportion to DG and therefore chain density. Generalized relationships incorporating galactose-dependent prefactors were therefore developed to model SEC fraction data of native galactomannans ([η]_GM = (1800 ± 200) × M_o^−1.61 × M^0.61±0.02, R_v,GM = 0.63 ± 0.05 × M_o^−0.54 × M^0.54±0.01) as well as lower-M fractions obtained by ultrasonication ([η]_GM = (730 ± 100) × M_o^−1.71 × M_w^0.71±0.02, R_v,GM = 0.49 ± 0.05 × M_o^−0.57 × M_w^0.57±0.01, M ≈ 1 × 10⁵-native). As a consequence of this dependence and the observed patterns in molar mass variation, [η] varies within a narrow range for galactomannans as a whole despite substantial M_w differences.     

Uptake and degradation of α2-macroglobulin-protease complexes in human cells in culture

Serum components, present intracellularly in cultured human fibroblasts, were identified as α₂-macroglobulin (α₂M), albumin, α₁-trypsin inhibitor, hemopexin and transferrin, among others. These components were shown to be taken up from the culture medium. Kinetic analysis of the uptake of α₂M-trypsin complexes by the cells showed the uptake to be of a high affinity mechanism (K_M = 6 × 10⁻⁸ M α₂M in the medium), with a high rate of internalization (V_max=1.03 × 10⁶ molecules α₂M/cell and α₂M per hour). The intracellular degradation of α₂M is rapid, as judged by the half-life of 1.6 h. Virus-transformed or tumor-derived cell lines showed low or undetectable levels of α₂M. The possible physiological significance of the described phenomena is discussed in relation to the in vivo situation.     

Cytochalasin B-induced alterations of insulin binding and microfilament organization in cultured fibroblasts

The effect of cytochalasin B (CB) on insulin binding has been investigated in confluent cultures of chick embryo fibroblasts. Time- and dose-dependent increases in binding of [¹²⁵I]insulin was observed after incubation of fibroblasts with CB. At 10 μg/ml, CB caused a 2-fold increase in binding, due to an increase in the number of binding sites from 9.3 × 10³ to 2.0 × 10⁴ per cell. Removal of CB from the growth medium was accompanied by a decrease in [¹²⁵I]insulin binding to control values in 24 h. Increase in the binding of insulin in CB-treated CEF was also accompanied by enhancement of insulin to stimulation of [³H]thymidine incorporation into acid-insoluble material. CB treatment also caused disorganization and disappearance of microfilament bundles and changes in cell shape from flat, with a few blebs and folds on the cell surface, to round with numerous blebs and folds. The data from this study suggest that changes in the number of surface insulin-binding sites may be related to the state of organization of cytoskeletal structures in chick embryo fibroblasts.     

Disruption of the Cytokeratin Cytoskeleton and Inhibition of Hepatocytic Autophagy by Okadaic Acid

To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 nM). Only small effects were seen with vinblastine (10 μM) or cytochalasin D (10 μM). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against α- and β-tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 μM), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 μM) and prunin (100 μM) as well as KN-62 (10 μM), a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 μM), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 μM), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process.     

Effect of dietary chitosans on trace iron, copper and zinc in mice

Dietary chitosans with different molecular weight M_w and the degree of deacetylation DDA (high molecular weight chitosan HCS with M_w 7.60 × 10⁵ and DDA 85.5%, middle molecular weight chitosan MCS with M_w 3.27 × 10⁴ and DDA 85.2%, chito-oligomer COS with M_w 0.99 × 10³ and DDA 85.7% and water-soluble chitosan WSC with M_w 3.91 × 10⁴ and DDA 52.6%) were used at the 1.05% level to feed mice for 90 days. Afterwards no pathological symptoms, clinical signs or deaths were observed. The body weight of mice in chitosan group and control group showed no significant difference. Although HCS, COS and WSC had no significant effect on the level of Fe, Zn and Cu in the tested mice’s liver, spleen, heart and kidney, MCS significantly increased the level of Fe, Zn and Cu in liver. Therefore dietary ingestion of chitosan did not depress the level of Fe, Zn and Cu in mice.     

Microtubule Integrity Is Necessary for the Epithelial Barrier Function of Cultured Thyroid Cell Monolayers

Vectorial transport in the thyroid epithelium requires an efficient barrier against passive paracellular flux, a role which is principally performed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton. Although the contribution of the actin cytoskeleton to tight junction physiology has been intensively studied, less is known about possible interactions with microtubules. In the present study we used electrophysiological and immunohistochemical approaches to investigate the contribution of microtubules to the paracellular barrier in cultured thyroid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm · cm²). Colchicine (1 μM) caused a progressive fall in electrical resistance to <10% of baseline after 6 h and depolarization of the transepithelial electrical potential difference consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhibit the major apical conductances of thyroid cells but was reversed upon removal of the drug. Immunofluorescent staining for tubulin combined with confocal laser scanning microscopy demonstrated that thyroid cells possessed a dense microtubule network extending throughout the cytoplasm which was destroyed by colchicine. Colchicine also produced changes in the localization of the tight junction-associated protein, ZO-1: its normally continuous junctional distribution was disrupted by striking discontinuities and the appearance of many fine strands which extended into the cytoplasm. A similar disruption in E-cadherin staining was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in this electrically tight, transporting epithelium.     

Conformation of (2→1)-β-d-fructan in aqueous solution

The conformation and dilute solution properties of (2→1)-β-d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights (M_w) from 1.49 × 10⁴ to 5.29 × 10⁶ were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M_w < 4 × 10⁴, the intrinsic viscosity [η] varies with M_w^0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M_w > 10⁵, [η] exhibits an unusually weak dependence on M_w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.     

The cytoprotective properties of prostaglandin E2 against the toxic effects of actinomycin C on embryonic neural retina cells

Prostaglandins (PGs) have been shown to cytoprotect various tissue types against the toxic effects of many chemicals. The mechanism of this protection is poorly understood, but the involvement of cAMP is often implied. Only one previous study examined nervous tissue and PG protection. The present study was designed to determine if PGE₂ affords cytoprotection to a more specific nervous tissue (embryonic neural retina) from the toxicity of actinomycin C (AMC) using a trypan blue exclusion assay. The lowest concentration of PGE₂ (2 × 10⁻⁵M) had no effect, but as the concentration increased (3 × 10⁻⁵M and 5 × 10⁻⁵M), PGE₂ did afford protection against AMC in a dose dependent fashion. Theophylline treated cells were not protected, suggesting that cAMP may not be the primary mechanism of protection.     

Rheological properties of concentrated solutions of gellan in an ionic liquid

The rheological properties of solutions of gellan were examined at high concentrations where there is entanglement coupling between gellan chains. An ionic liquid 1-butyl-3-methylimidazolium chloride (BmimCl) was used as a solvent. Concentrated solutions of gellan in BmimCl were obtained by using a hot-molding technique. The concentration of gellan was varied from 1.9 × 10² to 4.8 × 10² kg m⁻³. The measurement temperature ranged from 50 to 100 °C. The master curve of the angular frequency dispersion of the storage modulus for the gellan solutions showed a rubbery plateau at high angular frequency. The molecular weight between entanglements (M_e) for gellan was obtained from the plateau modulus. From the concentration dependence curve of M_e, M_e for gellan in the molten state was determined to be 2.3 × 10³.     

Molecular mass and antitumor activities of sulfated derivatives of α-glucan from Poria cocos mycelia

Two kinds of water-insoluble (1 → 3)-α-d-glucan samples, ab-PCM3-I and ac-PCM3-I, isolated from different Poria cocos mycelia were sulfated, to produce two series of water-soluble derivatives ab-PCM3-I-S1–S5 and ac-PCM3-I-S1–S5, respectively. The derivatives having different weight-average molecular mass (M_w) were produced by changing reaction temperature and time as well as molar ratios between chlorosulfonic acid and number of hydroxyl groups in the glucan. The degrees of substitution (DS) of the sulfated derivatives were analyzed by elemental analysis (EA) to be 0.39–0.67 for ab-PCM3-I-S and 0.73–0.96 for ac-PCM3-I-S, respectively. The M_w and the intrinsic viscosity ([η]) of the samples ab-PCM3-I-S and the ac-PCM3-I-S were measured by size exclusion chromatography combined with laser light scattering (SEC–LLS) and viscometry in phosphate buffer solution (PBS) at 37 °C. The results indicated that their M_w ranged from 2.0 to 11.3 × 10⁴ for the samples ab-PCM3-I-S, and 4.7 to 40.0 × 10⁴ for the samples ac-PCM3-I-S. Moreover, the antitumor activities of the sulfated derivatives ab-PCM3-I-S and ac-PCM3-I-S against Sarcoma 180 tumor cell tested both in vitro and in vivo are significantly higher than those of the native α-d-glucans. Therefore, a moderate range of molecular mass from 2.0 × 10⁴ to 40.0 × 10⁴, relatively high chain stiffness and good water solubility of the sulfated derivatives are beneficial to the enhancement of their antitumor activities.     

Specific binding of [H]heparin to human carcinoma SW-13 and other mammalian cells

This study reports on the specific binding of [³H]heparin to human adrenocortical carcinoma cell line SW-13. Heparin binding to SW-13 cells is specific, saturable, and time- and temperature-dependent with maximum binding occurring between 90 and 120 min at 22 °C. Scatchard analysis revealed two classes of binding sites. The apparent K_d for high-affinity receptors is 2.14 × 10⁻⁸ M with 1.48 × 10⁶ sites per cells. Six other tested mammalian cell lines also have specific binding sites for heparin.     

Methylmercury alters the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

In order to maintain normal functioning of the brain, glutamate homeostasis and extracellular levels of excitotoxic amino acids (EAA) must be tightly controlled. This is accomplished, in large measure, by the astroglial high-affinity Na⁺-dependent EAA transporters glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). Methylmercury (MeHg) is a potent neurotoxicant. Astrocytes are known targets for MeHg toxicity, representing a site for mercury localization. Mehg is known to cause astrocytic swelling, EAA release, and uptake inhibition in astrocytes, leading to increased extracellular glutamate levels and ensuing neuronal excitotoxicity and degeneration. However, the mechanisms and contribution of specific glutamate transporters to MeHg-induced glutamate dyshomeostasis remain unknown. Accordingly, the present study was carried out to investigate the effects of MeHg on the transport of [d-2, 3-³H]-d-aspartate, a nonmetabolizable glutamate analog in Chinese hamster ovary cells (CHO) transfected with the glutamate transporter subtypes GLAST or GLT-1. Additional studies examined the effects of MeHg on mRNA and protein levels of these transporters. Our results indicate the following (1) MeHg selectively affects glutamate transporter mRNA expression. MeHg treatment (6 h) led to no discernible changes in GLAST mRNA expression; however, GLT-1 mRNA expression significantly (p<0.001) increased following treatments with 5 or 10 μM MeHg. (2) Selective changes in the expression of glutamate transporter protein levels were also noted. GLAST transporter protein levels significantly (p<0.001, both at 5 and 10 μM MeHg) increased and GLT-1 transporter protein levels significantly (p<0.001) decreased followign MeHg exposure (5 μM). (3) MeHg exposure led to significant inhibition (p<0.05) of glutamate uptake by GLAST (both 5 and 10 μM MeHg), whereas GLT-1 transporter activity was significantly (p<0.01) increased following exposure to 5 and 10 μM MeHg. These studies suggest that MeHg contributes to the dysregulation of glutamate homeostasis and that its effects are distinct for GLAST and GLT-1.     

Influences of cell size and aging on l-[H]norepinephrine binding sites in rat epididymal fat cells

[³H]norepinephrine binding to isolated rat fat cells was studied as a function of adipose cell age and size. Rats aged from 4 to 78 weeks were used.Scatchard analysis of norepinephrine binding revealed in old fat cells like in young ones the existence of two orders of binding sites with respectively high and low affinity for norepinephrine. The apparent association constants Ka₁ and Ka₂ associated with these binding sites did not differ consistently in the different groups of fat cells studied (Ka₁ = 1.7 to 2.2 × 10⁶ × M⁻¹; Ka₂ = 1.9 to 2.5 × 10⁴ × M⁻¹), suggesting that age and cell size do not modify the apparent affinity of norepinephrine-binding sites in rat fat cells.On the contrary, the total amount of norepinephrine bound to each of these sites was dependent upon cell age and size. In fact, maximum binding of norepinephrine to the high affinity sites was 0.9 and 9 pmol/10⁵ cells in small (diameter: 35 μ) and large (diameter: 105 μ) adipocytes, respectively, the values found for the low affinity sites being 13 and 135 pmol/10⁵ cells. When expressed per unit of fat cell area, however, the total binding capacity for these sites appeared practically constant (2.4 — 2.8 pmol × 10⁻³/mm² and 34.2 — 38.2 pmol × 10⁻³/mm² for the high and low affinity sites respectively). These data suggest that the total norepinephrine binding capacity of the fat cell is directly proportional to its surface.     

Rapid method to determine the molecular weight of dextrins and dextrans

A rapid method was developed to determine the molecular weight (M_n) of β-limit dextrin and dextrans (Leuconostoc mesenteroides) using a reducing power approach. The M_n of the β-limit dextrin was also estimated from high performance liquid chromatography (HPLC). Chromatograms were pre-calibrated with the dextrans. The three dextrins had a M_n of 2.09, 2.40 and 2.63 × 10⁵ using the reducing method and 4.80, 5.90 and 2.80 × 10⁵ by HPLC. The method could be employed to estimate M_n of dextrins where chromatographic systems were not available.     

An influence of methane concentration on the methanotrophic activity of a model landfill cover

Evaluation of kinetic parameters of methane oxidation under various conditions, on the basis of an analysis of the literature and the authors’ own laboratory research, is presented. Variation in methanotrophic activity in the profile of a simulated landfill cover was observed. The greatest activity was found at a depth of 60 cm. A low affinity (1/K_M) and high potential activity (V_max) were observed. V_max values ranged from 0.11 × 10⁻³ to 0.86 × 10⁻³ units. The values of K_M ranged from 0.6 to 2.9% of CH₄ (v/v).