Effect of microorganisms on the phytotoxicity of herbicides. VI. Modification of venzar activity by bacterial cultures

Interactions between two bacterial strains and venzar were compared. It was found that the mechanism of interactions is various and causes the modification of herbicide phytotoxicity. Metabolites of Bacillus subtilis 72 interfered with herbicide by affecting physiological processes in plant tissues and enhancing its inhibitory influence. Arthrobacter sp. 18 strain decreased the phytoinhibitory effect of herbicide due to conjugation with the carrier from venzar.     

Antibacterial activity of plasma from crocodile (Crocodylus siamensis) against pathogenic bacteria

ABSTRACT: BACKGROUND: The Siamese crocodile (Crocodylus siamensis) is a critically endangered species of freshwater crocodiles. Crocodilians live with opportunistic bacterial infection but normally suffer no adverse effects. They are not totally immune to microbial infection, but their resistance thereto is remarkably effective. In this study, crude and purified plasma extracted from the Siamese crocodile were examined for antibacterial activity against clinically isolated, human pathogenic bacterial strains and the related reference strains. METHODS: Crude plasma was prepared from whole blood of the Siamese crocodile by differential sedimentation. The crude plasma was examined for antibacterial activity by the liquid growth inhibition assay. The scanning electron microscopy was performed to confirm the effect of crude crocodile plasma on the cells of Salmonella typhi ATCC 11778. Effect of crude crocodile plasma on cell viability was tested by MTT assay. In addition, the plasma was purified by anion exchange column chromatography with DEAE-Toyopearl 650M and the purified plasma was tested for antibacterial activity. RESULTS: Crude plasma was prepared from whole blood of the Siamese crocodile and exhibited substantial antibacterial activities of more than 40% growth inhibition against the six reference strains of Staphylococcus aureus, Salmonella typhi, Escherichia coli, Vibrio cholerae, Pseudomonas aeruginosa, and Staphylococcus epidermidis, and the four clinical isolates of Staphylococcus epidermidis, Pseudomonas aeruginosa, Salmonella typhi, and Vibrio cholerae. Especially, more than 80% growth inhibition was found in the reference strains of Salmonella typhi, Vibrio cholerae, and Staphylococcus epidermidis and in the clinical isolates of Salmonella typhi and Vibrio cholerae. The effect of the crude plasma on bacterial cells of Salmonella typhi, a certain antibacterial material probably penetrates progressively into the cytoplasmic space, perturbing and damaging bacterial membranes. The effect of the crude plasma was not toxic by the yellow tetrazolium bromide (MTT) assay using a macrophage-like cell, RAW 264.7. The pooled four fractions, designated as fractions D1-D4, were obtained by column chromatography, and only fraction D1 showed growth inhibition in the reference strains and the clinical, human pathogenic isolates. CONCLUSIONS: The crude and purified plasma from the Siamese crocodile significantly showed antibacterial activity against pathogenic bacteria and reference strains by damage cell membrane of target bacterial cells. From the MTT assay, the Siamese crocodile plasma was not cytotoxic to the cells.     

Effect of L-methionine and S-adenosylmethionine on growth of an adenine mutant of Saccharomyces cerevisiae

A pink, adenine-requiring yeast utilized adenine, hypoxanthine, or S-adenosylmethionine (SAM), in quantities up to 3 mumoles per 100 ml of medium, as equivalent sources of purine for cell growth, but not methylthioadenosine or S-adenosylhomocysteine. Utilization of SAM for growth was inhibited by the presence of l-methionine in quantities greater than 0.6 mumole per 100 ml of medium. However, 6 mumoles of l-methionine had no effect on growth when adenine or hypoxanthine was the source of purine. These sources also reversed the inhibitory effects of 6 mumoles of the amino acid on the utilization of SAM. The presence of 400 mumoles of the amino acid resulted in some inhibition of growth when the organisms were grown with adenine, hypoxanthine, or adenine plus SAM but had no effect on the total uptake of adenine-8-(14)C. Studies on the uptake of radioactivity from a mixture of SAM-adenine-8-(14)C and (3)H-labeled SAM-methyl indicated that these components were taken into the cells at different rates which were altered by the presence of l-methionine. The fixation of (35)S from (35)S-labeled adenosylmethionine into the cells was inhibited by the presence of the amino acid. The cells synthesized and accumulated SAM in the presence of 400 mumoles of l-methionine plus adenine even when exogenous SAM was supplied. Approximately 47% of radioactivity fixed from exogenous SAM-adenine-8-(14)C and 12% from (3)H-labeled SAM-methyl were found in reisolated SAM.     

Zinc does not inhibit the capacitation of human spermatozoa in vitro

The effects of zinc on human sperm motility, fertilizing capacity (as assessed by penetration of human spermatozoa into the zona pellucida-free hamster oocyte), and nuclear chromatin decondensation were investigated using spermatozoa from four fertile donors. Both sperm motility and the penetration of sperm into zona-free hamster ova were consistently impaired in media containing 1,000 μM zinc. Spermatozoa from one man were similarly affected at a concentration of 500 μM zinc, but no adverse effects were noted at this zinc concentration in experiments with other donors. Since decreased fertilizing capacity in response to zinc was always accompanied by a significant decline in both the percentage of motile cells and mean swimming speeds, it appears that all of these results reflect a general toxic effect on the cells. At lower concentrations (125–250 μM), zinc had no effect on human sperm motility nor their ability to undergo capacitation and penetrate zona-free hamster ova in vitro. For some donors, zinc (125–500 μM) stimulated both the attachment of spermatozoa to the hamster vitellus and the incorporation of spermatozoa into the hamster ooplasm. The decondensation of human sperm nuclear chromatin in sodium dodecyl sulfate was largely inhibited when zinc was added to the medium, but no significant changes in nuclear stability were apparent after capacitation in zinc-free medium. We conclude that zinc, when present in subtoxic concentrations, does not adversely affect the ability of human spermatozoa to undergo capacitation and penetrate zona-free hamster ova in vitro.     

Increased multiplication of dengue virus in mouse peritoneal macrophage cultures by treatment with extracts of Ascaris-Parascaris parasites

Methylcellulose-elicited peritoneal macrophages from BALB/c mice were cultivated in vitro and inoculated with dengue virus (DV). At intervals thereafter portions of the culture fluids were taken and titrated for viral infectivity. Extracts from Ascaris suum and Parascaris equorum, either crude or Sephadex G-100 fractionated, were examined for effects on the multiplication of DV. The macrophage cultures treated with the above substances produced larger amounts of DV compared with untreated control cultures. The enhancing effect of the substances depended on doses added and duration of treatment and was suppressed by co-treatment with carrageenan, a specific macrophage-inhibiting agent, but was not related to the viability of cultured cells. In fluorescent antibody (FA) as well as infectious center assay experiments, it was shown that the DV-infected cells were found more frequently in treated cultures than in untreated control cultures. In the treated cultures phagocytosis by cultured cells was also of a higher magnitude than that in untreated cultures. In cocultures of macrophages and splenocytes from the same line of mice, no additive effect of splenocytes was noted. The limulus amebocyte lysate clotting enzyme reaction (Limulus test) indicated that involvement of bacterial lipopolysaccharides in the enhancement phenomena was negligible. The data so far obtained suggest that the enhancing effect was due to direct action of the parasitic extracts on macrophages. Four Sephadex G-100 fractions from the crude extracts showed similar activities; however, the effects of fractions I and III appeared to be comparatively strong. Significance of the findings in relation to the pathogenesis of DV infection was discussed.     

Unoccupied binding sites for oestrogen in nuclei of a breast tumour cell line (MCF-7).

1. A method to measure both occupied and unoccupied oestrogen receptors directly in the crude nuclear fraction of the MCF-7 cells was developed. The receptors had high affinity for oestradiol (Kd approx. 0.7 nM) and binding specificity characteristics of oestrogen receptors. 2. A substantial amount of the unoccupied receptors were found in the crude nuclear fraction. 3. Several experiments excluded the possibility that the unoccupied nuclear receptor might be a cytoplasmic contaminant. (a) Multiple extractions with Tris buffer released about 75% of the total receptor content, leaving the rest unextractable in the crude nuclear fraction. (b) Nuclei purified by centrifugation through 1.8M-sucrose and treatment with 0.7% Triton X-100, or by centrifugation through 50% glycerol with 0.1% Triton X-100 contained similar amounts of unoccupied receptors to that found in the crude nuclear fraction. (c) In cells cultured during 5 days after preconfluency a 3-fold increase in the amount of unoccupied cytoplasmic receptors occurred, whereas the amount of unoccupied nuclear receptors did not change significantly and conversely in cells exposed to increasing concentrations of oestradiol the unoccupied cytoplasmic receptor was continuously depleted but no considerable change in the unoccupied nuclear receptor was found.     

Mitochondria transfection by oligonucleotides containing a signal peptide and vectorized by cationic liposomes.

The progress of research in gene therapy allows hope for treatment of mitochondrial genetic disorders provided that efficient methods for gene transfer into mitochondria can be found. In this work, we have used an oligonucleotide coupled covalently to a mitochondria-targeted peptide at one end and a cationic liposome prepared from trimethyl aminoethane carbamoyl cholesterol iodide (TMAEC-Chol) to carry it in living cells. With a fluorescent probe to label the oligonucleotide at the other end and by means of confocal microscopy, we show that such modified oligonucleotides complexed to liposomes enter into the cytoplasm of human fibroblasts in primary culture, and then, after dissociation from the complexes, they penetrate into the mitochondria. The fluorescence was still observed after 8 days, suggesting the continued presence of oligonucleotides. At the concentrations used for this study, the cationic liposomes have practically no effect on cell growth, as revealed by the MTT assay.     

Influence of fatty acids and bovine serum albumin on the growth of human hepatoma and immortalized human kidney epithelial cells

Summary The protective influence of bovine serum albumin against growth inhibition caused by fatty acids was studied in human hepatoma (HepG2) and immortalized human kidney epithelial (IHKE) cells. In general, growth inhibition by unsaturated fatty acids (0.15 mmol/liter) increased with increasing number of double bonds. For HepG2 cells crude albumin (1g/100 ml) did not greatly modify growth inhibition by arachidonic, eicosapentaenoic, and docosahexaenoic acid. With oleic, linoleic, and linolenic acids, crude and defatted albumin stimulated cell growth. In contrast, for IHKE cells both albumins counteracted growth inhibition by unsaturated fatty acids to approximately the same extent. When HepG2 cells were cultured in the presence of saturated fatty acids (0.3 mmol/liter), C2, C6, and C8 had no or little inhibitory effect. C10 and C12 inhibited cell growth appreciably, whereas C14, and especially C16, had poor inhibitory effects. Crude albumin counteracted growth inhibition by all these fatty acids. In contrast, defatted albumin had little or no effect (except against C10 and C12), and even increased the growth inhibition by C14 and C16. With unsaturated fatty acids there seemed to be an inverse relationship between cell growth and the concentration of thiobarbituric acid reactive substances (TBARS) in media. Vitamin E abolished growth inhibition (and the increase in TBARS concentration) by unsaturated fatty acids. The complex interaction between fatty acids and albumins calls for great caution when interpreting data on growth effects.     

人参皂苷粗提液对西洋参早期生长的化感效应

以西洋参幼苗为受体,采用玻璃砂培养方法研究不同浓度(25、50、100 mg·L-1)的人参皂苷粗提液对西洋参早期生长及生理代谢的影响,并用透射电镜观察幼苗根尖在处理过程中亚显微结构的变化.结果发现:(1)在不同浓度人参皂苷粗提液处理下,西洋参幼苗早期生长的各项生理指标均表现出不同程度的低促高抑现象.(2)各处理对西洋参幼根及幼苗的生物量无显著影响,但中高浓度处理对根长的抑制作用明显大于苗高.(3)中高浓度均对根系活力具有显著抑制作用(P<0.05);在高浓度下幼苗叶片中超氧阴离子自由基(O?2-)的产生速率和丙二醛(MDA)含量均显著增加,叶片及幼根中相对电导率也有明显增加,而叶片可溶性蛋白和叶绿素(Chl)合成受到轻微的促进作用,Chl含量随处理时间的延长虽呈逐渐下降趋势,但仍高于对照.(4)高浓度处理的幼苗根尖细胞的核膜膨胀、核仁变形,液泡膜解体,不能完成正常的生命活动.研究表明,不同浓度的人参皂苷粗提液对西洋参早期生长具有化感效应,且对受体地下部分的影响明显大于地上部分.     

Bruchid (Coleoptera: Bruchidae) ovicidal phenylbutanoid from Zingiber purpureum

The larvicidal activity of the dichloromethane extract of Zingiber purpureum Roscoe (Zingiberaceae) rhizome against the second instar of Aedes aegypti (L.) (Diptera: Culicidae) is shown to be due to 4-(3',4'-dimethoxyphenyl)buta-1,3-diene. The diene also showed ovicidal activity against the bruchid Callosobruchus maculatus (F.) (Coleoptera: Bruchidae). Most of the eggs laid by bruchids on treated cowpea seeds were transparent, and very few of them contained developing embryos. The few larvae produced from these embryos were unable to penetrate the seed coat and enter the seed. Similar effects were seen when adults were exposed to the compound and then placed on untreated cowpea seeds, suggesting that a new type of maternally mediated ovicidal effect was involved. Coated and impregnated granular formulations of the extract were evaluated for use in the control of bruchid infestation of stored cowpea seeds. Coated granules showed activity similar to that of the crude extract but were found to lose activity rapidly. Impregnated granules were found to be less active than the crude extract.     

Extracellular Matrix Penetration by Epithelial Cells Is Influenced by Quantitative Changes in Basement Membrane Components and Growth Factors

We have previously shown that isolated mouse fetal choroid plexus epithelial (CPE) cells penetrate a basement membrane matrix (Matrigel) substratein vitroto form single-layered epithelial vesicles embedded within the matrix. To determine which properties of the matrix are important for inducing or permitting cells to penetrate the substrate and organize into multicellular vesicles we have made quantitative changes to the basement membrane components and growth factors in cell cultures. Matrigel diluted to 33 or 10% with a collagen I gel was not permissive to cell invasion, and CPE cells formed a polarized epithelial monolayer on the substrate surface which had ultrastructural characteristics similar to those of CPE vesicles. Cells in these monolayers proliferated more rapidly than cells in epithelial vesicles. When deliberately embedded within a 33 or 10% Matrigel matrix, CPE cells were able to form vesicles, indicating that a dilute matrix is nonpermissive to cell invasion but promotes epithelial polarization and organization into vesicles. Cells embedded within a 100% collagen I matrix did not proliferate or form epithelial vesicles and the majority of cells did not remain viable. Addition of laminin to the collagen I gel promoted cell adhesion and cell survival, but did not promote the formation of extensive monolayers on the substrate nor the formation of epithelial vesicles within the matrix. Cell invasion into the 33% Matrigel matrix was induced by addition of laminin, nidogen, or a laminin–nidogen complex to the substrate or by addition of TGFβ2 to the culture medium, but not TGFβ1 or PDGF. These studies show that CPE cells are sensitive to quantitative changes in matrix composition, which influences their survival and proliferation and also their ability to penetrate the matrix and organize into multicellular epithelial vesicles.     

Predominant suppression of neutrophil colony growth by recombinant human tumor necrosis factor

To investigate the suppressive effect of recombinant human tumor necrosis factor (rH-TNF) on colony growth of human granulocyte-macrophage progenitor cells (CFU-GM), cytochemical examinations of CFU-GM colonies were performed by a triple staining method. Each colony was classified into five subtypes, and the effects of rH-TNF on each subtype were analyzed. Neutrophil colony growth was inhibited by rH-TNF in a dose-dependent manner, and it was almost completely suppressed at 100 U/ml. In contrast, no significant suppressive effect of rH-TNF was found on the growth of monocyte-macrophage and eosinophil colonies at 100 U/ml or less. When recombinant human granulocyte colony-stimulating factor which almost exclusively stimulates neutrophil colony formation was used as a source of colony-stimulating activity, the total colony growth was almost completely suppressed by 100 U/ml of rH-TNF. These results indicate predominant inhibition of neutrophil colony growth by rH-TNF.     

Impact of impurities in biodiesel-derived crude glycerol on the fermentation by <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis> ATCC 6013

During the production of biodiesel, crude glycerol is produced as a byproduct at 10% (w/w). Clostridium pasteurianum has the inherent potential to grow on glycerol and produce 1,3-propanediol and butanol as the major products. Growth and product yields on crude glycerol were reported to be slower and lower, respectively, in comparison to the results obtained from pure glycerol. In this study, we analyzed the effect of each impurity present in the biodiesel-derived crude glycerol on the growth and metabolism of glycerol by C. pasteurianum. The crude glycerol contains methanol, salts (in the form of potassium chloride or sulfate), and fatty acids that were not transesterified. Salt and methanol were found to have no negative effects on the growth and metabolism of the bacteria on glycerol. The fatty acid with a higher degree of unsaturation, linoleic acid, was found to have strong inhibitory effect on the utilization of glycerol by the bacteria. The fatty acid with lower or no degrees of unsaturation such as stearic and oleic acid were found to be less detrimental to substrate utilization. The removal of fatty acids from crude glycerol by acid precipitation resulted in a fermentation behavior that is comparable to the one on pure glycerol. These results show that the fatty acids in the crude glycerol have a negative effect by directly affecting the utilization of glycerol as the carbon source, and hence their removal from crude glycerol is an essential step towards the utilization of crude glycerol.     

Cyclic adenosine 3'',5''-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3'',5''-monophosphate phosphodiesterase in Pseudomonas and Bacteroides.

A modified Gilman assay was used to determine the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) in rapidly filtered cells and in the culture filtrates of Pseudomonas aeruginosa, Escherichia coli K-12, and Bacteroides fragilis. In P. aeruginosa cultures, levels of cAMP in the filtrate increased with the culture absorbance (3.5 to 19.8 X 10(-9) M) but did not vary significantly with the carbon source used to support growth. Intracellular concentrations (0.8 to 3.2 X 10(-5) M) were substantially higher and did not vary appreciably during growth or with carbon source. Sodium cAMP (5 mM) failed to reverse the catabolite repression of inducible glucose-6-phosphate dehydrogenase (EC 1.1.1.49) synthesis caused by the addition of 10 mM succinate. Exogenous cAMP also had no discernible effect on the catabolite repression control of inducible mannitol dehydrogenase (EC 1.1.1.67). P. aeruginosa was found to contain both soluble cAMP phosphodiesterase (EC 3.1.4.17) and membrane-associated adenylate cyclase (EC 4.6.1.1) activity, and these were compared to the activities detected in crude extracts of E. coli. B. fragilis crude cell extracts contain neither of these enzyme activities, and little or no cAMP was detected in cells or culture filtrates of this anaerobic bacterium.     

Characterization of cells isolated and cultured from human bone

Cells isolated from samples of human iliac crest and human femoral heads by collagenase digestion have been successfully cultured in Fitton-Jackson modified BGJb culture medium supplemented with penicillin (100 units/ml), streptomycin (100 micrograms/ml), and fetal calf serum (10%). Although only a low proportion of the cells survived the initial plating (less than 1%), cells established in culture were readily passaged. Examination of cells obtained at intervals during the collagenase digestion showed that the percentage of cells that attached increased with time of digestion. Rapid sample preparation of rat bone did not substantially increase the number of cells attaching. Thus, it seems unlikely that the low survival was due to loss of viability during sample transportation and preparation. Of several media tested BGJb supplemented with 10% fetal calf serum supported the best growth. Population doubling time averaged 104 hr. Cultured human bone cells were assayed for alkaline phosphatase activity using the azo dye method with naphthol ASTR phosphate as the substrate. A portion of the cells (19%) demonstrated high activity in all cultures examined regardless of the passage number of the culture. Autoradiography of cells exposed to [3H]thymidine showed incorporation of the label into both alkaline phosphate-positive and -negative cells. The stimulation of cell proliferation by growth factors was studied by determining the incorporation of [3H]thymidine into DNA. The specific skeletal growth factor from human bone stimulated cell proliferation several-fold with a half-maximal effect at 5 micrograms/ml. Insulin, epidermal growth factor, and a crude preparation of somatomedin C also stimulated cell proliferation.     

穴居狼蛛毒的某些组分对培养的人肺腺癌细胞(SPC-A1)的致伤作用

本实验观察了从新疆产穴居狼蛛（Lycosa singoriensis）的冻干毒腺中提取的粗毒及其经Sephadex G-25柱层析分离所得到的各组分对培养的人肺腺癌细胞的杀伤作用：①与对政党人胚的肺细胞、正常人淋巴细胞和红细胞相比,穴居狼蛛毒对培养的SPC-A1有明显的高杀伤作用。例如用于杀伤50%的SPC-A1细胞所需的粗毒浓度为25 μg/ml,而用于杀伤相同量正常人胚肺细胞和淋巴细胞所需的粗毒浓度分别为600-500 μg/ml,即使将粗毒浓度提高到2000 μg/ml,也只能杀伤40%左右的正常人的红细胞。②在粗毒的8个分离组分中,第Ⅲ、Ⅵ和Ⅷ组分表现出杀伤SPC-A1细胞的活性,尤以后两者为明显。③粗毒经100 ℃加热30分钟后,杀伤SPC-A1细胞的活力稍有下降,但组分Ⅳ和Ⅷ经同样的加温处理后,该活性不变,唯组分Ⅲ在加温后该活性完全丧失。     

Formate acts as a diffusible signal to induce Salmonella invasion

To infect an animal host, Salmonella enterica serovar Typhimurium must penetrate the intestinal epithelial barrier. This process of invasion requires a type III secretion system encoded within Salmonella pathogenicity island I (SPI1). We found that a mutant with deletions of the acetate kinase and phosphotransacetylase genes (ackA-pta) was deficient in invasion and SPI1 expression but that invasion gene expression was completely restored by supplying medium conditioned by growth of the wild-type strain, suggesting that a signal produced by the wild type, but not by the ackA-pta mutant, was required for invasion. This mutant also excreted 68-fold-less formate into the culture medium, and the addition of sodium formate to cultures restored both the expression of SPI1 and the invasion of cultured epithelial cells by the mutant. The effect of formate was pH dependent, requiring a pH below neutrality, and studies in mice showed that the distal ileum, the preferred site of Salmonella invasion in this species, had the appropriate formate concentration and pH to elicit invasion, while the cecum contained no detectable formate. Furthermore, we found that formate affected the major regulators of SPI1, hilA and hilD, but that the primary routes of formate metabolism played no role in its activity as a signal.     

Growth-inhibitory activity of lymphoid cell plasma membranes. I. Inhibition of lymphocyte and lymphoid tumor cell growth

Membranes isolated from normal spleen cells or lymphoid tumor cells were found to inhibit in vitro growth of several murine tumor cell lines including a B cell hybridoma, a thymoma, and a mastocytoma. 50% inhibition occurred at membrane protein concentrations of 60-100 micrograms/ml. A similar concentration dependence was found for inhibition of [3H]-thymidine incorporation by tumor cells and for the lipopolysaccharide-induced mitogenic response of normal spleen cells. The inhibitory activity co-purified with the plasma membrane upon fractionation of crude membranes. Membrane solubilization with deoxycholate followed by dialysis to remove the detergent gave good recovery of inhibitory activity in the resulting reconstituted membranes. Membrane-mediated growth inhibition resulted from a decreased rate of proliferation and not from increased cell death. A toxic effect of the membranes was further ruled out by the finding that increasing the fetal calf serum content of the medium could substantially reverse the growth inhibition. Thus, the plasma membrane of lymphoid cells contains a component that can slow or stop the growth of cells in culture. This membrane component may have a role in cell contact-mediated regulation of growth.     

ELECTRON MICROSCOPY OF ABSORPTION OF TRACER MATERIALS BY TOAD URINARY BLADDER EPITHELIUM

The absorption of Thorotrast and saccharated iron oxide by the epithelium of the toad urinary bladder was studied by electron microscopy. Whether the toads were hydrated, dehydrated, or given Pitressin, no significant differences in transport of colloidal particles by epithelial cells were observed. This implies that these physiological factors had little effect on the transport of the tracer particles. Tracer particles were encountered in three types of epithelial cells which line the bladder lumen, but most frequently in the mitochondria-rich cells. Tracer materials were incorporated into the cytoplasm of epithelial cells after being adsorbed to the coating layer covering the luminal surface of the cells. In the intermediate stage (1 to 3 hours after introducing tracer) particles were present in small vesicles, tubules, and multivesicular bodies. In the later stages (up to 65 hours), the particles were more commonly seen to be densely packed within large membrane-bounded bodies which were often found near the Golgi region. These large bodies probably were formed by the fusion of small vesicles. Irrespective of the stages of absorption, no particles were found in the intercellular spaces or in the submucosa. Particles apparently did not penetrate the intercellular spaces of the epithelium beyond the level of the tight junction.     

Proliferation of guinea-pig uterine epithelial cells in serum-free culture conditions: effect of 17 beta-estradiol, epidermal growth factor and insulin

The effects of 17 beta-estradiol (E2), epidermal growth factor (EGF) and insulin, alone or in association on guinea-pig uterine epithelial cell proliferation were examined in serum-free culture conditions. Primary cultures of epithelial cells were made quiescent by serum depletion, then incubated in a chemically defined medium. In this medium, insulin increased DNA synthesis but not in a dose-dependent manner for concentrations ranging from 0.2 to 10 micrograms/ml. A significant effect of EGF was found only for the highest concentration tested (100 ng/ml). E2 alone or in the presence of insulin (1 microgram/ml) had no effect whatsoever on the concentration tested (10(-10)-10(-5)M). Insulin (10 micrograms/ml) plus EGF (100 ng/ml) exerted on DNA synthesis and cell proliferation a significant additive effect which was identical to the growth stimulation induced by 10% fetal calf serum. The effects of insulin plus EGF were not modified by the addition of E2. These findings suggest that E2 is not directly mitogenic for uterine epithelial cells in defined culture conditions and that the mitogenic response to optimal concentration of insulin plus EGF is independent of E2.