In vitro effects of insulin on macromolecular events in newt limb regeneration blastemata

This work provides data demonstrating a stimulatory effect of insulin on macromolecular events occurring in cultured regeneration blastemata and demonstrates a synergistic interdependence between nerves and insulin in newt limb regeneration. The current experiments provide evidence for the following: (1) Insulin is paramount for expression of the mitogenic effect of nerves on cultures blastemata. (2) Insulin stimulates the incorporation of (3H)uridine into the acid-insoluble fraction of blastemal homogenates, but it does not alter the turnover rate of incorporated labeled uridine. (3) Insulin also stimulates the incorporation of 35SO4 and (3H)leucine into both chondroitinase-sensitive and chondroitinase-resistant blastemal proteoglycans. (4) Insulin increases the uptake of radiolabeled precursors by the blastemata, namely, (3H)leucine, (3H)uridine, 35SO4, (3H)alpha-aminoisobutyrate, and (3H)2-deoxy-D-glucose. The importance of insulin in the regulation of newt limb regeneration is discussed.     

In vitro and in vivo inhibition of LPS-induced tumor necrosis factor-alpha production by dimeric gallotannin analogues.

Designed dimeric gallotannin analogues featuring two tetragalloylglucopyranose cores connected by various hydrocarbon linkers inhibit tumor necrosis factor-alpha secretion from lipopolysaccharide-stimulated human peripheral blood mononuclear cells by up to 53% (5-24 microM concentration range) compared to control. Comparable suppression of tumor necrosis factor-alpha levels (approximately 50% vs control) was observed in the plasma of rats co-treated with lipopolysaccharide and specific tannin analogues selected for their lack of interleukin 1-beta stimulating activity.     

Inhibitory effects of pentamidine analogues on protein biosynthesis in vitro

Pentamidine despite its rather high toxicity, is currently in clinical use. For development of new drugs of this type it is important to know the mechanism of their action. Two new amidines (I and II) and 4',6-diamidino-2-phenylindole (DAPI) were found in preliminary experiments to inhibit protein synthesis in vitro in the cell-free rat liver system. The three compounds differed in the precise mode of action. The inhibitory effect of I on the activity of the eukaryotic elongation factor eEF-2 and ribosomes seems to suggest that the binding site of eEF-2 on the ribosome was blocked by this compound. eEF-2 has been identified as the primary target of II and eEF-1 as the primary target of DAPI in the system studied.     

Luliberin analogues exhibiting a cytotoxic effect on tumor cells in vitro

Luliberin analogues modified at the N-terminus were synthesized to search for drugs exerting a cytotoxic effect on cells of hormone-dependent tumors. A synthetic scheme effective in the preparation of analogues containing fatty acid residues was proposed. The cytotoxic effect of the peptides was studied on a number of cell lines of human tumors in vitro. The dependence of the antitumor effect on the length of peptide chain, amino acid sequence, and structure of the N-terminal group was demonstrated. Modification with palmitic acid was found to result in highly active compounds in the case of analogues containing more than ten aa, whereas modifications with lauric, caproic, or trimethylacetic acid led to compounds with significantly lower activities. Analogues of luliberin containing a palmitic acid residue and effectively inhibiting the growth of tumor cells in vitro were synthesized.     

In vitro polydeoxyribonucleotide effects on human pre-adipocytes

Abstract. Objectives: Adipose tissue is the most abundant and accessible source of adult stem cells. Human processed lipoaspirate contains pre‐adipocytes that possess one of the a characteristic pathways of multipotent adult stem cells and are able to differentiate in vitro into mesenchymal and also neurogenic lineages. Because stem cells have great potential for use in tissue repair and regeneration, it would be significant to be able to obtain large amounts of these cells in vitro. As demonstrated previously, purine nucleosides and nucleotides mixtures can act as mitogens for several cell types. The aim of this study was to evaluate the effects of polydeoxyribonucleotides (PDRN), at appropriate concentrations, on human pre‐adipocytes grown in a controlled medium, also using different passages, so as to investigate the relationship between the effect of this compound and cellular senescence, which is the phenomenon when normal diploid cells lose the ability to divide further. Materials and methods: Human pre‐adipocytes were obtained by liposuction. Cells from different culture passages (P6 and P16) were treated with PDRN at different experimental times. Cell number was evaluated for each sample by direct counting after trypan blue treatment. DNA assay and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide test were also carried out in all cases. Results and Conclusions: PDRN seemed to promote proliferation of human pre‐adipocytes at both passages, but cell population growth increased in pre‐adipocyte at P16, after 9 days as compared to control. Our data suggest that PDRN could act as a pre‐adipocyte growth stimulator.     

In vitro effects of monoterpenes on chloroplast membranes

A chloroplast preparation was extracted from squash (Cucurbita pepo (L.) var. Senator). Enrichment of intact chloroplasts was achieved by continuous free-flow electrophoresis. The addition of monoterpenes, detergent and free fatty acids changed the elecrophoretic separation pattern characteristically. Monoterpene-dependent degradation of envelope membranes could be prevented by addition of -tocopherol prior to monoterpene incubation.Photosynthetic electron transport of photosystem II was completely inhibited by -pinene, Triton X-100 and linolenic acid. Inhibition could be modulated by addition of -tocopherol or lecithin (phosphatidylcholine) either before or after inhibition by monoterpenes and detergent.Percentage reconstitution of photosynthetic electron transport inhibited by -pinene depended on light conditions and incubation time.     

In vitro modulation of human and murine melanoma growth by prostanoid analogues

The inhibitory effect of various prostaglandin analogues on the anchorage independent growth of murine and human melanoma cells was measured. PGA analogues (which were modified at C-16 and C-18) did not demonstrate any major improvement in activity over PGA alone. These included 16, 16-dimethyl PGA₁, 16,16-dimethyl-PGA₂, 16,16-dimethyl-18-oxa-PGA₂ and trans-δ-2-15-α acetoxy-16,16-dimethyl-18-oxa-11-deoxy-PGE₁-methylester. The thromboxane synthetase inhibitor, U51605, demonstrated weak anti-proliferative activity. PGD₂ (with a ketone at C-11 versus C-9 for PGA and PGE) was the most potent prostaglandin tested. Cells from melanoma lines displayed species differences in their sensitivities. PGA₁ and PGE₁ were the most potent inhibitors of the anchorage independent growth of murine melanoma cells. On human melanoma cells PGD₂ was the most active prostaglandin, 2–3 times more potent than PGA₁; PGE₁ was a very weak inhibitor.     

In vitro studies on the genotoxicity of the organophosphorus insecticide malathion and its two analogues

Malathion [S-(1,2-dicarboethoxyethyl)O,O-dimethyl phosphorodithioate] is a commonly used organophosphorus insecticide reported to be genotoxic both in vivo and in vitro, but the reports are conflicting. In order to elucidate the genotoxic potency of the main compounds present in commercial preparations of malathion, the DNA-damaging effect of this insecticide, its major metabolite malaoxon [S-(1,2-dicarboethoxyethyl)O,O-dimethyl phosphorothiolate] and its isomer isomalathion [S-(1,2-dicarboethoxyethyl)O,S-dimethyl phosphorodithioate], all at purity of at least 99.8%, was investigated by use of the alkaline single cell gel electrophoresis (comet assay). Freshly isolated human peripheral blood lymphocytes were incubated with 25, 75 and 200 microM of the chemicals for 1 h at 37 degrees C. The concentrations used are comparable to those found in blood following various non-lethal human exposures to pesticides. Malathion did not cause any significant changes in the comet length of the lymphocytes, throughout the range of concentrations tested. Malaoxon and isomalathion introduced damage to DNA in a dose-dependent manner. The effect induced by malaoxon was more pronounced than that caused by isomalathion. Treated cells were able to recover within a 60-min incubation in insecticide-free medium at 37 degrees C except the lymphocytes exposed to malaoxon at 200 microM, which did not show measurable DNA repair. The latter result suggests a considerable cytotoxic effect (cell death) of malaoxon at the highest concentration used. The reported genotoxicity of malathion might, therefore, be a consequence of its metabolic biotransformation to malaoxon or the presence of malaoxon and/or isomalathion as well as other unspecified impurities in commercial formulations of malathion. In this regard, the results of our study clearly indicate that malathion used as commercial product, i.e., containing malaoxon and isomalathion, can be considered as a genotoxic substance in vitro. This means that it may also produce DNA disturbances in vivo, such as DNA breakage at sites of oncogenes or tumor suppressor genes, thus playing a role in the induction of malignancies in individuals exposed to this agent. Therefore, malathion can be regarded as a potential mutagen/carcinogen and requires further investigation.     

In vitro effects of organophosphate pesticides on rat erythrocytes

In vitro effects of various organophosphate pesticides (dimethoate, chlorpyrifos, ethion and monocrotophos) were studied on hemolysis, K+ leakage and lipid peroxidation in rat erythrocytes. All the four pesticides increased hemolysis and K+ leakage from erythrocytes, that was concentration and time dependent. On the contrary, there was decrease in lipid peroxidation in erythrocyte membrane. Effect of pesticides on lipid peroxidation could be due to pesticide itself abstracting protons or interacting with free radicals rather than polyunsaturated fatty acids (PUFA), thereby protecting the latter against peroxidation.     

In vitro effects of leptin on human adipocyte metabolism

OBJECTIVE: Leptin receptors are expressed in adipocytes, suggesting potential autocrine/paracrine effects. Studies on the direct effects of leptin on adipose tissue metabolism in different species have yielded controversial data. To assess the in vitro effects of leptin on human adipocyte metabolism: lipolysis, the insulin-induced inhibition of lipolysis and lipogenesis were studied in adipocytes obtained from infants and adults. METHODS: Lipolysis was studied by incubating adipocytes with increasing concentrations of leptin or isoprenaline. Glycerol in the incubation medium was measured as an indicator of lipolysis. For the lipogenesis and insulin-induced inhibition of lipolysis experiments, the cells were preincubated with 0, 25, or 250 ng/ml of leptin for 2 h. RESULTS: Leptin did not stimulate lipolysis in human adipocytes, either in children or adults. Preincubation with leptin did not affect the insulin-induced inhibition of lipolysis, but decreased the insulin-induced lipogenesis (p < 0.05). CONCLUSIONS: This study shows that leptin has no direct lipolytic effect in human adipocytes. The lack of effect on the insulin-induced inhibition of lipolysis and the negative effect on lipogenesis indicates that the effect of leptin is not at the proximal insulin-signalling pathway but further downstream.     

In vitro study of gas effects on alveolar macrophages

Summary To evaluate the biological effects of gas pollutants on alveolar macrophages several in vitro systems ave been developed. We described here an original method of cell culture in aerobiosis, which permitted direct contact between the atmosphere and the target cells. We studied the long term (24 h) and short term (30 min) effects of NO₂ on alveolar macrophages. Our results demonstrated that exposure of alveolar macrophages to gas pollutants may be responsible for either cell injury or cell activation associated with the release of various bioactive mediators (superoxide anion, neutrophil chemotactic activity). Cell culture in aerobiosis opens new ways for the research on the biological effects of gas pollutants.Abbreviations AM alveolar macrophages - CL Chemiluminescence     

In vitro effects of citral on trypanosoma cruzi metacyclogenesis

Citral, the main constituent of lemongrass (Cymbopogon citratus) essential oil, was added to Trypanosoma cruzi cultures grown in TAU3AAG medium to observe the effect on the epimastigote-to-trypomastigote differentiation process (metacyclogenesis). Our results showed that citral (20 μg/mL) did not affect epimastigote viability or inhibit the differentiation process. Concentrations higher than 60 μg/mL, however, led to 100% cell death (both epimastigote and trypomastigote forms). Although epimastigotes incubated with 30 μg/mL citral were viable and able to adhere to the substrate, we observed around 50% inhibition in metacyclogenesis, with a calculated concentration that inhibited metacyclogenesis by 50% after 24 h (IC50/24 h) of about 31 μg/mL. Treatment with 30 μg/mL citral did not hinder epimastigote multiplication because epimastigote growth resumed when treated cells were transferred to a drug-free liver infusion tryptose culture medium. Metacyclogenesis was almost totally abolished at 40 μg/mL after 24 h of incubation. Furthermore, the metacyclic trypomastigotes obtained in vitro were similarly susceptible to citral, with an IC50/24 h, concentration that killed 50% of the cells after 24 h, of about 24.5 μg/mL. Therefore, citral appears to be a good candidate as an inhibitory drug for further studies analyzing the T. cruzi metacyclogenesis process.     

In vitro characteristics of metastatic variant subclones of restricted genetic origin

We have studied several metastatic variant cell lines derived from a common clonal origin and their transformed and untransformed parental cell lines. A number of in vitro characteristics were examined for each tumor line and these properties were correlated with the ability of the tumor cells to form pulmonary nodules in an experimental metastasis assay. Direct correlations with metastatic behavior in the lung colony assay were found to exist with the amount of cell-bound Concanavalin A and the procoagulant activities of cell lysates. In vitro parameters that did not correlate with the metastatic phenotype were: population doubling times in culture, saturation density achieved in culture, the number of colony-forming cells shed from confluent cultures, rates of cellular attachment to homotypic or heterotypic cell monolayers, plasminogen-activator production and procoagulant activity produced in serum-free conditioned medium.     

In vitro and in vivo antiherpetic activity of three new synthetic brassinosteroid analogues

Brassinosteroids are a novel group of steroids that appear to be ubiquitous in plants and are essential for normal plant growth and development. It has been previously reported that brassinosteroid analogues exert an antiviral activity against herpes simplex virus type 1 (HSV-1) and arenaviruses. In the present study, we report the chemical synthesis of compounds (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (2), (22S,23S)-5alpha-fluoro-3beta-22,23-trihydroxystigmastan-6-one (3), (22S,23S)-3beta,5alpha,22,23-tetrahydroxy-stigmastan-6-one (4) as well as their antiherpetic activity both in a human conjunctive cell line (IOBA-NHC) and in the murine herpetic stromal keratitis (HSK) experimental model. All compounds prevented HSV-1 multiplication in NHC cells in a dose dependent manner when added after infection with no cytotoxicity. Administration of compounds 2, 3, and 4 to the eyes of mice at 1, 2, and 3 days post-infection delayed and reduced the incidence of HSK, consisting mainly of inflammation, vascularization, and necrosis, compared to untreated, infected mice. However, viral titers of eye washes showed no differences among samples from treated and untreated mice. Since the decrease in the percentage of mice with ocular lesions occurred 5 days after treatment had ended, we suggest that brassinosteroids 2, 3, and 4 did not exert a direct antiviral effect in vivo, but rather may play a role in immune-mediated stromal inflammation, which would explain the improvement of the clinical signs of HSK observed.