Effects of Cymbopogon citratus L. essential oil on the growth, lipid content and morphogenesis of Aspergillus niger ML2-strain

The mycelial growth of Aspergillus niger van Tieghem was completely inhibited using 1.5 (microl/ml or 2.0 (microl/ml of Cymbopogon citratus essential oil applied by fumigation or contact method in Czapek liquid medium, respectively. This oil was found also to be fungicidal at the same concentrations. The sublethal doses 1.0 and 1.5 (microl/ml inhibited about 70% of fungal growth after five days of incubation and delayed conidiation as compared with the control. Microscopic observations using Light Microscope (LM), Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) were carried out to determine the ultra structural modifications of A. niger hyphae after treatment with C. citratus essential oil. The hyphal diameter and hyphal wall appeared markedly thinner. This oil also caused plasma membrane disruption and mitochondrial structure disorganization. Moreover, Ca+2, K+ and Mg+2 leakages increased from the fumigated mycelium and its total lipid content decreased, while the saturated fatty acids decreased and unsaturated fatty acids increased. These findings increase the possibility of exploiting C. citratus essential oil as an effective inhibitor of biodegrading and storage contaminating fungi and in fruit juice preservation.     

Effect of Cymbopogon citratus L. essential oil on growth and morphogenesis of Saccharomyces cerevisiae ML2-strain

The growth of Saccharomyces cerevisiae was completely inhibited using 2.0 microl/ml or 4.0 microl/ml of Cymbopogon citratus essential oil applied by fumigation or contact method in Sabouraud's broth medium, respectively. This oil was found also to be fungicidal at the same concentrations. The sublethal doses 1.0 and 3.0 microl/ml inhibited about 98% of yeast growth after 24 hr of incubation as compared with the control. Microscopic observations using Light Microscope (LM), Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) showed morphogenic and ultrastructure changes in the fumigated cells with 1.0 microl/ml of the oil. These changes including decrease in cell size, depressions on the surface of the cells, alteration in cell wall thickness and disruption of plasma membrane. Moreover, Ca(+2), K(+) and Mg(+2) leakages increased from the fumigated cells and its total lipid content decreased. Also, the fatty acid composition was altered with decrease in the amount of saturated fatty acids and increase in the amount of unsaturated fatty acids.     

The use of powder and essential oil of Cymbopogon citratus against mould deterioration and aflatoxin contamination of "egusi" melon seeds

Experiments were carried out to determine the potential of using the powder and essential oil from dried ground leaves of Cymbopogon citratus (lemon grass) to control storage deterioration and aflatoxin contamination of melon seeds. Four mould species: Aspergillus flavus, A. niger, A. tamarii and Penicillium citrinum were inoculated in the form of conidia suspension (approx. 10(6) conidia per ml) unto shelled melon seeds. The powdered dry leaves and essential oil from lemon grass were mixed with the inoculated seeds at levels ranging from 1-10 g/100 g seeds and 0.1 to 1.0 ml/100 g seeds respectively. The ground leaves significantly reduced the extent of deterioration in melon seeds inoculated with different fungi compared to the untreated inoculated seeds. The essential oil at 0.1 and 0.25 ml/100 g seeds and ground leaves at 10 g/100 g seeds significantly reduced deterioration and aflatoxin production in shelled melon seeds inoculated with toxigenic A. flavus. At higher dosages (0.5 and 1.0 ml/100 g seeds), the essential oil completely prevented aflatoxin production. After 6 months in farmers' stores, unshelled melon seeds treated with 0.5 ml/ 100 g seeds of essential oil and 10 g/100 g seeds of powdered leaves of C. citratus had significantly lower proportion of visibly diseased seeds and Aspergillus spp. infestation levels and significantly higher seed germination compared to the untreated seeds. The oil content, free fatty acid and peroxide values in seeds protected with essential oil after 6 months did not significantly differ from the values in seeds before storage. The efficacy of the essential oil in preserving the quality of melon seeds in stores was statistically at par with that of fungicide (iprodione) treatment.     

Anti-Candida albicans activity of essential oils including Lemongrass (Cymbopogon citratus) oil and its component, citral]

The effects of 12 essential oils, popularly used as antifungal treatments in aromatherapy, on growth of Candida albicans were investigated. Mycelial growth of C. albicans, which is known to give the fungus the capacity to invade mucosal tissues, was inhibited in the medium containing 100 micro g/ml of the oils: lemongrass (Cymbopogon citratus), thyme (Thymus vulgaris), patchouli (Pogostemon cablin) and cedarwood (Cedrus atlantica). Not only lemongrass oil but also citral, a major component of lemongrass oil (80%), in the range of 25 and 200 micro g/ml inhibited the mycelial growth but allowed yeast-form growth. More than 200 micro g/ml of citral clearly inhibited both mycelial and yeast-form growth of C. albicans. These results provide experimental evidence suggesting the potential value of lemongrass oil for the treatment of oral or vaginal candidiasis.     

Antifungal activity of Lavandula angustifolia essential oil against Candida albicans yeast and mycelial form.

The antifungal activity of the essential oil of Lavandula angustifolia Mill. (lavender oil) and its main components, linalool and linalyl acetate, was investigated against 50 clinical isolates of Candida albicans (28 oropharyngeal strains, 22 vaginal strains) and C. albicans ATCC 3153. Growth inhibition, killing time and inhibition of germ tube formation were evaluated. The chemical composition of the essential oil was determined by gas chromatography and mass spectrometry. Lavender oil inhibited C. albicans growth: mean minimum inhibitory concentration (MIC) of 0.69% (vol./vol.) (vaginal strains) and 1.04% (oropharyngeal strains); mean MFC of 1.1% (vaginal strains) and 1.8% (oropharyngeal strains). Linalool was more effective than essential oil: mean MIC of 0.09% (vaginal strains) and 0.29% (oropharyngeal strains); mean MFC of 0.1% (vaginal strains) and 0.3% (oropharyngeal strains). Linalyl acetate was almost ineffective. Lavender oil (2%) killed 100% of the C. albicans ATCC 3153 cells within 15 min; linalool (0.5%) killed 100% of the cells within 30 s. The essential oil inhibited germ tube formation (mean MIC of 0.09%), as did the main components (MIC of 0.11% for linalool and 0.08% for linalyl acetate). Both the essential oil and its main components inhibited hyphal elongation of C. albicans ATCC 3153 (about 50% inhibition at 0.016% with each substance). Lavender oil shows both fungistatic and fungicidal activity against C. albicans strains. At lower concentrations, it inhibits germ tube formation and hyphal elongation, indicating that it is effective against C. albicans dimorphism and may thus reduce fungal progression and the spread of infection in host tissues.     

Chemical composition and larvicidal properties of the essential oils from Drimys brasiliensis Miers (Winteraceae) on the cattle tick Rhipicephalus (Boophilus) microplus and the brown dog tick Rhipicephalus sanguineus

The essential oil obtained from leaves and stem barks of the Southern Brazilian native Drimys brasiliensis Miers, a tree with medicinal properties, was analyzed by gas chromatography (GC) and GC/mass spectrometry (MS). The oil was characterized by sesquiterpenoids (66%), cyclocolorenone being the most abundant (30.4%), followed by bicyclogermacrene (11.8%) and alpha-gurjunene (6.0%). Laboratory tests were carried out to determine the toxicity of the essential oil on larvae of the cattle tick Rhipicephalus (Boophilus) microplus and the brown dog tick Rhipicephalus sanguineus by the larval immersion test. It was observed that the oil was lethal, killing 100% of the larvae of both ticks at the doses of 25, 12.5, and 6.25 μl/ml. The lowest dose tested, 3.125 μl/ml, was also toxic, killing 95–98% of the larvae.     

Standardization of a hyphal inoculum of aspergilli for amphotericin B susceptibility testing.

Standardized, evenly dispersed hyphal suspensions served as the inoculum in a microtiter technique for amphotericin B antifungal susceptibility testing. Preliminary testing with six strains of Aspergillus fumigatus and A. flavus produced consistent and reproducible results at 30 degrees C over 24 h. The observed amphotericin B MICs required for hyphae (0.3 to 0.6 microgram/ml) were comparable to MICs required for conidia (0.16 to 0.6 microgram/ml). The results were evaluated and compared with previously published information.     

Piperitenone oxide as toxic, repellent, and reproduction retardant toward malarial vector Anopheles stephensi (Diptera: Anophelinae)

Anopheles stephensi (Liston) is a well-known vector of malarial parasite in tropical countries. The developing trend of resistance in mosquitoes toward synthetic mosquitocidal agents makes their management extremely difficult. Effectiveness of essential oils with aroma therapeutic values seems to be an emerging tool to combat this vector. Piperitenone oxide isolated from essential oil of a new genotype, Mentha spicata L. variety viridis, has been evaluated for larvicidal, ovicidal, oviposition-deterrent, developmental toxicity, and repellent properties against various stages of A. stephensi. The results indicated the higher efficacy of piperitenone oxide than the crude essential oil of M. spicata variety viridis in all the bioassay experiments. The lethal response of piperitenone oxide and the oil toward fourth instar larvae showed LD50 values of 61.64 and 82.95 microg/ml, respectively. Female adults of A. stephensi exposed to the oil laid approximately 42 times less number of eggs at the dose of 60.0 microg/ml as compared with control, whereas exposure of piperitenone oxide at the same dose completely inhibited the oviposition. Furthermore, piperitenone oxide also completely inhibited egg hatching at the dose of 75.0 microg/ml in ovicidal assay. Developmental toxicity studies showed the significant developmental inhibition potential of the compound and oil. Additionally, piperitenone oxide was found to be highly toxic and repellent toward adults of A. stephensi as compared with oil.     

Evaluation of miconazole activity contained in human serum to hypha of Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic pathogen, especially in an immunocompromised host. This fungus grows in a hyphal form in infected tissues; therefore, new tests to examine hyphal susceptibility are needed. In this study, we measured the mycotic activity of miconazole (MCZ) contained in human serum against A. fumigatus using the BioCell-Tracer method. Three serum samples were obtained from the same patient who was injected with 600 mg b.i.d. MCZ daily for 2 days. The concentrations of MCZ in the serum sample were 8.8, 3.5, and 1.6 micro g/ml, respectively. The serum containing 8.8 micro g/ml of MCZ inhibited hyphal growth 90 minutes after administration, and the hypha stopped growing. The serum containing 3.5 micro g/ml MCZ stopped hypha growth 100 minutes after administration, but re-growth of the hypha was observed at this concentration of MCZ. Serum containing 1.6 micro g/ml did not inhibite hyphal growth, nor did control serum have any inhibitory activity foward hyphae. Based on these results, we conclude that the BioCell-Tracer is a useful method for determining the effects on filamentous fungi of antifungal agents in the serum.     

Oxidative phosphorylation and the tricarboxylic acid cycle are essential for normal development of mouse ovarian follicles

BACKGROUND: Mouse ovarian follicles are typically grown in upright drops of culture medium. Recently we found that culture of follicles at the medium-gas interface in inverted drops markedly improved follicular development, possibly due to improved access of oxygen to the follicle. In this study, we examined the importance of aerobic energy metabolism for follicle development by culturing mouse follicles (198 6 16.5 initial microm diameter, mean 6 SD) in the presence of phosphorylation and tricarboxylic acid (TCA) cycle inhibitors. METHODS: All inhibitors were tested in the inverted system using 100 microl medium drops in 96-well plates; certain inhibitors were also tested in upright drops with or without an oil overlay. RESULTS: The oxidative phosphorylation inhibitor rotenone (0.1, 0.5 and 1 micromol/l) totally abolished follicle growth in the inverted system; cyanide (1 mmol/l) totally abolished growth in the upright with oil system but not in the inverted system (possibly due to loss of cyanide gas due to the absence of an oil overlay). The mitochondrial uncoupler 2,4-dinitrophenol (0.5 and 1 mmol/l) also abolished growth in the inverted system. The TCA cycle inhibitor monofluoroacetate (10 mmol/l), significantly inhibited growth in all three culture systems (P < 0.01) but malonate (10 mmol/l) had no effect. CONCLUSIONS: Aerobic metabolism and an adequate oxygen supply are essential for normal follicular development.     

Utilization of various concentrations of free cystine by the fungus Microsporum gypseum

The dermatophyte Microsporum gypseum was cultivated on two liquid media enriched with 50 to 1000 micrograms/ml free L-cystine. The presence of cystine in concentrations above 250 micrograms/ml (gelatin medium) or 500 micrograms/ml (glucose-glutamate medium) inhibited the growth. In all variants, however, cystine was utilized from the very beginning of growth and exhausted completely until stationary phase. The rate of cystine metabolization grew with its concentration to 500 micrograms/ml but decreased again with 1000 micrograms/ml. The excess sulfur was oxidized and excreted back into the medium mainly as inorganic sulfate. Moreover, sulfite was also produced which immediately reacted with the residual cystine in the medium giving rise to S-sulfocysteine. Sulfite excretion was higher in the initial phases of growth and on the medium with poorer growth (gelatin medium). The sulfate-to-sulfite ratio was different on the two media used but was little influenced by cystine concentration. The excretion of strongly acidic compounds (sulfate, sulfite, and S-sulfocysteine) reduced the usual alkalinization of the medium in the course of growth.     

Sirtuin 1 regulates skeletal myoblast survival and enhances differentiation in the presence of resveratrol

Sirtuin 1 also known as NAD-dependent deacetylase sirtuin 1, is a protein that in humans is encoded by the Sirt1 gene. Sirt1 is an enzyme that deacetylates proteins that contribute to cellular regulation and is a key regulator of cell defenses and survival in response to stress. Deletion of Sirt1 abolishes the increase in lifespan induced by calorie restriction or sublethal cytokine stress, indicating that Sirt1 promotes longevity and survival. We have demonstrated that administration of a sublethal dose of tumour necrosis factor-α (TNF-α; 1.25 ng ml(-1)) inhibits myotube formation, and co-incubation with insulin-like growth factor I (IGF-I; 1.5 ng ml(-1)) facilitates C2 myoblast death rather than rescuing differentiation. A higher dose of TNF-α (10 ng ml(-1)) resulted in significant apoptosis, which was rescued by IGF-I (1.5 ng ml(-1); 50% rescue; P < 0.05). We aimed to investigate the role of Sirt1 in the conflicting roles of IGF-I. Quantitative real-time PCR revealed that Sirt1 expression was elevated in myoblasts following incubation of 10 ng ml(-1) TNF-α or 1.25 ng ml(-1) TNF-α plus IGF-I (fivefold and 7.2-fold increases versus control, respectively; P < 0.05). A dose of 10 ng ml(-1) TNF-α induced ～21 ± 0.7% apoptosis, which was reduced (～50%; P < 0.05) when administered with IGF-I. Likewise, Sirt1 expression was elevated following 10 ng ml(-1) TNF-α administration, but was reduced (～30%; P < 0.05) in the presence of IGF-I. C2C12 myoblasts, a subclone of the C2 cell line produced for their differentiation potential and used to examine intrinsic ageing, unlike C2 cells, do not die in the presence of TNF-α and do not upregulate Sirt1. As conditions that induced the greatest myoblast stress/damage resulted in elevated Sirt1 expression, we investigated the effects of Sirt1 gene silencing. Treatment with 10 ng ml(-1) TNF-α or co-incubation with 1.25 ng ml(-1) TNF-α and 1.5 ng ml(-1) IGF-I resulted in apoptosis (20.33 ± 2.08 and 19 ± 2.65%, respectively), which was increased when myoblasts were pretreated with Sirt1 small interfering RNA (31 ± 2.65 and 27.33 ± 2.52%, respectively; P < 0.05) and was reduced (14.33 ± 3.05%, P < 0.05 and 12.78 ± 4.52%, P = 0.054) by resveratrol, which also significantly rescued the block on differentiation. In conclusion, Sirt1 expression increases in conditons of stress, potentially serving to reduce or dampen myoblast death.     

Antigiardial activity of <Emphasis Type="Italic">Ocimum basilicum</Emphasis> essential oil

In this study, we investigated the effects of Ocimum basilicum essential oil on Giardia lamblia and on the modulation of the interaction of these parasites by peritoneal mouse macrophage. The essential oil (2 mg/ml) and its purified substances demonstrated antigiardial activity. Linalool (300 μg/ml), however, was able to kill 100% parasites after 1 h of incubation, which demonstrates its high antigiardial potential. Pretreatment of peritoneal mouse macrophages with 2 mg/ml essential oil dilution reduced in 79% the association index between these macrophages and G. lamblia, with a concomitant increase by 153% on nitric oxide production by the G. lamblia-ingested macrophages. The protein profiles and proteolitic activity of these parasite trophozoites, previously treated or not with 2 mg/ml essential oil or with the purified fractions, were also determined. After 1 and 2 h of incubation, proteins of lysates and culture supernatants revealed significant differences in bands patterns when compared to controls. Besides, the proteolitic activity, mainly of cysteine proteases, was clearly inhibited by the essential oil (2 mg/ml) and the purified linalool (300 μg/ml). These results suggest that, with G. lamblia, the essential oil from O. basilicum and its purified compounds, specially linalool, have a potent antimicrobial activity. Igor de Almeida and Daniela Sales Alviano contributed equally to this study.     

Inhibition of multiplication of tobacco mosaic virus in protoplasts by antibiotics and its prevention by divalent metals.

At concentrations that inhibit bacterial growth, some antibiotics including gentamicin completely inhibited virus multiplication in protoplasts, and other antibiotics partially inhibited virus multiplication. The inhibition caused by each antibiotic was largely prevented by adding a divalent metal; MnC1(2) was more effective than CaC1(2) and other salts of divalent metals when added at 10 mM to the incubation medium. When added immediately after infection, 1 mug/ml of gentamicin halved the final virus concentration and 3 mug/ml completely inhibited virus multiplication, although 10 mug/ml was required to stop bacterial growth. Gentamicin inhibited virus multiplication even when added 24 h after virus inoculation. Also, when protoplasts were exposed to gentamicin for only 1 or 2 h, either immediately after inoculation or 2 h later, the virus concentration was considerably decreased. Gentamicin seemed not to affect virus multiplication in whole plants. Sap from Dianthus barbatus also strongly inhibited virus multiplication in protoplasts but, unlike gentamicin, it acted in the presence of MnC1(2). By contrast, chelating agents such as 1 mM-EDTA or 5 mM-potassium citrate was strong inhibitors of virus multiplication that were inactive in the presence of MnC1(2). It is suggested that gentamicin and other antibiotics may chelate metals from the protoplast membranes, thus disorganizing their function and affecting virus multiplication.     

Decontamination of gnotobiotic mice experimentally monoassociated with Candida albicans.

Gnotobiotic AKR mice, experimentally monoassociated with Candida albicans, were successfully decontaminated by oral treatment with amphotericin B incorporated in the drinking water. Germfree mice first were swabbed orally with viable C. albicans and then were allowed to acclimatize for 4 weeks. The log10 of number of C. albicans per gram of organ (with luminal contents) was 7.9 and 7.7 in the stomach and cecum, respectively. Direct fecal smears, as well as impresssion smears of stomach and cecum mucosal surfaces, revealed yeastphase cells, many with germ tubes, but no hyphal forms. No illness or mortality was observed over this period. The mice then were given amphotericin B DISsolved in the drinking water and offered ad libitum. At levels of 0.1 and 0.2 mg/ml, the number of fecal C. albicans was decreased but not eliminated completely. However, 0.3 mg/ml was sufficient to decontaminate the mice completely and return them to the germfree state. Residual amphotericin B was detected in the feces of the mice only while they were receiving the 0.3 mg/ml dose level. These mice remained germfree until the termination of the experiment, 10 weeks after the antibiotic had been discontinued and replaced by plain drinking water.     

Effect of oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> L.) and thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> L.) essential oils on <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> (Protozoa: Kinetoplastida) growth and ultrastructure

In the present work, we have investigated the effect of essential oils obtained from Origanum vulgare L. (oregano) and Thymus vulgaris L. (thyme) on growth and ultrastructure of diverse evolutive forms of Trypanosoma cruzi. Culture epimastigotes and bloodstream trypomastigotes were incubated for 24 h with different concentrations of oregano or thyme essential oils and with thymol (the main constituent of thyme), and the inhibitory concentration (IC)₅₀ was determined by cell counting. Crude extract of oregano essential oil inhibited epimastigote growth (IC₅₀/24 h = 175 μg/ml) and also induced trypomastigote lysis (IC₅₀/24 h = 115 μg/ml). Thyme essential oil presented IC₅₀/24 h values of 77 μg/ml for epimastigotes and 38 μg/ml for trypomastigotes, while treatment with thymol resulted in an IC₅₀/24 h of 62 μg/ml for epimastigotes and 53 μg/ml for trypomastigotes. Scanning electron microscopy of treated cells showed few morphological alterations at the plasma membrane. Observation by transmission electron microscopy showed cytoplasmic swelling with occasional morphological alterations in plasma and flagellar membrane. Our data indicate that oregano and thyme essential oils are effective against T. cruzi, with higher activity of thyme, and that thymol may be the main component responsible for the trypanocidal activity.     

Cinnamic aldehydes affect hydrolytic enzyme secretion and morphogenesis in oral Candida isolates

Effect of cinnamaldehyde (CD), 4-hydroxy-3-methoxy cinnamaldehyde (HMCD) and 3,5-dimethoxy-4-hydroxy cinnamaldehyde (HDMCD) on growth and virulence factors of standard (Candida albicans 90028) and 26 oral isolates of C. albicans has been investigated. Growth was significantly inhibited by all three compounds in both solid and liquid medium, no systematic difference was observed between various isolates. MIC₉₀ ranged from 125 to 450 μg/ml for CD, 100–250 μg/ml for HMCD and 62.5–125 μg/ml for HDMCD. All oral isolates were found to be proteinase and phospholipase secretors, both proteinase and phospholipase secretion was significantly inhibited by all the three tested molecules. No systematic difference in secretion or its inhibition was observed between standard and oral isolates as also between various isolates. Average drop in proteinase and phospholipase secretion caused by ½ MIC of CD was 33% and 28%, HMCD; 46% and 44%, HDMCD; 59% and 54%. The standard strain and all the 26 oral isolates displayed morphogenesis under triggering experimental conditions; no difference was seen between standard and various isolates. In the absence of test compounds hyphae development at 300 min was 83% for standard strain whereas average hyphae development for oral isolates was 85%. Average hyphal transition was suppressed by all tested compounds. At ½ MIC concentration at 300 min average hyphal transition of standard and oral isolates was CD; 49% and 57%, HMCD; 45% and 38%, HDMCD; 5% and 5%. Average haemolytic activity of the three tested compounds varied from 10 to 15% at their highest MIC compared to 20% shown by fluconazole at typical MIC of 30 μg/ml.     

Calcineurin is essential for virulence in Candida albicans

Calcineurin is a conserved Ca(2+)-calmodulin-activated, serine/threonine-specific protein phosphatase that regulates a variety of physiological processes, e.g., cell cycle progression, polarized growth, and adaptation to salt and alkaline pH stresses. In the pathogenic yeast Cryptococcus neoformans, calcineurin is also essential for growth at 37 degrees C and virulence. To investigate whether calcineurin plays a role in the virulence of Candida albicans, the major fungal pathogen of humans, we constructed C. albicans mutants in which both alleles of the CMP1 gene, encoding the calcineurin catalytic subunit, were deleted. The C. albicans Delta cmp1 mutants displayed hypersensitivity to elevated Na(+), Li(+), and Mn(2+) concentrations and to alkaline pH, phenotypes that have been described after calcineurin inactivation in the related yeast Saccharomyces cerevisiae. Unlike S. cerevisiae calcineurin mutants, which exhibit reduced susceptibility to high Ca(2+) concentrations, growth of C. albicans was inhibited in the presence of 300 mM CaCl(2) after the deletion of CMP1, demonstrating that there are also differences in calcineurin-mediated cellular responses between these two yeast species. In contrast to C. neoformans, inactivation of calcineurin did not cause temperature sensitivity in C. albicans. In addition, hyphal growth, an important virulence attribute of C. albicans, was not impaired in the Delta cmp1 mutants under a variety of inducing conditions. Nevertheless, the virulence of the mutants was strongly attenuated in a mouse model of systemic candidiasis, demonstrating that calcineurin signaling is essential for virulence in C. albicans.     

Stearate inhibition of breast cancer cell proliferation. A mechanism involving epidermal growth factor receptor and G-proteins.

Long chain saturated fatty acids are known to inhibit breast cancer cell proliferation; however, the mechanism of this inhibition is not known. Treatment of Hs578T breast cancer cells with long chain saturated fatty acids (0.15 mmol/L for 6 hours) before epidermal growth factor (EGF) treatment inhibited EGF-induced cell proliferation in a chain-length-dependent manner. Stearate (C:18) completely inhibited the EGF-induced cell proliferation, whereas palmitate (C:16) inhibited by 67 +/- 8% and myristate (C:14) had no effect. In contrast, stearate had little effect on insulin-like growth factor-1-stimulated cell proliferation. The inhibitory effect of stearate on cell proliferation was dose and time dependent and independent of EGF receptor (EGFR) tyrosine phosphorylation. Pretreatment of cells with pertussis toxin (0.1 microgram/ml for 24 hours) inhibited the EGF-induced cell growth by 50 +/- 8%, also independent of EGFR tyrosine phosphorylation. A pertussis-toxin-sensitive, 41-kd G-protein was specifically co-immunoprecipitated with the EGFR. Pretreatment of cells with 0.15 mmol/L stearate from 0 to 6 hours inhibits, in parallel, both the EGF-induced cell proliferation and pertussis-toxin-catalyzed ADP ribosylation of the G-protein associated with the EGFR. These studies suggest that long chain saturated fatty acids inhibit EGF-induced breast cancer cell growth via a mechanism involving an EGFR-G-protein signaling pathway.     

Modulation of lymphocyte proliferation by enzymes that degrade amino acids.

In a previous study we demonstrated thirteen amino acids to be essential and two to be partially essential for lymphocyte proliferation. Arginine is one of the essential amino acids, and the highly purified arginase strongly inhibited lymphocyte proliferation. The modulation of lymphocyte growth by various amino acid-degrading enzymes was studied. Peripheral lymphocytes were cultured in RPMI 1640 with or without amino acid-degrading enzyme for 72 h. A total of 17 commercial L-amino acid-degrading enzymes were studied. At 10 micrograms/ml, both lysine decarboxylase and asparaginase completely inhibited lymphocyte proliferation, arginase resulted in 78% inhibition and tyrosinase 57% inhibition. Other enzymes inhibited less than 20% lymphocyte proliferation; they included alanine dehydrogenase, arginine decarboxylase, aspartase, glutamic decarboxylase, glutamic dehydrogenase, glutaminase, histidase, histidine decarboxylase, leucine dehydrogenase, phenylalanine decarboxylase, phenylalanine hydroxylase, tryptophanase, and tyrosine decarboxylase. All four enzymes that strongly inhibited lymphocyte proliferation degraded amino acids that are essential for lymphocyte growth.