Adipocytes Isolated from Visceral and Subcutaneous Depots of Donors Differing in BMI Crosstalk with Colon Cancer Cells and Modulate their Invasive Phenotype.

PURPOSE: Mechanisms related the crosstalk between adipocytes and colon cancer cells are still not clear. We hypothesize that molecules and adipocytokines generated from the adipose tissue of obese individuals accentuate the effect on the metabolic reprogramming in colon cancer cells, i.e. induce disarray in energy metabolism networks of the targeted affected colonic epithelial cells, prompting their malignant phenotype. METHODS: To explore the mechanistic behind this crosstalk we conducted a co-culture model system using human colon cancer cells having different malignant abilities and adipocytes from different depots and subjects. RESULTS: The results demonstrate that co-culturing aggressive colon cancer cells such as HM-7 cells, with Visceral or Subcutaneous adipocytes (VA or SA respectively) from lean/obese subjects significantly up-regulate the secretion of the adipokines IL-8, MCP1, and IL-6 from the adipocytes. Surprisingly, the response of co-culturing HM-7 cells with obese SA was substantially more significant. In addition, these effects were significantly more pronounced when using HM-7 cells as compared to the less malignant HCT116 colon cancer cells. Moreover, the results showed that HM-7 cells, co-cultured with VA or SA from obese subjects, expressed higher levels of fatty acid binding protein 4; thus, the conditioned media obtained from the wells contained HM-7 cells and adipocytes from obese subjects was significantly more efficient in promoting invasion of HM-7 cells. CONCLUSIONS: We conclude that interaction between adipocytes and colon cancer cells, especially the highly malignant cells, results in metabolic alterations in colon cancer cells and in highly hypertrophy phenotype which characterized by increasing adipokines secretion from the adipocytes.

Galactomannan More than Pectin Exacerbates Liver Injury in Mice Fed with High-Fat, High-Cholesterol Diet.

SCOPE: Galactomannan and citrus pectin are considered 'super fibers' known for altering gut microbiota composition and improving glucose and lipid metabolism. The study aims to investigate the fiber's effect on a nonalcoholic steatohepatitis (NASH) model. METHODS AND RESULTS: Two feeding experiments are carried out using groups of 7-8 week-old male C57BL/6J mice. The diets used are based on a high cholesterol/cholate diet (HCD), such as a nutritional NASH model. Mice are fed a diet with or without 15% fiber-citrus pectin (HCD-CP) or galactomannan (HCD-G) together with the HCD (first experiment), which commenced 3 weeks prior to the HCD (second experiment). Liver damage is evaluated by histological and biochemical parameters. Galactomannan leads to lesser weight gain and improved glucose tolerance, but increased liver damage. This is shown by elevated levels of liver enzymes compared to that with HCD alone. Fibers induce higher steatosis, as evaluated by liver histology. This intriguing result is linked to various changes in the gut microbiota, such as elevated Proteobacteria levels in the galactomannan group, which are correlated with disturbed metabolism and dysbiosis. CONCLUSIONS: In a NASH mouse model, galactomannan increases liver damage but improves glucose metabolism. Changes in the microbiota composition may answer this enigmatic observation.

Ostreolysin induces browning of adipocytes and ameliorates hepatic steatosis.

BACKGROUND AND AIM: Non-alcoholic fatty liver disease (NAFLD) is associated with all features of the metabolic syndrome. Deposition of excess triglycerides in liver cells, a hallmark of NAFLD, is associated with loss of insulin sensitivity. Ostreolysin (Oly) is a 15-kDa fungal protein known to interact with cholesterol-enriched raft-like membrane domains. We aim to test whether a recombinant version of Oly (rOly) can induce functional changes in vitro in adipocytes or in vivo in mice fed a high-fat diet (HFD). METHODS: White preadipocyte 3T3-L1 cells or mouse primary adipocytes treated with rOly. Male C57BL/6 mice were fed a control or HFD and treated with saline or with rOly (1 mg/kg BW) every other day for 4 weeks. RESULTS: White preadipocyte 3T3-L1 cells or mouse primary adipocytes treated with rOly acquire a browning phenotype through activation of 5' adenosine monophosphate-activated protein kinase and downregulation of tumor necrosis factor α-mediated activation of IκB kinase ε and TANK-binding kinase 1. HFD-fed mice treated with rOly showed a 10% reduction in BW and improved glucose tolerance, which paralleled improved expression of liver and adipose functionality, metabolism, and inflammation status, mimicking the in vitro findings. CONCLUSION: This study provides first evidence of rOly's prevention of HFD-induced NAFLD by stimulating liver and adipose muscle tissue functionality and oxidative potential, improving glucose tolerance, and ameliorating the metabolic profile of diet-induced obese mice.

Recombinant ostreolysin induces brown fat-like phenotype in HIB-1B cells.

SCOPE: Brown adipose tissue (BAT) is the main regulator of thermogenesis by increasing energy expenditure through the uncoupling of oxidative metabolism from ATP synthesis. There is a growing body of evidence for BAT being the key responsible organ in combating obesity and its related disorders. Herein we propose the fungal protein ostreolysin (Oly), which has been previously shown to bind to cholesterol-enriched raft-like membrane domains (lipid rafts) of mammalian cells, as a suitable candidate for interaction with brown preadipocytes. The aim of the present study was therefore to characterize the mechanism by which a recombinant version of ostreolysin (rOly) induces brown adipocyte differentiation. METHODS AND RESULTS: Primary isolated brown preadipocytes or HIB-1B brown preadipocyte cells were treated with rOly and the effects on morphology, lipid accumulation, respiration rate, and associated gene and protein expression were measured. rOly upregulated mRNA and protein levels of factors related to brown adipocyte differentiation, induced lipid droplet formation, and increased cellular respiration rate due to expression of uncoupling protein 1. rOly also upregulated ß-tubulin expression, and therefore microtubules might be involved in its mechanism of action. CONCLUSION: rOly promotes brown adipocyte differentiation, suggesting a new mechanism for rOly's contribution to the battle against obesity.

A recombinant fungal compound induces anti-proliferative and pro-apoptotic effects on colon cancer cells.

Finding intracellular pathways and molecules that can prevent the proliferation of colon cancer cells can provide significant bases for developing treatments for this disease. Ostreolysin (Oly) is a protein found in the mushroom Pleurotus ostreatus, and we have produced a recombinant version of this protein (rOly).We measured the viability of several colon cancer cells treated with rOly. Xenografts and syngeneic colon cancer cells were injected into in vivo mouse models, which were then treated with this recombinant protein.rOly treatment induced a significant reduction in viability of human and mouse colon cancer cells. In contrast, there was no reduction in the viability of normal epithelial cells from the small intestine. In the search for cellular targets of rOly, we showed that it enhances the anti-proliferative activity of drugs targeting cellular tubulin. This was accompanied by a reduction in the weight and volume of tumours in mice injected with rOly as compared to their respective control mice in two in vivo models.Our results advance the functional understanding of rOly as a potential anti-cancer treatment associated with pro-apoptotic activities preferentially targeting colon cancer cells.

Mechanisms linking obesity to altered metabolism in mice colon carcinogenesis.

There are an increasing number of reports on obesity being a key risk factor for the development of colon cancer. Our goal in this study was to explore the metabolic networks and molecular signaling pathways linking obesity, adipose tissue and colon cancer. Using in-vivo experiments, we found that mice fed a high-fat diet (HFD) and injected with MC38 colon cancer cells develop significantly larger tumors than their counterparts fed a control diet. In ex-vivo experiments, MC38 and CT26 colon cancer cells exposed to conditioned media (CM) from the adipose tissue of HFD-fed mice demonstrated significantly lower oxygen consumption rate as well as lower maximal oxygen consumption rate after carbonyl cyanide-4-trifluoromethoxy-phenylhydrazone treatment. In addition, in-vitro assays showed downregulated expression of mitochondrial genes in colon cancer cells exposed to CM prepared from the visceral fat of HFD-fed mice or to leptin. Interestingly, leptin levels detected in the media of adipose tissue explants co-cultured with MC38 cancer cells were higher than in adipose tissue explants cultures, indicating cross talk between the adipose tissue and the cancer cells. Salient findings of the present study demonstrate that this crosstalk is mediated at least partially by the JNK/STAT3-signaling pathway.

Membrane-permeable tastants amplify ß2-adrenergic receptor signaling and delay receptor desensitization via intracellular inhibition of GRK2's kinase activity.

BACKGROUND: Amphipathic sweet and bitter tastants inhibit purified forms of the protein kinases GRK2, GRK5 and PKA activities. Here we tested whether membrane-permeable tastants may intracellularly interfere with GPCR desensitization at the whole cell context. METHODS: ß2AR-transfected cells and cells containing endogenous ß2AR were preincubated with membrane-permeable or impermeable tastants and then stimulated with isoproterenol (ISO). cAMP formation, ß2AR phosphorylation and ß2AR internalization were monitored in response to ISO stimulation. IBMX and H89 inhibitors and GRK2 silencing were used to explore possible roles of PDE, PKA, and GRK2 in the tastants-mediated amplification of cAMP formation and the tastant delay of ß2AR phosphorylation and internalization. RESULTS: Membrane-permeable but not impermeable tastants amplified the ISO-stimulated cAMP formation in a concentration- and time-dependent manner. Without ISO stimulation, amphipathic tastants, except caffeine, had no effect on cAMP formation. The amplification of ISO-stimulated cAMP formation by the amphipathic tastants was not affected by PDE and PKA activities, but was completely abolished by GRK2 silencing. Amphipathic tastants delayed the ISO-induced GRK-mediated phosphorylation of ß2ARs and GRK2 silencing abolished it. Further, tastants also delayed the ISO-stimulated ß2AR internalization. CONCLUSION: Amphipathic tastants significantly amplify ß2AR signaling and delay its desensitization via their intracellular inhibition of GRK2. GENERAL SIGNIFICANCE: Commonly used amphipathic tastants may potentially affect similar GPCR pathways whose desensitization depends on GRK2's kinase activity. Because GRK2 also modulates phosphorylation of non-receptor components in multiple cellular pathways, these gut-absorbable tastants may permeate into various cells, and potentially affect GRK2-dependent phosphorylation processes in these cells as well.

Prevention of diabetes-promoted colorectal cancer by (n-3) polyunsaturated fatty acids and (n-3) PUFA mimetic.

The global obesity / diabetes epidemic has resulted in robust increase in the incidence of colorectal cancer (CRC). Epidemiological, animal and human studies have indicated efficacy of (n-3) PUFA in chemoprevention of sporadic and genetic-driven CRC. However, diabetes-promoted CRC presents a treatment challenge that surpasses that of sporadic CRC. This report analyzes the efficacy of (n-3) PUFA generated by the fat-1 transgene that encodes an (n-6) to (n-3) PUFA desaturase, and of synthetic (n-3) PUFA mimetic (MEDICA analog), to suppress CRC development in carcinogen-induced diabetes-promoted animal model. Carcinogen-induced CRC is shown here to be promoted by the diabetes context, in terms of increased aberrant crypt foci (ACF) load, cell proliferation and epithelial dedifferentiation, being accompanied by increase in the expression of HNF4α, ß-catenin, and ß-catenin-responsive genes. Incorporating the fat-1 transgene in the diabetes context, or oral MEDICA treatment, resulted in ameliorating the diabetic phenotype and in abrogating CRC, with decrease in ACF load, cell proliferation and the expression of HNF-4α, ß-catenin, and ß-catenin-responsive genes. The specificity of (n-3) PUFA in abrogating CRC development, as contrasted with enhancing CRC by (n-6) PUFA, was similarly verified in CRC cell lines. These findings may indicate prospective therapeutic potential of (n-3) PUFA or MEDICA in the management of CRC, in particular diabetes-promoted CRC.

Epigenetic control of HNF-4α in colon carcinoma cells affects MUC4 expression and malignancy.

BACKGROUND: We previously found that enhanced expression of hepatocyte nuclear factor 4α (HNF-4α) is associated with hyper-proliferation of colon carcinoma cells. Here, the effect of histone deacetylase (HDAC) inhibitors on proliferation and the expression of HNF-4α and its downstream target genes were assessed in HM7, LS174T, HT29 and Caco-2 colon carcinoma cell lines. RESULTS: HNF-4α expression was found to vary in the different colon carcinoma cell lines tested, being highest in HM7. Additionally, a direct correlation with proliferation was observed. In HM7 cells, the weak HDAC inhibitor butyrate significantly inhibited the transcription of HNF-4α, its downstream target gene MUC4, and genes associated with proliferation, including the proliferating cell nuclear antigen gene PCNA. siRNA-mediated silencing of HNF-4α exerted an effect similar to butyrate on HM7 cell proliferation. The stronger HDAC inhibitor trichostatin A (TSA) exerted an effect similar to that of siRNA-mediated HNF-4α silencing and, concomitantly, inhibited the expression of the transcription factor gene SP1. Also, siRNA-mediated silencing of HDAC3 and HDAC4 reduced HNF-4α expression. Chromatin immunoprecipitation (ChIP) assays revealed that TSA induces hyperacetylation of histones H3 and H4 and, concomitantly, inhibits SP1 binding to the HNF-4α promoter. Subsequent electromobility shift assays supported these latter findings. CONCLUSIONS: HNF-4α transcriptional expression and activity are tightly controlled by epigenetic mechanisms. HDAC inhibitor targeting of HNF-4α may serve as an effective treatment for advanced colon carcinomas, since downstream cancer-associated target genes such as MUC4 are significantly down-regulated by this treatment.

Amelioration of diabesity-induced colorectal ontogenesis by omega-3 fatty acids in mice.

Postnatal intestinal ontogenesis in an animal model of diabesity may recapitulate morphological and transduction features of diabesity-induced intestinal dysplasia and its amelioration by endogenous (n-3) polyunsaturated fatty acids (PUFA). Proliferation, differentiation, and transduction aspects of intestinal ontogenesis have been studied here in obese, insulin-resistant db/db mice, in fat-1 transgene coding for desaturation of (n-6) PUFA into (n-3) PUFA, in db/db crossed with fat-1 mice, and in control mice. Diabesity resulted in increased colonic proliferation and dedifferentiation of epithelial colonocytes and goblet cells, with increased colonic ß-catenin and hepatocyte nuclear factor (HNF)-4α transcriptional activities accompanied by enrichment in HNF-4α-bound (n-6) PUFA. In contrast, in fat-1 mice, colonic proliferation was restrained, accompanied by differentiation of crypt stem cells into epithelial colonocytes and goblet cells and by decrease in colonic ß-catenin and HNF-4α transcriptional activities, with concomitant enrichment in HNF-4α-bound (n-3) PUFA at the expense of (n-6) PUFA. Colonic proliferation and differentiation, the profile of ß-catenin and HNF-4α-responsive genes, and the composition of HNF-4α-bound PUFA of db/db mice reverted to wild-type by introducing the fat-1 gene into the db/db context. Suppression of intestinal HNF-4α activity by (n-3) PUFA may ameliorate diabesity-induced intestinal ontogenesis and offer an effective preventive modality for colorectal cancer.

Glucans from the edible mushroom Pleurotus pulmonarius inhibit colitis-associated colon carcinogenesis in mice.

BACKGROUND: We have recently demonstrated that polysaccharides from fruiting body extract (FBE) or mycelia extract (ME) of the edible mushroom Pleurotus pulmonarius exert antiproliferative effects in intestinal cells and an anti-inflammatory effect in a dextran sulfate sodium (DSS) mouse model of acute colitis. The aim of this study was to assess the role of fungal FBE and ME in colon carcinogenesis. METHODS: In vitro, human colorectal cancer cells were treated with FBE and ME and analyzed for inflammation response, for markers of apoptosis, and for cell-cycle progression. In vivo, FBE and ME were tested in a mouse model of colitis-associated colorectal carcinogenesis induced by cyclic treatments with DSS and azoxymethane. Treated mice were fed a daily diet containing 2 or 20 mg FBE or ME per mouse for 80 days. RESULTS: In vitro, FBE and ME induced apoptosis in a dose-responsive manner and modulated the expression of Bcl-2, Bax, and cytochrome c, and blocked tumor necrosis factor (TNF)-α-induced inhibitor of nuclear factor (NF) (Iκ)-Bα degradation and NF-κB nuclear translocation. In vivo, dietary administration of FBE and ME significantly reduced the formation of aberrant crypt foci, which precedes colorectal cancer, and of microadenomas. The treatments significantly lowered the expression of proliferating cell nuclear antigen and increased the number of cells undergoing apoptosis in the colon. Additionally, FBE and ME inhibited the expression of the proinflammatory cytokine TNF-α in colonic tissue. CONCLUSIONS: We conclude that P. pulmonarius FBE and ME inhibit colitis-associated colon carcinogenesis induced in mice through the modulation of cell proliferation, induction of apoptosis, and inhibition of inflammation.

Improving bioavailability and stability of genistein by complexation with high-amylose corn starch.

Genistein, like other phytochemicals, has beneficial health effects, but its bioavailability is limited. This research studied the effect of complexation of genistein with starch on genistein bioavailability. Genistein release from these complexes was tested in vitro under simulated intestinal conditions and in vivo in rats fed high-amylose corn starch (HACS)-genistein complexes (experimental group) as compared to those fed a physical mixture of HACS and genistein (controls). In vitro results showed that genistein release is sustained and fits the normal transit time of food in the intestine. The genistein concentration in the plasma was twice as high in the experimental group versus controls; the genistein concentration in the urine was also higher in the experimental group but lower in the feces. These results indicate that starch-genistein complexes increase genistein bioavailability and suggest that starch can affect the bioavailability of additional food components.

Allicin purified from fresh garlic cloves induces apoptosis in colon cancer cells via Nrf2.

Allicin (diallyl thiosulfinate) is the best-known biologically active component in freshly crushed garlic extract. We developed a novel, simple method to isolate active allicin, which yielded a stable compound in aqueous solution amenable for use in in vitro and in vivo studies. We focused on the in vitro effects of allicin on cell proliferation of colon cancer cell lines HCT-116, LS174T, HT-29, and Caco-2 and assessed the underlying mechanisms. This allicin preparation exerted a time- and dose-dependent cytostatic effect on these cells at concentrations ranging from 6.2 to 310 µM. Treatment with allicin resulted in HCT-116 apoptotic cell death as demonstrated by enhanced hypodiploid DNA content, decreased levels of B-cell non-Hodgkin lymphoma-2 (Bcl-2), increased levels of bax and increased capability of releasing cytochrome c from mitochondria to the cytosol. Allicin also induced translocation of NF-E2-related factor-2 (Nrf2) to the nuclei of HCT-116 cells. Luciferase reporter gene assay showed that allicin induces Nrf2-mediated luciferase transactivation activity. SiRNA knock down of Nrf2 significantly affected the capacity of allicin to inhibit HCT-116 proliferation. These results suggest that Nrf2 mediates the allicin-induced apoptotic death of colon cancer cells.

Orally administered glucans from the edible mushroom Pleurotus pulmonarius reduce acute inflammation in dextran sulfate sodium-induced experimental colitis.

Polysaccharides are one of the most potent mushroom-derived substances exhibiting anti-inflammatory and immunomodulatory properties. The aims of the present study were to determine whether orally administered glucans from the edible mushroom Pleurotus pulmonarius could attenuate or prevent the development of experimental colitis in mice. Colonic inflammation was induced in mice by treatment with 3.5 % dextran sulfate sodium (DSS) for 18 d. Before or after DSS administration, mice were given hot water solubles (HWS) or mycelium extract (ME) (2 or 20 mg per mouse) daily in their food. Colonic damage was macroscopically and histologically evaluated. Inflammation was assessed by changes in colon length, TNF-alpha levels released by colonic samples in organ culture and myeloperoxidase (MPO) activity. mRNA levels of pro-inflammatory (IL-1beta) and anti-inflammatory (IL-10) cytokines in colonic samples were determined by quantitative real-time RT-PCR. P. pulmonarius glucans attenuated and prevented the development of symptoms associated with DSS-induced colitis. High doses of HWS and ME blocked colon shortening, suppressed MPO activity and improved macroscopic score in all treatment groups. In addition, histological damage from colitis was reduced by HWS and ME at all doses. The tissue levels of TNF-alpha protein were significantly decreased and correlated with degree of inflammation and macroscopic score. All treatments significantly attenuated the increased DSS-mediated expression levels of IL-1beta. We conclude that the different glucan preparations (HWS or ME) harvested from P. pulmonarius when orally administered to DSS-treated mice attenuate the development of colonic inflammation, suggesting putative clinical utility for these extracts in the treatment of colitis.

Chemical characterization, antiproliferative and antiadhesive properties of polysaccharides extracted from Pleurotus pulmonarius mycelium and fruiting bodies.

Mushroom polysaccharides are potent substances that exhibit antitumor and immunomodulatory properties. Studies comparing the chemical composition and antitumor-related activities of polysaccharides released by fungal strains under different growth conditions are not available. Thus, the present study compared polysaccharides extracts produced by Pleurotus pulmonarius from mycelium grown in liquid culture (ME) or fruiting bodies (FBE). Polysaccharides of both ME and FBE had a relatively high molecular mass. NMR spectroscopy indicated that ME glucan is an alpha-glucan whereas FBE glucan is a mixture of both alpha- and beta-glucans. Glucose and galactose where the most prominent monosaccharide in both glucans. Treatment of several colon cancer cell lines expressing varying amounts of galectin-3 with the two fungal glucans inhibited their viability and significantly reduced their ability to adhere to the key component of the extracellular matrix, fibronectin, and to a human umbilical vein endothelial cell monolayer, in a time- and dose-dependent manner mainly in those cell lines expressing high amounts of galectin-3. We conclude that ME and FBE glucans may exert a direct antiproliferative effect on cancer cells expressing high galectin-3 concentrations and concomitantly downregulate tumor cell adherence, the latter being directly related to cancer progression and metastasis.

Inhibition of colorectal cancer by targeting hepatocyte nuclear factor-4alpha.

Hepatocyte nuclear factor-4alpha (HNF-4alpha) serves as target for fatty acid nutrients and xenobiotic amphipathic carboxylates and may account for the differential effects of dietary fatty acids on colorectal cancer (CRC). The putative role played by HNF-4alpha in CRC has been verified here by evaluating the effect of HNF-4alpha antagonists and HNF-4alpha siRNA on CRC growth and proliferation in cultured CRC cells and xenotransplanted nude mice in vivo. HNF-4alpha ligand antagonists of the MEDICA series, namely, beta,beta'-tetramethylhexadecanedioic acid (M16betabeta) and gamma,gamma'-tetramethyloctadocanedioic acid (M18gammagamma) as well as HNF-4alpha siRNA are shown here to inhibit growth and proliferation of HT29 and Caco2 CRC cells, accompanied by increased subG1 cell population, downregulated PCNA, activation of caspase-3, upregulation of Bak and cytoplasmic cytochrome-c, and downregulation of Bcl-2 resulting in apoptotic death. Inhibition of CRC growth with concomitant apoptosis was further confirmed in nude mice xenotransplanted with HT29 CRC cells. CRC suppression by HNF-4alpha ligand antagonists and by HNF-4alpha siRNA was accounted for by suppression of HNF-4alpha transcription and protein expression. alpha,alpha'-tetrachlorotetradecanedioic acid (Cl-DICA), a MEDICA analogue that fails to suppress HNF-4alpha, was ineffective in suppressing growth of cultured or xenotransplanted HT29 CRC cells. Hence, increased transcriptional activity of HNF-4alpha converging onto genes coding for antiapoptotic oncogenes and cytokines may promote CRC development. Suppression of HNF-4alpha activity by natural or xenobiotic HNF-4alpha ligand antagonists or by HNF-4alpha siRNA may offer a treatment mode for CRC.

Inhibition of signal termination-related kinases by membrane-permeant bitter and sweet tastants: potential role in taste signal termination.

Sweet and bitter taste sensations are believed to be initiated by the tastant-stimulated T1R and T2R G protein-coupled receptor (GPCR) subfamilies, respectively, which occur in taste cells. Although such tastants, with their significantly diverse chemical structures (e.g., sugar and nonsugar sweeteners), may share the same or similar T1Rs, some nonsugar sweeteners and many bitter tastants are amphipathic and produce a significant delay in taste termination (lingering aftertaste). We report that such tastants may permeate rat taste bud cells rapidly in vivo and inhibit known signal termination-related kinases in vitro, such as GPCR kinase (GRK)2, GRK5, and PKA. GRK5 and perhaps GRK2 and GRK6 are present in taste cells. A new hypothesis is proposed in which membrane-permeant tastants not only interact with taste GPCRs but also interact intracellularly with the receptors' downstream shutoff components to inhibit signal termination.

Some sweet and bitter tastants stimulate inhibitory pathway of adenylyl cyclase via melatonin and alpha 2-adrenergic receptors in Xenopus laevis melanophores.

The sweeteners saccharin, D-tryptophan, and neohesperidin dihydrochalcone (NHD) and the bitter tastant cyclo(Leu-Trp) stimulated concentration-dependent pigment aggregation in a Xenopus laevis melanophore cell line similar to melatonin. Like melatonin, these tastants inhibited (by 45-92%) cAMP formation in melanophores; pertussis toxin pretreatment almost completely abolished the tastant-induced cAMP inhibition, suggesting the involvement of the inhibitory pathway (Gi) of adenylyl cyclase. The presence of luzindole (melatonin receptor antagonist) almost completely abolished the inhibition of cAMP formation induced by saccharin, D-tryptophan, and cyclo(Leu-Trp) but only slightly affected the inhibitory effect of NHD. In contrast, the presence of an alpha2-adrenergic receptor antagonist, yohimbine, almost completely abolished the inhibition of cAMP formation induced by NHD but had only a minor effect on that induced by the other tastants. Thus saccharin, D-tryptophan, and cyclo(Leu-Trp) are melatonin receptor agonists whereas NHD is an alpha2-adrenergic receptor agonist, but both pathways lead to the same transduction output and cellular response. Formation of D-myo-inositol 1,4,5-trisphosphate (IP3) in melanophores was reduced (15-58%, no concentration dependence) by saccharin, D-tryptophan, and cyclo(Leu-Trp) stimulation but increased by NHD stimulation. Tastant stimulation did not affect cGMP. Although some of the above tastants were found to be membrane permeant, their direct activation of downstream transduction components in this experimental system is questionable. MT1 and MT2 melatonin receptor mRNAs were identified in rat circumvallate papilla taste buds and nonsensory epithelium, suggesting the occurrence of MT1 and MT2 receptors in these tissues. Melatonin stimulation reduced the cellular content of cAMP in taste cells, which may or may not be related to taste sensation.

Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities.

The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.