酿酒酵母全基因组范围内筛选MTM1基因的抑制基因

MTM1 基因对于维持锰超氧化物歧化酶的活性和线粒体正常功能十分重要,MTM1 基因的缺失会严重影响酵母锰超氧化物歧化酶活性,并损伤线粒体功能,因此在非发酵培养基上不能生长．利用MTM1 基因缺失的突变体在非发酵培养基上的生长缺陷,转入酵母基因组文库筛选MTM1 抑制基因,发现MTM1基因缺失造成的损伤一旦形成不可逆转,重新引入MTM1 基因也无法挽救,直接筛选无法得到抑制基因．为了避免MTM1缺失造成的不可逆损伤,在野生型酵母中先转入带有MTM1 基因的质粒,再敲除染色体上的MTM1 基因,随后转入基因组文库,再利用药物5-氟乳清酸(5-FOA)迫使细胞丢失表达MTM1基因的外源质粒,再筛选能在非发酵培养基上生长的转化子,通过这种方法筛选发现,POR2等5个基因的过表达可以挽救MTM1 基因缺失造成的非发酵培养基上的生长缺陷,为深入了解MTM1基因的功能提供了线索,对筛选其他造成不可逆损伤的突变基因的抑制基因提供了一条可行的研究思路．     

A novel membrane protein capable of binding the Na+/H+ antiporter (Nha1p) enhances the salinity-resistant cell growth of Saccharomyces cerevisiae

The Na+/H+ antiporter Nha1p of Saccharomyces cerevisiae plays an important role in maintaining intracellular pH and Na+ homeostasis. Nha1p has a two-domain structure composed of integral membrane and hydrophilic tail regions. Overexpression of a peptide of approximately 40 residues (C1+C2 domains) that is localized in the juxtamembrane area of its cytoplasmic tail caused cell growth retardation in highly saline conditions, possibly by decreasing Na+/H+ antiporter activity. A multicopy suppressor gene of this growth retardation was identified from a yeast genome library. The clone encodes a novel membrane protein denoted as COS3 in the genome data base. Overexpression or deletion of COS3 increases or decreases salinity-resistant cell growth, respectively. However, in nha1Delta cells, overexpression of COS3 alone did not suppress the growth retardation. Cos3p and a hydrophilic portion of Cos3p interact with the C1+C2 peptide in vitro, and Cos3p is co-precipitated with Nha1p from yeast cell extracts. Cos3p-GFP mainly resides at the vacuole, but overexpression of Nha1p caused a portion of the Cos3p-GFP proteins to shift to the cytoplasmic membrane. These observations suggest that Cos3p is a novel membrane protein that can enhance salinity-resistant cell growth by interacting with the C1+C2 domain of Nha1p and thereby possibly activating the antiporter activity of this protein.     

Cellular processes and pathways that protect Saccharomyces cerevisiae cells against the plasma membrane-perturbing compound chitosan

Global fitness analysis makes use of a genomic library of tagged deletion strains. We used this approach to study the effect of chitosan, which causes plasma membrane stress. The data were analyzed using T-profiler, which was based on determining the sensitivities of groups of deletion strains to chitosan, as defined by Gene Ontology (GO) and by genomic synthetic lethality screens, in combination with t statistics. The chitosan-hypersensitive groups included a group of deletion strains characterized by a defective HOG (high-osmolarity glycerol) signaling pathway, indicating that the HOG pathway is required for counteracting chitosan-induced stress. Consistent with this, activation of this pathway in wild-type cells by hypertonic conditions offered partial protection against chitosan, whereas hypotonic conditions sensitized the cells to chitosan. Other chitosan-hypersensitive groups were defective in RNA synthesis and processing, actin cytoskeleton organization, protein N-glycosylation, ergosterol synthesis, endocytosis, or cell wall formation, predicting that these cellular functions buffer the cell against the deleterious effect of chitosan. These predictions were supported by showing that tunicamycin, miconazole, and staurosporine (which target protein N-glycosylation, ergosterol synthesis, and the cell wall integrity pathway, respectively) sensitized Saccharomyces cerevisiae cells to chitosan. Intriguingly, the GO-defined group of deletion strains belonging to the "cytosolic large ribosomal subunit" was more resistant to chitosan. We propose that global fitness analysis of yeast in combination with T-profiler is a powerful tool to identify specific cellular processes and pathways that are required for survival under stress conditions.     

Comprehensive phenotypic analysis for identification of genes affecting growth under ethanol stress in Saccharomyces cerevisiae

We quantified the growth behavior of all available single gene deletion strains of budding yeast under ethanol stress. Genome-wide analyses enabled the extraction of the genes and determination of the functional categories required for growth under this condition. Statistical analyses revealed that the growth of 446 deletion strains under stress induced by 8% ethanol was defective. We classified these deleted genes into known functional categories, and found that many were important for growth under ethanol stress including several categories that have not been characterized, such as peroxisome. We also performed genome-wide screening under osmotic stress and identified 329 osmotic-sensitive strains. We excluded these strains from the 446 ethanol-sensitive strains to extract the genes whose deletion caused sensitivity to ethanol-specific (359 genes), osmotic-specific (242 genes), and both stresses (87 genes). We also extracted the functional categories that are specifically important for growth under ethanol stress. The genes and functional categories identified in the analysis might provide clues to improving ethanol stress tolerance among yeast cells.     

Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis

Although activated Ras proteins are usually associated with driving growth and transformation, they may also induce senescence, apoptosis, and terminal differentiation. The subversion of these anti-neoplastic effects during Ras-dependent tumor development may be as important as the acquisition of the pro-neoplastic effects. None of the currently identified potential Ras effector proteins can satisfactorily explain the apoptotic action of Ras. Consequently, we have sought to identify novel Ras effectors that may be responsible for apoptosis induction. By examining the EST data base, we identified a potential Ras association domain in the tumor suppressor RASSF1. We now show that RASSF1 binds Ras in a GTP-dependent manner, both in vivo and directly in vitro. Moreover, activated Ras enhances and dominant negative Ras inhibits the cell death induced by transient transfection of RASSF1 into 293-T cells. This cell death appears to be apoptotic in nature, as RASSF1-transfected 293-T cells exhibit membrane blebbing and can be rescued by the addition of a caspase inhibitor. Thus, the RASSF1 tumor suppressor may serve as a novel Ras effector that mediates the apoptotic effects of oncogenic Ras.     

ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling

ARL4D is a developmentally regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of Ras-related GTPases. Although the primary structure of ARL4D is very similar to that of other ARF/ARL molecules, its function remains unclear. Cytohesin-2/ARF nucleotide-binding-site opener (ARNO) is a guanine nucleotide-exchange factor (GEF) for ARF, and, at the plasma membrane, it can activate ARF6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. Localization of ARL4D at the plasma membrane is GTP- and N-terminal myristoylation-dependent. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or small interfering RNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling.     

Chitosan oligosaccharides, dp 2-8, have prebiotic effect on the Bifidobacterium bifidium and Lactobacillus sp

In order to investigate the prebiotic potential of chitosan oligosaccharide (COS), prepared by enzymatic hydrolysis of fully deacetylated chitosan polymer, the effect of COS on bacterial growth was studied. The degree of polymerization (dp) of COS was determined by MALDI-ToF mass spectrometry, and the COS was found to be composed of dimer (33.6%), trimer (16.9%), tetramer (15.8%), pentamer (12.4%), hexamer (8.3%), heptamer (7.1%), and octamer (5.9%). The minimum inhibitory concentrations (MIC) of chitosan polymer against lactic acid bacteria and bifidobacteria were below 0.31%. However, this only applied to two strains, the other bacteria tested grew on MRS broth containing 5% COS. The effects of COS on the growth of bifidobacteria and lactic acid bacteria were compared with those of fructo-oligosaccharide (FOS). FOS was found to have a growth stimulatory effect on only three strains: Bifidobacterium bifidium, B. infantis and Lactobacillus casei. However, COS stimulated the growth of most Lactobacillus sp. and B. bifidium KCTC 3440. The amount of the growth and the specific growth rate of B. bifidium increased with increasing COS concentration. The cultivation time required to obtain maximum growth was reduced to about 25% in MRS broth supplemented with 0.2-0.4% COS. These results demonstrate that COS has considerable bifidogenic potential. Both cell growth and specific growth rates of L. brevis in MRS broth supplemented with 0.1% COS increased by 25%. The present study shows that COS stimulates the growth of some enteric bacteria, and that COS has potential use as a prebiotic health-food.     

Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of the Tok1 channel in membrane potential maintenance

K+ is one of the cations (besides protons) whose transport across the plasma membrane is believed to contribute to the maintenance of membrane potential. To ensure K+ transport, Saccharomyces cerevisiae cells possess several types of active and passive transporters mediating the K+ influx and efflux, respectively. A diS-C3(3) assay was used to compare the contributions of various potassium transporters to the membrane potential changes of S. cerevisiae cells in the exponential growth phase. Altogether, the contributions of six K+ transporters to the maintenance of a stable membrane potential were tested. As confirmed by the observed hyperpolarization of trk1 trk2 deletion strains, the diS-C3(3) assay is a suitable method for comparative studies of the membrane potential of yeast strains differing in the presence/absence of one or more cation transporters. We have shown that the presence of the Tok1 channel strongly influences membrane potential: deletion of the TOK1 gene results in significant plasma membrane depolarization, whereas strains overexpressing the TOK1 gene are hyperpolarized. We have also proved that plasma membrane potential is not the only parameter determining the hygromycin B sensitivity of yeast cells, and that the role of intracellular transporters in protecting against its toxic effects must also be considered.     

ARL1 participates with ATC1/LIC4 to regulate responses of yeast cells to ions

ATC1/LIC4, previously identified as a suppressor of the Li(+)-sensitive phenotype of calcineurin mutants, was also identified as a suppressor of the hygromycin B-sensitive phenotype of strains lacking the G protein gene, ARL1. Although loss of ARL1 confers several phenotypes, including sensitivity to hygromycin B and Li(+), reduced influx of K(+), and increased secretion of carboxypeptidase Y (CPY), loss of ATC1 was without effect by these and other measures. However, loss of ATC1 in an arl1 background exacerbated ion sensitivities, although not the CPY phenotype. Moreover, overexpression of ATC1 in an arl1 background partially suppressed ion sensitivities, but not the CPY phenotype. Additionally, expression of ENA1, the Na(+)/Li(+) efflux ATPase, and activated calcineurin, but not normal calcineurin, suppressed the Li(+)-sensitive phenotype of the arl1 atc1 double mutant. These results show ARL1 and ATC1 interact to control intracellular ion levels, but ATC1 has little influence on other functions of ARL1.     

Rom2p, the Rho1 GTP/GDP exchange factor of Saccharomyces cerevisiae, can mediate stress responses via the Ras-cAMP pathway

The Ras-cyclic AMP pathway is connected to other nutrient-regulated signaling pathways and mediates the global stress responses of Saccharomyces cerevisiae. Here, we show that Rom2p, the Rho1 GTP/GDP exchange factor, can mediate stress responses and cell growth via the Ras-cAMP pathways. ROM2 was isolated as a suppresser of heat and NaCl sensitivity caused by the lack of the Ras-GTPase activator Ira2p or of cAMP phosphodiesterases. Subsequent analysis of strains with a rom2 deletion showed that Rom2p is essential for resistance to a variety of stresses caused by freeze-thawing, oxidants, cycloheximide, NaCl, or cobalt ions. Stress sensitivity and the growth defect caused by the rom2 deletion could be suppressed by depleting Ras or protein kinase A (PKA) activity or by overexpressing the high affinity cAMP phosphodiesterase Pde2p. In addition, overexpression of ROM2 could not rescue cells lacking the regulatory subunit of PKA, indicating that the Ras-adenylate, cyclase-PKA cascade is essential for Rom2p-mediated stress responses and cell growth. Deletion of IRA2 exacerbated the freeze-thaw sensitivity and growth defect of the rom2 mutant, indicating that Rom2p signaling may control Ras independently of IRA2. Increases in cAMP levels were detected in the rom2 deletion mutants, and these were comparable with the effects of an ira2 mutation. The effects of the deletion of ROM2 on sensitivity to hydrogen peroxide, paraquat, and cobalt ions, but not to caffeine, were reduced when a constitutive allele of RHO1 was introduced on a single copy plasmid. However, the effects of the deletion of ROM2 on sensitivity to diamide and NaCl were exacerbated. Taken together, our data indicate that Rom2p can regulate PKA activity by controlling cAMP levels via the Ras-cAMP pathway and that for those stresses related to oxidative stress, this cross-talk is probably mediated via the Rho1p-activated MAPK pathway.     

Methyl β-cyclodextrin reduces accumulation of reactive oxygen species and cell death in yeast

Stabilized F-actin structures have been shown to be detrimental to both mammalian and yeast cells. In yeast, stabilization of actin caused by addition of jasplakinolide, by point mutations in the act1 gene, or by deletion of certain genes that regulate F-actin leads to cell death with hallmarks of apoptosis. In particular, there is an elevation in the levels of reactive oxygen species, and we have shown the importance of the Ras/cAMP pathway for this effect. Here we show that in yeast cells deleted for end3, which functions to regulate actin organization during endocytosis, treatment of cells with methyl β-cyclodextrin reduces levels of reactive oxygen species and inhibits cell death progression. Methyl β-cyclodextrin is widely used to disrupt lipid rafts that contain cholesterol. The mechanism through which the rescue is achieved was investigated and we demonstrate that methyl β-cyclodextrin reduces accumulation of Ras2 at the plasma membrane in Δend3 cells. We use FRAP and live cell imaging to determine the possible mechanism through which methyl β-cyclodextrin functions to elicit this effect on Ras2 localization. Finally, we demonstrate that addition of methyl β-cyclodextrin to wild-type cells can act to protect cells from acute oxidative stress caused by addition of hydrogen peroxide.     

Cellular response to oncogenic ras involves induction of the Cdk4 and Cdk6 inhibitor p15(INK4b)

The cell cycle inhibitor p15(INK4b) is frequently inactivated by homozygous deletion together with p16(INK4a) and p19(ARF) in some types of tumors. Although the tumor suppressor capability of p15(INK4b) is still questioned, it has been found to be specifically inactivated by hypermethylation in hematopoietic malignancies in the absence of p16(INK4a) alterations. Here we show that, in vitro, p15(INK4b) is a strong inhibitor of cellular transformation by Ras. Surprisingly, p15(INK4b) is induced in cultured cells by oncogenic Ras to an extent similar to that of p16(INK4a), and their expression is associated with premature G(1) arrest and senescence. Ras-dependent induction of these two INK4 genes is mediated mainly by the Raf-Mek-Erk pathway. Studies with activated and dominant negative forms of Ras effectors indicate that the Raf-Mek-Erk pathway is essential for induction of both the p15(INK4b) and p16(INK4a) promoters, although other Ras effector pathways can collaborate, giving rise to a stronger response. Our results indicate that p15(INK4b), by itself, is able to stop cell transformation by Ras and other oncogenes such as Rgr (a new oncogene member of the Ral-GDS family, whose action is mediated through Ras). In fact, embryonic fibroblasts isolated from p15(INK4b) knockout mice are susceptible to transformation by the Ras or Rgr oncogene whereas wild-type embryonic fibroblasts are not. Similarly, p15(INK4b)-deficient mouse embryo fibroblasts are more sensitive than wild-type cells to transformation by a combination of the Rgr and E1A oncogenes. The cell cycle inhibitor p15(INK4b) is therefore involved, at least in some cell types, in the tumor suppressor activity triggered after inappropriate oncogenic Ras activation in the cell.     

The cell factory approach toward biotechnological production of high-value chitosan oligomers and their derivatives: an update

Chitin is one of the most abundant renewable resources, and chitosans, the partially deacetylated derivatives of chitin, are among the most promising functional biopolymers, with superior material properties and versatile biological functionalities. Elucidating molecular structure–function relationships and cellular modes of action of chitosans, however, it is challenging due to the micro-heterogeneity and structural complexity of polysaccharides. Lately, it has become apparent that many of the biological activities of chitosan polymers, such as in agricultural plant disease protection or in mediating scar-free wound healing, may be attributed to oligomeric break-down products generated by the action of chitosanolytic hydrolases present in the target tissues, such as human chitotriosidase. Consequently, the focus of current research is shifting toward chitosan oligomers so that the availability of well-defined chitosan oligosaccharides (COS) becomes a bottleneck. Well-known ways of producing COS use physical and/or chemical means for the partial depolymerization of chitosan polymers, typically leading to broad mixtures of COS varying in their degrees of polymerization (DP) and acetylation (DA), and with more or less random patterns of acetylation (PAs). Even after chromatographic separation according to DP and DA, such mixtures are of limited value to elucidate structure–function relationships and modes of action. More recently, enzymatic means using chitinases and/or chitosanases, and sometimes chitin deacetylases, have been proposed as these can be more tightly controlled and yield slightly better defined mixtures of COS. An alternative would be chemical synthesis of COS which in principle would allow for full structural control, but protocols for it are lengthy, costly, and not yet well developed, and yields are low. Synthetic biology now allows to develop today’s in vitro bio-refinery approaches into in vivo cell factory approaches for the biotechnological production of defined COS using recombinant microbial strains expressing chitin oligomer synthases and chitin oligomer deacetylases. In this review, we will describe the state-of-the-art of this cell factory approach, as a basis for upcoming developments. We will briefly describe traditional chemical protocols and enzymatic production of COS as a background to the more detailed presentation of what has been achieved through in vivo biosynthesis. We will only briefly describe those as a background to the more detailed presentation of what has been achieved through in vivo biosynthesis. We will also touch on the production of COS derivatives that has been achieved in this way, as these oligomers open up another plethora of potential applications when used as building blocks for defined biomaterials.     

The possible mechanism of action of ciclopirox olamine in the yeast Saccharomyces cerevisiae

Ciclopirox olamine is a synthetic antifungal agent with a high affinity for trivalent metal cations. Ciclopirox olamine can be used to synchronize mammalian cells, but its mechanism of action is not understood well. In this study, we investigated the effect of ciclopirox olamine in yeast cells and used a genetic approach to identify potential ciclopirox olamine targets in yeast. Wild type strains of the yeast Saccharomyces cerevisiae were weakly sensitive to ciclopirox olamine, but high concentrations of the drug arrested their growth at many different stages. MMS-mutagenized yeast clones were screened for increased sensitivity to ciclopirox olamine. Fourteen mutants, cos101-cos114, were identified and characterized. The targets of ciclopirox olamine in S. cerevisiae appear to include multiple proteins that participate in various components of cellular metabolism, including DNA replication, DNA repair, and cellular transport. Three genes were cloned: a Fe/Cu reductase (FRE1/COS107), an oxidative stress response gene (YAP1/COS110), and a gene involved in signal transduction (YBR203W/COS111). These results suggest that CPO inhibits multiple aspects of cell growth and metabolism, possibly via multiple targets.     

Methylation of the RASSF1A gene in human cancers

Loss of genetic material from chromosome 3p21.3 is one of the most common and earliest events in the pathogenesis of lung cancer and many other solid tumors. The chromosomal area 3p21.3 is thought to harbor at least one important tumor suppressor gene, which, despite many years of investigation, has remained elusive. In our previous studies, we have identified and cloned a gene from the common homozygous deletion area at 3p21.3. The gene, named RASSF1A (Ras ASSociation domain Family 1A), has homology to a mammalian Ras effector. The RASSF1A gene is epigenetically inactivated in a large percentage of human lung cancers, in particular small cell carcinomas. A high frequency of methylation of RASSF1A is found also in breast cancers, renal cell carcinomas, ovarian, gastric and bladder cancers, and in neuroblastomas. The RASSF1A gene is a candidate for a tumor suppressor gene in 3p21.3.     

Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.     

c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.     

Neurofibromin can inhibit Ras-dependent growth by a mechanism independent of its GTPase-accelerating function.

The NF1 gene, which is altered in patients with type 1 neurofibromatosis, has been postulated to function as a tumor suppressor gene. The NF1 protein product neurofibromin stimulates the intrinsic GTPase activity of active GTP-bound Ras, thereby inactivating it. Consistent with a tumor suppressor function, we have found that the introduction of NF1 in melanoma cell lines that are deficient in neurofibromin inhibited their growth and induced their differentiation. In addition, overexpression of neurofibromin in NIH 3T3 cells was growth inhibitory but did not alter the level of GTP.Ras in the cells. Transformation by v-ras, whose protein product is resistant to GTPase stimulation by neurofibromin, was inhibited in a cell line overexpressing neurofibromin, while transformation by v-raf was not altered. The results demonstrate that NF1 is a tumor suppressor gene that can inhibit Ras-dependent growth by a regulatory mechanism that is independent of neurofibromin's ability to stimulate Ras GTPase.     

An in vitro cellular analysis of the radical scavenging efficacy of chitooligosaccharides

Despite extensive study on biological activities of chitosan and chitooligosaccharides (COS), there is no experimental evidence available as to COS mediated inhibition of free radical damage in cellular oxidizing systems. In this study, radical scavenging efficacies of different molecular weight bearing COS were assessed and their intracellular radical scavenging effects were tested employing B16F1, murine melanoma cell line. The results exhibited appreciable suppression in occurrence of intracellular radical species in the presence of low molecular weight bearing COS (<1 kDa) confirming low molecular weight is important for observed activities in biological systems. However, DNA oxidation carried out in the presence of COS clearly exhibited that COS exert protective effect on oxidative damage of purified genomic DNA regardless of molecular weight. Low molecular weight bearing COS was observed to be successively participated in suppression of NF-kappaB gene promoter activity suggesting its capability to prevent oxidative stress related disease complications. Moreover, induction of intracellular glutathione (GSH) level in the presence of COS promoted the effectiveness of COS to act against cellular oxidative stress.     

n-6 and n-3 polyunsaturated fatty acids differentially modulate oncogenic Ras activation in colonocytes

Ras proteins are critical regulators of cell function, including growth, differentiation, and apoptosis, with membrane localization of the protein being a prerequisite for malignant transformation. We have recently demonstrated that feeding fish oil, compared with corn oil, decreases colonic Ras membrane localization and reduces tumor formation in rats injected with a colon carcinogen. Because the biological activity of Ras is regulated by posttranslational lipid attachment and its interaction with stimulatory lipids, we investigated whether docosahexaenoic acid (DHA), found in fish oil, compared with linoleic acid (LA), found in corn oil, alters Ras posttranslational processing, activation, and effector protein function in young adult mouse colon cells overexpressing H-ras (YAMC-ras). We show here that the major n-3 polyunsaturated fatty acid (PUFA) constituent of fish oil, DHA, compared with LA (an n-6 PUFA), reduces Ras localization to the plasma membrane without affecting posttranslational lipidation and lowers GTP binding and downstream p42/44(ERK)-dependent signaling. In view of the central role of oncogenic Ras in the development of colon cancer, the finding that n-3 and n-6 PUFA differentially modulate Ras activation may partly explain why dietary fish oil protects against colon cancer development.