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Lentinus edodes (Shiitake) is a medicinal mushroom with a long tradition of use in Asia. The major active substance in L. edodes is a (1-6,1-3)-beta-glucan (lentinan). No clinical controlled studies have yet investigated the effect of orally administered lentinan on the immune response in healthy, elderly Caucasian subjects. We evaluated the effect and the safety of a beta-glucan from L. edodes mycelium, Lentinex, in healthy, elderly subjects in a double blind, crossover, placebo-controlled trial. Forty-two subjects were randomly allocated to two groups given orally either 2.5 mg/day Lentinex or placebo for 6 weeks; then after a washout period of 4 weeks, the alternate supplementation was given for 6 weeks. The changes in the number of B-cells were significantly different between the groups. The number ofNK cells increased significantly in both groups, but there was no significant difference between the groups. Other factors of the immune response (immunoglobulins, complement proteins, cytokines) were not altered. The safety blood variables (differential cell count, liver function, kidney function, and other blood chemistry) were not influenced by Lentinex, and the number, nature, and severity of adverse events were similar to placebo. Lentinex given orally to elderly subjects was safe and induced an increase in the number of circulating B-cells.

Purpose: Cytarabine (ara-C) has limited activity in solid tumours. CP-4055 (ELACYT) is a novel ara-C-5'-elaidic acid ester that may circumvent this limitation. CP-4055 maximum tolerated dose (MTD), pharmacokinetics and antitumor activity have been investigated in patients with solid tumours., Material and Methods: Thirty-four patients (19 malignant melanoma, 8 ovarian cancers and 7 NSCLC) received CP-4055 as a 30 min, or 2 hr intravenous (IV) infusion daily for 5 consecutive days every 3 or 4 weeks (D1-5 q3w or D1-5 q4w) in a dose escalation designed study with doses ranging from 30 to 240 mg/m(2)/day., Results: The most frequent CTC grade 1-2 adverse events (AEs) were nausea, fatigue, vomiting, anorexia and pyrexia. Most of the grade 3-4 AEs were neutropenia. The MTD was 200 mg/m(2)/day and 240 mg/m(2)/day for D1-5 q3w and D1-5 q4w, respectively. The MTD was independent of infusion time in the 4 week schedule. CP-4055 was maintained in plasma for up to 5-10 hr at dose levels >150 mg/m(2)/day. One objective partial response (PR) with time to progression (TTP) of 22 months was reported in an advanced malignant melanoma patient., Conclusion: CP-4055 was well tolerated; the majority of the AEs were of CTC grade 1. The 3 week schedule was not recommended due to neutropenic nadir between days 18-26. The recommended dose was 200 mg/m(2)/day in a D1-5 q4w schedule. Efficacy data suggest that CP-4055 might be active in treatment of solid tumours.

After 12 mo in a randomized, double-blind, placebo-controlled trial of conjugated linoleic acid (CLA) supplementation (2 groups received CLA as part of a triglyceride or as the free fatty acid, and 1 group received olive oil as placebo), 134 of the 157 participants who concluded the study were included in an open study for another 12 mo. The goals of the extension study were to evaluate the safety [with clinical chemistry analyses and reported adverse events (AEs)] and assess the effects of CLA on body composition [body fat mass (BFM), lean body mass (LBM), bone mineral mass (BMM)], body weight, and BMI. All subjects were supplemented with 3.4g CLA/d in the triglyceride form. Circulating lipoprotein(a) and thrombocytes increased in all groups. There was no change in fasting blood glucose. Aspartate amino transferase, but not alanine amino transferase, increased significantly. Plasma total cholesterol and LDL cholesterol were reduced, whereas HDL cholesterol and triglycerides were unchanged. The AE rate decreased compared with the first 12 mo of the study. Body weight and BFM were reduced in the subjects administered the placebo during the initial 12 mo study (-1.6 +/- 3.2 and -1.7 +/- 2.8 kg, respectively). No fat or body weight changes occurred in the 2 groups given CLA during the initial 12 mo. LBM and BMM were not affected in any of the groups. Changes in body composition were not related to diet and/or training. In conclusion, this study shows that CLA supplementation for 24 mo in healthy, overweight adults was well tolerated. It confirms also that CLA decreases BFM in overweight humans, and may help maintain initial reductions in BFM and weight in the long term.

Background: Short-term trials showed that conjugated linoleic acid (CLA) may reduce body fat mass (BFM) and increase lean body mass (LBM), but the long-term effect of CLA was not examined., Objective: The objective of the study was to ascertain the 1-y effect of CLA on body composition and safety in healthy overweight adults consuming an ad libitum diet., Design: Male and female volunteers (n = 180) with body mass indexes (in kg/m(2)) of 25-30 were included in a double-blind, placebo-controlled study. Subjects were randomly assigned to 3 groups: CLA-free fatty acid (FFA), CLA-triacylglycerol, or placebo (olive oil). Change in BFM, as measured by dual-energy X-ray absorptiometry, was the primary outcome. Secondary outcomes included the effects of CLA on LBM, adverse events, and safety variables., Results: Mean (+/- SD) BFM in the CLA-triacylglycerol and CLA-FFA groups was 8.7 +/- 9.1% and 6.9 +/- 9.1%, respectively, lower than that in the placebo group (P < 0.001). Subjects receiving CLA-FFA had 1.8 +/- 4.3% greater LBM than did subjects receiving placebo (P = 0.002). These changes were not associated with diet or exercise. LDL increased in the CLA-FFA group (P = 0.008), HDL decreased in the CLA-triacylglycerol group (P = 0.003), and lipoprotein(a) increased in both CLA groups (P < 0.001) compared with month 0. Fasting blood glucose concentrations remained unchanged in all 3 groups. Glycated hemoglobin rose in all groups from month 0 concentrations, but there was no significant difference between groups. Adverse events did not differ significantly between groups., Conclusion: Long-term supplementation with CLA-FFA or CLA-triacylglycerol reduces BFM in healthy overweight adults.

CLA mixtures are now commercially available. They differ from each other with respect to their content of CLA isomers and their degree of purification. As a group of natural FA, CLA have been widely assumed to be safe. However, the suspected presence of both impurities and particular isomers might induce undesirable side effects. Despite this potential health risk, only a few CLA preparations have been tested under rigorous conditions for clinical efficacy and safety. Based on the limited results available, it is possible to suggest that preparations enriched in c9,t11 and t10,c12 isomers are preferable for human consumption compared to preparations containing four isomers, in terms both of safety and efficacy.

Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.

FYVE zinc finger domains, which are conserved in multiple proteins from yeast to man, interact specifically with the membrane lipid phosphatidylinositol 3-phosphate (PtdIns(3)P). Here we have investigated the structural requirements for the interaction of the FYVE finger of the early endosome antigen EEA1 with PtdIns(3)P and early endosomes. The binding of the FYVE finger to PtdIns(3)P is Zn(2+)-dependent, and Zn(2+) could not be replaced by any other bivalent cations tested. By surface plasmon resonance, the wild-type FYVE finger was found to bind to PtdIns(3)P with an apparent K(D) of about 50 nm and a 1:1 stoichiometry. Mutagenesis of cysteines involved in Zn(2+) coordination, basic residues thought to be directly involved in ligand binding and other conserved residues, resulted in a 6- to >100-fold decreased affinity for PtdIns(3)P. A mutation in the putative PtdIns(3)P-binding pocket, R1375A, may prove particularly informative, because it led to a strongly decreased affinity for PtdIns(3)P without affecting the FYVE three-dimensional structure, as measured by fluorescence spectroscopy. Whereas the C terminus of EEA1 localizes to early endosomes when expressed in mammalian cells, all the FYVE mutants with reduced affinity for PtdIns(3)P were found to be largely cytosolic. Furthermore, whereas expression of the wild-type EEA1 C terminus interferes with early endosome morphology, the point mutants were without detectable effect. These results support recently proposed models for the ligand binding of the FYVE domain and indicate that PtdIns(3)P binding is crucial for the localization and function of EEA1.

The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.

Phosphatidylinositol 3-phosphate (PtdIns(3)P), generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3-kinase (PI 3-kinase), plays an essential role in intracellular membrane traffic. The underlying mechanism is still not understood in detail, but the recent identification of the FYVE finger as a protein domain that binds specifically to PtdIns(3)P provides a number of potential effectors for PtdIns(3)P. The FYVE finger (named after the first letter of the four proteins containing it; Fab1p, YOTB, Vac1p and EEA1) is a double-zinc binding domain that is conserved in more than 30 proteins from yeast to mammals. It is found in several proteins involved in intracellular traffic, and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins. The interaction of FYVE fingers with PtdIns(3)P may serve three alternative functions: First, to recruit cytosolic FYVE finger proteins to PtdIns(3)P-containing membranes (in concert with accessory molecules); second, to enrich for membrane bound FYVE finger proteins into PtdIns(3)P containing microdomains within the membrane; and third, to modulate the activity of membrane bound FYVE finger proteins.

EEA1, an early-endosomal protein originally identified as an autoantigen, is essential for endocytic membrane fusion. It interacts with early endosomes via binding to the membrane lipid phosphatidylinositol 3-phosphate (PtdIns3P) and the active form of the small GTPase Rab5. Most of the EEA1 sequence contains heptad repeats characteristic of proteins involved in coiled-coil protein-protein interactions. Here we have investigated the ability of EEA1 to self-interact. Crosslinking of cytosolic and recombinant EEA1 resulted in the disappearance of the 180-kDa monomer in SDS/PAGE and the strong appearance of a approximately 350-kDa crosslinked product. Glycerol gradient centrifugation experiments indicated that native EEA1 had the same hydrodynamic properties as the approximately 350-kDa crosslinked complex. Two-hybrid analysis indicated that N- and C-terminal fragments of EEA1 can interact with themselves, but not with each other, suggesting that EEA1 forms parallel coiled-coil dimers. The ability of the C-terminus of EEA1 to dimerize correlates with its ability to bind to Rab5 and early endosomes, whereas its binding to PtdIns3P is independent of dimerization. These data enable us to propose a model for the quaternary structure of EEA1.

GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion. The association of EEA1 with the endosomal membrane requires Rab5-GTP and PI(3)K activity, and excess Rab5-GTP stabilizes the membrane association of EEA1 even when PI(3)K is inhibited. The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

Human colon adenocarcinoma cells (WiDr) and Chinese hamster lung fibroblasts cells (V79) were incubated with different concentrations of 5-aminolevulinic acid (ALA), and the production of protoporphyrin IX (PpIX) was studied using several techniques. The amount of PpIX produced per cell increased with increasing ALA concentration according to different kinetics for the 2 cell lines. For both cell lines a cell density dependency of the PpIX synthesis was observed. For saturating ALA concentrations, 2-3 times more PpIX was produced per cell at a density of 5 x 10(4) than at a density of 5 x 10(3) cells/cm2. The photosensitivity of cells appeared to increase even more than the PpIX content, indicating a cooperative effect in inactivation. The PpIX production rate increased with cell size and was about 1.9 times higher for cells in the G2 + M phase than for cells in the G1 phase of the cell cycle. Neither cell size nor cell cycle distribution were significantly dependent on cell density.

A novel glutathione compound in which the amino group has been derivatized by a 2,5-dimethyl pyrrole is shown to be very effective against cell photosensitization in vitro. Protoporphyrin IX either added to the medium or produced endogenously by incubation of NCTC 2544 keratinocytes with 5-aminolevulinic acid has been chosen as the photosensitizer. The antioxidant effectiveness of glutathione-pyrrole derivatives against protoporphyrin photosensitization depends critically on the type of 2,5 substitution on the pyrrole ring. This structure-function relationship may be attributed to the difference in compartmentation and/or uptake of the various glutathione-pyrrole derivatives under study. The 2,5-dimethyl pyrrole derivative is much more effective than glutathione as a protective agent against phototoxic reactions induced by protoporphyrin IX.

Human tumor cells of the lines WiDr (adenocarcinoma of the rectosigmoid colon), NHIK 3025 (carcinoma of the cervix), and V79 Chinese hamster fibroblasts were treated with 5-aminolevulinic acid (ALA) and ALA esterified to C1-C3 and C6-C8 chained aliphatic alcohols (ALA-esters). In the human cell lines, esterification of ALA with the long-chain (C6-C8) alcohols was found to reduce 30-150-fold the amount of ALA needed to reach the same level of protoporphyrin IX (PpIX) accumulation as with non-esterified ALA. The long-chained ALA-esters were less efficient in stimulating PpIX formation in V79 cells, i.e., the same amount of PpIX was formed by a 1-2.6-fold lower concentration of long-chained ALA-esters than with ALA. Short-chained ALA-esters (C1-C3) induced 5 to 10 times lower PpIX accumulation than ALA in all of the cell lines. High-performance liquid chromatography and fluorescence microscopic studies indicated that esterification of ALA has neither impact on the fluorescing porphyrin species formed nor impact on their intracellular localization. The PpIX formed from ALA-esters and ALA was found to be equally efficient in sensitizing cells to photoinactivation. The present results indicate that esterified ALAs are new and promising drugs for use in photochemotherapy of cancer.

Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRH) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of lysosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal microenvironments.

The subcellular localization of protoporphyrin (PP) has been studied by microspectrofluorometric techniques in NCTC 2544 keratinocytes incubated with 5-aminolevulinic acid (ALA) for times up to 42 h. Whereas the plasma membrane shows strong staining, fluorescent spots are observed within the cytoplasm especially in the perinuclear region. Although the topographic pattern of the PP distribution does not change much with the incubation time with ALA, the fluorescence spectra suggest that the PP microenvironments are quite different at short and long incubation times. Addition of 18 microM desferrioxamine almost doubles the ALA-induced PP concentration. Colocalization experiments with rhodamine 123, a mitochondrial probe, and lucifer yellow (LY) or neutral red (NR), two lysosome probes, demonstrate that at least some of these spots are of lysosomal origin. Study of the time evolution of the NR fluorescence under irradiation with visible light in the presence and absence of ALA demonstrates that lysosomes are damaged cells that have synthesized PP. No PP fluorescence can be detected in mitochondria after incubation with ALA. However, photosensitization of mitochondria occurs under irradiation with visible light. Very little formation of lipofuscins by photosensitization with exogenous PP or ALA-induced PP is observed with the NCTC 2544 keratinocytes, as compared to normal human fibroblasts.

A new class of glutathione derivatives with antioxidant properties has been prepared by transformation of the NH2 group into a pyrrole ring with various substitutions at the 2 and 5 positions. Due to steric hindrance and/or hydrophobicity of the 2-5-disubstituted pyrrole ring, the reduced glutathione derivatives are poor substrates of the glutathione peroxidase and do not effectively compete with GSH. The oxidized glutathione derivatives are, in turn, relatively good substrates (Km = 1.5 mM) of the glutathione reductase as compared to natural oxidized glutathione (Km = 0.51 mM) but are not effective competitors of the enzyme. It can be considered that the new glutathione derivatives do not strongly interfere with the natural enzymatic defence against fatty acid hydroperoxides formed during an oxidative stress.