Natural 2-Amino-3-Methylhexanoic Acid as Plant Elicitor Inducing Resistance against Temperature Stress and Pathogen Attack.

2-Amino-3-methylhexanoic acid (AMHA) was synthetized as a non-natural amino acid more than 70 years ago; however, its possible function as an inducer of plant resistance has not been reported. Plant resistance inducers, also known as plant elicitors, are becoming a novel and important development direction in crop protection and pest management. We found that free AMHA accumulated in the mycelia but not in fermentation broths of four fungal species, Magnaporthe oryzae and three Alternaria spp. We unequivocally confirmed that AMHA is a naturally occurring endogenous (2S, 3S)-α-amino acid, based on isolation, purification and structural analyses. Further experiments demonstrated that AMHA has potent activity-enhancing resistance against extreme temperature stresses in several plant species. It is also highly active against fungal, bacterial and viral diseases by inducing plant resistance. AMHA pretreatment strongly protected wheat against powdery mildew, Arabidopsis against Pseudomonas syringae DC3000 and tobacco against Tomato spotted wilt virus. AMHA exhibits a great potential to become a unique natural elicitor protecting plants against biotic and abiotic stresses.

Indospicine combined with arginine deprivation triggers cancer cell death via caspase-dependent apoptosis.

Arginine-deprivation therapy is a rapidly developing metabolic anticancer approach. To overcome the resistance of some cancer cells to this monotherapy, rationally designed combination modalities are needed. In this report, we evaluated for the first time indospicine, an arginine analogue of Indigofera plant genus origin, as potential enhancer compound for the metabolic therapy that utilizes recombinant human arginase I. We demonstrate that indospicine at low micromolar concentrations is selectively toxic for human colorectal cancer cells only in the absence of arginine. In arginine-deprived cancer cells indospicine deregulates some prosurvival pathways (PI3K-Akt and MAPK) and activates mammalian target of rapamycin, exacerbates endoplasmic reticulum stress and triggers caspase-dependent apoptosis, which is reversed by the exposure to translation inhibitors. Simultaneously, indospicine is not degraded by recombinant human arginase I and does not inhibit this arginine-degrading enzyme at its effective dose. The obtained results emphasize the potential of arginine structural analogues as efficient components for combinatorial metabolic targeting of malignant cells.

Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart.

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects. Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRSL274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction. Methods and Results: Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton. Conclusions: MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

Blockage of O-linked GlcNAcylation induces AMPK-dependent autophagy in bladder cancer cells.

BACKGROUND: High levels of the post-translational modification O-GlcNAcylation (O-GlcNAc) are found in multiple cancers, including bladder cancer. Autophagy, which can be induced by stress from post-translational modifications, plays a critical role in maintaining cellular homeostasis and regulating tumorigenesis. The impact of O-GlcNAcylation on autophagy in bladder cancer remains unclear. Here, we evaluate the change in autophagic activity in response to O-GlcNAcylation and explore the potential mechanisms. METHODS: O-GlcNAcylation levels in bladder cancer cells were altered through pharmacological or genetic manipulations: treating with 6-diazo-5-oxo-norleucine (DON) or thiamet-G (TG) or up- and downregulation of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA). Autophagy was determined using fluorescence microscopy and western blotting. Co-immunoprecipitation (Co-IP) assays were performed to evaluate whether the autophagy regulator AMP-activated protein kinase (AMPK) was O-GlcNAc modified. RESULTS: Cellular autophagic flux was strikingly enhanced as a result of O-GlcNAcylation suppression, whereas it decreased at high O-GlcNAcylation levels. Phosphorylation of AMPK increased after the suppression of O-GlcNAcylation. We found that O-GlcNAcylation of AMPK suppressed the activity of this regulator, thereby inhibiting ULK1 activity and autophagy. CONCLUSION: We characterized a new function of O-GlcNAcylation in the suppression of autophagy via regulation of AMPK. GRAPHICAL ABSTRACT: Blockage of O-linked GlcNAcylation induces AMPK dependent autophagy in bladder cancer cells.

Adaptive landscape flattening allows the design of both enzyme: Substrate binding and catalytic power.

Designed enzymes are of fundamental and technological interest. Experimental directed evolution still has significant limitations, and computational approaches are a complementary route. A designed enzyme should satisfy multiple criteria: stability, substrate binding, transition state binding. Such multi-objective design is computationally challenging. Two recent studies used adaptive importance sampling Monte Carlo to redesign proteins for ligand binding. By first flattening the energy landscape of the apo protein, they obtained positive design for the bound state and negative design for the unbound. We have now extended the method to design an enzyme for specific transition state binding, i.e., for its catalytic power. We considered methionyl-tRNA synthetase (MetRS), which attaches methionine (Met) to its cognate tRNA, establishing codon identity. Previously, MetRS and other synthetases have been redesigned by experimental directed evolution to accept noncanonical amino acids as substrates, leading to genetic code expansion. Here, we have redesigned MetRS computationally to bind several ligands: the Met analog azidonorleucine, methionyl-adenylate (MetAMP), and the activated ligands that form the transition state for MetAMP production. Enzyme mutants known to have azidonorleucine activity were recovered by the design calculations, and 17 mutants predicted to bind MetAMP were characterized experimentally and all found to be active. Mutants predicted to have low activation free energies for MetAMP production were found to be active and the predicted reaction rates agreed well with the experimental values. We suggest the present method should become the paradigm for computational enzyme design.

Probing the Interactions of Sulfur-Containing Histidine Compounds with Human Gamma-Glutamyl Transpeptidase.

Gamma-glutamyl transpeptidase (GGT) is a cell surface enzyme involved in glutathione metabolism and maintenance of redox homeostasis. High expression of GGT on tumor cells is associated with an increase of cell proliferation and resistance against chemotherapy. GGT inhibitors that have been evaluated in clinical trials are too toxic for human use. We have previously identified ovothiols, 5(Nπ)-methyl-thiohistidines of marine origin, as non-competitive-like inhibitors of GGT that are more potent than the known GGT inhibitor, 6-diazo-5-oxo-l-norleucine (DON), and are not toxic for human embryonic cells. We extended these studies to the desmethylated form of ovothiol, 5-thiohistidine, and confirmed that this ovothiol derivative also acts as a non-competitive-like GGT inhibitor, with a potency comparable to ovothiol. We also found that both 5-thiohistidine derivatives act as reversible GGT inhibitors compared to the irreversible DON. Finally, we probed the interactions of 5-thiohistidines with GGT by docking analysis and compared them with the 2-thiohistidine ergothioneine, the physiological substrate glutathione, and the DON inhibitor. Overall, our results provide new insight for further development of 5-thiohistidine derivatives as therapeutics for GGT-positive tumors.

Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders.

HIV-associated neurocognitive disorders (HAND) have been linked to dysregulation of glutamate metabolism in the central nervous system (CNS) culminating in elevated extracellular glutamate and disrupted glutamatergic neurotransmission. Increased glutamate synthesis via upregulation of glutaminase (GLS) activity in brain immune cells has been identified as one potential source of excess glutamate in HAND. However, direct evidence for this hypothesis in an animal model is lacking, and the viability of GLS as a drug target has not been explored. In this brief report, we demonstrate that GLS inhibition with the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) can reverse cognitive impairment in the EcoHIV-infected mouse model of HAND. However, due to peripheral toxicity DON is not amenable to clinical use in a chronic disease such as HAND. We thus tested JHU083, a novel, brain penetrant DON prodrug predicted to exhibit improved tolerability. Systemic administration of JHU083 reversed cognitive impairment in EcoHIV-infected mice similarly to DON, and simultaneously normalized EcoHIV-induced increases in cerebrospinal fluid (CSF) glutamate and GLS activity in microglia-enriched brain CD11b + cells without observed toxicity. These studies support the mechanistic involvement of elevated microglial GLS activity in HAND pathogenesis, and identify JHU083 as a potential treatment option. Graphical Abstract Please provide Graphical Abstract caption.Glutamine Antagonist JHU083 Normalizes Aberrant Glutamate Production and Cognitive Deficits in the EcoHIV Murine Model of HIV-Associated Neurocognitive Disorders .

Indospicine cytotoxicity and transport in human cell lines.

Indospicine, a non-proteinogenic analogue of arginine, occurs only in Indigofera plant species and accumulates in the tissues of animals grazing on Indigofera. Canine deaths have resulted from the consumption of indospicine-contaminated meat but only limited information is available regarding indospicine toxicity in humans. In this study three human cell lines, Caco-2 (colorectal adenocarcinoma), HT29-MTX-E12 (colorectal adenocarcinoma) and HepG2 (hepatocellular carcinoma), were used to investigate the cytotoxicity of indospicine and its metabolite 2-aminopimelic acid in comparison to arginine. Indospicine and 2-aminopimelic acid were more cytotoxic than arginine, displaying the highest toxicity in HepG2 liver cells. Intestinal transport in vitro also revealed a 2-fold higher transport rate of indospicine compared to arginine. The sensitivity of HepG2 cells to indospicine is consistent with observed canine hepatotoxicity, and considering the higher in vitro transport of indospicine across an intestinal barrier, it is possible that similar ill effects could be seen in humans consuming contaminated meat.

Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.

Application of bio-orthogonal proteome labeling to cell transplantation and heterochronic parabiosis.

Studies of heterochronic parabiosis demonstrated that with age, the composition of the circulatory milieu changes in ways that broadly inhibit tissue regenerative capacity. In addition, local tissue niches have age-specific influences on their resident stem cells. Here we use bio-orthogonal proteome labeling for detecting in vivo proteins present only in transplanted myoblasts, but not in host tissue, and proteins exclusive to one young mouse and transferred during parabiosis to its old partner. We use a transgenic mouse strain that ubiquitously expresses a modified tRNA methionine synthase, metRS, which preferentially incorporates the methionine surrogate azido-nor-leucine (ANL) into newly generated proteins. Using click chemistry and a modified antibody array to detect ANL-labeled proteins, we identify several 'young' systemic factors in old regenerating muscle of the heterochronic parabiotic partners. Our approach enables the selective profiling of mammalian proteomes in mixed biological environments such as cell and tissue transplantation, apheresis or parabiosis.Clarifying the source of proteins in mixed biological environments, such as after transplantation or parabiosis, remains a challenge. Here, the authors address this need with a mouse strain that incorporates a methionine derivate into proteins, allowing for their detection using click chemistry and antibody arrays.

Evaluation of genetic toxicity of 6-diazo-5-oxo-l-norleucine (DON).

DON (6-diazo-5-oxo-l-norleucine), a glutamine antagonist, was demonstrated to exhibit analgesic, antibacterial, antiviral and anticancer properties. The study was performed to characterize its in vitro and in vivo genetic toxicity potential. DON was tested in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli tester strain (WP2 uvrA) with and without S9 and also with reductive S9. In addition, DON was tested for the chromosome aberrations in Chinese hamster ovary (CHO) cells with or without S9 to evaluate the clastogenic potential. Furthermore, DON was also evaluated for its in vivo clastogenic activity by detecting micronuclei in polychromatic erythrocyte (PCE) cells in bone marrow collected from the male mice dosed intravenously with 500, 100, 10, 1 and 0.1 mg/kg at 24 and 48-h post-dose. The Ames mutagenicity assay showed no positive mutagenic responses. However, the in vitro chromosome aberration assay demonstrated dose dependent statistically positive increase in structural aberrations at 4 and 20-h exposure without S9 and also at 4-h exposure with S9. The in vivo micronucleus assay also revealed a statistically positive response for micronucleus formation at 500, 100 and 10 mg/kg at 24 and 48-h post-dose. Thus, DON appears to be negative in the Ames test but positive in the in vitro chromosome aberration assay and in the in vivo micronucleus assay. In conclusion, the results indicate DON is a genotoxic compound with a plausible epigenetic mechanism.

Kinetic Study on Peptide-Bound Pyrraline Formation and Elimination in the Maillard Reaction Using Single- and Multiple-Response Models.

Pyrraline, an advanced glycation end product (AGE), is related to some chronic diseases and can be employed as an indicator for heat damage in food processing. In this study, the impact of changing the reactant concentration and ratio on the kinetic parameters describing peptide-bound pyrraline (pep-pyr) formation and elimination was evaluated in the Lys-Gly/glucose model systems, with microwave heating treatment ranging from 120 to 200 °C. The maximum pep-pyr concentration increased as follows: 200 °C ˂ 180 °C ˂ 160 °C ˂ 120 °C ˂ 140 °C. First, the pep-pyr formation and elimination was modeled by using a single-response modelling. The formation rate constant (kF ) of pep-pyr was independent of the initial concentration of the reactants and ratios. However, the elimination rate constant of pep-pyr (kE ) increased with increasing reactant concentrations. Second, a multiresponse modelling was performed to illustrate the pathways of pep-pyr formation and elimination. Two adapted models can fit to the experimental data with the goodness-of-fit ranging from 0.663 to 0.920. Glucose-to-fructose isomerization rather than glucose-to-mannose epimerization was detected in an equimolar model system and the model system with an excess of any of the reactants. The caramelization reaction was negligible in the equimolar systems and the model systems with an excess of peptide. The reaction rate constant of glucose-to-fructose isomerization was independent of the initial reactant ratios. It was more difficult for pep-pyr elimination in the model system with an excess of peptide than that in the other 2 model systems (the equimolar system and the system with an excess of glucose), whereas a reverse result in pep-pyr formation was obtained.

Accumulation, Persistence, and Effects of Indospicine Residues in Camels Fed Indigofera Plant.

Indospicine (l-2-amino-6-amidinohexanoic acid) is a natural hepatotoxin found in all parts of some Indigofera plants such as Indigofera linnaei and Indigofera spicata. Several studies have documented a susceptibility to this hepatotoxin in different species of animals, including cattle, sheep, dogs, and rats, which are associated with mild to severe liver disease after prolonged ingestion. However, there is little published data on the effects of this hepatotoxin in camels, even though Indigofera plants are known to be palatable to camels in central Australia. The secondary poisoning of dogs after prolonged dietary exposure to residual indospicine in camel muscle has raised additional food safety concerns. In this study, a feeding experiment was conducted to investigate the in vivo accumulation, excretion, distribution, and histopathological effects of dietary indospicine on camels. Six young camels (2-4 years old), weighing 270-390 kg, were fed daily a roughage diet consisting of Rhodes grass hay and lucerne chaff, supplemented with Indigofera and steam-flaked barley. Indigofera (I. spicata) was offered at 597 mg DM/kg body weight (bw)/day, designed to deliver 337 µg indospicine/kg bw/day, and fed for a period of 32 days. Blood and muscle biopsies were collected over the period of the study. Concentrations of indospicine in the plasma and muscle biopsy samples were quantitated by validated ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The highest concentrations in plasma (1.01 mg/L) and muscle (2.63 mg/kg fresh weight (fw)) were found at necropsy (day 33). Other tissues were also collected at necropsy, and analysis showed ubiquitous distribution of indospicine, with the highest indospicine accumulation detected in the pancreas (4.86 ± 0.56 mg/kg fw) and liver (3.60 ± 1.34 mg/kg fw), followed by the muscle, heart, and kidney. Histopathological examination of liver tissue showed multiple small foci of predominantly mononuclear inflammatory cells. After cessation of Indigofera intake, indospicine present in plasma in the remaining three camels had a longer terminal elimination half-life (18.6 days) than muscle (15.9 days), and both demonstrated monoexponential decreases.

Determination of Free-Form and Peptide Bound Pyrraline in the Commercial Drinks Enriched with Different Protein Hydrolysates.

Pyrraline, a causative factor for the recent epidemics of diabetes and cardiovascular disease, is also employed as an indicator to evaluate heat damage and formation of advanced glycation end-products (AGEs) in foods. Peptide-enriched drinks (PEDs) are broadly consumed worldwide due to rapid rate of absorption and perceived health effects. It can be hypothesized that PED is an important source of pyrraline, especially peptide bound pyrraline (Pep-Pyr). In this study we determined free-form pyrraline (Free-Pyr) and Pep-Pyr in drinks enriched with whey protein hydrolysate (WPH), soy protein hydrolysate (SPH) and collagen protein hydrolysate (CPH). A detection method was developed using ultrahigh-performance liquid chromatography with UV-visible detector coupled with tandem mass spectrometry after solid-phase extraction (SPE). The SPE led to excellent recovery rates ranging between 93.2% and 98.5% and a high reproducibility with relative standard deviations (RSD) of <5%. The limits of detection and quantification obtained were 30.4 and 70.3 ng/mL, respectively. Pep-Pyr was identified as the most abundant form (above 96 percent) of total pyrraline, whereas Free-Pyr was present in a small proportion (less than four percent) of total pyrraline. The results indicate that PED is an important extrinsic source of pyrraline, especially Pep-Pyr. As compared with CPH- and SPH-enriched drinks, WPH-enriched drinks contained high content of Pep-Pyr. The Pep-Pyr content is associated with the distribution of peptide lengths and the amino acid compositions of protein in PEDs.

Formation of Peptide Bound Pyrraline in the Maillard Model Systems with Different Lys-Containing Dipeptides and Tripeptides.

Peptide-bound advanced glycation end-products (peptide-bound AGEs) can be formed when peptides are heated with reducing saccharides. Pyrraline is the one of most commonly studied AGEs in foods, but the relative importance of the precursor peptide structure is uncertain. In the present study, model systems were prepared by heating peptides with glucose from 60 °C to 220 °C for up to 65 min, and the amounts of peptide-bound pyrraline formed were monitored to evaluate the effect of the neighboring amino acids on the peptide-bound pyrraline formation. The physico-chemical properties were introduced to explore the quantitative structure-reactivity relationships between physicochemical properties and peptide bound formation. 3-DG content in dipeptide-glucose model system was higher than that in the corresponding tripeptide-glucose model systems. Dipeptides produced higher amounts of peptide-bound pyrraline than the corresponding tripeptides. The peptide-bound pyrraline and 3-DG production were influenced by the physico-chemical properties of the side chain of amino acids adjacent to Lys in the following order: Lys-Leu/glucose > Lys-Ile/glucose > Lys-Val/ glucose > Lys-Thr/glucose > Lys-Ser/glucose > Lys-Ala/ glucose > Lys-Gly/glucose; Lys-Leu-Gly/glucose > Lys-Ile-Gly/glucose > Lys-Val-Gly/glucose > Lys-Thr-Gly/glucose > Lys-Ser-Gly/glucose > Lys-Ala-Gly/glucose > Lys-Gly-Gly/glucose. For the side chain of amino acids adjacent to Lys in dipeptides, residue volume, polarizability, molecular volume and localized electrical effect were positively related to the yield of peptide bound pyrraline, while hydrophobicity and pKb were negatively related to the yield of peptide bound pyrraline. In terms of side chain of amino acid adjacent to Lys in tripeptides, a similar result was observed, except hydrophobicity was positively related to the yield of peptide bound pyrraline.

On resin synthesis and cross-linking of collagen peptides containing the advanced glycation end-product pyrraline via Maillard condensation.

Glycation and its products cause a host of pathological conditions but their exact roles are yet to be determined. Pyrraline, a key product of glycation, and a novel pyrraline-derived cross-link have been incorporated into collagenous peptides via Maillard condensations performed on resin-bound peptide sequences.

Ovalbumin modified with pyrraline, a Maillard reaction product, shows enhanced T-cell immunogenicity.

The Maillard reaction (also referred to as "glycation") takes place between reducing sugars and compounds with free amino groups during thermal processing of foods. In the final stage of the complex reaction cascade, the so-called advanced glycation end products (AGEs) are formed, including proteins with various glycation structures. It has been suggested that some AGEs could have immunostimulatory effects. Here, we aimed to identify specific glycation structure(s) that could influence the T-cell immunogenicity and potential allergenicity of food allergens, using ovalbumin (OVA, an egg white allergen) as a model allergen. OVA was specifically modified with representative glycation structures: N(ε)-carboxymethyl lysine (CM-OVA), N(ε)-carboxyethyl lysine (CE-OVA), pyrraline (Pyr-OVA), or methylglyoxal-derived arginine derivatives (MGO-OVA). As well as AGE-OVA, a crude glycation product in thermal incubation of OVA with glucose, only Pyr-OVA, and not other modified OVAs, was efficiently taken up by bone marrow-derived murine dendritic cells (BMDCs). The uptake of Pyr-OVA was reduced in scavenger receptor class A (SR-A)-deficient BMDCs, but not in cells treated with inhibitors of scavenger receptor class B, galectin-3, or blocking antibodies against CD36, suggesting that pyrraline binds to SR-A. Compared with other modified OVAs, Pyr-OVA induced higher activation of OVA-specific CD4(+) T-cells in co-culture with BMDCs. Furthermore, compared with native OVA, AGE-OVA and Pyr-OVA induced higher IgE production in mice. Pyrraline could induce better allergen uptake by DCs via association with SR-A and subsequently enhance CD4(+) T-cell activation and IgE production. Our findings help us to understand how Maillard reaction enhances the potential allergenicity of food allergens.

Identification of patients with indolent B cell lymphoma sensitive to rituximab monotherapy.

The potential predictive value of tumor bulk, genetic, and immunological variants in patients with low-grade non-Hodgkin's lymphoma to respond to treatment with rituximab (RTX) monotherapy was evaluated. Thus, the value of assessing the effect of 18-fluoro-desoxy-D-glucose (FDG) uptake on PET scan, polymorphisms in Fc gamma receptor (FcγR) IIIa-158, FcγRIIa-131, and C1qA-276 genes in predicting the response to treatment were evaluated in 50 low-grade non-Hodgkin's lymphoma patients. The influence of RTX pharmacokinetics, plasma levels of the B cell-activating factor (BAFF), and human antichimeric antibodies was also investigated. The therapeutic response was evaluated 10 weeks after treatment using revised Cheson's criteria. Lower maximal standardized uptake values (SUV(max)) at baseline were predictive of complete response. FcγRIIIa-158 polymorphism was also associated with complete response to RTX confirming previous findings, whereas polymorphisms in the FcγRIIa-131 and C1qA-276 genes were not. Lower blood levels of RTX were observed in males, but the effectiveness of RTX in males and females was the same. BAFF was not detectable in plasma before or after treatment, and no patients developed human antichimeric antibodies. Low-grade non-Hodgkin's lymphoma patients with a low SUV(max) at baseline and an FcγRIIIa-158 V/V genotype generally had a complete response to RTX.

Synthesis and intestinal transport of the iron chelator maltosine in free and dipeptide form.

Maltosine, a 3-hydroxy-4-pyridinone derivative of lysine formed in the course of the advanced Maillard reaction, is an effective metal chelating agent. It therefore represents an interesting compound for the treatment of metal ion storage diseases. We synthesized 6-(3-hydroxy-4-oxo-2-methyl-4(1H)-pyridin-1-yl)-l-norleucine (free maltosine) and its dipeptide derivatives alanylmaltosine (Ala-Mal) and maltosinylalanine (Mal-Ala) and examined the transepithelial flux of these compounds across Caco-2 cells and their interaction with membrane transporters. Transepithelial flux of maltosine was significantly higher when added as Ala-Mal and Mal-Ala than in free form. Assays at Caco-2 cells and at HeLa cells expressing the human peptide transporter (hPEPT)1 revealed that Ala-Mal and Mal-Ala show medium to high affinity to the system. Only free but not peptide-bound maltosine inhibited the uptake of l-[(3)H]lysine in Caco-2 and OK cells. Maltosine dipeptides were transported by hPEPT1 across cell membranes and accumulated in hPEPT1-transfected HeLa cells. In electrophysiological measurements at hPEPT1-expressing Xenopus laevis oocytes, Ala-Mal and Mal-Ala induced significant inward directed currents. We conclude that Ala-Mal and Mal-Ala are transported by hPEPT1 into intestinal cells and then hydrolyzed to free maltosine and alanine. The results suggest that the oral bioavailability of maltosine can be increased significantly by applying this drug candidate in peptide-bound form.