Agouti signaling protein stimulates cell division in "viable yellow" (A(vy)/a) mouse liver.

Enhanced linear growth, hyperplasia, and tumorigenesis are well-known characteristics of "viable yellow" agouti A(vy)/- mice (Wolff GL, Roberts DW, Mountjoy KG. Physiol Genomics 1:151-163, 1999); however, the functional basis for this aspect of the phenotype is unknown. In the present study, we ascertained whether agouti signaling protein (ASIP) levels in A(vy)/a or a/a livers are associated with hepatocyte proliferation as a possible factor in promotion of hepatocellular tumor formation. Proliferating cell nuclear antigen (PCNA) assays and quantitative real-time reverse transcriptase polymerase chain reaction assays were performed on liver samples from mottled yellow A(vy)/a, pseudoagouti A(vy)/a, and black a/a VY mice to determine mitotic indices and expression levels of A(vy )and a in relation to the expression level of the housekeeping gene hprt. We found that ASIP levels were approximately 100-fold higher in yellow than in pseudoagouti or black mice and that the proportion of PCNA-positive hepatocytes was greater (P < 0.001) in yellow than in pseudoagouti or black mice.

Chondroitin sulfate glycosaminoglycans as major P-selectin ligands on metastatic breast cancer cell lines.

The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLe(x)). SLe(x) oligosaccharide on tumor cells can be recognized by E- and P-selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E-selectin reactivity, which was sLe(x) dependent, P-selectin reactivity with this cell line was sLe(x)-independent. The sLe(x)-Neg variant of the 4T1 cell line with markedly diminished expression of sLe(x) and lack of sLe(a), provided a unique opportunity to characterize P-selectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E-selectin binding. We observed that P-selectin binding was Ca(2+)-independent and sulfation-dependent. We found that P-selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P-selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P-selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P-selectin-reactive ligands on the surface of human MDA-MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease.

Allergenic characteristics of a modified peanut allergen.

Attempts to treat peanut allergy using traditional methods of allergen desensitization are accompanied by a high risk of anaphylaxis. The aim of this study was to determine if modifications to the IgE-binding epitopes of a major peanut allergen would result in a safer immunotherapeutic agent for the treatment of peanut-allergic patients. IgE-binding epitopes on the Ara h 2 allergen were modified, and modified Ara h 2 (mAra h 2) protein was produced. Wild-type (wAra h 2) and mAra h 2 proteins were analyzed for their ability to interact with T-cells, their ability to bind IgE, and their ability to release mediators from a passively sensitized RBL-2H3 cell line. Multiple T-cell epitopes were identified on the major peanut allergen, Ara h 2. Ara h 2 amino acid regions 11-35, 86-125, and 121-155 contained the majority of peptides that interact with T-cells from most patients. The wAra h 2 and mAra h 2 proteins stimulated proliferation of T-cells from peanut-allergic patients to similar levels. In contrast, the mAra h 2 protein exhibited greatly reduced IgE-binding capacity compared to the wild-type allergen. In addition, the modified allergen released significantly lower amounts of beta-hexosaminidase, a marker for IgE-mediated RBL-2H3 degranulation, compared to the wild-type allergen.

Synthesis of a rabbit polyclonal antibody to the human sodium-dependent multivitamin transporter.

In mammalian cells, biotin is covalently attached to carboxylases and histones and is required for cell proliferation and function. Cellular uptake of biotin (as well as pantothenic acid and lipoic acid) is mediated by the sodium-dependent multivitamin transporter, SMVT. Studies of cellular biotin homeostasis have been hampered by the lack of an antibody to SMVT. Here, we describe the synthesis of a rabbit polyclonal antibody to human SMVT. Using this antibody, SMVT has been identified in human peripheral blood mononuclear cells, Caco-2 cells, and HepG2 cells. Moreover, we observed that cells respond to proliferation with increased synthesis of SMVT.

Exposure to UV light causes increased biotinylation of histones in Jurkat cells.

Biotin in breakdown products of biotinylated carboxylases serves as substrate for biotinylation of histones by biotinidase. Here we determined whether biotinylation of histones might play a role in repair of damaged DNA and in apoptosis. Jurkat cells were exposed to UV light to induce DNA damage. Abundance of thymine dimers increased about three times in response to UV exposure, consistent with DNA damage. Biotin-containing carboxylases were degraded in response to UV exposure, as judged by Western blot analysis and carboxylase activities. Mitochondrial integrity decreased in response to UV exposure (as judged by confocal microscopy), facilitating the release of breakdown products of carboxylases from mitochondria. Biotinylation of histones increased in response to UV exposure; biotinylation of histones did not occur specifically at sites of newly repaired DNA. UV exposure triggered apoptosis, as judged by caspase-3 activity and analysis by confocal microscopy. In summary, this study provided evidence that increased biotinylation of histones in DNA-damaged cells might either be a side product of carboxylase degradation or a step during apoptosis.

Biotin uptake into human peripheral blood mononuclear cells increases early in the cell cycle, increasing carboxylase activities.

Cells respond to proliferation with increased accumulation of biotin, suggesting that proliferation enhances biotin demand. Here we determined whether peripheral blood mononuclear cells (PBMC) increase biotin uptake at specific phases of the cell cycle, and whether biotin is utilized to increase biotinylation of carboxylases. Biotin uptake was quantified in human PBMC that were arrested chemically at specific phases of the cell cycle, i.e., biotin uptake increased in the G1 phase of the cycle [658 +/- 574 amol biotin/(10(6) cells x 30 min)] and remained increased during phases S, G2, and M compared with quiescent controls [200 +/- 62 amol biotin/(10(6) cells x 30 min)]. The abundance of the sodium-dependent multivitamin transporter (SMVT, which transports biotin) was similar at all phases of the cell cycle, suggesting that transporters other than SMVT or splicing variants of SMVT may account for the increased biotin uptake observed in proliferating cells. Activities of biotin-dependent 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase were up to two times greater in proliferating PBMC compared with controls. The abundance of mRNA encoding 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase paralleled carboxylase activities, suggesting that PBMC respond to proliferation with increased expression of genes encoding carboxylases. Similarly, expression of the gene encoding holocarboxylase synthetase (which catalyzes binding of biotin to carboxylases) increased in response to proliferation, suggesting that cellular capacity to biotinylate carboxylases was increased. In summary, these findings suggest that PBMC respond to proliferation with increased biotin uptake early in the cell cycle, and that biotin is utilized to increase activities of two of the four biotin-requiring carboxylases.

Biotinidase catalyzes debiotinylation of histones.

BACKGROUND: Posttranslational modifications of histones play important roles in processes such as regulation of gene expression and DNA repair. Recently, evidence has been provided that histones in human cells are modified by covalent attachment of biotin. AIM OF THE STUDY To determine whether the reverse process (debiotinylation of histones) occurs in biological samples and whether debiotinylation is an enzyme-mediated process; and to characterize the enzyme that mediates debiotinylation of histones. METHODS: Plasma and lymphocytes from healthy adults and a biotinidase-deficient patient were used as sources of debiotinylating enzymes. Debiotinylation of histones by plasma and lymphocyte proteins was measured using a colorimetric 96-well plate assay. RESULTS: Histones were debiotinylated rapidly if incubated with human plasma or lysates of lymphocytes. The following observations are consistent with the hypothesis that debiotinylation is an enzyme-mediated process: (i) Hydrolysis was slower at 4 degrees C compared to 37 degrees C; (ii) debiotinylating activity was destroyed when biological samples were heated at 90 degrees C for 30 min preceding incubation with biotinylated histones; and (iii) rates of debiotinylation were pH dependent. Rates of histone debiotinylation were significantly decreased in biotinidase-deficient samples. CONCLUSION: Debiotinylation of histones in human samples is an enzyme-mediated process that is at least partly catalyzed by biotinidase.