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The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.

Background: Time-resolved fluorescence immunoassays (TR-FIAs) for phytoestrogens in biological samples are an alternative to mass spectrometric methods. These immunoassays were used to test urine and plasma samples from individuals in a dietary intervention trial aimed at determining the efficacy of dietary isoflavones in reducing the risk of coronary heart disease in postmenopausal women. Methods: We established murine monoclonal TR-FIA methods for daidzein, genistein, and equol. These assays could be performed manually or adapted to an automated analyzer for high throughput and increased accuracy. Analysis of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genistein and equol required diethyl-ether extraction. Results: Comparison of monoclonal TR-FIA, commercial polyclonal antibody–based TR-FIA, and gas chromatography–mass spectrometry showed correlations (r, 0.911–0.994) across the concentration range observed in the Isoheart study (50 mg/day isoflavones). The concentrations of urinary daidzein and genistein observed during intervention demonstrated good compliance, and a corresponding increase in serum daidzein and genistein confirmed bioavailability of the isoflavone-rich foods; 33 of the 117 volunteers (28.2%) were classified as equol producers on the basis of their urinary equol concentration (>936 nmol/L), and significant differences in the numbers of equol producers were observed between Berlin and the 3 other European cohorts studied. Conclusions: The validated monoclonal TR-FIA methods are applicable for use in large-scale human phytoestrogen intervention studies and can be used to monitor compliance, demonstrate bioavailability, and assess equol producer status.

The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.

This paper describes the application of two novel screening technologies, i.e. Domain Scan (24- and 30-mer peptides) and Matrix Scan (24-mer peptides) technology, in the mapping of a discontinuous epitope on FSH-beta for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan technology (12-mer peptides), showed selective binding to peptide-constructs corresponding to the beta3-loop of FSH in the Domain and/or Matrix Scan. Systematic replacement analysis studies with peptide-construct 57VYETVRVPGCAC-SAc-ADSLYTYPVATQ81 revealed that for most mAb's the amino acids R62, A70, D71, and L73 form the core of the epitope. A Domain Scan performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three beta1-beta3 peptide-constructs covering the residues 60TVRVPGCAHHADSLY74 in combination with 10IAIEKEECRFAI21, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues 97RGLGPSYCSFGEMKE114 in combination with the residues 10IAIEKEECRFAI21. A Matrix Scan of mAb 17 showed that peptides from four different regions on FSH (1st strand beta3-loop, alpha 1-loop, long alpha2-loop, det. loop) showed enhanced binding in combination with several 70ADSL73-containing peptides. BIACORE measurements with mAb's 1, 2, 13, and 17 using a set of 21 different peptide(-construct)s partially confirmed the Domain and Matrix Scan screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the beta3-loop showed measurable binding. Cyclic beta3-loop peptide mimics were found to bind significantly stronger (Kd approximately 5 microM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair beta1-peptide 1oIAIEKEECRFAI21 to the linear beta3-peptide *52TFKELVYETVRVPGCAHHADSLYTYPVATQAH83# via disulfide bond formation showed a 2-3 fold increase in Kd, thus conforming participation of the beta 1-loop in antibody binding for these mAb's.

Issue Title: Peptide Libraries and Peptide Drugs This paper describes the application of two novel screening technologies, i.e. Domain Scan(TM) (24- and 30-mer peptides) and Matrix Scan(TM) (24-mer peptides)technology, in the mapping of a discontinuous epitope on FSH-β for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan(TM) technology (12-merpeptides), showed selective binding to peptide-constructs corresponding to the β3-loop of FSH in the Domain(TM) and/or Matrix Scan(TM). Systematic replacement analysis studies with peptide-construct ^sub 57^VYETVRVPGCAC-SAc-ADSLYTYPVATQ^sub 81^ revealed that for most mAb's the amino acids R^sub 62^, A^sub 70^,D^sub 71^, and L^sub 73^ form the core of the epitope. A DomainScan(TM) performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three β1-β3 peptide-constructs covering the residues ^sub 60^TVRVPGCAHHADSLY^sub 74^ in combination with ^sub 10^IAIEKEECRFAI^sub 21^, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues^sub 97^RGLGPSYCSFGEMKE^sub 114^ in combination with the residues ^sub 10^IAIEKEECRFAI^sub 21^. A Matrix Scan(TM) of mAb 17 showed that peptides from four different regions on FSH (1st strand β3-loop, α1-loop, longα2-loop, det. loop) showed enhanced binding in combination with several ^sub 70^ADSL^sub 73^-containing peptides. BIACORE measurements with mAb's 1, 2, 13, and 17 using a set of 21 different peptide(-construct)s partially confirmed the Domain and MatrixScan(TM) screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the β3-loop showed measurable binding. Cyclic β3-loop peptide mimics were found to bind significantly stronger (K^sub d^ 5 μM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair β1-peptide ^sub 10^IAIEKEECRFAI^sub 21^to the linear β3-peptide*^sub 52^TFKELVYETVRVPGCAHHADSLYTYPVATQAH^sub 83^# via disulfide bond formation showed a 2-3 fold increase in K^sub d^, thus conforming participation of the β 1-loop in antibody binding for these mAb's.[PUBLICATION ABSTRACT]