Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells

Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells

In-Line-Test of Variability and Bit-Error-Rate of HfOx-Based Resistive Memory

Spatial and temporal variability of HfOx-based resistive random access memory (RRAM) are investigated for manufacturing and product designs. Manufacturing variability is characterized at different levels including lots, wafers, and chips. Bit-error-rate (BER) is proposed as a holistic parameter for the write cycle resistance statistics. Using the electrical in-line-test cycle data, a method is developed to derive BERs as functions of the design margin, to provide guidance for technology evaluation and product design. The proposed BER calculation can also be used in the off-line bench test and build-in-self-test (BIST) for adaptive error correction and for the other types of random access memories.Comment: 4 pages. Memory Workshop (IMW), 2015 IEEE Internationa

Decoupled CuO_2 and RuO_2 layers in superconducting and magnetically ordered RuSr_2GdCu_2O_8

Comprehensive measurements of dc and ac susceptibility, dc resistance, magnetoresistance, Hall resistivity, and microwave absorption and dispersion in fields up to 8 T have been carried out on RuSr_2GdCu_2O_8 with the aim to establish the properties of RuO_2 and CuO_2 planes. At ~130 K, where the magnetic order develops in the RuO_2 planes, one observes a change in the slope of dc resistance, change in the sign of magnetoresistance, and the appearance of an extraordinary Hall effect. These features indicate that the RuO_2 planes are conducting. A detailed analysis of the ac susceptibility and microwave data on both, ceramic and powder samples show that the penetration depth remains frequency dependent and larger than the London penetration depth even at low temperatures. We conclude that the conductivity in the RuO_2 planes remains normal even when superconducting order is developed in the CuO_2 planes below \~45 K. Thus, experimental evidence is provided in support of theoretical models which base the coexistence of superconductivity and magnetic order on decoupled CuO_2 and RuO_2 planes.Comment: 11 pages, 11 figures, submitted to PR

A common epitope on human tumor necrosis factor alpha and the autoantigen 'S-antigen/arrestin' induces TNF-alpha production

A common epitope on S-antigen (arrestin), a potent autoantigen inducing experimental autoimmune uveoretinitis (EAU), and on human tumor necrosis factor alpha (hTNF alpha) was revealed using two monoclonal antibodies to S-antigen which inhibit EAU induction. The minimal common sequence for monoclonal antibody recognition is GVxLxD in the S-antigen/hTNF alpha amino acid sequences. Peptides containing this sequence motif exhibited monocyte activating capacity similar to the autocrine stimulatory capacity of hTNF alpha itself. In the S-antigen this activity was located from residue 40 to 50, corresponding to the peptide PVDGVVLVDPE (epitope S2). In hTNF alpha, the monocyte activating capacity correlated to residue 31 to 53, corresponding to the peptide RRANALLANGVELRDNQLVVPSE (peptide RRAN). The identified regions define common functional structures in the autoantigen and in the hTNF alpha molecule. The data suggest a regulatory function of this particular structure in TNF alpha expression and in autoimmunity

Cytokine induction by immunomodulatory epitopes in S-antigen and tumor necrosis factor alpha

Common epitopes on S-antigen (arrestin), a potent autoantigen inducing experimental autoimmune uveoretinitis (EAU), and on human tumor necrosis factor alpha (hTNF alpha) are revealed with monoclonal antibodies (mAb) to S-antigen, which inhibit EAU induction. The minimal common sequence for mAb recognition is GVxLxD in the S-antigen/hTNF alpha amino acid (aa) sequences. Peptides containing this sequence motif exhibit monocyte activating capacity analogous to the autocrine stimulatory capacity of hTNF alpha itself. In S-antigen this activity is located at epitope S2 (aa residues 40 to 50), corresponding to the peptide PVDGVVLVDPE (peptide S2). In hTNF alpha the monocyte activating capacity correlates to aa residue 31 to 53, corresponding to the peptide RRANALLANGVELRDNQLVVPSE (peptide RRAN). Peptide S2 but not peptide RRAN is competing for mAbs S6H8 and S2D2 binding to S-antigen. Anti-idiotypic antibodies to S2D2 compete with peptide S2 but not peptide RRAN for binding to mAbs S2D2 and S6H8. In human retinal S-antigen epitope S2 is localized at the aa residues 44-54 and is cleaved in the human peptide 4 (aa 31-50). Competition experiments with peptide 4 (aa 31-50) and peptide 5 (aa 41-60) indicate that the C-terminal aa residues VDPD in the epitope S2 play an important role for internal image recognition of the anti-idiotypic antibodies. Peptide S2 and peptide RRAN define common functional structures in the autoantigen and hTNF alpha molecules. The data suggest regulatory functions of the peptides in cytokine expression, network regulation and in autoimmunity