Volume changes in the binding of lanthanides to peptide analogues of loop II of calmodulin

The solution expansion accompanying coordination of lanthanide ions to synthetic peptide analogues of a metal-binding loop in calmodulin was determined by a density method. This study was designed to further test the hypothesis that the nonlinear expansions observed upon sequential addition of Ca2+ to intracellular calcium-binding proteins reflect principally upon the coordination event at specific binding sequences. Three peptides of 13 residues each were synthesized as analogues of binding loop II in mammalian calmodulin: Peptide I was the native analogue; peptide II contained an aspartyl in place of an asparaginyl residue at position 5 from the N-terminus; for peptide III, the aspartyl residue in position 3 of the native analogue was interchanged with the asparaginyl residue in position 5. Thus, the number of charged-oxygen donor atoms for coordination was the same in I and in III, but the latter peptide could permit two pairs of acidic groups to converge toward the metal ion as in some loops of these proteins. The observed expansions with different lanthanide ions to the same peptide varied appreciably, suggesting dissimilar structures [Gariépy et al. (1983) Biochemistry 22, 1765-1772]; coordination to the simpler tetracarboxylate sequestrants, on the other hand, generated an expansion profile approximately as expected from the properties of the lanthanide series. The largest expansions were generated with peptide II (having the additional acidic group) for all lanthanides tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Behavioral response ofGraminella nigrifrons (Homoptera: Cicadellidae) to experimentally manipulated vibrational signals

Mate recognition for the leafhopper Graminella nigrifrons(Forbes) occurs when a male spontaneously emits a multisectional vibrational calling song to which females respond by emitting simple pulses. Significant differences were found among males in the duration, number of chirps, and chirp rate within sections of the song and the total song. Repeatability (proportion of total variation due to differences among males) of call features ranged from very low (0.04 for total chirps in song) to high (0.67 for section 3 chirp rate). However, song modification and playback experiments revealed that the variation in the measured song features was not important in determining whether a female will respond. Rather, female response depended only on the presence of two of the three types of pulses which comprise a chirp. These essential pulses were found within chirps of all call sections that contain chirps. Manipulation of chirp rates from 0.58 to 2.70 times the normal rate did not affect female response, nor did changing the period of silence between the essential pulse types from 0.25 to 1.75 times the normal period. These results suggest that components of the male calling song function in mate recognition but are not used by females to discriminate among conspecific males.     

Two new monoclonal antibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffin-embedded tissues with follicular center and mantle zone human B lymphocytes and derived tumors

Two monoclonal antibodies (LN-1, LN-2) reactive with B lymphocytes in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by utilizing cell extracts from pokeweed mitogen-stimulated peripheral blood lymphocytes and diffuse histiocytic lymphoma SU-DHL-4 cells, respectively. Both monoclonal antibodies were initially identified by indirect immunofluorescence screening techniques on paraformaldehyde-acetone-fixed cell preparations. Specificity screens with 36 well-characterized human lymphoma and leukemia cell lines showed that both LN-1 and LN-2 stained cell lines of B cell lineage but were unreactive with those of T cell or, with one exception, myeloid derivation. Null cell acute lymphoblastic leukemia cell lines were found to be LN-2+ but LN-1-. The B cell specificity of these reagents was confirmed on 15 lymphoma and 17 leukemia biopsy specimens by using indirect immunofluorescence techniques. Immunoperoxidase staining of sections from B5-fixed, paraffin-embedded human lymphoid tissues showed that LN-1 bound to the cell membrane and cytoplasm of germinal center cells whereas LN-2 stained the nuclear membrane and cytoplasm of germinal center and mantle zone B lymphocytes as well as interfollicular histiocytes and thymic medullary dendritic cells. Both monoclonal antibodies failed to stain cortical thymocytes, lymph node T cells, and peripheral blood T and myeloid cells. Immunoperoxidase staining of 20 nonlymphoid human organs and tissues revealed that LN-1 reacted positively with red blood cell precursors of the bone marrow, ciliated epithelial cells of the bronchus, distal tubular cells of the kidney, and ductal cells from several organs including the breast and prostate. In contrast, LN-2 was unreactive with all human nonlymphoid organs and tissues including the bone marrow. Indirect immunofluorescence staining of a panel of 26 solid tumor cells lines showed that LN-1 was reactive with the majority of epithelium-derived cell lines, glioblastomas, and astrocytomas but was unreactive with neuroblastomas, small cell carcinoma of the lung, and sarcomas. LN-2 was unreactive with 25 of 26 of the solid tumor cell lines by these techniques. Immunobiochemical studies have shown that LN-1 recognizes a cell surface sialoantigen whereas LN-2 is directed against a 35,000 dalton nuclear membrane protein. Because of their high specificity for B cell tumors and their ability to stain B5-fixed, paraffin-embedded tissues, LN-1 and LN-2 are useful reagents for the diagnosis and classification of the human lymphomas and leukemias.     

Purification and characterization of the adenosine A2-like binding site from human placental membrane

We have purified and characterized the adenosine A2-like binding site from human placental membranes. 5'-N-Ethylcarboxamido[2,8-3H]adenosine ([3H]NECA) binds to this site, with a Kd of 240 nM and a Bmax of 13.0 pmol/mg in human placental membranes. The adenosine A2-like binding site was purified after extraction from placental membranes with 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The purification included ammonium sulfate precipitation and concanavalin A, DEAE-Sephadex, and Sepharose 6B gel filtration chromatographies. The protein was purified 127-fold to homogeneity, with a final specific activity of 1.5-1.9 nmol/mg of protein and a 5.5-8.1% yield of binding activity from the membranes. The purified protein had similar binding properties and an identical potency order for displacement of [3H] NECA by adenosine analogs as the initial membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa which coeluted with [3H]NECA binding activity during Sepharose 6B gel filtration chromatography. In 0.1% Triton X-100, the binding complex has a Stokes radius of 70 A, a sedimentation coefficient of 6.9 S, and a partial specific volume of 0.698 ml/g. The detergent-protein complex has a calculated molecular mass of 230 kDa. The estimated frictional ratio is 1.5. The native binding complex appears to consist of a dimer of identical subunits. The function of this ubiquitous protein remains unclear.     

Molecular characterization of a major autoantibody-associated cross-reactive idiotype in Sjogren's syndrome

Primary Sjogren's syndrome is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, producing associated dry eyes (keratoconjunctivitis sicca), dry mouth, and intermittently swollen salivary glands. A high proportion of the infiltrating B lymphocytes express surface and cytoplasmic Ig bearing a kappa-L chain-associated CRI defined by reactivity with the murine mAb, 17.109. To determine the structural basis for CRI expression in this disease, we generated CRI+ lymphoblastoid cell lines and a cDNA library from lymphocytes extracted from Sjogren's syndrome patients' salivary gland biopsy specimens. Nucleic acid sequence analyses of the mRNA of one such 17.109-CRI+ lymphoblastoid cell line (NOV) reveals the expressed kappa light chain variable region gene (V kappa gene) to be homologous to Humkv325, a conserved V kappa gene used at relatively high frequency in certain B cell malignancies. In addition, synthetic oligonucleotides, corresponding to the first and third frameworks and the second complementarity determining region of the Humkv325 gene, were used to identify and isolate clones from a cDNA library generated from SS salivary gland lymphocytes. Clones annealing specifically with one or more of these oligonucleotide probes contained kappa light chain cDNA. The sequences corresponding to the variable region of two clones (Taykv320 and Taykv306) were homologous to Humkv325. The V kappa genes of four other cDNA clones (Taykv322, Taykv310, Taykv308, and Taykv312) most likely were generated somatically from the rearranged Humkv325 gene through a limited number of nucleic acid base substitutions. Our results suggest that the high frequency of 17.109-CRI expression in Sjogren's syndrome patients results from a multiclonal expansion of B cells using Humkv325, and that the expressed Humkv325 may undergo somatic diversification in an apparent Ag-driven response.