ArchaeobotanyofmoundstructuresinCampodelPucará,Catamarca, Argentina (1750–1450 b.p.): ceremonial use or rubbish dumps?

The archaeobotanical macroremains discussed in this study were recovered from six mound structures at Campo del Pucará (Andalgalá, Catamarca, northwest Argentina), a site inhabited between ca. 1750 and 1450 b.p. (a.d. 200–500). The most important identified taxon was Zea mays var. minima (maize), Acacia sp., Prosopis sp., P. nigra or P. alba, P. torquata (Leguminosae of the Mimosoideae subfamily), Phaseolus sp., P. vulgaris var. vulgaris, P. v. var. aborigineus, undetermined P. vulgaris (beans) and Cucurbita maxima (winter squash). The plant remains represent the leftovers of food, fuel and building materials. Plants were grown and gathered in the near surroundings. The material identified most probably represents waste, however it could not be unambiguously attributed either to household or ceremonial activities.     

Enzymatic mechanism controls redox-mediated protein-DNA interactions at the replication origin of kinetoplast DNA minicircles

Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.     

Expression of L-selectin and efficient binding to high endothelial venules do not modulate the dissemination potential of murine B-cell lymphoma

The homing receptor L-selectin is essential for the migration of naive lymphocytes into peripheral lymph nodes. In contrast to naive lymphocytes, activated and memory cells down-regulate L-selectin and enter peripheral lymph nodes by an L-selectin-independent mechanism. In view of the concept that lymphomas present the malignant counterparts of normal lymphocytes at a defined stage of differentiation, it has been suggested that in contrast to lymphomas with a memory/activated cell phenotype, L-selectin is essential for dissemination of lymphomas that represent naive cells. 38C-13 is a murine B-cell lymphoma with an immature naive cell phenotype. 38C-13 cells express high levels of L-selectin and bind to lymph node high endothelial venules in an L-selectin-dependent manner. In this study we demonstrate that treatment of 38C-13 tumor-bearing mice with anti-L-selectin antibodies did not inhibit tumor dissemination to peripheral lymph nodes. Moreover, L-selectin-negative 38C-13 variant cells disseminated as efficiently as wild-type cells. Thus, in spite of its expression, L-selectin is not required and does not affect the metastatic potential of the tumor. L-selectin of the malignant cells and of normal lymphocytes appears to be functionally different. Thus, whereas antibody cross-linking of L-selectin resulted in down-modulation of the receptor in normal lymphocytes, cross-linking had no effect on L-selectin expression in 38C-13 cells, suggesting that, in spite of comparable levels of surface expression in normal and malignant cells, L-selectin may be functionally impaired in some malignant cells. Received: 9 November 2000 / Accepted: 11 January 2001     

Anti-idiotype x anti-CD44 bispecific antibodies inhibit invasion of lymphoid organs by B cell lymphoma

The demonstration that Abs to adhesion molecules can block tumor metastasis suggested their use for therapy. However, such Abs affect nonmalignant cells as well. To circumvent this adverse effect, we proposed the use of bispecific Abs that bind simultaneously to an adhesion receptor and to a tumor-specific Ag. Such bifunctional Abs bind more avidly to tumor cells that coexpress both target Ags than to normal cells. The Id of the surface Ig of malignant B lymphocytes is a tumor-specific Ag. Therefore, we produced bispecific Abs with specificity to the adhesion molecule, CD44, and to an idiotypic determinant of the murine B cell lymphoma, 38C-13. These anti-Id x anti-CD44 bispecific Abs blocked 38C-13 cell adhesion to hyaluronic acid, while not affecting adhesion of Id-negative cells. In vivo studies demonstrated that the bispecific Abs inhibited lymphoma cell dissemination to the lymph nodes, bone marrow, and spleen, and prolonged survival of tumor-bearing mice. Migration of 38C-13 cells to the lymphoid organs was inhibited by the bispecific Abs. Thus, the bispecific Ab-mediated reduction in metastasis resulted, at least in part, from reduced homing to these organs. In contrast to anti-CD44 monospecific Abs, the anti-Id x anti-CD44 bispecific Abs did not affect immune responses such as delayed-type hypersensitivity. Hence, bispecific Abs against adhesion molecules and tumor-specific Ags may selectively block tumor metastasis in a way which may leave at least part of the immune system intact.     

Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes

Cytosolic phospholipase A2 (cPLA2)-generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.     

Molecular markers linked to papaya ring spot virus resistance and Fusarium race 2 resistance in melon

In melon, the Fom-1 gene confers monogenic resistance against the soil-borne fungus Fusarium oxysporum f. sp. melonis, races 0 and 2, while the closely linked Prv gene specifies resistance against the papaya ring spot virus. Markers linked to these resistance (R) genes were identified using two recombinant inbred line populations, derived from crosses between Cucumis melo Védrantais and C. melo PI 161375, and between C. melo Védrantais and C. melo PI 414723, respectively. Using bulked segregant analysis, as well as systematic scoring of the mapping populations, we developed two amplified fragment length polymorphism markers, two random amplified polymorphic DNA markers and five restriction fragment length polymorphism (RFLP) markers linked to this locus. Four of the RFLP sequences bear homology to nucleotide-binding site–leucine-rich repeat R genes, indicating the presence of a significant R-gene cluster in this locus. Our study provides the most closely linked markers published so far for these important traits. It also improves the resolution of the whole linkage group IX, which was difficult to order in our previous studies. Two of the markers were converted to cleaved amplified polymorphic sequence markers to facilitate their application in marker-assisted selection. Testing these two markers in several melon lines revealed different marker haplotypes in the melon germplasm and supported multiple, independent origin of the Fusarium races 0 and 2 resistance trait.     

Rejection of tumors of the B cell lineage by idiotype-vaccinated mice

Idiotypic determinants of immunoglobulins of malignant B lymphocytes and plasma cells are tumor-specific antigens and have been used extensively in immunotherapy studies. The mechanisms involved in resistance to tumor challenge following idiotype vaccination are poorly understood. Although a predominant role has been attributed to anti-idiotype antibodies, both humoral and cellular immune responses are probably involved. Cell-mediated responses may be particularly effective against tumor cell variants that do not express the idiotype on the cell surface and are therefore resistant to anti-idiotype antibodies but continue to produce one of the original immunoglobulin polypeptides that may be processed and presented to T cells. In this report we describe two experimental models of idiotype vaccination in which antibodies are unlikely to play a role, and hence tumor immunity is attributed to cell-mediated responses. One model consists of the murine B lymphocyte tumor 38C-13 and its idiotype-negative variant DB2, which has lost the idiotypic specificity of the parental 38C-13 cell line through the production of a different light chain but expresses the original heavy chain. Vaccination of mice with the purified IgM of 38C-13 induced resistance to 38C-13 tumor cells as well as to the variant cells. Although immunized mice produced high levels of anti-idiotype antibodies that bound to 38C-13 cells, no binding of antibodies to DB2 cells occurred. The finding that idiotype-vaccinated mice were resistant to idiotype-negative DB2 cells suggested that cellular mechanisms are involved in mediating resistance. The second model consists of the two plasma cell line JLμs and JLμm, which produce IgM with an identical specificity. Whereas one of them (JLμs) secretes the IgM, the other one(JLμm) can neither secrete nor deposit it on the cell surface. Immunization against JLμs IgM followed by tumor challenge resulted in prolonged survival of both JLμs- and JLμm-challenged mice. Although sera of immunized mice contained high levels of anti-idiotype antibodies, they did not react with the plasmacytoma cells. Similarly to the results obtained in the 38C-13 experimental model, these results suggest that a non-antibody-mediated mechanism was involved in the resistance of mice to tumor growth. Received: 11 June 1998 / Accepted: 26 November 1998     

Liposome-mediated introduction of the chloramphenicol acetyl transferase (CAT) gene and its expression in tobacco protoplasts

The expression plasmid vector pUC8CaMVCAT, containing the chloramphenicol acetyl transferase (CAT) gene, was encapsulated in large unilamellar vesicles (LUV) and introduced into tobacco protoplasts derived from either cell suspension culture or leaf mesophyll. Treatment with liposomes took place in a buffer containing either NaCl or CaCl₂, but no polyethylene glycol. The presence of polylysine in the incubation buffer increased the adsorption of liposomes to protoplasts but decreased the efficiency of CAT gene expression.The expression of the introduced CAT gene could be monitored for at least seven days, following the treatment (about 25% acetylation at day 3 as well as at day 7). Plasmid DNA sequences could be detected, apparently unmodified, for at least nine days in the plant cells, though unintegrated in the host genome.     

Phytochrome effects on K+ fluxes in guard cells of Commelina communis

The effect of phytochrome on K⁺ transport in guard cells of Commelina communis L. was studied following stomatal movement and using the K⁺−channel blockers tetraethylammonium (TEA), Cs⁺ and quinidine. TEA and quinidine prevented stomatal opening and closure in red light, but not when it was supplemented with far-red. This indicates that channels that can be blocked by TEA and quinidine are regulated by phytochrome. Evidence for a phytochrome effect on K⁺ leakage through other membranal compartments was also found. These phytochrome effects are modified by temperature. Elevated temperature decreases the involvement of channels and increases K⁺ transport through other membrane compartments, while low temperature causes channel opening and diminishes K⁺ leakage. The interaction between phytochrome effects and those of temperature is discussed.