Modelo multidimensional de los factores que afectan la interacción entre marketing y ventas en el lanzamiento de nuevos productos en Latino América

Esta tesis se centra en la identificación y análisis de los factores que pueden favorecer o actuar como barreras del éxito de la implementación de la innovación y las relaciones entre sí, desde el enfoque de la interface marketing-ventas. El trabajo empírico se enmarca en el vacío de investigación existente en el campo del proceso de lanzamiento de nuevos productos en los mercados donde operan subsidiarias de empresas multinacionales de consumo masivo (FMCG). Las empresas FMCG son altamente dependientes de la innovación como proceso clave determinante del crecimiento competitivo de mediano y largo plazo. En un contexto de acortamiento del ciclo de vida de los productos, como resultado del desarrollo tecnológico y científico que impactan en el comportamiento de los consumidores, las empresas invierten un mayor nivel de recursos en el desarrollo de nuevos productos, reingeniería y programas de innovación (Mundra, Gulati y Gupta, 2013). Sin embargo, a pesar del aumento en la inversión, las tasas de éxito de la innovación reportadas son inferiores al 25% (Evanschitzky, Eisend, Calantone y Jiang, 2012). Aumentar las tasas de éxito de los proyectos de innovación es reconocida en la literatura como un elemento clave para la supervivencia y competitividad de las empresas, para ser superiores a su competencia y desarrollar nuevos modelos de negocios. A pesar de la existencia de estudios que intentan comprender el proceso de lanzamiento de nuevos productos, no se ha identificado un claro prototipo de gestión de la innovación (Gupta et al, 2007). Profundizando en los factores de éxito, los autores Keupp, Palmié y Gassman (2012) reconocen que la innovación exitosa no depende solamente de la estrategia de selección de los proyectos de innovación, sino también la forma en que los mismos son implementados (Klein and Sorra, 1996; Repenning, 2002; Keupp, Palmié y Gassmann, 2012). Al analizar la implementación de los proyectos de lanzamiento de nuevos productos al mercado, en empresas FMCG, dicho proceso es responsabilidad principalmente de las funciones de marketing y ventas a través de la comunicación con los consumidores y los clientes respectivamente (Ernst, Hoyer y Rubsaamen, 2010). Es decir que el éxito en la implementación de la innovación requiere la gestión efectiva de la relación inter-funcional entre marketing y ventas (Ernst, Hoyer y Rubsaamen, 2010; Hughes, Le Bon y Malshe, 2012). A pesar de la importancia de la integración entre marketing y ventas en la conceptualización e implementación de la innovación, este tema no ha sido estudiado en profundidad (Hughes, Le Bon y Malshe, 2012; Keupp, Palmié y Gassmann, 2012). En las empresas multinacionales, está demostrado que el desempeño de las subsidiarias determinan el éxito competitivo de la empresa a nivel global. El desafío de dichas subsidiarias es conjugar el desarrollo global de innovación y comunicación con las características locales de comportamiento del consumidor y el mercado. Por lo tanto, esta investigación empírica responde a la pregunta académica y de gestión acerca de cómo mejorar las tasas de éxito de lanzamiento de nuevos productos al mercado en subsidiarias de empresas de consumo masivo, desde la perspectiva de la relación entre marketing y ventas. En particular analiza cómo afectan la formalización de los procesos y los mecanismos de comunicación a la confianza interpersonal y a la efectividad de la interface marketing-ventas y dichos factores a su vez sobre la planificación integrada de la implementación de la innovación. La determinación de los factores o ítems que conforman cada uno de los constructos del proceso de ejecución de la innovación, se llevó a cabo a partir de una revisión exhaustiva del estado del arte de la literatura sobre las interfaces funcionales y el proceso de innovación. Posteriormente, los ítems seleccionados (más de 50 en total) fueron validados por referentes de marketing y ventas de Argentina y Uruguay a través de entrevistas en profundidad. A partir de los factores identificados se construyeron dos modelos teóricos: • (1) relativo a la influencia de las dimensiones de confianza interpersonal sobre la efectividad de las uniones inter-funcionales y como los mecanismos organizacionales, tales como la frecuencia y la calidad de la comunicación entre las áreas, afectan la confianza y la relación entre ellas; • (2) relativo a la dimensión planificación integrada de la implementación de la innovación, ya que durante el lanzamiento de nuevos productos al mercado, marketing y ventas utilizan procesos formales que facilitan la comunicación frecuente y efectiva, desarrollando confianza inter-personal que no solamente afecta la efectividad de su relación sino también el desarrollo de planes integrados entre ambas áreas. El estudio fue llevado a cabo en una empresa multinacional de consumo masivo que integra la lista Global 500 (Fortune, 2015), presente en todo el mundo con más de 25 marcas participantes en más de 15 categorías, implementando 150 proyectos de innovación en el último año. El grupo de subsidiarias en estudio fue reconocido a nivel mundial por su desempeño en crecimiento competitivo y su alta contribución al crecimiento total. El modelo analizado en esta tesis fue expandido al resto de América Latina, tratándose entonces de un caso ejemplar que representa una práctica de excelencia en la implementación de la innovación en subsidiarias de una empresa multinacional. La recolección de los datos fue llevado a cabo a través de un cuestionario estructurado y confidencial, enviado a la base de datos de todo el universo de directores y gerentes de marketing y ventas. El nivel de respuesta fue muy elevado (70%), logrando 152 casos válidos. El análisis de datos comprendió el análisis descriptivo de los mismos, estimación de fiabilidad y análisis factorial exploratorio a través del software SPSS v.20. El análisis factorial confirmatorio y el análisis de senderos para examinar las relaciones entre los factores se estudiaron mediante el software R (Package 2.15.1., R Core Team, 2012) (Fox, 2006). Finalmente se llevaron a cabo entrevistas en profundidad a gerentes de marketing y ventas de cada uno de los seis países con el fin de profundizar en los constructos y sus relaciones. Los resultados de los modelos demuestran que la frecuencia de comunicación impacta positivamente en la calidad de la misma, que a su vez afecta directamente la relación entre marketing y ventas. Adicionalmente, la calidad de la comunicación impacta sobre la confianza cognitiva, que a su vez se relaciona no solamente con la confianza afectiva sino también con la relación entre ambas áreas. Esto significa que para mejorar la implementación de la innovación, los gerentes deberían orientarse a reforzar la relación entre marketing y ventas facilitando la construcción de confianza interpersonal primero cognitiva y luego afectiva, incrementando la frecuencia de la comunicación que alimenta la calidad de la comunicación entre ambas áreas. A través del segundo modelo se demuestra que durante el lanzamiento de nuevos productos al mercado, marketing y ventas necesitan emplear procesos formales que faciliten la comunicación frecuente y efectiva. De esta forma se contrarresta el efecto negativo de la formalización sobre la planificación integrada entre ambas áreas. Adicionalmente, los gerentes de ambos departamentos deberían promover la construcción de confianza interpersonal, no solamente para mejorar la efectividad de la relación, sino también para desarrollar planes integrados de implementación de nuevos productos. Finalmente, se valida que la frecuencia de la comunicación, la confianza afectiva y la relación marketing-ventas, se relacionan positivamente con la planificación integrada en la implementación de la innovación. El estudio contribuye a la comprensión de los factores que las empresas pueden emplear para mejorar la relación inter-funcional entre marketing y ventas y la implementación de la innovación en empresas de consumo masivo. El aporte de esta investigación puede ser valorado de dos maneras, los aportes a la gestión y a la academia. Desde el punto de vista empresarial, provee a los líderes al frente de empresas de consumo masivo, del conocimiento sobre los factores que afectan la implementación de la innovación y en definitiva el éxito del negocio a mediano y largo plazo. Desde el punto de vista académico aporta al conocimiento del proceso de implementación de la innovación y en la efectividad de la interface marketing y ventas en un caso de buenas prácticas en el mercado de consumo masivo. A su vez incorpora por primera vez un estudio empírico en geografías emergentes capaces de recuperar el camino de crecimiento posterior a una profunda crisis económica a través de la exitosa implementación de la innovación en sus mercados. ABSTRACT This thesis is focused on the identification, analysis and relationship study of factors which may benefit or hinder the successful deployment of innovation, from a marketing-sales interface perspective. Considering the non-existent investigation dedicated to the study of new products launches into markets in which Fast Moving Consumer Goods (FMCG) companies’ subsidiaries operate, it is that this investigation has been carried out. FMCG companies rely on innovation as their key process for a competitive growth on a medium and long term basis. Nowadays, the life-cycle of products is getting shorter as a result of new technological and scientific development, having impact on consumer behavior, and therefore companies are forced to dedicating more resources to the development of new products, reengineering and innovation programs (Mundra, Gulati and Gupta, 2013). However, in spite of the investment increase, the innovation success rates have been reported to be lower than 25% (Evanschitzky, Eisend, Calantone y Jiang, 2012). Increasing success rates on innovation processes has been considered as a key element on the survival and competitiveness of companies, outperforming competitors and developing new business models. Despite new studies which try to comprehend the process of new products launch, a prototype of innovation management has not yet been identified (Gupta et al, 2007). Emphasizing on success factors, authors Keupp, Palmié and Gassman (2012) recognize that successful innovation does not solely depend on innovation processes’ selection strategy, but it is also based on the way in which these are implemented (Klein and Sorra, 1996; Repenning, 2002; Keupp, Palmié y Gassmann, 2012). While analyzing the implementation of projects for new products releases on massive consumption companies, the two departments in charge of taking this forward are marketing and sales, by focusing on communication strategies with consumers and clients respectively (Ernst, Hoyer y Rubsaamen, 2010). This means that having success on innovation implementation requires an effective management of inter-functional relationship among marketing and sales (Ernst, Hoyer y Rubsaamen, 2010; Hughes, Le Bon y Malshe, 2012). In spite of the importance on the integration between marketing and sales on the conceptualization and implementation of innovation, this subject has not been studied in depth (Hughes, Le Bon y Malshe, 2012; Keupp, Palmié y Gassmann, 2012). In multinational companies, previous research has confirmed that the performance of their subsidiaries determine the competitive success of the company on a global scale. The challenge of said subsidiaries is to conjugate the global innovation development and communication with the local consumer and market behavior. Therefore, this empirical study aims to respond to the academic and management question of how to improve the success rates of new product launches into MNE subsidiary’ markets, from a marketing-sales relationship perspective. Particularly, this investigation analyses how the formalization of products and communication mechanisms affect interpersonal trust and marketing-sales interface effectiveness and also on how these factors affect the overall planning of the implementation of innovation. The determination of which factors build the hypothesis of the innovation execution process was taken forward through an extensive research on the extant literature on functional interfaces and innovation processes. More than 50 items were selected which were in turn validated by marketing and sales referents on Uruguay and Argentina through in depth interviews. Based on the identified factors, two theoretical models were proposed: (1) Relative to the influence that interpersonal trust dimensions have on inter functional linkages effectiveness and how organizational mechanisms such as frequency and quality of communication between departments affect trust and their relationship. (2) Relative to the integrated planning and innovation implementation dimensions. Marketing and sales department use formal process thus allowing inter-personal trust, which affects positively their relationship and also enables the development of integrated planning between them. The study was performed within a massive consumption company which is part of the “Global 500” (Fortune, 2015), with subsidiaries all over the world and more than 25 participant brands in 15 categories, having implemented over 150 innovation projects in the year under study. The analyzed subsidiary group has been awarded worldwide for their performance in competitive growth and their high contribution to the total growth. The model being analyzed in this thesis was implemented throughout Latin America, representing a remarkable case of innovation implementation excellence for subsidiaries of multinational companies. Data recollection was carried out through a structured and confidential questionnaire, sent to the universe of marketing-sales directors and managers’ database available with a high level of responsiveness of 70%, resulting in 152 valid cases. Data exploration involved a descriptive analysis, followed by a reliability estimation and an exploratory factorial analysis carried out through SPSS v.20. Confirmatory factorial analysis and path analysis (to examine relations between the different study factors) were studied using “R” software (Package 2.15.1., R Core Team, 2012) (Fox, 2006). Finally, in depth interviews were carried out to several marketing and sales managers in each of the six countries so as to further confirm the hypothesis and their relations. The models results prove that communication frequency has a positive impact on the quality of the same, which in turn has direct effects on the marketing-sales relations. Additionally, communication quality has an impact on the cognitive trust, which also relates not only to affective trust, but also to the relation between both areas. This means that in order to improve the implementation of innovation, managers should strive to enforce marketing-sales relations, facilitating the interpersonal trust construction (firstly cognitive, followed by affective trust), increasing the communication frequency, and therefore nurturing the communication quality among both areas. Through the second model, the results confirm the importance of creating effective relationships between sales and marketing to facilitate planning integrated new product implementations. While formalized new product development processes provide opportunities for sales and marketing to communicate, this does not directly influence the planning of integrated new product implementations. By using these formal opportunities to communicate to create information quality, it is possible to improve sales and marketing’s ability to integrate information during the planning process. Further, communication quality creates inter-personal trust in the other party’s competences (cognitive-based trust), leading to affect-based trust. Affect-based inter-personal trust, not only to improve the overall effectiveness of the sales and marketing relationship, but also helps in planning integrated new product implementations. This study contributes to the understanding of factors which enterprises can use to improve the inter-functional relations between marketing and sales, and the implementation of innovation in FMCG companies. The contribution of this investigation can be measured in two ways: enrichment of management and contribution to the academic area. From a business perspective, it provides massive consumption businesses leaders with knowledge on which factors affect innovation implementation, which results on mid and long-term success for the company. From an academic point of view, it provides knowledge on a prototype of successful innovation implementation management based on the marketing-sales interface effectiveness through a case study in the FMCG consumption market. Last but not least, it incorporates for the first time an empiric study on emerging geographies capable of recovery post a deep economic crisis through successful innovation implementation on their markets.

A cobalt complex that selectively disrupts the structure and function of zinc fingers

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.

GPIIIa-(49–66) is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa of HIV-1-related immunologic thrombocytopenia

High-affinity (Kd = 1 × 10−9 M) anti-platelet GPIIIa has been isolated from serum immune complexes of immunologic thrombocytopenic HIV-1-infected patients (HIV-1-ITP). Affinity-purified anti-platelet antibody reacted with a recombinant GPIIIa-(1–200) and -(1–66) fusion peptide and with an 18-mer GPIIIa-(49–66) peptide but not with seven other GPIIIa peptides spanning the length of GPIIIa. Most of the anti-platelet antibody (≈85%) could be adsorbed to and eluted from a GPIIIa-(49–66) affinity column. Binding of antibody to platelets could be inhibited by GPIIIa-(49–66) or an equimolar peptide-albumin conjugate (IC50 = 2 μM). Sera from 7 control subjects and 10 classic autoimmune thrombocytopenic patients gave background reactivity with GPIIIa-(49–66). HIV-1-ITP sera from 16 patients reacted with a mean OD 6-fold greater than background (range, 4- to 9-fold). Serum anti-GPIIIa-(49–66) concentration correlated inversely with platelet count, R2 = 0.51, n = 31, P < 0.0001. Because mouse platelet GPIIIa-(49–66) has 83% homology with human GPIIIa and mouse monocytes contain Fc receptors for the human IgG1-κ/λ antibody, we determined the in vivo effect of human anti-GPIIIa on mouse platelets. Affinity-purified antibody, 25–50 μg given i.p., resulted in a precipitous drop in platelet count to 30% of baseline, with nadir at 4 hr and return to normal in 36 hr. No effect was noted with control IgG. Acute thrombocytopenia could be prevented or reversed by the injection of the GPIIIa-(49–66) albumin conjugate at zero time or 2 hr after antibody, respectively, but not with a scrambled peptide-albumin conjugate. Thus HIV-1-ITP patients have high-affinity anti-platelet GPIIIa against a major antigenic determinant, GPIIIa-(49–66), which correlates inversely with platelet count and induces thrombocytopenia in mice.

Inactivation of the α C protein antigen gene, bca, by a novel shuttle/suicide vector results in attenuation of virulence and immunity in group B Streptococcus

The α C protein of group B Streptococcus (GBS) is a major surface-associated antigen. Although its role in the biology and virulence of GBS has not been defined, it is opsonic and capable of eliciting protective immunity. The α C protein is widely distributed among clinical isolates and is a potential protein carrier and antigen in conjugate vaccines to prevent GBS infections. The structural gene for the α C protein, bca, has been cloned and sequenced. The protein encoded by bca is related to a class of surface-associated proteins of Gram-positive cocci involved in virulence and immunity. To investigate the potential roles of the α C protein, bca null mutants were generated in which the bca gene was replaced with a kanamycin resistance cassette via homologous recombination using a novel shuttle/suicide vector. Studies of lethality in neonatal mice showed that the virulence of the bca null mutants was attenuated 5- to 7-fold when compared with the isogenic wild-type strain A909. Significant differences in mortality occurred in the first 24 h, suggesting that the role of the α antigen is important in the initial stages of the infection. In contrast to A909, bca mutants were no longer killed by polymorphonuclear leukocytes in the presence of α-specific antibodies in an in vitro opsonophagocytic assay. In contrast to previous studies, α antigen expression does not appear to play a role in resistance to opsonophagocytosis in the absence of α-specific antibodies. In addition, antibodies to the α C protein did not passively protect neonatal mice from lethal challenge with bca mutants, suggesting that these epitopes are uniquely present within the α antigen as expressed from the bca gene. Therefore, the α C protein is important in the pathogenesis of GBS infection and is a target for protective immunity in the development of GBS vaccines.

A cytosolic activity distinct from Crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells

The leucine-rich nuclear export signal (NES) is used by a variety of proteins to facilitate their delivery from the nucleus to the cytoplasm. One of the best-studied examples, protein kinase inhibitor (PKI), binds to the catalytic subunit of protein kinase A in the nucleus and mediates its rapid export to the cytoplasm. We developed a permeabilized cell assay that reconstitutes nuclear export mediated by PKI, and we used it to characterize the cytosolic factors required for this process. The two-step assay involves an import phase and an export phase, and quantitation is achieved by digital fluorescence microscopy. During the import phase, a fluorescent derivative of streptavidin is imported into the nuclei of digitonin-permeabilized HeLa cells. During the export phase, biotinylated PKI diffuses into the nucleus, binds to fluorescent streptavidin, and mediates export of the complex to the cytoplasm. Nuclear export of the PKI complex is cytosol dependent and can be stimulated by addition of the purified NES receptor, Crm1. HeLa cell cytosol treated with N-ethylmaleimide (NEM) or phenyl-Sepharose to inactivate or deplete Crm1, respectively, is still fully active in the PKI export assay. Significantly, the export activity can be depleted from cytosol by preadsorption with a protein conjugate that contains a functional NES. These data indicate that cytosol contains an export activity that is distinct from Crm1 and is likely to correspond to an NES receptor.

The Arabidopsis cullin AtCUL1 is modified by the ubiquitin-related protein RUB1

The ubiquitin-like protein RUB1 is conjugated to target proteins by a mechanism similar to that of ubiquitin conjugation. Genetic studies in Arabidopsis thaliana have implicated the RUB-conjugation pathway in auxin response. The first step in the pathway is RUB activation by a bipartite enzyme composed of the AXR1 and ECR1 proteins. Ubiquitin activation is an ATP-dependent process that involves the formation of an AMP-ubiquitin intermediate. Here we show that RUB activation by AXR1-ECR1 also involves formation of an AMP-RUB intermediate and that this reaction is catalyzed by the ECR1 subunit alone. In addition, we identified an Arabidopsis protein called RCE1 that is a likely RUB-conjugating enzyme. RCE1 works together with AXR1-ECR1 to promote formation of a stable RUB conjugate with the Arabidopsis cullin AtCUL1 in vitro. Using a tagged version of RUB1, we show that this modification occurs in vivo. Because AtCUL1 is a component of the ubiquitin protein ligase SCFTIR1, a complex that also functions in auxin response, we propose that RUB modification of AtCUL1 is important for auxin response.

Inhibition of mRNA export in vertebrate cells by nuclear export signal conjugates

Leucine-rich nuclear export signals (NESs) are recognized by the NES receptor exportin 1 and are central to the export of multiple shuttling proteins and RNAs. The export of messenger RNA in vertebrates was, however, thought to occur by a different pathway, because inhibition by injection of a synthetic Rev NES conjugate could not be demonstrated. Here we find that peptide conjugates composed of the NES of either protein kinase A inhibitor protein (PKI) or the HIV-1 Rev protein, when coupled to human serum albumin, are potent inhibitors of mRNA and small nuclear RNA export. These results provide direct evidence that mRNA export in vertebrates depends on interactions between an NES and its cognate NES receptors. PKI NES conjugates are significantly more efficient at inhibiting RNA export than are REV NES conjugates, indicating that different NESs may have different abilities to promote protein and RNA export. Surprisingly, an expected control conjugate containing the mutant Rev NES sequence M10 strongly inhibited the export of intronless dihydrofolate reductase mRNA. Nuclear injection of NES peptide conjugates led to mislocalization to the nucleus of 10–20% of the cytoplasmic Ran GTPase-binding protein (RanBP1) indicating that RanBP1 shuttles between the nucleus and the cytoplasm via an NES pathway. These results demonstrate that in vertebrates the export of mRNA, like that of small nuclear RNA, 5S rRNA, and transport factors such as RanBP1, employs NES-mediated molecular machinery.

Glucose-induced Autophagy of Peroxisomes in Pichia pastoris Requires a Unique E1-like Protein

Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Δ had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(ΔATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25–30 kDa protein. This conjugate was not observed in cells expressing Gsa7(ΔATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.

Apg7p/Cvt2p: A Novel Protein-activating Enzyme Essential for Autophagy

In the yeast Saccharomyces cerevisiae, the Apg12p–Apg5p conjugating system is essential for autophagy. Apg7p is required for the conjugation reaction, because Apg12p is unable to form a conjugate with Apg5p in the apg7/cvt2 mutant. Apg7p shows a significant similarity to a ubiquitin-activating enzyme, Uba1p. In this article, we investigated the function of Apg7p as an Apg12p-activating enzyme. Hemagglutinin-tagged Apg12p was coimmunoprecipitated with c-myc–tagged Apg7p. A two-hybrid experiment confirmed the interaction. The coimmunoprecipitation was sensitive to a thiol-reducing reagent. Furthermore, a thioester conjugate of Apg7p was detected in a lysate of cells overexpressing both Apg7p and Apg12p. These results indicated that Apg12p interacts with Apg7p via a thioester bond. Mutational analyses of Apg7p suggested that Cys507 of Apg7p is an active site cysteine and that both the ATP-binding domain and the cysteine residue are essential for the conjugation of Apg7p with Apg12p to form the Apg12p–Apg5p conjugate. Cells expressing mutant Apg7ps, Apg7pG333A, or Apg7pC507A showed defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These results indicated that Apg7p functions as a novel protein-activating enzyme necessary for Apg12p–Apg5p conjugation.

Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity

A covalent conjugate (NR-LU-10/SA) was prepared between streptavidin (SA) and NR-LU-10, a mAb that binds an antigen expressed on the surface of most human carcinomas. NR-LU-10/SA was injected into nude mice bearing human tumor xenografts. Injection of biotinylated galactosyl-human serum albumin reduced the circulating levels of conjugate by 95%. Subsequent administration of 90Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin achieved peak uptake at the tumor within 2 hr while >80% of the radioactivity was eliminated in the urine. A single dose of 600–800 μCi of 90Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin produced cures in 10/10 mice with established (>200 mm3) s.c. human small cell lung or colon cancer xenografts and 8/10 cures in mice with human breast cancer xenografts without significant toxicity.

Receptors for oxidized low-density lipoprotein on elicited mouse peritoneal macrophages can recognize both the modified lipid moieties and the modified protein moieties: Implications with respect to macrophage recognition of apoptotic cells

It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4°C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognize both the lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and/or covalently linked to the apoB of OxLDL, likely play a role in that recognition.

A bait and switch hapten strategy generates catalytic antibodies for phosphodiester hydrolysis

General base catalysis supplied by the histidine-12 (H-12) residue of ribonuclease (RNase) A has long been appreciated as a major component of the catalytic power of the enzyme. In an attempt to harness the catalytic power of a general base into antibody catalysis of phosphodiester bond hydrolysis, the quaternary ammonium phosphate 1 was used as a bait and switch hapten. Based on precedence, it was rationalized that this positively charged hapten could induce a counter-charged residue in the antibody binding site at a locus suitable for it to deprotonate the 2′-hydroxyl group of the anhydroribitol phosphodiester substrate 2. After murine immunization with hapten 1, mAb production yielded a library of 35 antibodies that bound to a BSA-1 conjugate. From this panel, two were found to catalyze the cyclization-cleavage of phosphodiester 2. Kinetic studies at pH 7.49 (Hepes, 20 mM) and 25°C showed that the most active antibody, MATT.F-1, obeyed classical Michaelis–Menten kinetics with a Km = 104 μM, a kcat = 0.44 min−1, and a kcat/kuncat = 1.7 × 103. Hapten 1 stoichiometrically inhibits the catalytic activity of the antibody. MATT.F-1 is the most proficient antibody–catalyst (1.6 × 107 M−1) yet generated for the function of phosphodiester hydrolysis and emphasizes the utility of the bait and switch hapten paradigm when generating antibody catalysts for processes for which general-base catalysis can be exploited.

Protein-free parallel triple-stranded DNA complex formation

A 14 nt DNA sequence 5′-AGAATGTGGCAAAG-3′ from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar–phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5′-end of the probe strand as donor and BODIPY-Texas Red on the 3′-amino group of either strand of the target duplex as acceptor. There was full protection from OsO4-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe–intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

Toward optimized carbohydrate-based anticancer vaccines: Epitope clustering, carrier structure, and adjuvant all influence antibody responses to Lewisy conjugates in mice

The feasibility of using carbohydrate-based vaccines for the immunotherapy of cancer is being actively explored at the present time. Although a number of clinical trials have already been conducted with glycoconjugate vaccines, the optimal design and composition of the vaccines has yet to be determined. Among the candidate antigens being examined is Lewisy (Ley), a blood group-related antigen that is overexpressed on the majority of human carcinomas. Using Ley as a model for specificity, we have examined the role of epitope clustering, carrier structure, and adjuvant on the immunogenicity of Ley conjugates in mice. A glycolipopeptide containing a cluster of three contiguous Ley-serine epitopes and the Pam3Cys immunostimulating moiety was found to be superior to a similar construct containing only one Ley-serine epitope in eliciting antitumor cell antibodies. Because only IgM antibodies were produced by this vaccine, the effect on immunogenicity of coupling the glycopeptide to keyhole limpet hemocyanin was examined; although both IgM and IgG antibodies were formed, the antibodies reacted only with the immunizing structure. Reexamination of the clustered Ley-serine Pam3Cys conjugate with the adjuvant QS-21 resulted in the identification of both IgG and IgM antibodies reacting with tumor cells, thus demonstrating the feasibility of an entirely synthetic carbohydrate-based anticancer vaccine in an animal model.

Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1

Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional odontoblast-like cells. Functional differentiation of odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of odontoblast-specific genes, indicating the involvement of other unidentified odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.