Colostrogenesis: IgG1 Transcytosis Mechanisms

Biological transport of intact proteins across epithelial cells has been documented for many absorptive and secretory tissues. Immunoglobulins were some of the earliest studied proteins in this category. The transcellular transport (transcytosis) of immunoglobulins in neonatal health and development has been recognized; the process is especially significant with ungulates because they do not transcytose immunoglobulins across the placenta to the neonate. Rather, they depend upon mammary secretion of colostrum and intestinal absorption of immunoglobulins in order to provide intestinal and systemic defense until the young ungulate develops its own humoral defense mechanisms. The neonatal dairy calf's ability to absorb immunoglobulins from colostrum is assisted by a ~24 h "open gut" phenomenon where large proteins pass the intestinal epithelial cells and enter the systemic system. However, a critical problem recognized for newborn dairy calves is that an optimum mass of colostrum Immunoglobulin G (IgG) needs to be absorbed within that 24 h window in order to provide maximal resistance to disease. Many calves do not achieve the optimum because of poor quality colostrum. While many studies have focused on calf absorption, the principal cause of the problem resides with the extreme variation (g to kg) in the mammary gland's capacity to transfer blood IgG1 into colostrum. Colostrum is a unique mammary secretory product that is formed during late pregnancy when mammary cells are proliferating and differentiating in preparation for lactation. In addition to the transcytosis of immunoglobulins, the mammary gland also concentrates a number of circulating hormones into colostrum. Remarkably, the mechanisms in the formation of colostrum in ungulates have been rather modestly studied. The mechanisms and causes of this variation in mammary gland transcytosis of IgG1 are examined, evaluated, and in some cases, explained

Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations

We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution to investigate interaction and binding of α-tochoperol transfer protein (α-TTP) to phosphatidylinositol phosphate lipids (PIPs). Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP-PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein/ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED) phenotypes. Specifically, R221 is main residue responsible for the stabilization of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP.

Solvation-Driven Charge Transfer and Localization in Metal Complexes

In any physicochemical process in liquids, the dynamical response of the solvent to the solutes out of equilibrium plays a crucial role in the rates and products: the solvent molecules react to the changes in volume and electron density of the solutes to minimize the free energy of the solution, thus modulating the activation barriers and stabilizing (or destabilizing) intermediate states. In charge transfer (CT) processes in polar solvents, the response of the solvent always assists the formation of charge separation states by stabilizing the energy of the localized charges. A deep understanding of the solvation mechanisms and time scales is therefore essential for a correct description of any photochemical process in dense phase and for designing molecular devices based on photosensitizers with CT excited states. In the last two decades, with the advent of ultrafast time-resolved spectroscopies, microscopic models describing the relevant case of polar solvation (where both the solvent and the solute molecules have a permanent electric dipole and the mutual interaction is mainly dipole−dipole) have dramatically progressed. Regardless of the details of each model, they all assume that the effect of the electrostatic fields of the solvent molecules on the internal electronic dynamics of the solute are perturbative and that the solvent−solute coupling is mainly an electrostatic interaction between the constant permanent dipoles of the solute and the solvent molecules. This well-established picture has proven to quantitatively rationalize spectroscopic effects of environmental and electric dynamics (time-resolved Stokes shifts, inhomogeneous broadening, etc.). However, recent computational and experimental studies, including ours, have shown that further improvement is required. Indeed, in the last years we investigated several molecular complexes exhibiting photoexcited CT states, and we found that the current description of the formation and stabilization of CT states in an important group of molecules such as transition metal complexes is inaccurate. In particular, we proved that the solvent molecules are not just spectators of intramolecular electron density redistribution but significantly modulate it. Our results solicit further development of quantum mechanics computational methods to treat the solute and (at least) the closest solvent molecules including the nonperturbative treatment of the effects of local electrostatics and direct solvent−solute interactions to describe the dynamical changes of the solute excited states during the solvent response.

Potential for virus transfer between the honey bees Apis mellifera and A. cerana

Viruses seem to play a key role in European honey bee, Apis mellifera health, and have a much broader host spectrum than previously thought. Few studies have investigated interspecific virus transfer within the genus Apis. The introduction of A. mellifera into Asia exposed endemic Apis species to the risk of obtaining new viruses or viral strains and vice versa. To investigate the potential for host shifts, virus prevalence and sequences were monitored over three years in single and mixed-species apiaries hosting introduced A. mellifera and endemic Apis cerana. Deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), and sacbrood virus (SBV) were found, but not KBV, VDV-1, ABPV, or CBPV. Virus infections and prevalence were generally lower in A. cerana compared to A. mellifera, and varied over the years. The sequence data provided evidence for interspecific transfer of IAPV, BQCV, and DWV, but SBV strains seem to be species specific. Prevalence and sequence results taken together indicate that interspecific transfers of viruses are rare, even if honey bees are kept in close proximity. We discuss the pattern observed in the context host specificity and resistance. Our understanding of the extent of these exchanges is limited by a lack of knowledge on the mechanisms of adaptation of viruses to different hosts.

Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 − T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

Cooperation in the knowledge transfer process: evidence from the demand side in Mexico

This paper presents the results of the analysis focused on scientific-technological KT in four Mexican firms and carried out by the case study approach. The analysis highlights the use of KT mechanisms as a means to obtain scientific-technological knowledge, learning, building S&T capabilities, and achieve the results of the R&D and innovation by firms.

Sensitization profiles and cross-reactivity among plant allergens. Molecular mechanisms of food allergy development and the role of enhancers

La prevalencia de las alergias está aumentando desde mediados del siglo XX, y se estima que actualmente afectan a alrededor del 2-8 % de la población, pero las causas de este aumento aún no están claras. Encontrar el origen del mecanismo por el cual una proteína inofensiva se convierte en capaz de inducir una respuesta alérgica es de vital importancia para prevenir y tratar estas enfermedades. Aunque la caracterización de alérgenos relevantes ha ayudado a mejorar el manejo clínico y a aclarar los mecanismos básicos de las reacciones alérgicas, todavía queda un largo camino para establecer el origen de la alergenicidad y reactividad cruzada. El objetivo de esta tesis ha sido caracterizar las bases moleculares de la alergenicidad tomando como modelo dos familias de panalergenos (proteínas de transferencia de lípidos –LTPs- y taumatinas –TLPs-) y estudiando los mecanismos que median la sensibilización y la reactividad cruzada para mejorar tanto el diagnóstico como el tratamiento de la alergia. Para ello, se llevaron a cabo dos estrategias: estudiar la reactividad cruzada de miembros de familias de panalérgenos; y estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas. Para estudiar la reactividad cruzada entre miembros de la misma familia de proteínas, se seleccionaron LTPs y TLPs, descritas como alergenos, tomando como modelo la alergia a frutas. Por otra parte, se estudiaron los perfiles de sensibilización a alérgenos de trigo relacionados con el asma del panadero, la enfermedad ocupacional más relevante de origen alérgico. Estos estudios se llevaron a cabo estandarizando ensayos tipo microarrays con alérgenos y analizando los resultados por la teoría de grafos. En relación al estudiar moléculas-co-adyuvantes que pudieran favorecer la capacidad alergénica de dichas proteínas, se llevaron a cabo estudios sobre la interacción de los alérgenos alimentarios con células del sistema inmune humano y murino y el epitelio de las mucosas, analizando la importancia de moléculas co-transportadas con los alérgenos en el desarrollo de una respuesta Th2. Para ello, Pru p 3(LTP y alérgeno principal del melocotón) se selección como modelo para llevarlo a cabo. Por otra parte, se analizó el papel de moléculas activadoras del sistema inmune producidas por patógenos en la inducción de alergias alimentarias seleccionando el modelo kiwi-alternaria, y el papel de Alt a 1, alérgeno mayor de dicho hongo, en la sensibilización a Act d 2, alérgeno mayor de kiwi. En resumen, el presente trabajo presenta una investigación innovadora aportando resultados de gran utilidad tanto para la mejora del diagnóstico como para nuevas investigaciones sobre la alergia y el esclarecimiento final de los mecanismos que caracterizan esta enfermedad. ABSTRACT Allergies are increasing their prevalence from mid twentieth century, and they are currently estimated to affect around 2-8% of the population but the underlying causes of this increase remain still elusive. The understanding of the mechanism by which a harmless protein becomes capable of inducing an allergic response provides us the basis to prevent and treat these diseases. Although the characterization of relevant allergens has led to improved clinical management and has helped to clarify the basic mechanisms of allergic reactions, it seems justified in aspiring to molecularly dissecting these allergens to establish the structural basis of their allergenicity and cross-reactivity. The aim of this thesis was to characterize the molecular basis of the allergenicity of model proteins belonging to different families (Lipid Transfer Proteins –LTPs-, and Thaumatin-like Proteins –TLPs-) in order to identify mechanisms that mediate sensitization and cross reactivity for developing new strategies in the management of allergy, both diagnosis and treatment, in the near future. With this purpose, two strategies have been conducted: studies of cross-reactivity among panallergen families and molecular studies of the contribution of cofactors in the induction of the allergic response by these panallergens. Following the first strategy, we studied the cross-reactivity among members of two plant panallergens (LTPs , Lipid Transfer Proteins , and TLPs , Thaumatin-like Proteins) using the peach allergy as a model. Similarly, we characterized the sensitization profiles to wheat allergens in baker's asthma development, the most relevant occupational disease. These studies were performed using allergen microarrays and the graph theory for analyzing the results. Regarding the second approach, we analyzed the interaction of plant allergens with immune and epithelial cells. To perform these studies , we examined the importance of ligands and co-transported molecules of plant allergens in the development of Th2 responses. To this end, Pru p 3, nsLTP (non-specific Lipid Transfer Protein) and peach major allergen, was selected as a model to investigate its interaction with cells of the human and murine immune systems as well as with the intestinal epithelium and the contribution of its ligand in inducing an allergic response was studied. Moreover, we analyzed the role of pathogen associated molecules in the induction of food allergy. For that, we selected the kiwi- alternaria system as a model and the role of Alt a 1 , major allergen of the fungus, in the development of Act d 2-sensitization was studied. In summary, this work presents an innovative research providing useful results for improving diagnosis and leading to further research on allergy and the final clarification of the mechanisms that characterize this disease.

Water option contracts for reducing water supply risks: an application to the Tagus-Segura Transfer

El agua es un recurso cada vez más escaso y valioso. Por ello, los recursos hídricos disponibles deben asignarse de una forma eficiente entre los diferentes usos. El cambio climático aumentará la frecuencia y severidad de los eventos extremos, y podría incrementar la demanda de agua de los cultivos. El empleo de mecanismos flexibles de asignación de agua puede ser imprescindible para hacer frente a este aumento en la variabilidad del balance hídrico y para asegurar que los riesgos de suministro, y no solo los recursos, son compartidos de manera eficiente entre los usuarios. Los mercados de agua permiten la reasignación de los recursos hídricos, favoreciendo su transferencia desde los usos de menor a los de mayor valor. Diferentes tipos de mercados de agua se han establecido en diferentes partes del mundo, ayudando a los participantes a afrontar los problemas de escasez de agua en esas zonas. En España, los intercambios de agua están permitidos desde 1999, aunque la participación de los usuarios en el mercado ha sido limitada. Hay varios aspectos de los mercados de agua en España que deben mejorarse. Esta tesis, además de proponer una serie de cambios en el marco regulatorio, propone la introducción de contratos de opción de agua como una posible mejora. La principal ventaja de este tipo de contratos es la estabilidad legal e institucional que éstos proporcionan tanto a compradores como vendedores. Para apoyar esta propuesta, se han llevado a cabo diferentes análisis que muestran el potencial de los contratos de opción como herramienta de reducción del riesgo asociado a una oferta de agua inestable. La Cuenca del Segura (Sureste de España), la Cuenca del Tajo y el Acueducto Tajo- Segura han sido seleccionados como casos de estudio. Tres análisis distintos aplicados a dicha región se presentan en esta tesis: a) una evaluación de los contratos de opción como mecanismo para reducir los riesgos de disponibilidad de agua sufridos por los regantes en la Cuenca del Segura; b) un marco teórico para analizar las preferencias de los regantes por diferentes mecanismos de gestión del riesgo de disponibilidad de agua, su disposición a pagar por ellos y los precios aproximados de estos instrumentos (seguro de sequía y contratos de opción de agua); y c) una evaluación del papel de los contratos de opción en las decisiones de aprovisionamiento de agua de una comunidad de regantes ante una oferta de agua incierta. Los resultados muestran el potencial de reducción del riesgo de los contratos de opción para regantes en España, pero pueden ser extrapolados a otros sectores o regiones. Las principales conclusiones de esta tesis son: a) la agricultura será uno de los sectores más afectados por el cambio climático. Si los precios del agua aumentan, la rentabilidad de los cultivos puede caer hasta niveles negativos, lo que podría dar lugar al abandono de cultivos de regadío en algunas zonas de España. Las políticas de cambio climático y de agua deben estar estrechamente coordinadas para asegurar un uso de agua eficiente y la rentabilidad de la agricultura; b) aunque los mercados de agua han ayudado a algunos usuarios a afrontar problemas de disponibilidad del recurso en momentos de escasez, hay varios aspectos que deben mejorarse; c) es necesario desarrollar mercados de agua más flexibles y estables para garantizar una asignación eficiente de los recursos entre los usuarios de agua; d) los resultados muestran los beneficios derivados del establecimiento de un contrato de opción entre usuarios de agua del Tajo y del Segura para reducir el riesgo de disponibilidad de agua en la cuenca receptora; e) la disposición a pagar de los regantes por un contrato de opción de agua o un seguro de sequía hidrológica, que representa el valor que tienen estos mecanismos para aquellos usuarios de agua que se enfrentan a riesgos relacionados con la disponibilidad del recurso, es consistente con los resultados obtenidos en estudios previos y superior al precio de mercado de estos instrumentos, lo que favorece la viabilidad de estos mecanismos de gestión del riesgo ; y f) los contratos de opción podrían ayudar a optimizar las decisiones de aprovisionamiento de agua bajo incertidumbre, proporcionando más estabilidad y flexibilidad que los mercados temporales de agua. ABSTRACT Water is becoming increasingly scarce and valuable. Thus, existing water resources need to be efficiently allocated among users. Climate change is expected to increase the frequency and severity of extreme events, and it may also increase irrigated crops' water demand. The implementation of flexible allocation mechanisms could be essential to cope with this increased variability of the water balance and ensure that supply risks, and not only water resources, are also efficiently shared and managed. Water markets allow for the reallocation of water resources from low to high value uses. Different water trading mechanisms have been created in different parts of the world and have helped users to alleviate water scarcity problems in those areas. In Spain, water trading is allowed since 1999, although market activity has been limited. There are several issues in the Spanish water market that should be improved. This thesis, besides proposing several changes in the legislative framework, proposes the introduction of water option contracts as a potential improvement. The main advantage for both buyer and seller derived from an option contract is the institutional and legal stability it provides. To support this proposal, different analyses have been carried out that show the potential of option contracts as a risk reduction tool to manage water supply instability. The Segura Basin (Southeast Spain), the Tagus Basin and the Tagus-Segura inter-basin Transfer have been selected as the case study. Three different analyses applied to this region are presented in this thesis: a) an evaluation of option contracts as a mechanisms to reduce water supply availability risks in the Segura Basin; b) a theoretical framework for analyzing farmer’s preferences for different water supply risk management tools and farmers’ willingness to pay for them, together with the assessment of the prices of these mechanisms (drought insurance and water option contracts); and c) an evaluation of the role of option contracts in water procurement decisions under uncertainty. Results show the risk-reduction potential of option contracts for the agricultural sector in Spain, but these results can be extrapolated to other sectors or regions. The main conclusions of the thesis are: a) agriculture would be one of the most affected sectors by climate change. With higher water tariffs, crop’s profitability can drop to negative levels, which may result in the abandoning of the crop in many areas. Climate change and water policies must be closely coordinated to ensure efficient water use and crops’ profitability; b) although Spanish water markets have alleviated water availability problems for some users during water scarcity periods, there are several issues that should be improved; c) more flexible and stable water market mechanisms are needed to allocate water resources and water supply risks among competing users; d) results show the benefits derived from the establishment of an inter-basin option contract between water users in the Tagus and the Segura basins for reducing water supply availability risks in the recipient area; e) irrigators’ willingness to pay for option contracts or drought insurance, that represent the value that this kind of trading mechanisms has for water users facing water supply reliability problems, are consistent with results obtained in previous works and higher than the prices of this risk management tools, which shows the feasibility of these mechanisms; and f) option contracts would help to optimize water procurement decisions under uncertainty, providing more flexibility and stability than the spot market.

The Ti plasmid increases the efficiency of Agrobacterium tumefaciens as a recipient in virB-mediated conjugal transfer of an IncQ plasmid

The T-DNA transfer apparatus of Agrobacterium tumefaciens mediates the delivery of the T-DNA into plant cells, the transfer of the IncQ plasmid RSF1010 into plant cells, and the conjugal transfer of RSF1010 between Agrobacteria. We show in this report that the Agrobacterium-to-Agrobacterium conjugal transfer efficiencies of RSF1010 increase dramatically if the recipient strain, as well as the donor strain, carries a wild-type Ti plasmid and is capable of vir gene expression. Investigation of possible mechanisms that could account for this increased efficiency revealed that the VirB proteins encoded by the Ti plasmid were required. Although, with the exception of VirB1, all of the proteins that form the putative T-DNA transfer apparatus (VirB1–11, VirD4) are required for an Agrobacterium strain to serve as an RSF1010 donor, expression of only a subset of these proteins is required for the increase in conjugal transfer mediated by the recipient. Specifically, VirB5, 6, 11, and VirD4 are essential donor components but are dispensable for the increased recipient capacity. Defined point mutations in virB9 affected donor and recipient capacities to the same relative extent, suggesting that similar functions of VirB9 are important in both of these contexts.

Evidence for the recent horizontal transfer of long terminal repeat retrotransposon

The evolutionary dynamics existing between transposable elements (TEs) and their host genomes have been likened to an “arms race.” The selfish drive of TEs to replicate, in turn, elicits the evolution of host-mediated regulatory mechanisms aimed at repressing transpositional activity. It has been postulated that horizontal (cross-species) transfer may be one effective strategy by which TEs and other selfish genes can escape host-mediated silencing mechanisms over evolutionary time; however, to date, the most definitive evidence that TEs horizontally transfer between species has been limited to class II or DNA-type elements. Evidence that the more numerous and widely distributed retroelements may also be horizontally transferred between species has been more ambiguous. In this paper, we report definitive evidence for a recent horizontal transfer of the copia long terminal repeat retrotransposon between Drosophila melanogaster and Drosophila willistoni.

Charge transfer and transport in DNA

We explore charge migration in DNA, advancing two distinct mechanisms of charge separation in a donor (d)–bridge ({Bj})–acceptor (a) system, where {Bj} = B1,B2, … , BN are the N-specific adjacent bases of B-DNA: (i) two-center unistep superexchange induced charge transfer, d*{Bj}a → d∓{Bj}a±, and (ii) multistep charge transport involves charge injection from d* (or d+) to {Bj}, charge hopping within {Bj}, and charge trapping by a. For off-resonance coupling, mechanism i prevails with the charge separation rate and yield exhibiting an exponential dependence ∝ exp(−βR) on the d-a distance (R). Resonance coupling results in mechanism ii with the charge separation lifetime τ ∝ Nη and yield Y ≃ (1 + δ̄ Nη)−1 exhibiting a weak (algebraic) N and distance dependence. The power parameter η is determined by charge hopping random walk. Energetic control of the charge migration mechanism is exerted by the energetics of the ion pair state d∓B1±B2 … BNa relative to the electronically excited donor doorway state d*B1B2 … BNa. The realization of charge separation via superexchange or hopping is determined by the base sequence within the bridge. Our energetic–dynamic relations, in conjunction with the energetic data for d*/d− and for B/B+, determine the realization of the two distinct mechanisms in different hole donor systems, establishing the conditions for “chemistry at a distance” after charge transport in DNA. The energetic control of the charge migration mechanisms attained by the sequence specificity of the bridge is universal for large molecular-scale systems, for proteins, and for DNA.

The R1 component of mammalian ribonucleotide reductase has malignancy-suppressing activity as demonstrated by gene transfer experiments

Our recent studies have shown that deregulated expression of R2, the rate-limiting component of ribonucleotide reductase, enhances transformation and malignant potential by cooperating with activated oncogenes. We now demonstrate that the R1 component of ribonucleotide reductase has tumor-suppressing activity. Stable expression of a biologically active ectopic R1 in ras-transformed mouse fibroblast 10T½ cell lines, with or without R2 overexpression, led to significantly reduced colony-forming efficiency in soft agar. The decreased anchorage independence was accompanied by markedly suppressed malignant potential in vivo. In three ras-transformed cell lines, R1 overexpression resulted in abrogation or marked suppression of tumorigenicity. In addition, the ability to form lung metastases by cells overexpressing R1 was reduced by >85%. Metastasis suppressing activity also was observed in the highly malignant mouse 10T½ derived RMP-6 cell line, which was transformed by a combination of oncogenic ras, myc, and mutant p53. Furthermore, in support of the above observations with the R1 overexpressing cells, NIH 3T3 cells cotransfected with an R1 antisense sequence and oncogenic ras showed significantly increased anchorage independence as compared with control ras-transfected cells. Finally, characteristics of reduced malignant potential also were demonstrated with R1 overexpressing human colon carcinoma cells. Taken together, these results indicate that the two components of ribonucleotide reductase both are unique malignancy determinants playing opposing roles in its regulation, that there is a novel control point important in mechanisms of malignancy, which involves a balance in the levels of R1 and R2 expression, and that alterations in this balance can significantly modify transformation, tumorigenicity, and metastatic potential.

Large kinetic isotope effects in enzymatic proton transfer and the role of substrate oscillations

We propose an interpretation of the experimental findings of Klinman and coworkers [Cha, Y., Murray, C. J. & Klinman, J. P. (1989) Science 243, 1325–1330; Grant, K. L. & Klinman, J. P. (1989) Biochemistry 28, 6597–6605; and Bahnson, B. J. & Klinman, J. P. (1995) Methods Enzymol. 249, 373–397], who showed that proton transfer reactions that are catalyzed by bovine serum amine oxidase proceed through tunneling. We show that two different tunneling models are consistent with the experiments. In the first model, the proton tunnels from the ground state. The temperature dependence of the kinetic isotope effect is caused by a thermally excited substrate mode that modulates the barrier, as has been suggested by Borgis and Hynes [Borgis, D. & Hynes, J. T. (1991) J. Chem. Phys. 94, 3619–3628]. In the second model, there is both over-the-barrier transfer and tunneling from excited states. Finally, we propose two experiments that can distinguish between the possible mechanisms.

Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos in Xenopus Oocytes in Response to Progesterone

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34cdc2 could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34cdc2, and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor–induced feedback. We report here that the cdk inhibitor p21cip1, when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21cip1, progesterone fails to induce the activation of MAPK or p34cdc2, and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.