Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively reflecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.

Cellular responses to psychomotor stimulant and neuroleptic drugs are abnormal in mice lacking the D1 dopamine receptor

Stimulation of dopamine D1 receptors has profound effects on addictive behavior, movement control, and working memory. Many of these functions depend on dopaminergic systems in the striatum and D1–D2 dopamine receptor synergies have been implicated as well. We show here that deletion of the D1 dopamine receptor produces a neural phenotype in which amphetamine and cocaine, two addictive psychomotor stimulants, can no longer stimulate neurons in the striatum to express cFos or JunB or to regulate dynorphin. By contrast, haloperidol, a typical neuroleptic that acts preferentially at D2-class receptors, remains effective in inducing catalepsy and striatal Fos/Jun expression in the D1 mutants, and these behavioral and neural effects can be blocked by D2 dopamine receptor agonists. These findings demonstrate that D2 dopamine receptors can function without the enabling role of D1 receptors but that D1 dopamine receptors are essential for the control of gene expression and motor behavior by psychomotor stimulants.

Agrin in Alzheimer’s disease: Altered solubility and abnormal distribution within microvasculature and brain parenchyma

Agrin is a heparan sulfate proteoglycan that is widely expressed in neurons and microvascular basal lamina in the rodent and avian central nervous system. Agrin induces the differentiation of nerve-muscle synapses, but its function in either normal or diseased brains is not known. Alzheimer’s disease (AD) is characterized by loss of synapses, changes in microvascular architecture, and formation of neurofibrillary tangles and senile plaques. Here we have asked whether AD causes changes in the distribution and biochemical properties of agrin. Immunostaining of normal, aged human central nervous system revealed that agrin is expressed in neurons in multiple brain areas. Robust agrin immunoreactivity was observed uniformly in the microvascular basal lamina. In AD brains, agrin is highly concentrated in both diffuse and neuritic plaques as well as neurofibrillary tangles; neuronal expression of agrin also was observed. Furthermore, patients with AD had microvascular alterations characterized by thinning and fragmentation of the basal lamina. Detergent extraction and Western blotting showed that virtually all the agrin in normal brain is soluble in 1% SDS. In contrast, a large fraction of the agrin in AD brains is insoluble under these conditions, suggesting that it is tightly associated with β-amyloid. Together, these data indicate that the agrin abnormalities observed in AD are closely linked to β-amyloid deposition. These observations suggest that altered agrin expression in the microvasculature and the brain parenchyma contribute to the pathogenesis of AD.

Memory landscapes of single-enzyme molecules

Immobilized single horseradish peroxidase enzymes were observed by confocal fluorescence spectroscopy during catalysis of the oxidation reaction of the nonfluorescent dihydrorhodamine 6G substrate into the highly fluorescent product rhodamine 6G. By extracting only the non-Markovian behavior of the spectroscopic two-state process of enzyme-product complex formation and release, memory landscapes were generated for single-enzyme molecules. The memory landscapes can be used to discriminate between different origins of stretched exponential kinetics that are found in the first-order correlation analysis. Memory landscapes of single-enzyme data shows oscillations that are expected in a single-enzyme system that possesses a set of transient states. Alternative origins of the oscillations may not, however, be ruled out. The data and analysis indicate that substrate interaction with the enzyme selects a set of conformational substates for which the enzyme is active.

Cadherin Sequences That Inhibit β-Catenin Signaling: A Study in Yeast and Mammalian Cells

Drosophila Armadillo and its mammalian homologue β-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/β-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/β-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or β-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/β-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited β-catenin–mediated signaling. This suggests that the interaction between β-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

Regulation of the myeloperoxidase enhancer binding proteins Pu1, C-EBP alpha, -beta, and -delta during granulocyte-lineage specification.

We have compared the molecular architecture and function of the myeloperoxidase upstream enhancer in multipotential versus granulocyte-committed hematopoietic progenitor cells. We show that the enhancer is accessible in multipotential cell chromatin but functionally incompetent before granulocyte commitment. Multipotential cells contain both Pu1 and C-EBP alpha as enhancer-binding activities. Pu1 is unphosphorylated in both multipotential and granulocyte-committed cells but is phosphorylated in B lymphocytes, raising the possibility that differential phosphorylation may play a role in specifying its lymphoid versus myeloid functions. C-EBP alpha exists as multiple phosphorylated forms in the nucleus of both multipotential and granulocyte-committed cells. C-EBP beta is unphosphorylated and cytoplasmically localized in multipotential cells but exists as a phosphorylated nuclear enhancer-binding activity in granulocyte-committed cells. Granulocyte colony-stimulating factor-induced granulocytic differentiation of multipotential progenitor cells results in activation of C-EBP delta expression and functional recruitment of C-EBP delta and C-EBP beta to the nucleus. Our results implicate Pu1 and the C-EBP family as critical regulators of myeloperoxidase gene expression and are consistent with a model in which a temporal exchange of C-EBP isoforms at the myeloperoxidase enhancer mediates the transition from a primed state in multipotential cells to a transcriptionally active configuration in promyelocytes.

Reconstitution of the hippocampal mossy fiber and associational-commissural pathways in a novel dissociated cell culture system.

Synapses of the hippocampal mossy fiber pathway exhibit several characteristic features, including a unique form of long-term potentiation that does not require activation of the N-methyl-D-aspartate receptor by glutamate, a complex postsynaptic architecture, and sprouting in response to seizures. However, these connections have proven difficult to study in hippocampal slices because of their relative paucity (<0.4%) compared to commissural-collateral synapses. To overcome this problem, we have developed a novel dissociated cell culture system in which we have enriched mossy fiber synapses by increasing the ratio of granule-to-pyramidal cells. As in vivo, mossy fiber connections are composed of large dynorphin A-positive varicosities contacting complex spines (but without a restricted localization). The elementary synaptic connections are glutamatergic, inhibited by dynorphin A, and exhibit N-methyl-D-aspartate-independent long-term potentiation. Thus, the simplicity and experimental accessibility of this enriched in vitro mossy fiber pathway provides a new perspective for studying nonassociative plasticity in the mammalian central nervous system.

Conscious recollection and the human hippocampal formation: evidence from positron emission tomography.

We used positron emission tomography (PET) to examine the role of the hippocampal formation in implicit and explicit memory. Human volunteers studied a list of familiar words, and then they either provided the first word that came to mind in response to three-letter cues (implicit memory) or tried to recall studied words in response to the same cues (explicit memory). There was no evidence of hippocampal activation in association with implicit memory. However, priming effects on the implicit memory test were associated with decreased activity in extrastriate visual cortex. On the explicit memory test, subjects recalled many target words in one condition and recalled few words in a second condition, despite trying to remember them. Comparisons between the two conditions showed that blood-flow increases in the hippocampal formation are specifically associated with the conscious recollection of studied words, whereas blood-flow increases in frontal regions are associated with efforts to retrieve target words. Our results help to clarify some puzzles concerning the role of the hippocampal formation in human memory.

Learning about categories in the absence of memory.

A fundamental question about memory and cognition concerns how information is acquired about categories and concepts as the result of encounters with specific instances. We describe a profoundly amnesic patient (E.P.) who cannot learn and remember specific instances--i.e., he has no detectable declarative memory. Yet after inspecting a series of 40 training stimuli, he was normal at classifying novel stimuli according to whether they did or did not belong to the same category as the training stimuli. In contrast, he was unable to recognize a single stimulus after it was presented 40 times in succession. These findings demonstrate that the ability to classify novel items, after experience with other items in the same category, is a separate and parallel memory function of the brain, independent of the limbic and diencephalic structures essential for remembering individual stimulus items (declarative memory). Category-level knowledge can be acquired implicitly by cumulating information from multiple training examples in the absence of detectable conscious memory for the examples themselves.

Adenovirus-mediated urokinase gene transfer induces liver regeneration and allows for efficient retrovirus transduction of hepatocytes in vivo.

Retrovirus-mediated gene transfer into hepatocytes in vivo results in long-term gene expression. Limitations include the need to remove two-thirds of the liver and the relatively low frequency of gene transfer. To increase gene transfer without surgical hepatectomy, mouse hepatocytes were transduced in vivo with a recombinant adenovirus that transiently expressed urokinase, resulting in high rates of asynchronous liver regeneration. During the regenerative phase, in vivo retroviral-mediated gene transfer in hepatocytes resulted in 5- to 10-fold greater transduction efficiencies than that obtained by conventional partial hepatectomy. In 3-4 weeks, the architecture and microscopic structure of the recipient livers were normal. The two-viral system of achieving permanent transgene expression from hepatocytes in vivo offers an alternative approach to current ex vivo and in vivo gene-transfer models.

Lina Bo Bardi: as moradas da alma

Esta pesquisa se detém em verificar a inserção do tema da habitação denominada por Lina Bo Bardi de \"espontânea\", \"primitiva\', \"popular\", na sua obra, no período de 1939 a 1958. Inclui tempos de formação e de primeiras atividades profissionais em solo italiano e os anos iniciais de vida no Brasil, quando ela reside em São Paulo. O núcleo central do estudo desenvolve-se com duas abordagens distintas para o tema: uma teórica, que contempla os escritos de Lina Bo Bardi - ensaios, trabalhos editoriais e jornalísticos - publicados em revistas especializadas; e outra prática, que analisa dois projetos da arquiteta: a Casa de Vidro e a Casa Valéria Piacentini Cirell. Na primeira abordagem, realiza-se um levantamento inicial de conceitos que seriam, na visão de Lina Bo Bardi, inerentes ao trabalho popular, para, no segundo momento, verificar a possível presença desses conceitos na sua prática projetual. Nesse sentido, além de elencar os textos escritos por Lina Bo Bardi no período, procede-se a um levantamento minucioso dos documentos referentes aos projetos das duas casas, incluindo os desenhos e a fortuna crítica de cada uma delas. Cumpre-se a tarefa inicial de organizar em uma seqüência lógica os croquis e os desenhos de projeto, que se encontravam sem data ou anotação de seriação, a qual possibilita a compreensão do procedimento projetual adotado por Lina Bo Bardi. Pretende-se, ao construir essa trajetória de pesquisa, trazer a tona elementos de análise, influências e mananciais de inspiração, que possam contribuir para desvendar os princípios que guiam Lina Bo Bardi ao realizar projetos com resultados tão contrastantes entre si como as duas casas em estudo. Essa Arquiteta que, pela extrema personalidade de sua linguagem, é considerada por alguns historiadores e críticos como \"mestra de si mesma\", e que é alvo de qualificações que situam sua obra como impossível de se moldar a correntes estéticas estabelecidas, concebe uma arquitetura que não privilegia a questão formal e sim princípios previamente definidos.

"Another Way": The Pneumatology of Deleuzean Minoritarian Communal Interpretation in Scripture, the 16th Century Radical Reformation, and Alternative 21st Century Anabaptist Community

This dissertation proposes a constructive theology of the Holy Spirit called the "pneumatology of minoritarian communal interpretation," the alternative creation of meaning within an oppressive majority context. It illustrates the convergence of Deleuzean philosophy with Anabaptist pneumatology and media communal interpretation theory in three particular locations: 1) selected mentions of the Holy Spirit in the Hebrew Bible and Christian New Testament; 2) the 16th century Radical Reformation; and 3) "Another Way," a 21st century alternative Anabaptist group focused around the spiritual discussion of art and popular media. Chapter One outlines the three theories. Chapter Two examines the Holy Spirit in the Hebrew Bible, particularly 1 Samuel 8, the book of Ezekiel, and the Gospel narratives. Chapter Three examines the pneumatological writings of the Radical Reformers, concentrating particularly on their theologies of the intersection between church and the surrounding majoritarian culture. Chapter Four outlines my original field research with Another Way, and examines the tension between minoritarian communal interpretation and the 21st century semiotic regime. Chapter Five then summarizes the conversations between theory and illustration to propose the pneumatology of minoritarian communal interpretation for Christian theology.

Chinese Intellectuals and China`s Policy toward Japan

This dissertation aims at integrating two scholarships: state-society relation studies and Chinese foreign policy analysis. I created Two-level Perception Gap Model to analyze different intellectual groups' relations with party-state by confirming Chinese intellectuals play a role in CFP making in general, China's Japan policy in particular. This model is an alternative approach, instead of conventional wisdom patron-client approach, to explain and analyze the pluralized intellectual-state relations in China. This model first analyzed the role of two intellectual groups, namely think tank scholars and popular nationalist, in China's Japan policy making, and then based on these analyses it explains the interactional patterns between these two intellectual groups and party-state. I used three case studies, which represented different types of issue, Chinese attitude toward the U.S.-Japan alliance and the Japanese defense policy, the controversy over the Yasukuni Shrine Visit, and the territorial dispute over the Diaoyu/Senkaku Islands, to examine this model. First, I examined think tank scholar groups and the extent they influenced "core interest issue and sensitive issue (Issue 1)," Chinese attitude toward the U.S.-Japan alliance and the Japanese defense policy, and their international patterns with party-state. Chapter 3 compares the responses of Chinese officials to the changes in the defense policy of Japan to the analyses from the think tank scholars. As the model assumes, results show that think tank scholars' analyses are consistent with China's policy position; nevertheless, it is difficult to confirm their analyses have influence on Chinese attitude toward the U.S.-Japan alliance and the Japanese defense policy. Based on the analysis of journal articles, most articles do not provide policy suggestions or simply provide suggestions that do not deviate from the policy. As Gu's theory of pluralist institutionalism and my hypothesis points out, most think tank scholars are establishment intellectuals so they tend to be self-disciplined. Second, this model provide a new concept "patriotic dilemma" for analyzing the challenge and constraints brought by popular nationalist discourses and public mobilization to Chinese foreign policy decision makers. Chapter 4 investigated the cases study of the controversy over the Yasukuni Shrine Visit, defined as "major/minor interest issue/ sensitive issue (Issue 3)," and the discourses from the popular nationalist, mainly focusing on anti-Japanese activists. The chapter also observes their influence on nationalist public opinions and analyzes how the nationalist public opinions constrain the policy choices among decision makers. Results strongly supported the hypothesis of patriotic dilemma that, although the popular nationalist group and public opinions constrained the policy choices of Chinese decision makers in the short term, they were unable to change the fundamental policy direction. Third, chapter 5 also focuses on anti-Japanese activists and examines the model with the case of the territorial dispute over the Diaoyu/Senkaku Islands. The result supported that hypothesis that China's policy change was not because of the influence from popular nationalist's discourses or public opinions but because of the change of priority of this issue, from major/minor interest issue to core interest issue. These two chapters also indicate that the patron-client model is unable to describe the popular nationalist. An alternative approach, such as the concept "patriotic dilemma" is needed to describe the relations between the popular nationalist and the government.