Reelin controls position of autonomic neurons in the spinal cord

Mutation of the reeler gene (Reln) disrupts neuronal migration in several brain regions and gives rise to functional deficits such as ataxic gait and trembling in the reeler mutant mouse. Thus, the Reln product, reelin, is thought to control cell–cell interactions critical for cell positioning in the brain. Although an abundance of reelin transcript is found in the embryonic spinal cord [Ikeda, Y. & Terashima, T. (1997) Dev. Dyn. 210, 157–172; Schiffmann, S. N., Bernier, B. & Goffinet, A. M. (1997) Eur. J. Neurosci. 9, 1055–1071], it is generally thought that neuronal migration in the spinal cord is not affected by reelin. Here, however, we show that migration of sympathetic preganglionic neurons in the spinal cord is affected by reelin. This study thus indicates that reelin affects neuronal migration outside of the brain. Moreover, the relationship between reelin and migrating preganglionic neurons suggests that reelin acts as a barrier to neuronal migration.

Footprints on the viral DNA ends in Moloney murine leukemia virus preintegration complexes reflect a specific association with integrase

Retroviral DNA integration is mediated by the preintegration complex, a large nucleoprotein complex derived from the core of the infecting virion. We previously have used Mu-mediated PCR to probe the nucleoprotein organization of Moloney murine leukemia virus preintegration complexes. A region of protection spans several hundred base pairs at each end of the viral DNA, and strong enhancements are present near the termini. Here, we show that these footprints reflect a specific association between integrase and the viral DNA ends in functional preintegration complexes. Barrier-to-autointegration factor, a cellular protein that blocks autointegration of Moloney murine leukemia virus DNA, also plays an indirect role in generating the footprints at the ends of the viral DNA. We have exploited Mu-mediated PCR to examine the effect of mutations at the viral DNA termini on complex formation. We find that a replication competent mutant with a deletion at one end of the viral DNA still exhibits a strong enhancement about 20 bp from the terminus of the mutant DNA end. The site of the enhancement therefore appears to be at a fixed distance from the ends of the viral DNA. We also find that a mutation at one end of the viral DNA, which renders the virus incompetent for replication, abolishes the enhancements and protection at both the U3 and U5 ends. A pair of functional viral DNA ends therefore are required to interact before the chemical step of 3′ end processing.

Streptogramin- and tetracycline-responsive dual regulated expression of p27Kip1 sense and antisense enables positive and negative growth control of Chinese hamster ovary cells

We constructed a dual regulated expression vector cassette (pDuoRex) whereby two heterologous genes can be independently regulated via streptogramin- and tetracycline-responsive promoters. Two different constructs containing growth-promoting and growth-inhibiting genes were stably transfected in recombinant Chinese hamster ovary (CHO) cells that express the streptogramin- and tetracycline-dependent transactivators in a dicistronic configuration. An optimally balanced heterologous growth control scenario was achieved by reciprocal expression of the growth-inhibiting human cyclin-dependent kinase inhibitor p27Kip1 in sense (p27Kip1S) and antisense (p27Kip1AS) orientation. Exclusive expression of p27Kip1S resulted in complete G1-phase-specific growth arrest, while expression of only p27Kip1AS showed significantly increased proliferation compared to control cultures (both antibiotics present), presumably by decreasing host cell p27Kip1 expression. In a second system, a derivative of pDuoRex encoding streptogramin-responsive expression of the growth-promoting SV40 small T antigen (sT) and tetracycline-regulated expression of p27Kip1 was stably transfected into CHO cells. Expression of sT alone resulted in an increase in cell proliferation, but the expression of p27Kip1 failed to provide the expected G1-specific growth arrest despite having demonstrated expression of the protein. This illustrates the difficulty in balancing the complex pathways underlying cell proliferation control through the expression of two functionally distinct genes involved in those pathways, and how a single-gene sense/antisense approach using pDuoRex can overcome this barrier to complete metabolic engineering control.

The pKa of His-24 in the folding transition state of apomyoglobin

In native apomyoglobin, His-24 cannot be protonated, although at pH 4 the native protein forms a molten globule folding intermediate in which the histidine residues are readily protonated. The inability to protonate His-24 in the native protein dramatically affects the unfolding/refolding kinetics, as demonstrated by simulations for a simple model. Kinetic data for wild type and for a mutant lacking His-24 are analyzed. The pKa values of histidine residues in native apomyoglobin are known from earlier studies, and the average histidine pKa in the molten globule is determined from the pH dependence of the equilibrium between the native and molten globule forms. Analysis of the pH-dependent unfolding/refolding kinetics reveals that the average pKa of the histidine residues, including His-24, is closely similar in the folding transition state to the value found in the molten globule intermediate. Consequently, protonation of His-24 is not a barrier to refolding of the molten globule to the native protein. Instead, the normal pKa of His-24 in the transition state, coupled with its inaccessibility in the native state, promotes fast unfolding at low pH. The analysis of the wild-type results is confirmed and extended by using the wild-type parameters to fit the unfolding kinetics of a mutant lacking His-24.

Kinetic stability as a mechanism for protease longevity

The folding of the extracellular serine protease, α-lytic protease (αLP; EC 3.4.21.12) reveals a novel mechanism for stability that appears to lead to a longer functional lifetime for the protease. For αLP, stability is based not on thermodynamics, but on kinetics. Whereas this has required the coevolution of a pro region to facilitate folding, the result has been the optimization of native-state properties independent of their consequences on thermodynamic stability. Structural and mutational data lead to a model for catalysis of folding in which the pro region binds to a conserved β-hairpin in the αLP C-terminal domain, stabilizing the folding transition state and the native state. The pro region is then proteolytically degraded, leaving the active αLP trapped in a metastable conformation. This metastability appears to be a consequence of pressure to evolve properties of the native state, including a large, highly cooperative barrier to unfolding, and extreme rigidity, that reduce susceptibility to proteolytic degradation. In a test of survival under highly proteolytic conditions, homologous mammalian proteases that have not evolved kinetic stability are much more rapidly degraded than αLP. Kinetic stability as a means to longevity is likely to be a mechanism conserved among the majority of extracellular bacterial pro-proteases and may emerge as a general strategy for intracellular eukaryotic proteases subject to harsh conditions as well.

Effects of Oxygen on Nodule Physiology and Expression of Nodulins in Alfalfa1

Early nodulin 2 (ENOD2) transcripts and protein are specifically found in the inner cortex of legume nodules, a location that coincides with the site of a barrier to O2 diffusion. The extracellular glycoprotein that binds the monoclonal antibody MAC236 has also been localized to this site. Thus, it has been proposed that these proteins function in the regulation of nodule permeability to O2 diffusion. It would then be expected that the levels of ENOD2 mRNA/protein and MAC236 antigen would differ in nodules with different permeabilities to O2. We examined the expression of ENOD2 and other nodule-expressed genes in Rhizobium meliloti-induced alfalfa nodules grown under 8, 20, or 50% O2. Although there was a change in the amount of MAC236 glycoprotein, the levels of ENOD2 mRNA and protein did not differ significantly among nodules grown at the different [O2], suggesting that neither ENOD2 transcription nor synthesis is involved in the long-term regulation of nodule permeability. Moreover, although nodules from all treatments reduced their permeability to O2 as the partial pressure of O2 (pO2) was increased to 100%, the levels of extractable ENOD2 and MAC236 proteins did not differ from those measured at the growth pO2, further suggesting that if these proteins are involved in a short-term regulation of the diffusion barrier, they must be involved in a way that does not require increased transcription or protein synthesis.

Tobamovirus and Potyvirus Accumulation in Minor Veins of Inoculated Leaves from Representatives of the Solanaceae and Fabaceae1

Virus invasion of minor veins in inoculated leaves of a host is the likely prelude to systemic movement of the pathogen and to subsequent yield reduction and quality loss. In this study we have analyzed the cell number and arrangement in minor veins within mature leaves of various members of the Solanaceae and Fabaceae families. We then monitored the accumulation pattern of several tobamoviruses and potyviruses in these veins at the time of rapid, phloem-mediated movement of viruses. Vascular parenchyma cells were the predominant and sometimes only cells to become visibly infected among the cells surrounding the sieve elements in minor veins containing 9 to 12 cells. In no instance did we observe a companion cell infected without a vascular parenchyma cell also being infected in the same vein. This suggests that the viruses used in this study first enter the vascular parenchyma cells and then the companion cells during invasion. The lack of detectable infection of smooth-walled companion or transfer cells, respectively, from inoculated leaves of bean (Phaseolus vulgaris) and pea (Pisum sativum) during a period of known rapid, phloem-mediated movement suggests that some viruses may be able to circumvent these cells in establishing phloem-mediated infection. The cause of the barrier to virus accumulation in the companion or transfer cells, the relationship of this barrier to previously identified barriers for virus or photoassimilate transport, and the relevance of these findings to photoassimilate transport models are discussed.

Polarity and permeation profiles in lipid membranes

The isotropic 14N-hyperfine coupling constant, a\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{o}^{N}}}\end{equation*}\end{document}, of nitroxide spin labels is dependent on the local environmental polarity. The dependence of a\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{o}^{N}}}\end{equation*}\end{document} in fluid phospholipid bilayer membranes on the C-atom position, n, of the nitroxide in the sn-2 chain of a spin-labeled diacyl glycerophospholipid therefore determines the transmembrane polarity profile. The polarity variation in phospholipid membranes, with and without equimolar cholesterol, is characterized by a sigmoidal, trough-like profile of the form {1 + exp [(n − no)/λ]}−1, where n = no is the point of maximum gradient, or polarity midpoint, beyond which the free energy of permeation decreases linearly with n, on a characteristic length-scale, λ. Integration over this profile yields a corresponding expression for the permeability barrier to polar solutes. For fluid membranes without cholesterol, no ≈ 8 and λ ≈ 0.5–1 CH2 units, and the permeability barrier introduces an additional diffusive resistance that is equivalent to increasing the effective membrane thickness by 35–80%, depending on the lipid. For membranes containing equimolar cholesterol, no ≈ 9–10, and the total change in polarity is greater than for membranes without cholesterol, increasing the permeability barrier by a factor of 2, whereas the decay length remains similar. The permeation of oxygen into fluid lipid membranes (determined by spin-label relaxation enhancements) displays a profile similar to that of the transmembrane polarity but of opposite sense. For fluid membranes without cholesterol no ≈ 8 and λ ≈ 1 CH2 units, also for oxygen. The permeation profile for polar paramagnetic ion complexes is closer to a single exponential decay, i.e., no lies outside the acyl-chain region of the membrane. These results are relevant not only to the permeation of water and polar solutes into membranes and their permeabilities, but also to depth determinations of site-specifically spin-labeled protein residues by using paramagnetic relaxation agents.

Subcutaneous vaccination with irradiated, cytokine-producing tumor cells stimulates CD8+ cell-mediated immunity against tumors located in the "immunologically privileged" central nervous system.

Vaccination with cytokine-producing tumor cells generates potent immune responses against tumors outside the central nervous system (CNS). The CNS, however, is a barrier to allograft and xenograft rejection, and established tumors within the CNS have failed to respond to other forms of systemic immunotherapy. To determine what barriers the "immunologically privileged" CNS would pose to cytokine-assisted tumor vaccines and what cytokines would be most efficacious against tumors within the CNS, we irradiated B16 murine melanoma cells producing murine interleukin 2 (IL-2), IL-3, IL-4, IL-6, gamma-interferon, or granulocyte-macrophage colony stimulating factor (GM-CSF) and used these cells as subcutaneous vaccines against tumors within the brain. Under conditions where untransfected B16 cells had no effect, cells producing IL-3, IL-6, or GM-CSF increased the survival of mice challenged with viable B16 cells in the brain. Vaccination with B16 cells producing IL-4 or gamma-interferon had no effect, and vaccination with B16 cells producing IL-2 decreased survival time. GM-CSF-producing vaccines were also able to increase survival in mice with pre-established tumors. The response elicited by GM-CSF-producing vaccines was found to be specific to tumor type and to be abrogated by depletion of CD8+ cells. Unlike the immunity generated against subcutaneous tumors by GM-CSF, however, the effector responses generated against tumors in the CNS were not dependent on CD4+ cells. These data suggest that cytokine-producing tumor cells are very potent stimulators of immunity against tumors within the CNS, but effector responses in the CNS may be different from those obtained against subcutaneous tumors.

Negative electrostatic surface potential of protein sites specific for anionic ligands.

Determination of the crystal structure of an "open" unliganded active mutant (T141D) form of the Escherichia coli phosphate receptor for active transport has allowed calculation of the electrostatic surface potential for it and two other comparably modeled receptor structures (wild type and D137N). A discovery of considerable implication is the intensely negative potential of the phosphate-binding cleft. We report similar findings for a sulfate transport receptor, a DNA-binding protein, and, even more dramatically, redox proteins. Evidently, for proteins such as these, which rely almost exclusively on hydrogen bonding for anion interactions and electrostatic balance, a noncomplementary surface potential is not a barrier to binding. Moreover, experimental results show that the exquisite specificity and high affinity of the phosphate and sulfate receptors for unions are insensitive to modulations of charge potential, but extremely sensitive to conditions that leave a hydrogen bond donor or acceptor unpaired.

Targeting p53 as a general tumor antigen.

A major barrier to the design of immunotherapeutics and vaccines for cancer is the idiosyncratic nature of many tumor antigens and the possibility that T cells may be tolerant of broadly distributed antigens. We have devised an experimental strategy that exploits species differences in protein sequences to circumvent tolerance of high-affinity T cells. HLA transgenic mice were used to obtain cytotoxic T lymphocytes specific for peptides from the human p53 tumor-suppressor molecule presented in association with HLA-A2.1. Although such p53-specific cytotoxic T cells did not recognize nontransformed human cells, they were able to lyse a wide variety of human tumor cells lines, thus confirming the existence of broadly distributed determinants that may serve as targets for immunotherapy.

Homeologous recombination and mismatch repair during transformation in Streptococcus pneumoniae: saturation of the Hex mismatch repair system.

The ability of the Hex generalized mismatch repair system to prevent recombination between partially divergent (also called homeologous) sequences during transformation in Streptococcus pneumoniae was investigated. By using as donor in transformation cloned fragments 1.7-17.5% divergent in DNA sequence from the recipient, it was observed that the Hex system prevents chromosomal integration of the least and the most divergent fragments but frequently fails to do so for other fragments. In the latter case, the Hex system becomes saturated (inhibited) due to an excess of mismatches: it is unable to repair a single mismatch located elsewhere on the chromosome. Further investigation with chromosomal donor DNA, carrying only one genetically marked divergent region, revealed that a single divergent fragment can lead to saturation of the Hex system. Increase in cellular concentration of either HexA, the MutS homologue that binds mismatches, or HexB, the MutL homologue for which the essential role in repair as yet remains obscure, was shown to restore repair ability in previously saturating conditions. Investigation of heterospecific transformation by chromosomal DNA from two related streptococcal species, Streptococcus oralis and Streptococcus mitis, also revealed complete saturation of the Hex system. Therefore the Hex system is not a barrier to interspecies recombination in S. pneumoniae. These results are discussed in light of those described for the Mut system of Escherichia coli.

Oligogalacturonide defense signals in plants: large fragments interact with the plasma membrane in vitro.

Oligogalacturonides are plant cell wall-derived regulatory molecules which stimulate defense gene expression during pathogenesis. In vitro, these compounds enhance the phosphorylation of an approximately 34-kDa protein (pp34) in purified plasma membranes from potato and tomato leaves. We now show that polygalacturonate-enhanced phosphorylation of pp34 occurs in plasma membranes purified from tomato roots, hypocotyls, and stems and from undifferentiated potato cells. Furthermore, a similar phosphorylation is detected in leaf plasma membranes from soybean, a plant distantly related to tomato. Purified oligogalacturonides 13 to at least 26 residues long stimulate pp34 thiophosphorylation in vitro. This stimulation pattern differs from the induction of many known defense responses in vivo, where a narrower range of smaller fragments, between approximately 10 and 15 residues long, are active. On the basis of these differences we suggest that observed effects of applied exogenous oligogalacturonides on defense responses may not necessarily reflect the situation during pathogenesis. The cell wall could act as a barrier to many exogenous oligo- and polygalacturonides as well as other large regulatory ligands.

Teste anti-HIV entre homens que fazem sexo com homens em São Paulo: busca espontânea rotineira e episódica

Introdução No contexto da valorização crescente do teste anti-HIV como estratégia de prevenção programática, a promoção do teste anti-HIV como estratégia de prevenção entre homens que fazem sexo com homens (HSH) é fundamental. Objetivo - Analisar os fatores associados tanto ao uso rotineiro como episódico do teste anti-HIV. Métodos - Os participantes foram 946 HSH entrevistados pelo Projeto SampaCentro em locais de sociabilidade HSH da região central de São Paulo entre novembro de 2011 e janeiro de 2012, nunca testados ou que procuraram o teste espontaneamente. A metodologia de amostragem foi a time-space-sampling e foram utilizados protocolos do Stata 12.0 para análise de amostras complexas. Os homens que se testaram por rotina ou episodicamente foram comparados aos nunca testados. As variáveis analisadas nos dois modelos de regressão de Poisson foram divididas em três níveis: características sociodemográficas (primeiro nível); socialização na comunidade gay e exposição da orientação sexual, discriminação e opiniões e atitudes em relação ao HIV/Aids e ao teste (segundo nível); percepção de risco, estratégias de prevenção e práticas e parcerias sexuais (terceiro nível). Resultados Os homens que se testaram rotineiramente eram mais velhos e moradores no Centro de SP. Além disso, tinham exposto a orientação sexual para profissional de saúde, sido discriminados em serviços de saúde mas não por amigos e/ou vizinhos (em razão da sexualidade) e não mencionaram medo do resultado do teste como motivo para HSH não se testarem. Também tinham maior probabilidade de conhecer pessoa soropositiva e de ter parcerias estáveis sem sexo anal desprotegido nas casuais (comparado a ter apenas parcerias casuais protegidas). Os homens que se testaram episodicamente eram mais velhos, residentes do Centro de SP, não moravam com parentes, expuseram sua orientação sexual para profissional de saúde, não reportaram medo do resultado do teste como barreira, conheciam pessoa soropositiva e mencionaram parceria estável sem sexo desprotegido com parceiro casual ou então sexo desprotegido em parcerias casuais (comparado a ter apenas parcerias casuais protegidas). Conclusões Os mais jovens, os que moram fora do centro de São Paulo, e os que expões menos sua orientação sexual são os segmentos que menos se testam rotineira ou episodicamente. Assim, dependem de ações para que seu direito seja protegido e assegurado. A estigmatização e a discriminação da homossexualidade deve ser combatida para que não impeça o acesso ao teste e a outros serviços de saúde. Disseminar informações e socializar os mais jovens para o diálogo sobre as estratégias de prevenção biomédicas e estratégias comunitárias de prevenção é necessário. Para ampliar o acesso e qualidade da testagem como recurso fundamental de programas de prevenção permanece o desafio de sustentar o debate sobre sexualidade e prevenção a cada geração, assim como nos programas de formação de educadores e de profissionais de saúde de todas as áreas.

Comportamento social na adolescência: as normas e a rotina na regulação da conduta delituosa

O envolvimento de adolescentes com a prática de atos infracionais, no Brasil, ocupa espaço significativo no debate público. No entanto, tal debate carece de profundidade, pois pouco se relaciona ao conhecimento cientificamente produzido sobre o fenômeno. De acordo com a literatura acadêmica especializada no tema, um melhor conhecimento dos fatores associados à pratica de atos infracionais permitiria não só auxiliar na proposição de políticas públicas voltadas à prevenção deste problema, mas também no desenvolvimento de formas mais eficientes de intervenção, baseadas nas necessidades específicas apresentadas pelos adolescentes em conflito com a lei. Em meio aos diferentes fatores que devem ser pesquisados, no presente trabalho focalizam-se especificamente aqueles subentendidos sob o conceito de Normas e de Rotina, no referencial da Teoria da Regulação Social e Pessoal da Conduta, cujo autor principal é Marc Le Blanc. Divide-se assim o presente trabalho em dois estudos. O Estudo 1 trata de regulação normativa que opera por meio do mecanismo de socialização, e se refere à internalização, pelo adolescente, das normas sociais de conduta tidas como convencionais, o que promoveria um nível de constrangimento interno capaz de atuar como barreira ao envolvimento em atividades delituosas. Nesse sentido, maior adesão às normas, menos atitudes favoráveis ao comportamento divergente, mais atitudes de respeito a figuras de autoridade, maior percepção de risco de apreensão e menor utilização de técnicas de neutralização das barreiras psicológicas à emissão do comportamento indicariam um maior índice de constrangimento interno e, portanto, uma probabilidade reduzida de se engajar persistentemente em atividades divergentes/infracionais. O objetivo geral deste Estudo foi caracterizar a regulação da conduta em adolescentes pelas normas, no contexto sociocultural brasileiro. Utilizou-se o questionário de Normas proposto por Le Blanc, um questionário de caracterização sociodemográfica e a Entrevista de Delinquência Autorrevelada. Os dados foram coletados junto a 48 adolescentes Infratores e a 102 Escolares. Os resultados reforçam a importância do aspecto normativo para o melhor entendimento acerca dos fatores que explicam a conduta divergente em adolescentes. No Estudo 2 focalizou-se as atividades de rotina que podem se associar ao comportamento delituoso por meio do mecanismo de aprendizagem, na medida em que as diversas atividades nas quais o adolescente investe seu tempo constituem-se em contexto onde o comportamento divergente/infracional pode ser adquirido e reforçado. De acordo com a literatura, as atividades sem objetivos específicos, acompanhadas por pares de idade e que ocorrem na ausência de alguma figura de autoridade são aquelas que melhor explicam o comportamento delituoso de um adolescente. O objetivo geral deste Estudo foi caracterizar a regulação da conduta pela rotina em adolescentes, no contexto sociocultural brasileiro. Foram utilizados 3 instrumentos: o questionário de Rotina proposto por Le Blanc, um questionário de caracterização sociodemográfica e a Entrevista de Delinquência Autorrevelada. As análises foram feitas com base nas respostas de 102 adolescentes recrutados em escolas públicas. Os resultados comprovam a relevância das Atividades de Rotina como fator explicativo para o comportamento delituoso, com ênfase para os efeitos provocados pelos Pares, pela Família e pela frequentação de Lugares destinados aos adultos. Em síntese, ambos estudos reforçam a importância dos sistemas de regulação estudados e colocam em pauta a necessidade de outros trabalhos, que possam avançar nas questões apontadas dentro da Regulação pela Rotina e pelas Normas.