Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

Amphi-Eomes/Tbr1: An amphioxus cognate of vertebrate Eomesodermin and T-Brain1 genes whose expression reveals evolutionarily distinct domain in amphioxus development

A cDNA for a novel T-box containing gene was isolated from the amphioxus Branchiostoma belcheri. A molecular phylogenetic tree constructed from the deduced amino acid sequence of the isolated cDNA indicates that this gene belongs to the T-Brain subfamily. In situ hybridization reveals that the expression is first detected in the invaginating archenteron at the early gastrula stage and this expression is down-regulated at the neurula stage. In early larvae, the expression appears again and transcripts are detected exclusively in the pre-oral pit (wheel organ-Hatschek's pit of the adult). In contrast to the vertebrate counterparts, no transcripts are detected in the brain vesicle or nerve cord throughout the development. These results are interpreted to mean that a role of T-Brain products in vertebrate forebrain development was acquired after the amphioxus was split from the lineage leading to the vertebrates. On the other hand, comparison of the tissue-specific expression domain of T-Brain genes and other genes between amphioxus and vertebrates revealed that the pre-oral pit of amphioxus has several molecular features which are comparable to those of the vertebrate olfactory and hypophyseal placode. (C) 2002 Wiley-Liss, Inc.

情绪应激对大鼠体液免疫功能的影响及其机制

This study was undertaken to investigate the effect of emotional stress on humoral immunoactivity and to examine whether the sympathetic nervous system was involved in the immunomodulation. In the present study, two types of emotional stressors were used. One was footshock apparatus used to cause the rats which were given footshock before, emotional stressed; the other was an empty water bottle used to cause the rats which were trained to drink water at two set times each day, emotional stressed. The effect of emotional stress on the primary immune function (anti-ovallum antibody level and spleen index), the endocrine response (corticosterone level, epinephrine and norepinephrine level), the behavioral changes (freezing, defecation, grooming and attacking behavior) were investigated. The main results were: 1. Two types of emotional stress significantly increased the level of plasma corticosterone, norepinephrine and epinephrine, as well as freezing, defecation and attacking behavior. 2. Two types of emotional stress significantly decreased the level of anti-ovallum antibody. A negative correlation between catecholamine level (epinephrine and norepinephrine) and antibody level or spleen index was found. 3. β-adrenergic receptor antagonist propranolol could reverse the immunomodulation induced by emotional stress. 4. After two types of emotional stress, c-fos expression was observed in the following brain areas or nucleus; arcuate nucleus, anterior commissure nucleus, diffuse part of dorsalmedial nucleus hypothalamus, lateral dorsal nucleus thalamus, medial nucleus amygdala, solitary nucleus, frontal cortex and cingulum. These brain areas and nucleus are involved in the central modulation of the autonomic nervous system. Taken together, these findings demonstrate that emotional stress can suppress humoral immunity and the activation of the sympathetic nervous system is involved in the humoral immunomodulation induced by emotional stress.

Neonatal exposure to estradiol in female rats influences neuroactive steroid concentrations and behaviour in the adult rat

The gonadal steroids, in particular estradiol, exert an important action during perinatal period in the regulation of sexual dimorphism and neuronal plasticity, and in the growth and development of nervous system. Exposure of the developing female to estrogens during perinatal period may have long-lasting effects that are now regarded as “programming” the female neuroendocrine axis to malfunction in adulthood. The purpose of this study was to describe the effect of a single administration of a low dose (10 μg) of β-estradiol 3-benzoate (EB) to female rats on the day of birth on brain and plasma concentrations of the neuroactive steroid allopregnanolone, general behaviours and behavioral sensitivity to benzodiazepines. Neonatal administration of EB induces a dramatic reduction in the cerebrocortical and plasma levels of allopregnanolone and progesterone that were apparent in both juvenile (21 days) and adult (60 days). In contrast, this treatment did not affect 17β-estradiol levels. Female rats treated with β-estradiol 3-benzoate showed a delay in vaginal opening, aciclicity characterized by prolonged estrus, and ovarian failure. Given that allopregnanolone elicits anxiolytic, antidepressive, anticonvulsant, sedative-hypnotic effects and facilitates social behaviour, we assessed whether this treatment might modify different emotional, cognitive and social behaviours. This treatment did not affect locomotor activity, anxiety- and mood-related behaviours, seizures sensitivity and spatial memory. In contrast, neonatal β-estradiol 3-benzoate-treated rats showed a dominant, but not aggressive, behaviour and an increase in body investigation, especially anogenital investigation, characteristic of male appetitive behaviour. On the contrary, neonatal administration of β-estradiol 3-benzoate to female rats increases sensitivity to the anxiolytic, sedative, and amnesic effects of diazepam in adulthood. These results indicate that the marked and persistent reduction in the cerebrocortical and peripheral concentration of the neuroactive steroid allopregnanolone induced by neonatal treatment with β-estradiol 3-benzoate does not change baseline behaviours in adult rats. On the contrary, the low levels of allopregnanolone seems to be associated to changes in the behavioural sensitivity to the positive allosteric modulator of the GABAA receptor, diazepam. These effects of estradiol suggest that it plays a major role in pharmacological regulation both of GABAergic transmission and of the abundance of endogenous modulators of such transmission during development of the central nervous system.

Progettazione e sintesi di nuovi antagonisti CB1 a struttura pirazolica come potenziali radiofarmaci per la PET imaging

Cannabinoid receptors are members of the large family of G-protein coupled receptors. Two types of cannabinoid receptor have been discovered: CB1 and CB2. CB1 receptors are localised predominantly in the brain whereas CB2 receptors are more abundant in peripheral nervous system cells. CB1 receptors have been related with a number of disorders, including depression, anxiety, stress, schizophrenia, chronic pain and obesity. For this reason, several cannabinoid ligands were developed as drug candidates. Among these ligands, a prominent position is occupied by SR141716 (Rimonabant), which is a pyrazole derivative with inverse agonist activity discovered by Sanofi-Synthelabo in 1994. This compound was marketed in Europe as an anti-obesity drug, but subsequently withdrawn due to its side-effects. Since the relationship between the CB1 receptors’ functional modification, density and distribution, and the beginning of a pathological state is still not well understood, the development of radio-ligands suitable for in vivo PET (Positron Emission Tomography) functional imaging of CB1 receptors remains an important area of research in medicine and drug development. To date, a few radiotracers have been synthesised and tested in vivo, but most of them afforded unsatisfactory brain imaging results. A handful of radiolabelled CB1 PET ligands have also been submitted to clinical trials in humans. In this PhD Thesis the design, synthesis and characterization of three new classes of potential high-affinity CB1 ligands as candidate PET tracers is described.

Characterisation of the non-muscle α-actinins

Actinins are cytoskeleton proteins that cross-link actin filaments. Evolution of the actinin family resulted in the formation of Ca++-insensitive muscle isoforms (actinin-2 and- 3) and Ca++-sensitive non-muscle isoforms (actinin-1 and -4) with regard to their actin-binding function. Despite high sequence similarity, unique properties have been ascribed to actinin-4 compared with actinin-1. Actinin-4 is the predominant isoform reported to be associated with the cancer phenotype. Actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney and and is able to translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions I have comprehensively compared these proteins in terms of localisation, migration, alternative splicing, actin-binding properties, heterodimer formation and molecular interactions for the first time. This work characterises a number of commercially available actinin antibodies and in doing so, identifies actinin-1, -2 and -4 isoform-specific antibodies that enabled studies of actinin expression and localisation. This work identifies the actinin rod domain as the predominant domain that influences actinin localisation however localisation is likely to be effected by the entire actinin protein. si-RNA- mediated knockdown of actinin-1 and -4 did not affect migration in a number of cell lines highlighting that migration may only require a fraction of total non-muscle actinin levels. This work finds that the Ca++-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca++-insensitive variant of actinin-1. This work also identifies a previously unreported exon 19a+19b expressing variant of actinin-4 in human skeletal muscle. This work finds that alternative splice variants of actinin-1 and -4 are co-expressed in a number of tissues, in particular the brain. In contrast to healthy brain, glioblastoma cells express Ca++-sensitive variants of both actinin-1 and -4. Actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, this work reveals that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cancer cell lines. Taken together this data suggests that actinin-1 and -4 cannot be viewed as distinct entities from each other but rather as proteins that can exist in both homodimeric and heterodimeric forms. Finally, this work employs yeast two-hybrid and proteomic approaches to identify actinin-interacting proteins. In doing so, this work identifies a number of putative actinin-4 specific interacting partners that may help to explain some of the unique functions attributed the actinin-4. The observation of alternative splice variants of actinin-1 and -4 combined with the observed potential of these proteins to form homodimers and heterodimers suggests that homodimers and heterodimers with novel actin-binding properties and interaction networks may exist. The ability to behave in this manner may have functional implications. This may be of importance considering that these proteins are central to such processes as cell migration and adhesion. This significantly alters our view of the non-muscle actinins.

Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.

Serotonin synthesis, release and reuptake in terminals: a mathematical model.

BACKGROUND: Serotonin is a neurotransmitter that has been linked to a wide variety of behaviors including feeding and body-weight regulation, social hierarchies, aggression and suicidality, obsessive compulsive disorder, alcoholism, anxiety, and affective disorders. Full understanding of serotonergic systems in the central nervous system involves genomics, neurochemistry, electrophysiology, and behavior. Though associations have been found between functions at these different levels, in most cases the causal mechanisms are unknown. The scientific issues are daunting but important for human health because of the use of selective serotonin reuptake inhibitors and other pharmacological agents to treat disorders in the serotonergic signaling system. METHODS: We construct a mathematical model of serotonin synthesis, release, and reuptake in a single serotonergic neuron terminal. The model includes the effects of autoreceptors, the transport of tryptophan into the terminal, and the metabolism of serotonin, as well as the dependence of release on the firing rate. The model is based on real physiology determined experimentally and is compared to experimental data. RESULTS: We compare the variations in serotonin and dopamine synthesis due to meals and find that dopamine synthesis is insensitive to the availability of tyrosine but serotonin synthesis is sensitive to the availability of tryptophan. We conduct in silico experiments on the clearance of extracellular serotonin, normally and in the presence of fluoxetine, and compare to experimental data. We study the effects of various polymorphisms in the genes for the serotonin transporter and for tryptophan hydroxylase on synthesis, release, and reuptake. We find that, because of the homeostatic feedback mechanisms of the autoreceptors, the polymorphisms have smaller effects than one expects. We compute the expected steady concentrations of serotonin transporter knockout mice and compare to experimental data. Finally, we study how the properties of the the serotonin transporter and the autoreceptors give rise to the time courses of extracellular serotonin in various projection regions after a dose of fluoxetine. CONCLUSIONS: Serotonergic systems must respond robustly to important biological signals, while at the same time maintaining homeostasis in the face of normal biological fluctuations in inputs, expression levels, and firing rates. This is accomplished through the cooperative effect of many different homeostatic mechanisms including special properties of the serotonin transporters and the serotonin autoreceptors. Many difficult questions remain in order to fully understand how serotonin biochemistry affects serotonin electrophysiology and vice versa, and how both are changed in the presence of selective serotonin reuptake inhibitors. Mathematical models are useful tools for investigating some of these questions.

Cryptococcal cell morphology affects host cell interactions and pathogenicity.

Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 microm. Cell enlargement was observed in vivo, producing cells up to 100 microm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aDelta pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.

Monitoring maternal Beta carotene and retinol consumption may decrease the incidence of neurodevelopmental disorders in offspring.

Retinoic acids (13-cis and 13-trans) are known teratogens, and their precursor is retinol, a form of vitamin A. In 1995, Rothman et al demonstrated an association between excessive vitamin A, >10,000 IU/day, during the first trimester of pregnancy and teratogenic effects, particularly in the central nervous system. However, vitamin A deficiency has long been known to be deleterious to the mother and fetus. Therefore, there may be a narrow therapeutic ratio for vitamin A during pregnancy that has not previously been fully appreciated. Neurodevelopmental disorders may not be apparent by macroscopic brain examination or imaging, and proving the existence of a behavioral teratogen is not straightforward. However, an excess of retinoic acid and some neurodevelopmental disorders are both associated with abnormalities in cerebellar morphology. Physical and chemical evidence strongly supports the notion that beta carotene crosses the placenta and is metabolized to retinol. Only very limited amounts of beta carotene are stored in fetal fat cells as evidenced by the fact that maternal fat is yellow from beta carotene, whereas non-brown neonatal fat is white. Furthermore, newborns of carotenemic mothers do not share the yellow complexion of their mothers. The excess 13-trans retinoic acid derived from metabolized beta carotene in the fetus increases the concentration of the more teratogenic 13-cis retinoic acid since the isomerization equilibrium is shifted to the left. Therefore, this paper proposes that consideration be given to monitoring all potential sources of fetal 13-cis and 13-trans retinoic acid, including nutritional supplements, dietary retinol, and beta carotene, particularly in the first trimester of pregnancy.

Chronic opioid therapy and opioid tolerance: a new hypothesis.

Opioids are efficacious and cost-effective analgesics, but tolerance limits their effectiveness. This paper does not present any new clinical or experimental data but demonstrates that there exist ascending sensory pathways that contain few opioid receptors. These pathways are located by brain PET scans and spinal cord autoradiography. These nonopioid ascending pathways include portions of the ventral spinal thalamic tract originating in Rexed layers VI-VIII, thalamocortical fibers that project to the primary somatosensory cortex (S1), and possibly a midline dorsal column visceral pathway. One hypothesis is that opioid tolerance and opioid-induced hyperalgesia may be caused by homeostatic upregulation during opioid exposure of nonopioid-dependent ascending pain pathways. Upregulation of sensory pathways is not a new concept and has been demonstrated in individuals impaired with deafness or blindness. A second hypothesis is that adjuvant nonopioid therapies may inhibit ascending nonopioid-dependent pathways and support the clinical observations that monotherapy with opioids usually fails. The uniqueness of opioid tolerance compared to tolerance associated with other central nervous system medications and lack of tolerance from excess hormone production is discussed. Experimental work that could prove or disprove the concepts as well as flaws in the concepts is discussed.

Induction of anti-myelin antibodies in EAE and their possible role in demyelination.

Experimental allergic encephalomyelitis is characterized by invasion of lymphocytes and macrophages into the central nervous system resulting in inflammation, edema, and demyelination. Sera from Lewis rats from 7-95 days after immunization with purified guinea pig CNS myelin were examined with respect to their ability to opsonize myelin. This was correlated with the appearance of antibody components and the relative amounts of antibody to myelin basic protein (MBP) and proteolipid protein (PLP). Sera from rats 10-95 days after immunization preincubated with purified myelin induced phagocytosis of myelin by cultured macrophages with the resulting production of cholesterol ester. This opsonization activity as measured by the percentage of cholesterol esterified reached a peak at 26-27 days after immunization but remained significantly elevated up to 95 days post-immunization compared to the activity of serum from the Freund's adjuvant-injected controls. Immunoblots of the sera revealed a gradual increase in antibody activity against myelin components. ELISA assays for MBP and PLP antibody showed a similar pattern. Antibody to galactocerebroside (GC) was not detected by immunostains nor by the ELISA assay. Areas of demyelination were observed histologically by luxol-fast blue stained spinal cords up to 60 days post-immunization. These results indicate that antibodies to myelin protein when given access to myelin through or within the blood brain barrier could initiate or enhance the phagocytic response by peripheral or resident macrophages.

Low temperature X-ray structures of two crystalline forms (I) and (II) of the pyrimidine derivative and sodium channel blocker BW1003C87: 5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine

The X-ray crystal structures of two crystalline forms of 5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine, C10H7Cl3N4 (code name BW1003C87) (I) and (II), have been carried out at liquid nitrogen temperature. A detailed comparison of the two structures is given. Both are centrosymmetric, with structure (I) in the triclinic space group P (1) over bar unit cell a = 6.4870(10), b = 9.216(2), c = 12.016(2) angstrom, alpha = 75.78(3)degrees, beta = 89.95(3)degrees, gamma = 83.45(3)degrees, V = 691.5(2) angstrom(3), Z = 2 and density (calculated) = 1.544 Mg/m(3); and (II) in the monoclinic space group P2(1)/c, unit cell a = 12.000(2), b = 7.518(2), c = 13.450(3) angstrom, beta = 97.87(3)degrees, V = 1202.0(5) angstrom(3), Z = 4, Density (calculated) = 1.600 Mg/m(3). Structure (I) includes a solvated CH3OH in the lattice. Final R indices [I > 2sigma(I)] are R1 = 0.0427, wR2 = 0.1075 for (I) and R1 = 0.0487, wR2 = 0.1222 for (II). R indices (all data) are R1 = 0.0470, wR2 = 0.1118 for (I) and R1 = 0.0623, wR2 = 0.1299 for (II). 5-Phenyl-2,4 diaminopyrimidine and 6-phenyl-1,2,4 triazine derivatives, which include lamotrigine (3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine), have been investigated for some time for their effects on the central nervous system. Both lamotrigine and 5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine (code name BW1003C87), the subject of the present study, are anticonvulsant as well as neuroprotective in models of brain ischaemia and in a model of white matter ischaemia. BW1003C87 is a sodium channel blocker which also reduces the release of the neurotransmitter glutamate. The three dimensional structures reported here form part of a newly developed data base for the detailed investigation of members of this drug family and their biological activities.

Predisposition to arrhythmia and autonomic dysfunction in Nhlh1-deficient mice.

Nhlh1 is a basic helix-loop-helix transcription factor whose expression is restricted to the nervous system and which may play a role in neuronal differentiation. To directly study Nhlh1 function, we generated null mice. Homozygous mutant mice were predisposed to premature, adult-onset, unexpected death. Electrocardiograms revealed decreased total heart rate variability, stress-induced arrhythmia, and impaired baroreceptor sensitivity. This predisposition to arrhythmia is a likely cause of the observed death in the mutant mice. Heterozygosity for the closely related transcription factor Nhlh2 increased the severity of the Nhlh1-null phenotype. No signs of primary cardiac structural or conduction abnormalities could be detected upon necropsy of the null mice. The pattern of altered heart rhythm observed in basal and experimental conditions (stress and pharmacologically induced) suggests that a deficient parasympathetic tone may contribute to the arrhythmia in the Nhlh1-null mouse. The expression of Nhlh1 in the developing brain stem and in the vagal nuclei in the wild-type mouse further supports this hypothesis. The Nhlh1 mutant mouse may thus provide a model to investigate the contribution of the autonomic nervous system to arrhythmogenesis.

Monocyte-derived dendritic cells: a potential target for therapy in multiple sclerosis (MS)

Monocytes can differentiate into dendritic cells (DC), cells with a pivotal role in both protective immunity and tolerance. Defects in the maturation or function of DC may be important in the development of autoimmune disease. We sought to establish if there were differences in the cytokine (granulocyte-macrophage colony-stimulating factor and IL-4)-driven maturation of monocytes to DC in patients with MS and whether drugs used to treat MS affected this process in vitro. We have demonstrated that there is no defect in the ability of magnetic activated cell sorting (MACS)-purified monocytes from patients with MS to differentiate to DC, but equally they show no tendency to acquire a DC phenotype without exogenous cytokines. Interferon-beta1a prevents the acquisition of a full DC phenotype as determined by light and electron microscopy and by flow cytometry. Methylprednisolone not only prevents the development of monocyte-derived DC but totally redirects monocyte differentiation towards a macrophage phenotype. Evidence is evolving for a role for DC in central nervous system immunity, either within the brain or in cervical lymph nodes. The demonstrated effect of both drugs on monocyte differentiation may represent an important site for immune therapy in MS.