Showing papers by "Karolinska Institutet published in 1997"

Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta

Abstract: The rat estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes. Saturation ligand binding analysis of in vitro synthesized human ER alpha and rat ER beta protein revealed a single binding component for 16 alpha-iodo-17 beta-estradiol with high affinity [dissociation constant (Kd) = 0.1 nM for ER alpha protein and 0.4 nM for ER beta protein]. Most estrogenic substances or estrogenic antagonists compete with 16 alpha-[125I]iodo-17 beta-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol > hexestrol > dienestrol > 4-OH-tamoxifen > 17 beta-estradiol > coumestrol, ICI-164384 > estrone, 17 alpha-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3 beta, 17 beta-diol, genistein for the ER alpha protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17 beta-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3 beta, 17 beta-diol > 17 alpha-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ER beta protein. The rat tissue distribution and/or the relative level of ER alpha and ER beta expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER alpha and prostate, ovary, lung, bladder, brain, uterus, and testis for ER beta. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.

Molecular basis of agonism and antagonism in the oestrogen receptor.

Abstract: Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17beta-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 A, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.

Dopamine neuron agenesis in Nurr1-deficient mice.

Abstract: Dopamine neurons of the substantia nigra and ventral tegmental area regulate movement and affective behavior and degenerate in Parkinson's disease. The orphan nuclear receptor Nurr1 was shown to be expressed in developing dopamine neurons before the appearance of known phenotypic markers for these cells. Mice lacking Nurr1 failed to generate midbrain dopaminergic neurons, were hypoactive, and died soon after birth. Nurr1 expression continued into adulthood, and brains of heterozygous animals, otherwise apparently healthy, contained reduced dopamine levels. These results suggest that putative Nurr1 ligands may be useful for treatment of Parkinson's disease and other disorders of midbrain dopamine circuitry.

Human estrogen receptor beta-gene structure, chromosomal localization, and expression pattern.

Abstract: The estrogen receptor (ER) is a ligand-activated transcription factor that mediates the effects of the steroid hormone 17 beta-estradiol, in both males and females. Since the isolation and cloning of ER, the consensus has been that only one such receptor exists. The finding of a second subtype of ER (ER beta) has caused considerable excitement amongst endocrinologists. In this article, we present data regarding the genomic structure and chromosomal localization of the human ER beta gene, demonstrating that two independent ER genes do exist in the human. Furthermore, we present data regarding the tissue distribution of human ER beta, showing that this receptor is expressed in multiple tissues. For instance, ER beta is found in developing spermatids of the testis, a finding of potential relevance for the ongoing debate on the effects of environmental estrogens on sperm counts. In addition, we find ER beta in ovarian granulosa cells, indicating that estrogens also participate in the regulation of follicular growth in the human.

Tissue Distribution and Quantitative Analysis of Estrogen Receptor-α (ERα) and Estrogen Receptor-β (ERβ) Messenger Ribonucleic Acid in the Wild-Type and ERα-Knockout Mouse

Abstract: Until recently, only a single type of estrogen receptor (ER) was thought to exist and mediate the genomic effects of the hormone 17beta-estradiol in mammalian tissues. However, the cloning of a gene encoding a second type of ER, termed ERbeta, from the mouse, rat, and human has prompted a reevaluation of the estrogen signaling system. Based on in vitro studies, the ERbeta protein binds estradiol with an affinity similar to that of the classical ER (now referred to as ERalpha) and is able to mediate the effects of estradiol in transfected mammalian cell lines. Essential to further investigations of the possible physiological roles of ERbeta, and its possible interactions with ERalpha, are data on the tissue distribution of the two ER types. Herein, we have described the optimization and use of an RNase protection assay able to detect and distinguish messenger RNA (mRNA) transcripts from both the ERalpha and ERbeta genes in the mouse. Because this assay is directly quantitative, a comparison of the levels of expression within various tissues was possible. In addition, the effect of disruption of the ERalpha gene on the expression of the ERbeta gene was also investigated using the ERalpha-knockout (ERKO) mouse. Transcripts encoding ERalpha were detected in all the wild-type tissues assayed from both sexes. In the female reproductive tract, the highest expression of ERbeta mRNA was observed in the ovary and showed great variation among individual animals; detectable levels were observed in the uterus and oviduct, whereas mammary tissue was negative. In the male reproductive tract, significant expression of ERbeta was seen in the prostate and epididymis, whereas the testes were negative. In other tissues of both sexes, the hypothalamus and lung were clearly positive for both ERalpha and ERbeta mRNA. The ERKO mice demonstrated slightly reduced levels of ERbeta mRNA in the ovary, prostate, and epididymis. These data, in combination with the several described phenotypes in both sexes of the ERKO mouse, suggest that the biological functions of the ERbeta protein may be dependent on the presence of ERalpha in certain cell types and tissues. Further characterization of the physiological phenotypes in the ERKO mice may elucidate possible ERbeta specific actions.

Proteolytic processing regulates receptor specificity and activity of VEGF-C

Abstract: The recently identified vascular endothelial growth factor C (VEGF-C) belongs to the platelet-derived growth factor (PDGF)/VEGF family of growth factors and is a ligand for the endothelial-specific receptor tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF homology domain spans only about one-third of the cysteine-rich VEGF-C precursor. Here we have analysed the role of post-translational processing in VEGF-C secretion and function, as well as the structure of the mature VEGF-C. The stepwise proteolytic processing of VEGF-C generated several VEGF-C forms with increased activity towards VEGFR-3, but only the fully processed VEGF-C could activate VEGFR-2. Recombinant 'mature' VEGF-C made in yeast bound VEGFR-3 (K[D] = 135 pM) and VEGFR-2 (K[D] = 410 pM) and activated these receptors. Like VEGF, mature VEGF-C increased vascular permeability, as well as the migration and proliferation of endothelial cells. Unlike other members of the PDGF/VEGF family, mature VEGF-C formed mostly non-covalent homodimers. These data implicate proteolytic processing as a regulator of VEGF-C activity, and reveal novel structure-function relationships in the PDGF/VEGF family.

Comparative pharmacology of human adenosine receptor subtypes – characterization of stably transfected receptors in CHO cells

Abstract: Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A1, A2A, A2B and A3 are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B). The A1 subtype showed the typical pharmacological profile with 2-chloro-N6-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N6-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadeno sine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC50-value of 2.3 microM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine) as the most potent antagonist at this receptor subtype. The A3 receptor was characterized utilizing the nonselective agonist [3H]NECA. The N6-benzyl substituted derivatives of adenosine-5'-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A3 receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands.

Enamel matrix, cementum development and regeneration

Abstract: Studies during the last 20 years have indicated that enamel-related proteins are involved in the formation of cementum. In the present article, this relation is further explored. Attention is called to the fact that coronal acellular extrinsic fiber cementum is formed on the enamel surface in a number of species. The composition of the enamel matrix proteins and the expression of these proteins during root formation are briefly reviewed. The dominating constituent of the enamel matrix, amelogenin, is shown by means of immunohistochemistry to be expressed in human teeth during root formation. Amelogenin was also found to be present in Tomes' granular layer of human teeth. When mesenchymal cells of the dental follicle were exposed to the enamel matrix a non-cellular hard tissue matrix was formed at the enamel surface. Application of porcine enamel matrix in experimental cavities in the roots of incisors of monkeys induced formation of acellular cementum that was well attached to the dentin. In control cavities without enamel matrix, a cellular, poorly attached hard tissue was formed. The present studies provide additional support to the idea that enamel matrix proteins are involved in the formation of acellular cementum and also that they have the potential to induce regeneration of the same type of cementum.

In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression.

Abstract: Following the identification of the C-C chemokines RANTES, MIP-1alpha and MIP-1beta as major human immunodeficiency virus (HIV)-suppressive factors produced by CD8+ T cells, several chemokine receptors were found to serve as membrane co-receptors for primate immunodeficiency lentiretroviruses. The two most widely used co-receptors thus far recognized, CCR5 and CXCR4, are expressed by both activated T lymphocytes and mononuclear phagocytes. CCR5, a specific RANTES, MIP-1alpha and MIP-1 receptor, is used preferentially by non-MT2-tropic HIV-1 and HIV-2 strains and by simian immunodeficiency virus (SIV), whereas CXCR4, a receptor for the C-X-C chemokine SDF-1, is used by MT2-tropic HIV-1 and HIV-2, but not by SIV. Other receptors with a more restricted cellular distribution, such as CCR2b, CCR3 and STRL33, can also function as co-receptors for selected viral isolates. The third variable region (V3) of the gp120 envelope glycoprotein of HIV-1 has been fingered as a critical determinant of the co-receptor choice. Here, we document a consistent pattern of evolution of viral co-receptor usage and sensitivity to chemokine-mediated suppression in a longitudinal follow-up of children with progressive HIV-1 infection. Viral isolates obtained during the asymptomatic stages generally used only CCR5 as a co-receptor and were inhibited by RANTES, MIP-1alpha and MIP-1beta, but not by SDF-1. By contrast, the majority of the isolates derived after the progression of the disease were resistant to C-C chemokines, having acquired the ability to use CXCR4 and, in some cases, CCR3, while gradually losing CCR5 usage. Surprisingly, most of these isolates were also insensitive to SDF-1, even when used in combination with RANTES. An early acquisition of CXCR4 usage predicted a poor prognosis. In children who progressed to AIDS without a shift to CXCR4 usage, all the sequential isolates were CCR5-dependent but showed a reduced sensitivity to C-C chemokines. Discrete changes in the V3 domain of gp120 were associated with the loss of sensitivity to C-C chemokines and the shift in co-receptor usage. These results suggest an adaptive evolution of HIV-1 in vivo, leading to escape from the control of the antiviral C-C chemokines.

Hospital Anxiety and Depression Scale (HAD): some psychometric data for a Swedish sample

Abstract: The Hospital Anxiety and Depression Scale (HAD) was evaluated in a Swedish population sample. The purpose of the study was to compare the HAD with the Beck Depression Inventory (BDI) and Spielberger's State Trait Anxiety Inventory (STAI). A secondary aim was to examine the factor structure of the HAD. The results indicated that the factor structure was quite strong, consistently showing two factors in the whole sample as well as in different subsamples. The correlations between the total HAD scale and BDI and STAI, respectively, were stronger than those obtained using the different subscales of the HAD (the anxiety and depression subscales). As expected, there was also a stronger correlation between the HAD and the non-physical items of the BDI. It was somewhat surprising that the factor analyses were consistently extracting two factors, 'depression' and 'anxiety', while on the other hand both BDI and STAI tended to correlate more strongly with the total HAD score than with the specific depression and anxiety HAD subscales. Nevertheless, the HAD appeared to be (as was indeed originally intended) a useful clinical indicator of the possibility of depression and clinical anxiety.

Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype.

Abstract: The biological phenotype of primary human immunodeficiency virus type 1 (HIV-1) isolates varies according to the severity of the HIV infection. Here we show that the two previously described groups of rapid/high, syncytium-inducing (SI) and slow/low, non-syncytium-inducing (NSI) isolates are distinguished by their ability to utilize different chemokine receptors for entry into target cells. Recent studies have identified the C-X-C chemokine receptor CXCR4 (also named fusin or Lestr) and the C-C chemokine receptor CCR5 as the principal entry cofactors for T-cell-line-tropic and non-T-cell-line-tropic HIV-1, respectively. Using U87.CD4 glioma cell lines, stably expressing the chemokine receptor CCR1, CCR2b, CCR3, CCR5, or CXCR4, we have tested chemokine receptor specificity for a panel of genetically diverse envelope glycoprotein genes cloned from primary HIV-1 isolates and have found that receptor usage was closely associated with the biological phenotype of the virus isolate but not the genetic subtype. We have also analyzed a panel of 36 well-characterized primary HIV-1 isolates for syncytium induction and replication in the same series of cell lines. Infection by slow/low viruses was restricted to cells expressing CCR5, whereas rapid/high viruses could use a variety of chemokine receptors. In addition to the regular use of CXCR4, many rapid/high viruses used CCR5 and some also used CCR3 and CCR2b. Progressive HIV-1 infection is characterized by the emergence of viruses resistant to inhibition by beta-chemokines, which corresponded to changes in coreceptor usage. The broadening of the host range may even enable the use of uncharacterized coreceptors, in that two isolates from immunodeficient patients infected the parental U87.CD4 cell line lacking any engineered coreceptor. Two primary isolates with multiple coreceptor usage were shown to consist of mixed populations, one with a narrow host range using CCR5 only and the other with a broad host range using CCR3, CCR5, or CXCR4, similar to the original population. The results show that all 36 primary HIV-1 isolates induce syncytia, provided that target cells carry the particular coreceptor required by the virus.

Mouse estrogen receptor beta forms estrogen response element-binding heterodimers with estrogen receptor alpha.

Abstract: The recent discovery that an additional estrogen receptor subtype is present in various rat tissues has advanced our understanding of the mechanisms underlying estrogen signaling. Here we report on the cloning of the cDNA encoding the mouse homolog of estrogen receptor-beta (ER beta) and the functional characterization of mouse ER beta protein. ER beta is shown to have overlapping DNA-binding specificity with that of the estrogen receptor-alpha (ER alpha) and activates transcription of reporter gene constructs containing estrogen-response elements in transient transfections in response to estradiol. Using a mammalian two-hybrid system, the formation of heterodimers of the ER beta and ER alpha subtypes was demonstrated. Furthermore, ER beta and ER alpha form heterodimeric complexes with retained DNA-binding ability and specificity in vitro. In addition, DNA binding by the ER beta/ER alpha heterodimer appears to be dependent on both subtype proteins. Taken together these results suggest the existence of two previously unrecognized pathways of estrogen signaling; I, via ER beta in cells exclusively expressing this subtype, and II, via the formation of heterodimers in cells expressing both receptor subtypes.

Gender differences in episodic memory

Abstract: The relationship between gender and memory has been largely neglected by research, despite occasional studies reporting gender differences in episodic memory performance. The present study examined potential gender differences in episodic memory, semantic memory, primary memory, and priming. Five hundred thirty women and 470 men, randomly sampled from the city of Umea, Sweden, 35–80 years of age, participated in the study. There were no differences between men and women with regard to age or education, or on a measure of global intellectual functioning. As has been demonstrated previously, men outperformed women on a visuospatial task and women outperformed men on tests of verbal fluency. In addition, the results demonstrated that women consistently performed at a higher level than did men on the episodic memory tasks, although there were no differences between men and women on the tasks assessing semantic memory, primary memory, or priming. The women’s higher level of performance on the episodic memory tasks could not be fully explained by their higher verbal ability.

The betula prospective cohort study: Memory, health, and aging

Abstract: The objective of this article is to present an overview of a prospective cohort study involving a total of 3,000 subjects whose ages were 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80 years when first tested. the design of the study includes three waves of data collection. the first of these waves was conducted in 1988-1990, the second in 1993-1995, and the third will be conducted in 1998-2000. One sample of 1,000 subjects in these age cohorts underwent testing in 1988-1990 (100 subjects per cohort). This sample and two additional samples were tested in 1993-1995 and will be tested again in 1998-2000. Subjects take part in extensive health and memory examinations, and interviews about social factors. the memory testing covers a wide range of memory functions. the chief objectives of the study are to (a) examine the development of health and memory in adulthood and old age; (b) determine early preclinical signs of dementia; (c) determine risk factors for dementia; and (d) assess premorbid memory func...

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1

Abstract: The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins.

Abstract: There is increasing evidence that cells of the epithelial root sheath synthesize enamel matrix proteins and that these proteins play a fundamental role in the formation of acellular cementum, the key tissue in the development of a functional periodontium. The purpose of the present study was to explore the effect of locally applied enamel matrix and different protein fractions of the matrix on periodontal regeneration in a buccal dehiscence model in monkeys. Buccal, mucoperiosteal flaps were raised from the canine to the 1st molar on each side of the maxilla. The buccal alveolar bone plate, the exposed periodontal ligament and cementum were removed. Various preparations of porcine enamel matrix with or without vehicles were applied before the flaps were repositioned and sutured. After 8 weeks, the healing was evaluated in the light microscope, and morphometric comparisons were made. Application of homogenized enamel matrix or an acidic extract of the matrix containing the hydrophobic, low molecular weight proteins, amelogenins, resulted in an almost complete regeneration of acellular cementum, firmly attached to the dentin and with collagenous fibers extending over to newly formed alveolar bone. After application of fractions obtained by neutral EDTA extraction containing the acidic, high molecular weight proteins of the enamel matrix, very little new cementum was formed and hardly any new bone. The results of the controls in which no test substance was applied before the repositioning of the flap, were very similar to those obtained with the EDTA extracted material. Propylene glycol alginate (PGA), hydroxyethyl cellulose and dextran were tried as vehicles for the enamel matrix preparations. Only PGA in combination with the amelogenin fraction resulted in significant regeneration of the periodontal tissues.

Modulatory influence on somatosensory perception from vibration and heterotopic noxious conditioning stimulation (HNCS) in fibromyalgia patients and healthy subjects

Abstract: In order to assess the function of endogenous mechanisms modulating somatosensory input in fibromyalgia (FM), the effect of vibratory stimulation (VS) and heterotopic noxious conditioning stimulation (HNCS) on perception of various somatosensory modalities was assessed. Ten female FM patients and 10 healthy, age-matched, females participated. VS (100 Hz) was applied to the left forearm for 45 min and quantitative sensory testing (QST) was performed within the vibrated area and in the right thigh before, during and 45 min following vibration. Pressure pain thresholds (PPTs) were assessed by pressure algometry. Perception thresholds to non-painful cold (CT) and warmth (WT), heat pain thresholds (HPTs), cold pain thresholds (CPTs) and stimulus-response curves of pain intensity as a function of graded nociceptive heat stimulation were assessed using a Peltier element based thermal stimulator. The effects of HNCS were tested using the upper extremity submaximal effort tourniquet test. Subjects rated tourniquet induced pain intensity on a visual analogue scale (VAS). QST was performed in the right thigh before, during and 60 min following the tourniquet. FM patients did not differ from controls in the response to VS. There was a local increase of PPTs during vibration (P < 0.001) and of WTs following vibration (P < 0.001). HPTs increased in the forearm and in the thigh (P < 0.009) during vibration. CTs and sensitivity to suprathreshold heat pain were not influenced by VS. The intensity of pain induced by the tourniquet did not differ between groups. PPTs increased during the tourniquet in controls (P < 0.001) but not in FM patients (difference between groups P < 0.001). Decreased sensitivity to non-painful cold (P < 0.001) and non-painful warmth (P < 0.001) was seen during and following (P < 0.001; P < 0.05, respectively) the tourniquet in both groups alike. HPTs and perception of suprathreshold heat pain remained unaffected in both groups. In conclusion, FM patients did not differ from healthy controls in their response to vibration, but no modulation of pressure pain was induced by HNCS, as opposed to controls, suggesting a dysfunction in systems subserving 'diffuse noxious inhibitory controls' (DNIC).

Complementary and overlapping expression of glial cell line-derived neurotrophic factor (GDNF), c-ret proto-oncogene, and GDNF receptor-alpha indicates multiple mechanisms of trophic actions in the adult rat CNS.

Abstract: Glial cell line-derived neurotrophic factor (GDNF), the most potent trophic factor yet described for both dopaminergic neurons of the substantia nigra and spinal motorneurons, has recently been shown to signal through a multireceptor complex composed of a novel glycosylphosphatidylinositol-anchored GDNF receptor-alpha (GDNFR-alpha) and the receptor tyrosine kinase product of the c-ret proto-oncogene (RET). Despite its importance, the individual expression patterns and the relationships between domains of expression of the different components of this trophic system are not understood. We show here by in situ hybridization that GDNF mRNA is expressed in the normal adult rat brain in several targets of substantia nigra neurons, including striatum, nucleus accumbens, thalamic nuclei, olfactory tubercle, hippocampus, cerebellum, and cingulate cortex as well as in the internal granular cell layer of the olfactory bulb. Within the basal ganglia we observe a pronounced segregation of regions expressing GDNF from those expressing GDNF receptors, suggesting that within these structures GDNF is functioning in its anticipated role as a target-derived trophic factor. In addition, the expression of GDNF and both GDNF receptors within the cerebellum, hippocampus, and olfactory bulb may indicate a paracrine mode of action. Importantly, we also see expression of RET mRNA in cellular populations within the cerebellum and the glomerular layer of the olfactory bulb, as well as in the subthalamic nucleus, which lack GDNFR-alpha expression, indicating that RET functions either independently of GDNFR-alpha or with GDNFR-alpha presented in trans. Conversely, GDNFR-alpha is widely expressed in many regions in which RET expression is absent, suggesting that GDNFR-alpha may associate with additional signaling receptors. Finally, RET and GDNFR-alpha show distinct patterns of regulated expression in the brain after kainic acid stimulation and in the sciatic nerve after nerve transection. Taken together these findings indicate that GDNF, RET, and GDNFR-alpha utilize multiple mechanisms to comprise physiologically relevant trophic circuits for different neuronal populations.

Ablation of the CDK inhibitor p57Kip2 results in increased apoptosis and delayed differentiation during mouse development.

Abstract: p57(Kip2) is a paternally imprinted gene that encodes a potent inhibitor of several cyclin/Cdk complexes. p57(Kip2) is primarily expressed in terminally differentiated cells, associates with G1 Cdks, and can cause cell cycle arrest in G1 phase. To investigate the role of p57(Kip2) in vivo, we have ablated the p57(Kip2) gene by homologous recombination in ES cells and generated mice devoid of p57(Kip2) expression. Most p57(Kip2) null mice die after birth and display severe developmental defects with varying degrees of penetrance. As expected, heterozygous mice that inherit a maternal, but not a paternal, targeted allele exhibit similar deficiencies and neonatal death. Developmental defects of p57(Kip2) mutant mice include cleft palate and gastrointestinal abnormalities ranging from an inflated GI tract to loss of the jejunum and ileum. These tissues display a significant increase of apoptotic cells in the absence of p57(Kip2). Most p57(Kip2) mutant mice have short limbs, a defect attributable to abnormal endochondral ossification caused by delayed cell cycle exit during chondrocyte differentiation. A similar defect has been observed in mice lacking p107 and p130, thus suggesting that p57(Kip2) might be an upstream regulator of these Rb-related proteins. The p57(Kip2) locus has been implicated in the Beckwith-Wiedemann syndrome and in the development of sporadic Wilms' tumors and lung carcinomas. To date, we have not observed neoplastic development even in those p57(Kip2) mutant mice that have survived for >5 months of age. These findings indicate that p57(Kip2) has an important role during mouse development that cannot be compensated by other Cdk inhibitors.

Aryl hydrocarbon receptor-mediated signal transduction.

Abstract: The aryl hydrocarbon (or dioxin) receptor (AhR) is a ligand-activated basic helix-loop-helix (bHLH) protein that heterodimerizes with the bHLH protein ARNT (aryl hydrocarbon nuclear translocator) forming a complex that binds to xenobiotic regulatory elements in target gene enhancers. Genetic, biochemical, and molecular biology studies have revealed that the AhR mediates the toxic and biological effects of environmentally persistent dioxins and related compounds. Cloning of the receptor and its DNA-binding partner, ARNT, has facilitated detailed efforts to understand the mechanisms of AhR-mediated signal transduction. These studies have determined that this unique receptor consists of several functional domains and belongs to a subfamily of bHLH proteins that share a conserved motif termed the PAS domain. In addition, recent genetic studies have revealed that expression of the AhR is a requirement for proper embryonal development, which appears to be a common function shared by many other bHLH proteins. This review is a summary of recent molecular studies of AhR-mediated gene regulation.

Volume and number of neurons of the human hippocampal formation in normal aging and Alzheimer's disease

Abstract: In order to observe changes owing to aging and Alzheimer's disease (AD) in the volumes of subdivisions of the hippocampus and the number of neurons of the hippocampal formation, 18 normal brains from subjects who died of nonneurological causes and had no history of long-term illness or dementia (ten of these brains comprised the aged control group) and 13 AD brains were analyzed. An optimized design for sampling, measuring volume by using the Cavalieri principle, and counting the number of neurons by using the optical disector was implemented on 50 microns-thick cresyl-violet sections. The mean total volume of the principal subdivisions of the hippocampal formation (fascia dentata, hilus, CA3-2, CA1, and subiculum) showed a negative correlation with age in normal subjects (r = -0.56, 2P < 0.05), and a 32% mean reduction in the AD group compared with controls (P < 0.001). This finding supports the measurement of the coronal cross-sectional area and the volume of the hippocampal formation in the clinical diagnosis of AD. There was an inverse relationship between the age of normal subjects and the number of neurons in CA1 (r = -0.84, 2P < 0.0001) and subiculum (r = -0.49, 2P < 0.05) but not in other subdivisions. Pronounced AD-related reductions in neuron number were found only in the subiculum and the fascia dentata. Compared with controls, both losses represented 23% of neurons (P < 0.05). These results 1) confirm that AD is a qualitatively different process from normal aging and 2) reveal the regional selectivity of neuron loss within the hippocampal formation in aging and AD, which may be relevant to understanding the mechanisms involved in the neuron loss associated with the two processes.

Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions.

Abstract: The proline-rich COOH-terminal region of dynamin binds various Src homology 3 (SH3) domain–containing proteins, but the physiological role of these interactions is unknown. In living nerve terminals, the function of the interaction with SH3 domains was examined. Amphiphysin contains an SH3 domain and is a major dynamin binding partner at the synapse. Microinjection of amphiphysin’s SH3 domain or of a dynamin peptide containing the SH3 binding site inhibited synaptic vesicle endocytosis at the stage of invaginated clathrin-coated pits, which resulted in an activity-dependent distortion of the synaptic architecture and a depression of transmitter release. These findings demonstrate that SH3-mediated interactions are required for dynamin function and support an essential role of clathrin-mediated endocytosis in synaptic vesicle recycling.

Learning Deficit in BDNF Mutant Mice

Abstract: Brain-derived neurotrophic factor (BDNF) has been implicated in the regulation of high-frequency synaptic transmission and long-term potentiation in the hippocampus, processes that are also thought to be involved in the learning of spatial tasks such as the Morris water maze. In order to determine whether BDNF is required for normal spatial learning, mice carrying a deletion in one copy of the BDNF gene were subjected to the Morris water maze task. Young adult BDNF mutant mice were significantly impaired compared with wild-type mice, requiring twice the number of days to reach full performance. Aged wild-type mice performed significantly worse than young wild-type mice and the effect was even more pronounced in the BDNF mutant mice, which did not learn at all. Although there was no difference in mean swimming speed between BDNF mutant and wild-type mice, we cannot exclude the possibility that developmental or peripheral deficits also contribute to the learning deficits in these mice. In situ hybridization and RNase protection analysis revealed that BDNF mRNA expression was indeed decreased in BDNF mutant mice. Furthermore, a pronounced effect of age on BDNF mRNA expression was seen, displayed as both a reduced level of mRNA expression and a reduced or entirely absent layer-specific expression pattern in the cerebral cortex of aged animals. Thus, our data suggest that BDNF expression may be linked to learning.

Pesticides and cancer

Abstract: Epidemiologic evidence on the relationship between chemical pesticides and cancer is reviewed. In animal studies, many pesticides are carcinogenic, (e.g., organochlorines, creosote, and sulfallate) while others (notably, the organochlorines DDT, chlordane, and lindane) are tumor promoters. Some contaminants in commercial pesticide formulations also may pose a carcinogenic risk. In humans, arsenic compounds and insecticides used occupationally have been classified as carcinogens by the International Agency for Research on Cancer. Human data, however, are limited by the small number of studies that evaluate individual pesticides. Epidemiologic studies, although some-times contradictory, have linked phenoxy acid herbicides or contaminants in them with soft tissue sarcoma (STS) and malignant lymphoma; organochlorine insecticides are linked with STS, non-Hodgkin's lymphoma (NHL), leukemia, and, less consistently, with cancers of the lung and breast; organophosphorous compounds are linked with NHL and leukemia; and triazine herbicides with ovarian cancer. Few, if any, of these associations can be considered established and causal. Hence, further epidemiologic studies are needed with detailed exposure assessment for individual pesticides, taking into consideration work practices, use of protective equipment, and other measures to reduce risk.

N-acetylcysteine: pharmacological considerations and experimental and clinical applications.

Abstract: The diversity of application of the thiol drug NAC in both the experimental setting, as a tool for the study of the mechanisms and consequences of oxidative stress, and the clinical setting, as a therapeutic agent, clearly reflects the central role played by the redox chemistries of the group XVI elements, oxygen and sulfur, in biology. As our understanding of such redox processes increases, particularly their roles in specific pathophysiological processes, new avenues will open for the use of NAC in the clinical setting. As a drug, NAC represents perhaps the ideal xenobiotic, capable of directly entering endogenous biochemical processes as a result of its own metabolism. Thus, it is hoped that the experience gained with this unique agent will help in future efforts to design antioxidants and chemoprotective principles which are able to more accurately utilize endogenous biochemical processes for cell- or tissue-specific therapy.