Development and characterization of a rodent model of immune-mediated cholangitis.

The cholangiopathies are a group of hepatobiliary diseases in which intrahepatic bile duct epithelial cells, or cholangiocytes, are the target for a variety of destructive processes, including immune-mediated damage. We tested the hypothesis that cholangitis could be induced in rodents by immunization with highly purified cholangiocytes. Inbred Wistar rats were immunized with purified hyperplastic cholangiocytes isolated after bile duct ligation from either syngeneic Wistar or allogeneic Fischer 344 rats; control rats were immunized with bovine serum albumin (BSA) or hepatocytes. After immunization with cholangiocytes, recipient animals developed histologic evidence of nonsuppurative cholangitis without inflammation in other organs; groups immunized with BSA or hepatocytes showed no cholangitis. Immunohistochemical studies revealed that portal tract infiltrates around bile ducts consisted of CD3-positive lymphocytes, some of which expressed major histocompatibility complex class II antigen; B cells and exogenous monocytes/macrophages were essentially absent. Transfer of unfractionated ConA-stimulated spleen cells from cholangiocyte-immunized (but not BSA-immunized) rats into recipients also caused nonsuppurative cholangitis. Moreover, these splenocytes from cholangiocyte-immunized (but not BSA-immunized) rats were cytotoxic in vitro for cultured rodent cholangiocytes; no cytotoxicity was observed against a rat hepatocyte cell line. Also, a specific antibody response in sera of cholangiocyte-immunized rats was demonstrated by immunoblots against cholangiocyte proteins. Finally, cholangiograms in cholangiocyte-immunized rats showed distortion and tortuosity of the entire intrahepatic biliary ductal system. This unique rodent model of experimental cholangitis demonstrates the importance of immune mechanisms in the pathogenesis of cholangitis and will prove useful in exploring the mechanisms by which the immune system targets and damages cholangiocytes.

Autoradiographic characterization of [3H]-5-HT-moduline binding sites in rodent brain and their relationship to 5-HT1B receptors

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of β-particles (β-imager) indicated that [3H]-5-HT-moduline bound specifically to rat brain sections with high affinity (Kd = 0.77 nM and Bmax = 0.26 dpm/mm2). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT1B receptor knock-out mice, the specific binding of [3H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.

Prion protein NMR structure and species barrier for prion diseases

The structural basis of species specificity of transmissible spongiform encephalopathies, such as bovine spongiform encephalopathy or “mad cow disease” and Creutzfeldt–Jakob disease in humans, has been investigated using the refined NMR structure of the C-terminal domain of the mouse prion protein with residues 121–231. A database search for mammalian prion proteins yielded 23 different sequences for the fragment 124–226, which display a high degree of sequence identity and show relevant amino acid substitutions in only 18 of the 103 positions. Except for a unique isolated negative surface charge in the bovine protein, the amino acid differences are clustered in three distinct regions of the three-dimensional structure of the cellular form of the prion protein. Two of these regions represent potential species-dependent surface recognition sites for protein–protein interactions, which have independently been implicated from in vitro and in vivo studies of prion protein transformation. The third region consists of a cluster of interior hydrophobic side chains that may affect prion protein transformation at later stages, after initial conformational changes in the cellular protein.

Transcriptional regulation of the rat Müllerian inhibiting substance type II receptor in rodent Leydig cells

Müllerian inhibiting substance (MIS) causes regression of the fetal Müllerian duct on binding a heteromeric complex of types I and II cell-surface receptors in the fetal urogenital ridge. The MIS type II receptor (MISRII), which provides specificity for MIS, is also expressed in the adult testis, ovary, and uterus. The rat MISRII promoter was cloned to study the molecular mechanisms underlying its temporal and cell-specific expression. The 1.6-kilobase (kb) promoter contained no recognizable TATA or CAAT box, but there was a consensus Sp1 site upstream of the transcription initiation site. Two binding sites for the orphan nuclear receptor steroidogenic factor-1 (SF-1) are occupied in vitro by using nuclear extracts from R2C cells, an MIS-responsive rat Leydig cell line that expresses endogenous MISRII, with differing affinities, indicating that the distal SF-1 site is bound more avidly than is the proximal SF-1 site. R2C cells transfected with MISRII promoter/luciferase reporter constructs show a 12-fold induction with the 1.6-kb fragment and deletion of sequences upstream of −282-bp lowered luciferase expression to one-third. Mutation of both SF-1 sites greatly inhibited luciferase expression, whereas mutation of either site alone resulted in continuing activation by endogenous SF-1, indicating redundancy. In vitro binding and transcriptional analyses suggest that a proximal potential Smad-responsive element and an uncharacterized element also contribute to activation of the MISRII gene. R2C cells and MISRII promoter regulation can now be used to uncover endogenous transcription factors responsible for receptor expression or repression.

An alternative pathway for rod signals in the rodent retina: Rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors

In the mammalian retina, extensive processing of spatiotemporal and chromatic information occurs. One key principle in signal transfer through the retina is parallel processing. Two of these parallel pathways are the ON- and OFF-channels transmitting light and dark signals. This dual system is created in the outer plexiform layer, the first relay station in retinal signal transfer. Photoreceptors release glutamate onto ON- and OFF-type bipolar cells, which are functionally distinguished by their postsynaptic expression of different types of glutamate receptors, namely ionotropic and metabotropic glutamate receptors. In the current concept, rod photoreceptors connect only to rod bipolar cells (ON-type) and cone photoreceptors connect only to cone bipolar cells (ON- and OFF-type). We have studied the distribution of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunits at the synapses in the outer plexiform layer of the rodent retina by immunoelectron microscopy and serial section reconstruction. We report a non-classical synaptic contact and an alternative pathway for rod signals in the retina. Rod photoreceptors made synaptic contact with putative OFF-cone bipolar cells that expressed the AMPA glutamate receptor subunits GluR1 and GluR2 on their dendrites. Thus, in the retina of mouse and rat, an alternative pathway for rod signals exists, where rod photoreceptors bypass the rod bipolar cell and directly excite OFF-cone bipolar cells through an ionotropic sign-conserving AMPA glutamate receptor.

Homeobox gene Prx3 expression in rodent brain and extraneural tissues

Different cDNA clones encoding a rat homeobox gene and the mouse homologue OG-12 were cloned from adult rat brain and mouse embryo mRNA, respectively. The predicted amino acid sequences of the proteins belong to the paired-related subfamily of homeodomain proteins (Prx homeodomains). Hence, the gene was named Prx3 and the mouse and rat genes are indicated as mPrx3 and rPrx3, respectively. In the mouse as well as in the rat, the predicted Prx3 proteins share the homeodomain but have three different N termini, a 12-aa residue variation in the C terminus, and contain a 14-aa residue motif common to a subset of homeodomain proteins, termed the “aristaless domain.” Genetic mapping of Prx3 in the mouse placed this gene on chromosome 3. In situ hybridization on whole mount 12.5-day-old mouse embryos and sections of rat embryos at 14.5 and 16.5 days postcoitum revealed marked neural expression in discrete regions in the lateral and medial geniculate complex, superior and inferior colliculus, the superficial gray layer of the superior colliculus, pontine reticular formation, and inferior olive. In rat and mouse embryos, nonneuronal structures around the oral cavity and in hip and shoulder regions also expressed the Prx3 gene. In the adult rat brain, Prx3 gene expression was restricted to thalamic, tectal, and brainstem structures that include relay nuclei of the visual and auditory systems as well as other ascending systems conveying somatosensory information. Prx3 may have a role in specifying neural systems involved in processing somatosensory information, as well as in face and body structure formation.

Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1 complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

Neonatal lead exposure impairs development of rodent barrel field cortex

Childhood exposure to low-level lead can permanently reduce intelligence, but the neurobiologic mechanism for this effect is unknown. We examined the impact of lead exposure on the development of cortical columns, using the rodent barrel field as a model. In all areas of mammalian neocortex, cortical columns constitute a fundamental structural unit subserving information processing. Barrel field cortex contains columnar processing units with distinct clusters of layer IV neurons that receive sensory input from individual whiskers. In this study, rat pups were exposed to 0, 0.2, 1, 1.5, or 2 g/liter lead acetate in their dam's drinking water from birth through postnatal day 10. This treatment, which coincides with the development of segregated columns in the barrel field, produced blood lead concentrations from 1 to 31 μg/dl. On postnatal day 10, the area of the barrel field and of individual barrels was measured. A dose-related reduction in barrel field area was observed (Pearson correlation = −0.740; P < 0.001); mean barrel field area in the highest exposure group was decreased 12% versus controls. Individual barrels in the physiologically more active caudoventral group were affected preferentially. Total cortical area measured in the same sections was not altered significantly by lead exposure. These data support the hypothesis that lead exposure may impair the development of columnar processing units in immature neocortex. We demonstrate that low levels of blood lead, in the range seen in many impoverished inner-city children, cause structural alterations in a neocortical somatosensory map.

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.

Estrogen receptors α and β in the rodent mammary gland

An obligatory role for estrogen in growth, development, and functions of the mammary gland is well established, but the roles of the two estrogen receptors remain unclear. With the use of specific antibodies, it was found that both estrogen receptors, ERα and ERβ, are expressed in the rat mammary gland but the presence and cellular distribution of the two receptors are distinct. In prepubertal rats, ERα was detected in 40% of the epithelial cell nuclei. This decreased to 30% at puberty and continued to decrease throughout pregnancy to a low of 5% at day 14. During lactation there was a large induction of ERα with up to 70% of the nuclei positive at day 21. Approximately 60–70% of epithelial cells expressed ERβ at all stages of breast development. Cells coexpressing ERα and ERβ were rare during pregnancy, a proliferative phase, but they represented up to 60% of the epithelial cells during lactation, a postproliferative phase. Western blot analysis and sucrose gradient centrifugation confirmed this pattern of expression. During pregnancy, the proliferating cell nuclear antigen was not expressed in ERα-positive cells but was observed in 3–7% of ERβ-containing cells. Because more than 90% of ERβ-bearing cells do not proliferate, and 55–70% of the dividing cells have neither ERα nor ERβ, it is clear that the presence of these receptors in epithelial cells is not a prerequisite for estrogen-mediated proliferation.

Growth factor-mediated Fyn signaling regulates α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression in rodent neocortical neurons

Src-family protein tyrosine kinases (PTKs) transduce signals to regulate neuronal development and synaptic plasticity. However, the nature of their activators and molecular mechanisms underlying these neural processes are unknown. Here, we show that brain-derived neurotrophic factor (BDNF) and platelet-derived growth factor enhance expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor 1 and 2/3 proteins in rodent neocortical neurons via the Src-family PTK(s). The increase in AMPA receptor levels was blocked in cultured neocortical neurons by addition of a Src-family-selective PTK inhibitor. Accordingly, neocortical cultures from Fyn-knockout mice failed to respond to BDNF whereas those from wild-type mice responded. Moreover, the neocortex of young Fyn mutants exhibited a significant in vivo reduction in these AMPA receptor proteins but not in their mRNA levels. In vitro kinase assay revealed that BDNF can indeed activate the Fyn kinase: It enhanced tyrosine phosphorylation of Fyn as well as that of enolase supplemented exogenously. All of these results suggest that the Src-family kinase Fyn, activated by the growth factors, plays a crucial role in modulating AMPA receptor expression during brain development.