Adenosine signaling contributes to ethanol-induced fatty liver in mice.

Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5'-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5'-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver.

Synaptic vesicle dynamics in mouse rod bipolar cells.

To better understand synaptic signaling at the mammalian rod bipolar cell terminal and pave the way for applying genetic approaches to the study of visual information processing in the mammalian retina, synaptic vesicle dynamics and intraterminal calcium were monitored in terminals of acutely isolated mouse rod bipolar cells and the number of ribbon-style active zones quantified. We identified a releasable pool, corresponding to a maximum of 7 s. The presence of a smaller, rapidly releasing pool and a small, fast component of refilling was also suggested. Following calcium channel closure, membrane surface area was restored to baseline with a time constant that ranged from 2 to 21 s depending on the magnitude of the preceding Ca2+ transient. In addition, a brief, calcium-dependent delay often preceded the start of onset of membrane recovery. Thus, several aspects of synaptic vesicle dynamics appear to be conserved between rod-dominant bipolar cells of fish and mammalian rod bipolar cells. A major difference is that the number of vesicles available for release is significantly smaller in the mouse rod bipolar cell, both as a function of the total number per neuron and on a per active zone basis.

Application of in vivo induced antigen technology (IVIAT) to Bacillus anthracis.

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets.

Cyan fluorescent protein expression in ganglion and amacrine cells in a thy1-CFP transgenic mouse retina.

PURPOSE: To characterize cyan fluorescent protein (CFP) expression in the retina of the thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) transgenic mouse line. METHODS: CFP expression was characterized using morphometric methods and immunohistochemistry with antibodies to neurofilament light (NF-L), neuronal nuclei (NeuN), POU-domain protein (Brn3a) and calretinin, which immunolabel ganglion cells, and syntaxin 1 (HPC-1), glutamate decarboxylase 67 (GAD(67)), GABA plasma membrane transporter-1 (GAT-1), and choline acetyltransferase (ChAT), which immunolabel amacrine cells. RESULTS: CFP was extensively expressed in the inner retina, primarily in the inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer, and optic nerve. CFP fluorescent cell bodies were in all retinal regions and their processes ramified in all laminae of the IPL. Some small, weakly CFP fluorescent somata were in the inner nuclear layer (INL). CFP-containing somata in the GCL ranged from 6 to 20 microm in diameter, and they had a density of 2636+/-347 cells/mm2 at 1.5 mm from the optic nerve head. Immunohistochemical studies demonstrated colocalization of CFP with the ganglion cell markers NF-L, NeuN, Brn3a, and calretinin. Immunohistochemistry with antibodies to HPC-1, GAD(67), GAT-1, and ChAT indicated that the small, weakly fluorescent CFP cells in the INL and GCL were cholinergic amacrine cells. CONCLUSIONS: The total number and density of CFP-fluorescent cells in the GCL were within the range of previous estimates of the total number of ganglion cells in the C57BL/6J line. Together these findings suggest that most ganglion cells in the thy1-CFP mouse line 23 express CFP. In conclusion, the thy1-CFP mouse line is highly useful for studies requiring the identification of ganglion cells.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

Population structure, inbreeding and local adaptation within an endangered riverine specialist: the nase (Chondrostoma nasus)

Chondrostoma nasus is a cyprinid fish with highly specialized, ecologically and geographically distinct, ontogenetic trophic niches. Nase population numbers across their Swiss range have shown massive declines and many localized extinctions. Here we integrate data from different genetic markers with phenotypic and demographic data to survey patterns of neutral and adaptive genetic diversity in all extant (and one extinct) Swiss nase populations, with the aim to delineate intraspecific conservation units (CUs) and to inform future population management strategies. We discovered two major genetically and geographically distinct population groupings. The first population grouping comprises nase inhabiting rivers flowing into Lake Constance; the second comprises nase populations from Rhine drainages below Lake Constance. Within these clusters there is generally limited genetic differentiation among populations. Genomic outlier scans based on 256–377 polymorphic AFLP loci revealed little evidence of local adaptation both within and among population clusters, with the exception of one candidate locus identified in scans involving the inbred Schanzengraben population. However, significant phenotypic differentiation in body shape between certain populations suggests a need for more intensive future studies of local adaptation. Our data strongly suggests that the two major population groups should be treated as distinct CUs, with any supplemental stocking and reintroductions sourced only from within the range of the CU concerned.

Quantitative diffusion tensor imaging detects dopaminergic neuronal degeneration in a murine model of Parkinson's disease.

Early diagnosis of Parkinson's disease (PD) is required to improve therapeutic responses. Indeed, a clinical diagnosis of resting tremor, rigidity, movement and postural deficiencies usually reflect >50% loss of the nigrostriatal system in disease. In a step to address this, quantitative diffusion tensor magnetic resonance imaging (DTI) was used to assess nigrostriatal degeneration in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication model of dopaminergic nigral degeneration. We now demonstrate increased average diffusion (p<0.005) and decreased fractional anisotropy (p<0.03) in the substantia nigra (SN) of 5- to 7-day MPTP-treated animals when compared to saline controls. Transverse diffusivity demonstrated the most significant differences (p < or = 0.002) and correlated with the numbers of SN dopaminergic neurons (r=-0.75, p=0.012). No differences were found in the striatum, corpus callosum, cerebral cortex, or ventricles. These results demonstrate that DTI may be used as a surrogate biomarker of nigral dopaminergic neuronal degeneration.

Neural reprogramming in retinal degeneration.

PURPOSE: Early visual defects in degenerative diseases such as retinitis pigmentosa (RP) may arise from phased remodeling of the neural retina. The authors sought to explore the functional expression of ionotropic (iGluR) and group 3, type 6 metabotropic (mGluR6) glutamate receptors in late-stage photoreceptor degeneration. METHODS: Excitation mapping with organic cations and computational molecular phenotyping were used to determine whether retinal neurons displayed functional glutamate receptor signaling in rodent models of retinal degeneration and a sample of human RP. RESULTS: After photoreceptor loss in rodent models of RP, bipolar cells lose mGluR6 and iGluR glutamate-activated currents, whereas amacrine and ganglion cells retain iGluR-mediated responsivity. Paradoxically, amacrine and ganglion cells show spontaneous iGluR signals in vivo even though bipolar cells lack glutamate-coupled depolarization mechanisms. Cone survival can rescue iGluR expression by OFF bipolar cells. In a case of human RP with cone sparing, iGluR signaling appeared intact, but the number of bipolar cells expressing functional iGluRs was double that of normal retina. CONCLUSIONS: RP triggers permanent loss of bipolar cell glutamate receptor expression, though spontaneous iGluR-mediated signaling by amacrine and ganglion cells implies that such truncated bipolar cells still release glutamate in response to some nonglutamatergic depolarization. Focal cone-sparing can preserve iGluR display by nearby bipolar cells, which may facilitate late RP photoreceptor transplantation attempts. An instance of human RP provides evidence that rod bipolar cell dendrite switching likely triggers new gene expression patterns and may impair cone pathway function.

Selective elevation of c-{\it myc} RNA transcripts during clonal evolution of N-methyl-N-nitrosourea induced thymic lymphomas in AKR/J mice

Untreated AKR mice develop spontaneous thymic lymphomas by 6-12 months of age. Lymphoma development is accelerated when young mice are injected with the carcinogen N-methyl-N-nitrosourea (MNU). Selected molecular and cellular events were compared during the latent period preceding "spontaneous" (retrovirally-induced) and MNU-induced thymic lymphoma development in AKR mice. These studies were undertaken to test the hypothesis that thymic lymphomas induced in the same inbred mouse strain by endogenous retroviruses and by a chemical carcinogen develop by different mechanisms.^ Immunofluorescence analysis of differentiation antigens showed that most MNU-induced lymphomas express an immature CD4-8+ profile. In contrast, spontaneous lymphomas represent each of the major lymphocyte subsets. These data suggest involvement of different target populations in MNU-induced and spontaneous lymphomas. Analyses at intervals after MNU treatment revealed selective expansion of the CD4-8+ J11d+ thymocyte subset at 8-10 weeks post-MNU in 68% of the animals examined, suggesting that these cells are targets for MNU-induced lymphomagenesis. Untreated age-matched animals showed no selective expansion of thymocyte subsets.^ Previous data have shown that both spontaneous and MNU-induced lymphomas are monoclonal or oligoclonal. Distinct rearrangement patterns of the J$\sb2$ region of the T-cell receptor $\beta$-chain showed emergence of clonal thymocyte populations beginning at 6-7 weeks after MNU treatment. However, lymphocytes from untreated animals showed no evidence of clonal expansion at the time intervals investigated.^ Activation of c-myc frequently occurs during development of B- and T- cell lymphomas. Both spontaneous and MNU-induced lymphomas showed increased c-myc transcript levels. Increased c-myc transcription was first detected at 6 weeks post-MNU, and persisted throughout the latent period. However, untreated animals showed no increases in c-myc transcripts at the time intervals examined. Another nuclear oncogene, c-fos, did not display a similar change in RNA transcription during the latent period.^ These results supports the hypothesis that MNU-induced and spontaneous tumors develop by multi-step pathways which are distinct with respect to the target cell population affected. Clonal emergence and c-myc deregulation are important steps in the development of both MNU-induced and spontaneous tumors, but the onset of these events is later in spontaneous tumor development. ^

B cell memory and idiotype network regulation

This investigation examined the clonal dynamics of B-cell expression and evaluated the role of idiotype network interactions in shaping the expressed secondary B-cell repertoire. Three interrelated experimental approaches were applied. The first approach was designed to distinguish between regulatory influences controlled by the major histocompatibility complex (MHC) and regulatory influences controlled by non-MHC factors including the idiotype network. This approach consisted of studies on the clonal dynamics and heterogeneity of the expressed IgG antibody repertoire of BALB/c mice. The second approach involved the analysis of the clonal dynamics of antibody responses of outbred rabbits. This analysis was coupled with studies to detect the occurrence and activity of constituents of the idiotype network. In the third approach the transfer of rabbit lymphocytes from immunized donors to MHC matched naive recipients was used to examine the effects of recipient non-MHC immunoregulatory influences on the expression of donor memory B-cells. Although many memory B cells were unaffected by non-MHC influences, these data show that non-MHC immunoregulatory influences can affect the expression of B-cells in the secondary response of inbred mice and outbred rabbits. The results also indicate that most IgG antibody responses are heterogeneous and are characterized by a stable group of dominant clonotypes. Clonal dominance and B-cell memory were found to be established early in an immune response. The expression of B memory clones appeared to be favored over the expression of virgin B cells. The injection of anti-tetanus antibody induced the antigen independent production of anti-tetanus antibody, probably through idiotypic mechanisms. These results demonstrate that both antibody and antigen can affect the expressed B-ceIl repertoire. Thus, idiotypic interactions are capable of influencing the expression of B-cells and these findings support the existence and function of an idiotype network with strong immunoregulatory potential. ^

The transcriptional repressor CDP (Cutl1) is essential for epithelial cell differentiation of the lung and the hair follicle

The mammalian Cutl1 gene codes for the CCAAT displacement protein (CDP), which has been implicated as a transcriptional repressor in diverse processes such as terminal differentiation, cell cycle progression, and the control of nuclear matrix attachment regions. To investigate the in vivo function of Cutl1, we have replaced the C-terminal Cut repeat 3 and homeodomain exons with an in-frame lacZ gene by targeted mutagenesis in the mouse. The CDP-lacZ fusion protein is retained in the cytoplasm and fails to repress gene transcription, indicating that the Cutl1(lacZ) allele corresponds to a null mutation. Cutl1 mutant mice on inbred genetic backgrounds are born at Mendelian frequency, but die shortly after birth because of retarded differentiation of the lung epithelia, which indicates an essential role of CDP in lung maturation. A less pronounced delay in lung development allows Cutl1 mutant mice on an outbred background to survive beyond birth. These mice are growth-retarded and develop an abnormal pelage because of disrupted hair follicle morphogenesis. The inner root sheath (IRS) is reduced, and the transcription of Sonic hedgehog and IRS-specific genes is deregulated in Cutl1 mutant hair follicles, consistent with the specific expression of Cutl1 in the progenitors and cell lineages of the IRS. These data implicate CDP in cell-lineage specification during hair follicle morphogenesis, which resembles the role of the related Cut protein in specifying cell fates during Drosophila development.

Genetic analysis of tumor progression susceptibility in the mouse skin model

In the field of chemical carcinogenesis the use of animal models has proved to be a useful tool in dissecting the multistage process of tumor formation. In this regard the outbred SENCAR mouse has been the strain of choice in the analysis of skin carcinogenesis given its high sensitivity to the chemically induced acquisition of premalignant lesions, papillomas, and the later progression of these lesions into squamous cell carcinomas (SCC).^ The derivation of an inbred strain from the SENCAR stock called SSIN, that in spite of a high sensitivity to the development of papillomas lack the ability to transform these premalignant lesions into SCC, suggested that tumor promotion and progression were under the genetic control of different sets of genes.^ In the present study the nature of susceptibility to tumor progression was investigated. Analysis of F1 hybrids between the outbred SENCAR and SSIN mice suggested that there is at least one dominant gene responsible for susceptibility to tumor progression.^ Later development of another inbred strain from the outbred SENCAR stock, that had sensitivity to both tumor promotion and progression, allowed the formulation of a more accurate genetic model. Using this newly derived line, SENCAR B/Pt. and SSIN it was determined that there is one dominant tumor progression susceptibility gene. Linkage analysis showed that this gene maps to mouse chromosome 14 and it was possible to narrow the region to a 16 cM interval.^ In order to better characterize the nature of the progression susceptibility differences between these two strains, their proliferative pattern was investigated. It was found that SENCAR B/Pt, have an enlarged proliferative compartment with overexpression of cyclin D1, p16 and p21. Further studies showed an aberrant overexpression of TGF-$\beta$ in the susceptible strain, an increase in apoptosis, p53 protein accumulation and early loss of connexin 26. These results taken together suggest that papillomas in the SENCAR B/Pt. mice have higher proliferation and may have an increase in genomic instability, these two factors would contribute to a higher sensitivity to tumor progression. ^

CYTOPLASMIC MALATE DEHYDROGENASE IS THE AROMATIC ALPHA-KETO ACID REDUCTASE IN MAN (PHENYLKETONURIA, PKU, TYROSINE)

This dissertation presents evidence to support the hypothesis that cytoplasmic malate dehydrogenase (MDH-1) is the enzyme in humans which catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, and the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; E.C. 1.1.1.96) is actually a secondary activity of cytoplasmic malate dehydrogenase.^ Purified MDH and purified KAR have the same molecular weight, subunit structure, heat-inactivation profile and tissue distribution. After starch gel electrophoresis, and using p-hydroxyphenylpyruvic acid (HPPA) as substrate, KAR activity co-migrates with MDH-1 in all species studied except some marine animals. Inhibition with malate, the end-product of malate dehydrogenase, substantially reduces or totally eliminates KAR activity. Purified cytoplasmic MDH from human erythrocytes has an alpha-keto acid reductase activity with identical mobility. All electrophoretic variants of MDH-1 seen in the fresh-water bony fish Xiphophorus, the amphibians Rana and humans exhibited identical variation for KAR, and the two traits co-segregated in the small group of offspring from one Rana heterozygote studied. Both enzymes show almost no electrophoretic variation among humans from many ethnic groups, and among several inbred strains of mice both MDH-s and KAR co-migrate with no variation. MDH-1 and KAR in mouse and Chinese hamster fibroblasts show identical mobility differences between species. Antisera raised against purified chicken cytoplasmic MDH totally inhibited both MDH-1 and KAR in chickens and humans. Mitochondrial MDH from tissue homogenates has no detectable KAR activity but purified MDH-2 does.^ The previous claim that the gene for KAR is on human chromosome 12 is disputed because both MDH-1 and LDH bands appear with slightly different mobility approximately midway between the human and hamster controls in somatic cell hybrid studies, and the meaning of this artifact is discussed. ^

LINKAGE RELATIONSHIPS AMONG PROTEIN-CODING LOCI IN FISHES OF THE GENUS XIPHOPHORUS

Inbred strains of three species of fishes of the genus Xiphophorus (platyfish and swordtails) were crossed to produce intra- and interspecific F(,1) hybrids, which were then backcrossed to one or both parental stocks. Backcross hybrids were used for the analysis of segregation and linkage of 33 protein-coding loci (whose products were visualized by starch gel electrophoresis) and a sex-linked pigment pattern gene. Segregation was Mendelian for all loci with the exception of one instance of segregation distortion. Six linkage groups of enzyme-coding loci were established: LG I, ADA --6%-- G(,6)PD --24%-- 6PGD; LG II, Est-2 --27%-- Est-3 --0%-- Est-5 --23%-- LDH-1 --16%-- MPI; LG III, AcPh --38%-- G(,3)PD-1 (GUK-2 --14%-- G(,3)PD-1 is also in LG III, but the position of GUK-2 with respect to AcPh has not yet been determined); LG IV, GPI-1 --41%-- IDH-1; LG V, Est-1 --38%-- MDH-2; and LG VI, P1P --7%-- UMPK-1 (P1P is a plasma protein, very probably transferrin).^ Sex-specific recombination appeared absent in LG II and LG IV locus pairs; significantly higher male recombination was demonstrated in LG I but significantly higher female recombination was detected in LG V. Only one significant population-specific difference in recombination was detected, in the G(,6)PD - 6PGD region of LG I; the notable absence of such effects implies close correspondence of the genomes of the species used in the study. Two cases of possible evolutionary conservation of linkage groups in fishes and mammals were described, involving the G(,6)PD - 6PGD linkage in LG I and the cluster of esterase loci in LG II. One clear case of divergence was observed, that of the linkage of ADA in LG I. It was estimated that a minimum of (TURN)50% of the Xiphophorus genome was marked by the loci studied. Therefore, the prior probability that a new locus will assort independently from the markers already established is estimated to be less than 0.5. A maximum of 21 of the 24 pairs of chromosomes could be marked with at least one locus.^ Only the two LG V loci showed a significant association with a postulated gene controlling the severity of a genetically controlled melanoma caused by abnormal proliferation of macromelanophore pigment pattern cells. The independence of melanotic severity from all other informative markers implies that one or at most a few major genes are involved in control of melanotic severity in this system. ^

Stable two-element control of dTph1 transposition in mutator strains of Petunia by an inactive ACT1 introgression from a wild species

The high copy dTph1 transposon system of Petunia (Solanaceae) is one of the most powerful insertion mutagens in plants, but its activity cannot be controlled in the commonly used mutator strains. We analysed the regulation of dTph1 activity by QTL analysis in recombinant inbred lines of the mutator strain W138 and a wild species (P. integrifolia spp. inflata). Two genetic factors were identified that control dTph1 transposition. One corresponded to the ACT1 locus on chromosome I. A second, previously undescribed locus ACT2 mapped on chromosome V. As a 6-cM introgression in W138, the P. i. inflata act1(S6) allele behaved as a single recessive locus that fully eliminated transposition of all dTph1 elements in all stages of plant development and in a heritable fashion. Weak dTph1 activity was restored in act1(S6)/ACT2(S6) double introgression lines, indicating that the P. i. inflata allele at ACT2 conferred a low level of transposition. Thus, the act1(S6) allele is useful for simple and predictable control of transposition of the entire dTph1 family when introgressed into an ultra-high copy W138 mutator strain. We demonstrate the use of the ACT1(W138)/act1(S6) allele pair in a two-element dTph1 transposition system by producing 10 000 unique and fixed dTph1 insertions in a population of 1250 co-isogenic lines. This Petunia system produces the highest per plant insertion number of any known two-element system, providing a powerful and logistically simple tool for transposon mutagenesis of qualitative as well as quantitative traits.