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Biomedical subjects

G Eichele

Publications and source records attributed to G Eichele.

At least 19 recordsLinked to original sources
PubMed2001

Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock.

Mice carrying a null mutation in the Period 1 (mPer1) gene were generated using embryonic stem cell technology. Homozygous mPer1 mutants display a shorter circadian period with reduced precision and stability. Mice deficient in both mPer1 and mPer2 do not express circadian rhythms. While mPER2 regulates clock gene expression at the transcriptional level, mPER1 is dispensable for the rhythmic RNA expression of mPer1 and mPer2 and may instead regulate mPER2 at a posttranscriptional level. Studies of clock-controlled genes (CCGs) reveal a complex pattern of regulation by mPER1 and mPER2, suggesting independent controls by the two proteins over some output pathways. Genes encoding key enzymes in heme biosynthesis are under circadian control and are regulated by mPER1 and mPER2. Together, our studies show that mPER1 and mPER2 have distinct and complementary roles in the mouse clock mechanism.

Animals↗
PubMed2001

Development of high-throughput tools to unravel the complexity of gene expression patterns in the mammalian brain.

Genomes of animals contain between 15000 (e.g. Drosophila) and 50000 (human, mouse) genes, many of which encode proteins involved in regulatory processes. The availability of sequence data for many of these genes opens up opportunities to study complex genetic and protein interactions that underlie biological regulation. Many examples demonstrate that an understanding of regulatory networks consisting of multiple components is significantly advanced by a detailed knowledge of the spatiotemporal expression pattern of each of the components. Gene expression patterns can readily be determined by RNA in situ hybridization. The unique challenge emerging from the knowledge of the sequence of entire genomes is that assignment of biological functions to genes needs to be carried out on an appropriately large scale. In terms of gene expression analysis by RNA in situ hybridization, efficient technologies need to be developed that permit determination and representation of expression patterns of thousands of genes within an acceptable time-scale. We set out to determine the spatial expression pattern of several thousand genes encoding putative regulatory proteins. To achieve this goal we have developed high-throughput technologies that allow the determination and visualization of gene expression patterns by RNA in situ hybridization on tissue sections at cellular resolution. In particular, we have invented instrumentation for robotic in situ hybridization capable of carrying out in a fully automated fashion, all steps required for detecting sites of gene expression in tissue sections. In addition, we have put together hardware and software for automated microscopic scanning of gene expression data that are produced by RNA in situ hybridization. The potential and limitations of these techniques and our efforts to build a Web-based database of gene expression patterns are discussed.

Animals↗
PubMed2001

NudE-L, a novel Lis1-interacting protein, belongs to a family of vertebrate coiled-coil proteins.

The LIS1-encoded protein (Lis1) plays a role in brain development because a hemizygous deletion or mutation of the human gene causes neuronal migration disorders, such as Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS). Using a yeast two-hybrid screen, we have isolated a novel protein that interacts with mouse Lis1 (mLis1) which is termed mouse NudE-like protein (mNudE-L) because of its 49% amino acid conservation with NudE, a protein involved in nuclear migration in Aspergillus nidulans. GST pull-down assays and co-immunoprecipitation of fusion proteins expressed in mammalian cells confirmed the interaction of mLis1 and mNudE-L. mNudE-L gives rise to a approximately 2.3 kb mRNA and encodes an ORF corresponding to approximately 38 kDa protein. The overall amino acid sequence of mNudE-L is 49-95% identical to proteins found in a variety of organisms, thus establishing mNudE-L as a new member of a protein family. The hallmark of this family is an N-terminal region predicted to form a coiled-coil domain. We show that mNudE-L and mLis1 are coexpressed in the postnatal and adult cerebral cortices and in the Purkinje neurons of the cerebellum. In contrast to mLis1, mNudE-L transcripts are absent in the mitral cell layer of the olfactory bulb and in the inward migrating granular neurons of the developing cerebellum. Mutant mLis1 proteins modelling mutations found in human lissencephaly patients fail to interact with mNudE-L, raising the possibility that phenotypic changes result, in part, from the inability of mutant Lis1 proteins to interact with the human NudE-L polypeptide.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2001

Evidence for a role of protein kinase C in FGF signal transduction in the developing chick limb bud.

In developing limbs, numerous signaling molecules have been identified but less is known about the mechanisms by which such signals direct patterning. We have explored signal transduction pathways in the chicken limb bud. A cDNA encoding RACK1, a protein that binds and stabilizes activated protein kinase C (PKC), was isolated in a screen for genes induced by retinoic acid (RA) in the chick wing bud. Fibroblast growth factor (FGF) also induced RACK1 and such induction of RACK1 expression was accompanied by a significant augmentation in the number of active PKC molecules and an elevation of PKC enzymatic activity. This suggests that PKCs mediate signal transduction in the limb bud. Application of chelerythrine, a potent PKC inhibitor, to the presumptive wing region resulted in buds that did not express sonic hedgehog (Shh) and developed into wings that were severely truncated. This observation suggests that the expression of Shh depends on PKCs. Providing ectopic SHH protein, RA or ZPA grafts overcome the effects of blocking PKC with chelerythrine and resulted in a rescue of the wing morphology. Taken together, these findings suggest that the responsiveness of Shh to FGF is mediated, at least in part, by PKCs.

Alkaloids↗
PubMed2000

Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: evidence for BMP2 induction of Tbx2.

Expression patterns of Tbx2, -3, and -5 genes were analyzed during chick embryonic heart development. Transcripts of these three cTbx genes were detected in overlapping patterns in the early cardiac crescent. cTbx2 and cTbx3 expression patterns closely overlapped with that of bmp2. cTbx5 expression diverged from cTbx2 and bmp2 during the elaboration and folding of the heart tube. In comparison, cTbx2 expression overlapped significantly with that of bmp2 and bmp4 during all stages of heart development and during later embryonic stages, suggestive of a specialized role for Tbx2 in septation. Coexpression of cTbx 2 and cTbx3 genes with bmp2 transcripts raised the possibility that these cTbx genes might be downstream bmp2 targets. Application of bmp2 selectively induced cTbx2 and cTbx3 expression in noncardiogenic embryonic tissue, and the bmp antagonist Noggin down-regulated cTbx2 gene activity. Moreover, the appearance of murine mTbx2 was blocked in bmp2 null mouse embryos. cTbx2 and to a lesser extent cTbx3 gene activity appears to be directed by BMPs during early cardiogenesis.

Animals↗
PubMed2000

Fenretinide therapy in prostate cancer: effects on tissue and serum retinoid concentration.

PURPOSE: To examine the feasibility of using fenretinide (4-HPR) for the prevention and treatment of prostate cancer. MATERIALS AND METHODS: We measured the impact of 4-HPR therapy on retinoid concentrations in vivo, in a mouse model of prostate cancer and clinically, in patients with prostate cancer who were given oral 4-HPR (200 mg/d) or placebo for 4 weeks before undergoing a radical prostatectomy. RESULTS: Prostate tumors in mice treated with 4-HPR contained high levels of 4-HPR and of all-trans-retinoic acid (RA) and reduced levels of retinol (ROH). Patients given 4-HPR were found to have significantly higher concentrations of 4-HPR in the cancerous prostate as compared with the serum levels (463 nmol/L v 326 nmol/L; P =.049), but they were only 1/10 the levels found in mice and were far below the concentrations reported in human breast tissue. Serum and tissue ROH levels were reduced to less than half the concentrations found in untreated controls. RA concentrations in human serum and in cancerous prostates were not significantly affected by 4-HPR treatment, in contrast with the findings in mice. CONCLUSION: The standard oral dose of 4-HPR proposed for breast cancer (200 mg/d) achieved only modest drug levels in the prostate and is unlikely to be effective for prostate cancer prevention or treatment. Higher doses need to be explored.

Aged↗
PubMed2000

Lissencephaly associated mutations suggest a requirement for the PAFAH1B heterotrimeric complex in brain development.

Human brain malformations, such as Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) may result from abnormal neuronal migration during brain development. MDS and ILS patients have a hemizygous deletion or mutation in the LIS1 gene (PAFAH1B1), therefore, the LIS1 encoded protein (Lis1) may play a role in neuronal migration. Lis1 is a subunit of a brain platelet-activating factor acetylhydrolase (PAFAH1B) where it forms a heterotrimeric complex with two hydrolase subunits, referred to as 29 kDa (PAFAH1B3) and 30 kDa (PAFAH1B2). In order to determine whether this heterotrimer is required for the developmental functions of PAFAH1B, we examined the binding properties of 29 and 30 kDa subunits to mutant Lis1 proteins. The results defined the critical regions of Lis1 for PAFAH1B complex formation and demonstrated that all human LIS1 mutations examined resulted in abolished or reduced capacity of Lis1 to interact with the 29 and 30 kDa subunits, suggesting that the PAFAH1B complex participates in the process of neuronal migration.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1999

Complementary domains of retinoic acid production and degradation in the early chick embryo.

Excess retinoids as well as retinoid deprivation cause abnormal development, suggesting that retinoid homeostasis is critical for proper morphogenesis. RALDH-2 and CYP26, two key enzymes that carry out retinoic acid (RA) synthesis and degradation, respectively, were cloned from the chick and show significant homology with their orthologs in other vertebrates. Expression patterns of RALDH-2 and CYP26 genes were determined in the early chick embryo by in situ hybridization. During gastrulation and neurulation RALDH-2 and CYP26 were expressed in nonoverlapping regions, with RALDH-2 transcripts localized to the presumptive presomitic and lateral plate mesoderm and CYP26 mRNA to the presumptive mid- and forebrain. The two domains of expression were separated by an approximately 300-micrometer-wide gap, encompassing the presumptive hindbrain. In the limb region, a similar spatial segregation of RALDH-2 and CYP26 expression was found at stages 14 and 15. Limb region mesoderm expressed RALDH-2, whereas the overlying limb ectoderm expressed CYP26. RA-synthesizing and -degrading enzymatic activities were measured biochemically in regions expressing RALDH-2 or CYP26. Regions expressing RALDH-2 generated RA efficiently from precursor retinal but degraded RA only inefficiently. Conversely, tissue expressing CYP26 efficiently degraded but did not synthesize RA. Localized regions of RA synthesis and degradation mediated by these two enzymes may therefore provide a mechanism to regulate RA homeostasis spatially in vertebrate embryos.

Aldehyde Oxidoreductases↗
PubMed1999

Evidence for a role of Smad6 in chick cardiac development.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are obligatory growth factors for early embryogenesis and heart formation. SMAD proteins transduce signals of the TGF-beta superfamily. We isolated chicken Smad6 (cSmad6), a member of inhibitory SMADs, and found its expression to be remarkably restricted to the developing heart, eyes, and limbs. cSmad6 expression was detected in the cardiogenic region of stage 5 embryos and overlapped Nkx2-5 and bmp-2, -4, and -7 expression. Throughout development, cSmad6 was expressed strongly in the heart, primarily in the myocardium, endocardium, and endocardial cushion tissue. Myocardial expression of cSmad6 was stronger in the forming septum, where highly localized expression of bmp-2 and -4 was also observed. Ectopically applied BMP-2 protein induced the expression of cSmad6, a putative negative regulator of BMP-signaling pathway, in anterior medial mesoendoderm of stage 4-5 embryos. In addition, blocking of BMP signaling using Noggin downregulated cSmad6 in cardiogenic tissue. cSmad1, one of the positive mediators of BMP signaling, was also expressed in cardiogenic region, but was not BMP-2 inducible. Our data suggest that cSmad6 has a role in orchestrating BMP-mediated cardiac development. We propose the possible mechanism of action of cSmad6 as modulating BMP signal by keeping a balance between constitutively expressed pathway-specific cSmad1 and ligand-induced inhibitory cSmad6 in the developing heart.

Amino Acid Sequence↗
PubMed1999

Expression of the Fanconi anemia group A gene (Fanca) during mouse embryogenesis.

About 80% of all cases of Fanconi anemia (FA) can be accounted for by complementation groups A and C. To understand the relationship between these groups, we analyzed the expression pattern of the mouse FA group-A gene (Fanca) during embryogenesis and compared it with the known pattern of the group-C gene (Fancc). Northern analysis of RNA from mouse embryos at embryonic days 7, 11, 15, and 17 showed a predominant 4.5 kb band in all stages. By in situ hybridization, Fanca transcripts were found in the whisker follicles, teeth, brain, retina, kidney, liver, and limbs. There was also stage-specific variation in Fanca expression, particularly within the developing whiskers and the brain. Some tissues known to express Fancc (eg, gut) failed to show Fanca expression. These observations show that (1) Fanca is under both tissue- and stage-specific regulation in several tissues; (2) the expression pattern of Fanca is consistent with the phenotype of the human disease; and (3) Fanca expression is not necessarily coupled to that of Fancc. The presence of distinct tissue targets for FA genes suggests that some of the variability in the clinical phenotype can be attributed to the complementation group assignment.

Ameloblasts↗
PubMed1999

The mPer2 gene encodes a functional component of the mammalian circadian clock.

Circadian rhythms are driven by endogenous biological clocks that regulate many biochemical, physiological and behavioural processes in a wide range of life forms. In mammals, there is a master circadian clock in the suprachiasmatic nucleus of the anterior hypothalamus. Three putative mammalian homologues (mPer1, mPer2 and mPer3) of the Drosophila circadian clock gene period (per) have been identified. The mPer genes share a conserved PAS domain (a dimerization domain found in Per, Arnt and Sim) and show a circadian expression pattern in the suprachiasmatic nucleus. To assess the in vivo function of mPer2, we generated and characterized a deletion mutation in the PAS domain of the mouse mPer2 gene. Here we show that mice homozygous for this mutation display a shorter circadian period followed by a loss of circadian rhythmicity in constant darkness. The mutation also diminishes the oscillating expression of both mPer1 and mPer2 in the suprachiasmatic nucleus, indicating that mPer2 may regulate mPer1 in vivo. These data provide evidence that an mPer gene functions in the circadian clock, and define mPer2 as a component of the mammalian circadian oscillator.

Animals↗
PubMed1998

The lissencephaly gene product Lis1, a protein involved in neuronal migration, interacts with a nuclear movement protein, NudC.

Important clues to how the mammalian cerebral cortex develops are provided by the analysis of genetic diseases that cause cortical malformations [1-5]. People with Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) have a hemizygous deletion or mutation in the LIS1 gene [3,6]; both conditions are characterized by a smooth cerebral surface, a thickened cortex with four abnormal layers, and misplaced neurons [7,8]. LIS1 is highly expressed in the ventricular zone and the cortical plate [9,10], and its product, Lis1, has seven WD repeats [3]; several proteins with such repeats have been shown to interact with other polypeptides, giving rise to multiprotein complexes [11]. Lis1 copurifies with platelet-activating factor acetylhydrolase subunits alpha 1 and alpha 2 [12], and with tubulin; it also reduces microtubule catastrophe events in vitro [13]. We used a yeast two-hybrid screen to isolate new Lis1-interacting proteins and found a mammalian ortholog of NudC, a protein required for nuclear movement in Aspergillus nidulans [14]. The specificity of the mammalian NudC-Lis1 interaction was demonstrated by protein-protein interaction assays in vitro and by co-immunoprecipitation from mouse brain extracts. In addition, the murine mNudC and mLis1 genes are coexpressed in the ventricular zone of the forebrain and in the cortical plate. The interaction of Lis1 with NudC, in conjunction with the MDS and ILS phenotypes, raises the possibility that nuclear movement in the ventricular zone is tied to the specification of neuronal fates and thus to cortical architecture.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1998

Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.

The E6-AP ubiquitin ligase (human/mouse gene UBE3A/Ube3a) promotes the degradation of p53 in association with papilloma E6 protein, and maternal deficiency causes human Angelman syndrome (AS). Ube3a is imprinted with silencing of the paternal allele in hippocampus and cerebellum in mice. We found that the phenotype of mice with maternal deficiency (m-/p+) for Ube3a resembles human AS with motor dysfunction, inducible seizures, and a context-dependent learning deficit. Long-term potentiation (LTP) was severely impaired in m-/p+ mice despite normal baseline synaptic transmission and neuroanatomy, indicating that ubiquitination may play a role in mammalian LTP and that LTP may be abnormal in AS. The cytoplasmic abundance of p53 was increased in postmitotic neurons in m-/p+ mice and in AS, providing a potential biochemical basis for the phenotype through failure to ubiquitinate and degrade various effectors.

Acoustic Stimulation↗
PubMed1997

A differential response of two putative mammalian circadian regulators, mper1 and mper2, to light.

A mouse gene, mper1, having all the properties expected of a circadian clock gene, was reported recently. This gene is expressed in a circadian pattern in the suprachiasmatic nucleus (SCN). mper1 maintains this pattern of circadian expression in constant darkness and can be entrained to a new light/dark cycle. Here we report the isolation of a second mammalian gene, mper2, which also has these properties and greater homology to Drosophila period. Expression of mper1 and mper2 is overlapping but asynchronous by 4 hr. mper1, unlike period and mper2, is expressed rapidly after exposure to light at CT22. It appears that mper1 is the pacemaker component which responds to light and thus mediates photic entrainment.

Amino Acid Sequence↗
PubMed1997

RIGUI, a putative mammalian ortholog of the Drosophila period gene.

The molecular components of mammalian circadian clocks are elusive. We have isolated a human gene termed RIGUI that encodes a bHLH/PAS protein 44% homologous to Drosophila period. The highly conserved mouse homolog (m-rigui) is expressed in a circadian pattern in the suprachiasmatic nucleus (SCN), the master regulator of circadian clocks in mammals. Circadian expression in the SCN continues in constant darkness, and a shift in the light/dark cycle evokes a proportional shift of m-rigui expression in the SCN. m-rigui transcripts also appear in a periodic pattern in Purkinje neurons, pars tuberalis, and retina, but with a timing of oscillation different from that seen in the SCN. Sequence homology and circadian patterns of expression suggest that RIGUI is a mammalian ortholog of the Drosophila period gene, raising the possibility that a regulator of circadian clocks is conserved.

Adaptation, Ocular↗
PubMed1997

Chicken Nkx-2.8: a novel homeobox gene expressed in early heart progenitor cells and pharyngeal pouch-2 and -3 endoderm.

Members of the NK family of homeobox transcription factors regulate critical steps of organogenesis during vertebrate development. In the studies described in this report, we have isolated and functionally characterized the chicken Nkx-2.8 (cNkx-2.8) cDNA and protein and defined the temporal and spatial pattern of cNkx-2.8 gene expression during chicken development. cNkx-2.8 transcripts are first detectable at HH stage 7 in the splanchnopleura. At stage 10(+), the cNkx-2.8 gene is expressed in the linear heart tube and the dorsal half of the vitelline vein. However, after looping, HH stage 13, cNkx-2.8 is no longer expressed in the looped heart tube, but is expressed in the ventral pharyngeal endoderm. At stage 15, in addition to the pharyngeal expression pattern, cNkx-2.8 is expressed in the ectoderm of the pharyngeal arches and the aortic sac. By HH Stage 17, cNkx-2.8 expression is detectable in lateral endoderm of the second and third pharyngeal pouches, the posterior portion of the aortic sac, and the sinus venosus. cNkx-2.8 binds to previously characterized Nkx2-1 and Nkx2-5 DNA-binding sites and overexpression of cNkx-2.8 transactivates a minimal promoter which contains multimerized Nkx-2 DNA-binding sites. In addition, cNkx-2.8 and serum response factor can coactivate a minimal cardiac alpha-actin promoter. These data are consistent with a model in which cNkx-2.8 performs a unique temporally and spatially restricted function in the developing embryonic heart and pharyngeal region. Moreover, the coexpression of cNkx-2.5 and -2.8 raises the possibility that cNkx-2. 8 may have a redundant role with cNkx-2.5 in the coalescing heart tube and may play an important role in the transcriptional program(s) that underlies thymus formation. The existence of multiple NK-2 family members and their partially overlapping patterns of expression are discussed within the framework of a "Nkx code."

Amino Acid Sequence↗