Biomedical subjects

David M Valenzuela

Publications and source records attributed to David M Valenzuela.

16 recordsLinked to original sources

PubMed2006

F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.

A useful approach for exploring gene function involves generating mutant mice from genetically modified embryonic stem (ES) cells. Recent advances in genetic engineering of ES cells have shifted the bottleneck in this process to the generation of mice. Conventional injections of ES cells into blastocyst hosts produce F0 generation chimeras that are only partially derived from ES cells, requiring additional breeding to obtain mutant mice that can be phenotyped. The tetraploid complementation approach directly yields mice that are almost entirely derived from ES cells, but it is inefficient, works only with certain hybrid ES cell lines and suffers from nonspecific lethality and abnormalities, complicating phenotypic analyses. Here we show that laser-assisted injection of either inbred or hybrid ES cells into eight cell-stage embryos efficiently yields F0 generation mice that are fully ES cell-derived and healthy, exhibit 100% germline transmission and allow immediate phenotypic analysis, greatly accelerating gene function assignment.

Animals↗

PubMed2006

SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells.

Sir2 is an NAD-dependent deacetylase that connects metabolism with longevity in yeast, flies, and worms. Mammals have seven Sir2 homologs (SIRT1-7). We show that SIRT4 is a mitochondrial enzyme that uses NAD to ADP-ribosylate and downregulate glutamate dehydrogenase (GDH) activity. GDH is known to promote the metabolism of glutamate and glutamine, generating ATP, which promotes insulin secretion. Loss of SIRT4 in insulinoma cells activates GDH, thereby upregulating amino acid-stimulated insulin secretion. A similar effect is observed in pancreatic beta cells from mice deficient in SIRT4 or on the dietary regimen of calorie restriction (CR). Furthermore, GDH from SIRT4-deficient or CR mice is insensitive to phosphodiesterase, an enzyme that cleaves ADP-ribose, suggesting the absence of ADP-ribosylation. These results indicate that SIRT4 functions in beta cell mitochondria to repress the activity of GDH by ADP-ribosylation, thereby downregulating insulin secretion in response to amino acids, effects that are alleviated during CR.

Adenosine Diphosphate Ribose↗

PubMed2006

Cutting edge: IL-23 cross-regulates IL-12 production in T cell-dependent experimental colitis.

Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. Finally, in vivo blockade of IL-12 p40 in IL-23-deficient mice rescued mice from lethal colitis. Taken together, our data identify cross-regulation of IL-12 expression by IL-23 as novel key regulatory pathway during initiation of T cell dependent colitis.

Animals↗

PubMed2006

Peripheral myelin protein 22 is in complex with alpha6beta4 integrin, and its absence alters the Schwann cell basal lamina.

Peripheral myelin protein 22 (PMP22) is a tetraspan membrane glycoprotein, the misexpression of which is associated with hereditary demyelinating neuropathies. Myelinating Schwann cells (SCs) produce the highest levels of PMP22, yet the function of the protein in peripheral nerve biology is unresolved. To investigate the potential roles of PMP22, we engineered a novel knock-out (-/-) mouse line by replacing the first two coding exons of pmp22 with the lacZ reporter. PMP22-deficient mice show strong beta-galactosidase reactivity in peripheral nerves, cartilage, intestines, and lungs, whereas phenotypically they display the characteristics of tomaculous neuropathy. In the absence of PMP22, myelination of peripheral nerves is delayed, and numerous axon-SC profiles show loose basal lamina, suggesting altered interactions of the glial cells with the extracellular matrix. The levels of beta4 integrin, a molecule involved in the linkage between SCs and the basal lamina, are severely reduced in nerves of PMP22-deficient mice. During early stages of myelination, PMP22 and beta4 integrin are coexpressed at the cell surface and can be coimmunoprecipitated together with laminin and alpha6 integrin. In agreement, in clone A colonic carcinoma cells, epitope-tagged PMP22 forms a complex with beta4 integrin. Together, these data indicate that PMP22 is a binding partner in the integrin/laminin complex and is involved in mediating the interaction of SCs with the extracellular environment.

Adenocarcinoma↗

PubMed2006

Resistin-like molecule beta regulates innate colonic function: barrier integrity and inflammation susceptibility.

BACKGROUND: Resistin-like molecule (RELM) beta is a cysteine-rich cytokine expressed in the gastrointestinal tract and implicated in insulin resistance and gastrointestinal nematode immunity; however, its function primarily remains an enigma. OBJECTIVE: We sought to elucidate the function of RELM-beta in the gastrointestinal tract. METHODS: We generated RELM-beta gene-targeted mice and examined colonic epithelial barrier function, gene expression profiles, and susceptibility to acute colonic inflammation. RESULTS: We show that RELM-beta is constitutively expressed in the colon by goblet cells and enterocytes and has a role in homeostasis, as assessed by alterations in colon mRNA transcripts and epithelial barrier function in the absence of RELM-beta. Using acute colonic inflammatory models, we demonstrate that RELM-beta has a central role in the regulation of susceptibility to colonic inflammation. Mechanistic studies identify that RELM-beta regulates expression of type III regenerating gene (REG) (REG3beta and gamma), molecules known to influence nuclear factor kappaB signaling. CONCLUSIONS: These data define a critical role for RELM-beta in the maintenance of colonic barrier function and gastrointestinal innate immunity. CLINICAL IMPLICATIONS: These findings identify RELM-beta as an important molecule in homeostatic gastrointestinal function and colonic inflammation, and as such, these results have implications for a variety of human inflammatory gastrointestinal conditions, including allergic gastroenteropathies.

Animals↗

PubMed2005

Leucine aminopeptidase is not essential for trimming peptides in the cytosol or generating epitopes for MHC class I antigen presentation.

To detect viral infections and tumors, CD8+ T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Leucine aminopeptidase (LAP) is an IFN-inducible cytosolic aminopeptidase that can trim precursor peptides to mature epitopes and has been thought to play an important role in Ag presentation. To examine the role of LAP in generating MHC class I peptides in vivo, we generated LAP-deficient mice and LAP-deficient cell lines. These mutant mice and cells are viable and grow normally. The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full-length Ag constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. These data demonstrate that LAP is not an essential enzyme for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases.

Amino Acid Sequence↗

PubMed2005

Lhx6 delineates a pathway mediating innate reproductive behaviors from the amygdala to the hypothalamus.

In mammals, innate reproductive and defensive behaviors are mediated by anatomically segregated connections between the amygdala and hypothalamus. This anatomic segregation poses the problem of how the brain integrates activity in these circuits when faced with conflicting stimuli eliciting such mutually exclusive behaviors. Using genetically encoded and conventional axonal tracers, we have found that the transcription factor Lhx6 delineates the reproductive branch of this pathway. Other Lhx proteins mark neurons in amygdalar nuclei implicated in defense. We have traced parallel projections from the posterior medial amygdala, activated by reproductive or defensive olfactory stimuli, respectively, to a point of convergence in the ventromedial hypothalamus. The opposite neurotransmitter phenotypes of these convergent projections suggest a "gate control" mechanism for the inhibition of reproductive behaviors by threatening stimuli. Our data therefore identify a potential neural substrate for integrating the influences of conflicting behavioral cues and a transcription factor family that may contribute to the development of this substrate.

Aggression↗

PubMed2005

Functional characterization of mouse RDH11 as a retinol dehydrogenase involved in dark adaptation in vivo.

We previously cloned mouse RDH11 (mRDH11) as a gene regulated by the transcription factor sterol regulatory element-binding proteins and showed that it is a retinol dehydrogenase expressed in non-ocular tissues such as the liver and testis and in the retina (Kasus-Jacobi, A., Ou, J., Bashmakov, Y. K., Shelton, J. M., Richardson, J. A., Goldstein, J. L., and Brown, M. S. (2003) J. Biol. Chem. 278, 32380-32389). It was proposed to function in the recycling of the visual chromophore 11-cis-retinal after photoisomerization by a bleaching light, a pathway referred to as the visual cycle. In this work, we describe our studies on the ocular function of mRDH11. We created a knockout mouse by replacing the mrdh11 coding sequence with the lacZ reporter gene for expression profiling. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining demonstrated active transcription of this gene in photoreceptor cells. We show by immunoblot analysis that mRDH11 is associated with retinal membranes purified from a non-outer segment fraction of the retina. No obvious retinal defect was found during development and aging of RDH11-deficient mice. The functional consequences of mRDH11 disruption were investigated by electroretinography. Dark adaptation was delayed by a factor of 2.5-3 compared with wild-type mice. However, the kinetics of 11-cis-retinal recycling during dark adaptation was not affected, suggesting that mRDH11 is not involved in the visual cycle. We propose that mRDH11 disruption affects retinoid metabolism in photoreceptor inner segments and delays the kinetics of dark adaptation through modulation of calcium homeostasis.

Aging↗

PubMed2005

Resistance to diet-induced obesity in mice globally overexpressing OGH/GPB5.

We identified a glycoprotein hormone beta-subunit (OGH, also called GPB5) that, as a heterodimer with the alpha-subunit GPA2, serves as a second ligand for the thyroid-stimulating hormone receptor. Mice in which the OGH gene is deleted (OGH-/-) are indistinguishable from WT littermates in body weight, response to high-fat diet, metabolic parameters, body composition, and insulin tolerance. Mice engineered to transgenically globally overexpress OGH (OGH-TG) develop approximately 2-fold elevations in their basal thyroid levels and weigh slightly less than WT littermates despite increased food intake because of an increase in their metabolic rates. Moreover, when OGH-TG mice are challenged with a high-fat diet, they gain significantly less weight and body fat than their WT littermates. The OGH-TG mice also have reduced blood glucose, insulin, cholesterol, and triglycerides. In contrast to other approaches in which the thyroid axis is activated, OGH-TG mice exhibit only minor changes in heart rate and blood pressure. Our findings suggest that constitutive low-level activation of the thyroid axis (via OGH or other means) may provide a beneficial therapeutic approach for combating diet-induced obesity.

Animals↗

PubMed2004

Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development.

Vascular development depends on the highly coordinated actions of a variety of angiogenic regulators, most of which apparently act downstream of vascular endothelial growth factor (VEGF). One potential such regulator is delta-like 4 ligand (Dll4), a recently identified partner for the Notch receptors. We generated mice in which the Dll4 gene was replaced with a reporter gene, and found that Dll4 expression is initially restricted to large arteries in the embryo, whereas in adult mice and tumor models, Dll4 is specifically expressed in smaller arteries and microvessels, with a striking break in expression just as capillaries merge into venules. Consistent with these arterial-specific expression patterns, heterozygous deletion of Dll4 resulted in prominent albeit variable defects in arterial development (reminiscent of those in Notch knockouts), including abnormal stenosis and atresia of the aorta, defective arterial branching from the aorta, and even arterial regression, with occasional extension of the defects to the venous circulation; also noted was gross enlargement of the pericardial sac and failure to remodel the yolk sac vasculature. These striking phenotypes resulting from heterozygous deletion of Dll4 indicate that vascular development may be as sensitive to subtle changes in Dll4 dosage as it is to subtle changes in VEGF dosage, because VEGF accounts for the only other example of haploid insufficiency, resulting in obvious vascular abnormalities. In summary, Dll4 appears to be a major trigger of Notch receptor activities previously implicated in arterial and vascular development, and it may represent a new opportunity for pro- and anti-angiogenic therapies.

Animals↗

PubMed2003

High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.

One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive-negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5-1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.

Animals↗

PubMed1996

The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo.

Formation of neuromuscular synapses requires a series of inductive interactions between growing motor axons and differentiating muscle cells, culminating in the precise juxtaposition of a highly specialized nerve terminal with a complex molecular structure on the postsynaptic muscle surface. The receptors and signaling pathways mediating these inductive interactions are not known. We have generated mice with a targeted disruption of the gene encoding MuSK, a receptor tyrosine kinase selectively localized to the postsynaptic muscle surface. Neuromuscular synapses do not form in these mice, suggesting a failure in the induction of synapse formation. Together with the results of an accompanying manuscript, our findings indicate that MuSK responds to a critical nerve-derived signal (agrin), and in turn activates signaling cascades responsible for all aspects of synapse formation, including organization of the postsynaptic membrane, synapse-specific transcription, and presynaptic differentiation.

Agrin↗

PubMed1996

Agrin acts via a MuSK receptor complex.

Formation of th neuromuscular junction depends upon reciprocal inductive interactions between the developing nerve and muscle, resulting in the precise juxtaposition of a differentiated nerve terminal with a highly specialized patch on the muscle membrane, termed the motor endplate. Agrin is a nerve-derived factor that can induced molecular reorganizations at the motor endplate, but the mechanism of action of agrin remains poorly understood. MuSK is a receptor tyrosine kinase localized to the motor endplate, seemingly well positioned to receive a key nerve-derived signal. Mice lacking either agrin or MuSK have recently been generated and exhibit similarly profound defects in their neuromuscular junctions. Here we demonstrate that agrin acts via a receptor complex that includes MuSK as well as a myotube-specific accessory component.

Agrin↗

PubMed1996

Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis.

We report that the many Eph-related receptor tyrosine kinases, and their numerous membrane-bound ligands, can each be grouped into only two major specificity subclasses. Receptors in a given subclass bind most members of a corresponding ligand subclass. The physiological relevance of these groupings is suggested by viewing the collective distributions of all members of a subclass. These composite distributions, in contrast with less informative patterns seen with individual members of the family, reveal that the developing embryo is subdivided into domains defined by reciprocal and apparently mutually exclusive expression of a receptor subclass and its corresponding ligands. Receptors seem to encounter their ligands only at the interface between these domains. This reciprocal compartmentalization implicates the Eph family in the formation of spatial boundaries that may help to organize the developing body plan.

Animals↗

PubMed1993

Alternative forms of rat TrkC with different functional capabilities.

We have identified transcripts encoding several different forms of rat TrkC, a member of the Trk family of receptor tyrosine kinases that serves as a receptor for neurotrophin-3. Some forms of TrkC lack the intracytoplasmic kinase domain and thus resemble previously defined truncated variants of TrkB. Other forms of TrkC contain variable-sized amino acid insertions within the tyrosine kinase domain. Transcripts encoding all forms of TrkC can be detected throughout the nervous system, displaying substantial overlap as well as mutually exclusive distribution patterns with transcripts for TrkB. Strikingly, only transcripts encoding the truncated forms of TrkB and TrkC are found in astrocytes, peripheral nerve, and nonneural tissues. Finally, forms of TrkC containing insertions within the kinase domain retain their ability to autophosphorylate in response to neurotrophin-3, but cannot mediate proliferation in fibroblasts or neuronal differentiation in PC12 cells.

Amino Acid Sequence↗

PubMed1991

trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor.

A variety of findings seem to functionally link brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), while distinguishing both of these factors from the third member of the neurotrophin family, nerve growth factor (NGF). Here we demonstrate that all three of these neuronal survival molecules bind similarly to the low affinity NGF receptor, but that BDNF and NT-3, unlike NGF, do not act via the high affinity NGF receptor. However, both BDNF and NT-3, but not NGF, bind to full-length and truncated forms of a receptor-like tyrosine kinase, trkB, for which no ligand had previously been identified. In addition to binding BDNF and NT-3, trkB can mediate functional responses to both of these neurotrophins when it is expressed in PC12 cells, although BDNF appears to be the more effective ligand. Thus trkB encodes an essential component of a functional receptor for BDNF and NT-3, but not for NGF. Further evidence predicts the existence of additional functional receptors for the neurotrophins.

Adrenal Gland Neoplasms↗