Obesity and mild hyperinsulinemia found in neuropeptide Y-Y1 receptor-deficient mice

To elucidate the role of neuropeptide Y (NPY)-Y1 receptor (Y1-R) in food intake, energy expenditure, and other possible functions, we have generated Y1-R-deficient mice (Y1-R−/−) by gene targeting. Contrary to our hypothesis that the lack of NPY signaling via Y1-R would result in impaired feeding and weight loss, Y1-R−/− mice showed a moderate obesity and mild hyperinsulinemia without hyperphagia. Although there was some variation between males and females, typical characteristics of Y1-R−/− mice include: greater body weight (females more than males), an increase in the weight of white adipose tissue (WAT) (approximately 4-fold in females), an elevated basal level of plasma insulin (approximately 2-fold), impaired insulin secretion in response to glucose administration, and a significant changes in mitochondrial uncoupling protein (UCP) gene expression (up-regulation of UCP1 in brown adipose tissue and down-regulation of UCP2 in WAT). These results suggest either that the Y1-R in the hypothalamus is not a key molecule in the leptin/NPY pathway, which controls feeding behavior, or that its deficiency is compensated by other receptors, such as NPY-Y5 receptor. We believe that the mild obesity found in Y1-R−/− mice (especially females) was caused by the impaired control of insulin secretion and/or low energy expenditure, including the lowered expression of UCP2 in WAT. This model will be useful for studying the mechanism of mild obesity and abnormal insulin metabolism in noninsulin-dependent diabetes mellitus.

Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: The effects of ionizing radiation on growth, survival, and development

We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination. These defects may be related to abnormalities in DNA metabolism that arise from the inability of Ku80 mutant cells to process DNA double-strand breaks. To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation. Ku80−/− embryonic stem cells are more sensitive than controls to γ-irradiation, and pre-B cells derived from Ku80 mutant mice display enhanced spontaneous and γ-ray-induced apoptosis. We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development in Ku80-deficient mice. Ku80−/− mice display a hypersensitivity to γ-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality. Exposure of newborn Ku80−/− mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation. However, unlike severe combined immunodeficient mice, radiation-induced T cell development in Ku80−/− mice is not accompanied by extensive thymocyte proliferation. The response of Ku80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair.

Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms, GAD65 and GAD67. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that GAD65 may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding GAD65 and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to GAD67−/− animals, which are born with developmental abnormalities and die shortly after birth, GAD65−/− mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5′ phosphate-inducible apo-enzyme reservoir is significantly decreased. GAD65−/− mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in GAD65−/− mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the GAD65−/− mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by GAD65 is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present GAD65−/− animals as a model of epilepsy involving GABA-ergic pathways.

Recovery of hearing and vocal behavior after hair-cell regeneration

Postmitotic hair-cell regeneration in the inner ear of birds provides an opportunity to study the effect of renewed auditory input on auditory perception, vocal production, and vocal learning in a vertebrate. We used behavioral conditioning to test both perception and vocal production in a small Australian parrot, the budgerigar. Results show that both auditory perception and vocal production are disrupted when hair cells are damaged or lost but that these behaviors return to near normal over time. Precision in vocal production completely recovers well before recovery of full auditory function. These results may have particular relevance for understanding the relation between hearing loss and human speech production especially where there is consideration of an auditory prosthetic device. The present results show, at least for a bird, that even limited recovery of auditory input soon after deafening can support full recovery of vocal precision.

Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1, has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

Defective localization of the NADPH phagocyte oxidase to Salmonella-containing phagosomes in tumor necrosis factor p55 receptor-deficient macrophages

Tumor necrosis factor receptor (TNFR) p55-knockout (KO) mice are susceptible profoundly to Salmonella infection. One day after peritoneal inoculation, TNFR-KO mice harbor 1,000-fold more bacteria in liver and spleen than wild-type mice despite the formation of well organized granulomas. Macrophages from TNFR-KO mice produce abundant quantities of reactive oxygen and nitrogen species in response to Salmonella but nevertheless exhibit poor bactericidal activity. Treatment with IFN-γ enhances killing by wild-type macrophages but does not restore the killing defect of TNFR-KO cells. Bactericidal activity of macrophages can be abrogated by a deletion in the gene encoding TNFα but not by saturating concentrations of TNF-soluble receptor, suggesting that intracellular TNFα can regulate killing of Salmonella by macrophages. Peritoneal macrophages from TNFR-KO mice fail to localize NADPH oxidase-containing vesicles to Salmonella-containing vacuoles. A TNFR-KO mutation substantially restores virulence to an attenuated mutant bacterial strain lacking the type III secretory system encoded by Salmonella pathogenicity island 2 (SPI2), suggesting that TNFα and SPI2 have opposing actions on a common pathway of vesicular trafficking. TNFα–TNFRp55 signaling plays a critical role in the immediate innate immune response to an intracellular pathogen by optimizing the delivery of toxic reactive oxygen species to the phagosome.

Cardiovascular abnormalities with normal blood pressure in tissue kallikrein-deficient mice

Tissue kallikrein is a serine protease thought to be involved in the generation of bioactive peptide kinins in many organs like the kidneys, colon, salivary glands, pancreas, and blood vessels. Low renal synthesis and urinary excretion of tissue kallikrein have been repeatedly linked to hypertension in animals and humans, but the exact role of the protease in cardiovascular function has not been established largely because of the lack of specific inhibitors. This study demonstrates that mice lacking tissue kallikrein are unable to generate significant levels of kinins in most tissues and develop cardiovascular abnormalities early in adulthood despite normal blood pressure. The heart exhibits septum and posterior wall thinning and a tendency to dilatation resulting in reduced left ventricular mass. Cardiac function estimated in vivo and in vitro is decreased both under basal conditions and in response to βadrenergic stimulation. Furthermore, flow-induced vasodilatation is impaired in isolated perfused carotid arteries, which express, like the heart, low levels of the protease. These data show that tissue kallikrein is the main kinin-generating enzyme in vivo and that a functional kallikrein–kinin system is necessary for normal cardiac and arterial function in the mouse. They suggest that the kallikrein–kinin system could be involved in the development or progression of cardiovascular diseases.

FHIT gene therapy prevents tumor development in Fhit-deficient mice

The tumor suppressor gene FHIT spans a common fragile site and is highly susceptible to environmental carcinogens. FHIT inactivation and loss of expression is found in a large fraction of premaligant and malignant lesions. In this study, we were able to inhibit tumor development by oral gene transfer, using adenoviral or adenoassociated viral vectors expressing the human FHIT gene, in heterozygous Fhit+/− knockout mice, that are prone to tumor development after carcinogen exposure. We therefore suggest that FHIT gene therapy could be a novel clinical approach not only in treatment of early stages of cancer, but also in prevention of human cancer.

Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice

Induction of phase 2 enzymes, which neutralize reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through the antioxidant response element (ARE) found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to the ARE, appears to be essential for the induction of prototypical phase 2 enzymes such as glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase (NQO1). Constitutive hepatic and gastric activities of GST and NQO1 were reduced by 50–80% in nrf2-deficient mice compared with wild-type mice. Moreover, the 2- to 5-fold induction of these enzymes in wild-type mice by the chemoprotective agent oltipraz, which is currently in clinical trials, was almost completely abrogated in the nrf2-deficient mice. In parallel with the enzymatic changes, nrf2-deficient mice had a significantly higher burden of gastric neoplasia after treatment with benzo[a]pyrene than did wild-type mice. Oltipraz significantly reduced multiplicity of gastric neoplasia in wild-type mice by 55%, but had no effect on tumor burden in nrf2-deficient mice. Thus, Nrf2 plays a central role in the regulation of constitutive and inducible expression of phase 2 enzymes in vivo and dramatically influences susceptibility to carcinogenesis. Moreover, the total loss of anticarcinogenic efficacy of oltipraz in the nrf2-disrupted mice highlights the prime importance of elevated phase 2 gene expression in chemoprotection by this and similar enzyme inducers.

CA1-specific N-methyl-d-aspartate receptor knockout mice are deficient in solving a nonspatial transverse patterning task

In both humans and animals, the hippocampus is critical to memory across modalities of information (e.g., spatial and nonspatial memory) and plays a critical role in the organization and flexible expression of memories. Recent studies have advanced our understanding of cellular basis of hippocampal function, showing that N-methyl-d-aspartate (NMDA) receptors in area CA1 are required in both the spatial and nonspatial domains of learning. Here we examined whether CA1 NMDA receptors are specifically required for the acquisition and flexible expression of nonspatial memory. Mice lacking CA1 NMDA receptors were impaired in solving a transverse patterning problem that required the simultaneous acquisition of three overlapping odor discriminations, and their impairment was related to an abnormal strategy by which they failed to adequately sample and compare the critical odor stimuli. By contrast, they performed normally, and used normal stimulus sampling strategies, in the concurrent learning of three nonoverlapping concurrent odor discriminations. These results suggest that CA1 NMDA receptors play a crucial role in the encoding and flexible expression of stimulus relations in nonspatial memory.

Defect in regulated secretion of P-selectin affects leukocyte recruitment in von Willebrand factor-deficient mice

Stimulation of endothelial cells by various inflammatory mediators leads to release of Weibel–Palade bodies and therefore to exocytosis of both P-selectin (adhesion receptor for leukocytes) and von Willebrand factor (vWf) (platelet ligand). The potential role of vWf in leukocyte recruitment was investigated with the use of vWf-deficient mice. We report a strong reduction of leukocyte rolling in venules of vWf-deficient mice. Similarly, vWf deficiency led to a decrease in neutrophil recruitment in a cytokine-induced meningitis model as well as in early skin wounds. In all instances with an antibody that preferentially recognizes plasma membrane P-selectin, we observed a dramatic reduction in P-selectin expression at the cell surface of vWf-deficient endothelium. With confocal microscopy, we found that the typical rodlike shape of the Weibel–Palade body is missing in vWf −/− endothelial cells and that part of the P-selectin content in the vWf −/− cells colocalized with LAMP-1, a lysosomal marker. However, intracellular P-selectin levels were similar in tumor necrosis factor α- and lipopolysaccharide-activated cells of both genotypes. We conclude that the absence of vWf, as found in severe von Willebrand disease, leads to a defect in Weibel–Palade body formation. This defect results in decreased P-selectin translocation to the cell surface and reduced leukocyte recruitment in early phases of inflammation.

Characterization of N-Glycans from Arabidopsis. Application to a Fucose-Deficient Mutant1

The structures of glycans N-linked to Arabidopsis proteins have been fully identified. From immuno- and affinodetections on blots, chromatography, nuclear magnetic resonance, and glycosidase sequencing data, we show that Arabidopsis proteins are N-glycosylated by high-mannose-type N-glycans from Man5GlcNAc2 to Man9GlcNAc2, and by xylose- and fucose (Fuc)-containing oligosaccharides. However, complex biantenary structures containing the terminal Lewis a epitope recently reported in the literature (A.-C. Fitchette-Lainé, V. Gomord, M. Cabanes, J.-C. Michalski, M. Saint Macary, B. Foucher, B. Cavalier, C. Hawes, P. Lerouge, and L. Faye [1997] Plant J 12: 1411–1417) were not detected. A similar study was done on the Arabidopsis mur1 mutant, which is affected in the biosynthesis of l-Fuc. In this mutant, one-third of the Fuc residues of the xyloglucan has been reported to be replaced by l-galactose (Gal) (E. Zablackis, W.S. York, M. Pauly, S. Hantus, W.D. Reiter, C.C.S. Chapple, P. Albersheim, and A. Darvill [1996] Science 272: 1808–1810). N-linked glycans from the mutant were identified and their structures were compared with those isolated from the wild-type plants. In about 95% of all N-linked glycans from the mur1 plant, l-Fuc residues were absent and were not replaced by another monosaccharide. However, in the remaining 5%, l-Fuc was found to be replaced by a hexose residue. From nuclear magnetic resonance and mass spectrometry data of the mur1 N-glycans, and by analogy with data reported on mur1 xyloglucan, this subpopulation of N-linked glycans was proposed to be l-Gal-containing N-glycans resulting from the replacement of l-Fuc by l-Gal.

Feedback Inhibition of Chlorophyll Synthesis in the Phytochrome Chromophore-Deficient aurea and yellow-green-2 Mutants of Tomato

The aurea (au) and yellow-green-2 (yg-2) mutants of tomato (Solanum lycopersicum L.) are unable to synthesize the linear tetrapyrrole chromophore of phytochrome, resulting in plants with a yellow-green phenotype. To understand the basis of this phenotype, we investigated the consequences of the au and yg-2 mutations on tetrapyrrole metabolism. Dark-grown seedlings of both mutants have reduced levels of protochlorophyllide (Pchlide) due to an inhibition of Pchlide synthesis. Feeding experiments with the tetrapyrrole precursor 5-aminolevulinic acid (ALA) demonstrate that the pathway between ALA and Pchlide is intact in au and yg-2 and suggest that the reduction in Pchlide is a result of the inhibition of ALA synthesis. This inhibition was independent of any deficiency in seed phytochrome, and experiments using an iron chelator to block heme synthesis demonstrated that both mutations inhibited the degradation of the physiologically active heme pool, suggesting that the reduction in Pchlide synthesis is a consequence of feedback inhibition by heme. We discuss the significance of these results in understanding the chlorophyll-deficient phenotype of the au and yg-2 mutants.

Electric Signaling and Pin2 Gene Expression on Different Abiotic Stimuli Depend on a Distinct Threshold Level of Endogenous Abscisic Acid in Several Abscisic Acid-Deficient Tomato Mutants1

Experiments were performed on three abscisic acid (ABA)-deficient tomato (Lycopersicon esculentum Mill.) mutants, notabilis, flacca, and sitiens, to investigate the role of ABA and jasmonic acid (JA) in the generation of electrical signals and Pin2 (proteinase inhibitor II) gene expression. We selected these mutants because they contain different levels of endogenous ABA. ABA levels in the mutant sitiens were reduced to 8% of the wild type, in notabilis they were reduced to 47%, and in flacca they were reduced to 21%. In wild-type and notabilis tomato plants the induction of Pin2 gene expression could be elicited by heat treatment, current application, or mechanical wounding. In flacca and sitiens only heat stimulation induced Pin2 gene expression. JA levels in flacca and sitiens plants also accumulated strongly upon heat stimulation but not upon mechanical wounding or current application. Characteristic electrical signals evolved in the wild type and in the notabilis and flacca mutants consisting of a fast action potential and a slow variation potential. However, in sitiens only heat evoked electrical signals; mechanical wounding and current application did not change the membrane potential. In addition, exogenous application of ABA to wild-type tomato plants induced transient changes in membrane potentials, indicating the involvement of ABA in the generation of electrical signals. Our data strongly suggest the presence of a minimum threshold value of ABA within the plant that is essential for the early events in electrical signaling and mediation of Pin2 gene expression upon wounding. In contrast, heat-induced Pin2 gene expression and membrane potential changes were not dependent on the ABA level but, rather, on the accumulation of JA.

Naturally variant autosomal and sex-linked loci determine the severity of iron overload in β2-microglobulin-deficient mice

Hereditary hemochromatosis (HH) is a common chronic human genetic disorder whose hallmark is systemic iron overload. Homozygosity for a mutation in the MHC class I heavy chain paralogue gene HFE has been found to be a primary cause of HH. However, many individuals homozygous for the defective allele of HFE do not develop iron overload, raising the possibility that genetic variation in modifier loci contributes to the HH phenotype. Mice deficient in the product of the β2-microglobulin (β2M) class I light chain fail to express HFE and other MHC class I family proteins, and they have been found to manifest many characteristics of the HH phenotype. To determine whether natural genetic variation plays a role in controlling iron overload, we performed classical genetic analysis of the iron-loading phenotype in β2M-deficient mice in the context of different genetic backgrounds. Strain background was found to be a major determinant in iron loading. Sex played a role that was less than that of strain background but still significant. Resistance and susceptibility to iron overload segregated as complex genetic traits in F1 and back-cross progeny. These results suggest the existence of naturally variant autosomal and Y chromosome-linked modifier loci that, in the context of mice genetically predisposed by virtue of a β2M deficiency, can profoundly influence the severity of iron loading. These results thus provide a genetic explanation for some of the variability of the HH phenotype.