Impaired eyeblink conditioning in 78 kDa-glucose regulated protein (GRP78)/immunoglobulin binding protein (BiP) conditional knockout mice

Evidence grows that the cerebellum and its associated circuitry are the essential neural substrates for standard delay classical eyeblink conditioning. To further investigate the relative roles of the cerebellar cortex and nuclei in eyeblink conditioning, a novel mouse model with Purkinje cell atrophy was studied. The 78 kDa-glucose regulated protein, a chaperone molecule, was knocked out leading to postnatal Purkinje cell degeneration (Wang et al., 2010), and standard delay eyeblink conditioning was performed in the conditional knockout mice. Learning was impaired, yet not completely prevented. Histological studies showed a reduction in the cell number and the size of the anterior interpositus nucleus. When the anterior interpositus nucleus was lesioned bilaterally, eyeblink conditioning was completely prevented. The important roles of both cerebellar cortex and AIP nucleus in eyeblink conditioning were seen.     

Prenatal expression of growth hormone receptor/binding protein and insulin-like growth factor-I (IGF-I) in the enamel organ

Immunohistochemistry was used to study the ontogeny of GH receptor/binding protein (GHR/BP) and IGF-I from the 13-day-old embryo (E13) to the E19 rat fetus in the developing incisor and molar. Analysis of serial sections revealed diffuse staining of GHR/BP and IGF-I at the bud and early cap stages within both the mesenchyme of the dental papilla and the ectodermal-erived enamel organ. Just before transition to the cap stage, immunoreactivity of GHR/BP and IGF-I increased in the epithelial bud and extended to the condensed dental mesenchyme. At the cap stage, the dental epithelium showed an intense expression of GHR/BP and IGF-I, whereas the dental mesenchymal cells showed very weak staining. The inner enamel epithelium and the outer enamel epithelium were positive for both GHR/BP and IGF-I in the bell stage. Differentiating ameloblasts, odontoblasts and the secretory ameloblasts and odontoblasts continued to express GHR/BP and IGF-I in incisors. These findings support the premise that growth hormone and IGF-I may play a role in embryonic tooth development by regulating the epithelial-mesenchymal interactions that influence events in growth and cytodifferentiation.     

Fertility defects in Surfactant associated protein D knockout female mice: altered ovarian hormone profile

Surfactant associated protein D (SFTPD, also known as SP-D), a pattern recognition molecule, is an integral component of the mucosal immune system of female reproductive tract (FRT). In addition to host defense functions in the FRT, recent evidences indicate immunomodulatory role of SFTPD in parturition and pre-term labor. Regulation of SFTPD expression by ovarian hormones in the mouse uterus implicates SFTPD of FRT in pregnancy establishment and maintenance. In the current study, we attempted to decipher the functional relevance of SFTPD in FRT by characterizing the fertility parameters of surfactant associated protein D knockout (Sftpd^tm1Jhf/Sftpd^tm1Jhf) female mice. Knockout female mice exhibited extended estrous cycle with altered serum profile of ovarian hormones. We also demonstrate altered expression of ovarian hormone receptors and hormone responsive genes ITGB1, LIF and HOXA10 in uteri of these mice. Knockout females mated with wild type males had significantly smaller litter size due to increased pre-implantation embryo loss. We also observed an altered immune profile in knockout mice uteri with elevated levels of inflammatory cytokines, increased numbers of pro-inflammatory monocytes/macrophages and lower FOXP3 levels during the pre-implantation period. LPS administration to pregnant knockout mice did not result in any increase in embryo implantation loss and was associated with a blunted uterine pro-inflammatory response, plausibly due to higher levels of serum progesterone. Taken together, our results demonstrate that SFTPD deficiency affects female fertility, highlighting roles for SFTPD in ovarian and uterine physiology.     

Actin binding LIM protein 3 (abLIM3)

LIM domain proteins were demonstrated to play key roles in various biological processes such as embryonic development, cell lineage determination, and cancer differentiation. Actin binding LIM protein 1 (abLIM1) was reported to be localized in a genomic region often deleted in human cancers and suggested to be involved in axon guidance. Recently, existence of a second family member was reported, actin binding LIM protein 2. By means of computational biology and comparative genomics, we now characterized an additional, third member of the actin binding LIM protein subgroup, actin binding LIM protein 3 (abLIM3). The human mRNA sequence was previously annotated as differentially regulated in hepatoblastoma compared to normal livers. Conservation of key structural features of abLIM1 and abLIM2, four LIM domains and a VHD domain, suggested comparable biological function of abLIM3 as a linker between actin cytoskeleton and cell signaling pathways. AbLIM3 was found to be conserved in vertebrates, as orthologous sequences were characterized for mouse, fish, and frog. In addition, we report the existence of abLIM2 orthologs in fish and frog, suggesting a similar degree of evolutionary conservation. The intracellular localization of the abLIM3 protein was predicted to be nuclear by means of Reinhardt's neural network and the k-nearest neighbor algorithm. The corresponding abLIM3 gene was localized to chromosome 5q32 and spanned 119 kb, organized in 24 exons. An RT-PCR based expression profile available from the human unidentified gene-encoded (HUGE) database demonstrated highest expression for abLIM3 in heart, lung, liver, and brain/cerebellum accompanied by lower expression in multiple other tissues. Furthermore, abLIM3 was expressed in fetal liver, CNS, and spinal cord.     

Prostanoids and inflammation: a new concept arising from receptor knockout mice

Prostanoids including various types of prostaglandins and thromboxanes are arachidonate metabolites produced and released in response to a variety of physiological and pathological stimuli and function to maintain the body homeostasis. Since cyclooxygenase, the enzyme initiating their biosynthesis, is inhibited by aspirin-like antipyretic, anti-inflammatory, and analgesic drugs, contribution of prostanoids to acute inflammation such as fever generation, pain sensitization, and inflammatory swelling has been recognized very early. On the other hand, since aspirin-like drugs generally show little effects on allergy and immunity, it has been believed that prostanoids play little roles in these processes. Prostanoids act on a family of G-protein-coupled receptors designated PGD receptor, PGE receptor subtypes EP1–EP4, PGF receptor, PGI receptor, and TX receptor to elicit their actions. Studies using mice deficient in each of these receptors have revealed that prostanoids indeed function in the above aspirin-sensitive processes. However, these studies have also revealed that prostanoids exert both pro-inflammatory and anti-inflammatory actions not only by acting as mediators of acute inflammation but also by regulating gene expression in mesenchymal and epithelial cells at inflammatory site. Such dual actions of prostanoids are frequently seen in immune and allergic reactions, where different type of prostanoids and their receptors often exert opposite actions in a single process. Thus, a new concept on the role of prostanoids in inflammation has arisen from studies using the receptor knockout mice.     

Lipoatrophic diabetes in Irs1(-/-)/Irs3(-/-) double knockout mice

Based on the phenotypes of knockout mice and cell lines, as well as pathway-specific analysis, the insulin receptor substrates IRS-1, IRS-2, IRS-3, and IRS-4 have been shown to play unique roles in insulin signal transduction. To investigate possible functional complementarity within the IRS family, we generated mice with double knockout of the genes for IRS-1/IRS-3 and IRS-1/IRS-4. Mice with a combined deficiency of IRS-1 and IRS-4 showed no differences from Irs1(-/-) mice with respect to growth and glucose homeostasis. In contrast, mice with a combined deficiency of IRS-1 and IRS-3 developed early-onset severe lipoatrophy associated with marked hyperglycemia, hyperinsulinemia, and insulin resistance. However, in contrast to other models of lipoatrophic diabetes, there was no accumulation of fat in liver or muscle. Furthermore, plasma leptin levels were markedly decreased, and adenovirus-mediated expression of leptin in liver reversed the hyperglycemia and hyperinsulinemia. The results indicate that IRS-1 and IRS-3 play important complementary roles in adipogenesis and establish the Irs1(-/-)/Irs3(-/-) double knockout mouse as a novel model of lipoatrophic diabetes.     

Behavioral and neurochemical characterization of alpha(2A)-adrenergic receptor knockout mice

Genetic manipulation of mice now provides new tools to evaluate the biological functions of the alpha(2)-adrenergic receptor (alpha(2)-AR) subtypes (alpha(2A), alpha(2B), and alpha(2C)). To investigate the role of the alpha(2A)-AR in the modulation of mouse primary behavioral characteristics and brain neurochemistry, mice with targeted inactivation of the gene for the alpha(2A)-AR were compared with wild-type C57BL/6 control animals. First, a comprehensive behavioral screen was employed to provide a detailed characterization of basic neurologic functions. Thereafter, the mice were analyzed in three models of anxiety, i.e. the elevated-plus maze test, the marble burying test and the open field test. The diurnal activity pattern of the mice was assessed in a 24-h locomotor activity test. Furthermore, receptor autoradiography of the brain was performed using the subtype-non-selective alpha(2)-AR antagonist radioligand [(3)H]RS-79948-197. Lack of the alpha(2A)-AR was associated with alterations in autonomic functions, including increased heart rate and piloerection. The mutant mice also exhibited impaired motor coordination skills, increased anxiety-like behavior and an abnormal diurnal activity pattern. In addition, neurochemical analysis of monoamine neurotransmitters revealed a considerable increase in brain norepinephrine turnover in mice lacking alpha(2A)-AR. Our results provide further support for the crucial role of the alpha(2A)-AR in modulating brain noradrenergic neurotransmission and many aspects of mouse behavior and physiology.     

Glucocorticoid-induced TNF receptor family gene (GITR) knockout mice exhibit a resistance to splanchnic artery occlusion (SAO) shock

In the present study, we used glucocorticoid-induced tumor necrosis factor (TNF) receptor family gene knockout (GITR-KO) mice to evaluate a possible role of GITR on the pathogenesis of splanchnic artery occlusion (SAO) shock, which was induced in mice by clamping the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. There was a marked increase in the lipid peroxidation in the ileum of the SAO-shocked, GITR wild-type (WT) mice after reperfusion. The absence of GITR significantly reduced the lipid peroxidation in the intestine. SAO-shocked WT mice developed a significant increase of ileum tissue, TNF-alpha, and myeloperoxidase activity and marked histological injury. SAO shock was also associated with a significant mortality (5% survival at 24 h after reperfusion). Reperfused ileum tissue sections from SAO-shocked WT mice showed positive staining for P-selectin, intercellular adhesion molecule 1 (ICAM-1), and E-selectin. The intensity and degree of P-selectin, E-selectin, and ICAM-1 were markedly reduced in tissue section from SAO-shocked, GITR-KO mice. SAO-shocked, GITR-KO mice also showed a significant reduction of the TNF-alpha production and neutrophil infiltration into the reperfused intestine, an improved histological status of the reperfused tissues, and an improved survival. Taken together, our results clearly demonstrate that GITR plays an important role in the ischemia and reperfusion injury and put forward the hypothesis that modulation of GITR expression may represent a novel and possible strategy.     

Repeated stress in young and old 5-HT(2C) receptor knockout mice

Serotonin (5-HT)(2C) receptor null mutant (knockout, KO) mice develop hyperphagia and midlife obesity. Based upon previous observations indicating altered responsiveness to stressful environmental conditions in these mice, we hypothesized that this KO mouse was hyperresponsive to repeated stress. To test this, we examined the effect of two intensities of repeated stress on food intake and body weight in 5-HT(2C) receptor KO and wild-type (WT) mice. The stressors involved daily cage change (including handling) for 3 days then daily restraint for 4 days. On the final day, mice were immediately decapitated after restraint to assess levels of plasma hormones. Two ages were used: young (12 weeks) and old (32-34 weeks). Basally, young KO were prehyperphagic and weighed the same as WT. In the old mice, KO were frankly hyperphagic and heavier than WT. In response to repeated cage change alone, the genotype-specific difference in food intake in the young group was enhanced, whereas in the old group it was diminished. This stressor did not significantly affect body weight change or caloric efficiency with respect to age or genotype. Repeated restraint had little effect on the young mice. However, in the old mice, KO had decreases in relative body weight and caloric efficiency compared with WT. In the old KO mice, adrenocorticotrophic hormone (ACTH), corticosterone and insulin were increased compared with WT mice. Together, these findings indicate that 5-HT(2C) receptor KO mice are hyperresponsive to repeated stress and this effect is influenced by stressor intensity and initial metabolic state of the mouse.     

Altered photic and non-photic phase shifts in 5-HT(1A) receptor knockout mice

The mammalian circadian clock located in the suprachiasmatic nucleus (SCN) is thought to be modulated by 5-HT. 5-HT is though to inhibit photic phase shifts by inhibiting the release of glutamate from retinal terminals, as well as by decreasing the responsiveness of retinorecipient cells in the SCN. Furthermore, there is also evidence that 5-HT may underlie, in part, non-photic phase shifts of the circadian system. Understanding the mechanism by which 5-HT accomplishes these goals is complicated by the wide variety of 5-HT receptors found in the SCN, the heterogeneous organization of both the circadian clock and the location of 5-HT receptors, and by a lack of sufficiently selective pharmacological agents for the 5-HT receptors of interest. Genetically modified animals engineered to lack a specific 5-HT receptor present an alternative avenue of investigation to understand how 5-HT regulates the circadian system. Here we examine behavioral and molecular responses to both photic and non-photic stimuli in mice lacking the 5-HT(1A) receptor. When compared with wild-type controls, these mice exhibit larger phase advances to a short late-night light pulse and larger delays to long 12 h light pulses that span the whole subjective night. Fos and mPer1 expression in the retinorecipient SCN is significantly attenuated following late-night light pulses in the 5-HT(1A) knockout animals. Finally, non-photic phase shifts to (+/-)-8-hydroxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT) are lost in the knockout animals, while attenuation of the phase shift to the long light pulse due to rebound activity following a wheel lock is unaffected. These findings suggest that the 5-HT(1A) receptor plays an inhibitory role in behavioral phase shifts, a facilitatory role in light-induced gene expression, a necessary role in phase shifts to 8-OH-DPAT, and is not necessary for activity-induced phase advances that oppose photic phase shifts to long light pulses.     

Radioligand binding analysis of knockout mice reveals 5-hydroxytryptamine(7) receptor distribution and uncovers 8-hydroxy-2-(di-n-propylamino)tetralin interaction with alpha(2) adrenergic receptors

In the present autoradiographic study, we took advantage of 5-hydroxytryptamine(7) (5-HT(7)) receptor knockout mice to analyze the brain distribution of 5-HT(7) receptor binding sites using [(3)H]5-carboxamidotryptamine (5-CT; a 5-HT(1A/1B/1D/5/7) receptor ligand) and [(3)H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; a 5-HT(1A/7) receptor ligand). Low to moderate densities of [(3)H]5-CT (2 nM) binding sites insensitive to pindolol (10 microM, for 5-HT(1A/1B) receptor blockade) and GR-127935 (1 microM; for 5-HT(1D) receptor blockade) were observed in wild-type mice (mainly in thalamus and hypothalamus) but not in 5-HT(7) receptor knockout mice. Surprisingly, moderate to high densities of [(3)H]8-OH-DPAT (10 nM) binding sites insensitive to pindolol (10 microM) remained in 5-HT(7) receptor knockout mouse brain. These non-5-HT(1A), non-5-HT(7) binding sites were found to be adrenergic alpha(2A) receptor binding sites. In alpha(2A) receptor knockout mice low to moderate densities of [(3)H]8-OH-DPAT binding sites insensitive to pindolol but sensitive to the selective 5-HT(7) receptor antagonist SB-269970 (300 nM) were observed mainly in thalamus and hypothalamus. Therefore, in addition to 5-HT(1A) and 5-HT(7) binding sites, [(3)H]8-OH-DPAT also binds to alpha(2A) receptor binding sites in wild-type mouse brain. [(3)H]8-OH-DPAT (in the presence of pindolol and 1 microM RX-821002 for alpha(2) receptor blockade) and [(3)H]5-CT (in the presence of pindolol and GR-127935) bind to a similar receptor binding population corresponding to 5-HT(7) binding sites. Detailed anatomical mapping of 5-HT(7) receptor binding sites in wild-type mouse brain was then performed using both radioligands in the presence of suitable pharmacological agents for non-5-HT(7) receptor binding sites blockade. The mapping revealed binding sites consistent with the mRNA distribution with the highest densities found in anterior thalamic nuclei.     

Isolation and characterization of a novel HS1 SH3 domain binding protein, HS1BP3

We have isolated a novel gene, HS1BP3, which encodes an HS1 binding protein. Analysis of HS1BP3 cDNA indicates several potentially important segments, including a PX domain, a leucine zipper, immunoreceptor tyrosine-based inhibitory motif-like motifs and proline-rich regions. HS1BP3 associates with HS1 proteins in vivo as confirmed by immunoprecipitation in B and T cell lines. HS1BP3 preferentially associates with the HS1 SH3 domains rather than with other SH3 molecules, suggesting a role of HS1BP3 as an HS1 signaling mediator. Overexpression of mutant HS1BP3 protein in T cell lines results in decreased IL-2 production. Our data suggest a novel role for HS1BP3 in lymphocyte activation.     

Importance of CCK-A receptor for gallbladder contraction and pancreatic secretion: a study in CCK-A receptor knockout mice.

Bile and pancreatic secretions were determined in a CCK-A receptor deficient mouse mutant generated by gene targeting in embryonic stem cells. The targeting vector contained lacZ and neo insertions in exon 2. Under the urethane anesthesia, the common bile duct was cannulated, and the mixture of bile-pancreatic juice was collected every 30 min. After the 1 h basal secretion, CCK-8 (0.5 and 1.0 nmol/kg), acetylcholine (500 nmol/kg), and neuromedin C (1.0 micromol/kg) were injected subcutaneously, and the secretions were collected following 1 h. Amylase and bile acid outputs were determined as parameters of pancreatic secretion and gallbladder contraction, respectively. In some CCK-A receptor (+/-) animals, LacZ staining was performed. CCK-8 significantly increased amylase and bile acid outputs in CCK-A receptor (+/+) and (+/-) mice, whereas no response was observed in (-/-) mice. Neuromedin C and acetylcholine increased amylase secretion in CCK-A receptor (-/-) mice similar to (+/-) and (+/+) mice. The same doses of neuromedin C and acetylcholine could not increase bile acid secretion. The gallbladder smooth muscles, pancreatic acinar cells, duct cells, and islets were stained by LacZ. CCK and CCK-A receptor are important for pancreatic secretion and gallbladder contraction. Neuromedin C and acetylcholine may compensate pancreatic function, but not gallbladder contraction.     

Transforming growth factor-beta(1) overexpression in tumor necrosis factor-alpha receptor knockout mice induces fibroproliferative lung disease

Tumor necrosis factor-alpha receptor knockout (TNF-alphaRKO) mice have homozygous deletions of the genes that code for both the 55- and 75-kD receptors. The mice are protected from the fibrogenic effects of bleomycin, silica, and inhaled asbestos. The asbestos-exposed animals exhibit reduced expression of other peptide growth factors such as transforming growth factor (TGF)-alpha, platelet-derived growth factors, and TGF-beta. In normal animals, these and other cytokines are elaborated at high levels during the development of fibroproliferative lung disease, but there is little information available that has allowed investigators to establish the role of the individual growth factors in disease pathogenesis. Here, we show that overexpression of TGF-beta(1) by means of a replication-deficient adenovirus vector induces fibrogenesis in the lungs of the fibrogenic-resistant TNF-alphaRKO mice. The fibrogenic lesions developed in both the KO and background controls within 7 d, and both types of animals exhibited similar incorporation of bromodeoxyuridine. Interestingly, airway epithelial cell proliferation appeared to be suppressed, perhaps due to the presence of the TGF-beta(1), a well-known inhibitor of epithelial mitogenesis. Before these experiments, there was no information available that would provide a basis for predicting whether or not TGF-beta(1) expression induces fibroproliferative lung disease in fibrogenic-resistant TNF-alphaRKO mice, an increasingly popular animal model.     

Growth hormone deficiency (GHD): a new association in Peters' Plus Syndrome (PPS)

We report a case of a girl with clinical features of Peters' Plus Syndrome (PPS) (association of anterior eye chamber defects; peculiar facies; cleft lip/palate; brachymelia; developmental delay; growth retardation) and documented growth hormone deficiency (height -3.5 SDS at chronological age 5 years 8 months; low growth factors; bone age delay; growth velocity 4.4 cm/year (<3rd centile); and peak growth hormone levels of 1.7 and 4.7 ng/ml by clonidine and insulin provocative testing, respectively). Treatment with recombinant human growth hormone (0.3 mg/kg/week) resulted in a dramatic increase in growth velocity, increasing the height from -3.5 to -1.5 SDS over 2.3 years of therapy, indicative of an excellent response. Growth retardation is a known association in PPS: a condition that includes other midline facial defects. This case supports a role for GHD in the pathogenesis of the short stature observed in these children; demonstrates the efficacy of GH treatment; and further reinforces the relationship of pituitary anomalies with common congenital defects.     

Role of dopamine D(1) receptors for kappa-opioid-mediated locomotor activity and antinociception during the preweanling period: a study using D(1) receptor knockout mice

kappa-Opioid receptor agonists both increase the locomotor activity of preweanling rats and induce antinociception. To determine whether dopamine (DA) D(1) receptors are necessary for either of these kappa-opioid-mediated effects we used D(1) (D(1A)) receptor knockout mice (i.e., D(1)-deficient mice). Heterozygous, wild-type, and D(1)-deficient mice (13 days old at testing) were injected with the kappa-opioid receptor agonist U-50,488 methanesulfonate (0.0, 0.2, 1. 0, 2.5, or 5.0 mg/kg, s.c.) and locomotor activity was measured for 60 min. In a separate experiment, tail-flick latencies of heterozygous, wild-type, and D(1)-deficient 13-day-old mice were assessed both before and after treatment with U-50,488 (0.0, 1.0, 2. 5, 5.0, or 10.0 mg/kg, s.c.). Results showed that lower doses of U-50,488 (0.2 and 1.0 mg/kg) increased the locomotor activity of 13-day-old mice regardless of genotype. Besides affecting locomotion, kappa-opioid receptor stimulation induced antinociception in preweanling mice, as U-50,488 caused a dose-dependent increase in the tail-flick latencies of heterozygous, wild-type, and D(1)-deficient mice. U-50,488's locomotor activating and analgesic effects did not differ according to genotype, thus suggesting that D(1) receptors are not necessary for kappa-opioid-mediated locomotor activity and antinociception during the preweanling period.     

Heart fatty-acid binding protein (h-FABP): a new cardiac marker

Heart Fatty-Acid Binding Protein (h-FABP) is a small cytosolic protein that is abundant in the heart and found at lower concentrations in muscle or in the brain. h-FABP is released into the circulation shortly after the onset of ischemia. Several studies indicate its usefulness in cardiology: exclusion of acute myocardial infarction, detection of reperfusion, prognostic value... A rapid immuno-chromatographic assay (Cardiodetect) was recently commercialized in France with a result obtainable within 15 minutes. We review the strengths and weakness of h-FABP for detecting myocardial injury.     

The androgen receptor gene mutations database (ARDB): 2004 update

The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 374 to 605, and the number of AR-interacting proteins described has increased from 23 to 70, both over the past 3 years. A 3D model of the AR ligand-binding domain (AR LBD) has been added to give a better understanding of gene structure-function relationships. In addition, silent mutations have now been reported in both androgen insensitivity syndrome (AIS) and prostate cancer (CaP) cases. The database also now incorporates information on the exon 1 CAG repeat expansion disease, spinobulbar muscular atrophy (SBMA), as well as CAG repeat length variations associated with risk for female breast, uterine endometrial, colorectal, and prostate cancer, as well as for male infertility. The possible implications of somatic mutations, as opposed to germline mutations, in the development of future locus-specific mutation databases (LSDBs) is discussed. The database is available on the Internet (http://www.mcgill.ca/androgendb/).