Search

We previously identified a SNF1/AMPK-related protein kinase, Hunk, from a mammary tumor arising in an MMTV-neu transgenic mouse. The function of this kinase is unknown. Using targeted deletion in mice, we now demonstrate that Hunk is required for the metastasis of c-myc-induced mammary tumors, but is dispensable for normal development. Reconstitution experiments revealed that Hunk is sufficient to restore the metastatic potential of Hunk-deficient tumor cells, as well as defects in migration and invasion, and does so in a manner that requires its kinase activity. Consistent with a role for this kinase in the progression of human cancers, the human homologue of Hunk is overexpressed in aggressive subsets of carcinomas of the ovary, colon, and breast. In addition, a murine gene expression signature that distinguishes Hunk-wild type from Hunk-deficient mammary tumors predicts clinical outcome in women with breast cancer in a manner consistent with the pro-metastatic function of Hunk in mice. These findings identify a direct role for Hunk kinase activity in metastasis and establish an in vivo function for this kinase. breast cancer | knockout mice | mouse models | protein kinase

We have recently reported that transgenic (mRen2)27 rats (Ren2 rats) exhibit pulmonary arterial hypertension (PAH), which is, in part, mediated by oxidative stress. Since 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) exhibit beneficial vascular effects independent of cholesterol synthesis, we hypothesized that rosuvastatin (RSV) treatment ameliorates PAH and pulmonary vascular remodeling in Ren2 rats. in part, by reducing oxidative stress. Six-week-old male Reh2 and Sprague-Dawley rats received RSV (10 mg x [kg.sup.-1] x [day.sup.-1] ip) or vehicle for 3 wk. After treatment, right ventricular systolic pressure (RVSP) and mean arterial pressure (MAP) were measured. To evaluate treatment effects on pulmonary arteriole remodeling, morphometric analyses were performed to quantitate medial thickening and cell proliferation, whereas whole lung samples were used to quantitate the levels of 3-nitro-tyrosine, superoxide, stable nitric oxide (NO)metabolites [nitrates and nitrites N[O.sub.x], and expression of NO synthase isoforms. In the Ren2 rat, RVSP is normal at 5 wk of agc, PAH dexelops between 5 and 7 wk of age, and the elevated pressure is maintained with little variation through 13 wk. At 8 wk of age, left ventricular function and blood gases were normal in the Reh2 rat. Ren2 rats exhibited elevations in medial hypertrophy due to smooth muscle cell proliferation, 3-nitro-tyrosine, NO, NADPH oxidase activity, and endothelial NO synthase expression compared with Sprague-Dawley rats. RSV significantly blunted the increase in RVSP but did not reduce MAP in the Ren2 rat; additionally, RSV significantly attenuated the elevated parameters examined in the Ren2 rat. These data suggest that statins may be a clinically viable adjunct treatment of PAH through reducing peroxy-nitrite formation. oxidative stress; NADPH oxidase

Vascular dysfunction occurs with aging. We hypothesized that oxidative stress and ANG II [acting via ANG II type 1 ([AT.sub.1] receptors] promotes cerebral vascular dysfunction with aging. We studied young (5-6 mo), old (17-19 mo), and very old (23 [+ or -] 1 mo) mice. In basilar arteries in vitro, acetylcholine (an endothelium-dependent agonist) produced dilation in young wild-type mice that was reduced by ~60 and 90% (P < 0.05) in old and very old mice, respectively. Similar effects were seen using A23187, a second endothelium-dependent agonist. The vascular response to acetylcholine in very old mice was almost completely restored with tempol (a scavenger of superoxide) and partly restored by PJ34, an inhibitor of poly(ADP-ribose) polymerase (PARP). We used mice deficient in Mn-SOD (Mn-[SOD.sup.+/-]) to test whether this form of SOD protected during aging but found that age-induced endothelial dysfunction was not altered by Mn-SOD deficiency. Cerebral vascular responses were similar in young mice lacking AT; receptors ([AT.sub.1.sup.-/-]) and wild-type mice. Vascular responses to acetylcholine and A23187 were reduced by ~50% in old wild-type mice (P < 0.05) but were normal in old [AT.sub.1]-deficient mice. Thus, aging produces marked endothelial dysfunction in the cerebral artery that is mediated by ROS, may involve the activation of PARP, but was not enhanced by Mn-SOD deficiency. Our findings suggest a novel and fundamental role for ANG II and [AT.sub.1] receptors in age-induced vascular dysfunction. basilar artery; endothelium, genetically altered mice; acetylcholine; angiotensin II; A23187

Intracerebroventricular (ICV) infusion of NaCl mimics the effects of a high-salt diet in salt-sensitive hypertension, raising the sodium concentration in the cerebrospinal fluid (CSF [Na]) and subsequently increasing the concentration of an endogenous ouabain-like substance (OLS) in the brain. The OLS, in turn, inhibits the brain [Na.sup.+]-[K.sup.+]-ATPase, causing increases in the activity of the brain renin-angiotensin system (RAS) and blood pressure. The [Na.sup.+]-[K.sup.+]-ATPase [alpha] (catalytic)-isoform(s) that mediates the pressor response to increased CSF [Na] is unknown, but it is likely that one or more isoforms that bind ouabain with high affinity are involved (e.g., the [Na.sup.+]-[K.sup.+]-ATPase [[alpha].sub.2]- and/or [[alpha].sub.3]-subunits). We hypothesize that OLS-induced inhibition of the [[alpha].sub.2]-subunit mediates this response. Therefore, a chronic reduction in [[alpha].sub.2] expression via a heterozygous gene knockout ([[alpha].sub.2] +/-) should enhance the pressor response to increased CSF [Na]. Intracerebroventricular (ICV) infusion of artificial CSF containing 0.225 M NaCl increased mean arterial pressure (MAP) in both wild-type (+/+) and [[alpha].sub.2] +/- mice, hut to a greater extent in [[alpha].sub.2] +/-. Likewise, the pressor response to ICV ouabain was enhanced in [[alpha].sub.2] +/- mice, demonstrating enhanced sensitivity to brain [Na.sup.+]-[K.sup.+]-ATPase inhibition per se. The pressor response to ICV ANG I but not ANG II was also enhanced in [[alpha].sub.2] +/- vs. [[alpha].sub.2]+/+ mice, suggesting an enhanced brain RAS activity that may be mediated by increased brain angiotensin converting enzyme (ACE). The latter hypothesis is supported by enhanced ACE ligand binding in the organum vasculosum laminae terminalis. These studies demonstrate that chronic downregulation of [Na.sup.+]-[K.sup.+]-ATPase [[alpha].sub.2]-isoform expression by heterozygous knockout increases the pressor response to increased CSF [Na] and activates the brain RAS. Since these changes mimic those produced by the endogenous brain OLS, the brain [[alpha].sub.2]-isoform may be a target for the brain OLS during increases in CSF [Na], such as in salt-dependent hypertension. [[alpha].sub.2]-isoform; gene knockout; sodium chloride; brain ouabain-like substance; brain renin-angiotensin system; intracerebroventricular infusion

Chronic wasting disease (CWD) of deer, elk, and moose is the only recognized prion disease of wild animals. To date, 15 US states and 2 Canada provinces have reported CWD [...]

Sweet taste transduction involves taste receptor type 1, member 2 (T1R2), taste receptor type 1, member 3 (T1R3), gustducin, and TRPM5. Because knockout (KO) mice lacking T1R3, gustducin's G[alpha] subunit (G[alpha]gust), or TRPM5 exhibited greatly reduced, but not abolished responses of the chorda tympani (CT) nerve to sweet compounds, it is likely that multiple sweet transduction pathways exist. That gurmarin (Gur), a sweet taste inhibitor, inhibits some but not all mouse CT responses to sweet compounds supports the existence of multiple sweet pathways. Here, we investigated Gur inhibition of CT responses to sweet compounds as a function of temperature in KO mice lacking T1R3, G[alpha]gust, or TRPM5. In T1R3-KO mice, responses to sucrose and glucose were Gur sensitive (GS) and displayed a temperature-dependent increase (TDI). In G[alpha]gust-KO mice, responses to sucrose and glucose were Gur-insensitive (GI) and showed a TDI. In TRPM5-KO mice, responses to glucose were GS and showed a TDI. All three KO mice exhibited no detectable responses to SC45647, and their responses to saccharin displayed neither GS nor a TDI. For all three KO mice, the lingual application of pronase, another sweet response inhibitor, almost fully abolished responses to sucrose and glucose but did not affect responses to saccharin. These results provide evidence for 1) the existence of multiple transduction pathways underlying responses to sugars: a T1R3-independent GS pathway for sucrose and glucose, and a TRPM5-independent temperature sensitive GS pathway for glucose; 2) the requirement for G[alpha]gust in GS sweet taste responses; and 3) the existence of a sweet independent pathway for saccharin, in mouse taste cells on the anterior tongue. sweet taste transduction; gurmarin sensitivity; temperature-dependent increase; knockout mice; chorda tympani nerve

The increase in adiponectin levels in obese patients with untreated dyslipidemia and its mRNA expression in adipose tissue of obese animals are one of the most interesting consequences of rimonabant treatment. Thus, part of rimonabant's metabolic effects could be related to an enhancement of adiponectin secretion and its consequence on the modulation of insulin action, as well as energy homeostasis. The present study investigated the effects of rimonabant in adiponectin knockout mice ([Ad.sup.-/-]) exposed to diet-induced obesity conditions. Six-week-old [Ad.sup.-/-] male mice and their wild-type littermate controls ([Ad.sup.+/+]) were fed a high-fat diet for 7 mo. During the last month, animals were administered daily either with vehicle or rimonabant by mouth (10 mg/kg). High-fat feeding induced weight gain by about 130% in both wild-type and [Ad.sup.-/-] mice. Obesity was associated with hyperinsulinemia and insulin resistance. Treatment with rimonabant led to a significant and similar decrease in body weight in both [Ad.sup.+/+] and [Ad.sup.-/-] mice compared with vehicle-treated animals. In addition, rimonabant significantly improved insulin sensitivity in [Ad.sup.+/+] mice compared with [Ad.sup.+/+] vehicle-treated mice by decreasing hepatic glucose production and increasing glucose utilization index in both visceral and subcutaneous adipose tissue. In contrast, rimonabant failed to improve insulin sensitivity in [Ad.sup.-/-] mice, despite the loss in body weight. Rimonabant's effect on body weight appeared independent of the adiponectin pathway, whereas adiponectin seems required to mediate rimonabant-induced improvement of insulin sensitivity in rodents. endocannabinoid system; cannabinoid receptor 1 antagonist; metabolic syndrome

We have previously reported that a 2.1-kb homology (H) sequence, conserved between mouse and human, regulates the odorant receptor (OR) gene MOR28 in transgenic mice. Here, we narrowed down the essential sequences of the H to a core of 124 bp by using a transient expression system in zebrafish embryos. Transgenic experiments in mice demonstrated that the core-H sequence is sufficient to endow expression of the MOR28 minigene. Deletion and mutation analyses of the core-H region revealed two homeodomain sequences to be essential for the H enhancer activity. Targeted deletion of the core-H abolished expression of three proximal OR genes, MOR28, MOR10, and MOR83, in cis, indicating the presence of another locus control region/enhancer in the downstream region, that regulates four distal OR genes in the same MOR28 cluster. In the heterozygous mice, the [H.sup.-] phenotype of the mutant allele was not rescued by the wild-type [H.sup.+] allele in trans. olfactory sensory neuron | gene targeting | locus control region | transgenic mouse | zebrafish

In mammals, male fertility is a quantitative feature determined by numerous genes. Until now, several wide chromosomal regions involved in fertility have been defined by genetic mapping approaches; unfortunately, the underlying genes are very difficult to identify. Here, 53 interspecific recombinant congenic mouse strains (IRCSs) bearing 1-2% SEG/Pas (Mus spretus) genomic fragments disseminated in a C57B1/ 6J (Mus domesticus) background were used to systematically analyze male fertility parameters. One of the most prominent advantages of this model is the possibility of analyzing stable phenotypes in living animals. Here, we demonstrate the possibility in one-step fine mapping for several fertility traits. Focusing on strains harboring a unique spretus fragment, we could unambiguously localize two testis and one prostate weight-regulating QTL (Ltw1, Ltw2, and Lpw1), four QTL controlling the sperm nucleus shape (Sh1, Sh2, Sh3, and Sh4), and one QTL influencing sperm smwival (Dss1). In several cases, the spretus DNA fragment was small enough to propose sound candidates. For instance, Spatal, Capza, and TubaTare very strong candidates for influencing the shape of the sperm head. Identifying new genes implied in mammalian fertility pathways is a necessary prerequisite for clarifying their molecular grounds and for proposing diagnostic tools for masculine infertilities.

Single gene mutations in [beta] integrins can account for functional defects of individual cells of the hematopoietic system. In humans, mutations in [[beta].sub.2] integrin lead to leukocyte adhesion deficiency (LAD) [...]

The effects of Hippi expression on Sonic hedgehog pathway and huntingtin signaling, using knock out mice studies, are presented.

The effects of Notch 1 signaling on development of male reproductive organs of transgenic mice are analyzed.

The effects of distant downstream regulatory enhancers on paired box gene 6 expression, using transgenic mouse studies, are presented.

Genetically modified mouse models have been used widely to advance our knowledge in the field of endocrinology and metabolism. A number of different approaches to generate genetically modified mice are now available, which provide the power to analyze the role of individual proteins in vivo. However, there are a number of points to be considered in the use and interpretation of these models. This review discusses the advantages and disadvantages involved in the generation and use of different genetically modified mouse models in endocrine research, including conventional techniques (e.g., overexpression, knockout, and knock-in models), tissue- and/or time-specific deletion of target genes [e.g., Cre-loxP and short interfering (si)RNA transgenic approaches], and gene-trap approaches to undertake functional genomics. This review also highlights the many factors that should be considered when assessing the phenotype of these mouse models, many of which are relevant to all murine physiological studies. These approaches are a powerful means by which to dissect the function of genes and are revolutionizing our understanding of endocrine physiology and metabolism. transgenic; knockout; knock-in; cre-loxp; functional genomics; physiology doi:10.1152/ajpendo.00124.2006

Regulation of slow fiber-specific genes by muscle TF II-In like repeat domain 1?1 gene, using transgenic mice studies, is presented.

Byline: Rolf Eckey (1), Mario Menschikowski (1), Peter Lattke (1), Werner Jaross (1) Keywords: phospholipase A.sub.2; inflammation; cholesterol; transgenic mice Abstract: It has been demonstrated in transgenic mice that the overexpression of human phospholipase A.sub.2 group IIA (sPLA.sub.2), an acute-phase reactant, is associated with depressed plasma cholesterol levels, altered lipoprotein compositions, and increased lipid depositions in aortic walls. It was the aim of the present study to investigate whether the reduced plasma cholesterol levels in sPLA.sub.2-transgenic mice may be due to an increased transfer of lipids from sPLA.sub.2-modified lipoproteins to the liver and/or other nonvascular tissues. Ten sPLA.sub.2-transgenic mice and an equal number of nontransgenic littermates were fed a cholesterol-enriched (1%) diet for 13 weeks. After autopsy, cholesterol and triglyceride concentrations were measured in homogenates of liver, spleen, kidney, and myocardial tissues. Compared to the nontransgenic controls, the sPLA.sub.2-transgenic mice exhibited signi- ficantly lower plasma cholesterol levels, which was due to a reduction in both HDL and [beta]-lipoprotein (LDL+[beta]-VLDL) cholesterol. Liver tissues from the transgenic mice were found to contain signi- ficantly increased concentrations of free and esterified cholesterol, which was not associated with increased triglyceride concentrations. Spleen, kidney, and heart tissues of the two animal groups showed no significant differences in cholesterol or triglyceride concentrations. The findings suggest that the overexpression of human secretory phospholipase A.sub.2 group IIA leads to an enhanced delivery of cholesterol from phospholipolysed lipoproteins to the liver. This mechanism is likely to contribute to the development of hypocholesterolemia observed in patients with inflammatory diseases. Author Affiliation: (1) Institute for Clinical Chemistry and Laboratory Medicine, Medical Faculty, Technical University of Dresden, Dresden, Germany Article History: Registration Date: 28/09/2004

Chemoprevention through nutritional and dietary changes may offer an important means of inhibiting the development and progression of breast cancer, which would have a major impact on public health. Studies to assess the efficacy of potential chemopreventive compounds are difficult to perform in large human populations, whereas the use of genetically engineered mice (GEM) for preclinical testing offers several advantages. GEM models can be utilized to assess the inhibitory effects of nutritional and chemopreventive agents on well-defined oncogenic signaling pathways. Because several transgenic mouse models progress through a well-defined temporal series of stages leading to invasive carcinoma formation, they may be particularly useful for determining cancer stage-specific responses to nutritional and chemopreventive agents. The C3(1)SV40 T/t-antigen transgenic mouse mammary cancer model has been utilized for chemopreventive research in which mammary tumors develop over a well-characterized time course. Several compounds have been shown to inhibit mammary tumor development in this model, including retinoids, difluoromethylomithine (DFMO), dehydroepiandrosterone (DHEA), antiangiogenic compounds and nonsteroidal anti-inflammatory drugs (NSAID). All of the chemopreventive agents used in the C3(1)Tag mammary mouse model appear to affect the promotion stage of tumorigenesis, suggesting that these agents may be useful in inhibiting the transition of human ductal carcinoma in situ (DCIS) to invasive carcinoma. Selective combinations of chemopreventive agents may be particularly useful for targeting multiple signaling pathways involved in cancer development and progression leading to improved clinical responses. The application of gene expression profiling to chemopreventive studies will aid in the selection of appropriate models for preclinical testing and further define mechanisms of action. KEY WORDS: * genetically engineered mice * chemoprevention * breast cancer

Objective Extensive WHO collaborative studies were performed to evaluate the suitability of transgenic mice susceptible to poliovirus (TgPVR mice, strain 21, bred and provided by the Central Institute for Experimental Animals, Japan) as an alternative to monkeys in the neurovirulence test (NVT) of oral poliovirus vaccine (OPV). Methods Nine laboratories participated in the collaborative study on testing neurovirulence of 94 preparations of OPV and vaccine derivatives of all three serotypes in TgPVR21 mice. Findings Statistical analysis of the data demonstrated that the TgPVR21 mouse NVT was of comparable sensitivity and reproducibility to the conventional WHO NVT in simians. A statistical model for acceptance/rejection of OPV lots in the mouse test was developed, validated, and shown to be suitable for all three vaccine types. The assessment of the transgenic mouse NVT is based on clinical evaluation of paralysed mice. Unlike the monkey NVT, histological examination of central nervous system tissue of each mouse offered no advantage over careful and detailed clinical observation. Conclusions Based on data from the collaborative studies the WHO Expert Committee for Biological Standardization approved the mouse NVT as an alternative to the monkey test for all three OPV types and defined a standard implementation process for laboratories that wish to use the test. This represents the first successful introduction of transgenic animals into control of biologicals. Keywords Poliovirus vaccine, Oral/toxicity; Mice, Transgenic/physiology; Macaca mulatta; Nervous system/virology; Virulence; Sensitivity and specificity; Reproducibility of results; World Health Organization; Comparative study; Validation studies (source: MESH, NLM). Mots cles Vaccin antipoliomyelitique Sabin/toxicite; Souris transgeniques/physiologie; Macaca mulatta; Systeme nerveux/virologie; Virulence; Sensibilite et specificite (Epidemiologie); Reproductibilite des resultats; Organisation mondiale de la Sante; Etude comparative; Etude validation (source: MESH, INSERM). Palabras clave Vacuna antipolio oral/toxicidad; Ratones transgenicos/fisiologia; Macaca mulatta; Sistema nervioso/virologia; Virulencia; Sensibilidad y especificidad; Reproducibilidad de resultados; Organizacion Mundial de la Salud; Estudio comparativo; Estudios de validacion (fuente: DeCS, BIREME)., Introduction The neurovirulence test (NVT) for oral poliovirus vaccine (OPV) is a key test for monitoring the consistency of vaccine production (1), and following WHO guidelines is required for each [...]

High-resolution magnetic resonance microscopy (MRM) was used to determine regional brain volumetric changes in a mouse model of Alzheimer's disease. These transgenic (Tg) mice over-express human mutant amyloid precursor protein (APP) V717F under control of platelet-derived growth factor promoter (PDAPP mice), and cortical and hippocampal [beta]-amyloid (A[beta]) deposits accumulate in heterozygotes after 8-10 mos. We used MRM to obtain 3D volumetric data on mouse brains imaged in their skulls to define genotype- and age-related changes. Hippocampal, cerebellar, and brain volumes and corpus callosum length were quantified in 40-, 100-, 365-, and 630-day-old mice. Measurements taken at age 100 days, before A[beta] deposition, revealed a 12.3% reduction of hippocampus volume in Tg mice compared with WT controls. This reduction persisted without progression to age 21 mos. A significant 18% increase in hippocampal volume occurred between 40 and 630 days in WT mice, and no corresponding significant increase occurred in Tg mice. Cavalieri volume estimates of hippocampal subfields from 100-day-old Tg mice further localized a 28% volume deficit in the dentate gyrus. In addition, corpus callosum length was reduced by [approximately equal to] 25% in Tg mice at all ages analyzed. In summary, reduced hippocampal volume and corpus callosum length can be detected by MRM before A[beta] deposition. We conclude that over-expression of APP and amyloid may initiate pathologic changes before the appearance of plaques, suggesting novel targets for the treatment of Alzheimer's disease and further reinforcing the need for early diagnosis and treatment.

Hepatitis B virus (HBV) infects humans and certain nonhuman primates. Viral clearance and acute disease are associated with a strong, polyclonal, multispecific cytotoxic T lymphocyte response. Infiltrating T cells, as well as other activated inflammatory cells, produce cytokines that can regulate hepatocellular gene expression. Using an HBV transgenic mouse model, our laboratory has previously demonstrated that adoptive transfer of HBV-specific cytotoxic T lymphocytes or injection of IL-2 can noncytopathically inhibit HBV gene expression by a posttranscriptional IFN-[gamma]- and/or tumor necrosis factor [alpha]-dependent mechanism. Here, we report that HBV gene expression can also be controlled at the posttranscriptional level during persistent lymphocytic choriomeningitis virus infection. In contrast, it is controlled at the transcriptional level during acute murine cytomegalovirus infection or after repetitive polyinosinic-polycytidylic acid injection. Finally, we show that transcriptional inhibition of HBV is associated with changes in liver-specific gene expression. These results elucidate pathways that regulate the viral life cycle and suggest additional approaches for the treatment of chronic HBV infection.

Mammalian [beta]-globin loci contain multiple [beta]-like genes that are expressed at different times during development. The murine [beta]-globin locus contains two genes expressed during the embryo stage, Ey and/[beta]h1, and two genes expressed at both the fetal and postnatal stages,[beta]-major and [beta]-minor. Studies of transgenic human [beta]-like globin loci in mice have suggested that expression of one gene at the locus will suppress expression of other genes at the locus. To test this hypothesis we produced mouse lines with deletions of either the Ey or [beta]h1 promoter in the endogenous murine [beta]-globin locus. Promoter deletion eliminated expression of the mutant gene but did not affect expression of the remaining embryonic gene or the fetal/adult [beta]-globin genes on the mutant allele. These results demonstrate a lack of competitive effects between individual mouse embryonic [beta]-globin gene promoters and other genes in the locus. The implication of these findings for models of [beta]-globin gene expression are discussed.

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhP[G.sub.1-80]) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1-1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CC[K.sub.1] and CC[K.sub.2] receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CC[K.sub.1]-R and CC[K.sub.2]-R on IEC cells. High-affinity ([K.sub.d] = 0.5-1.0 nM) binding sites for [sup.I]-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrinlike peptides in the order PG [greater than or equal to] COOH-terminally extended G17 [greater than or equal to] G-Gly > G17 > *CCK-8 (* significant difference; P < 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells. progastrin-preferring receptors; confocal microscopy; in vitro growth effects; Gly-gastrin

This study characterized the cardiac contractile function and IGF-I response in a transgenic diabetic mouse model. Mechanical properties were evaluated in cardiac myocytes from OVE26 diabetic and FVB wild-type mice, including peak shortening (PS), time to PS (TPS), time to 90% relengthening (T[R.sub.90]) and maximal velocity of shortening/relengthening ([+ or -]dL/dt). Intracellular [Ca.sup.2+] was evaluated as [Ca.sup.2+]-induced [Ca.sup.2+] release [difference in fura 2 fluorescent intensity ([DELTA]FFI)] and fluorescence decay rate ([tau]). Sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase (SERCA)2a, phospholamban (PLB), [Na.sup.+]-[Ca.sup.2+] exchanger (NCX), GLUT4, and the serine-threonine kinase Akt were assessed by Western blot. RhoA and IGF-I/IGF-I receptor mRNA levels were determined by RT-PCR and Northern blot. OVE26 myocytes displayed decreased PS, [+ or -]dL/dt, and ([DELTA]FFI associated with prolonged TPS, T[R.sub.90], and [tau]. SERCA2a, NCX, and Akt activation were reduced, whereas PLB and RhoA were enhanced in OVE26 hearts. GLUT4 was unchanged. IGF-I enhanced PS and [DELTA]FFI in FVB but not OVE26 myocytes. IGF-I mRNA was increased, but IGF-I receptor mRNA was reduced in OVE26 hearts and livers. These results validate diabetic cardiomyopathy in OVE26 mice due to reduced SERCA2, NCX, IGF-I response, and Akt activation associated with enhanced RhoA level, suggesting a therapeutic potential for Akt and RhoA. diabetic mouse; ventricular myocyte; excitation-contraction coupling; insulin-like growth factor I; sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase; sodium-calcium exchanger

The role of the [Na.sup.+] pump [[alpha].sub.2]-subunit in [Ca.sup.2+] signaling was examined in primary cultured astrocytes from wild-type ([[alpha].sub.2.sup.+/+] = WT) mouse fetuses and those with a null mutation in one [[[alpha].sub.2.sup.+/-] = heterozygote (Het)] or both [[[alpha].sub.2.sup.-/-] = knockout (KO)] [[alpha].sub.2] genes. [Na.sup.+] pump catalytic ([alpha]) subunit expression was measured by immunoblot; cytosol [[Na.sup.+]] ([[[Na.sup.+]].sub.cyt]) and [[Ca.sup.2+]] ([[[Ca.sup.2+]].sub.cyt]) were measured with sodium-binding benzofuran isophthalate and fura 2 by using digital imaging. Astrocytes express [Na.sup.+] pumps with both [[alpha].sub.1-] ([approximately equal to] 80% of total [alpha]) and [[alpha].sub.2-] ([approximately equal to] 20% of total [alpha]) subunits. Het astrocytes express [approximately equal to] 50% of normal [[alpha].sub.2]; those from KO express none. Expression of [[alpha].sub.1] is normal in both Het and KO cells. Resting [[[Na.sup.+]].sub.cyt] = 6.5 mM in WT, 6.8 mM in Het (P > 0.05 vs. WT), and 8.0 mM in KO cells (P < 0.001); 500 nM ouabain (inhibits only [[alpha].sub.2]) equalized [[[Na.sup.+]].sub.cyt] at 8 mM in all three cell types. Resting [[[Ca.sup.2+]].sub.cyt] = 132 nM in WT, 162 nM in Het, and 196 nM in KO cells (both P < 0.001 vs. WT). Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) [Ca.sup.2+] pumps and unloads the ER, induces transient (in [Ca.sup.2+]-free media) or sustained (in [Ca.sup.2+]-replete media) elevation of [[[Ca.sup.2+]].sub.cyt]. These [Ca.sup.2+] responses to 10 [micro]M CPA were augmented in Het as well as KO cells. When CPA was applied in [Ca.sup.2+]-free media, the reintroduction of [Ca.sup.2+] induced significantly larger transient rises in [[[Ca.sup.2+]].sub.cyt] (due to [Ca.sup.2+] entry through store-operated channels) in Het and KO cells than in WT cells. These results correlate with published evidence that [[alpha].sub.2] [Na.sup.+] pumps and [Na.sup.+]/[Ca.sup.2+] exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of [[alpha].sub.2] [Na.sup.+] pump activity can elevate local [[Na.sup.+]] and, via [Na.sup.+]/[Ca.sup.2+] exchange, [[Ca.sup.2+]] in the tiny volume of cytosol between the plasma membrane and ER. This, in turn, augments adjacent ER [Ca.sup.2+] stores and thereby amplifies [Ca.sup.2+] signaling without elevating bulk [[[Na.sup.+]].sub.cyt]. astrocytes; catalytic subunit; fura 2; sodium-binding benzofuran isophthalate; sodium-potassium-adenosine 5'-triphosphatase isoforms; transgenic mice

Here, we provide evidence for the neural crest origin of mammalian Merkel cells. Together with nerve terminals, Merkel cells form slowly adapting cutaneous mechanoreceptors that transduce steady indentation in hairy and glabrous skin. We have determined the ontogenetic origin of Merkel cells in Wnt1-cre/R26R compound transgenic mice, in which neural crest cells are marked indelibly. Merkel cells in whiskers and interfollicular locations express the transgene, [beta]-galactosidase, identifying them as neural crest descendants. We thus conclude that murine Merkel cells originate from the neural crest. Keywords: Neural crest; Merkel cell; Mechanoreceptor; Wnt1; R26R; Cre; [beta]-Galactosidase; Mouse; Mammalian; Whisker

The adult mouse intestine contains an intricate vascular network. The factors that control development of this network are poorly understood. Quantitative three-dimensional imaging studies revealed that a plexus of branched interconnected vessels developed in small intestinal villi during the period of postnatal development that coincides with assembly of a complex society of indigenous gut microorganisms (microbiota). To investigate the impact of this environmental transition on vascular development, we compared the capillary networks of germ-free mice with those of ex-germ-free animals colonized during or after completion of postnatal gut development. Adult germ-free mice had arrested capillary network formation. The developmental program can be restarted and completed within 10 days after colonization with a complete microbiota harvested from conventionally raised mice, or with Bacteroides thetaiotaomicron, a prominent inhabitant of the normal mouse/human gut. Paneth cells in the intestinal epithelium secrete antibacterial peptides that affect luminal microbial ecology. Comparisons of germ-free and B. thetaiotaomicron-colonized transgenic mice lacking Paneth cells established that microbial regulation of angiogenesis depends on this lineage. These findings reveal a previously unappreciated mechanism of postnatal animal development, where microbes colonizing a mucosal surface are assigned responsibility for regulating elaboration of the underlying microvasculature by signaling through a bacteria-sensing epithelial cell. small intestine | gnotobiotics | ecology | symbiosis

Networks of [gamma]-aminobutyric acid (GABA)ergic interneurons connected via electrical and chemical synapses are thought to play an important role in detecting and promoting synchronous activity in the cerebral cortex. Although the properties of electrical and chemical synaptic interactions among inhibitory interneurons are critical for their function as a network, they have only been studied systematically in juvenile animals. Here, we have used transgenic mice expressing the enhanced green fluorescent protein in cells containing parvalbumin (PV) to study the synaptic connectivity among fast-spiking (FS) cells in slices from adult animals (2-7 months old). We have recorded from pairs of PV-FS cells and found that the majority of them were electrically coupled (61%, 14 of 23 pairs). In addition, 78% of the pairs were connected via GABAergic chemical synapses, often reciprocally. The average coupling coefficient for step injections was 1.5% (n = 14), a smaller value than that reported in juvenile animals. GABA-mediated inhibitory postsynaptic currents and potentials decayed with exponential time constants of 2.6 and 5.9 ms, respectively, and exhibited paired-pulse depression (50-ms interval). The inhibitory synaptic responses in the adult were faster than those observed in young animals. Our results indicate that PV-FS cells are highly interconnected in the adult cerebral cortex by both electrical and chemical synapses, establishing networks that can have important implications for coordinating activity in cortical circuits.

Phenotypic knockout of nerve growth factor (NGF) activity in transgenic anti-NGF mice (AD11 mice) results in a progressive neurodegenerative phenotype resembling Alzheimer's disease. In this article, we examine whether and how the progressive neurodegenerative phenotype of AD11 mice could be prevented or ameliorated by pharmacological treatments with NGF or the cholinergic agonist galantamine, at a relatively early phase of Alzheimer's disease-like neurodegeneration. We demonstrate that the neurodegeneration induced by the expression of anti-NGF antibodies in AD11 mice can be largely reversed by NGF delivery through an olfactory route.

The serine-threonine kinase Akt seems to be central in mediating stimuli from different classes of receptors. In fact, both IGF-1 and IL6-like cytokines induce hypertrophic and antiapoptotic signals in cardiomyocytes through PI3K-dependent Akt activation. More recently, it was shown that Akt is involved also in the hypertrophic and antiapoptotic effects of [beta]-adrenergic stimulation. Thus, to determine the effects of Akt on cardiac function in vivo, we generated a model of cardiac-specific Akt overexpression in mice. Transgenic mice were generated by using the E40K, constitutively active mutant of Akt linked to the rat [alpha]-myosin heavy chain promoter. The effects of cardiac-selective Akt overexpression were studied by echocardiography, cardiac catheterization, histological and biochemical techniques. We found that Akt overexpression produced cardiac hypertrophy at the molecular and histological levels, with a significant increase in cardiomyocyte cell size and concentric LV hypertrophy. Akt-transgenic mice also showed a remarkable increase in cardiac contractility compared with wild-type controls as demonstrated by the analysis of left ventricular ([dP/dt.sub.max]) in an invasive hemodynamic study, although with graded dobutamine infusion, the maximum response was not different from that in controls. Diastolic function, evaluated by left ventricular [dP/dt.sub.min], was not affected at rest but was impaired during graded dobutamine infusion. Isoproterenol-induced cAMP levels, [beta]-adrenergic receptor ([beta]-AR) density, and [beta]-AR affinity were not altered compared with control mice. Moreover, studies on signaling pathway activation from myocardial extracts demonstrated that glycogen synthase kinase3-[beta] is phosphorylated, whereas p42/44 mitogen--activated protein kinases is not, indicating that Akt induces hypertrophy in vivo by activating the glycogen synthase kinase3-[beta]/GATA 4 pathway. In summary, our results not only demonstrate that Akt regulates cardiomyocyte cell size in vivo, but, importantly, show that Akt modulates cardiac contractility in vivo without directly affecting [beta]-AR signaling capacity.

To investigate the role of the HOX-like homeoprotein PDX1 in the formation and maintenance of the pancreas, we have genetically engineered mice so that the only source of PDX1 is a transgene that can be controlled by the application of tetracycline or its analogue doxycycline. In these mice the coding region for the tetracycline-regulated transactivator (tT[A.sub.off]) has replaced the coding region of the endogenous Pdx1 gene to ensure correct temporal and spatial expression of the regulatable transactivator. In the absence of doxycycline, tT[A.sub.off] activates the transcription of a bicistronic transgene encoding PDX1 and an enhanced green fluorescent protein reporter, which acts as a visual marker of transgene expression in living cells. Expression of the transgene-encoded PDX1 rescues the Pdx1-null phenotype; the pancreata of these mice develop and function normally. The rescue is conditional; doxycycline-mediated repression of the transgenic Pdx1 throughout gestation recapitulates the Pdx1 null phenotype. Moreover, application of doxycycline at mid-pancreogenesis blocks further development. Adult animals of the rescue genotype that were treated with doxycycline for 3 weeks shut off Pdx1 expression, decreased insulin production, and lost the ability to maintain glucose homeostasis. These results demonstrate the feasibility of controlling the formation of an organ during embryogenesis in utero and the maintenance of the mature organ through the experimental manipulation of a key developmental regulator.

Transforming growth factor-[[beta].sub.1] (TGF-[[beta].sub.1]) promotes or inhibits cell proliferation and induces fibrotic processes and extracellular matrix production in numerous cell types. Several cardiac diseases are associated with an increased expression of TGF-[[beta].sub.1] mRNA, particularly during the transition from stable cardiac hypertrophy to heart failure. In vitro studies suggest a link between TGF-[[beta].sub.1] signaling and the [[beta].sub.1]-adrenergic system. However, the in vivo effects of this growth factor on myocardial tissue have been poorly identified. In transgenic mice overexpressing TGF-[[beta].sub.1] (TGF-[beta]), we investigated the in vivo effects on cardiac morphology, [beta]-adrenergic signaling, and contractile function. When compared with nontransgenic controls (NTG), TGF-[beta] mice revealed significant cardiac hypertrophy (heart weight, 164 [+ or -] 7 vs. 130 [+ or -] 3 mg, P < 0.01; heart weight-to-body weight ratio, 6.8 [+ or -] 0.3 vs. 5.1 [+ or -] 0.1 mg/g, P < 0.01), accompanied by interstitial fibrosis. These morphological changes correlated with an increased expression of hypertrophy-associated proteins such as atrial natriuretic factor (ANF). Furthermore, overexpression of TGF-[[beta].sub.1] led to alterations of [beta]-adrenergic signaling as myocardial [beta]-adrenoceptor density increased from 7.3 [+ or -] 0.3 to 11.2 [+ or -] 1.1 fmol/mg protein (P < 0.05), whereas the expression of [beta]-adrenoceptor kinase-1 and inhibitory G proteins decreased by 56 [+ or -] 9.7% and 58 [+ or -] 7.6%, respectively (P < 0.05). As a consequence of altered [beta]-adrenergic signaling, hearts from TGF-[beta] showed enhanced contractile responsiveness to isoproterenol stimulation. In conclusion, we conclude that TGF-[[beta].sub.1] induces cardiac hypertrophy and enhanced [beta]-adrenergic signaling in vivo. The morphological alterations are either induced by direct effects of TGF-[[beta].sub.1] or may at least in part result from increased [beta]-adrenergic signaling, which may contribute to excessive catecholamine stimulation during the transition from compensated hypertrophy to heart failure. cardiac hypertrophy; heart failure; G proteins

[[alpha].sub.2]-Adrenergic receptors mediate part of the diverse biological effects of the endogenous catecholamines epinephrine and norepinephrine. Three distinct subtypes of [[alpha].sub.2]-adrenergic receptors, [[alpha].sub.2A], [[alpha].sub.2B], [[alpha].sub.2C] have been identified from multiple species. Because of the lack of sufficiently subtype-selective ligands, the specific biological functions of these receptor subtypes were largely unknown until recently. Gene-targeted mice carrying deletions in the genes encoding for individual [[alpha].sub.2]-receptor subtypes have added important new insight into the physiological significance of adrenergic receptor diversity. Two different strategies have emerged to regulate adrenergic signal transduction. Some biological functions are controlled by two counteracting [[alpha].sub.2]-receptor subtypes, e.g., [[alpha].sub.2A]-receptors decrease sympathetic outflow and blood pressure, whereas the [[alpha].sub.2B]-subtype increases blood pressure. Other biological functions are regulated by synergistic [[alpha].sub.2]-receptor subtypes. The inhibitory presynaptic feedback loop that tightly regulates neurotransmitter release from adrenergic nerves also requires two receptor subtypes, [[alpha].sub.2A] and [[alpha].sub.2C]. Similarly, nociception is controlled at several levels by one of the three [[alpha].sub.2]-receptor subtypes. Further investigation of the specific function of [[alpha].sub.2]-subtypes will greatly enhance our understanding of the relevance of closely related receptor proteins and point out novel therapeutic strategies for subtype-selective drug development. adrenergic receptors; transgenic mice; gene targeting

The individual functional significance of the various creatine kinase (CK) isoenzymes for myocardial energy homeostasis is poorly understood. Whereas transgenic hearts lacking the M subunit of CK (M-CK) show unaltered cardiac energetics and left ventricular (LV) performance, deletion of M-CK in combination with loss of sarcomeric mitochondrial CK (ScCKmit) leads to significant alterations in myocardial high-energy phosphate metabolites. To address the question as to whether this alteration is due to a decrease in total CK activity below a critical threshold or due to the specific loss of ScCKmit, we studied isolated perfused hearts with selective loss of ScCKmit ([ScCKmit.sup.-/-], remaining total CK activity ~70%) using [31.sup]P NMR spectroscopy at two different workloads. LV performance in [ScCKmit.sup.-/-] hearts (n = 11) was similar compared with wild-type hearts (n = 9). Phosphocreatine/ATP, however, was significantly reduced in [ScCKmit.sup.-/-] compared with wild-type hearts (1.02 [+ or -] 0.05 vs. 1.54 [+ or -] 0.07, P < 0.05). In parallel, free [ADP] was higher (144 [+ or -] 11 vs. 67 [+ or -] 7 [micro] M, P < 0.01) and free energy release for ATP hydrolysis ([DELTA][G.sub.ATP]) was lower (-55.8 [+ or -] 0.5 vs. -58.5 [+ or -] 0.5 kJ/mol, P < 0.01) in [ScCKmit.sup.-/-] compared with wild-type hearts. These results demonstrate that M- and B-CK containing isoenzymes are unable to fully substitute for the loss of ScCKmit. We conclude that ScCKmit, in contrast to M-CK, is critically necessary to maintain normal high-energy phosphate metabolite levels in the heart. creatine kinase; energy metabolism; nuclear magnetic resonance spectroscopy; transgenic mouse

Despite the high prevalence of age-related cataracts, there are currently no known therapies to delay or prevent their occurrence. Studies in humans and rodent models suggest that estrogen may provide protection against age-related cataracts. The discovery of ocular estrogen receptors (ERs) indicates that estrogen protection may result from direct interactions with its receptors in the eye, instead an indirect consequence from effects on another tissue. Studies in our transgenic mouse model validate the concept that estrogen is beneficial for the eye. These mice express ER[DELTA]3, a dominant-negative form of ER[alpha] that inhibits ER[alpha] function. In the ER[DELTA]3 transgenic mice, cortical cataracts spontaneously form in ER[DELTA]3 females after puberty and progress with age. The cataracts initiate in the equatorial region of the lens where the epithelial cells differentiate into elongating fiber cells. Cataract formation can be prevented if the females are ovariectomized before, but not after, sexual maturity. Both male and female ER[DELTA]3 mice develop cataracts after neonatal treatment with the potent estrogen diethylstilbestrol (DES). The incidence of spontaneous and DES-induced cataracts in ER[DELTA]3 mice is 100%, yet these cataracts are absent from the wild-type mice. These data suggest that repression of estrogen action induces cortical cataract formation because estrogen is required to activate the ER[DELTA]3 repressor. Evidence of DES-induced cataracts in the ER[DELTA]3 males as well as the females suggests that estrogen is important in lens physiology in both sexes. The ER[DELTA]3 mice provide a powerful model for assessing the role of estrogen in maintaining the transparency of the lens.

CD[25.sup.+] [4.sup.+] regulatory T cells ([T.sub.reg]) play an indispensable role in preventing autoimmunity. Little is known, however, about the antigen specificities required for their development and effector functions. Mice transgenic for an anti-myelin basic protein (MBP) T cell antigen receptor (TCR) spontaneously develop experimental autoimmune encephalomyelitis (EAE) when deficient for the RAG-1 gene (T/[R.sup.-]), whereas RAG-1-competent transgenic animals (T/[R.sup.+]) remain healthy, protected by CD[4.sup.+] [T.sub.reg]-expressing endogenous TCRs. We have now investigated the role and specificity of CD[25.sup.+] [4.sup.+] [T.sub.reg] in this system. The results show that T/[R.sup.+] animals contain MBP-specific suppressive CD[25.sup.+] [4.sup.+] cells, whereas T/[R.sup.-] do not. Adoptive transfer of CD[25.sup.+] [4.sup.+] cells from nontransgenic or T/[R.sup.+] donors into T/[R.sup.-] mice prevented the development of EAE. Surprisingly, transfer of nontransgenic CD[25.sup.+] [4.sup.+] cells purified from T/[R.sup.+] donors conferred only a limited protection, possibly because of their restricted repertoire diversity that we demonstrate here. Absence of transgenic CD[25.sup.+] [4.sup.+] cells in animals deficient for endogenous TCR[alpha] chains and analyses of endogenous TCR gene expression in subsets of CD[4.sup.+] cells from T/[R.sup.+] mice demonstrate that development of transgenic MBP-specific CD[25.sup.+] [4.sup.+] [T.sub.reg] depends on the coexpression of endogenous TCR[alpha] chains. Taken together, these results indicate that specificity to MBP is required for effector functions but is not sufficient for thymic selection/ commitment of CD[25.sup.+] [4.sup.+] [T.sub.reg] preventing EAE.

The role of the hepatitis B virus X protein (HBx) in the pathogenesis of hepatitis B virus (HBV) infection remains unclear. HBx exhibits pleiotropic biological effects, whose in vivo relevance is a matter for debate. In the present report, we have used a combination of HBx-expressing transgenic mice and liver cell transplantation to investigate the in vivo impact of HBx expression on liver cell proliferation and viability in a regenerative context. We show that moderate HBx expression inhibits liver regeneration after partial hepatectomy in HBx-expressing transgenic mice. We also demonstrate that the transplantation of HBx-expressing liver cells, isolated from HBx transgenic mice, is sufficient to inhibit overall recipient liver regeneration after partial hepatectomy. Moreover, the injection of serum samples drawn from HBx-expressing transgenic mice mimicked the inhibitory effect of HBx on liver regeneration. Finally, the incubation of primary rat hepatocytes with the supernatant of HBx-expressing liver cells inhibits cellular DNA synthesis. Taken together, our results demonstrate a paracrine inhibitory effect of HBx on liver cell proliferation and lead us to propose HBV as one of the few viruses implicated in human cancer which act, at least in part, through paracrine biological pathways.

Fibroproliferative processes are a group of disorders in which there is excessive proliferation of spindle (mesenchymal fibroblast-like) cells. They range from hypertrophic scars to neoplasms such as aggressive fibromatosis. Cells from these disorders share cytologic similarity with fibroblasts present during the proliferative phase of wound healing, suggesting that they represent a prolonged wounding response. A critical role for [beta]-catenin in mesenchymal cells in fibroproliferative processes is suggested by its high rate of somatic mutation in aggressive fibromatosis. Using a Tcf-reporter mouse we found that [beta]-catenin protein level and Tcf-transcriptional activity are elevated in fibroblasts during the proliferative phase of healing. We generated a transgenic mouse in which stabilized [beta]-catenin is expressed in mesenchymal cells under control of a tetracycline-regulated promoter. Fibroblasts from the transgenic mice exhibited increased proliferation, motility, and invasiveness when expressing stabilized [beta]-catenin and induced tumors after induction of the transgene when grafted into nude mice. Mice developed aggressive fibromatoses and hyperplastic gastrointestinal polyps after 3 months of transgene induction and healed with hyperplastic cutaneous wounds compared with control mice, which demonstrates an important function for [beta]-catenin in mesenchymal cells and shows a central role for [beta]-catenin in wound healing and fibroproliferative disorders.

The ATP-binding cassette transporter 1 (ABCA1) has recently been identified as a key regulator of high-density lipoprotein (HDL) metabolism, which is defective in familial HDL-deficiency syndromes such as Tangier disease. ABCA1 functions as a facilitator of cellular cholesterol and phospholipid efflux, and its expression is induced during cholesterol uptake in macrophages. To assess the role of macrophage ABCA1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDL[r.sup.-/-]) mice that are selectively deficient in leukocyte ABCA1 (ABCA[1.sup.-/-]) by using bone marrow transfer (ABCA[1.sup.-/-] [right arrow] LDL[r.sup.-/-]). Here we demonstrate that ABCA[1.sup.-/-] [right arrow] LDL[r.sup.-/-] chimeras develop significantly larger and more advanced atherosclerotic lesions compared with chimeric LDL[r.sup.-/-] mice with functional ABCA1 in hematopoietic cells. Targeted disruption of leukocyte ABCA1 function did not affect plasma HDL cholesterol levels. The amount of macrophages in liver and spleen and peripheral blood leukocyte counts is increased in the ABCA[1.sup.-/-] [right arrow] LDL[r.sup.-/-] chimeras. Our results provide evidence that leukocyte ABCA1 plays a critical role in the protection against atherosclerosis, and we identify ABCA1 as a leukocyte factor that controls the recruitment of inflammatory cells.

Multiple cytokines, including IL-2, can affect T cell proliferation and survival. However, IL-2 can lead to apoptosis as well as proliferation, making unclear whether IL-2 receptor (IL-2R) signals ultimately have a predominantly positive or negative effect. To address this issue, we examined the effect of enhancing IL-2R signals in CD[8.sup.+] T cells after antigen stimulation by engineering a transgenic (Tg) mouse strain with CD[8.sup.+] T cells capable of augmented, regulated, autocrine IL-2R signaling after target recognition by means of expression of a chimeric granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-2R. The Tg CD[8.sup.+] T cells can bind the granulocyte-macrophage colony-stimulating GM-CSF produced by antigen stimulation, but the GM-CSF binding results in delivery of an IL-2R signal. After antigen stimulation in vivo, the Tg T cells demonstrated marked increases in the initial proliferative response and cell expansion and displayed continued increases in cell expansion after repeated antigen exposure. These data suggest that the predominant role of IL-2R signals delivered to responding CD[8.sup.+] T cells is to set the size of the initial response to antigen by promoting T cell proliferation and survival and not cell death.

The Cre/IoxP system is increasingly showing promise for investigating genes involved in neural function. Here, we demonstrate that in vivo modification of genes in the mouse brain can be accomplished in a spatial- and temporal-specific manner by targeted delivery of an adeno-associated virus (AAV) encoding a green fluorescent protein/Cre recombinase (GFP/Cre) fusion protein. By using a reporter mouse, in which Cre recombinase activates [beta]-galactosidase expression, we demonstrate long-term recombination of neurons in the hippocampus, striatum, and septum as early as 7 days after stereotaxic injection of virus. Recombined cells were observed for at least 6 months postinjection without evidence of cell loss or neural damage. AAV-mediated delivery of GFP/Cre provides a valuable approach to alter the mouse genome, as AAV delivers genes efficiently to neurons with low toxicity. This approach will greatly facilitate the study of genetic modifications in the mouse brain. gene transfer | Cre recombinase | transgenic mouse | IoxP | ROSA26

Results indicate that the chemical structure of the amyloid peptides, generated in transgenic mice, are chemically different from those occuring in Alzheimer's disease plaues, suggesting that in drug evaluation studies, using transgenic animals, results interpretation must take this into consideration.

The adult myocardium responds to a variety of pathologic stimuli by hypertrophic growth that frequently progresses to heart failure. The calcium/calmodulin-dependent protein phosphatase calcineurin is a potent transducer of hypertrophic stimuli. Calcineurin dephosphorylates members of the nuclear factor of activated T cell (NFAT) family of transcription factors, which results in their translocation to the nucleus and activation of calcium-dependent genes. Glycogen synthase kinase-3 (GSK-3) phosphorylates NFAT proteins and antagonizes the actions of calcineurin by stimulating NFAT nuclear export. To determine whether activated GSK-3 can act as an antagonist of hypertrophic signaling in the adult heart in vivo, we generated transgenic mice that express a constitutively active form of GSK-3[beta] under control of a cardiac-specific promoter. These mice were physiologically normal under nonstressed conditions, but their ability to mount a hypertrophic response to calcineurin activation was severely impaired. Similarly, cardiac-specific expression of activated GSK-3[beta] diminished hypertrophy in response to chronic [beta]-adrenergic stimulation and pressure overload. These findings reveal a role for GSK-3[beta] as an inhibitor of hypertrophic signaling in the intact myocardium and suggest that elevation of cardiac GSK-3[beta] activity may provide clinical benefit in the treatment of pathologic hypertrophy and heart failure.

Transgenic mice expressing platelet-derived growth factor A chain (PDGF-A) in the distal lung epithelium from the surfactant protein C (SPC) promoter were generated to investigate the role of this growth factor in lung development. Expression of the SPC-PDGFA transgene resulted in an enlarged, nonfunctional lung and perinatal lethality caused by failure to initiate ventilation. Histologic analysis of embryonic day (E) 16.5 lungs revealed increased mesenchymal cells and acinar buds and decreased bronchioles and dilated airspaces in SPC-PDGFA transgenic mice. At E18.5, nontransgenic lungs exhibited lung morphology typical of the saccular stage of lung development, including dilated airspaces, thin respiratory epithelium and mesenchyme, and elastin fiber deposition in primary septa. In contrast, E18.5 transgenic lungs retained many features of the canalicular stage of lung development, including undilated airspaces, cuboidal respiratory epithelium, thickened mesenchyme, and lack of parenchymal elastin deposition. These results indicate that PDGF-A is a potent growth factor for mesenchymal cells in the developing lung and that the downregulation of PDGF-A expression that normally occurs in the lung during late gestation is required for transition from the canalicular to the saccular stage of lung development.

Mice that express hypoxia-inducible factor-1alpha (HIF-1alpha)(Delta)ODD developed a microvasculature with biology different from that described before for either vascular endothelial growth factor (VEGF) or angiopoietin-1 and -2. Hypervascularity induced by HIF-1alpha could improve tissue ischemia therapy.

Functional inactivation of the IGF-I and insulin receptors (IGF-IR and IR) in striated muscle has been found to cause type 2 diabetes. A transgenic mouse with a dominant-negative insulin-like growth factor-1 receptor specifically targeted to the striated muscle was developed for study. The mice, MKR mice, were excellent as a model for this type of study. They showed striking loss of function of the IR and the IGF-IR