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Background: Theanine, an amino acid in tea, has significant anti-stress effects on animals and humans. However, the anti-stress effects of drinking green tea have not yet been elucidated. Hypothesis/purpose: The [...]

Author(s): Xiao Han[sup.1] , Tianren Huang[sup.1] and Junqing Han[sup.2] Introduction In accordance with latest statistics, hepatocellular carcinoma (HCC) is the third cause of cancer death in the world [1]. Hepatitis [...], Background Recently, intestinal microbiome has been involved in hepatic diseases due to the immunologic and metabolic communication between liver and intestine. Initiation of hepatocellular carcinoma (HCC) frequently attributes to conspiracy between immune cells and infectious carcinogens. Here, the hypothesis that the tumorigenesis of HCC with HBV infection will be aggravated by specific intestinal bacteria was verified in viral transgenic mouse models. Methods Comparative 16S rRNA sequencing was adopted to observe the intestinal enrichment of Helicobacter hepaticus in HCC. Oral administration of Helicobacter hepaticus was carried out to evaluate its hepatic carcinogenic effect in HBV transgenic mice or wildtype C57BL/6. The livers of experimental mice were collected and examined for the degree of tumorigenesis. Results We found that Helicobacter hepaticus more likely colonized at lower colon of HBV-infected mice with HCC, compared with C57BL/6 and HBV-infected mice without neoplasm. Pretreatment of Helicobacter hepaticus in transgenic mice aggravated tumor formation, with higher incidence, more tumor nodule and higher serum AFP. Then, a cytokines expression patterns with inclined IFN-[gamma], IFN-[gamma]R1, IL-17 and IL-23 was found in HBV-infected mice with Helicobacter hepaticus. Furthermore, innate lymphoid cells, especially Th17 and NK cells which can secret IL-17 and IFN-[gamma] respectively, might be recruited by Helicobacter hepaticus cooperated with HBV. Besides, increased expression of CD69, NKG2D and IFN-[gamma] showed activation of cytokine production in intrahepatic NK cells. Finally, IFN-[gamma] decreased E-cadherin expression through p-STAT1 pathway, resulting in epithelial-mesenchymal transition with inclined expression of Snail2, SIP1 and CXCR4 in vitro. p-STAT1 inhibitor was able to reverse the expression of E-cadherin and EMT resulted from IFN-[gamma] function on HBsAg-positive hepatocytes. Conclusions Helicobacter hepaticus generate a detrimental immune microenvironment by IFN-[gamma]/p-STAT1 axis which can promote the tumorigenesis of hepatitis B via recruiting innate lymphoid cells. Keywords: Helicobacter hepaticus, Hepatitis B virus, Hepatocellular carcinoma, Innate lymphoid cells, IFN-[gamma]/p-STAT1 axis

Author(s): Zhen-Zhong Xu [1, 2]; Yong Ho Kim [1, 2, 3]; Sangsu Bang [1, 2]; Yi Zhang [2]; Temugin Berta [1, 2]; Fan Wang [2]; Seog Bae Oh [3, 4]; [...]

Author(s): Evelyn Schmid [1]; Stefan Neef [2]; Christopher Berlin [1]; Angela Tomasovic [1]; Katrin Kahlert [1]; Peter Nordbeck [3, 4]; Katharina Deiss [1]; Sabrina Denzinger [1]; Sebastian Herrmann [3, 4]; [...], In heart failure therapy, it is generally assumed that attempts to produce a long-term increase in cardiac contractile force are almost always accompanied by structural and functional damage. Here we show that modest overexpression of the Raf kinase inhibitor protein (RKIP), encoded by Pebp1 in mice, produces a well-tolerated, persistent increase in cardiac contractility that is mediated by the [beta][sub.1]-adrenoceptor ([beta][sub.1]AR). This result is unexpected, as [beta][sub.1]AR activation, a major driver of cardiac contractility, usually has long-term adverse effects. RKIP overexpression achieves this tolerance via simultaneous activation of the [beta][sub.2]AR subtype. Analogously, RKIP deficiency exaggerates pressure overload-induced cardiac failure. We find that RKIP expression is upregulated in mouse and human heart failure, indicative of an adaptive role for RKIP. Pebp1 gene transfer in a mouse model of heart failure has beneficial effects, suggesting a new therapeutic strategy for heart failure therapy.

Author(s): David L Waning [1]; Khalid S Mohammad [1]; Steven Reiken [2]; Wenjun Xie [2]; Daniel C Andersson [2]; Sutha John [1]; Antonella Chiechi [1]; Laura E Wright [1]; Alisa [...], Cancer-associated muscle weakness is a poorly understood phenomenon, and there is no effective treatment. Here we find that seven different mouse models of human osteolytic bone metastases--representing breast, lung and prostate cancers, as well as multiple myeloma--exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that transforming growth factor (TGF)-[beta], released from the bone surface as a result of metastasis-induced bone destruction, upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor and calcium (Ca[sup.2+]) release channel (RyR1). The oxidized RyR1 channels leaked Ca[sup.2+], resulting in lower intracellular signaling, which is required for proper muscle contraction. We found that inhibiting RyR1 leakage, TGF-[beta] signaling, TGF-[beta] release from bone or Nox4 activity improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, increased Nox4 binding to RyR1 and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a nonmalignant metabolic bone disorder associated with increased TGF-[beta] activity. Thus, pathological TGF-[beta] release from bone contributes to muscle weakness by decreasing Ca[sup.2+]-induced muscle force production.

Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues (1).Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers [...]

The most interesting results in experimental biology are those that are unexpected. Never has this been truer than in the age of transgenic mice. Now that we can manipulate the [...], Proteins that end with a CAAX sequence are targeted to cellular membranes by a series of posttranslational modifications that include prenylation, proteolysis, and carboxyl methylation. Two prenyltransferases modify CAAX proteins: farnesyltransferase and geranylgeranyltransferase type I (GGTase-I). Rho family GTPases that control the actin cytoskeleton and are therefore critical to inflammatory cell function are substrates forGGTase-I. In this issue of the JCI, Khan et al. examined mice in which GGTase-I was conditionally deleted in macrophages. Rather than obtunded cells, the authors found activated Rho proteins in fully functional macrophages that hypersecreted inflammatory cytokines and induced an erosive, inflammatory arthritis. This surprising result calls into question the role of protein geranylgeranylation in inflammatory cell signaling.

Mice that are transgenic for rearranged antigen-specific T ceil receptors (TCRs) are essential tools to study T cell development and function. Such TCRs are usually isolated from the relevant T cells after long-term culture, often after repeated antigen stimulation, which unavoidably skews the T cell population used. Random genomic integration of the TCR a and 13 chain and expression from nonendogenous promoters represent additional drawbacks of transgenics. Using epigenetic reprogramming via somatic cell nuclear transfer, we demonstrated that T cells with predefined specificities against Toxoplasma gondii can be used to generate mouse models that express the TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the cnstruction of other disease models. 10.1126/science.1178590

Hippocampal theta (5-10 Hz) and gamma (35-85 Hz) oscillations depend on an inhibitory network of GABAergic intemeurons. However, the lack of methods for direct and cell-type-specific interference with inhibition has prevented better insights that help link synaptic and cellular properties with network function. Here, we generated genetically modified mice (PV-[DELTA][[gamma].sub.2]) in which synaptic inhibition was ablated in parvalbumin-positive (PV+) interneurons. Hippocampal local field potential and unit recordings in the CA1 area of freely behaving mice revealed that theta rhythm was strongly reduced in these mice. The characteristic coupling of theta and gamma oscillations was strongly altered in PV-[DELTA][[gamma].sub.2] mice more than could be accounted for by the reduction in theta rhythm only. Surprisingly, gamma oscillations were not altered. These data indicate that synaptic inhibition onto PV+ interneurons is indispensable for theta- and its coupling to gamma oscillations but not for rhythmic gamma-activity in the hippocampus. Similar alterations in rhythmic activity were obtained in a computational hippocampal network model mimicking the genetic modification, suggesting that intrahippocampal networks might contribute to these effects. compartmental model GABA | [GABA.sub.A] receptor | knockout | network synchrony

Meprin metalloproteases, composed of a and/or [beta] subunits, consist of membrane-bound and secreted forms that are abundantly expressed in proximal tubules of the kidney as well as secreted into the urinary tract. Previous studies indicated that meprin metalloproteases play a role in pathological conditions such as ischemic acute renal failure and urinary tract infection. The aim of this work was to examine the role of meprins in endotoxemic acute renal failure using meprin [alpha] knockout ([alpha]KO), meprin [beta] knockout ([beta]KO), and wild-type (WT) mice. Differences among the responses of the genotypes were observed as early as 1 h after challenge with 2.5 mg/kg ip Escherichia coli LPS, establishing roles for meprins in the endotoxemic response. Meprin [alpha]KO mice displayed lower blood urea nitrogen levels and decreased nitric oxide levels, indicative of a decreased systemic response to LPS compared with WT and meprin [beta]KO mice. Serum cytokine profiles showed lower levels of IL-1[beta] and TNF-[alpha] in the meprin [alpha]KO mice within 3 h after LPS challenge and confirmed a role for meprins in the early phases of the host response. Meprin [alpha]KO mice were also hyporesponsive to LPS administered to the bladder, exhibiting significantly less bladder edema, leukocyte infiltration, and bladder permeability than WT mice. These data indicate that meprin A contributes to the renal and urogenital pathogenesis of endotoxicity. knockout mice; lipopolysaccharide; kidney; metalloproteinase

Cloning animals by nuclear transfer provides an opportunity to preserve endangered mammalian species. However, it has been suggested that the 'resurrection' of frozen extinct species (such as the woolly mammoth) is impracticable, as no live cells are available, and the genomic material that remains is inevitably degraded. Here we report production of cloned mice from bodies kept frozen at -20[degrees]C for up to 16 years without any cryoprotection. As all of the cells were ruptured after thawing, we used a modified cloning method and examined nuclei from several organs for use in nuclear transfer attempts. Using brain nuclei as nuclear donors, we established embryonic stem cell lines from the cloned embryos. Healthy cloned mice were then produced from these nuclear transferred embryonic stem cells by serial nuclear transfer. Thus, nuclear transfer techniques could be used to 'resurrect' animals or maintain valuable genomic stocks from tissues frozen for prolonged periods without any cryopreservation. brain | cloning | cryopreservation | nuclear transfer | reprogramming

Prediction of individual life span based on characteristics evaluated at middle-age represents a challenging objective for aging research. In this study, we used machine learning algorithms to construct models that predict life span in a stock of genetically heterogeneous mice. Life-span prediction accuracy of 22 algorithms was evaluated using a cross-validation approach, in which models were trained and tested with distinct subsets of data. Using a combination of body weight and T-cell subset measures evaluated before 2 years of age, we show that the life-span quartile to which an individual mouse belongs can be predicted with an accuracy of 35.3% ([+ or -] 0.10%). This result provides a new benchmark for the development of life-span--predictive models, but improvement can be expected through identification of new predictor variables and development of computational approaches. Future work in this direction can provide tools for aging research and will shed light on associations between phenotypic traits and longevity. Key Words: Aging--Classification--Longevity--Shrunken centroid--T-cell subset--Weight.

A-Kinase Anchoring Proteins (AKAPs) ensure the fidelity of second messenger signaling events by directing protein kinases and phosphatases toward their preferred substrates. AKAP150 brings protein kinase A (PKA), the calcium/calmodulin dependent phosphatase PP2B and protein kinase C (PKC) to postsynaptic membranes where they facilitate the phosphorylation dependent modulation of certain ion channels. Immunofluorescence and electrophysiological recordings were combined with behavioral analyses to assess whether removal of AKAP150 by gene targeting in mice changes the signaling environment to affect excitatory and inhibitory neuronal processes. Mislocalization of PKA in AKAP150 null hippocampal neurons alters the bidirectional modulation of postsynaptic AMPA receptors with concomitant changes in synaptic transmission and memory retention. AKAP150 null mice also exhibit deficits in motor coordination and strength that are consistent with a role for the anchoring protein in the cerebellum. Loss of AKAP150 in sympathetic cervical ganglion (SCG) neurons reduces muscarinic suppression of inhibitory M currents and provides these animals with a measure of resistance to seizures induced by the non-selective muscarinic agonist pilocarpine, These studies argue that distinct AKAP150-enzyme complexes regulate context-dependent neuronal signaling events in vivo. AMPA | behavior | KCNQ | knockout

The feasibility of gene therapy in congenital erthropoietic porphyria (CEP) is tested using a murine model. It is demonstrated that transplantation of genetically modified hematpoietic stem cells at a moderate transproduction level supports the proof of concept of a gene therapy in CEP.

The last 2007 Nobel Prize in Medicine went to three great scientists, who discovered a new technique of introducing gene modifications in mice by using the embryonic stem cells. The research is found to be very useful for human clinical practices.

K-Cl cotransport activity in rbc is a major determinant of rbc volume and density. Pathologic activation of erythroid K-Cl cotransport activity in sickle cell disease contributes to rbc dehydration and [...]

To permit rapid optical control of brain activity, we have engineered multiple lines of transgenic mice that express the light-activated cation channel Channelrhodopsin-2 (ChR2) in subsets of neurons. Illumination of ChR2-positive neurons in brain slices produced photocurrents that generated action potentials within milliseconds and with precisely timed latencies. The number of light-evoked action potentials could be controlled by varying either the amplitude or duration of illumination. Furthermore, the frequency of light-evoked action potentials could be precisely controlled up to 30 Hz. Photostimulation also could evoke synaptic transmission between neurons, and, by scanning with a small laser light spot, we were able to map the spatial distribution of synaptic circuits connecting neurons within living cerebral cortex. We conclude that ChR2 is a genetically based photostimulation technology that permits analysis of neural circuits with high spatial and temporal resolution in transgenic mammals. brain networks | cortical circuitry | synaptic transmission

The prion protein (PrP) level in muscle has been reported to be elevated in patients with inclusion-body myositis, polymyositis, dermatomyositis, and neurogenic muscle atrophy, but it is not clear whether the elevated PrP accumulation in the muscles is sufficient to cause muscle diseases. We have generated transgenic mice with muscle-specific expression of PrP under extremely tight regulation by doxycycline, and we have demonstrated that doxycycline-induced overexpression of PrP strictly limited to muscles leads to a myopathy characterized by increased variation of myofiber size, centrally located nuclei, and endomysial fibrosis, in the absence of intracytoplasmic inclusions, rimmed vacuoles, or any evidence of a neurogenic disorder. The PrP-induced myopathy correlates with accumulation of an N-terminal truncated PrP fragment in the muscle, and the muscular PrP displayed consistent mild resistance to protease digestion. Our findings indicate that overexpression of wild-type PrP in skeletal muscles is sufficient to cause a primary myopathy with no signs of peripheral neuropathy, possibly due to accumulation of a cytotoxic truncated form of PrP and/or PrP aggregation. doxycycline | inducible transgenic mice | muscle disease

While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice, generated to overexpress sarco(endo) plasmic reticulum calcium-ATPase (SERCA), which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabeled glucose and palmitate. We also examined oxygen consumption to see whether SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte, we examined cytosolic calcium transients in isolated myocytes using indo-1, and mitochondrial calcium levels using pericam, an adenovirally expressed, mitochondrially targeted ratiometric calcium indicator. Oxygen consumption did not differ between wildtype and SERCA groups; however, we were able to show an increased utilization of glucose for oxidative metabolism and a corresponding decreased utilization of fatty acids in the SERCA group. Cytosolic calcium transients were increased in myocytes isolated from SERCA mice, and they show a faster rate of decay of the calcium transient. With these observations we noted increased levels of mitochondrial calcium in the SERCA group, which was associated with an increase in the active form of the pyruvate dehydrogenase complex. Since an increase in mitochondrial calcium levels leads to activation of the pyruvate dehydrogenase complex (the rate-limiting step for carbohydrate oxidation), the increased glucose utilization observed in isolated perfused hearts in the SERCA group may reflect a higher level of mitochondrial calcium. mitochondrial calcium; pyruvate dehydrogenase; cardiac energetics; sarco(endo)plasmic reticulum calcium-adenosine 5'-triphosphatase

Gene knockout of the KCNJ11-encoded Kir6.2 ATP-sensitive [K.sup.+] ([K.sub.ATP]) channel implicates this stress-response element in the safeguard of cardiac homeostasis under imposed demand. [K.sub.ATP] channels are abundant in ventricular sarcolemma, where subunit expression appears to vary between the sexes. A limitation, however, in establishing the full significance of [K.sub.ATP] channels in the intact organism has been the inability to monitor in vivo the contribution of the channel to intracellular calcium handling and the superimposed effect of sex that ultimately defines heart function. Here, in vivo manganese-enhanced cardiac magnetic resonance imaging revealed, under dobutamine stress, a significantly greater accumulation of calcium in both male and female [K.sub.ATP] channel knockout (Kir6.2-KO) mice compared with sex- and age-matched wild-type (WT) counterparts, with greatest calcium load in Kir6.2-KO females. This translated, poststress, into a sustained contracture manifested by reduced end-diastolic volumes in [K.sub.ATP] channel-deficient mice. In response to ischemia-induced stunning, male and female Kir6.2-KO hearts demonstrated accelerated time to contracture and increased peak contracture compared with WT. The outcome on reperfusion, in both male and female Kir6.2-KO hearts, was a transient reduction in systolic performance, measured as rate-pressure product compared with WT, with protracted increase in left ventricular end-diastolic pressure, exaggerated in female knockout hearts, despite comparable leakage of creatine kinase across groups. Kir6.2-KO hearts were rescued from diastolic dysfunction by agents that target alternative pathways of calcium handling. Thus [K.sub.ATP] channel deficit confers a greater susceptibility to calcium overload in vivo, accentuated in female hearts, impairing contractile recovery under various conditions of high metabolic demand. ATP-sensitive [K.sup.+] channel; Kir6.2; magnetic resonance imaging; myocardium; sex

The FRK tyrosine kinase has previously been shown to transduce [beta]-cell cytotoxic signals in response to cytokines and streptozotocin and to promote [beta]-cell proliferation and an increased [beta]-cell mass. We therefore aimed to further evaluate the effects of overexpression of FRK tyrosine kinase in [beta]-cells. A transgenic mouse expressing kinase-active FRK under control of the insulin promoter (RIP-FRK) was studied with regard to islet endocrine function and vascular morphology. Mild glucose intolerance develops in RIP-FRK male mice of at least 4 mo of age. This effect is accompanied by reduced glucose-stimulated insulin secretion in vivo and reduced second-phase insulin secretion in response to glucose and arginine upon pancreas perfusion. Islets isolated from the FRK transgenic mice display a glucose-induced insulin secretory response in vitro similar to that of control islets. However, islet blood flow per islet volume is decreased in the FRK transgenic mice. These mice also exhibit a reduced islet capillary lumen diameter as shown by electron microscopy. Total body weight and pancreas weight are not significantly affected, but the [beta]-cell mass is increased. The data suggest that long-term expression of active FRK in [beta]-cells causes an in vivo insulin-secretory defect, which may be the consequence of islet vascular abnormalities that yield a decreased islet blood flow. [beta]-cells; insulin secretion

Constitutive myostatin gene knockout in mice causes excessive muscle growth during development. To examine the effect of knocking out the myostatin gene after muscle has matured, we generated mice in which myostatin exon 3 was flanked by loxP sequences (Mstn[f/f]) and crossed them with mice bearing a tamoxifen-inducible, ubiquitously expressed Cre recombinase transgene. At 4 mo of age, Mstn[f/f]/Cre+ mice that had not received tamoxifen had a 50-90% reduction in myostatin expression due to basal Cre activity but were not hypermuscular relative to Mstn[w/w]/Cre+ mice (homozygous for wild-type myostatin gene). Three months after tamoxifen treatment (initiated at 4 mo of age), muscle mass had not changed from the pretreatment level in Mstn[w/w]/Cre+ control mice. Tamoxifen administration to 4-mo-old Mstn[f/f]/Cre+ mice reduced myostatin mRNA expression to less than 1% of normal, which increased muscle mass ~25% over the following 3 mo in both male and female mice (P < 0.005 vs. control). Fiber hypertrophy appeared to be sufficient to explain the increase in muscle mass. The pattern of expression of genes encoding the various myosin heavy-chain isoforms was unaffected by postdevelopmental myostatin knockout. We conclude that, even after developmental muscle growth has ceased, knockout of the myostatin gene induces a significant increase in muscle mass. Cre recombinase; tamoxifen; muscle fiber hypertrophy; myosin heavy chains; conditional knockout

The mechanism of biotin uptake in human intestine has been well characterized and involves the human sodium-dependent multivitamin transporter (hSMVT), yet little is known about the molecular/transcriptional regulation of the system. Previous investigations cloned the 5' regulatory region of the hSMVT gene and identified the minimal promoter. To expand these investigations, we compared activity of the hSMVT promoter in three human intestinal epithelial cell lines (NCM460, Caco-2, and HuTu-80) and contrasted a renal epithelial cell line (HEK-293). We analyzed the role of putative cis-elements in regulating promoter activity and confirmed activity of the cloned hSMVT promoter in vivo. In vitro studies demonstrated that all cell lines utilized the same minimal promoter region, and mutation of specific cis-regulatory elements [Kruppel-like factor 4 (KLF-4) and activator protein-2 (AP-2)] led to a decrease in promoter activity in all intestinal cell types but not in renal cells. Using electrophoretic mobility shift assays, we identified two specific DNA/protein complexes. Using oligonucleotide competition and antibody supershift analysis, we determined that KLF-4 and AP-2 were involved in forming the complexes. In HEK-293 cells, overexpressing KLF-4 increased the endogenous hSMVT message levels threefold and activated a cotransfected hSMVT promoter-reporter construct. In vivo studies using hSMVT promoter-luciferase transgenic mice established physiological relevance and showed the pattern of hSMVT promoter expression to be similar to endogenous mouse SMVT mRNA expression. The results demonstrate, for the first time, the importance of KLF-4 and AP-2 in regulating the activity of the hSMVT promoter in the intestine and provide direct in vivo confirmation of hSMVT promoter activity. human sodium-dependent multivitamin transporter; promoter analysis in vitro and in vivo; transgenic mice

Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the CNS that is used to model certain parameters of multiple sclerosis. To establish the relative contributions of T cell reactivity, the loss of blood-brain barrier (BBB) integrity, CNS inflammation, and lesion formation toward the pathogenesis of EAE, we assessed the incidence of EAE and these parameters in mice lacking NF-[kappa]B, TNF-[alpha], IFN-[alpha][beta] receptors, IFN-[gamma] receptors, and inducible nitric oxide synthase. Although increased myelin oligodendrocyte glycoprotein-specific T cell reactivity was generally associated with a more rapid onset or increased disease severity, the loss of BBB integrity and cell accumulation in spinal cord tissues was invariably associated with the development of neurological disease signs. Histological and real-time RT-PCR analyses revealed differences in the nature of immune/inflammatory cell accumulation in the spinal cord tissues of the different mouse strains. On the other hand, disease severity during the acute phase of EAE directly correlated with the extent of BBB permeability. Thus, the loss of BBB integrity seems to be a requisite event in the development of EAE and can occur in the absence of important inflammatory mediators. gene knockout mice | multiple sclerosis

The transcriptional coactivator peroxisome proliferator-activated receptor-[gamma] coactivator 1[beta] (PGC-1[beta]) is believed to control mitochondrial oxidative energy metabolism by activating specific target transcription factors including estrogen-related receptors and nuclear respiratory factor 1, yet its physiological role is not yet clearly understood. To define its function in vivo, we generated and characterized mice lacking the functional PGC-1[beta] protein [PGC-1[beta] knockout (KO) mice]. PGC-1[beta] KO mice are viable and fertile and show no overt phenotype under normal laboratory conditions. However, the KO mice displayed an altered expression in a large number of nuclear-encoded genes governing mitochondrial and metabolic functions in multiple tissues including heart, skeletal muscle, brain, brown adipose tissue, and liver. In contrast to PGC-1[alpha] KO mice that are reportedly hyperactive, PGC-1[beta] KO mice show greatly decreased activity during the dark cycle. When acutely exposed to cold, the KO mice developed abnormal hypothermia and morbidity. Furthermore, high-fat feeding induced hepatic steatosis and increased serum triglyceride and cholesterol levels in the KO mice. These results suggest that PGC-1[beta] in mouse plays a nonredundant role in controlling mitochondrial oxidative energy metabolism. knockout mice | oxidative metabolism | energy metabolism | lipogenesis

In mice that express a transgene for the 2C T cell antigen-receptor (TCR) and lack a recombinase-activating gene (2[C.sup.+]RA[G.sup.-/-] mice) most of the peripheral T cells are CD[8.sup.+], a few are CD[4.sup.+], and a significant fraction are CD4-CD8- [double negative (DN)]. The DN 2C cells, like DN T cells that are abundant in various other [alpha][beta] TCR-transgenic mice, appear to be derived directly from DN thymocytes that prematurely express the TCR transgene. The DN 2C cells are virtually absent in mice deficient in major histocompatibility complex class II (MHC-II) but more abundant in mice deficient in MHC-I, suggesting that the DN 2C thymocytes are positively selected by self-peptide--MHC-II (pMHC-II) complexes and negatively selected by self-pMHC-I complexes. The pMHC-I complexes, however, positively select CD[8.sup.+] 2C T cells in the same mice. The different effects of thymic pMHC-I on DN and CD[8.sup.+] thymocytes are consistent with the finding that DN 2C thymocytes are more sensitive than more mature CD[4.sup.+]CD[8.sup.+] [double positive (DP)] thymocytes to a weak pMHC-I agonist for the 2C TCR. Together with previous evidence that DP thymocytes respond more sensitively than T cells in the periphery to weak pMHC agonists, the findings suggest progressive decreases in responsiveness to self-pMHC-I complexes as thymocytes develop from DN to DP thymocytes and then to mature naive T cells in the periphery. antigen recognition degeneracy | development | lineage | positive and negative selection | T cell antigen receptor

Aminopeptidase N (APN, CD13; EC 3.4.11.2) is a transmembrane metalloprotease with several functions, depending on the cell type and tissue environment. In tumor vasculature, APN is overexpressed in the endothelium and promotes angiogenesis. However, there have been no reports of in vivo inactivation of the APN gene to validate these findings. Here we evaluated, by targeted disruption of the APN gene, whether APN participates in blood vessel formation and function under normal conditions. Surprisingly, APN-null mice developed with no gross or histological abnormalities. Standard neurological, cardiovascular, metabolic, locomotor, and hematological studies revealed no alterations. Nonetheless, in oxygen-induced retinopathy experiments, APN-deficient mice had a marked and dose-dependent deficiency of the expected retinal neovascularization. Moreover, gelfoams embedded with growth factors failed to induce functional blood vessel formation in APN-null mice. These findings establish that APN-null mice develop normally without physiological alterations and can undergo physiological angiogenesis but show a severely impaired angiogenic response under pathological conditions. Finally, in addition to vascular biology research, APN-null mice may be useful reagents in other medical fields such as malignant, cardiovascular, immunological, or infectious diseases. CD13 | knockout mice | retinopathy | vasculogenesis

Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical [Cl.sup.-]/HC[O.sup.-.sub.3] exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, [J.sub.Cl] in mouse CCD through a pendrin-dependent mechanism. [J.sub.Cl] and transepithelial voltage, [V.sub.T], were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCl-replete diet or a NaCl-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaClreplete diet, [V.sub.T] and [J.sub.Cl] were not different from zero either in the presence or absence of angiotensin II ([10.sup.-8] M) in the bath. Thus further experiments employed mice given the high-NaCl diet and furosemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative [V.sub.T] and absorbed [Cl.sup.-]. With angiotensin II in the bath, [Cl.sup.-] absorption doubled although [V.sub.T] did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, [Cl.sup.-] secretion and a [V.sub.T] of ~0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished [V.sub.T] although [J.sub.Cl] fell only ~50%. Thus substantial [Cl.sup.-] absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the [H.sup.+]-ATPase inhibitor bafilomycin abolished benzamil-insensitive [Cl.sup.-] absorption. In conclusion, angiotensin II increases transcellular [Cl.sup.-] absorption in the CCD through a pendrin- and [H.sup.+]-ATPase-dependent process. transepithelial voltage; bafilomycin; [H.sup.+]-ATPase; knockout mice; Slc26a4; intercalated cell

Aquaporin-9 (AQP9) is an aquaglyceroporin membrane channel shown biophysically to conduct water, glycerol, and other small solutes. Because the physiological role/s of AQP9 remain undefined and the expression sites of AQP9 remain incomplete and conflicting, we generated AQP9 knockout mice. In the absence of physiological stress, knockout mice did not display any visible behavioral or severe physical abnormalities. Immunohistochemical analyses using multiple antibodies revealed AQP9 specific labeling in hepatocytes, epididymis, vas deferens, and in epidermis of wild type mice, but a complete absence of labeling in AQP[9.sup.-/-] mice. In brain, no detectable labeling was observed. Compared with control mice, plasma levels of glycerol and triglycerides were markedly increased in AQP[9.sup.-/-] mice, whereas glucose, urea, free fatty acids, alkaline phosphatase, and cholesterol were not significantly different. Oral administration of glycerol to fasted mice resulted in an acute rise in blood glucose levels in both AQP[9.sup.-/-] and AQP[9.sup.+/-] mice, revealing no defect in utilization of exogenous glycerol as a gluconeogenic substrate and indicating a high gluconeogenic capacity in nonhepatic organs. Obese [Lepr.sup.db]/ [Lepr.sup.db] AQP[9.sup.-/-] and obese [Lepr.sup.db]/[Lepr.sup.db] AQP[9.sup.+/-] mice showed similar body weight, whereas the glycerol levels in obese [Lepr.sup.db]/[Lepr.sup.db] AQP[9.sup.-/-] mice were dramatically increased. Consistent with a role of AQP9 in hepatic uptake of glycerol, blood glucose levels were significantly reduced in [Lepr.sup.db]/[Lepr.sup.db] AQP[9.sup.-/-] mice compared with [Lepr.sup.db]/ [Lepr.sup.db] AQP[9.sup.+/-] in response to 3 h of fasting. Thus, AQP9 is important for hepatic glycerol metabolism and may play a role in glycerol and glucose metabolism in diabetes mellitus. aquaglyceroporin | diabetes meliitus | leptin receptor

Heterotrimeric G proteins of the [G.sub.i] class have been implicated in signaling pathways regulating growth and metabolism under physiological and pathophysiological conditions. Knockout mice carrying inactivating mutations in both of the widely expressed G[[alpha].sub.i] class genes, G[[alpha].sub.i2] and G[[alpha].sub.i3], demonstrate shared as well as genespecific functions. The presence of a single active allele of G[[alpha].sub.i3] is sufficient for embryonic development, whereas at least one allele of G[[alpha].sub.i2] is required for extrauterine life. Mice lacking both G[[alpha].sub.i2] and G[[alpha].sub.i3] are massively growth-retarded and die in utero. We have used biochemical and cell biological methods together with in situ liver perfusion experiments to study G[[alpha].sub.i] isoform-specific functions in G[[alpha].sub.i2]- and G[[alpha].sub.i3]-deficient mice. The subcellular localization of G[[alpha].sub.i3] in isolated mouse hepatocytes depends on the cellular metabolic status. G[[alpha].sub.i3] localizes to autophagosomes upon starvation-induced autophagy and distributes to the plasma membrane upon insulin stimulation. Analysis of autophagic proteolysis in perfused mouse livers showed that mice lacking G[[alpha].sub.i3] are deficient in the inhibitory action of insulin. These data indicate that G[[alpha].sub.i3] is crucial for the antiautophagic action of insulin and suggest an as-yet-unrecognized function for G[[alpha].sub.i3] on autophagosomal membranes. anticatabolic actions | autophagy | mouse knockout | pertussis toxin-sensitive G proteins

The joining of DNA ends during Ig class-switch recombination (CSR) is thought to involve the same nonhomologous end-joining pathway as used in V(D)J recombination. However, we reported earlier that CSR can readily occur in Ig transgenic SCID mice lacking DNA-dependent protein kinase (DNA-PK) activity, a critical enzymatic activity for V(D)J recombination. We were thus led to question whether the catalytic subunit of DNA-PK (DNA-PKcs) is essential for CSR. To address this issue, we asked whether class switching to different Ig isotypes could occur in a line of Ig transgenic mice lacking detectable DNA-PKcs protein. The answer was affirmative. We conclude that joining of DNA ends during CSR does not require DNA-PKcs and can occur by an alternative repair pathway to that used for V(D)J recombination. Ig transgenic mice | V(D)J recombination

The [alpha]B-crystallin and HspB2 genes are located [approximately equal to] 0.9 kb apart in a head-to-head arrangement in mammals. Previous experiments have shown that a truncated -668/+45 [alpha]B-crystallin enhancer/promoter fragment from blind mole rats (Spalax ehrenbergl), which have nonfunctional lenses, lacks lens activity and has enhanced muscle activity in transgenic mice. Here we show that the full-length mole rat [alpha]B-crystallin intergenic region behaves similarly in transgenic mice. A two-nucleotide mutation ([sup.-273]CA [right arrow] G) in the mouse [alpha]B-crystallin enhancer/promoter fragment mimicking the wild-type mole rat sequence functionally converted the mouse promoter fragment to that of the wild-type mole rat promoter when tested in transgenic mice. The reciprocal mutation in the mole rat promoter fragment ([sup.-272]G [right arrow] CA) did not affect its activity. Oligonucleotides from the wild-type mouse and mole rat [alpha]B-crystallin promoter region under study formed distinct complexes with nuclear proteins from cultured cells. The mouse mutant sequence lost binding ability, whereas the mutated mole rat sequence gained the ability to form a complex similar in size to that of the wild-type mouse oligonucleotide. Our data support the idea that blind mole rats' [alpha]B-crystallin promoter activity was modified during the evolution of subterranean life and shows that tissue-specific promoter activity can be modulated by changing as few as two apparently neutral nucleotides in the mouse [alpha]B-crystallin enhancer region, implying the importance of the context of regulatory sequences for promoter activity. evolution | gene expression | lens | muscle

Protein phosphorylation is a complex network of signaling and regulatory events that affects virtually every cellular process. Our understanding of the nature of this network as a whole remains limited, largely because of an array of technical challenges in the isolation and high-throughput sequencing of phosphorylated species. In the present work, we demonstrate that a combination of tandem phosphopeptide enrichment methods, high performance MS, and optimized database search/data filtering strategies is a powerful tool for surveying the phosphoproteome. Using our integrated analytical platform, we report the identification of 5,635 nonredundant phosphorylation sites from 2,328 proteins from mouse liver. From this list of sites, we extracted both novel and known motifs for specific Ser/Thr kinases including a 'dipolar' motif. We also found that C-terminal phosphorylation was more frequent than at any other location and that the distribution of potential kinases for these sites was unique. Finally, we identified double phosphorylation motifs that may be involved in ordered phosphorylation. mass spectrometry | proteomics

Mitochondrial dysfunction is implicated in the pathophysiology of Parkinson's disease (PD), a common age-associated neurode-generative disease characterized by intraneuronal inclusions (Lewy bodies) and progressive degeneration of the nigrostriatal dopamine (DA) system. It has recently been demonstrated that midbrain DA neurons of PD patients and elderly humans contain high levels of somatic mtDNA mutations, which may impair respiratory chain function. However, clinical studies have not established whether the respiratory chain deficiency is a primary abnormality leading to inclusion formation and DA neuron death, or whether generalized metabolic abnormalities within the degenerating DA neurons cause secondary damage to mitochondria. We have used a reverse genetic approach to investigate this question and created conditional knockout mice (termed MitoPark mice), with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. The knockout mice have reduced mtDNA expression and respiratory chain deficiency in midbrain DA neurons, which, in turn, leads to a parkinsonism phenotype with adult onset of slowly progressive impairment of motor function accompanied by formation of intraneuronal inclusions and dopamine nerve cell death. Confocal and electron microscopy show that the inclusions contain both mitochondrial protein and membrane components. These experiments demonstrate that respiratory chain dysfunction in DA neurons may be of pathophysiological importance in PD. inclusion | rnitochondria | mtDNA | neurodegeneration | Parkinson

The current testing of anti-HIV drugs is hampered by the lack of a small animal that is readily available and easy to handle; can be infected systemically with HIV type 1 (HIV-1); harbors the major HIV-1 target cells in a physiological frequency, organ distribution, and activation state; and is established as a pharmacological model. Here, we explored the potential of outbred Sprague-Dawley rats that transgenically express the HIV-1 receptor complex on CD4 T cells and macrophages as a model for the preclinical evaluation of inhibitors targeting virus entry or reverse transcription. The concentrations of the peptidic fusion inhibitor enfuvirtide or the nonnucleoside reverse transcriptase inhibitor efavirenz required to inhibit HIV-1 infection of cultured primary CD4 T cells and macrophages from human CD4 and CCR5-transgenic rats differed by no more than 3-fold from those required for human reference cultures. Prophylactic treatment of double-transgenic rats with a weight-adapted pediatric dosing regimen for either enfuvirtide (s.c., twice-daily) or efavirenz (oral, once-daily) achieved a 92.5% or 98.8% reduction, respectively, of the HIV-1 cDNA load in the spleen 4 days after i.v. HIV-1 challenge. Notably, a once-daily dosing regimen for enfuvirtide resulted in a [approximately equal to] 5-fold weaker inhibition of infection, unmasking the unfavorable pharmacokinetic characteristics of the synthetic peptide in the context of an efficacy trial. This work provides proof of principle that HIV-susceptible transgenic rats can allow a rapid and predictive preclinical evaluation of the inhibitory potency and of the pharmacokinetic properties of antiviral compounds targeting early steps in the HIV replication cycle. animal model | drugs | efficacy trial

Ovulation rarely occurs in mice in which the estrogen receptor [beta] (ER[beta]) gene has been inactivated (ER[[beta].sup.-/-] mice). Here, we investigated whether this subfertility is due to a defect in the ovary itself or to more general endocrine changes in ER[[beta].sup.-/-] mice. We transplanted ER[[beta].sup.-/-] ovaries into WT mice and WT ovaries into ER[[beta].sup.-/-] mice. Upon mating with ER[[beta].sup.-/-] males, fertility increased from 20% in control intact ER[[beta].sup.-/-] group to 40% in the WT recipients with ER[[beta].sup.-/-] ovaries. The transplantation procedure was not efficient, and when WT ovaries were transplanted into WT mice, fertility was only 36%. Surgical ovarian wedge resection, a procedure which induces ovulation in anovulatory women with polycystic ovarian syndrome, resulted in 100% fertility of ER[[beta].sup.-/-] mice. In ER[[beta].sup.-/-] mice, as the follicles enlarged, the thecal layer remained very compact (revealed by H&E and collagen staining), and there was no increase in vascularization (measured as smooth muscle actin). In addition, there was an increase in PDGF receptor [alpha] (PDGFR[alpha]) and a decrease in PDGF[beta] expression in the granulosa cells, similar to what has been found in follitropin receptor knockout mice. After wedge resection, expression of both smooth muscle actin and PDGFRs was normalized. During normal follicular development, increased vascularization of the thecal layer is a prerequisite for further follicular growth. We suggest that the defect in ER[[beta].sup.-/-] mouse ovaries is a failure of communication between the granulosa and thecal layers. The follicles do not mature because of insufficient blood supply. This problem is overcome by stimulating neovascularization by simple wedge resection of the ovaries. estrogen receptor | follitropin or follicle-stimulating hormone | thecal | granulosa | neovascularization

Recent progresses in signal transduction have revealed that [beta]-catenin signaling controls embryonic development, tumorigenesis, cell shape, and polarity. The role of this pathway in myocyte shape regulation during cardiac hypertrophy and failure is, however, not clearly defined. Since homozygous knockout of [beta]-catenin is embryonically lethal, we have deleted [beta]-catenin genes specifically in the heart of adult mice by crossing loxP-flanked [beta]-catenin mice with transgenic mice expressing tamoxifen-activated MerCreMer protein (MCM) driven by the [alpha]-myosin heavy chain promoter. Administration of tamoxifen to homozygous loxP-flanked [beta]-catenin mice positive for MCM induces the deletion of [beta]-catenin only in cardiomyocytes. Immunolabeling with [beta]-catenin antibody demonstrates that 90% of cardiomyocytes completely lose their [beta]-catenin expression but maintain normal rod-shaped morphology. The intercalated disk of cardiomyocytes lacking [beta]-catenin is morphologically unremarkable with normal distribution of vinculin, N-cadherin, desmoplakin, ZO-1, connexin43, and [alpha]-, [gamma]-, and p120 catenins. The expression level of these proteins, except that of-[gamma]-catenin, is also similar in tamoxifen-treated and control mice with both homozygous loxP-flanked [beta]-catenin genes and the MCM transgene. Western blot analyses reveal that [gamma]-catenin increases in the heart of [beta]-catenin knockout mice compared with controls. Confocal microscopy also demonstrates that [gamma]-catenin has significantly increased in the intercalated disk of cardiomyocytes lacking [beta]-catenin. Echocardiographic data indicate that the knockout mice maintain normal ventricular geometry and cardiac function. The results suggest that upregulation of [gamma]-catenin can compensate for the loss of [beta]-catenin in cardiomyocytes to maintain normal cardiac structure and function. catenin; heart; intercalated disk; cardiomyocyte; knockout

LKB1 has been identified as a component of the major upstream kinase of AMP-activated protein kinase (AMPK) in skeletal muscle. To investigate the roles of LKB1 in skeletal muscle, we used muscle-specific LKB1 knockout (MLKB1KO) mice that exhibit low expression of LKB1 in heart and skeletal muscle, but not in other tissues. The importance of LKB1 in muscle physiology was demonstrated by the observation that electrical stimulation of the muscle in situ increased AMPK phosphorylation and activity in the wild-type (WT) but not in the muscle-specific LKB1KO mice. Likewise, phosphorylation of acetyl-CoA carboxylase (ACC) was markedly attenuated in the KO mice. The LKB1KO mice had difficulty running on the treadmill and exhibited marked reduction in distance run in voluntary running wheels over a 3-wk period (5.9 [+ or -] 0.9 km/day for WT vs. 1.7 [+ or -] 0.7 km/day for MLKB1KO mice). The MLKB1KO mice anesthetized at rest exhibited significantly decreased phospho-AMPK and phospho-ACC compared with WT mice. KO mice exhibited lower levels of mitochondrial protein expression in the red and white regions of the quadriceps. These observations, along with previous observations from other laboratories, clearly demonstrate that LKB1 is the major upstream kinase in skeletal muscle and that it is essential for maintaining mitochondrial marker proteins in skeletal muscle. These data provide evidence for a critical role of LKB1 in muscle physiology, one of which is maintaining basal levels of mitochondrial oxidative enzymes. Capacity for voluntary running is compromised with muscle and heart LKB1 deficiency. adenosine 3'-cyclic monophosphate-activated protein kinase; muscle specific LKB1 knockout mouse; muscle mitochondria; citrate synthase

Using a Cre-Lox conditional knockout strategy, we generated a germ cell-specific androgen receptor (AR) knockout mouse (G[AR.sup.-/y]) with normal spermatogenesis. Sperm count and motility in epididymis from [AR.sup.-/y] mice are similar to that of WT (G-[AR.sup.-/y]) mice. Furthermore, fertility tests show there was no difference in fertility, and almost 100% of female pups sired by G-[AR.sup.-/y] males younger than 15 weeks carried the deleted AR allele, suggesting the efficient AR knockout occurred in germ cells during meiosis. Together, these data provide in vivo evidence showing male mice without AR in germ cells can still have normal spermatogenesis and fertility, suggesting the essential roles of AR during spermatogenesis might come from indirect cell-cell communication in a paracrine fashion. We then compared the consequences of AR loss in the spermatogenesis and fertility of G-[AR.sup.-/y] mice with two other testicular cell-specific [AR.sup.-/y] mice and total AR knockout male mice. The results provide clear in vivo evidence that androgen/AR signaling in Sertoli cells plays a direct important role in spermatogenesis and in Leydig cells plays an autocrine regulatory role to modulate Leydig cell steroidogenic function. Total AR knockout male mice have the most severe defects among these mice. These contrasting data with G-[AR.sup.-/y] mice suggest AR might have different roles in the various cells within testis to contribute to normal spermatogenesis and male fertility in mice. germ cell | knockout | Sertoli cells | Leydig cells

Long interspersed element type 1 (L1) retrotransposons are ubiquitous mammalian mobile elements and potential tools for in vivo mutagenesis; however, native L1 elements are relatively inactive in mice when introduced as transgenes. We have previously described a synthetic L1 element, ORFeus, containing two synonymously recoded ORFs relative to mouse L1. It is significantly more active for retrotransposition in cell culture than all native L1 elements tested. To study its activity in vivo, we developed a transgenic mouse model in which ORFeus expression was controlled by a constitutive heterologous promoter, and we established definitive evidence for ORFeus retrotransposition activity both in germ line and somatic tissues. Germ line retrotransposition frequencies resulting in 0.33 insertions per animal are seen among progeny of ORFeus donor element heterozygotes derived from a single founder, representing a >20-fold increase over native L1 elements. We observe somatic transposition events in 100% of the ORFeus donor-containing animals, and an average of 17 different insertions are easily recovered from each animal; modeling suggests that the number of somatic insertions per animal exceeds this number by perhaps several orders of magnitude. Nearly 200 insertions were precisely mapped, and their distribution in the mouse genome appears random relative to transcription units and guanine-cytosine content. The results suggest that ORFeus may be developed into useful tools for in vivo mutagenesis. gene trap | integration site preference | LINE-1 | retrotransposon | transgenic mouse

Genetic mouse models provide a unique opportunity to investigate gene function in the natural course of the disease. Although diabetic nephropathy (DN) in models of type II diabetes has been well characterized, diabetic renal disease in hypoinsulinemic diabetic mice is still incompletely understood. Here, we characterized renal changes in the [pdx1.sup.PB]-HNF6 transgenic mouse that exhibits [beta]-cell dysfunction and nonobese hypoinsulinemic diabetes. Male transgenic mice developed hyperglycemia by the age of 7 wk and survived for over 1 yr without insulin treatment. Diabetes ensued earlier and progressed more severely in the HNF6 males than the females. The HNF6 males exhibited albuminuria as early as 10 wk of age, and the urinary albumin excretion increased with age, exceeding 150 [micro]g/24 h at 11 mo of age. Diabetic males developed renal hypertrophy after 7 wk of age, whereas glomerular hyperfiltration was not observed in the mice. Hypertension and hyperlipidemia were not observed in the diabetic mice. Histological analysis of the HNF6 kidneys displayed diabetic glomerular changes, including glomerular enlargement, diffuse mesangial proliferation and matrix expansion, thickened glomerular basement membrane, and arteriolar hyalinosis. Mesangial matrix accumulation increased with age, resulting in nodular lesions by 44 wk of age. Immunohistochemistry showed accumulation of type IV collagen and TGF-[beta]1 in the mesangial area. No significant immune complex deposition was observed in the HNF6 glomeruli. Thus the HNF6 mouse exhibits diabetic renal changes that parallel the early phase of human DN. The model should facilitate studies of genetic and environmental factors that may affect DN in hypoinsulinemic diabetes. animal model; diabetic renal disease; hepatocyte nuclear factor-6; transgenic mice

Imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (MMPs/TIMPs) takes part in age-related renal fibrosis; so does molecular inflammation. As several inflammatory mediators including intercellular adhesion molecule-1 (ICAM-1) are substrates of MMPs, we speculated that TIMP-1 might affect ICAM-I through MMPs and subsequently promote age-related renal fibrosis. Then, we observed changes of kidney in human TIMP-1 transgenic mice and wild-type mice of different ages. It was found that the expressions and activities of gelatinases were downregulated; the expressions of ICAM-1, collagen III, collagen IV, and transforming growth factor (TGF)-[beta] were upregulated; and the number of infiltrating macrophages was increased in kidneys of 24-month-old TIMP-1 transgenic mice with high expressions of TIMP-1, compared with wild-type mice. Our results indicated that TIMP-1 could promote age-related renal fibrosis, which was partly attributed to enhancing inflammation through upregulation of ICAM-1.

Much research has implicated the striatum in motor learning, but the underlying mechanisms have not been identified. Although NMDA receptor (NMDAR)-dependent long-term potentiation has been observed in the striatum, its involvement in motor learning remains unclear. To examine the role of striatal NMDAR in motor learning, we created striatum-specific NMDAR1 subunit knockout mice, analyzed the striatal anatomy and neuronal morphology of these mice, evaluated their performance on well established motor tasks, and performed electrophysiological recordings to assay striatal NMDAR function and long-term synaptic plasticity. Our results show that deleting the NMDAR1 subunit of the NMDAR specifically in the striatum, which virtually abolished NMDAR-mediated currents, resulted in only small changes in striatal neuronal morphology but severely impaired motor learning and disrupted dorsal striatal long-term potentiation and ventral striatal long-term depression. long-term potentiation | NMDA receptor | knockout | RGS9-2

Transgenic mice overexpressing PKCα in the epidermis (K5-PKCα mice) exhibit an inducible severe intraepidermal neutrophilic inflammation and systemic neutrophilia when PKCα is activated by topical 12-O-tetradecanoylphorbol-13-acetate (TPA). This inducible model [...]

Our previous studies demonstrated that, in gracilis muscle arterioles of male mice deficient in the gene for endothelial nitric oxide synthase (eNOS), flow-induced dilation (FID) is mediated by endothelial PGs. Thus the present study aimed to identify the specific isoform of cyclooxygenase (COX) responsible for the compensatory mediation of FID in arterioles of eNOS-knockout (KO) mice. Experiments were conducted on gracilis muscle arterioles of male eNOS-KO and wild-type (WT) mice. Basal tone and magnitude of FID of arterioles were comparable in the two strains of mice. A role for COX isoforms in the mediation of the responses was assessed by use of valeryl salicylate (3 mM) and NS-398 (10 [micro]M), inhibitors of COX-1 and COX-2, respectively. In eNOS-KO arterioles, valeryl salicylate or NS-398 alone inhibited FID (at maximal flow rate) by ~51% and ~58%, respectively. Administration of both inhibitors eliminated the dilation. In WT arterioles, inhibition of COX-2 did not significantly affect FID, whereas inhibition of COX-1 decreased the dilation by ~57%. The residual portion of the response was abolished by additional administration of [N.sup.[omega]-nitro-L-arginine methyl ester. Western blot analysis indicated a comparable content of COX-1 protein in arterioles of WT and eNOS-KO mice. COX-2 protein, which was not detectable in arterioles of WT mice, was strongly expressed in arterioles of eNOS-KO mice, together with an upregulation of COX-2 gene expression. Immunohistochemical staining confirmed the presence of COX-2 in the endothelium of eNOS-KO arterioles. In conclusion, COX-2-derived PGs are the mediators responsible for maintenance of FID in arterioles of eNOS-deficient mice. nitric oxide; endothelium doi: 10.1152/ajpheart.01130.2005

Blockade of NF-[kappa]B is a key transcription factor that regulates inflammatory processes. In the present study, we tested the hypothesis that blockade of NF-[kappa]B ameliorates cardiac remodeling and failure after myocardial infarction (MI). Knockout mice with targeted disruption of the p50 subunit of NF-[kappa]B (KO) were used to block the activation of NF-[kappa]B. MI was induced by ligation of the left coronary artery in male KO and age-matched wild-type (WT) mice. NF-[kappa]B was activated in noninfarct as well as infarct myocardium in WT + MI mice, while the activity was completely abolished in KO mice. Blockade of NF-[kappa]B significantly reduced early ventricular rupture after MI and improved survival by ameliorating congestive heart failure. Echocardiographic and pressure measurements revealed that left ventricular fractional shortening and maximum rate of rise of left ventricular pressure were significantly increased and end-diastolic pressure was significantly decreased in KO + MI mice compared with WT + MI mice. Histological analysis demonstrated significant suppression of myocyte hypertrophy as well as interstitial fibrosis in the noninfarct myocardium of KO + MI mice. Blockade of NF-[kappa]B did not ameliorate expression of proinflammatory cytokines in infarct or noninfarct myocardium. In contrast, phosphorylation of c-Jun N[H.sub.2]-terminal kinase was almost completely abolished in KO + MI mice. The present study demonstrates that targeted disruption of the p50 subunit of NF-[kappa]B reduces ventricular rupture as well as improves cardiac function and survival after MI. Blockade of NF-[kappa]B might be a new therapeutic strategy to attenuate cardiac remodeling and failure after MI. cardiac remodeling; inflammation; mitogen-activated protein kinases doi:10.1152/ajpheart.01175.2005