Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.

Rhodopsin, encoded by the gene Rhodopsin (RH1), is extremely sensitive to light, and is responsible for dim-light vision. Bats are nocturnal mammals that inhabit poor light environments. Megabats (Old-World fruit bats) generally have well-developed eyes, while microbats (insectivorous bats) have developed echolocation and in general their eyes were degraded, however, dramatic differences in the eyes, and their reliance on vision, exist in this group. In this study, we examined the rod opsin gene (RH1), and compared its evolution to that of two cone opsin genes (SWS1 and M/LWS). While phylogenetic reconstruction with the cone opsin genes SWS1 and M/LWS generated a species tree in accord with expectations, the RH1 gene tree united Pteropodidae (Old-World fruit bats) and Yangochiroptera, with very high bootstrap values, suggesting the possibility of convergent evolution. The hypothesis of convergent evolution was further supported when nonsynonymous sites or amino acid sequences were used to construct phylogenies. Reconstructed RH1 sequences at internal nodes of the bat species phylogeny showed that:(1) Old-World fruit bats share an amino acid change (S270G) with the tomb bat;(2) Miniopterus share two amino acid changes (V104I, M183L) with Rhinolophoidea;(3) the amino acid replacement I123V occurred independently on four branches, and the replacements L99M, L266V and I286V occurred each on two branches. The multiple parallel amino acid replacements that occurred in the evolution of bat RH1 suggest the possibility of multiple convergences of their ecological specialization (i.e., various photic environments) during adaptation for the nocturnal lifestyle, and suggest that further attention is needed on the study of the ecology and behavior of bats.

Endogenous cannabinoids acting at CB(1) receptors stimulate appetite, and CB(1) antagonists show promise in the treatment of obesity. CB(1)(-/-) mice are resistant to diet-induced obesity even though their caloric intake is similar to that of wild-type mice, suggesting that endocannabinoids also regulate fat metabolism. Here, we investigated the possible role of endocannabinoids in the regulation of hepatic lipogenesis. Activation of CB(1) in mice increases the hepatic gene expression of the lipogenic transcription factor SREBP-1c and its targets acetyl-CoA carboxylase-1 and fatty acid synthase (FAS). Treatment with a CB(1) agonist also increases de novo fatty acid synthesis in the liver or in isolated hepatocytes, which express CB(1). High-fat diet increases hepatic levels of the endocannabinoid anandamide (arachidonoyl ethanolamide), CB(1) density, and basal rates of fatty acid synthesis, and the latter is reduced by CB(1) blockade. In the hypothalamus, where FAS inhibitors elicit anorexia, SREBP-1c and FAS expression are similarly affected by CB(1) ligands. We conclude that anandamide acting at hepatic CB(1) contributes to diet-induced obesity and that the FAS pathway may be a common molecular target for central appetitive and peripheral metabolic regulation.

Cannabinoids and ethanol can activate the same reward pathways, which could suggest endocannabinoid involvement in the rewarding effects of ethanol. The high ethanol preference of young (6-10 weeks) C57BL6J mice is reduced by the cannabinoid receptor 1 (CB1) antagonist SR141716A to levels observed in their CB1 knockout littermates or in old (26-48 weeks) wild-type mice, in both of which ethanol preference is unaffected by SR141716A. Similarly, SR141716A inhibits food intake in food-restricted young, but not old, wild-type mice. There are no age-dependent differences in the tissue levels of the endocannabinoids anandamide and 2-arachidonoylglycerol or the density of CB1 in the hypothalamus, limbic forebrain, amygdala, and cerebellum. CB1-stimulated guanosine 5'-[gamma-thio]triphosphate (GTP[gammaS]) binding is selectively reduced in the limbic forebrain of old compared with young wild-type mice. There is no age-dependent difference in G(i) or G(o) subunit protein expression in the limbic forebrain, and the selective reduction in GTP[gammaS] labeling in tissue from old mice is maintained in a receptorG protein reconstitution assay by using functional bovine brain G protein. These findings suggest that endocannabinoids acting at CB1 contribute to ethanol preference, and decreased coupling of CB1 to G proteins in the limbic forebrain by mechanisms other than altered receptor or G protein levels may be involved in the age-dependent decline in the appetite for both ethanol and food.

Minocycline has been demonstrated to be neuroprotective after spinal cord injury (SCI). However, the cellular consequences of minocycline treatment on the secondary injury response are poorly understood. We examined the ability of minocycline to reduce oligodendrocyte apoptosis, microglial/macrophage activation, corticospinal tract (CST) dieback, and lesion size and to improve functional outcome after SCI. Adult rats were subjected to a C7-C8 dorsal column transection, and the presence of apoptotic oligodendrocytes was assessed within the ascending sensory tract (AST) and descending CST in segments (3-7 mm) both proximal and distal to the injury site. Surprisingly, the numbers of dying oligodendrocytes in the proximal and distal segments were comparable, suggesting more than the lack of axon-cell body contiguity played a role in their demise. Minocycline or vehicle control was injected into the intraperitoneal cavity 30 min and 8 hr after SCI and thereafter twice daily for 2 d. We report a reduction of apoptotic oligodendrocytes and microglia within both proximal and distal segments of the AST after minocycline treatment, using immunostaining for active caspase-3 and Hoechst 33258 staining in combination with cell-specific markers. Activated microglial/macrophage density was reduced remote to the lesion as well as at the lesion site. Both CST dieback and lesion size were diminished after minocycline treatment. Footprint analysis revealed improved functional outcome after minocycline treatment. Thus, minocycline ameliorates multiple secondary events after SCI, rendering this clinically used drug an attractive candidate for SCI treatment trials.

Stem cells play a critical role in normal tissue maintenance, and mutations in these stem cells may give rise to cancer. We hypothesize that melanoma develops from a mutated stem cell and therefore residual stem cell characteristics should be able to be identified in melanoma cell lines. We studied three metastatic melanoma cell lines that exhibited multiple morphologic forms in culture and demonstrated the capacity to pigment. We used the ability to efflux Hoechst 33342 dye, a technique known to enrich for stem cells in many tissues, to segregate cell populations. The cells with the greatest ability to efflux the dye were (1) small in size,(2) had the capacity to give rise to larger cell forms, and (3) had the greatest ability to expand in culture. The small cells were found to have a decreased proliferative rate and were less melanized. Large dendritic cells that appeared to be nonproliferative were identified in cultures. Treatment with cytosine beta-D-arabinofuranoside hydrochloride (Ara-C) expanded the large cell population but the residual proliferative capacity, both in vitro and in vivo, remained concentrated in the smaller cell fraction. Antigenic staining patterns were variable and heterogeneous. Nestin (a neural stem cell marker) and gp100 (premelanosomal marker) favored the smaller cell population, while nerve growth factor receptor often labeled larger cells. Morphologic and antigenic heterogeneity remained intact after clonal purification. These findings are consistent with the behavior expected for a tumor based on stem cell biology; this finding has diagnostic and therapeutic implications for melanocytic neoplasias.

Phosphoinositide 3-kinase (PI3K) is thought to contribute to the pathogenesis of asthma by effecting the recruitment, activation, and apoptosis of inflammatory cells. We examined the role of class IA PI3K in antigen-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of Deltap85 protein, a dominant negative form of the class IA PI3K regulatory subunit, p85alpha, which was fused to HIV-TAT (TAT-Deltap85). Intraperitoneal administration of TAT-Deltap85 caused time-dependent transduction into blood leukocytes, and inhibited activated phosphorylation of protein kinase B (PKB), a downstream target of PI3K, in lung tissues in mice receiving intranasal FMLP. Antigen challenge elicited pulmonary infiltration of lymphocytes, eosinophils and neutrophils, increase in mucus-containing epithelial cells, and airway hyperresponsiveness to methacholine. Except for modest airway neutrophilia, these effects all were blocked by treatment with 3-10 mg/kg of TAT-Deltap85. There was also significant reduction in IL-5 and IL-4 secretion into the BAL. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Deltap85. We conclude that PI3K has a regulatory role in Th2-cell cytokine secretion, airway inflammation, and airway hyperresponsiveness in mice.

Even though sequencing of the mammalian genome has led to the discovery of a large number of ionic channel genes, identification of the molecular determinants of cellular electrical properties in different regions of the heart has been rarely obtained. We developed a high-throughput approach capable of simultaneously assessing the expression pattern of ionic channel repertoires from different regions of the mouse heart. By using large-scale real-time RT-PCR, we have profiled 71 channels and related genes in the sinoatrial node (SAN), atrioventricular node (AVN), the atria (A) and ventricles (V). Hearts from 30 adult male C57BL/6 mice were microdissected and RNA was isolated from six pools of five mice each. TaqMan data were analysed using the threshold cycle (C(t)) relative quantification method. Cross-contamination of each region was checked with expression of the atrial and ventricular myosin light chains. Two-way hierarchical clustering analysis of the 71 genes successfully classified the six pools from the four distinct regions. In comparison with the A, the SAN and AVN were characterized by higher expression of Nav beta 1, Nav beta 3, Cav1.3, Cav3.1 and Cav alpha 2 delta 2, and lower expression of Kv4.2, Cx40, Cx43 and Kir3.1. In addition, the SAN was characterized by higher expression of HCN1 and HCN4, and lower expression of RYR2, Kir6.2, Cav beta 2 and Cav gamma 4. The AVN was characterized by higher expression of Nav1.1, Nav1.7, Kv1.6, Kvbeta1, MinK and Cav gamma 7. Other gene expression profiles discriminate between the ventricular and the atrial myocardium. The present study provides the first genome-scale regional ionic channel expression profile in the mouse heart.

Preconditioning with sublethal ischemia results in natural tolerance to ischemic stress, where multiple mediators of ischemic damage are simultaneously counteracted. Tumor necrosis factor alpha (TNF-alpha) has been implicated in development of ischemic tolerance. Using cellular models of ischemic tolerance, we have demonstrated that an effector of TNF-alpha-induced preconditioning is ceramide, a sphingolipid messenger in TNF-alpha signaling. TNF-alpha/ceramide-induced preconditioning protected cultured neurons against ischemic death and cultured astrocytes against proinflammatory effects of TNF-alpha. TNF-alpha activates a transcription factor NF-kappaB that binds promoters of multiple genes, thus ensuring pleiotropic effects of TNF-alpha. We describe here a mechanism that allows selective suppression of TNF-alpha/NF-kappaB-induced harmful genes in preconditioned cells while preserving cytoprotective responses. We demonstrate that in astrocytes activation of an adhesion molecule ICAM-1 by TNF-alpha is regulated through association of the phosphorylated p65 subunit of NF-kappaB with an adapter protein, p300, and that in preconditioned cells p65 remains unphosphorylated and ICAM-1 transcription is inhibited. However, TNF-alpha-activated transcription of a protective enzyme, MnSOD, does not depend on p300 and does not become inhibited in preconditioned cells. This new understanding of TNF-alpha-induced adaptation to ischemic stress and inflammation could suggest novel avenues for clinical intervention during ischemic and inflammatory diseases.

In expression systems and in yeast, Na/H exchanger regulatory factor (NHERF)-1 and NHERF-2 have been demonstrated to interact with the renal brush border membrane proteins NHE3 and Npt2. In renal tissue of mice, however, NHERF-1 is required for cAMP regulation of NHE3 and for the apical targeting of Npt2 despite the presence of NHERF-2, suggesting another order of specificity. The present studies examine the subcellular location of NHERF-1 and NHERF-2 and their interactions with target proteins including NHE3, Npt2, and ezrin. The wild-type mouse proximal tubule expresses both NHERF-1 and NHERF-2 in a distinct pattern. NHERF-1 is strongly expressed in microvilli in association with NHE3, Npt2, and ezrin. Although NHERF-2 can be detected weakly in the microvilli, it is expressed predominantly at the base of the microvilli in the vesicle-rich domain. NHERF-2 appears to associate directly with ezrin and NHE3 but not Npt2. NHERF-1 is involved in the apical expression of Npt2 and the presence of other Npt2-binding proteins does not compensate totally for the absence of NHERF-1 in NHERF-1-null mice. Although NHERF-1 links NHE3 to the actin cytoskeleton through ezrin, the absence of NHERF-1 does not result in a generalized disruption of the architecture of the cell. Thus the mistargeting of Npt2 seen in NHERF-1-null mice likely represents a specific disruption of pathways mediated by NHERF-1 to achieve targeting of Npt2. These findings suggest that the organized subcellular distribution of the NHERF isoforms may play a role in the specific interactions mediating physiological control of transporter function.

The stimulatory G protein alpha-subunit G(s)alpha couples receptors to adenylyl cyclase and is required for hormone-stimulated cAMP generation. In Albright hereditary osteodystrophy, heterozygous G(s)alpha null mutations only lead to PTH, TSH, and gonadotropin resistance when inherited maternally [pseudohypoparathyroidism type 1A;(PHP1A)]. Maternal-specific expression of G(s)alpha in specific hormone targets could explain this observation. Using hot-stop PCR analysis on total RNA from six normal human thyroid specimens, we showed that the majority of the G(s)alpha mRNA (72 +/- 3%) was derived from the maternal allele. This is consistent with the presence of TSH resistance in patients with maternal G(s)alpha null mutations (PHP1A) and the absence of TSH resistance in patients with paternal G(s)alpha mutations (pseudopseudohypoparathyroidism). Patients with PTH resistance in the absence of Albright hereditary osteodystrophy (PHP1B) have an imprinting defect of the G(s)alpha gene resulting in both alleles having a paternal epigenotype, which would lead to a more moderate level of thyroid-specific G(s)alpha deficiency. We found evidence of borderline TSH resistance in 10 of 22 PHP1B patients. This study provides further evidence for tissue-specific imprinting of G(s)alpha in humans and provides a potential mechanism for mild to moderate TSH resistance in PHP1A and borderline resistance in some patients with PHP1B.