Predation on eggs by conspecific males in two Japanese damselfishes,Pomacentrus nagasakiensis andChromis notatus notatus,after removal of egg-guarding males

Egg predation by conspecific males of damselfishes,Pomacentrus nagasakiensis andChromis notatus notatus, was studied at Bohnotsu (31°15′ N, 131°15′ E) and Tsuyazaki (33°47′ N, 130°29′ E), respectively. InP. nagasakiensis, 7 egg-guarding males were removed from nests, and intrusion and egg predation by conspecific males took place in all of the nests within an average time of 12.2 min. Frequency of attacks on eggs by other reef fishes during the period between the removal of egg-guarding males and intrusion of conspecific males was lower than expected, considering the frequency of aggressive displays of the egg-guarding males. The results of male removal experiments forChromis notatus notatus were almost the same, with intrusion and egg predation by conspecific males occurring in all 12 of the nests from which egg-guarding males had been removed. SixP. nagasakiensis and 15C. notatus notatus which intruded into the nests and preyed on eggs were collected. All were males except for 1C. notatus notatus female. Results suggest that these males which intruded into vacant nests to eat eggs did not have clutches of their own during the spawning bouts when the experiments were done. Results also showed that the intruding males were not inferior to egg-guarding males in standard length, gonadosomatic index, and condition factor. It is clear that males which do not have their own clutches during at least 1 spawning bout and prey on others' eggs when egg-guarding males disappear are common in these 2 gregariously nesting damselfishes. It is discussed that conspecific eggs are potentially a good food source not only for other species of reef fishes but also for conspecific males which do not have their own eggs during the spawning bouts. The origin of males wich prey on eggs of other conspecifics is also considered. Some aspects of egg-guarding and aggressive behavior of males in damselfishes are discussed.     

Division of labor in the monogamous goby,Valenciennea longipinnis, in relation to burrowing behavior

Feeding and burrowing behavior of the monogamous gobiid fish,Valenciennea longipinnis, were studied on the coral reef at Sesoko Island, Okinawa, Japan. These fish usually live in pairs, the male and female feeding in close proximity to one another upon benthic animals and constructing several burrows cooperatively for purposes of shelter or spawning. Paired females fed more and burrowed less frequently than their mates. Because burrow maintenance was mostly conducted by the latter, the paired females performed work much less frequently than solitary females. Thus, the paired females may be able to allocate more energy toward egg production. The division of labor related to burrowing behavior in this species may be an effective way to increase reproductive success for both sexes. Moreover, the fameles burrowed even less frequently when paired with larger males, probably because burrowing ability may be correlated with mouth size in males. This is a likely reason for the preference of females to mate with larger males.     

Cellobiose Transport by Mixed Ruminal Bacteria from a Cow

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 μM for the K_m and 10 nmol/min/mg of protein for the V_max. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher V_max values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.     

Evolution of Otx paralogue usages in early patterning of the vertebrate head

To assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates.     

Highly purified eicosapentaenoic acid prevents the progression of hepatic steatosis by repressing monounsaturated fatty acid synthesis in high-fat/high-sucrose diet-fed mice

Eicosapentaenoic acid (EPA) is a member of the family of n-3 polyunsaturated fatty acids (PUFAs) that are clinically used to treat hypertriglyceridemia. The triglyceride (TG) lowering effect is likely due to an alteration in lipid metabolism in the liver, but details have not been fully elucidated. To assess the effects of EPA on hepatic TG metabolism, mice were fed a high-fat and high-sucrose diet (HFHSD) for 2 weeks and were given highly purified EPA ethyl ester (EPA-E) daily by gavage. The HFHSD diet increased the hepatic TG content and the composition of monounsaturated fatty acids (MUFAs). EPA significantly suppressed the hepatic TG content that was increased by the HFHSD diet. EPA also altered the composition of fatty acids by lowering the MUFAs C16:1 and C18:1 and increasing n-3 PUFAs, including EPA and docosahexaenoic acid (DHA). Linear regression analysis revealed that hepatic TG content was significantly correlated with the ratios of C16:1/C16:0, C18:1/C18:0, and MUFA/n-3 PUFA, but was not correlated with the n-6/n-3 PUFA ratio. EPA also decreased the hepatic mRNA expression and nuclear protein level of sterol regulatory element binding protein-1c (SREBP-1c). This was reflected in the levels of lipogenic genes, such as acetyl-CoA carboxylase α (ACCα), fatty acid synthase, stearoyl-CoA desaturase 1 (SCD1), and glycerol-3-phosphate acyltransferase (GPAT), which are regulated by SREBP-1c. In conclusion, oral administration of EPA-E ameliorates hepatic fat accumulation by suppressing TG synthesis enzymes regulated by SREBP-1 and decreases hepatic MUFAs accumulation by SCD1.     

Molecular Basis for E-cadherin Recognition by Killer Cell Lectin-like Receptor G1 (KLRG1)

The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca²⁺-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K_d ∼7–12 μm), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1⁺NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.Natural killer (NK)³ cells play a critical role in the innate immune system because of their ability to kill other cells. For example, NK cells can kill virus-infected cells and tumor cells without presensitization to a specific antigen, and they produce various cytokines, including interferon-γ and tumor necrosis factor-α (1). NK cells are controlled by both inhibitory and activating receptors that are expressed on their surfaces (2). The killer cell Ig-like receptor, Ly49, CD94/NKG2, and paired Ig-like type 2 receptor families include both inhibitory and activating members and thus are designated as paired receptor families. On the other hand, some inhibitory receptors, including KLRG1 (killer cell lectin-like receptor G1), and activating receptors, such as NKG2D, also exist. The integration of the signals from these receptors determines the final functional outcome of NK cells.These inhibitory and activating receptors can also be divided into two structurally different groups, the Ig-like receptors and the C-type lectin-like receptors, based on the structural aspects of their extracellular regions. The Ig-like receptors include killer cell Ig-like receptors and the leukocyte Ig-like receptors, and the C-type lectin-like receptors include CD94/NKG2(KLRD/KLRC), Ly49(KLRA), NKG2D(KLRK), NKR-P1(KLRB), and KLRG1. Many of these immune receptors recognize major histocompatibility complex class I molecules or their relatives (2–4), but there are still many orphan receptors expressed on NK cells. KLRG1 was one such orphan receptor; however, E-cadherin was recently found to be a ligand of KLRG1 (5, 6). Although major histocompatibility complex-receptor interactions have been extensively examined, the molecular basis of non-major histocompatibility complex ligand-receptor recognition is poorly understood.KLRG1 is a type II membrane protein, with one C-type lectin domain in the extracellular region, one transmembrane region, and one immunoreceptor tyrosine-based inhibitory motif. KLRG1 is expressed on a subset of mature NK cells in spleen, lungs, and peripheral blood during normal development. KLRG1 expression is induced on the surface of NK cells during viral responses (7, 8). NK cells expressing KLRG1 produce low levels of interferon-γ and cytokines and have a slow in vivo turnover rate and low proliferative responsiveness to interleukin-15 (9). Furthermore, KLRG1 is recognized as a marker of some T cell subsets, as follows. KLRG1 defines a subset of T cells, short lived effector CD8 T cells (SLECs), which are mature effector cells that express high levels of KLRG1 and cannot be differentiated into long lived memory CD8 T cells. In addition, memory precursor effector cells express low levels of KLRG1 and harbor the potential to become long lived memory CD8 T cells (10). Since SLECs exhibit stronger effector function than memory precursor effector cells, it is potentially beneficial, in terms of preventing harmful excess cytotoxicity, that SLECs express KLRG1 at a higher level to inhibit the immune response. Taken together, the expression of KLRG1 during the viral response and normal development might confer the inhibition of effector function and the regulation of NK and T cell proliferation (9).E-cadherin plays a pivotal role in Ca²⁺-dependent cell-cell adhesion and also contributes to tissue organization and development (11–14). E-cadherin is primarily expressed on epithelial cells, and its extracellular region consists of several domains that include cadherin motifs (15, 16). These domains mediate Ca²⁺-dependent homophilic interactions to facilitate cell adhesion. When E-cadherins form cis- or trans-homodimers, they utilize their N-terminal regions as an interface, which can dock with domain 1 of another E-cadherin to form strand exchange (17). Therefore, the N-terminal region plays important roles in homophilic binding and cell adhesion.KLRG1 recognizes E-cadherins (and other class I cadherins), which are widely expressed in tissues and form tight adhesive cell-cell junctions, and Ito et al. (5) demonstrated that E-cadherin binding by KLRG1 inhibits NK cytotoxicity. Further, Gründermann et al. (6) showed that the E-cadherin-KLRG1 interaction inhibits the antigen-induced proliferation and induction of the cytolytic activity of CD8 T cells. Therefore, it is plausible that E-cadherin recognition by KLRG1, expressed on the surfaces of NK cells and T cells, may raise their activation thresholds by transducing inhibitory signals. Such an inhibition would prevent the excess injury of normal cells, which might result in inflammatory autoimmune diseases. KLRG1 may also have an important role in monitoring and removing cancer cells that lose E-cadherin expression. A recent report demonstrated that N-terminal domains 1 and 2 of E-cadherin are critical for KLRG1 recognition (18); however, despite accumulating evidence supporting the functional importance of the E-cadherin-KLRG1 interaction, the molecular basis of this interaction is poorly understood. Here, we report that the N-terminal region of E-cadherin, comprising the dimer interface, is the binding site for KLRG1. This suggests that KLRG1 does not recognize the dimeric form of E-cadherin but rather recognizes the monomeric form, which is exposed on the cell surfaces of disrupted or infected cells. This may suppress excess immune responses.     

Suppression of hepatic fat accumulation by highly purified eicosapentaenoic acid prevents the progression of d-galactosamine-induced hepatitis in mice fed with a high-fat/high-sucrose diet

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis. BALB/cA mice were fed with a standard diet (STD) or a high-fat and high-sucrose diet (HFHSD) for 14 days followed by intraperitoneal injection of d-galactosamine (DGalN) or vehicle. After 20–21 h, plasma and liver tissue were collected and analyzed. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma were increased significantly in HFHSD-fed mice treated with DGalN compared to STD-fed mice treated with DGalN. This exacerbation by the HFHSD was also observed in the plasma soluble tumor necrosis factor receptor (sTNFR) levels, and hepatic levels of reactive oxygen species (ROS) and the fibrogenic gene expression, such as tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), connective tissue growth factor (CTGF) and osteopontin (OPN) in HFHSD-fed mice treated with DGalN. The triglyceride contents of the liver were significantly increased by the HFHSD. When eicosapentaenoic acid (EPA), a suppressor of sterol regulatory element binding protein 1 (SREBP-1), was administered to HFHSD-fed mice, the sensitivity of DGalN, as a result of plasma ALT and AST levels, was suppressed accompanied by reduced plasma sTNFR2 level and hepatic levels of triglyceride, ROS, and fibrogenic parameters, and by increased plasma adiponectin levels. These data suggest that the progression of steatotic liver injury closely depends on the accumulation of fat in the liver and is prevented by EPA through the suppression of the fatty liver change.     

Regional stiffening of the mitral valve anterior leaflet in the beating ovine heart

Left atrial muscle extends into the proximal third of the mitral valve (MV) anterior leaflet and transient tensing of this muscle has been proposed as a mechanism aiding valve closure. If such tensing occurs, regional stiffness in the proximal anterior mitral leaflet will be greater during isovolumic contraction (IVC) than isovolumic relaxation (IVR) and this regional stiffness difference will be selectively abolished by β-receptor blockade. We tested this hypothesis in the beating ovine heart. Radiopaque markers were sewn around the MV annulus and on the anterior MV leaflet in 10 sheep hearts. Four-dimensional marker coordinates were obtained from biplane videofluoroscopy before (CRTL) and after administration of esmolol (ESML). Heterogeneous finite element models of each anterior leaflet were developed using marker coordinates over matched pressures during IVC and IVR for CRTL and ESML. Leaflet displacements were simulated using measured left ventricular and atrial pressures and a response function was computed as the difference between simulated and measured displacements. Circumferential and radial elastic moduli for ANNULAR, BELLY and EDGE leaflet regions were iteratively varied until the response function reached a minimum. The stiffness values at this minimum were interpreted as the in vivo regional material properties of the anterior leaflet. For all regions and all CTRL beats IVC stiffness was 40–58% greater than IVR stiffness. ESML reduced ANNULAR IVC stiffness to ANNULAR IVR stiffness values. These results strongly implicate transient tensing of leaflet atrial muscle during IVC as the basis of the ANNULAR IVC–IVR stiffness difference.     

Anticonvulsant profile and teratogenic evaluation of potent new analogues of a valproic acid urea derivative in NMRI mice

BACKGROUND: Valproic acid (VPA) is used to treat epilepsy and bipolar disorders, as well as for migraine prophylaxis. However, its clinical use is limited by two life-threatening side effects: hepatotoxicity and teratogenicity. To develop a more potent and safer second-generation VPA drug, the urea derivatives of four VPA analogs (2-ethyl-3-methylpentanoyl urea, 2-ethylhexanoyl urea, 2-ethyl-4-methylpentanoyl urea, and 2-methylbutanoyl urea) were synthesized. METHODS: Four CNS-active analogs of a VPA urea derivative testedthe anticonvulsant activity in the maximal electroshock seizure test (MES) and subcutaneous metrazol seizure threshold test (scMet). Teratogenic effects of these compounds were evaluated in NMRI mice susceptible to VPA-induced teratogenicity by comparison with VPA. RESULTS: All four VPA analogs showed superior anticonvulsant activity over VPA. Compared with VPA, which induced neural tube defects (NTDs) in fetuses at 1.8 and 3.6 mmol/kg, the analog derivatives induced no NTDs at any concentration up to 4.8 mmol/kg (except for a single abnormality at 3.6 mmol/kg with 2-ethyl-3-methylpentanoyl urea). Skeletal examination also revealed that the acylurea derivatives induced vertebral and rib abnormalities in fetuses markedly less frequently than VPA. Our results confirmed that the analogue derivatives are significantly less teratogenic than VPA in NMRI mice. CONCLUSIONS: The CNS-active VPA analogs containing a urea moiety, which have better anticonvulsant potency and lack teratogenicity, are good potential candidates as second-generation VPA antiepileptic drugs. Birth Defects Res (Part B) 86:394–401, 2009. © 2009 Wiley-Liss, Inc.