Genetic reevaluation of the role of F-box proteins in cyclin D1 degradation

D-type cyclins play a pivotal role in G(1)-S progression of the cell cycle, and their expression is frequently deregulated in cancer. Cyclin D1 has a half-life of only ~30 min as a result of its ubiquitylation and proteasomal degradation, with various F-box proteins, including Fbxo4, Fbxw8, Skp2, and Fbxo31, having been found to contribute to its ubiquitylation. We have now generated Fbxo4-deficient mice and found no abnormalities in these animals. Cyclin D1 accumulation was thus not observed in Fbxo4(-/-) mouse tissues. The half-life of cyclin D1 in mouse embryonic fibroblasts (MEFs) prepared from Fbxo4(-/-), Fbxw8(-/-), and Fbxo4(-/-); Fbxw8(-/-) mice also did not differ from that in wild-type MEFs. Additional depletion of Skp2 and Fbxo31 in Fbxo4(-/-); Fbxw8(-/-) MEFs by RNA interference did not affect cyclin D1 stability. Although Fbxo31 depletion in MEFs increased cyclin D1 abundance, this effect appeared attributable to upregulation of cyclin D1 mRNA. Furthermore, abrogation of the function of the Skp1-Cul1-F-box protein (SCF) complex or the anaphase-promoting complex/cyclosome (APC/C) complexes did not alter the half-life of cyclin D1, whereas cyclin D1 degradation was dependent largely on proteasome activity. Our genetic analyses thus do not support a role for any of the four F-box proteins examined in cyclin D1 degradation during normal cell cycle progression. They suggest the existence of other ubiquitin ligases that target cyclin D1 for proteolysis.     

IL-17 is a critical component of vaccine-induced protection against lung infection by lipopolysaccharide-heterologous strains of Pseudomonas aeruginosa

In a murine model of acute fatal pneumonia, we previously showed that nasal immunization with a live-attenuated aroA deletant of Pseudomonas aeruginosa strain PAO1 elicited LPS serogroup-specific protection, indicating that opsonic Ab to the LPS O Ag was the most important immune effector. Because P. aeruginosa strain PA14 possesses additional virulence factors, we hypothesized that a live-attenuated vaccine based on PA14 might elicit a broader array of immune effectors. Thus, an aroA deletant of PA14, denoted PA14DeltaaroA, was constructed. PA14DeltaaroA-immunized mice were protected against lethal pneumonia caused not only by the parental strain but also by cytotoxic variants of the O Ag-heterologous P. aeruginosa strains PAO1 and PAO6a,d. Remarkably, serum from PA14DeltaaroA-immunized mice had very low levels of opsonic activity against strain PAO1 and could not passively transfer protection, suggesting that an antibody-independent mechanism was needed for the observed cross-serogroup protection. Compared with control mice, PA14DeltaaroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa DeltaaroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14DeltaaroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1DeltaaroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14DeltaaroA vaccine's protection against lethal pneumonia. These data show that IL-17 plays a critical role in antibody-independent vaccine-induced protection against LPS-heterologous strains of P. aeruginosa in the lung.     

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.     

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.