A novel function for Sam68: enhancement of HIV-1 RNA 3' end processing

Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3' splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3' end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3' end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3' end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression.     

Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition

MEK1 and MEK2 are closely related, dual-specificity tyrosine/threonine protein kinases found in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway. Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK1 results in cellular transformation. Here we present the X-ray structures of human MEK1 and MEK2, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 A and 3.2 A, respectively. The structures reveal that MEK1 and MEK2 each have a unique inhibitor-binding pocket adjacent to the MgATP-binding site. The presence of the potent inhibitor induces several conformational changes in the unphosphorylated MEK1 and MEK2 enzymes that lock them into a closed but catalytically inactive species. Thus, the structures reported here reveal a novel, noncompetitive mechanism for protein kinase inhibition.     

Natal homing in juvenile loggerhead turtles (Caretta caretta)

Juvenile loggerhead turtles (Caretta caretta) from West Atlantic nesting beaches occupy oceanic (pelagic) habitats in the eastern Atlantic and Mediterranean, whereas larger juvenile turtles occupy shallow (neritic) habitats along the continental coastline of North America. Hence the switch from oceanic to neritic stage can involve a trans-oceanic migration. Several researchers have suggested that at the end of the oceanic phase, juveniles are homing to feeding habitats in the vicinity of their natal rookery. To test the hypothesis of juvenile homing behaviour, we surveyed 10 juvenile feeding zones across the eastern USA with mitochondrial DNA control region sequences (N = 1437) and compared these samples to potential source (nesting) populations in the Atlantic Ocean and Mediterranean Sea (N = 465). The results indicated a shallow, but significant, population structure of neritic juveniles (PhiST = 0.0088, P = 0.016), and haplotype frequency differences were significantly correlated between coastal feeding populations and adjacent nesting populations (Mantel test R2 = 0.52, P = 0.001). Mixed stock analyses (using a Bayesian algorithm) indicated that juveniles occurred at elevated frequency in the vicinity of their natal rookery. Hence, all lines of evidence supported the hypothesis of juvenile homing in loggerhead turtles. While not as precise as the homing of breeding adults, this behaviour nonetheless places juvenile turtles in the vicinity of their natal nesting colonies. Some of the coastal hazards that affect declining nesting populations may also affect the next generation of turtles feeding in nearby habitats.     

Microampullary organs of a freshwater eel-tailed catfish,Plotosus (tandanus) tandanus

Whole body studies of Plotosus tandanus revealed that ampullary pores occur over the entire body of the fish, but are in higher concentrations in the head region. These pores give rise to a short canal (50-60 microm) produced by columnar epithelial cells bound together by tight junctions and desmosomes. At the junction of the canal and the ampulla, cuboidal epithelial cells make up the wall. The ampulla consists of layers of collagen fibers that surround flattened epithelial cells in the lateral regions and give rise to supportive cells that encase a small number of receptor cells (10-15). The ampullary wall comprises several types of cells that are adjoined via tight junctions and desmosomes between cell types. The ovoid receptor cells possess microvilli along the luminar apical area. Beneath this area, the cells are rich in mitochondria and rough endoplasmic reticulum. An unmyelinated neuron adjoins with each receptor cell opposite multiple presynaptic bodies. This form of microampulla has not been previously described within the Family Plotosidae.     

17 beta-Estradiol inhibits angiotensin II activation of area postrema neurons

It is well established that the area postrema, as a circumventricular organ, is susceptible to modulation by circulating hormones and peptides. Furthermore, activation of the area postrema has been shown to modulate central neurons involved in the regulation of cardiovascular function and blood pressure. In particular, the vasoactive peptide angiotensin II (ANG II) has been shown to inhibit baroreflex regulation of heart rate and increase sympathetic outflow and blood pressure via activation of area postrema neurons. Estrogen is thought to protect against hypertension in both humans and animal models and has been shown in a number of systems to alter the effects of ANG II. The purpose of the present study was to determine the effects of estrogen on ANG II activation of area postrema neurons. In this study, the effects of ANG II and KCl on fura 2-measured cytosolic Ca2+ concentration ([Ca2+]i) responses in cultured area postrema neurons in the presence and absence of 12-h exposure to 100 nM 17 beta-estradiol (E2) were evaluated. In neurons incubated in control vehicle media, 50 nM ANG II increased [Ca2+]i by 92 +/- 12%. In neurons preincubated with 100 nM E2, ANG II increased [Ca2+]i by only 68 +/- 11%, for a total inhibition of the ANG II-evoked response of 24%. Coapplication of the estrogen receptor antagonist ICI-182,780 did not inhibit the effects of E2. In the same cells in which the effects of E2 on ANG II-evoked responses were tested, the effects of incubation in E on the depolarization-induced increased [Ca2+2]i due to 60 mM KCl were also tested. Incubation of the cells with 100 nM E increased the KCl-evoked [Ca2+2]i response, and this response was blocked by ICI-182,780. These results suggest that in the area postrema, estrogen may utilize multiple pathways to modulate neural activity and responses to ANG II.     

Tumor necrosis factor regulates intestinal epithelial cell migration by receptor-dependent mechanisms

Altered mucosal integrity andincreased cytokine production, including tumor necrosis factor (TNF),are the hallmarks of inflammatory bowel disease (IBD). In this study,we addressed the role of TNF receptors (TNFR) on intestinal epithelialcell migration in an in vitro wound closure model. With mouse TNFR1 orTNFR2 knockout intestinal epithelial cells, gene transfection, andpharmacological inhibitors, we show a concentration-dependentreceptor-mediated regulation of intestinal cell migration by TNF. Aphysiological TNF level (1 ng/ml) enhances migration through TNFR2,whereas a pathological level (100 ng/ml) inhibits wound closure through TNFR1. Increased rate of wound closure by TNFR2 or inhibition by TNFR1cannot be explained by either increased proliferation orapoptosis, respectively. Furthermore, inhibiting Src tyrosine kinase decreases TNF-induced focal adhesion kinase (FAK) tyrosine phosphorylation and cellular migration. We therefore conclude thatTNFR2 activates a novel Src-regulated pathway involving FAK tyrosinephosphorylation that enhances migration of intestinal epithelial cells.

     

Vocal clans in sperm whales (Physeter macrocephalus)

Cultural transmission may be a significant source of variation in the behaviour of whales and dolphins, especially as regards their vocal signals. We studied variation in the vocal output of 'codas' by sperm whale social groups. Codas are patterns of clicks used by female sperm whales in social circumstances. The coda repertoires of all known social units (n = 18, each consisting of about 11 females and immatures with long-term relationships) and 61 out of 64 groups (about two social units moving together for periods of days) that were recorded in the South Pacific and Caribbean between 1985 and 2000 can be reliably allocated into six acoustic 'clans', five in the Pacific and one in the Caribbean. Clans have ranges that span thousands of kilometres, are sympatric, contain many thousands of whales and most probably result from cultural transmission of vocal patterns. Units seem to form groups preferentially with other units of their own clan. We suggest that this is a rare example of sympatric cultural variation on an oceanic scale. Culture may thus be a more important determinant of sperm whale population structure than genes or geography, a finding that has major implications for our understanding of the species' behavioural and population biology.     

A live,attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3' untranslated region is highly attenuated and immunogenic in monkeys

The Delta30 deletion mutation, which was originally created in dengue virus type 4 (DEN4) by the removal of nucleotides 172 to 143 from the 3' untranslated region (3' UTR), was introduced into a homologous region of wild-type (wt) dengue virus type 1 (DEN1). The resulting virus, rDEN1Delta30, was attenuated in rhesus monkeys to a level similar to that of the rDEN4Delta30 vaccine candidate. rDEN1Delta30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rDEN1mutF, which also contains mutations in the 3' UTR, and both vaccines were highly protective against challenge with wt DEN1. Both rDEN1Delta30 and rDEN1mutF were also attenuated in HuH-7-SCID mice. However, neither rDEN1Delta30 nor rDEN1mutF showed restricted replication following intrathoracic inoculation in the mosquito Toxorhynchites splendens. The ability of the Delta30 mutation to attenuate both DEN1 and DEN4 viruses suggests that a tetravalent DEN vaccine could be generated by introduction of the Delta30 mutation into wt DEN viruses belonging to each of the four serotypes.     

Interaction between human respiratory syncytial virus (RSV) M2-1 and P proteins is required for reconstitution of M2-1-dependent RSV minigenome activity

We have investigated protein-protein interactions among the respiratory syncytial virus (RSV) RNA polymerase subunits using affinity chromatography. Here we demonstrate a novel interaction of P and M2-1 proteins. Phosphorylation of either M2-1 or P appears to be dispensable for this interaction. Internal deletions within P mapped the M2-1-binding domain to a region between residues 100 and 120. Alanine-scanning mutagenesis within this region of P revealed that substitution of any one of the three residues, L101, Y102, and F109, prevented both M2-1 and P binding and expression of an M2-1-dependent luciferase reporter gene. However, these same mutations did not prevent the activity of an M2-1-independent chloramphenicol acetyltransferase minigenome, suggesting that these residues of P specifically affect M2-1-P interaction. On the basis of these observations, it is possible that the interaction between RSV M2-1 and P proteins is important for viral replication.