The G glycoprotein of respiratory syncytial virus depresses respiratory rates through the CX3C motif and substance P

Respiratory syncytial virus (RSV) infection in the neonate can alter respiratory rates, i.e., lead to episodes of apnea. We show that RSV G glycoprotein reduces respiratory rates associated with the induction of substance P (SP) and G glycoprotein-CX3CR1 interaction, an effect that is inhibited by treatment with anti-G glycoprotein, anti-SP, or anti-CX3CR1 monoclonal antibodies. These data suggest new approaches for treating some aspects of RSV disease.     

Kinetic mechanisms of IkappaB-related kinases (IKK) inducible IKK and TBK-1 differ from IKK-1/IKK-2 heterodimer

Nuclear factor-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB. The phosphorylation of IkappaBalpha on Ser(32) and Ser(36) is initiated by an IkappaB kinase (IKK) complex that includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK-1) and IkappaB kinase 2 (IKK-2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. Recently, two related IkappaB kinases, TBK-1 and IKK-i, have been described. TBK-1 and IKK-i show sequence and structural homology to IKK-1 and IKK-2. TBK-1 and IKK-i phosphorylate Ser(36) of IkappaBalpha. We describe the kinetic mechanisms in terms of substrate and product inhibition of the recombinant human (rh) proteins, rhTBK-1, rhIKK-I, and rhIKK-1/rhIKK-2 heterodimers. The results indicate that although each of these enzymes exhibits a random sequential kinetic mechanism, the effect of the binding of one substrate on the affinity of the other substrate is significantly different. ATP has no effect on the binding of an IkappaBalpha peptide for the rhIKK-1/rhIKK-2 heterodimer (alpha = 0.99), whereas the binding of ATP decreased the affinity of the IkappaBalpha peptide for both rhTBK-1 (alpha = 10.16) and rhIKK-i (alpha = 62.28). Furthermore, the dissociation constants of ATP for rhTBK-1 and rhIKK-i are between the expected values for kinases, whereas the dissociation constants of the IkappaBalpha peptide for each IKK isoforms is unique with rhTBK-1 being the highest (K(IkappaBalpha) = 69.87 microm), followed by rhIKK-i (K(IkappaBalpha) = 5.47 microm) and rhIKK-1/rhIKK-2 heterodimers (K(IkappaBalpha) = 0.12 microm). Thus this family of IkappaB kinases has very unique kinetic properties.     

Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the beta- and gamma-class

Catalysis of the dehydration of HCO(3)(-) by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the alpha, beta, and gamma classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the alpha class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (beta class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (gamma class). A series of structurally homologous imidazole and pyridine buffers were used as proton acceptors in the activation of CO(2) hydration at steady state and as proton donors of the exchange of (18)O between CO(2) and water at chemical equilibrium. Free energy plots of the rate constants for this intermolecular proton transfer as a function of the difference in pK(a) of donor and acceptor showed extensive curvature, indicating a small intrinsic kinetic barrier for the proton transfers. Application of Marcus rate theory allowed quantitative estimates of the intrinsic kinetic barrier which were near 0.3 kcal/mol with work functions in the range of 7-11 kcal/mol for mutants in the beta and gamma class, similar to results obtained for mutants of carbonic anhydrase in the alpha class. The low values of the intrinsic kinetic barrier for all three classes of carbonic anhydrase reflect proton transfer processes that are consistent with a model of very rapid proton transfer through a flexible matrix of hydrogen-bonded solvent structures sequestered within the active sites of the carbonic anhydrases.     

Density and distribution of water boatmen and brine shrimp at a major shorebird wintering area in Puerto Rico

The Cabo Rojo salt flats are an important wintering area for migratory shorebirds. Their quality is intimately related to prey availability, as prey are needed to meet energetic requirements. Understanding prey dynamics is, therefore, a key element of shorebird conservation plans. To this end, we monitored the density and distribution of water-boatmen (Trichocorixa spp.) and brine shrimp (Artemia spp.) in relation to water salinity from September to November of 1994 and 1995. Salinity ranged from 4 to 292 ppt, and gradients were related to hydrological alterations (e.g., salt extraction) and connection to the ocean. Brine shrimp were restricted to areas of highest salinity ( 106 ppt), whereas water-boatmen to areas of lowest salinity (< 65 ppt). We used aquaria experiments to discern potential mechanisms influencing density and distribution of water boatmen. We focused on this species because its caloric value is similar to the brine shrimp's, but it occurs in areas of lower salinity where shorebirds are less prone to hyperosmotic stress. We hypothesized that areas devoid of water boatmen exceeded their tolerance limit, and that these limits could hamper survival as individuals move among areas. Experiments showed that an increase of 8.5 ± 2.1 ppt, when the base salinity was 40 ppt, induced a 50% mortality rate. From a base salinity of 55 ppt, median survival time decreased curvilinearly across salinity concentrations of 65 to 195 ppt. Median survival was lowest > 100 ppt. Lowering water salinity did not result in osmolal related mortality. Results underscored the sensitivity of water boatmen to high salinity, particularly when the difference in salinity between the source and destination localities widened. Water boatmen density increased in one lagoon as salinity decreased from 65 to 47 ppt. On the basis of our experiments, local adult survivorship improved and immigration and subsequent survival of adults, if any, was not hindered. The density of nymphs also suggested that hatching occurred concurrently. The foraging value of the salt flats can be enhanced by maintaining salinity at < 65 ppt in selected management units and minimizing differences in salinity concentrations among them.     

Influence of somatotropin and nutrition on bovine oocyte retrieval and in vitro development

The objective of this study was to determine the effects of supplemental bovine somatotropin (bST) and limit feeding on follicular growth and oocyte competence in yearling beef heifers. Sixteen growing heifers (424+/-4 kg) were randomly assigned to 1 of 4 treatments in a 2 x 2 factorial arrangement, with main effects of bST (0 or 33 microg/kg BW/d) and feeding regimen (ad libitum or 0.75 ad libitum intake). Animals were treated for 100 d prior to follicular aspiration, and treatments continued for the 42-d period that follicles were aspirated. Follicles were observed ultrasonically then aspirated, and recovered oocytes were matured, fertilized and developed in vitro. The number of follicles observed ultrasonically was greater with bST treatment (P<0.01) but was unchanged by plane of nutrition. The number and quality of recovered oocytes were similar among treatments, as was the number of oocytes resulting in blastocyst formation.     

Developmental competence of juvenile calf oocytes in vitro and in vivo: influence of donor animal variation and repeated gonadotropin stimulation

Juvenile calf oocytes represent an untapped source of germ plasm for reproduction. Reports on the developmental competence of calf oocytes have been controversial. In this research, oocytes were recovered after gonadotropin stimulation from Holstein calves (N = 10) at 2-3 mo of age (2-mo cycle) and again at 4-5 mo of age (4-mo cycle). The in vitro developmental competence was measured, and prestimulation follicle numbers (for 2-mo cycle) and poststimulation follicle numbers (both cycles) were obtained. The number of antral follicles doubled after stimulation (23.4 +/- 6.1 vs. 55.1 +/- 16.1) for the 2-mo cycle and for the 4-mo cycle (47.4 +/- 12.4). The number of follicles observed prior to stimulation in the 2-mo cycle was found to be highly correlated with the poststimulation oocyte recovery for both collection cycles (r = 0.95, 2-mo cycle; r = 0.81, 4-mo cycle). The majority (90-96%) of recovered oocytes were found to be usable for in vitro maturation and fertilization; of these, 41-42% cleaved and 10-11% developed to morulae or blastocysts. Eighty-four in vitro-produced embryos were transferred to synchronized recipients and resulted in 11 pregnancies, leading to 7 live (4 males, 3 females) and 2 dead (one male, one female) calves at full term. No significant differences were observed between the 2-mo and 4-mo collection cycles; however, 73% of the total pregnancies resulted from the 2-mo cycle. All pregnancies resulted from embryos of high-responding donors. The high correlation between the number of follicles prior to stimulation and the poststimulation response suggests the possibility of screening calves prior to stimulation for routine embryo production.     

Respiratory syncytial virus infection and G and/or SH protein expression contribute to substance P, which mediates inflammation and enhanced pulmonary disease in BALB/c mice

A distinct clinical presentation of respiratory syncytial virus (RSV) infection of humans is bronchiolitis, which has clinical features similar to those of asthma. Substance P (SP), a tachykinin neuropeptide, has been associated with neurogenic inflammation and asthma; therefore, we chose to examine SP-induced inflammation with RSV infection. In this study, we examined the production of pulmonary SP associated with RSV infection of BALB/c mice and the effect of anti-SP F(ab)(2) antibodies on the pulmonary inflammatory response. The peak production of pulmonary SP occurred between days 3 and 5 following primary RSV infection and day 1 after secondary infection. Treatment of RSV-infected mice with anti-SP F(ab)(2) antibodies suggested that SP may alter the natural killer cell response to primary and secondary infection. In mice challenged after formalin-inactivated RSV vaccination, SP appears to markedly enhance pulmonary eosinophilia as well as increase polymorphonuclear cell trafficking to the lung. Based on studies with a strain of RSV that lacks the G and SH genes, the SP response to RSV infection appears to be associated with G and/or SH protein expression. These data suggest that SP may be an important contributor to the inflammatory response to RSV infection and that anti-SP F(ab)(2) antibodies might be used to ameliorate RSV-associated disease.