Organ tropism of Sendai virus in mice: proteolytic activation of the fusion glycoprotein in mouse organs and budding site at the bronchial epithelium.

Wild-type Sendai virus is exclusively pneumotropic in mice, while a host range mutant, F1-R, is pantropic. The latter was attributed to structural changes in the fusion (F) glycoprotein, which was cleaved by ubiquitous proteases present in many organs (M. Tashiro, E. Pritzer, M. A. Khoshnan, M. Yamakawa, K. Kuroda, H.-D. Klenk, R. Rott, and J. T. Seto, Virology 165:577-583, 1988). These studies were extended by investigating, by use of an organ block culture system of mice, whether differences exist in the susceptibility of the lung and the other organs to the viruses and in proteolytic activation of the F protein of the viruses. Block cultures of mouse organs were shown to synthesize the viral polypeptides and to support productive infections by the viruses. These findings ruled out the possibility that pneumotropism of wild-type virus results because only the respiratory organs are susceptible to the virus. Progeny virus of F1-R was produced in the activated form as shown by infectivity assays and proteolytic cleavage of the F protein in the infected organ cultures. On the other hand, much of wild-type virus produced in cultures of organs other than lung remained nonactivated. The findings indicate that the F protein of wild-type virus was poorly activated by ubiquitous proteases which efficiently activated the F protein of F1-R. Thus, the activating protease for wild-type F protein is present only in the respiratory organs. These results, taken together with a comparison of the predicted amino acid substitutions between the viruses, strongly suggest that the different efficiencies among mouse organs in the proteolytic activation of F protein must be the primary determinant for organ tropism of Sendai virus. Additionally, immunoelectron microscopic examination of the mouse bronchus indicated that the budding site of wild-type virus was restricted to the apical domain of the epithelium, whereas budding by F1-R occurred at the apical and basal domains. Bipolar budding was also observed in MDCK monolayers infected with F1-R. The differential budding site at the primary target of infection may be an additional determinant for organ tropism of Sendai virus in mice.     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

Prebiotic synthesis of diaminopyrimidine and thiocytosine

The reaction of guanidine hydrochloride with cyanoacetaldehyde gives high yields (40–85%) of 2,4-diaminopyrimidine under the concentrated conditions of a drying lagoon model of prebiotic synthesis, in contrast to the low yields previously obtained under more dilute conditions. The prebiotic source of cyanoacetaldehyde, cyanoacetylene, is produced from electric discharges under reducing conditions. The effect of pH and concentration of guanidine hydrochloride on the rate of synthesis and yield of diaminopyrimidine were investigated, as well as the hydrolysis of diaminopyrimidine to cytosine, isocytosine, and uracil. Thiourea also reacts with cyanoacetaldehyde to give 2-thiocytosine, but the pyrimidine yields are much lower than with guanidine hydrochloride or urea. Thiocytosine hydrolyzes to thiouracil and cytosine and then to uracil. This synthesis would have been a significant prebiotic source of 2-thiopyrimidines and 5-substituted derivatives of thiouracil, many of which occur in tRNA. The applicability of these results to the drying lagoon model of prebiotic synthesis was tested by dry-down experiments where dilute solutions of cyanoacetaldehyde, guanidine hydrochloride, and 0.5m NaCl were evaporated over varying periods of time. The yields of diaminopyrimidine varied from 1 to 7%. These results show that drying lagoons and beaches may have been major sites of prebiotic syntheses.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

Involvement of the mutated M protein in altered budding polarity of a pantropic mutant, F1-R, of Sendai virus.

Wild-type Sendai virus buds at the apical plasma membrane domain of polarized epithelial MDCK cells, whereas a pantropic mutant, F1-R, buds at both the apical and basolateral domains. In F1-R-infected cells, polarized protein transport and the microtubule network are impaired. It has been suggested that the mutated F and/or M proteins in F1-R are responsible for these changes (M. Tashiro, J. T. Seto, H.-D. Klenk, and R. Rott, J. Virol. 67:5902-5910, 1993). To clarify which gene or mutation(s) was responsible for the microtubule disruption which leads to altered budding of F1-R, MDCK cell lines containing the M gene of either the wild type or F1-R were established. When wild-type M protein was expressed at a level corresponding to that synthesized in virus-infected cells, cellular polarity and the integrity of the microtubules were affected to some extent. On the other hand, expression of the mutated F1-R M protein resulted in the formation of giant cells about 40 times larger than normal MDCK cells. Under these conditions, the effects on the microtubule network were enhanced. The microtubules were disrupted and polarized protein transport was impaired as indicated by the nonpolarized secretion of gp80, a host cell glycoprotein normally secreted from the apical domain, and bipolar budding of wild-type and F1-R Sendai viruses. The mutated F glycoprotein of F1-R was transported bipolarly in cells expressing the F1-R M protein, whereas it was transported predominantly to the apical domain when expressed alone or in cells coexpressing the wild-type M protein. These findings indicate that the M protein of F1-R is involved in the disruption of the microtubular network, leading to impairment of cellular polarity, bipolar transport of the F glycoprotein, and bipolar budding of the virus.     

Inbreeding and incompatibility in Trichogramma nr. brassicae: evidence and implications for quality control

Inbreeding effects and incompatibility relationships were examined in strains of the egg parasitoid Trichogramma nr brassicae (Hymenoptera: Trichogrammatidae) from southeastern Australia. Crosses between strains provided weak evidence of incompatibility in a few cases. However sex ratio in crosses within strains tended to be more female-biased than in crosses between strains. Inbreeding was imposed for four generations (F>0.59) of sib mating. The fitness of inbred strains was compared to that of outbred strains generated by crossing the inbred strains. No effects of inbreeding were found for any of the four female traits examined (fecundity, body length, head width and hind tibia length), indicating that T. nr. brassicae is not subjected to inbreeding depression. Inbreeding effects were also not found for male mating success as expected for the haploid sex. There were differences among strains for all traits apart from fecundity, indicating heritable variation. Strain differences for fitness measures were uncorrelated with wasp size. The potential use of inbreeding in the quality control of Trichogramma for mass-release is discussed. Inbreeding may be a useful tool in minimising the effects of laboratory adaptation, thereby extending the useful life of a strain.     

LIFE-HISTORY ADAPTATION AND REPRODUCTIVE ISOLATION IN A GRASSHOPPER HYBRID ZONE

Patterns of life-history adaptation and reproductive isolation were investigated in the acridid grasshoppers Melanoplus sanguinipes and M. devastator, which hybridize along an altitudinal gradient in the Sierra Nevada of California. Melanoplus sanguinipes females crossed with M. devastator males produced eggs that were approximately half as viable as eggs from other crosses. Diminished viability was not attributable either to infection by Wolbachia pipientis or to failure of sperm transfer. When offered an opportunity to choose a mate, females from all populations discriminated against males of the other species, whereas in no-choice tests measuring copulation duration only females from the tails of the clines showed preferences. Melanoplus sanguinipes, found at high elevations where the growing season is short, exhibited faster egg hatch, faster larval development, smaller adult body sizes, and smaller clutch sizes than M. devastator. Melanoplus devastator, from California's Central Valley, endured a hot and dry summer in a reproductive diapause that was absent in M. sanguinipes. Clines in reproductive diapause and clutch size coincided with the region of reproductive incompatibility. Development time, body size, and hatch time also changed across the hybrid zone, but the regions of largest transitions in these traits were either difficult to locate using the limited populations studied here or were not coincident with the zone's center. A method is described for combining ecological and phylogenetic analyses to address the unknown issue of whether life-history divergence has conributed to reproductive isolation in this system.     

Computed three-dimensional structures for theras-binding domain of theraf-p74 protein complexed withras-p21 and with its suppressor protein, rap-1A

The three-dimensional structures of theras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to theras-binding domain, RBD (residues 55–131), of theraf-p74 protein, a critical target protein ofras-p21 in theras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of bothras- p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of theras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains ofras-p21 and rap-1a, including residues 32–47, a domain that directly interacts with RBD, 60–66, 96–110, involved in the interaction ofras-p21 withjun kinase (JNK) andjun protein, and 115–126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its-2 binding loop (residues 63–71) and residues 89–91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK andjun.     

Water relations and leaf chemistry of Chrysothamnus nauseosus ssp. consimilis (Asteraceae) and Sarcobatus vermiculatus (Chenopodiaceae)

At Mono Lake, California, we investigated field water relations, leaf and xylem chemistry, and gas exchange for two shrub species that commonly co-occur on marginally saline soils, and have similar life histories and rooting patterns. Both species had highest root length densities close to the surface and have large tap roots that probably reach ground water at 3.4-5.0 m on the study site. The species differed greatly in leaf water relations and leaf chemistry. Sarcobatus vermiculatus had a seasonal minimum predawn xylem pressure potential (ψ_pd) of -2.7 MPa and a midday potential (ψ_md) of -4.1 MPa. These were significantly lower than for Chrysothamnus nauseosus, which had a minimum ψ_pd of -1.0 MPa and ψ_md of -2.2 MPa. Sarcobatus had leaf Na of up to 9.1 % and K up to 2.7 % of dry mass, and these were significantly higher than for Chrysothamnus which had seasonal maxima of 0.4% leaf Na and 2.4 % leaf K. The molar ratios of leaf K/Na, Ca/Na, and Mg/Na were substantially lower for Sarcobatus than for Chrysothamnus. Xylem ionic contents indicated that both species excluded some Na at the root, but that Chrysothamnus was excluding much more than Sarcobatus. The higher Na content of Sarcobatus leaves was associated with greater leaf succulence, lower calculated osmotic potential, and lower xylem pressure potentials. Despite large differences in water relations and leaf chemistry, these species maintained similar diurnal patterns and rates of photosynthesis and stomatal conductance to water vapor diffusion. Sarcobatus ψ_pd may not reflect soil moisture availability due to root osmotic and hydraulic properties.     

Effects of chlorogenic acid-and tomatine-fed caterpillars on the behavior of an insect predator

If generalist insect predators are a selective force contributing to patterns of feeding specialization by insect herbivores, then predators should be deterred from eating allelochemical-fed prey. The attack and feeding behaviors of naive predators (Podisus maculiventris stinkbugs) reared on control caterpillars (Manduca sexta) fed plain diet were compared to experienced predators reared on caterpillars fed tomato allelochemicals. Tomatine-fed prey were found more quickly by both naive and tomatine-experienced predators, and chlorogenic acid-experienced predators were more stimulated to begin searching for prey. However, experienced predators were less likely to attack both chlorogenic acidfed and tomatine-fed caterpillars than were naive predators. These results indicate that allelochemical-fed prey were easier for predators to locate, but allelochemical-containing prey often deterred predation by experienced predtors.