Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor

Addition of elicitor, cell wall fragments of the fungus Phytophthora parasitica, to tobacco cell suspension cultures (Nicotiana tabacum) resulted in the rapid synthesis and secretion of large amounts of antibiotic sesquiterpenoids. Pulse-labeling experiments with [¹⁴C]acetate and [³H] mevalonate demonstrated that the induction of sesquiterpenoid biosynthesis, maximal by 6 to 9 hours after elicitor addition to the cell cultures, was paralleled by a rapid and large decline in the incorporation rate of radioactivity into sterols. Consequently, sterol accumulation was also inhibited upon addition of elicitor to the cell cultures. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 10 hours of elicitor addition to the cell cultures. The cyclase activity remained elevated for an additional 30 hours before declining. In contrast, squalene synthetase activity was suppressed to less than 15% of that found in control cells within 7 hours of elicitor addition. Our results suggest that the channeling of isoprenoid intermediates, and especially farnesyl diphosphate, into sesquiterpenoids occurred by a coordinated increase in the sesquiterpene cyclase and a decrease in the squalene synthetase enzyme activities. A reexamination of the data pertaining to the transient induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (EC 1.1.1.34) in elicitor-treated cells suggested that, while the reductase activity was necessary for sesquiterpenoid biosynthesis, it functioned more to maintain a sufficient level of intermediates between mevalonate and farnesyl diphosphate rather than as a rate limiting step controlling the synthesis rate of any one class of isoprenoids.     

Characterization by high performance liquid chromatography of angiotensin peptides in the plasma and cerebrospinal fluid of the dog

A high performance liquid chromatography (HPLC) method is described for the separation of angiotensin (Ang) peptides and their subsequent quantification by radioimmunoassay in plasma and cerebrospinal fluid (CSF). The use of the ion-pair solvent heptafluorobutyric acid in gradient HPLC achieves baseline resolution of Ang I, Ang II, and the C-terminal fragments des-[Asp1]-Ang I, des-[Asp1]-Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in approximately 25 min. Recovery of synthetic Ang standards after phenylsilica extraction and HPLC separation was greater than 70% for each peptide in both plasma and CSF. Ang I and Ang II were shown to be the major immunoreactive Ang components in plasma, and Ang II, des-[Asp1,Arg2]-Ang II and des-[Asp1,Arg2,Val3]-Ang II in CSF.     

ABSOLUTE CONFIGURATION ANALYSIS OF THE FLAGELLAR APPARATUS IN CLADOPHORA AND CHAETOMORPHA MOTILE CELLS,WITH AN ASSESSMENT OF THE PHYLOGENETIC POSITION OF THE CLADOPHORACEAE (ULVOPHYCEAE,CHLOROPHYTA)

Absolute configurational analyses of flagellar apparatus components were performed on the motile cells produced by three species of Cladophora, Cl. dalmatica Kütz., Cl. flexuosa (Dillw.) Harv., and Cl. glomerata (L.) Kütz., and by Chaetomorpha aerea (Dillw.) Kütz. There was little variation among the species. All of the flagellar apparatuses demonstrated the ulvophyceous features of 180° rotational symmetry, counterclockwise absolute orientation, and basal body overlap, as well as the alignment of the basal bodies perpendicular to the long axis of the cell. Diagnostic features included the nearly complete absence of C tubules from the basal bodies and the presence of a coarsely striated component dorsal to the two-membered rootlets in all cells, as well as, in quadriflagellate cells, a tetralobate distal fiber, the coaxial arrangement of the lowermost pair of basal bodies, and the presence of a characteristic array of basal-body-associated striated bands. The distal fiber architecture, the presence of a “wing” in the X-membered rootlets, and the “flattening” of the flagellar apparatus components suggests a close relationship of the Cladophoraceae to the Dasycladales, and indicates that these two groups may have shared a common ancestor, possibly ancient in terms of the geological time scale but relatively recent in the context of ulvophyceous evolution. A sizeable phylogenetic gap exists between the Cladophoraceae and uninucleate-celled, presumably primitive members of the Ulvophyceae.     

Global identifiability of the parameters of nonlinear systems with specified inputs: a comparison of methods

The two methods available for analyzing the global structural identifiability of the parameters of a nonlinear system with a specified input function, the Taylor series approach and the similarity transformation approach, are compared and contrasted through application to three examples. It is shown that, as for linear systems, it is very difficult to predict which of the available methods will result in the least effort for a particular example. The role of modern symbolic manipulation packages in the analysis is assessed. The third example proves intractable using the similarity transformation approach as originally formulated, but the analysis is completed using a reformulation that exploits the polynominal form of the system equations in the example.     

Spotted fever group rickettsiae in immature and adult ticks (Acari: Ixodidae) from a focus of Rocky Mountain spotted fever in Connecticut

Immature and adult ixodid ticks were collected during 1983 and 1984 in Newtown, Connecticut, an area endemic for Rocky Mountain spotted fever (RMSF), to determine prevalence of infection by spotted fever group (SFG) rickettsiae. Direct fluorescent-antibody (FA) staining revealed SFG organisms in 6 (1.8%) of 332 Dermacentor variabilis larvae, 5 (7.8%) of 64 D. variabilis nymphs, and in 2 (40%) of 5 Ixodes cookei nymphs removed from small- and medium-sized mammals. Hemolymph tests detected rickettsia-like organisms in 15 (8.8%) of 170 D. variabilis adults; 8 specimens retested by direct FA were negative. In contrast, hemocytes from 5 (8.6%) of 58 Ixodes texanus females contained organisms that stained positively in both hemolymph and direct FA tests. An indirect microimmunofluorescence test identified specific antibodies to Rickettsia rickettsii, the etiologic agent of RMSF, in serum samples from a chipmunk, raccoons, and white-footed mice. Results indicate that immature or adult ticks of at least three species may be involved in the maintenance and transmission of SFG rickettsiae at Newtown.     

Dynamics of skate horizontal cells

The all-rod retina of the skate (Raja erinacea or R. oscellata) is known to have the remarkable capability of responding to incremental flashes superimposed on background intensities that initially block all light-evoked responses and are well above the level at which rods saturate in mixed rod/cone retinas. To examine further the unusual properties of the skate visual system, we have analyzed responses of their horizontal cells to intensity-modulated step, sinusoidal, and white-noise stimuli. We found that during exposures to mean intensities bright enough to block responses to incremental stimuli, decremental stimuli were also initially blocked. Thereafter, the horizontal cells underwent a slow recovery phase during which there was marked nonlinearity in their response properties. The cell first (within 2-3 min) responded to decrements in intensity and later (after greater than 10 min) became responsive to incremental stimuli. After adaptation to a steady state, however, the responses to intensity modulation were nearly linear over a broad range of modulation depths even at the brightest mean levels of illumination. Indeed, examination of the steady-state responses over a 5-log-unit range of mean intensities revealed that the amplitude of the white noise-evoked responses depended solely on contrast, and was independent of the retinal irradiance as the latter was increased from 0.02 to 20 muW/cm2; i.e., contrast sensitivity remained unchanged over this 1,000-fold increase in mean irradiance. A decrement from the mean as brief as 2 s, however, disturbed the steady state. Another unexpected finding in this all-rod retina concerns surround-enhancement, a phenomenon observed previously for cone-mediated responses of horizontal cells in the retinas of turtle and catfish. While exposure to annular illumination induced response compression and a pronounced sensitivity loss in response to incremental light flashes delivered to the dark central region, the cell's sensitivity showed a significant increase when tested with a white noise or sinusoidally modulated central spot. Unlike horizontal cells in other retinas studied thus far, however, response dynamics remained unchanged. Responses evoked either by a small spot (0.25-mm diam) or by a large field light covering the entire retina were almost identical in time course. This is in contrast with past findings from cone-driven horizontal cells whose response waveform (dynamics) was dependent upon the size of the retinal area stimulated.     

Infectious subvirion particles of reovirus type 3 Dearing exhibit a loss in infectivity and contain a cleaved sigma 1 protein.

Mammalian reoviruses exhibit differences in the capacity to grow in intestinal tissue: reovirus type 1 Lang (T1L), but not type 3 Dearing (T3D), can be recovered in high titer from intestinal tissue of newborn mice after oral inoculation. We investigated whether in vitro protease treatment of virions of T1L and T3D, using conditions to generate infectious subvirion particles (ISVPs) as occurs in the intestinal lumen of mice (D. K. Bodkin, M. L. Nibert, and B. N. Fields, J. Virol. 63:4676-4681, 1989), affects viral infectivity. Chymotrypsin treatment of T1L was associated with a 2-fold increase in viral infectivity, whereas identical treatment of T3D resulted in a 10-fold decrease in infectivity. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we found that loss of T3D infectivity was correlated with cleavage of its sigma 1 protein. We used reassortant viruses to identify viral determinants of infectivity loss and sigma 1 cleavage and found that both phenotypes segregate with the sigma 1-encoding S1 gene. Comparable results were obtained when trypsin treatment of virions of T1L and T3D was used. In experiments to determine the fate of sigma 1 fragments following cleavage, the capacity of anti-sigma 1 monoclonal antibody G5 to neutralize infectivity of T3D ISVPs was significantly decreased in comparison with its capacity to neutralize infectivity of virions, suggesting that a sigma 1 domain bound by G5 is lost from viral particles after proteolytic digestion. In contrast to the decrease in infectivity, chymotrypsin treatment of T3D virions leading to generation of ISVPs resulted in a 10-fold increase in their capacity to produce hemagglutination, indicating that a domain of sigma 1 important for binding to sialic acid remains associated with viral particles after sigma 1 cleavage. Neuraminidase treatment of L cells substantially decreased the yield of T3D ISVPs in comparison with the yield of virions, indicating that a sigma 1 domain important for binding sialic acid also can mediate attachment of T3D ISVPs to L cells and lead to productive infection. These results suggest that cleavage of T3D sigma 1 protein following oral inoculation of newborn mice is at least partly responsible for the decreased growth of T3D in the intestine and provide additional evidence that T3D sigma 1 contains more than a single receptor-binding domain.     

Amplitude and frequency modulation of pulsatile luteinizing hormone-releasing hormone release

Summary 1. A variety of neuroendocrine approaches has been used to characterize cellular mechanisms governing luteinizing hormone-releasing hormone (LHRH) pulse generation. We review recentin vivo microdialysis,in vitro superfusion, andin situ hybridization experiments in which we tested the hypothesis that the amplitude and frequency of LHRH pulses are subject to independent regulation via distinct and identifiable cellular pathways.2. Augmentation of LHRH pulse amplitude is proposed as a central feature of preovulatory LHRH surges. Three mechanisms are described which may contribute to this increase in LHRH pulse amplitude: (a) increased LHRH gene expression, (b) augmentation of facilitatory neurotransmission, and (c) increased responsiveness of LHRH neurons to afferent synaptic signals. Neuropeptide Y (NPY) is examined as a prototypical afferent transmitter regulating the generation of LHRH surges through the latter two mechanisms.3. Retardation of LHRH pulse generator frequency is postulated to mediate negative feedback actions of gonadal hormones. Evidence supporting this hypothesis is reviewed, including results ofin vivo monitoring experiments in which LHRH pulse frequency, but not amplitude, is shown to be increased following castration. A role for noradrenergic neurons as intervening targets of gonadal hormone negative feedback actions is discussed.4. Future directions for study of the LHRH pulse generator are suggested.     

Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.