Markers for trans-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells

Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650-6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying trans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.     

Peripheral chemoreflex responsiveness is increased at elevated levels of carbon dioxide after episodic hypoxia in awake humans.

We hypothesized that the acute ventilatory response to hypoxia is enhanced after exposure to episodic hypoxia in awake humans. Eleven subjects completed a series of rebreathing trials before and after exposure to eight 4-min episodes of hypoxia. During the rebreathing trials, subjects initially hyperventilated to reduce the partial pressure of carbon dioxide (Pet(CO(2))) below 25 Torr. Subjects then breathed from a bag containing normocapnic (42 Torr), low (50 Torr), or high oxygen (140 Torr) gas mixtures. During the trials, Pet(CO(2)) increased while a constant oxygen level was maintained. The point at which ventilation began to rise in a linear fashion as Pet(CO(2)) increased was considered to be the ventilatory recruitment threshold. The ventilatory response below and above the recruitment threshold was determined. Ventilation did not persist above baseline values immediately after exposure to episodic hypoxia; however, Pet(CO(2)) levels were reduced compared with baseline. In contrast, compared with baseline, the ventilatory response to progressive increases in carbon dioxide during rebreathing trials in the presence of low but not high oxygen levels was increased after exposure to episodic hypoxia. This increase occurred when carbon dioxide levels were above but not below the ventilatory recruitment threshold. We conclude that long-term facilitation of ventilation (i.e., increases in ventilation that persist when normoxia is restored after episodic hypoxia) is not expressed in awake humans in the presence of hypocapnia. Nevertheless, despite this lack of expression, the acute ventilatory response to hypoxia in the presence of hypercapnia is increased after exposure to episodic hypoxia.     

Enzymic activity of salivary amylase when bound to the surface of oral streptococci

The enzymatic activity of salivary amylase bound to the surface of several species of oral streptococci was determined by the production of acid from starch and by the degradation of maltotetraose to glucose in a coupled, spectrophotometric assay. Most strains able to bind amylase exhibited functional enzyme on their surface and produced acid from the products of amylolytic degradation. These strains were unable to utilise starch in the absence of salivary amylase. Two strains failed to produce acid from starch, despite the presence of functional salivary amylase, because they could not utilise maltose. Strains that could not bind salivary amylase failed to produce acid from starch. In no case was all the bound salivary amylase active, and two strains of Streptococcus mitis which bound amylase did not exhibit any enzyme activity on their cell surface. The ability to bind amylase may confer a survival advantage on oral bacteria which inhabit hosts that consume diets containing starch.     

Effects of urbanization on streams of the Melbourne region, Victoria, Australia. I. Benthic macroinvertebrate communities

1. Macroinvertebrate community composition was assessed in small streams of the Melbourne region to test the effects of (a) urban density (catchment imperviousness 0–51%) and (b) stormwater drainage intensity (comparing the intensively drained metropolitan area with urban areas of the hinterland, which had open drains and some localized stormwater drainage).
2. Hinterland communities separated into two groups of sites correlating strongly with patterns of electrical conductivity (EC), basalt geology and annual rainfall. Community composition varied little in the high-EC, western group (imperviousness 0.2–1.2%), but in the eastern group it was strongly correlated with catchment imperviousness (0–12%), with lower taxon richness in more impervious catchments.
3. Metropolitan communities (imperviousness 1–51%) were all severely degraded, with high abundances of a few tolerant taxa. Community composition was poorly correlated with patterns of geology, rainfall or imperviousness. Differences between metropolitan and hinterland communities were well explained by patterns of biochemical oxygen demand and electrical conductivity, which were postulated to indicate the more efficient transport of pollutants to receiving streams by the metropolitan stormwater drainage system.
4. Degradation of macroinvertebrate community composition was well explained by urban density but intensive urban drainage increased degradation severely at even low urban densities. Quantification of relationships between imperviousness, drainage intensity and stream degradation can better inform the assessment, conservation and restoration of urban streams.     

A generic algorithm for finding restriction sites within DNA sequences

This paper describes a generic algorithm for finding restrictionsites within DNA sequences. The ‘genericity’ ofthe algorithm is made possible through the use of set theory.Basic elements of DNA sequences, i.e. nucleotides (bases), arerepresented in sets, and DNA sequences, whether specific, ambiguousor even protein-coding, are represented as sequences of thosesets. The set intersection operation demonstrates its abilityto perform pattern-matching correctly on various DNA sequences.The performance analysis showed that the degree of complexityof the pattern matching is reduced from exponential to linear.An example is given to show the actual and potential restrictionsites, derived by the generic algorithm, in the DNA sequencetemplate coding for a synthetic calmodulin. Received on October 2, 1990; accepted on December 18, 1990     

Latitudinal variation in population structure of wintering Pacific Black Brant

ABSTRACT.   Latitudinal variation in population structure during the winter has been reported in many migratory birds, but has been documented in few species of waterfowl. Variation in environmental and social conditions at wintering sites can potentially influence the population dynamics of differential migrants. We examined latitudinal variation in sex and age classes of wintering Pacific Black Brant ( Branta bernicla nigricans ). Brant are distributed along a wide latitudinal gradient from Alaska to Mexico during the winter. Accordingly, migration distances for brant using different wintering locations are highly variable and winter settlement patterns are likely associated with a spatially variable food resource. We used resightings of brant banded in southwestern Alaska to examine sex and age ratios of birds wintering at Boundary Bay in British Columbia, and at San Quintin Bay, Ojo de Liebre Lagoon, and San Ignacio Lagoon in Baja California from 1998 to 2000. Sex ratios were similar among wintering locations for adults and were consistent with the mating strategy of geese. The distribution of juveniles varied among wintering areas, with greater proportions of juveniles observed at northern (San Quintin Bay and Ojo de Liebre Lagoon) than at southern (San Ignacio Lagoon) locations in Baja California. We suggest that age-related variation in the winter distribution of Pacific Black Brant is mediated by variation in productivity among individuals at different wintering locations and by social interactions among wintering family groups.     

Synthesis of a tetrasaccharide unit of the capsular polysaccharide of Streptococcus pneumoniae type 9V

In the synthesis of 8-methoxycarbonyloctyl O-(alpha-D-galactopyranosyl)-(1----3)-O-(2-acetamido-2-deoxy-beta-D- mannopyranosyl)-(1----4)-O-(beta-D-glucopyranosyl)-(1----4)-alpha-D- glucopyranoside, which represents a component of the capsular polysaccharide of Streptococcus pneumoniae type 9V, the key step was the coupling of alpha-D-Galp-(1----3)-beta-D-ManpNAc-(1----4)-D-Glc as glycosyl donor with 8-ethoxy-carbonyloctyl 6-O-acetyl-2,3-di-O-benzyl-alpha-D-glucopyranoside as glycosyl acceptor by use of the imidate method. Only the beta-imidate of the trisaccharide could be employed in this glycosidation reaction to give stereoselectively the tetrasaccharide in high yield. The alpha-imidate of the trisaccharide led to hydrolysis of the imidate group.     

Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D

The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover ( Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.     

Cortical microtubules in sweet clover columella cells developed in microgravity

Electron micrographs of columella cells from sweet clover seedlings grown and fixed in microgravity revealed longitudinal and cross sectioned cortical microtubules. This is the first report demonstrating the presence and stability of this network in plants in microgravity.     

Effects of ophiobolin a on ion leakage and hexose uptake by maize roots

Ophiobolin A, a sesterterpene metabolite of Helminthosporium maydis, Nisikado and Miyake, stimulates net leakage of electrolytes and glucose from maize (Zea mays L.) seedling roots. Treatment of the roots with ophiobolin A at a concentration of 10 mug/ml (25 mum) inhibits uptake of 10 mm 2-deoxyglucose by 50% and of 0.5 mm 2-deoxyglucose by 85%. Compartmental analysis of the efflux of 3-O-methylglucose failed to show a similar effect of ophiobolin A on the rate of efflux of hexose. The inhibition of uptake is not reversible by washing. There is no difference in the effects on roots from cytoplasmic male sterile or normal cytoplasm plants, and exposure of carrot (Daucus carota L.) root discs to ophiobolin A also causes inhibition of 2-deoxyglucose uptake by this tissue.