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.     

The topology of genome-scale metabolic reconstructions unravels independent modules and high network flexibility

The topology of metabolic networks is recognisably modular with modules weakly connected apart from sharing a pool of currency metabolites. Here, we defined modules as sets of reversible reactions isolated from the rest of metabolism by irreversible reactions except for the exchange of currency metabolites. Our approach identifies topologically independent modules under specific conditions associated with different metabolic functions. As case studies, the E.coli iJO1366 and Human Recon 2.2 genome-scale metabolic models were split in 103 and 321 modules respectively, displaying significant correlation patterns in expression data. Finally, we addressed a fundamental question about the metabolic flexibility conferred by reversible reactions: “Of all Directed Topologies (DTs) defined by fixing directions to all reversible reactions, how many are capable of carrying flux through all reactions?”. Enumeration of the DTs for iJO1366 model was performed using an efficient depth-first search algorithm, rejecting infeasible DTs based on mass-imbalanced and loopy flux patterns. We found the direction of 79% of reversible reactions must be defined before all directions in the network can be fixed, granting a high degree of flexibility.     

Exercise increases the release of NAMPT in extracellular vesicles and alters NAD + activity in recipient cells

Aging is associated with a loss of metabolic homeostasis, with cofactors such as nicotinamide adenine dinucleotide (NAD⁺) declining over time. The decrease in NAD⁺ production has been linked to the age‐related loss of circulating extracellular nicotinamide phosphoribosyltransferase (eNAMPT), the rate‐limiting enzyme in the NAD⁺ biosynthetic pathway. eNAMPT is found almost exclusively in extracellular vesicles (EVs), providing a mechanism for the distribution of the enzyme in different tissues. Currently, the physiological cause for the release of eNAMPT is unknown, and how it may be affected by age and physical exercise. Here, we show that release of small EVs into the bloodstream is stimulated following moderate intensity exercise in humans. Exercise also increased the eNAMPT content in EVs, most prominently in young individuals with higher aerobic fitness. Both mature fit and young unfit individuals exhibited a limited increase in EV‐eNAMPT release following exercise, indicating that this mechanism is related to both the age and physical fitness of a person. Notably, unfit mature individuals were unable to increase the release of eNAMPT in EVs after exercise, suggesting that lower fitness levels and aging attenuate this important signalling mechanism in the body. EVs isolated from exercising humans containing eNAMPT were able to alter the abundance of NAD⁺ and SIRT1 activity in recipient cells compared to pre‐exercise EVs, indicating a pathway for inter‐tissue signalling promoted through exercise. Our results suggest a mechanism to limit age‐related NAD⁺ decline, through the systemic delivery of eNAMPT via EVs released during exercise.     

Detecting Invasions of Marine Organisms: Kamptozoan Case Histories

Detecting marine invasions can be challenging, especially for lesser-known taxa, and requires (a) thorough field surveys of the region of interest for members of the taxon, (b) systematic analyses to identify all species found, (c) literature searches for the worldwide distribution of these species and for previous records of the taxon in this region, and (d) application of rigorous criteria to assess whether each species found is native or introduced. We carried out these steps in order to detect and document kamptozoan (entoproct) invasions on the American mid-Atlantic coast. We report on the occurrence of two colonial kamptozoans (Barentsia benedeni, Loxosomatoides laevis) in Chesapeake Bay (Maryland and Virginia, USA). On the American Atlantic coast, B. benedeni had previously only been reported from Massachusetts, although this species has a worldwide distribution in bays and harbors. The genus Loxosomatoides had not previously been reported from North America and L. laevis was known only from India. Since the genus Loxosomatoides was very poorly characterized, we briefly review all four of its species, which differ only slightly from each other. We have also synonymized L. japonicum with L. laevis. We did not find any of the kamptozoan species previously recorded in surveys of Chesapeake Bay and the American Atlantic coast. This is the first detailed consideration of anthropogenic influences on kamptozoan distributions, and we emphasize that most kamptozoan species are cryptogenic pending further investigation.     

Snow depth manipulation and its influence on soil frost and water dynamics in a northern hardwood forest

Climate change will likelyresult in warmer winter temperatures leading toless snowfall in temperate forests. Thesechanges may lead to increases in soil freezingbecause of lack of an insulating snow cover andchanges in soil water dynamics during theimportant snowmelt period. In this study, wemanipulated snow depth by removing snow for twowinters, simulating the late development of thesnowpack as may occur with global warming, toexplore the relationships between snow depth,soil freezing, soil moisture, and infiltration.We established four sites, each with two pairedplots, at the Hubbard Brook Experimental Forest(HBEF) in New Hampshire, U.S.A. and instrumentedall eight plots with soil and snow thermistors,frost tubes, soil moisture probes, and soillysimeters. For two winters, we removed snowfrom the designated treatment plots untilFebruary. Snow in the reference plots wasundisturbed. The treatment winters (1997/1998 and1998/1999) were relatively mild, withtemperatures above the seasonal norm and snowdepths below average. Results show the treatedplots accumulated significantly less snow andhad more extensive soil frost than referenceplots. Snow depth was a strong regulator ofsoil temperature and frost depth at all sites.Soil moisture measured by time domainreflectometry probes and leaching volumescollected in lysimeters were lower in thetreatment plots in March and April compared tothe rest of the year. The ratio of leachatevolumes collected in the treatment plots tothat in the reference plots decreased as thesnow ablation seasons progressed. Our data showthat even mild winters with low snowfall,simulated by snow removal, will result inincreased soil freezing in the forests at theHBEF. Our results suggest that a climate shifttoward less snowfall or a shorter duration ofsnow on the ground will produce increases insoil freezing in northern hardwood forests.Increases in soil freezing will haveimplications for changes in soil biogeochemicalprocesses.