RELATIONSHIPS OF SEED BANKS TO PLANT DISTRIBUTION PATTERNS IN A FRESHWATER TIDAL WETLAND

Study of seed banks, field seedling emergence, and survival of macrophytes in four zones (steep bank—SB; gentle bank—GB; midbank—MB; high marsh —HM) along transects perpendicular to a stream channel in a freshwater tidal wetland showed that many species are widely distributed. Of the 35 species in the seed bank, 50% were common to all zones; of the 20 species emerging in the field, 77% were observed in all zones. Density of seeds, seedlings, and mature plants of most species, however, varied significantly with habitat. The seed bank of each zone reflected the dominant vegetation of that zone. Most species, even those with high potential for water dispersal, were not evenly distributed. Reciprocal transplants and survival persistence data of dominants corresponded with their habitat preferences. Seed bank densities differed from zone to zone (SB 1,717 m^-2; GB 1,645; MB 2,730; HM 3,620). In all zones the maximum field seedling density was less than the comparable seed bank one (SB 38% less; GB 33%; MB 46%; and HM 10%). These data, coupled with the higher proportion of the total seed bank and total field seedlings occurring in the HM, suggest that the stream channel sites were more stressful early in the growing season than the HM. Because of differential establishment and survival, importance of a species relative to the rest of the vegetation may change with time and occurrence of a species in the vegetation may greatly outweigh its importance in the seed bank or even the seedling stage. Although seeds of annual species were numerous with seven species making up 85% of the seed bank, annual species comprised only about half of the species recorded in the seed bank of each zone. It is not possible at our present level of understanding of seed banks in the freshwater tidal marsh to predict vegetation change. Various combinations of species attributes contribute to the zonation patterns observed in the freshwater tidal wetland.     

Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours

Background

Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.

Methods

100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (10⁵ CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (T_RLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of T_RLU values was determined. External validation was further done using 50 additional CR-GNB.

Results

Predictive accuracies of T_RLU were high for when all antibiotic combinations and species were collectively analyzed (T_RLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (T_RLU = 0.81, UA = 91%), and lowest for AB (T_RLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.

Conclusion

We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.     

Incidence of Neoplasms in Children Born after Influenza Epidemics

Following a recent report that neoplasia of the lymphatic and haematopoietic tissues is commoner than average in children whose mothers have had influenza in pregnancy, the incidence of neoplasms in 1954-68 in children of the Manchester Hospital Region was examined in relation to date of birth. There were no significant differences between cohorts born in different quinquennia. Incidence among children born after six influenza epidemics in 1951-68 was no higher than among other children born in these years. It is concluded that if there is an association between maternal influenza and childhood neoplasia it is probably due to factors such as immunological deficiencies which may predispose independently to both conditions.     

Limited Role for the Bilirubin-Biliverdin Redox Amplification Cycle in the Cellular Antioxidant Protection by Biliverdin Reductase

In mammalian cells, heme is degraded by heme oxygenase to biliverdin, which is then reduced to bilirubin by biliverdin reductase (BVR). Both bile pigments have reducing properties, and bilirubin is now generally considered to be a potent antioxidant, yet it remains unclear how it protects cells against oxidative damage. A presently popular explanation for the antioxidant function of bilirubin is a redox cycle in which bilirubin is oxidized to biliverdin and then recycled by BVR. Here, we reexamined this putative BVR-mediated redox cycle. We observed that lipid peroxidation-mediated oxidation of bilirubin in chloroform, a model of cell membrane-bound bilirubin, did not yield biliverdin, a prerequisite for the putative redox cycle. Similarly, H₂O₂ did not oxidize albumin-bound bilirubin to biliverdin, and in vitro oxidation of albumin or ligandin-bound bilirubin by peroxyl radicals gave modest yields of biliverdin. In addition, decreasing cellular BVR protein and activity in HeLa cells using RNA interference did not alter H₂O₂-mediated cell death, just as BVR overexpression failed to enhance protection of these cells against H₂O₂-mediated damage, irrespective of whether bilirubin or biliverdin were added to the cells as substrate for the putative redox cycle. Similarly, transformation of human BVR into hmx1 (heme oxygenase) mutant yeast did not provide protection against H₂O₂ toxicity above that seen in hmx1 mutant yeast expressing human heme oxygenase-1. Together, these results argue against the BVR-mediated redox cycle playing a general or important role as cellular antioxidant defense mechanism.Biliverdin reductase (BVR)³ forms part of the major pathway for the disposition of cellular heme in mammalian cells. This pathway is initiated by heme oxygenase, which converts heme to carbon monoxide, iron, and biliverdin, which in turn is reduced to bilirubin by BVR at the expense of NADPH. Because of its intramolecular hydrogen bonding, the bilirubin produced is sparingly soluble in water at physiological pH and ionic strength (1). Hence, bilirubin is usually tightly bound to albumin in order to be transported within the blood circulation (2), from which it is removed mainly through uptake by hepatocytes. Once bilirubin is transferred across the cell membrane of hepatocytes, it binds glutathione S-transferases before being transformed to water-soluble derivatives by conjugation of one or both of its propionyl groups before its excretion into bile and then the intestine (3).Under physiological conditions, plasma bilirubin concentrations in humans range from ∼5 to 20 μm, practically all of which is unconjugated pigment bound to albumin (1). Abnormally high plasma concentrations are associated with the risk of developing neurologic dysfunction due to preferential deposition of bilirubin in brain and its toxic effects on cell functions. In fact for many years, biliverdin and bilirubin were generally regarded as waste products of heme metabolism in higher animals, although earlier work suggested that these bile pigments might play a role as natural antioxidants, since small quantities of the pigment stabilize vitamin A and β-carotene during intestinal uptake, and animals with low plasma bilirubin showed early signs of vitamin E deficiency (4, 5).In a series of in vitro studies, Stocker et al. (6–8) demonstrated that unconjugated bilirubin, at micromolar concentrations, efficiently scavenged peroxyl radicals in homogenous solution or multilamellar liposomes. At physiologically relevant oxygen tension, bilirubin surpassed α-tocopherol as an antioxidant in liposomes (8), and it is thought to protect plasma proteins and lipids from many but not all oxidants (9). However, it is less clear whether this antioxidant activity extends to in vivo situations or protection of cells from oxidative stress. Although produced in essentially all cells, the normal range of cellular bilirubin concentrations is unknown. However, it is probably in the low nanomolar range, well below that of established cellular antioxidants, such as glutathione and ascorbate, arguing against bilirubin being an important cellular antioxidant. Nonetheless, in vitro studies with rat neuronal cultures showed that the presence of 10 nm bilirubin in the culture medium protected cells against 10,000-fold higher concentrations of hydrogen peroxide (10). Later, Barañano et al. (11) confirmed such observations in HeLa cells and demonstrated that BVR depletion increased reactive oxygen species (ROS) and cell death. This led to the following proposal of the BVR-amplified redox cycle. While acting as an antioxidant, bilirubin is oxidized to biliverdin that is then reduced back to bilirubin by the ubiquitous and abundant BVR.An important underlying assumption of this amplification cycle is that ROS-mediated bilirubin oxidation in cells is specific and yields substantial if not stoichiometric amounts of biliverdin. Inconsistent with this assumption, however, earlier studies showed that high yields of biliverdin formation are limited to certain oxidants (i.e. peroxyl radicals) and albumin-bound bilirubin. In cells, bilirubin is probably present in membranes, bound to proteins other than albumin, or present in conjugated form. Therefore, we reexamined the putative redox amplification cycle. Our results show that reaction of these forms of bilirubin with 1e- or 2e-oxidants at best generates modest amounts of biliverdin. Furthermore, overexpression of BVR does not protect mammalian or yeast cells from hydrogen peroxide-mediated damage, thereby casting doubt on the importance of the putative BVR redox cycle for cellular antioxidant protection.     

Purification and characterization of the L-fucose transporter

L-Fucose is a monosaccharide present in low levels in the serum. It is, however, a common structural component of glycoproteins. L-Fucose is accumulated in eukaryotic cells by a specific, facilitative diffusion transport system which has been designated the fucose transporter. In this study, purification of the transporter from mouse brain was performed by detergent extraction followed by ion-exchange and reactive dye ligand column chromatography. Purification was followed using a transport assay into reconstituted liposomes. A 111-fold purification with 5% yield was achieved from the crude homogenate. The apparent molecular weight of the protein was 57 kDa. Transport was found to be saturable. The K(m) and V(max) values are estimated at 3 microM and 275 pmol/min/mg, respectively. The tissue distribution of fucose transport was examined in liver, kidney, heart, lung, spleen, brain, muscle, adipose, ovary, pancreas, and thymus. Some fucose transport was found in all tissues examined. Very low levels were observed in the liver relative to all other tissues examined. The only monosaccharide which could inhibit the uptake of L-[5,6-(3)H]fucose was fucose itself.     

Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity

Cullin RING ligases (CRLs) are the largest family of cellular E3 ubiquitin ligases and mediate polyubiquitination of a number of cellular substrates. CRLs are activated via the covalent modification of the cullin protein with the ubiquitin-like protein Nedd8. This results in a conformational change in the cullin carboxy terminus that facilitates the ubiquitin transfer onto the substrate. COP9 signalosome (CSN)-mediated cullin deneddylation is essential for CRL activity in vivo. However, the mechanism through which CSN promotes CRL activity in vivo is currently unclear. In this paper, we provide evidence that cullin deneddylation is not intrinsically coupled to substrate polyubiquitination as part of the CRL activation cycle. Furthermore, inhibiting substrate-receptor autoubiquitination is unlikely to account for the major mechanism through which CSN regulates CRL activity. CSN also did not affect recruitment of the substrate-receptor SPOP to Cul3, suggesting it may not function to facilitate the exchange of Cul3 substrate receptors. Our results indicate that CSN binds preferentially to CRLs in the neddylation-induced, active conformation. Binding of the CSN complex to active CRLs may recruit CSN-associated proteins important for CRL regulation. The deneddylating activity of CSN would subsequently promote its own dissociation to allow progression through the CRL activation cycle.     

Biochemical and cellular effects of inhibiting Nedd8 conjugation

The conjugation of proteins with the ubiquitin-like protein Nedd8 is an essential cellular process and an important anti-cancer therapeutic target. The major known role of Nedd8 is the attachment to and activation of Cullin RING E3 ubiquitin ligases (CRL). The attachment of Nedd8 to its substrates occurs via a process analogous to ubiquitin transfer, involving a Nedd8 E1 activating enzyme and a Nedd8 E2 conjugating enzyme, Ubc12, which transfers Nedd8 onto lysine residues of target proteins. In this study, we utilize dominant-negative Ubc12 (dnUbc12) and the Nedd8 E1 inhibitor MLN4924 to inhibit cellular neddylation. We demonstrate that dnUbc12 functions by depleting cellular Nedd8 concentrations. Inhibition of cellular neddylation leads to rapid accumulation of CRL substrates and an enlarged and flattened morphology in HEK293 cells. Inhibiting Nedd8 conjugation also causes abnormalities in the actin cytoskeleton. This is likely at least partially mediated via accumulation of the small GTPase RhoA, a recently identified CRL substrate. We indeed found that siRNA mediated knockdown of RhoA can reverse the morphological changes observed upon inhibition of cellular neddylation. In conclusion, the Nedd8 pathway plays an important role in regulating the actin cytoskeleton and cellular morphology. Dysfunction of the actin cytoskeleton may contribute to the anti-cancer effect of Nedd8 inhibition.