The interaction of nucleotides and yeast hexokinase

The kinetics of ethenoadenosine triphosphate (?ATP) as the phosphate donor in the phosphoryl transfer reaction of hexokinase were examined to obtain the K_m′s, V's, and K_α's for the nucleotide and sugar. Dissociation constants for eATP and ?ADP with hexokinase were obtained from fluorometric measurements and compared with similar constants obtained kinetically. Other selected nucleoside triphosphates were used as phosphate donors in the hexokinase reaction and their kinetic constants were obtained. Reactions were also performed using two nucleotides simultaneously as phosphorylating substrates for the hexokinase reaction in an attempt to find the individual dissociation constants, K_m′s and K_i′s. These were compared with the K_m′s obtained from using the nucleotides separately in the hexokinase reaction. From these kinetic and fluorescence binding studies, evidence is presented supporting the postulate that the K_m′s are primarily dissociation constants in a random bi-bi mechanism. Analysis of the K_m values provides additional evidence to support the importance of the amino group in position 6 on the purine ring as a hydrogen-bond acceptor during binding. It was found that ?CTP was a much better hexokinase substrate than CTP. These observations suggest that the V for this reaction is highly dependent upon the size of the nucleotide.     

Heterospecific pollen transfer between sympatric species in a midsuccessional old-field community

The cumulative (season-long) incidence of heterospecific pollen transfer (HPT) was examined using nine sympatric species in a midsuccessional old field. Inflorescences were collected weekly during the flowering season, and the proportion of foreign pollen/stigma was recorded. Flowering phenologies of sympatric species and ovule and seed counts of study species were also recorded. Heterospecific pollen was detected on some stigmas of each species. Medicago sativa (Fabaceae) received the most foreign pollen; in some cases, all of the grains on a stigma were heterospecific. Lotus corniculatus (Fabaceae) received the least amount of foreign pollen; the incidence of heterospecific pollen was near zero in most cases. The mean and range of foreign pollen received varied by as much as an order of magnitude between species. The six species with zygomorphic flowers, all Fabaceae, received more heterospecific pollen than the three species with actinomorphic flowers, Potentilla recta and P. simplex (Rosaceae) and Ranunculus acris (Ranunculaceae). This probably reflects a bias because our data were analyzed on a cumulative basis and the Fabaceae had longer flowering phenologies. HPT was not correlated with the species' relative abundance within the community. Proportion of foreign pollen received varied temporally within species, and this variation generally was not related to phenology of any sympatric taxa or the species' own phenology. Pollen grain diameter was positively related to levels of foreign pollen received by species. This might be caused by poor adhesion of large pollen grains to small stigmatic papillae or if generalist pollinators carrying large amounts of heterospecific pollen visit the large-grained species and specialists with little foreign pollen visit the small-grained species. The large proportions of heterospecific pollen on stigmas of many species indicate that HPT occurs frequently in the community we studied and the implications may include reduced seed set because of occlusion by foreign grains. As yet, however, it is unclear how important a factor HPT is in mediating pollen limitation of reproductive success.     

24-methylenepollinastanol and 24-dehydropollinastanol—two sterols of pollen

The esterified and unesterified sterol fractions of bee-gathered mixed pollens were examined, and total sterol composition was determined. Two new sterols of pollens, 14α-methyl-9β,19-cyclo-5α-cholest-24-en-3β-ol (24-dehydropollinastanol) and 14α-methyl-5α-ergost-24(28)-en-3β-ol (24-methylenepollinastanol) were isolated and identified. Both sterols were found primarily in the esterified sterol fraction, and 24-methylenepollinastanol accounted for 43% of the sterols of this fraction. 24-Dehydropollinastanol and four other sterols which also contain a 9β,19-cyclopropane ring were found only in the esterified sterol fraction. 24-Methylenecholesterol was the major sterol of the unesterified sterol fraction.     

Modulating the Intrinsic Disorder in the Cytoplasmic Domain Alters the Biological Activity of the N-Methyl-d-aspartate-sensitive Glutamate Receptor

The NMDA-sensitive glutamate receptor is a ligand-gated ion channel that mediates excitatory synaptic transmission in the nervous system. Extracellular zinc allosterically regulates the NMDA receptor by binding to the extracellular N-terminal domain, which inhibits channel gating. Phosphorylation of the intrinsically disordered intracellular C-terminal domain alleviates inhibition by extracellular zinc. The mechanism for this functional effect is largely unknown. Proline is a hallmark of intrinsic disorder, so we used proline mutagenesis to modulate disorder in the cytoplasmic domain. Proline depletion selectively uncoupled zinc inhibition with little effect on receptor biogenesis, surface trafficking, or ligand-activated gating. Proline depletion also reduced the affinity for a PDZ domain involved in synaptic trafficking and affected small molecule binding. To understand the origin of these phenomena, we used single molecule fluorescence and ensemble biophysical methods to characterize the structural effects of proline mutagenesis. Proline depletion did not eliminate intrinsic disorder, but the underlying conformational dynamics were changed. Thus, we altered the form of intrinsic disorder, which appears sufficient to affect the biological activity. These findings suggest that conformational dynamics within the intrinsically disordered cytoplasmic domain are important for the allosteric regulation of NMDA receptor gating.     

Bioinformatic Identification and Analysis of Extensins in the Plant Kingdom

Extensins (EXTs) are a family of plant cell wall hydroxyproline-rich glycoproteins (HRGPs) that are implicated to play important roles in plant growth, development, and defense. Structurally, EXTs are characterized by the repeated occurrence of serine (Ser) followed by three to five prolines (Pro) residues, which are hydroxylated as hydroxyproline (Hyp) and glycosylated. Some EXTs have Tyrosine (Tyr)-X-Tyr (where X can be any amino acid) motifs that are responsible for intramolecular or intermolecular cross-linkings. EXTs can be divided into several classes: classical EXTs, short EXTs, leucine-rich repeat extensins (LRXs), proline-rich extensin-like receptor kinases (PERKs), formin-homolog EXTs (FH EXTs), chimeric EXTs, and long chimeric EXTs. To guide future research on the EXTs and understand evolutionary history of EXTs in the plant kingdom, a bioinformatics study was conducted to identify and classify EXTs from 16 fully sequenced plant genomes, including Ostreococcus lucimarinus, Chlamydomonas reinhardtii, Volvox carteri, Klebsormidium flaccidum, Physcomitrella patens, Selaginella moellendorffii, Pinus taeda, Picea abies, Brachypodium distachyon, Zea mays, Oryza sativa, Glycine max, Medicago truncatula, Brassica rapa, Solanum lycopersicum, and Solanum tuberosum, to supplement data previously obtained from Arabidopsis thaliana and Populus trichocarpa. A total of 758 EXTs were newly identified, including 87 classical EXTs, 97 short EXTs, 61 LRXs, 75 PERKs, 54 FH EXTs, 38 long chimeric EXTs, and 346 other chimeric EXTs. Several notable findings were made: (1) classical EXTs were likely derived after the terrestrialization of plants; (2) LRXs, PERKs, and FHs were derived earlier than classical EXTs; (3) monocots have few classical EXTs; (4) Eudicots have the greatest number of classical EXTs and Tyr-X-Tyr cross-linking motifs are predominantly in classical EXTs; (5) green algae have no classical EXTs but have a number of long chimeric EXTs that are absent in embryophytes. Furthermore, phylogenetic analysis was conducted of LRXs, PERKs and FH EXTs, which shed light on the evolution of three EXT classes.     

Disentangling the effects of drought,salinity, and sulfate on baldcypress growth in a coastal plain restored wetland

Because of their dominance in swamps of the southeastern United States, baldcypress (Taxodium distichum) trees are commonly used in wetland restoration. Though baldcypress are known to tolerate moderate flooding and salinity, their growth has been shown to decrease when they experience drought and high salinity. This study examined the effects of drought and elevated salinity on the growth of baldcypress seedlings and saplings. In a restored wetland in North Carolina, we examined the growth of 8‐year‐old baldcypress by measuring height and diameter at breast height (DBH) along salinity, nutrient, and flooding gradients. In a greenhouse, we placed 1‐year‐old baldcypress seedlings in either drought or saturated conditions and applied different water treatments: fresh water, sulfate, and artificial salt water (5 ppt [parts per thousand]). Over 26 weeks, we measured diameter at root collar, height, and biomass. In the field, chloride concentrations in soil solution had a negative effect on DBH and height (51 and 36% decrease, respectively); high water levels had a negative effect on height (47% decrease) and DBH (46% decrease). In the greenhouse, both drought and salinity decreased diameter growth (43 and 61% decline, respectively) and height (64 and 43% decline, respectively). Sulfate did not have a significant effect on diameter growth, but caused a 14% decrease in height. Our results suggest that both drought and salinity (even as low as 1 ppt) can lead to a 20–60% decline in baldcypress growth. Restoration practitioners should consider the negative consequences of both drought and increased salinity on baldcypress growth when planning for wetland restoration.