Interrogating accessibility of telomeric sequences with FRET-PAINT: evidence for length-dependent telomere compaction

Single-stranded telomeric overhangs are ∼200 nucleotides long and can form tandem G-quadruplex (GQ) structures, which reduce their accessibility to nucleases and proteins that activate DNA damage response. Whether these tandem GQs further stack to form compact superstructures, which may provide better protection for longer telomeres, is not known. We report single-molecule measurements where the accessibility of 24–144 nucleotide long human telomeric DNA molecules is interrogated by a short PNA molecule that is complementary to a single GGGTTA repeat, as implemented in the FRET-PAINT method. Binding of the PNA strand to available GGGTTA sequences results in discrete FRET bursts which were analyzed in terms of their dwell times, binding frequencies, and topographic distributions. The binding frequencies were greater for binding to intermediate regions of telomeric DNA compared to 3′- or 5′-ends, suggesting these regions are more accessible. Significantly, the binding frequency per telomeric repeat monotonically decreased with increasing telomere length. These results are consistent with telomeres forming more compact structures at longer lengths, reducing accessibility of these critical genomic sites.     

Comamonas testosteronan synthase, a bifunctional glycosyltransferase that produces a unique heparosan polysaccharide analog

Glycosaminoglycans (GAGs) are linear hexosamine-containing polysaccharides. These polysaccharides are synthesized by some pathogenic bacteria to form an extracellular coating or capsule. This strategy forms the basis of molecular camouflage since vertebrates possess naturally occurring GAGs that are essential for life. A recent sequence database search identified a putative protein from the opportunistic pathogen Comamonas testosteroni that exhibits similarity with the Pasteurella multocida GAG synthase PmHS1, which is responsible for the synthesis of a heparosan polysaccharide capsule. Initial supportive evidence included glucuronic acid (GlcUA)-containing polysaccharides extracted from C. testosteroni KF-1. We describe here the cloning and analysis of a novel Comamonas GAG synthase, CtTS. The GAG produced by CtTS in vitro consists of the sugars d-GlcUA and N-acetyl-D-glucosamine, but is insensitive to digestion by GAG digesting enzymes, thus has distinct glycosidic linkages from vertebrate GAGs. The backbone structure of the polysaccharide product [-4-D-GlcUA-α1,4-D-GlcNAc-α1-](n) was confirmed by nuclear magnetic resonance. Therefore, this novel GAG, testosteronan, consists of the same sugars as the biomedically relevant GAGs heparosan (N-acetyl-heparosan) and hyaluronan but may have distinct properties useful for future medical applications.     

Retention of the mitochondrial probe rhodamine 123 in normal lymphocytes and leukemic cells in relation to the cell cycle

The cationic fluorochrome rhodamine 123 (R123) is specifically taken up by mitochondria of live cells where it is retained due to the mitochondrial transmembrane potential. After pulse exposure of human normal quiescent or proliferating lymphocytes, human lymphocytic leukemic MOLT cells, and mice leukemic L1210 cells to 10 micrograms/ml of R123, the dye release was studied using flow cytometry. Two distinct phases of R123 release, each following first-order kinetics, were apparent; the half-time of retention for the rapidly and slowly released fractions of R123 was 0.8-1.1 and 2.8-4.2 h, respectively. Simultaneous supravital cell staining with R123 and Hoechst 33342 made it possible to correlate retention of R123 with cell position in the cell cycle. No significant differences were observed in the rate of R123 release from cells in G1 vs S or vs G2 + M phases of the cycle. The data rule out a possibility that the release of R123 is due to periodic depolarization of the mitochondria in the cell as may be postulated by cell cycle models that assume a transient passage of cells through resting phase following division. The observed similar rates of R123 release regardless of cell type or cell cycle phase suggest that the factors affecting the exchange are similar in normal lymphocytes vs leukemic cells and unrelated to cell proliferation rate or phase of the cell cycle. Two distinct rates of R123 release indicate the presence of two kinds of binding sites differing in affinity to the dye.     

Modelling the Effects of Seasonality and Socioeconomic Impact on the Transmission of Rift Valley Fever Virus

Rift Valley fever (RVF) is an important mosquito-borne viral zoonosis in Africa and the Middle East that causes human deaths and significant economic losses due to huge incidences of death and abortion among infected livestock. Outbreaks of RVF are sporadic and associated with both seasonal and socioeconomic effects. Here we propose an almost periodic three-patch model to investigate the transmission dynamics of RVF virus (RVFV) among ruminants with spatial movements. Our findings indicate that, in Northeastern Africa, human activities, including those associated with the Eid al Adha feast, along with a combination of climatic factors such as rainfall level and hydrological variations, contribute to the transmission and dispersal of the disease pathogen. Moreover, sporadic outbreaks may occur when the two events occur together: 1) abundant livestock are recruited into areas at risk from RVF due to the demand for the religious festival and 2) abundant numbers of mosquitoes emerge. These two factors have been shown to have impacts on the severity of RVF outbreaks. Our numerical results present the transmission dynamics of the disease pathogen over both short and long periods of time, particularly during the festival time. Further, we investigate the impact on patterns of disease outbreaks in each patch brought by festival- and seasonal-driven factors, such as the number of livestock imported daily, the animal transportation speed from patch to patch, and the death rate induced by ceremonial sacrifices. In addition, our simulations show that when the time for festival preparation starts earlier than usual, the risk of massive disease outbreaks rises, particularly in patch 3 (the place where the religious ceremony will be held).     

Irreversible Heavy Chain Transfer to Chondroitin

We have recently demonstrated that the transfer of heavy chains (HCs) from inter-α-inhibitor, via the enzyme TSG-6 (tumor necrosis factor-stimulated gene 6), to hyaluronan (HA) oligosaccharides is an irreversible event in which subsequent swapping of HCs between HA molecules does not occur. We now describe our results of HC transfer experiments to chondroitin sulfate A, chemically desulfated chondroitin, chemoenzymatically synthesized chondroitin, unsulfated heparosan, heparan sulfate, and alginate. Of these potential HC acceptors, only chemically desulfated chondroitin and chemoenzymatically synthesized chondroitin were HC acceptors. The kinetics of HC transfer to chondroitin was similar to HA. At earlier time points, HCs were more widely distributed among the different sizes of chondroitin chains. As time progressed, the HCs migrated to lower molecular weight chains of chondroitin. Our interpretation is that TSG-6 swaps the HCs from the larger, reversible sites on chondroitin chains, which function as HC acceptors, onto smaller chondroitin chains, which function as irreversible HC acceptors. HCs transferred to smaller chondroitin chains were unable to be swapped off the smaller chondroitin chains and transferred to HA. HCs transferred to high molecular weight HA were unable to be swapped onto chondroitin. We also present data that although chondroitin was a HC acceptor, HA was the preferred acceptor when chondroitin and HA were in the same reaction mixture.     

Testing for adaptive evolution of the female reproductive protein ZPC in mammals,birds and fishes reveals problems with the M7-M8 likelihood ratio test

Background  

Adaptive evolution appears to be a common feature of reproductive proteins across a very wide range of organisms. A promising way of addressing the evolutionary forces responsible for this general phenomenon is to test for adaptive evolution in the same gene but among groups of species, which differ in their reproductive biology. One can then test evolutionary hypotheses by asking whether the variation in adaptive evolution is consistent with the variation in reproductive biology. We have attempted to apply this approach to the study of a female reproductive protein, zona pellucida C (ZPC), which has been previously shown by the use of likelihood ratio tests (LRTs) to be under positive selection in mammals.     

Topological organization of the hyaluronan synthase from Streptococcus pyogenes

Since we first reported (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184) the cloning of the hyaluronan (HA) synthase from Streptococcus pyogenes (spHAS), numerous membrane-bound HA synthases have been discovered in both prokaryotes and eukaryotes. The HASs are unique among enzymes studied to date because they mediate 6-7 discrete functions in order to assemble a polysaccharide containing hetero-disaccharide units and simultaneously effect translocation of the growing HA chain through the plasma membrane. To understand how the relatively small spHAS performs these various functions, we investigated the topological organization of the protein utilizing fusion analysis with two reporter enzymes, alkaline phosphatase and beta-galactosidase, as well as several other approaches. From these studies, we conclude that the NH2 terminus and the COOH terminus, as well as the major portion of a large central domain are localized intracellularly. The first two predicted membrane domains were confirmed to be transmembrane domains and give rise to a very small extracellular loop that is inaccessible to proteases. Several regions of the large internal central domain appear to be associated with, but do not traverse, the membrane. Following the central domain, there are two additional transmembrane domains connected by a second small extracellular loop that also is inaccessible to proteases. The COOH-terminal approximately 25% of spHAS also contains a membrane domain that does not traverse the membrane and may contain extensive re-entrant loops or amphipathic helices. Numerous membrane associations of this latter COOH-terminal region and the central domain may be required to create a pore-like structure through which a growing HA chain can be extruded to the cell exterior. Based on the high degree of similarity among Class I HAS family members, these enzymes may have a similar topological organization for their spHAS-related domains.     

Single species models with logistic growth and dissymmetric impulse dispersal

In this paper, two classes of single-species models with logistic growth and impulse dispersal (or migration) are studied: one model class describes dissymmetric impulsive bi-directional dispersal between two heterogeneous patches; and the other presents a new way of characterizing the aggregate migration of a natural population between two heterogeneous habitat patches, which alternates in direction periodically. In this theoretical study, some very general, weak conditions for the permanence, extinction of these systems, existence, uniqueness and global stability of positive periodic solutions are established by using analysis based on the theory of discrete dynamical systems. From this study, we observe that the dynamical behavior of populations with impulsive dispersal differs greatly from the behavior of models with continuous dispersal. Unlike models where the dispersal is continuous in time, in which the travel losses associated with dispersal make it difficult for such dispersal to evolve e.g., [25], [26], [28], in the present study it was relatively easy for impulsive dispersal to positively affect populations when realistic parameter values were used, and a rich variety of behaviors were possible. From our results, we found impulsive dispersal seems to more nicely model natural dispersal behavior of populations and may be more relevant to the investigation of such behavior in real ecological systems.     

Synchronized chemoenzymatic synthesis of monodisperse hyaluronan polymers

The length of the hyaluronan (HA) polysaccharide chain dictates its biological effects in many cellular and tissue systems. Long and short HA polymers often appear to have antagonistic or inverse effects. However, no source of very defined, uniform HA polymers with sizes greater than 10 kDa is currently available. We present a method to produce synthetic HA with very narrow size distributions in the range of approximately 16 kDa to approximately 2 MDa. The Pasteurella HA synthase enzyme, pmHAS, catalyzes the synthesis of HA polymer utilizing monosaccharides from UDP-sugar precursors. Recombinant pmHAS will also elongate exogenously supplied HA oligosaccharide acceptors in vitro in a nonprocessive fashion. As a result of bypassing the slow initiation step in vitro, the elongation process is synchronized in the presence of acceptor; thus all of polymer products are very similar in length. In contrast, without the use of an acceptor, the final polymer size range is difficult to predict and the products are more polydisperse. HA polymers of a desired size are constructed by controlling the reaction stoichiometry (i.e. molar ratio of precursors and acceptor molecules). The use of modified acceptors allows the synthesis of HA polymers containing tags (e.g. fluorescent, radioactive). In this scheme, each molecule has a single foreign moiety at the reducing terminus. Alternatively, the use of radioactive UDP-sugar precursors allows the synthesis of uniformly labeled native HA polymers. Overall, synthetic HA reagents with monodisperse size distributions and defined structures should assist in the elucidation of the numerous roles of HA in health and disease.