Alpha-synuclein aggregation

Alpha-synuclein is a major component of Lewy bodies in Parkinson's disease and is found associated with several other forms of dementia. As with other neurodegenerative diseases, the ability of alpha-synuclein to aggregate and form fibrillar deposits seems central to its pathology. We have defined a sequence within the NAC region of alpha-synuclein that is necessary for aggregation. Exploitation of chemically modified analogues of this peptide may produce inhibitors of aggregation.     

Heterologous complementation of the exopolysaccharide synthesis and carbon utilization phenotypes of Sinorhizobium meliloti Rm1021 polyhydroxyalkanoate synthesis mutants

A reduced exopolysaccharide phenotype is associated with inability to synthesize polyhydroxyalkanaote (PHA) stores in Sinorhizobium meliloti strain Rm1021. Loss of function mutations in phbB and phbC result in non-mucoid colony morphology on Yeast Mannitol Agar, compared to the mucoid phenotype exhibited by the parental strain. This phenotype is attributed to reduction in succinoglycan synthesis. We have used complementation of this phenotype and the previously described D-3-hydroxybutyrate/acetoacetate utilization phenotype to isolate a heterologous clone containing a Bradyrhizobium japonicum phbC gene. Sequence analysis confirmed that this clone contains one of the five predicted phbC genes in the B. japonicum genome. The described phenotypic complementation strategy should be useful for isolation of novel PHA synthesis genes of diverse origin.     

Comparison of functional aspects of the coagulation cascade in human and sea turtle plasmas

Functional hemostatic pathways are critical for the survival of all vertebrates and have been evolving for more than 400 million years. The overwhelming majority of studies of hemostasis in vertebrates have focused on mammals with very sparse attention paid to reptiles. There have been virtually no studies of the coagulation pathway in sea turtles whose ancestors date back to the Jurassic period. Sea turtles are often exposed to rapidly altered environmental conditions during diving periods. This may reduce their blood pH during prolonged hypoxic dives. This report demonstrates that five species of turtles possess only one branch of the mammalian coagulation pathway, the extrinsic pathway. Mixing studies of turtle plasmas with human factor-deficient plasmas indicate that the intrinsic pathway factors VIII and IX are present in turtle plasma. These two factors may play a significant role in supporting the extrinsic pathway by feedback loops. The intrinsic factors, XI and XII are not detected which would account for the inability of reagents to induce coagulation via the intrinsic pathway in vitro. The analysis of two turtle factors, factor II (prothrombin) and factor X, demonstrates that they are antigenically/functionally similar to the corresponding human factors. The turtle coagulation pathway responds differentially to both pH and temperature relative to each turtle species and relative to human samples. The coagulation time (prothrombin time) increases as the temperature decreases between 37 and 15 °C. The increased time follows a linear relationship, with similar slopes for loggerhead, Kemps ridley and hawksbill turtles as well as for human samples. Leatherback turtle samples show a dramatic nonlinear increased time below 23 °C, and green turtle sample responses were similar but less dramatic. All samples also showed increased prothrombin times as the pH decreased from 7.8 to 6.4, except for three turtle species. The prothrombin times decreased, to varying extents, in a linear fashion relative to reduced pH with the rate of change greatest in leatherbacks>greenloggerhead turtles. All studies were conducted with reagents developed for human samples which would impact on the quantitative results with the turtle samples, but are not likely to alter the qualitative results. These comparative studies of the coagulation pathway in sea turtles and humans could enhance our knowledge of structure/function relationships and evolution of coagulation factors.     

Major histocompatibility complex variation at three class II loci in the northern elephant seal

Northern elephant seals were hunted to near extinction in the 19th century, yet have recovered remarkably and now number around 175,000. We surveyed 110 seals for single-strand conformation polymorphism (SSCP) and sequence variation at three major histocompatibility (MHC) class II loci (DQA, DQB and DRB) to evaluate the genetic consequences of the population bottleneck at these loci vs. other well-studied genes. We found very few alleles at each MHC locus, significant variation among breeding sites for the DQA locus, and linkage disequilibrium between the DQB and DRB loci. Northern elephant seals are evidently inbred, although there is as yet no evidence of correlative reductions in fitness.     

Relationship between two proprioceptive measures and stiffness at the ankle.

Previous research has investigated the role of proprioception and stiffness in the control of joint stability. However, to date, no research has been done on the relationship between proprioception and stiffness. Therefore, the purpose of this study was to determine the relationship between force sense, joint reposition sense, and stiffness at the ankle. A heterogeneous sample was obtained for this study; 20 of the 40 participants had a history of ankle sprains, and 13 of the 20 had been diagnosed by a physician (two mild ankle sprains, seven moderate sprains, four severe sprains). All subjects were asymptomatic and active at the time of the study. Active joint reposition sense was measured using a custom-built ankle goniometer, force sense was measured unilaterally and contralaterally with a load cell, and ankle muscle stiffness was measured via transient oscillation using a custom-built inversion-eversion cradle. We found no significant correlations between stiffness and joint reposition sense, with values of r ranging from 0.01 to 0.21. Significant correlations were found between stiffness and force sense. Specifically, contralateral force sense reproduction was significantly correlated to stiffness in the injured or "involved" ankle (r's ranging from 0.47 to 0.65; P< or =0.008). Whether the decreased ability to appropriately sense force (increased error) sends information to the central nervous system to increase muscle stiffness in response to an unexpected loss of stability, or whether these two phenomena function independently and both change concurrently as a result of injury to the system requires further investigation.     

Tumor necrosis factor regulates intestinal epithelial cell migration by receptor-dependent mechanisms

Altered mucosal integrity andincreased cytokine production, including tumor necrosis factor (TNF),are the hallmarks of inflammatory bowel disease (IBD). In this study,we addressed the role of TNF receptors (TNFR) on intestinal epithelialcell migration in an in vitro wound closure model. With mouse TNFR1 orTNFR2 knockout intestinal epithelial cells, gene transfection, andpharmacological inhibitors, we show a concentration-dependentreceptor-mediated regulation of intestinal cell migration by TNF. Aphysiological TNF level (1 ng/ml) enhances migration through TNFR2,whereas a pathological level (100 ng/ml) inhibits wound closure through TNFR1. Increased rate of wound closure by TNFR2 or inhibition by TNFR1cannot be explained by either increased proliferation orapoptosis, respectively. Furthermore, inhibiting Src tyrosine kinase decreases TNF-induced focal adhesion kinase (FAK) tyrosine phosphorylation and cellular migration. We therefore conclude thatTNFR2 activates a novel Src-regulated pathway involving FAK tyrosinephosphorylation that enhances migration of intestinal epithelial cells.

     

Changes in juvenile hormone biosynthetic rate and whole body content in maturing virgin queens of Solenopsis invicta

Studies were conducted on the physiological and hormonal changes following the release of alates from developmentally suppressive pheromones produced by mature queens of the fire ant Solenopsis invicta Buren. Winged virgin queens were removed from the pheromonal signal and placed in colony fragments. The time for dealation, degree of ovarian development, and biosynthesis rate and whole body content of juvenile hormone (JH) were measured. The production rate and content of JH were highly correlated. Dealation and the initiation of oviposition corresponded to peak production of JH. JH production rose sharply following separation from the natal nest, peaking after 3 days. After 8 days of isolation, JH production gradually subsided to levels similar to that found in pre-release queens, but began to increase again after 12 days. Mature queens had highly elevated levels of JH relative to recently dealate females, probably reflecting the increased reproductive capability of these older females. The results support the hypothesis that the pheromone released by functional queens inhibits reproduction in virgin alates by suppressing corpora allata activity and the production of JH.     

Rapid responses to high temperature and desiccation but not to low temperature in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera,Tineidae)

A broad definition of rapid cold hardening (RCH) is that it is the process whereby insects increase their survival of a sub-zero temperature after a brief (h) pre-exposure to a less severe low temperature. The effects of various pre-treatments on survival of two h at -7.9 degrees C were investigated in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera: Tineidae), the first time RCH has been investigated in a freeze tolerant arthropod. All caterpillars froze when exposed to -7.9 degrees C, and none of the low temperature pre-treatments (-5, 0, 5 and 15 degrees C, as well as -5 degrees C and 0 degrees C with a delay before freezing) nor slow cooling (0.1 degrees C/min) elicited any improvement in survival of -7.9 degrees C as compared to controls. However, high temperature treatments (25, 30 and 35 degrees C), desiccation and acclimation for 5 days at 0 degrees C did result in significant increases in survival of the test temperature, possibly as a result of heat shock protein production. Haemolymph osmolality was elevated only by the 35 degrees C pre-treatment. It is suggested that the unpredictable environment of Marion Island means that P. marioni must always be physiologically prepared to survive cold snaps, and that this year-round cold hardiness therefore supersedes a rapid cold hardening response.