Pseudopterosin biosynthesis—pathway elucidation,enzymology, and a proposed production method for anti-inflammatory metabolites from<Emphasis Type="Italic"> Pseudopterogorgia elisabethae</Emphasis>

The pseudopterosins are a family of diterpene pentosides isolated from the marine octocoral, Pseudopterogorgia elisabethae. These compounds possess non-steroidal anti-inflammatory and analgesic properties which have been shown to be greater than the industry standard, indomethacin. In our investigations, we are interested in examining the biosynthesis and enzymology of these compounds for the development of a biotechnological production method. We have isolated the pseudopterosin diterpene cyclase product, elisabethatriene, using a radioactivity-guided isolation. This has provided us with an assay to isolate the diterpene cyclase enzyme. The amino acid sequence of the purified diterpene cyclase will facilitate cloning and expression of the gene in a suitable host. In addition, we have identified over 25 novel diterpenes from one of our collections of P. elisabethae. Several of these compounds appear to be involved in pseudopterosin biosynthesis and are presently being evaluated as potential intermediates. These compounds have also been evaluated for anti-inflammatory activity and some possess greater activity than that of the pseudopterosins. We therefore propose a production method utilizing a combination of recombinant enzyme technology and synthetic methods/biocatalysis in order to produce one or more anti-inflammatory metabolites in P. elisabethae.     

Leucine-enkephalin interrupts sympathetically mediated tachycardia prejunctionally in the canine sinoatrial node

This study examined the role of leucine-enkephalin (LE) in the sympathetic regulation of the cardiac pacemaker. LE was administered by microdialysis into the interstitium of the canine sinoatrial node during either sympathetic nerve stimulation or norepinephrine infusion. In study one, the right cardiac sympathetic nerves were isolated as they exit the stellate ganglion and were stimulated to produce graded (low, 20-30 bpm; high 40-50 bpm) increases in heart rate (HR). LE (1.5 nmoles/min) was added to the dialysis inflow and the sympathetic stimulations were repeated after 5 and 20 min of LE infusion. After 5 min, LE reduced the tachycardia during sympathetic stimulation at both low (18.2 +/- 1.3 bpm to 11.4 +/- 1.4 bpm) and high (45 +/- 1.5 bpm to 22.8 +/- 1.5 bpm) frequency stimulations. The inhibition was maintained during 20 min of continuous LE exposure with no evidence of opioid desensitization. The delta-opioid antagonist, naltrindole (1.1 nmoles/min), restored only 30% of the sympathetic tachycardia. Nodal delta-receptors are vagolytic and vagal stimulations were included in the protocol as positive controls. LE reduced vagal bradycardia by 50% and naltrindole completely restored the vagal bradycardia. In Study 2, additional opioid antagonists were used to determine if alternative opioid receptors might be implicated in the sympatholytic response. Increasing doses of the kappa-antagonist, norbinaltorphimine (norBNI), were combined with LE during sympathetic stimulation. NorBNI completely restored the sympathetic tachycardia with an ED50 of 0.01 nmoles/min. A single dose of the micro -antagonist, CTAP (1.0 nmoles/min), failed to alter the sympatholytic effect of LE. Study 3 was conducted to determine if the sympatholytic effect was prejunctional or postjunctional in character. Norepinephrine was added to the dialysis inflow at a rate (30-45 pmoles/min) sufficient to produce intermediate increases (35.2 +/- 1.8 bpm) in HR. LE was then combined with norepinephrine and responses were recorded at 5-min intervals for 20 min. The tachycardia mediated by added norepinephrine was unaltered by LE or LE plus naltrindole. At the same 5-min intervals, LE reduced vagal bradycardia by more than 50%. This vagolytic effect was again completely reversed by naltrindole. Collectively, these observations support the hypothesis that the local nodal sympatholytic effect of LE was mediated by kappa-opioid receptors that reduced the effective interstitial concentration of norepinephrine and not the result of a postjunctional interaction between LE and norepinephrine.     

Social learning of a novel foraging patch in families of free-living Florida scrub-jays

Free-living juvenile Florida scrub-jays, Aphelocoma coerulescens, learned to forage in a novel patch (the centre of a ring) when in proximity to other family members that foraged successfully. We were able to distinguish the contributions of social learning and of individual learning, and to show that social learning occurred. The foraging task required individual jays to dig for peanut bits (chopped fragments) buried in sand in the centre of a 33-cm plastic ring. Jays were trained in their family groups to perform the task during a summer season, and were allowed to perform the task in the presence of juveniles (aged 40-85 days) in later years. Jays living in 18 control families received partial exposure to the training situation, but received no exposure to the ring before being presented with the task in the presence of their young. Juveniles in 16 families with trained jays were able to witness demonstrations and to scrounge peanut pieces from the models as they completed the task. These 41 juveniles learned more of the task than the 33 juveniles in control families. Seven juvenile jays and two older, nonbreeding jays in the trained families completed the task at least once, whereas no jays in control families completed the task. A modified task that prevented snatching also prevented transmission of the complete task, although the 22 juveniles that observed the modified task learned more of the task than the 33 control juveniles. Further analysis indicated that demonstrations had their greatest effect in increasing the probability that juveniles would enter the ring. Copyright 2000 The Association for the Study of Animal Behaviour.     

Herbal Supplement Extends Life Span Under Some Environmental Conditions and Boosts Stress Resistance

Genetic studies indicate that aging is modulated by a great number of genetic pathways. We have used Drosophila longevity and stress assays to test a multipath intervention strategy. To carry out this strategy, we supplemented the flies with herbal extracts (SC100) that are predicted to modulate the expression of many genes involved in aging and stress resistance, such as mTOR, NOS, NF-KappaB, and VEGF. When flies were housed in large cages with SC100 added, daily mortality rates of both male and female flies were greatly diminished in mid to late life. Surprisingly, SC100 also stabilized midlife mortality rate increases so as to extend the maximum life span substantially beyond the limits previously reported for D. melanogaster. Under these conditions, SC100 also promoted robust resistance to partial starvation stress and to heat stress. Fertility was the same initially in both treated and control flies, but it became significantly higher in treated flies at older ages as the fertility of control flies declined. Mean and maximum life spans of flies in vials at the same test site were also extended by SC100, but the life spans were short in absolute terms. In contrast, at an independent test site where stress was minimized, the flies exhibited much longer mean life spans, but the survival curves became highly rectangular and the effects of SC100 on both mean and maximum life spans declined greatly or were abolished. The data indicate that SC100 is a novel herbal mix with striking effects on enhancing Drosophila stress resistance and life span in some environments, while minimizing mid to late life mortality rates. They also show that the environment and other factors can have transformative effects on both the length and distribution of survivorship, and on the ability of SC100 to extend the life span.     

Dynamic Disturbance Processes Create Dynamic Lek Site Selection in a Prairie Grouse

It is well understood that landscape processes can affect habitat selection patterns, movements, and species persistence. These selection patterns may be altered or even eliminated as a result of changes in disturbance regimes and a concomitant management focus on uniform, moderate disturbance across landscapes. To assess how restored landscape heterogeneity influences habitat selection patterns, we examined 21 years (1991, 1993–2012) of Greater Prairie-Chicken (Tympanuchus cupido) lek location data in tallgrass prairie with restored fire and grazing processes. Our study took place at The Nature Conservancy’s Tallgrass Prairie Preserve located at the southern extent of Flint Hills in northeastern Oklahoma. We specifically addressed stability of lek locations in the context of the fire-grazing interaction, and the environmental factors influencing lek locations. We found that lek locations were dynamic in a landscape with interacting fire and grazing. While previous conservation efforts have treated leks as stable with high site fidelity in static landscapes, a majority of lek locations in our study (i.e., 65%) moved by nearly one kilometer on an annual basis in this dynamic setting. Lek sites were in elevated areas with low tree cover and low road density. Additionally, lek site selection was influenced by an interaction of fire and patch edge, indicating that in recently burned patches, leks were located near patch edges. These results suggest that dynamic and interactive processes such as fire and grazing that restore heterogeneity to grasslands do influence habitat selection patterns in prairie grouse, a phenomenon that is likely to apply throughout the Greater Prairie-Chicken’s distribution when dynamic processes are restored. As conservation moves toward restoring dynamic historic disturbance patterns, it will be important that siting and planning of anthropogenic structures (e.g., wind energy, oil and gas) and management plans not view lek locations as static points, but rather as sites that shift around the landscape in response to shifting vegetation structure. Acknowledging shifting lek locations in these landscapes will help ensure conservation efforts are successful by targeting the appropriate areas for protection and management.     

Exploring Genomic,Geographic and Virulence Interactions among Epidemic and Non-Epidemic St. Louis Encephalitis Virus (Flavivirus) Strains

St. Louis encephalitis virus (SLEV) is a re-emerging arbovirus in South America. In 2005, an encephalitis outbreak caused by SLEV was reported in Argentina. The reason for the outbreak remains unknown, but may have been related to virological factors, changes in vectors populations, avian amplifying hosts, and/or environmental conditions. The main goal of this study was to characterize the complete genome of epidemic and non-epidemic SLEV strains from Argentina. Seventeen amino acid changes were detected; ten were non-conservative and located in proteins E, NS1, NS3 and NS5. Phylogenetic analysis showed two major clades based on geography: the North America and northern Central America (NAnCA) clade and the South America and southern Central America (SAsCA) clade. Interestingly, the presence of SAsCA genotype V SLEV strains in the NAnCA clade was reported in California, Florida and Texas, overlapping with known bird migration flyways. This work represents the first step in understanding the molecular mechanisms underlying virulence and biological variation among SLEV strains.     

A Mutation in SLC24A1 Implicated in Autosomal-Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder that can be associated with impaired night vision. The last decade has witnessed huge progress in ophthalmic genetics, including the identification of three genes implicated in the pathogenicity of autosomal-recessive CSNB. However, not all patients studied could be associated with mutations in these genes and thus other genes certainly underlie this disorder. Here, we report a large multigeneration family with five affected individuals manifesting symptoms of night blindness. A genome-wide scan localized the disease interval to chromosome 15q, and recombination events in affected individuals refined the critical interval to a 10.41 cM (6.53 Mb) region that harbors SLC24A1, a member of the solute carrier protein superfamily. Sequencing of all the coding exons identified a 2 bp deletion in exon 2: c.1613_1614del, which is predicted to result in a frame shift that leads to premature termination of SLC24A1 (p.F538CfsX23) and segregates with the disorder under an autosomal-recessive model. Expression analysis using mouse ocular tissues shows that Slc24a1 is expressed in the retina around postnatal day 7. In situ and immunohistological studies localized both SLC24A1 and Slc24a1 to the inner segment, outer and inner nuclear layers, and ganglion cells of the retina, respectively. Our data expand the genetic basis of CSNB and highlight the indispensible function of SLC24A1 in retinal function and/or maintenance in humans.