Model organisms: new insights into ion channel and transporter function. L-type calcium channels regulate epithelial fluid transport in Drosophila melanogaster

The neuropeptide CAP2b stimulates fluid transport obligatorily via calcium entry, nitric oxide, and cGMP in Drosophila melanogaster Malpighian (renal) tubules. We have shown by RT-PCR that the Drosophila L-type calcium channel alpha1-subunit genes Dmca1D and Dmca1A (nbA) are both expressed in tubules. CAP2b-stimulated fluid transport and cytosolic calcium concentration ([Ca2+]i) increases are inhibited by the L-type calcium channel blockers verapamil and nifedipine. cGMP-stimulated fluid transport is verapamil and nifedipine sensitive. Furthermore, cGMP induces a slow [Ca2+]i increase in tubule principal cells via verapamil- and nifedipine-sensitive calcium entry; RT-PCR shows that tubules express Drosophila cyclic nucleotide-gated channel (cng). Additionally, thapsigargin-induced [Ca2+]i increase is verapamil sensitive. Phenylalkylamines bind with differing affinities to the basolateral and apical surfaces of principal cells in the main segment; however, dihydropyridine binds apically in the tubule initial segment. Immunocytochemical evidence suggests localization of alpha1-subunits to both basolateral and apical surfaces of principal cells in the tubule main segment. We suggest roles for L-type calcium channels and cGMP-mediated calcium influx in both calcium signaling and fluid transport mechanisms in Drosophila.     

Monoamine Changes in the Brain of BALB/c Mice Following Sub-Lethal Infection with Nocardia asteroides (GUH-2)

BALB/c mice injected intravenously with a single, sub-lethal dose of Nocardia asteroides GUH-2 develop several levodopa responsive movement disorders. These included head-shake, stooped posture, bradykinesia, and hesitation to forward movement (6). The changes in monoamine levels in the brain of these mice were determined. There was a significant loss of dopamine with greatly increased dopamine turnover in the neostriatum 7 to 29 days after infection. These effects were specific for dopaminergic neurons since minimal changes were found in neostriatal norepinephrine and serotonin even though serotonin turnover was increased. Changes in monoamine metabolism were not limited to the neostriatum. There were reduced levels of serotonin and norepinephrine with increased serotonin turnover in the cerebellum. One year after infection, dopamine metabolism had returned to near normal levels, but many of the movement disorders persisted. Specific changes in neurochemistry did not always appear to correspond with these impairments. Nevertheless, these data are similar to those reported in MPTP treated BALB/c mice.     

Enhanced neointimal growth in cultured rabbit aorta following in vivo balloon angioplasty

Summary We have used in vivo balloon catheterization in combination with in vitro organ culture to develop a model system for vascular neointima formation. A Fogarty balloon catheter was used to deendothelialize and rupture the internal elastic lamina of aortae in adult rabbits. After three d of recovery, aortae were harvested, divided into segments, and placed into organ culture. We obtained a daily index of cell proliferation in cultured vessels using [³H]thymidine incorporation into DNA. Also, segments were collected and processed for routine histology or immunohistochemistry. Aortic segments that had undergone ballooning 3 d before harvest and then cultured exhibited diffuse neointimal growth after several d in vitro, whereas those from sham-operated (nonballooned) rabbits showed generally only a single endothelial cell layer that is characteristic of normal intima. Aortae that were harvested, balloon-damaged in vitro, and then cultured exhibited no neointimal growth. The neointima that developed in cultured segments from in vivo ballooned rabbits was primarily of smooth muscle cell origin as determined by positive immunostaining for α-smooth muscle actin. The intima:media thickness ratios were significantly higher in aortic segments from ballooned rabbits at harvest and after 4 or 7 d in culture compared with those from nonballooned rabbits. Also, the [³H]thymidine index was higher in the in vivo ballooned aorta compared to non-ballooned or in vitro ballooned vessel. We conclude that ballooning in vivo followed by exposure to blood-borne elements produces an enhanced proliferative response in cultured vessels that is distinct from other in vitro models of neointimal growth.     

Habitat quality mediates personality through differences in social context

Assessing the stability of animal personalities has become a major goal of behavioral ecologists. Most personality studies have utilized solitary individuals, but little is known on the extent that individuals retain their personality across ecologically relevant group settings. We conducted a field survey which determined that mud crabs, Panopeus herbstii, remain scattered as isolated individuals on degraded oyster reefs while high quality reefs can sustain high crab densities (>10 m^?2). We examined the impact of these differences in social context on personality by quantifying the boldness of the same individual crabs when in isolation and in natural cohorts. Crabs were also exposed to either a treatment of predator cues or a control of no cue throughout the experiment to assess the strength of this behavioral reaction norm. Crabs were significantly bolder when in groups than as solitary individuals with predator cue treatments exhibiting severally reduced crab activity levels in comparison to corresponding treatments with no predator cues. Behavioral plasticity depended on the individual and was strongest in the presence of predator cues. While bold crabs largely maintained their personality in isolation and group settings, shy crabs would become substantially bolder when among conspecifics. These results imply that the shifts in crab boldness were a response to changes in perceived predation risk, and provide a mechanism for explaining variation in behavioral plasticity. Such findings suggest that habitat degradation may produce subpopulations with different behavioral patterns because of differing social interactions between individual animals.     

Personality interacts with habitat quality to govern individual mortality and dispersal patterns

Individual phenotypic differences are increasingly recognized as key drivers of ecological processes. However, studies examining the relative importance of these differences in comparison with environmental factors or how individual phenotype interacts across different environmental contexts remain lacking. We performed two field experiments to assess the concurrent roles of personality differences and habitat quality in mediating individual mortality and dispersal. We quantified the predator avoidance response of mud crabs, Panopeus herbstii, collected from low‐ and high‐quality oyster reefs and measured crab loss in a caging experiment. We simultaneously measured the distance crabs traveled as well as the stability of personalities across reef quality in a separate reciprocal transplant experiment. Habitat quality was the primary determinant of crab loss, although the distance crabs traveled was governed by personality which interacted with habitat quality to control the fate of crabs. Here, crabs on low‐quality reefs rapidly emigrated, starting with the boldest individuals, and experienced modest levels of predation regardless of personality. In contrast, both bold and shy crabs would remain on high‐quality reefs for months where bolder individuals experienced higher predation risk. These findings suggest that personalities could produce vastly different population dynamics across habitat quality and govern community responses to habitat degradation.     

Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS

Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.     

Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing

Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.     

Sprouty2 in the Dorsal Hippocampus Regulates Neurogenesis and Stress Responsiveness in Rats

Both the development and relief of stress-related psychiatric conditions such as major depression (MD) and post-traumatic stress disorder (PTSD) have been linked to neuroplastic changes in the brain. One such change involves the birth of new neurons (neurogenesis), which occurs throughout adulthood within discrete areas of the mammalian brain, including the dorsal hippocampus (HIP). Stress can trigger MD and PTSD in humans, and there is considerable evidence that it can decrease HIP neurogenesis in laboratory animals. In contrast, antidepressant treatments increase HIP neurogenesis, and their efficacy is eliminated by ablation of this process. These findings have led to the working hypothesis that HIP neurogenesis serves as a biomarker of neuroplasticity and stress resistance. Here we report that local alterations in the expression of Sprouty2 (SPRY2), an intracellular inhibitor of growth factor function, produces profound effects on both HIP neurogenesis and behaviors that reflect sensitivity to stressors. Viral vector-mediated disruption of endogenous Sprouty2 function (via a dominant negative construct) within the dorsal HIP of adult rats stimulates neurogenesis and produces signs of stress resilience including enhanced extinction of conditioned fear. Conversely, viral vector-mediated elevation of SPRY2 expression intensifies the behavioral consequences of stress. Studies of these manipulations in HIP primary cultures indicate that SPRY2 negatively regulates fibroblast growth factor-2 (FGF2), which has been previously shown to produce antidepressant- and anxiolytic-like effects via actions in the HIP. Our findings strengthen the relationship between HIP plasticity and stress responsiveness, and identify a specific intracellular pathway that could be targeted to study and treat stress-related disorders.     

Structure–activity relationships amongst 4-position quinoline methanol antimalarials that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum

Utilizing mefloquine as a scaffold, a next generation quinoline methanol (NGQM) library was constructed to identify early lead compounds that possess biological properties consistent with the target product profile for malaria chemoprophylaxis while reducing permeability across the blood–brain barrier. The library of 200 analogs resulted in compounds that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum. Herein we report selected chemotypes and the emerging structure–activity relationship for this library of quinoline methanols.