Inhibition of Dopamine Transporter Activity by G Protein βγ Subunits

Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.     

Spatial variation in the ongoing and widespread decline of a keystone plant species

Extensive dieback in dominant plant species in response to climate change is increasingly common. Climatic conditions and related variables, such as evapotranspiration, vary in response to topographical complexity. This complexity plays an important role in the provision of climate refugia. In 2008/2009, an island‐wide dieback event of the keystone cushion plant Azorella macquariensis Orchard (Apiaceae) occurred on sub‐Antarctic Macquarie Island. This signalled the start of a potential regime shift, suggested to be driven by increasing vapour pressure deficit. Eight years later, we quantified cover and dieback across the range of putative microclimates to which the species is exposed, with the aim of explaining dieback patterns. We test for the influence of evapotranspiration using a suite of topographic proxies and other variables as proposed drivers of change. We found higher cover and lower dieback towards the south of the island. The high spatial variation in A. macquariensis populations was best explained by latitude, likely a proxy for macroscale climate gradients and geology. Dieback was best explained by A. macquariensis cover and latitude, increasing with cover and towards the north of the island. The effect sizes of terrain variables that influence evapotranspiration rates were small. Island‐wide dieback remains conspicuous. Comparison between a subset of sites and historical data revealed a reduction of cover in the north and central regions of the island, and a shift south in the most active areas of dieback. Dieback remained comparatively low in the south. The presence of seedlings was independent of dieback. This study provides an empirical baseline for spatial variation in the cover and condition of A. macquariensis, both key variables for monitoring condition and ‘cover‐debt’ in this critically endangered endemic plant species. These findings have broader implications for understanding the responses of fellfield ecosystems and other Azorella species across the sub‐Antarctic under future climates.     

Altered mRNA binding activity and decreased translational initiation in a nuclear mutant lacking translation of the chloroplast psbA mRNA.

Translational regulation has been identified as one of the key steps in chloroplast-encoded gene expression. Genetic and biochemical analysis with Chlamydomonas reinhardtii has implicated nucleus-encoded factors that interact specifically with the 5' untranslated region of chloroplast mRNAs to mediate light-activated translation. F35 is a nuclear mutation in C. reinhardtii that specifically affects translation of the psbA mRNA (encoding D1, a core polypeptide of photosystem II), causing a photosynthetic deficiency in the mutant strain. The F35 mutant has reduced ribosome association of the psbA mRNA as a result of decreased translation initiation. This reduction in ribosome association correlates with a decrease in the stability of the mRNA. Binding activity of the psbA specific protein complex to the 5' untranslated region of the mRNA is diminished in F35 cells, and two members of this binding complex (RB47 and RB55) are reduced compared with the wild type. These data suggest that alteration of members of the psbA mRNA binding complex in F35 cells results in a reduction in psbA mRNA-protein complex formation, thereby causing a decrease in translation initiation of this mRNA.     

Skewed X Chromosome Inactivation and Trisomic Spontaneous Abortion: No Association

Several studies suggest that highly skewed X chromosome inactivation (HSXI) is associated with recurrent spontaneous abortion. We hypothesized that this association reflects an increased rate of trisomic conceptions due to anomalies on the X chromosome that lead both to HSXI and to a diminished oocyte pool. We compared the distribution of X chromosome inactivation (XCI) skewing percentages (range: 50%–100%) among women with spontaneous abortions in four karyotype groups—trisomy (n = 154), chromosomally normal male (n = 43), chromosomally normal female (n = 38), nontrisomic chromosomally abnormal (n = 61)—to the distribution for age-matched controls with chromosomally normal births (n = 388). In secondary analyses, we subdivided the nontrisomic chromosomally abnormal group, divided trisomies by chromosome, and classified women by reproductive history. Our data support neither an association of HSXI with all trisomies nor an association of HSXI with chromosomally normal male spontaneous abortions. We also find no association between HSXI and recurrent abortion (n = 45).     

Crop Pollination Exposes Honey Bees to Pesticides Which Alters Their Susceptibility to the Gut Pathogen Nosema ceranae

Recent declines in honey bee populations and increasing demand for insect-pollinated crops raise concerns about pollinator shortages. Pesticide exposure and pathogens may interact to have strong negative effects on managed honey bee colonies. Such findings are of great concern given the large numbers and high levels of pesticides found in honey bee colonies. Thus it is crucial to determine how field-relevant combinations and loads of pesticides affect bee health. We collected pollen from bee hives in seven major crops to determine 1) what types of pesticides bees are exposed to when rented for pollination of various crops and 2) how field-relevant pesticide blends affect bees’ susceptibility to the gut parasite Nosema ceranae. Our samples represent pollen collected by foragers for use by the colony, and do not necessarily indicate foragers’ roles as pollinators. In blueberry, cranberry, cucumber, pumpkin and watermelon bees collected pollen almost exclusively from weeds and wildflowers during our sampling. Thus more attention must be paid to how honey bees are exposed to pesticides outside of the field in which they are placed. We detected 35 different pesticides in the sampled pollen, and found high fungicide loads. The insecticides esfenvalerate and phosmet were at a concentration higher than their median lethal dose in at least one pollen sample. While fungicides are typically seen as fairly safe for honey bees, we found an increased probability of Nosema infection in bees that consumed pollen with a higher fungicide load. Our results highlight a need for research on sub-lethal effects of fungicides and other chemicals that bees placed in an agricultural setting are exposed to.     

Aberrant DNA Methylation: Implications in Racial Health Disparity

BackgroundIncidence and mortality rates of colorectal carcinoma (CRC) are higher in African Americans (AAs) than in Caucasian Americans (CAs). Deficient micronutrient intake due to dietary restrictions in racial/ethnic populations can alter genetic and molecular profiles leading to dysregulated methylation patterns and the inheritance of somatic to germline mutations.ResultsDNA from the tumor of AA CRC patients, compared to adjacent normal tissues, contained 1,588 hypermethylated and 100 hypomethylated differentially methylated regions (DMRs). Whereas, 109 hypermethylated and 4 hypomethylated DMRs were observed in DNA from the tumor of CA CRC patients; representing a 14.6-fold and 25-fold change, respectively. Specifically; CHL1, 4 anti-inflammatory genes (i.e., NELL1, GDF1, ARHGEF4, and ITGA4), and 7 miRNAs (of which miR-9-3p and miR-124-3p have been implicated in CRC) were hypermethylated in DNA samples from AA patients with CRC. From the same sample set, RNAseq analysis revealed 108 downregulated genes (including 14 ribosomal proteins) and 34 upregulated genes (including POLR2B and CYP1B1 [targets of miR-124-3p]) in AA patients with CRC versus CA patients.ConclusionDNA methylation profile and/or products of its downstream targets could serve as biomarker(s) addressing racial health disparity.     

Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of photosystem II particles in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii the oxygen evolving enhancer protein 1 (OEE1), which is part of the oxygen evolving complex of photosystem II (PS II), is coded for by a single nuclear gene (psb1). The nuclear mutant FuD44 specifically lacks the OEE1 polypeptide and is completely deficient in photosynthetic oxygen evolution. In this mutant a 5 kb DNA insertion into the 5' region of the psb1 gene results in the complete absence of OEE1 mRNA and protein. A revertant, FuD44-R 2, which is capable of 30% of the photosynthetic oxygen evolution of wild-type cells, has lost 4 kb of the 5 kb DNA insert, and accumulates both OEE1 mRNA and protein, although at levels somewhat less than those of wild-type cells. Absence of the OEE1 protein in the FuD44 mutant does not affect the accumulation of other nuclear encoded PS II peripheral polypeptides. OEE1 absence does, however, result in a more rapid turnover of the chloroplast encoded PS II core polypeptides, thus resulting in a substantial deficiency of PS II core polypeptides in FuD44 cells. These PS II core proteins again accumulate in revertant FuD44-R2 cells.     

RNA and β-Hemolysin of Group B Streptococcus Induce Interleukin-1β (IL-1β) by Activating NLRP3 Inflammasomes in Mouse Macrophages

The inflammatory cytokine IL-1β is critical for host responses against many human pathogens. Here, we define Group B Streptococcus (GBS)-mediated activation of the Nod-like receptor-P3 (NLRP3) inflammasome in macrophages. NLRP3 activation requires GBS expression of the cytolytic toxin, β-hemolysin, lysosomal acidification, and leakage. These processes allow the interaction of GBS RNA with cytosolic NLRP3. The present study supports a model in which GBS RNA, along with lysosomal components including cathepsins, leaks out of lysosomes and interacts with NLRP3 to induce IL-1β production.