HIV-1 Nef binds PACS-2 to assemble a multikinase cascade that triggers major histocompatibility complex class I (MHC-I) down-regulation: analysis using short interfering RNA and knock-out mice

Human immunodeficiency virus, type 1, negative factor (Nef) initiates down-regulation of cell-surface major histocompatibility complex-I (MHC-I) by assembling an Src family kinase (SFK)-ZAP70/Syk-phosphoinositide 3-kinase (PI3K) cascade through the sequential actions of two sites, Nef EEEE(65) and PXXP(75). The internalized MHC-I molecules are then sequestered in endosomal compartments by a process requiring Nef Met(20). How Nef assembles the multikinase cascade to trigger the MHC-I down-regulation pathway is unknown. Here we report that EEEE(65)-dependent binding to the sorting protein PACS-2 targets Nef to the paranuclear region, enabling PXXP(75) to bind and activate a trans-Golgi network (TGN)-localized SFK. This SFK then phosphorylates ZAP-70 to recruit class I PI3K by interaction with the p85 C-terminal Src homology 2 domain. Using splenocytes and embryonic fibroblasts from PACS-2(-/-) mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system.     

Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks

In an effort to build a comprehensive genomic approach to food safety challenges, the FDA has implemented a whole genome sequencing effort, GenomeTrakr, which involves the sequencing and analysis of genomes of foodborne pathogens. As a part of this effort, we routinely sequence whole genomes of Listeria monocytogenes (Lm) isolates associated with human listeriosis outbreaks, as well as those isolated through other sources. To rapidly establish genetic relatedness of these genomes, we evaluated tetranucleotide frequency analysis via the JSpecies program to provide a cursory analysis of strain relatedness. The JSpecies tetranucleotide (tetra) analysis plots standardized (z-score) tetramer word frequencies of two strains against each other and uses linear regression analysis to determine similarity (r²). This tool was able to validate the close relationships between outbreak related strains from four different outbreaks. Included in this study was the analysis of Lm strains isolated during the recent caramel apple outbreak and stone fruit incident in 2014. We identified that many of the isolates from these two outbreaks shared a common 4b variant (4bV) serotype, also designated as IVb-v1, using a qPCR protocol developed in our laboratory. The 4bV serotype is characterized by the presence of a 6.3 Kb DNA segment normally found in serotype 1/2a, 3a, 1/2c and 3c strains but not in serotype 4b or 1/2b strains. We decided to compare these strains at a genomic level using the JSpecies Tetra tool. Specifically, we compared several 4bV and 4b isolates and identified a high level of similarity between the stone fruit and apple 4bV strains, but not the 4b strains co-identified in the caramel apple outbreak or other 4b or 4bV strains in our collection. This finding was further substantiated by a SNP-based analysis. Additionally, we were able to identify close relatedness between isolates from clinical cases from 1993–1994 and a single case from 2011 as well as links between two isolates from over 30 years ago. The identification of these potential links shows that JSpecies Tetra analysis can be a useful tool in rapidly assessing genetic relatedness of Lm isolates during outbreak investigations and for comparing historical isolates. Our analyses led to the identification of a highly related clonal group involved in two separate outbreaks, stone fruit and caramel apple, and suggests the possibility of a new genotype that may be better adapted for certain foods and/or environment.     

The Effects of Sub-Regional Climate Velocity on the Distribution and Spatial Extent of Marine Species Assemblages

Many studies illustrate variable patterns in individual species distribution shifts in response to changing temperature. However, an assemblage, a group of species that shares a common environmental niche, will likely exhibit similar responses to climate changes, and these community-level responses may have significant implications for ecosystem function. Therefore, we examine the relationship between observed shifts of species in assemblages and regional climate velocity (i.e., the rate and direction of change of temperature isotherms). The assemblages are defined in two sub-regions of the U.S. Northeast Shelf that have heterogeneous oceanography and bathymetry using four decades of bottom trawl survey data and we explore temporal changes in distribution, spatial range extent, thermal habitat area, and biomass, within assemblages. These sub-regional analyses allow the dissection of the relative roles of regional climate velocity and local physiography in shaping observed distribution shifts. We find that assemblages of species associated with shallower, warmer waters tend to shift west-southwest and to shallower waters over time, possibly towards cooler temperatures in the semi-enclosed Gulf of Maine, while species assemblages associated with relatively cooler and deeper waters shift deeper, but with little latitudinal change. Conversely, species assemblages associated with warmer and shallower water on the broad, shallow continental shelf from the Mid-Atlantic Bight to Georges Bank shift strongly northeast along latitudinal gradients with little change in depth. Shifts in depth among the southern species associated with deeper and cooler waters are more variable, although predominantly shifts are toward deeper waters. In addition, spatial expansion and contraction of species assemblages in each region corresponds to the area of suitable thermal habitat, but is inversely related to assemblage biomass. This suggests that assemblage distribution shifts in conjunction with expansion or contraction of thermal habitat acts to compress or stretch marine species assemblages, which may respectively amplify or dilute species interactions to an extent that is rarely considered. Overall, regional differences in climate change effects on the movement and extent of species assemblages hold important implications for management, mitigation, and adaptation on the U.S. Northeast Shelf.     

Ospemifene inhibits the growth of dimethylbenzanthracene-induced mammary tumors in Sencar mice

Ospemifene is a new selective estrogen receptor modulator (SERM) that is being developed for the treatment of urogenital atrophy and osteoporosis. Similarly to other SERMs, ospemifene exhibits antiestrogenic effects in breast tissue, which led to the hypothesis that it may be a potential breast cancer chemopreventive agent. We first assessed the ability of ospemifene, compared to tamoxifen and raloxifene, to prevent dimethylbenzanthracene (DMBA)-induced mammary tumors in female Sencar mice. Ospemifene (N = 18), tamoxifen (N = 20) and raloxifene (N = 17), each dosed at 50 mg/kg, were administered daily by oral gavage, in combination with 20 microg DMBA for the first 6 weeks. Control mice (N = 21) received vehicle plus DMBA only for the first 6 weeks. Daily treatment then continued for 37 weeks. As hypothesized, ospemifene greatly reduced the incidence of mammary carcinomas compared to control mice (p = 0.003), similar to tamoxifen (p = 0.0004); however, in the raloxifene group, no significant effect was seen in mammary tumor prevention (p = 0.20). A follow-up study comparing ospemifene (N = 20) to tamoxifen (N = 20) in the same model was then performed to confirm the results of the first study. The results of the follow-up study, which extended the treatment to 52 weeks, confirmed the results of our previous study, with ospemifene (p = 0.01) and tamoxifen (p = 0.004) significantly decreasing mammary carcinomas compared to controls. The results of these two studies suggest that women taking ospemifene for osteoporosis and/or urogenital atrophy may further benefit from ospemifene's breast cancer chemopreventive effects.     

Changes in replication, nuclear location, and expression of the Igh locus after fusion of a pre-B cell line with a T cell line

We have previously observed that replication and nuclear location of the murine Igh locus are developmentally regulated during B cell differentiation. In non-B, B, and plasma cells, sequences near the 3' end of the Igh locus replicate early in S while upstream Vh sequences replicate late in S, and the Igh locus is located near the nuclear periphery. In fact, in MEL non-B cells, replication of a 500-kb segment containing Igh-C and flanking sequences occurs progressively later throughout S by 3' to 5' unidirectional fork movement. In contrast, in pro- and pre-B cells, the entire 3-Mb Igh locus is located away from the nuclear periphery and replicates early in S by forks progressing in both directions. In this study, using an 18-81 (pre-B) x BW5147 (T) cell fusion system in which Igh expression is extinguished, we found that in all Igh alleles, Vh sequences replicated later in S than 3' Igh sequences (similar to that detected in BW5147), but the Igh locus was situated away from the nuclear periphery (similar to that observed in 18-81). Thus, pre-B cell-derived Igh genes had changes in replication timing, but not in nuclear location, whereas T cell-derived Igh genes changed their nuclear location but not their replication timing. These data are consistent with the silencing of a pre-B cell-specific replication program in the fusion hybrid cells and independent regulation of the nuclear location of Igh loci.     

Monoclonal antibodies to ThB detect close linkage ofLy-6 and a gene regulating ThB expression

We have generated three hybridomas producing rat monoclonal antibodies to a surface antigen, ThB, that is shared by murine B lymphocytes and approximately 50 percent of murine thymocytes. These antibodies, produced by immunizations with MOPC-104E cells, appear to recognize the same antigen that was previously detected by rabbit and goat antisera to MOPC-104E cells (Yutoku et al. 1974, Yutoku et al. 1976).Using these antibodies, we have studied a genetic polymorphism that is associated with the level of ThB expression on B lymphocytes but not with the antigen's expression on thymocytes. We present evidence that this trait is controlled by one gene,Thb, which we find to be very closely linked to the gene or genes controlling the Ly-6, Ly-8, DAG, and Ala 1 antigen(s). While the latter four antigens were described as markers on mature T (or activated T and B) lymphocytes, ThB is restricted to immature thymocytes and all B cells. ThB is not expressed on kidney, although some investigators (McKenzie et al. 1977 a, Halloran et al. 1978) report Ly-6 expression on that tissue. SJL/J, C57BL/10JHz, DBA/2J, and AKR/J are among the mouse strains carrying theThb ^h allele, while BALB/cN, CBA/J, C3H.SW/SnHz, and A/J carry theThb ^l allele. The ThB antigen has not yet been identified as a glycoprotein after cell-surface iodination, NP-40 solubilization, and immunoprecipitation.This work was supported in part by grants from the National Institutes of Health (AI-08917, CA-04681, GM-17367).     

Comparison of the 13C-n.m.r. spectra of gangliosides GM1 with those of GM1-oligosaccharide and asialo-GM1

The ¹³C-n.m.r. spectra of asialo-G_M1 and G_M1-oligosaccharide are completely assigned and compared to those previously found for intact G_M1 and for the series G_M4, G_M3, G_M2, G_M1, G_D1a, G_D1b, and G_T1b. Removal of the ceramide residue from G_M1 liberated a free, reducing aldehyde group, which was reflected in a doubling of the ¹³C-n.m.r. signals assignable to the d-glucose residue because of α,β equilibrium. The spectrum of asialo-G_M1 lacks the resonances from the sialic acid residue, as expected; in addition, several resonances from the neutral gangliotetraglycosyl residue shifted to different field positions after removal of sialic acid from G_M1. These resonances include that of C-4 of the inner β-d-galactosyl residue, and C-1 of the 2-acetamido-2-deoxy-d-galactosyl residue that is near the site of attachment of the sialosyl residue. The differences between the chemical shifts of the carbon resonances of oligomeric and monomeric saccharides, termed linkage shifts, provide a quantitative assignment aid. They are ～ $\text{1}\text{3}$ of those for residues linked to sialic acid than those for residues linked to the neutral hexose chain. Correlations among linkage shifts for pairs of glycosidically-linked carbon atoms for asialo-G_M1 and G_M1-oligosaccharide were compared with those for the series of gangliosides G_M4 to G_T1b, and differences are noted for resonances for carbon atoms near the sialic acid residue. The spectrum of ganglioside G_M1b, a positional isomer of G_M1 whose ¹³C-n.m.r. spectrum has not yet been observed, is predicted.     

The acute effect of 30 min of moderate exercise on high density lipoprotein cholesterol in untrained middle-aged men

Fifteen middle-aged, untrained (defined as no regular exercise) men (mean age 49.9 years, range 42-67) cycled on a cycle ergometer at 50 rpm for 30 min at an intensity producing 60% predicted maximum heart rate [(fc,max), where fc,max = 220 - age]. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride (Tg) concentrations were measured from fasting fingertip capillary blood samples collected at rest, after 15 and 30 min of exercise, and at 15 min post-exercise. The mean HDL-C level increased significantly from the resting level of 0.85 mmol.l-1 to 0.97 mmol.l-1 (P < 0.05) after 15 min of exercise, increased further to 1.08 mmol.l-1 (P < 0.01) after 30 min of exercise and remained elevated at 1.07 mmol.l-1 (P < 0.01) at 15 min post-exercise. These increases represented changes above the mean resting level of 14.1%, 27.1% and 25.9% respectively. The HDL-C/LDL-C ratio increased significantly from a resting ratio of 0.20 to 0.26 after 30 min of exercise (P < 0.01) and to 0.24 at 15 min post-exercise (P < 0.05). The mean Tg level increased significantly from a resting level of 0.88 mmol.l-1 to 1.05 mmol.l-1 after 15 min, and to 1.06 mmol.l-1 after 30 min of exercise (P < 0.05 at each time). The TC/HDL-C ratio decreased significantly (P = 0.05) after 30 min of exercise and at 15 min post-exercise by 18.8% and 14%, respectively. No significant changes were observed in the levels of TC or LDL-C over time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1–43)) and Ser¹⁹-phosphorylated (THp-(1–43)) states by surface plasmon resonance. THp-(1–43) showed high affinity for 14-3-3 proteins (K_d ∼ 0.5 μm for 14-3-3γ and -ζ and 7 μm for 14-3-3η). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S_0.5 = 25–58 μm (TH-(1–43)) and S_0.5 = 135–475 μm (THp-(1–43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3γ showed a preferential binding to membranes, compared with 14-3-3ζ, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3γ for negatively charged membranes (S_0.5 = 1–9 μm) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser¹⁹-phosphorylated TH, 14-3-3γ, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of l-DOPA and dopamine synthesis.     

Increased fat deposition in injured skeletal muscle is regulated by sex-specific hormones

Sex differences in skeletal muscle regeneration are controversial; comparisons of regenerative events between sexes have not been rigorously defined in severe injury models. We comprehensively quantified inflammation and muscle regeneration between sexes and manipulated sex-specific hormones to determine effects on regeneration. Cardiotoxin injury was induced in intact, castrated and ovariectomized female and male mice; ovariectomized mice were replaced with low- or high-dose 17-β estradiol (E(2)) or progesterone (P4). Extent of injury was comparable between intact mice, but females were more efficient in removal of necrotic debris, despite similar tissue levels of inflammatory cells and chemokines. Myofiber size during regeneration was equivalent between intact mice and after castration or ovariectomy (OVX) but was decreased (P < 0.001) in ovariectomized mice with high-dose E(2) replacement. Intermuscular adipocytes were absent in uninjured muscle, whereas adipocyte area was increased among regenerated myofibers in all groups. Interestingly, intermuscular fat was greater (P = 0.03) in intact females at day 14 compared with intact males. Furthermore, castration increased (P = 0.01) and OVX decreased adipocyte accumulation. After OVX, E(2), but not P4, replacement decreased (P ≤ 0.03) fat accumulation. In conclusion, sex-dependent differences in regeneration consisted of more efficient removal of necrosis and increased fat deposition in females with similar injury, inflammation, and regenerated myofiber size; high-dose E(2) decreased myofiber size and fat deposition. Adipocyte accumulation in regenerating muscle was influenced by sex-specific hormones. Recovery following muscle injury was different between males and females, and sex-specific hormones contributed to these differences, suggesting that sex-specific treatments could be beneficial after injury.