Glucocorticoid receptor binding to calf thymus DNA. 1. Identification and characterization of a macromolecular factor involved in receptor-steroid complex binding to DNA.

Activation of receptor-steroid complexes to a form with high affinity for DNA is a poorly understood process involving multiple components in addition to the holoreceptor. Employing rat HTC cells as the source of glucocorticoid receptor, we show that maximal receptor binding to calf thymus DNA is mediated by a previously unknown small molecular weight factor. This factor can be removed from cytosolic preparations of receptor by gel filtration chromatography. Salt extraction of crude nuclear pellets afforded much larger amounts of a similar DNA-binding activity factor. The cytoplasmic factor and the more abundant nuclear factor were identical on the basis of their similar physical properties. The factor was precipitable in the crude state with (NH4)2SO4 and stable to heat as well as freezing and thawing. Chromatography on DNA-cellulose revealed that the factor itself did not bind to DNA. The factor could be filtered through a Centricon C-3 microconcentrator (molecular weight cutoff approximately 3000) but was excluded from Sephadex G-10 columns. These parameters enable us to determine an apparent molecular weight of 700-3000 for this factor. The presence of large amounts of this factor in nuclei accounts for the previously unexplained observation that, following size exclusion chromatography, more activated complexes bind to nuclei than to DNA. These data indicate that some, but not all, of the activated complexes require factor to be able to bind to DNA. The predominantly nuclear localization of this factor, coupled with its ability to increase DNA binding, attests to the biological relevance of this factor in the whole cell action of receptor-glucocorticoid complexes.     

Interaction between pathogenic and saprobic fungi isolated from soybean roots and seeds

A variety of interactions was recorded in culture between 11 saprobic fungi isolated from soybean (Glycine max) roots and seeds and the soybean pathogens Cercospora sojina, Colletotrichum truncatum, Macrophomina phaseolina, Phomopsis sojae, and Septoria glycines. The most active saprobes were Aspergillus terreus, Chaetomium cupreum, Epicoccum nigrum, Gliocladium roseum, Myrothecium roridum, Penicillium thomii, and Trichothecium roseum. Hyphal lysis of several fungal pathogens by Acremonium sp., C. cupreum and P. thomii was recorded perhaps because of parasitism by G. roseum and T. roseum. In greenhouse studies, seeds coated with G. roseum, P. thomii, and T. harzianum emerged significantly (P=0.05) more than those coated with A. terreus and the control. In field studies, seeds coated with a conidial suspension of A. terreus, G. roseum, P. thomii or Trichoderma harzianum produced a significantly greater stand than the control. The area of cotyledons covered with lesions caused by C. truncatum was significantly less on seeds coated with G. roseum, P. thomii and T. harzianum than the control.     

Identification and characterization of a calcium-binding protein in the mouse chorioallantoic placenta.

Mouse chorioallantoic placenta contains a specific calcium-binding protein (MCaBP). A procedure involving gel filtration and ion-exchange chromatography was developed to purify the MCaBP. The MCaBP activity increased as a function of embryonic gestation and was highly specific for Ca2+. The MCaBP is a monomeric protein of Mr 57000, with pI 4.7. Specific antibodies were prepared against the MCaBP and were used to localize the MCaBP to syncytiotrophoblasts of the chorionic villi of mouse chorioallantoic placenta. These properties suggest that the MCaBP may be involved in transplacental calcium transport.     

Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses. Correspondence to: D.L. Distel     

Interleukin-12 inhibits hepatitis B virus replication in transgenic mice.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by antigen-presenting cells that has the ability to induce gamma interferon (IFN-gamma) secretion by T and natural killer cells and to generate normal Th1 responses. These properties suggest that IL-12 may play an important role in the immune response to many viruses, including hepatitis B virus (HBV). Recently, we have shown that HBV-specific cytotoxic T lymphocytes inhibit HBV replication in the livers of transgenic mice by a noncytolytic process that is mediated in part by IFN-gamma. In the current study, we demonstrated that the same antiviral response can be initiated by recombinant murine IL-12 and we showed that the antiviral effect of IL-12 extends to extrahepatic sites such as the kidney. Southern blot analyses revealed the complete disappearance of HBV replicative intermediates from liver and kidney tissues at IL-12 doses that induce little or no inflammation in these tissues. In addition, immunohistochemical analysis demonstrated the disappearance of cytoplasmic hepatitis B core antigen from both tissues after IL-12 treatment, suggesting that IL-12 either prevents the assembly or triggers the degradation of the nucleocapsid particles within which HBV replication occurs. Importantly, we demonstrated that although IFN-gamma, tumor necrosis factor alpha, and IFN-alpha/beta mRNA are induced in the liver and kidney after IL-12 administration, the antiviral effect of IL-12 is mediated principally by its ability to induce IFN-gamma production in this model. These results suggest that IL-12, through its ability to induce IFN-gamma, probably plays an important role in the antiviral immune response to HBV during natural infection. Further, since relatively nontoxic doses of recombinant IL-12 profoundly inhibit HBV replication in the liver and extrahepatic sites in this model, IL-12 may have therapeutic value as an antiviral agent for the treatment of chronic HBV infection.     

Pharmacological inhibition of the MEK5/ERK5 and PI3K/Akt signaling pathways synergistically reduces viability in triple-negative breast cancer

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.     

Functional and anatomical variations in retinorecipient brain areas in Arvicanthis niloticus and Rattus norvegicus: implications for the circadian and masking systems

ABSTRACT

Daily rhythms in light exposure influence the expression of behavior by entraining circadian rhythms and through its acute effects on behavior (i.e., masking). Importantly, these effects of light are dependent on the temporal niche of the organism; for diurnal organisms, light increases activity, whereas for nocturnal organisms, the opposite is true. Here we examined the functional and morphological differences between diurnal and nocturnal rodents in retinorecipient brain regions using Nile grass rats (Arvicanthis niloticus) and Sprague-Dawley (SD) rats (Rattus norvegicus), respectively. We established the presence of circadian rhythmicity in cFOS activation in retinorecipient brain regions in nocturnal and diurnal rodents housed in constant dark conditions to highlight different patterns between the temporal niches. We then assessed masking effects by comparing cFOS activation in constant darkness (DD) to that in a 12:12 light/dark (LD) cycle, confirming light responsiveness of these regions during times when masking occurs in nature. The intergeniculate leaflet (IGL) and olivary pretectal nucleus (OPN) exhibited significant variation among time points in DD of both species, but their expression profiles were not identical, as SD rats had very low expression levels for most timepoints. Light presentation in LD conditions induced clear rhythms in the IGL of SD rats but eliminated them in grass rats. Additionally, grass rats were the only species to demonstrate daily rhythms in LD for the habenula and showed a strong response to light in the superior colliculus. Structurally, we also analyzed the volumes of the visual brain regions using anatomical MRI, and we observed a significant increase in the relative size of several visual regions within diurnal grass rats, including the lateral geniculate nucleus, superior colliculus, and optic tract. Altogether, our results suggest that diurnal grass rats devote greater proportions of brain volume to visual regions than nocturnal rodents, and cFOS activation in these brain regions is dependent on temporal niche and lighting conditions.     

Evaluating regional resiliency of coastal wetlands to sea level rise through hypsometry‐based modeling

Sea level rise (SLR) threatens coastal wetlands worldwide, yet the fate of individual wetlands will vary based on local topography, wetland morphology, sediment dynamics, hydrologic processes, and plant‐mediated feedbacks. Local variability in these factors makes it difficult to predict SLR effects across wetlands or to develop a holistic regional perspective on SLR response for a diversity of wetland types. To improve regional predictions of SLR impacts to coastal wetlands, we developed a model that addresses the scale‐dependent factors controlling SLR response and accommodates different levels of data availability. The model quantifies SLR‐driven habitat conversion within wetlands across a region by predicting changes in individual wetland hypsometry. This standardized approach can be applied to all wetlands in a region regardless of data availability, making it ideal for modeling SLR response across a range of scales. Our model was applied to 105 wetlands in southern California that spanned a broad range of typology and data availability. Our findings suggest that if wetlands are confined to their current extents, the region will lose 12% of marsh habitats (vegetated marsh and unvegetated flats) with 0.6 m of SLR (projected for 2050) and 48% with 1.7 m of SLR (projected for 2100). Habitat conversion was more drastic in wetlands with larger proportions of marsh habitats relative to subtidal habitats and occurred more rapidly in small lagoons relative to larger sites. Our assessment can inform management of coastal wetland vulnerability, improve understanding of the SLR drivers relevant to individual wetlands, and highlight significant data gaps that impede SLR response modeling across spatial scales. This approach augments regional SLR assessments by considering spatial variability in SLR response drivers, addressing data gaps, and accommodating wetland diversity, which will provide greater insights into regional SLR response that are relevant to coastal management and restoration efforts.