Autoradiographic method to screen for soil microorganisms which accumulate zinc.

An autoradiographic method was developed to screen for and isolate soil microorganisms which accumulate zinc (Zn). Diluted soil samples (Rubicon fine sand, Entic Haplorthods [pH 5.9]) were plated on soil extract-glucose agar containing radioactive 65Zn. After 7 days of incubation, individual colonies which accumulated sufficient 65Zn could be detected by autoradiography. These colonies were isolated and confirmed as Zn accumulators in pure culture by using the autoradiographic plate technique. Most Zn accumulators were filamentous fungi, identified as Penicillium janthinellum, Aspergillus fumigatus, and Paecilomyces sp. Isolates of Penicillium janthinellum were the most common Zn accumulators. The most abundant Zn-accumulating bacteria were Bacillus spp. The validity of the autoradiographic plate technique to differentiate soil microbes which accumulate Zn was examined independently by energy dispersive X-ray analysis in a scanning electron microscope. This method confirmed that fungal isolates which gave positive autoradiographic responses in the plate assay bioaccumulated more Zn in their biomass than fungal isolates from the same soil sample which gave negative autoradiographic responses. Thus, this technique can be applied to specifically screen for and isolate microbes from the environment which bioaccumulate Zn.     

Autoradiographic method to screen for soil microorganisms which accumulate zinc

An autoradiographic method was developed to screen for and isolate soil microorganisms which accumulate zinc (Zn). Diluted soil samples (Rubicon fine sand, Entic Haplorthods [pH 5.9]) were plated on soil extract-glucose agar containing radioactive 65Zn. After 7 days of incubation, individual colonies which accumulated sufficient 65Zn could be detected by autoradiography. These colonies were isolated and confirmed as Zn accumulators in pure culture by using the autoradiographic plate technique. Most Zn accumulators were filamentous fungi, identified as Penicillium janthinellum, Aspergillus fumigatus, and Paecilomyces sp. Isolates of Penicillium janthinellum were the most common Zn accumulators. The most abundant Zn-accumulating bacteria were Bacillus spp. The validity of the autoradiographic plate technique to differentiate soil microbes which accumulate Zn was examined independently by energy dispersive X-ray analysis in a scanning electron microscope. This method confirmed that fungal isolates which gave positive autoradiographic responses in the plate assay bioaccumulated more Zn in their biomass than fungal isolates from the same soil sample which gave negative autoradiographic responses. Thus, this technique can be applied to specifically screen for and isolate microbes from the environment which bioaccumulate Zn.     

Perfusion of hearts with triglyceride-rich particles reproduces the metabolic abnormalities in lipotoxic cardiomyopathy

Hearts with overexpression of anchored lipoprotein lipase (LpL) by cardiomyocytes (hLpL(GPI) mice) develop a lipotoxic cardiomyopathy. To characterize cardiac fatty acid (FA) and triglyceride (TG) metabolism in these mice and to determine whether changes in lipid metabolism precede cardiac dysfunction, hearts from young mice were perfused in Langendorff mode with [14C]palmitate. In hLpL(GPI) hearts, FA uptake and oxidation were decreased by 59 and 82%, respectively. This suggests reliance on an alternative energy source, such as TG. Indeed, these hearts oxidized 88% more TG. Hearts from young hLpL(GPI) mice also had greater uptake of intravenously injected cholesteryl ester-labeled Intralipid and VLDL. To determine whether perfusion of normal hearts would mimic the metabolic alterations found in hLpL(GPI) mouse hearts, wild-type hearts were perfused with [14C]palmitate and either human VLDL or Intralipid (0.4 mM TG). Both sources of TG reduced [14C]palmitate uptake (48% with VLDL and 45% with Intralipid) and FA oxidation (71% with VLDL and 65% with Intralipid). Addition of either heparin or LpL inhibitor P407 to Intralipid-containing perfusate restored [14C]palmitate uptake and confirmed that Intralipid inhibition requires local LpL. Our data demonstrate that reduced FA uptake and oxidation occur before mechanical dysfunction in hLpL(GPI) lipotoxicity. This physiology is reproduced with perfusion of hearts with TG-containing particles. Together, the results demonstrate that cardiac uptake of TG-derived FA reduces utilization of albumin-FA.     

Perkinsus olseni detected in Vietnamese aquacultured reef clams Tridacna crocea imported to the USA, following a mortality event

Morbidity and mortality were observed in a group of 30 reef clams Tridacna crocea that were imported to Florida, USA, from a Vietnamese culture facility and held in research facility aquaria. Clinical signs included an incompletely extended mantle, slow mantle responses to stimuli, and sloughing of byssal tissue beginning 2 to 5 d prior to death. Necropsy findings included emaciation, visceral mass edema, and rare multifocal 1 mm off-white to light-tan gill nodules. Histopathology revealed marked inflammation and necrosis within the visceral mass and gills, with interstitial edema and atrophy of glandular, gonadal, and muscular tissues. Inflamed tissues contained large numbers of 10 to 15 microm extracellular round organisms consistent with Perkinsus sp. trophozoites. The organisms often formed clusters of 1 to 4 cells and were surrounded by a 1 to 3 microm rim of eosinophilic material variably forming a radiating corona pattern and by 3 to 4 host hemocytes with dense round nuclei. Polymerase chain reaction assays indicated the presence of Perkinsus sp. DNA in these animals, and species-specific assays indicated the presence of P. olseni, and possibly other Perkinsus spp., but not P. marinus. Identification of Perkinsus spp. other than P. marinus in T. crocea imported from Vietnam confirms that importation of untested and unquarantined ornamental reef clams has possibly allowed incursion of P. olseni into the USA.     

Cardiac and CNS defects in a mouse with targeted disruption of suppressor of fused

The hedgehog (Hh) pathway is conserved from Drosophila to humans and plays a key role in embryonic development. In addition, activation of the pathway in somatic cells contributes to cancer development in several tissues. Suppressor of fused is a negative regulator of Hh signaling. Targeted disruption of the murine suppressor of fused gene (Sufu) led to a phenotype that included neural tube defects and lethality at mid-gestation (9.0-10.5 dpc). This phenotype resembled that caused by loss of patched (Ptch1), another negative regulator of the Hh pathway. Consistent with this finding, Ptch1 and Sufu mutants displayed excess Hh signaling and resultant altered dorsoventral patterning of the neural tube. Sufu mutants also had abnormal cardiac looping, indicating a defect in the determination of left-right asymmetry. Marked expansion of nodal expression in 7.5 dpc embryos and variable degrees of node dysmorphology in 7.75 dpc embryos suggested that the pathogenesis of the cardiac developmental abnormalities was related to node development. Other mutants of the Hh pathway, such as Shh, Smo and Shh/Ihh compound mutants, also have laterality defects. In contrast to Ptch1 heterozygous mice, Sufu heterozygotes had no developmental defects and no apparent tumor predisposition. The resemblance of Sufu homozygotes to Ptch1 homozygotes is consistent with mouse Sufu being a conserved negative modulator of Hh signaling.     

Loss of lipoprotein lipase-derived fatty acids leads to increased cardiac glucose metabolism and heart dysfunction

Long-chain fatty acids (FAs) are the predominant energy substrate utilized by the adult heart. The heart can utilize unesterified FA bound to albumin or FA obtained from lipolysis of lipoprotein-bound triglyceride (TG). We used heart-specific lipoprotein lipase knock-out mice (hLpL0) to test whether these two sources of FA are interchangeable and necessary for optimal heart function. Hearts unable to obtain FA from lipoprotein TG were able to compensate by increasing glucose uptake, glycolysis, and glucose oxidation. HLpL0 hearts had decreased expression of pyruvate dehydrogenase kinase 4 and increased cardiomyocyte expression of glucose transporter 4. Conversely, FA oxidation rates were reduced in isolated perfused hLpL0 hearts. Following abdominal aortic constriction expression levels of genes regulating FA and glucose metabolism were acutely up-regulated in control and hLpL0 mice, yet all hLpL0 mice died within 48 h of abdominal aortic constriction. Older hLpL0 mice developed cardiac dysfunction characterized by decreased fractional shortening and interstitial and perivascular fibrosis. HLpL0 hearts had increased expression of several genes associated with transforming growth factor-beta signaling. Thus, long term reduction of lipoprotein FA uptake is associated with impaired cardiac function despite a compensatory increase in glucose utilization.     

Effects of lipoprotein lipase and statins on cholesterol uptake into heart and skeletal muscle

Regulation of cholesterol metabolism in cultured cells and in the liver is dependent on actions of the LDL receptor. However, nonhepatic tissues have multiple pathways of cholesterol uptake. One possible pathway is mediated by LPL, an enzyme that primarily hydrolyzes plasma triglyceride into fatty acids. In this study, LDL uptake and tissue cholesterol levels in heart and skeletal muscle of wild-type and transgenic mice with alterations in LPL expression were assessed. Overexpression of a myocyte-anchored form of LPL in heart muscle led to increased uptake of LDL and greater heart cholesterol levels. Loss of LDL receptors did not alter LDL uptake into heart or skeletal muscle. To induce LDL receptors, mice were treated with simvastatin. Statin treatment increased LDL receptor expression and LDL uptake by liver and skeletal muscle but not heart muscle. Plasma creatinine phosphokinase as well as muscle mitochondria, cholesterol, and lipid droplet levels were increased in statin-treated mice overexpressing LPL in skeletal muscle. Thus, pathways affecting cholesterol balance in heart and skeletal muscle differ.     

Multiple CCR5 conformations on the cell surface are used differentially by human immunodeficiency viruses resistant or sensitive to CCR5 inhibitors

Resistance to small-molecule CCR5 inhibitors arises when HIV-1 variants acquire the ability to use inhibitor-bound CCR5 while still recognizing free CCR5. Two isolates, CC101.19 and D1/85.16, became resistant via four substitutions in the gp120 V3 region and three in the gp41 fusion peptide (FP), respectively. The binding characteristics of a panel of monoclonal antibodies (MAbs) imply that several antigenic forms of CCR5 are expressed at different levels on the surfaces of U87-CD4-CCR5 cells and primary CD4(+) T cells, in a cell-type-dependent manner. CCR5 binding and HIV-1 infection inhibition experiments suggest that the two CCR5 inhibitor-resistant viruses altered their interactions with CCR5 in different ways. As a result, both mutants became generally more sensitive to inhibition by CCR5 MAbs, and the FP mutant is specifically sensitive to a MAb that stains discrete cell surface clusters of CCR5 that may correspond to lipid rafts. We conclude that some MAbs detect different antigenic forms of CCR5 and that inhibitor-sensitive and -resistant viruses can use these CCR5 forms differently for entry in the presence or absence of CCR5 inhibitors.     

BOBBER1 Is a Noncanonical Arabidopsis Small Heat Shock Protein Required for Both Development and Thermotolerance

Plants have evolved a range of cellular responses to maintain developmental homeostasis and to survive over a range of temperatures. Here, we describe the in vivo and in vitro functions of BOBBER1 (BOB1), a NudC domain containing Arabidopsis (Arabidopsis thaliana) small heat shock protein. BOB1 is an essential gene required for the normal partitioning and patterning of the apical domain of the Arabidopsis embryo. Because BOB1 loss-of-function mutants are embryo lethal, we used a partial loss-of-function allele (bob1-3) to demonstrate that BOB1 is required for organismal thermotolerance and postembryonic development. Recombinant BOB1 protein functions as a molecular chaperone and prevents the aggregation of a model protein substrate in vitro. In plants, BOB1 is cytoplasmic at basal temperatures, but forms heat shock granules containing canonical small heat shock proteins at high temperatures. In addition to thermotolerance defects, bob1-3 exhibits pleiotropic development defects during all phases of development. bob1-3 phenotypes include decreased rates of shoot and root growth as well as patterning defects in leaves, flowers, and inflorescence meristems. Most eukaryotic chaperones play important roles in protein folding either during protein synthesis or during cellular responses to denaturing stress. Our results provide, to our knowledge, the first evidence of a plant small heat shock protein that has both developmental and thermotolerance functions and may play a role in both of these folding networks.Plants are autotrophic sessile organisms that depend on sunlight for their energetic needs. One consequence of this lifestyle is that plants are often subjected to high temperature stress, especially in dry conditions when transpirational cooling is limited. At a cellular level, elevated temperatures result in changes in protein structure that can result in the exposure of normally buried hydrophobic residues. As a consequence of thermal denaturation, proteins may aggregate and cease to function normally. A universal response to temperature-induced protein unfolding in all living organisms is the production of heat shock proteins (HSPs). HSPs are molecular chaperones that provide organismal thermotolerance by preventing the denaturation and aggregation of target proteins as well as facilitating protein refolding. Highly conserved HSPs are found in all organisms and include the small HSP (sHSP) as well as the Hsp60, Hsp70, Hsp90, and Hsp100 families (Baniwal et al., 2004; Taiz and Zeiger, 2006). Members of the sHSP family are defined by their small size (12–43 kD), their ability to prevent protein aggregation, and by a conserved α-crystallin domain (ACD). Plants are unusual in the large number of ACD-containing sHSPs encoded by their genomes: Arabidopsis (Arabidopsis thaliana) has 19 compared to 10 in humans, four in Drosophila melanogaster, and one or two in bacteria (Haslbeck et al., 2005).Although the biochemical activity of plant sHSPs has been well characterized (Lee et al., 1995, 1997; Basha et al., 2004; Siddique et al., 2008), little is known about the in vivo functions of plant sHSPs, perhaps due to functional redundancies in this large gene family. Apart from temperature-dependent changes in hypocotyl elongation, which reflects the ability of cells to expand, no developmental roles for a sHSP have been reported in plants (Jenks and Hasegawa, 2005; Dafny-Yelin et al., 2008). In addition to redundancy, a lack of known developmental functions for plant sHSPs may also be a result of the fact that most are only expressed in response to heat or other stresses. Exceptions include a subset of sHSPs expressed during seed and pollen maturation, developmental stages that involve desiccation (Wehmeyer and Vierling, 2000; Dafny-Yelin et al., 2008). However, since most plant sHSPs are not expressed under nonstress conditions, they are unlikely to affect normal growth and development (Swindell et al., 2007).BOBBER1 (BOB1; At5g53400) is an essential gene required for the normal partitioning and patterning of the apical domain of the Arabidopsis embryo. In bob1-1 and bob1-2 null mutants, meristematic identity is expanded into the portion of the embryo that would normally form the seedling leaves (cotyledons), which in turn are never established. Auxin gradients are never established in bob1 mutant embryos. However, since there are multiple feedback loops involved in auxin signaling and transport, it is unclear whether the lack of auxin maxima in bob1 mutants is a direct or indirect result of a lack of BOB1 activity (Jurkuta et al., 2009). BOB1 encodes a protein with C-terminal homology to NudC, a protein identified in a screen for genes required for nuclear migration in Aspergillus nidulans. Genes with homology to NudC have been shown to interact with dynein microtubule motors. In mammalian tissue culture systems, interference with NudC-like gene function results in defects in chromosome segregation and cytokinesis (Aumais et al., 2003; Nishino et al., 2006; Zhou et al., 2006). The NudC domain has predicted structural homology with the α-crystallin/p23 protein families (Garcia-Ranea et al., 2002), which includes the ACD-containing sHSPs. The ACD, originally identified in the α-crystallin chaperone of the vertebrate eye lens, forms a structure consisting of two antiparallel β-sheets in a sandwich (Scharf et al., 2001; Haslbeck et al., 2005). The NMR structure of the mouse NudC homolog (PDB 1wfi) has the same β-sheet sandwich structure that provides support for the predicted structural homology between NudC domains and ACDs. These observations suggest that NudC domain proteins might share conserved functions with sHSPs. Support for this hypothesis comes from Caenorhabditis elegans where the NudC homolog NUD-1, an essential gene, displays protein chaperone activity in vitro (Faircloth et al., 2009).Here, we use bob1-3, a partial loss-of-function allele, to show that BOB1 is required for normal development and meristem function after embryogenesis. To determine whether BOB1 functions as a protein chaperone, we characterized the in vitro activity of BOB1 protein. We also investigated the thermotolerance functions of BOB1 using bob1-3 and used a BOB1:GFP line that is biologically active to document that BOB1 protein is incorporated into heat shock granules (HSGs) at high temperatures. All of these data suggest that BOB1 encodes a novel sHSP with dual functions in development and thermotolerance. To our knowledge, this is the first demonstration of a developmental patterning function for a plant sHSP.     

IL-1 induces p62/SQSTM1 and autophagy in ERα+/PR+ BCa cell lines concomitant with ERα and PR repression,conferring an ERα−/PR− BCa-like phenotype

Estrogen receptor α (ERα)^low/− tumors are associated with breast cancer (BCa) endocrine resistance, where ERα low tumors show a poor prognosis and a molecular profile similar to triple negative BCa tumors. Interleukin-1 (IL-1) downregulates ERα accumulation in BCa cell lines, yet the cells can remain viable. In kind, IL-1 and ERα show inverse accumulation in BCa patient tumors and IL-1 is implicated in BCa progression. IL-1 represses the androgen receptor hormone receptor in prostate cancer cells concomitant with the upregulation of the prosurvival, autophagy-related protein, Sequestome-1 (p62/SQSTM1; hereinafter, p62); and given their similar etiology, we hypothesized that IL-1 also upregulates p62 in BCa cells concomitant with hormone receptor repression. To test our hypothesis, BCa cell lines were exposed to conditioned medium from IL-1-secreting bone marrow stromal cells (BMSCs), IL-1, or IL-1 receptor antagonist. Cells were analyzed for the accumulation of ERα, progesterone receptor (PR), p62, or the autophagosome membrane protein, microtubule-associated protein 1 light chain 3 (LC3), and for p62-LC3 interaction. We found that IL-1 is sufficient to mediate BMSC-induced ERα and PR repression, p62 and autophagy upregulation, and p62-LC3 interaction in ERα⁺/PR⁺ BCa cell lines. However, IL-1 does not significantly elevate the high basal p62 accumulation or high basal autophagy in the ERα⁻/PR⁻ BCa cell lines. Thus, our observations imply that IL-1 confers a prosurvival ERα⁻/PR⁻ molecular phenotype in ERα⁺/PR⁺ BCa cells that may be dependent on p62 function and autophagy and may underlie endocrine resistance.