Ceramide activates a mitochondrial p38 mitogen-activated protein kinase: A potential mechanism for loss of mitochondrial transmembrane potential and apoptosis

This study examined the impact of ceramide, an intracellular mediator of apoptosis, on the mitochondria to test the hypothesis that ceramide utilized p38 MAPK in the mitochondria to alter mitochondrial potential and induce apoptosis. The capacity of ceramide to adversely affect mitochondria was demonstrated by the significant loss of mitochondrial potential (ΔΨ_m), indicated by a J-aggregate fluorescent probe, after embryonic chick cardiomyocytes were treated with the cell permeable ceramide analogue C₂-ceramide. p38 MAPK was identified in the mitochondrial fraction of the cell and p38 MAPK phosphorylation in this mitochondrial fraction of the cell occurred with ceramide treatment. In addition, SAPK phosphorylation and a decrease in ERK phosphorylation occurred in whole cell lysates after ceramide treatment. The p38 MAPK inhibitor SB 202190 but not the MEK inhibitor PD 98059 significantly inhibited ceramide-induced apoptosis and loss of ΔΨ_m. These data suggest that p38 MAPK is present in the mitochondria and its activation by ceramide indicates local signaling more directly coupled to the mitochondrial pathway in apoptosis. (Mol Cell Biochem 278: 39–51, 2005)     

A target function for quaternary structural refinement from small angle scattering and NMR orientational restraints

We present a novel target function based on atomic coordinates that permits quaternary structural refinement of multi-domain protein–protein or protein–RNA complexes. It requires that the high-resolution structures of the individual domains are known and that small angle scattering (SAS) data as well as NMR orientational restraints from residual dipolar couplings (RDCs) of the complex are available. We show that, when used in combination, the translational and rotational restraints contained in SAS intensities and RDCs, respectively, define a target potential function that permits to determine the overall topology of complexes made up of domains with low internal symmetry. We apply the target function on a modestly anisotropic model system, the Barnase/Barstar complex, and discuss factors that influence the structural refinement such as data errors and the geometrical properties of the individual domains.     

Human macrophages do not require phagosome acidification to mediate fungistatic/fungicidal activity against Histoplasma capsulatum

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mphi). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mphi that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of approximately 6.4-6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. Mphi that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by Mphi, also maintained an intraphagosomal pH of approximately 6.5 over a period of several hours. Stimulation of human Mphi fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin, an inhibitor of the vacuolar ATPase. Human Mphi cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal Mphi digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse Mphi fungistatic activity induced by IFN-gamma. Thus, human Mphi do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse Mphi do require acidification for fungicidal but not fungistatic activity.     

The functional and numerical response of Typhlodromips swirskii (Acari: Phytoseiidae) to the factitious prey Suidasia medanensis (Acari: Suidasidae) in the context of a breeding sachet

The whitefly and thrips predator Typhlodromips swirskii (Athias-Henriot) (Acari: Phytoseiidae) can be reared on the factitious astigmatid mite Suidasia medanensis (Oudemans) (Acari: Suidasiidae). The predator–prey relationship allows the system to be incorporated into a breeding sachet which releases predators into a crop over several weeks ensuring predator presence on arrival of the target pests and increased predator numerical response on the crop through immigration from the breeding sachet. This study investigated whether the prey preference and functional and numerical response of T. swirskii to different development stages of S. medanensis can provide understanding of the predator–prey interactions sustaining such a breeding sachet. T. swirskii elicited a strong preference to egg stages of S. medanensis, exhibited a Type II functional response and increased oviposition rate with increasing prey density. The relevance of these attributes to a balanced breeding sachet is discussed.     

Resorption of mandibular alveolar bone following loss of molar teeth and its relationship to age at death in a human skeletal population

Estimating adult age at death in skeletal remains is problematic, particularly in older adults. Molar wear is arguably the most reliable ageing technique for palaeopopulations, but many older adult skeletons have lost their molar teeth ante mortem, precluding its application. Resorption of the alveolar process occurs following tooth loss, and this appears to continue for a prolonged period. The current work investigates the relationship of this process to individual age in a nineteenth century AD European archaeological skeletal series of known age at death (N = 92 individuals), and discusses its potential as an age indicator. Mandibular corpus height was measured at the different molar positions. In females, reduction of corpus height with age was found at molar positions showing ante mortem loss. In both sexes, a relationship was found between age and a simple composite measure of corpus height in the molar region in those showing ante mortem loss of one or more mandibular molars. The correlation was stronger in females (r = ?0.74) than in males (r = ?0.49), appeared approximately linear, and continued into the ninth decade, the oldest age group in the study material. The results suggest that investigation of height of the posterior part of the mandibular corpus as a skeletal age indicator for individuals that have lost one or more molar teeth is merited in other palaeopopulations. Am J Phys Anthropol 153:643–652, 2014. © 2014 Wiley Periodicals, Inc.     

In situ formation of blends by photopolymerization of poly(ethylene glycol) dimethacrylate and polylactide

Blends of cross-linked poly(ethylene glycol) dimethacrylate (PEGDMA) and poly(d,l-lactide) (PLA) were prepared by mixing photoactive PEGDMA (molecular mass: 875 g/mol) and PLA, and subsequently photopolymerizing the mixture with visible light. The effects of PLA molecular mass and mass fraction on the rheological properties of the PEGDMA/PLA mixtures, and on the degree of methacrylate vinyl conversion (DC), as well as blend miscibility, microstructure, mechanical properties, in vitro swelling behavior, and cell responses were studied. PLA-2K (molecular mass: 2096 g/mol) and PLA-63K (molecular mass: 63 000 g/mol) formed miscible and partially miscible blends with cross-linked PEGDMA, respectively. The addition of the PLA-2K did not affect the immediate or post-cure (>24 h) DC of the PEGDMA upon photopolymerization. However, the addition of PLA-63K decreased the immediate DC of the PEGDMA, which can be increased through extending the curing time or post-curing period. Compared to the cross-linked neat PEGDMA and PLA-2K/PEGDMA blends, PLA-63K/PEGDMA blends were significantly stronger, stiffer, and tougher. Both types of blends and the cross-linked PEGDMA swelled when soaked in a phosphate buffered saline (PBS) solution. The attachment and spreading of MCT3-E1 cells increased with increasing PLA-63K content in the blends. The facile and rapid formation of PEGDMA/PLA blends by photopolymerization represents a simple and efficient approach to a class of biomaterials with a broad spectrum of properties.     

ERbeta protein expression in female cynomolgus monkey and CF-1 mouse brain: Western analysis

In humans and rodents, multiple ERbeta variants with sizes ranging from 477-549 amino acids (aa) have been described. The identification of these variants in target tissues has important implications for estrogen signaling and cellular responsiveness. Western blot analysis using two anti-ERbeta antibodies specific for mammalian ERbeta sequences (PA1-310B and PA1-311) was employed to examine ERbeta protein expression in neural tissues from ovariectomized (OVX) cynomolgus macaques and CF-1 mice as well as to assess potential regulatory effects of acute and extended estradiol (E(2)) treatment. In hypothalamic extracts from both species, a single ERbeta immunoreactive (ERbeta-ir) band was detected at approximately 54 kDa, corresponding to the expected molecular weight for ERbeta477 and/or 485. In cynomolgus females, oral E(2) administration for 16 weeks had no apparent effect on hypothalamic ERbeta protein expression. In mouse, a single injection of E(2) did not change hypothalamic ERbeta protein levels 1.5, 4, 8, 16, or 24 h after injection. Extending the hormonal treatment to 4 or 21 days in OVX female mice also had no effect on the level of hypothalamic ERbeta protein. Additional regional analyses in female mouse brain with PA1-310B antibody showed that a second, 59 kDa ERbeta-ir band was present in cortex, striatum, hippocampus, and amygdala that could represent one or both of the larger ERbeta variants (530 and 549aa). The expression level of the second ERbeta isoform exhibited regional variation, with the strongest immunoreactivity detected in cortex and amygdala. Elucidating the functions of these ERbeta isoforms in the CNS will facilitate our understanding of the tissue- and promoter-specific actions of estrogen.     

Self-malonylation is an intrinsic property of a chemically synthesized type II polyketide synthase acyl carrier protein

During polyketide biosynthesis, malonyl groups are transferred to the acyl carrier protein (ACP) component of the polyketide synthase (PKS), and it has been shown that a number of type II polyketide ACPs undergo rapid self-acylation from malonyl-CoA in the absence of a malonyl-CoA:holo-acyl carrier protein transacylase (MCAT). More recently, however, the observation of self-malonylation has been ascribed to contamination with Escherichia coli MCAT (FabD) rather than an intrinsic property of the ACP. The wild-type apo-ACP from the actinorhodin (act) PKS of Streptomyces coelicolor (synthetic apo-ACP) has therefore been synthesized using solid-state peptide methods and refolded using the GroEL/ES chaperone system from E. coli. Correct folding of the act ACP has been confirmed by circular dichroism (CD) and 1H NMR. Synthetic apo-ACP was phosphopantetheinylated to 100% by S. coelicolor holo-acyl carrier protein synthase (ACPS), and the resultant holo-ACP underwent self-malonylation in the presence of malonyl-CoA. No malonylation of negative controls was observed, confirming that the use of ACPS and GroEL/ES did not introduce contamination with E. coli MCAT. This result proves unequivocally that self-malonylation is an inherent activity of this PKS ACP in vitro.