The Ca2+ homeostasis defects in a pgm2Delta strain of Saccharomyces cerevisiae are caused by excessive vacuolar Ca2+ uptake mediated by the Ca2+-ATPase Pmc1p

Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2Delta strain also exhibits a severe imbalance in intracellular Ca(2+) homeostasis when grown under these conditions. In the present study, we examined how the pgm2Delta mutation alters yeast Ca(2+) homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca(2+) uptake in wild-type cells, whereas Ca(2+) uptake increased 8-fold in the pgm2Delta mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca(2+)-ATPase Pmc1p, suppressed the Ca(2+)-related phenotypes observed in the pgm2Delta strain. This suggests that excessive vacuolar Ca(2+) uptake is tightly coupled to these defects in Ca(2+) homeostasis. An in vitro assay designed to measure Ca(2+) sequestration into intracellular compartments confirmed that the pgm2Delta mutant contained a higher level of Pmc1p-dependent Ca(2+) transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca(2+) uptake also coincided with a large induction of the unfolded protein response in the pgm2Delta mutant, suggesting that Ca(2+) uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca(2+) uptake and accumulation previously shown to be associated with the pgm2Delta mutation are due to a severe imbalance in the distribution of cellular Ca(2+) into different intracellular compartments.     

HLA-DQ8-associated T cell responses to the diabetes autoantigen phogrin (IA-2 beta) in human prediabetes

Susceptibility to type 1A autoimmune diabetes is linked to expression of particular MHC class II molecules, notably HLA-DQ8 in man and the orthologous I-Ag7 in the nonobese diabetic mouse. In the present study, we analyzed two peptide epitopes (peptides 2 and 7) from the diabetes autoantigen phogrin (IA-2beta), in the context of their presentation by the I-Ag7 and HLA-DQ8 molecules and their role as potential T cell antigenic epitopes in human diabetes. Both of these peptides are targets of diabetogenic CD4+ T cell clones in the nonobese diabetic mouse. Transgenic mice expressing HLA-DQ8 as the sole class II molecule generated a robust T cell-proliferative response when primed with peptide 2 or peptide 7 in CFA. Analysis of the IL-2 secretion from peptide 2-reactive T cell hybridomas stimulated with alanine-substituted peptides identified three residues that were crucial to the response. Among 41 islet cell Ag-positive prediabetic human subjects, 36.5% showed PBMC-proliferative responses to peptide 7, 17.1% to peptide 2, and 17.1% to both peptides; no response was seen among 20 matched healthy controls. Stratification of the data based upon HLA haplotype suggested that peptide 7 could be presented by at least one HLA-DR molecule in addition to HLA-DQ8, a finding that was supported by blocking studies with monomorphic mAbs. The results indicate that common phogrin peptides are targeted by autoreactive T cells in human and murine type 1A diabetes, and that the responses may in part be associated with the similar peptide-binding specificities of I-Ag7 and HLA-DQ8.     

Neonatal handling increases sensitivity to acute neurodegeneration in adult rats

Environmental stimuli during the perinatal period can result in persistent individual differences in neural viability and cognitive functions. Earlier studies have shown that brief daily maternal separation and/or handling of rat pups during the first weeks of life reduces stress reactivity during adulthood and attenuates neuronal loss and cognitive decline during aging. In the present study we examined whether neonatal handling also affects the sensitivity of the adult brain to an acute neurotoxic insult. Postnatally handled and nonhandled control rats were left undisturbed from weaning onwards until the age of 11 months. At this age, the animals were subjected to a neurotoxic challenge by unilateral infusion of 60 mM of the glutamate analogue N-methyl-D-aspartate (NMDA) into the nucleus basalis magnocellularis (NBM). The brains were collected to measure cholinergic cell and fiber loss. In the nonlesioned side of the brain, cholinergic cell number in the NBM and fiber density in the cortex were not different between postnatally handled and control rats. However, in the lesioned hemisphere handled animals exhibited a significantly higher loss of choline-acetyltransferase-immunoreactive and acetylcholinesterase-positive fibers in the somatosensory cortex. The present results provide evidence for an enhanced vulnerability of postnatally handled rats to acute neurodegeneration in contrast to the previously reported attenuation of spontaneous aging-related neurodegenerative processes.     

Nucleoside Map of the Human Central Nervous System

Nucleosides are neuromodulators that have a wide range of biological roles in the brain. In order to better understand the function of nucleosides in the human central nervous system (CNS), we constructed a nucleoside map showing the concentration of various nucleosides and their metabolites using post mortem samples from 61 human brain areas and 4 spinal cord areas. We evaluated in vivo tissue levels of four nucleosides (uridine, inosine, guanosine, and adenosine) and three of their metabolites (uracil, hypoxanthine, and xanthine). The concentrations of nucleosides were unevenly distributed across different brain regions, where the highest levels were found in the cerebral cortex and basal ganglia, whereas the lowest concentrations were located in the locus coeruleus, the zona incerta, the substantia nigra, and the inferior colliculus. The regional differences in nucleoside levels in the CNS may reflect the distinct physiological functions adopted by these compounds in different brain areas.     

Progesterone-induced blocking factor activates STAT6 via binding to a novel IL-4 receptor

Progesterone-induced blocking factor (PIBF) induces Th2-dominant cytokine production. Western blotting and EMSA revealed phosphorylation as well as nuclear translocation of STAT6 and inhibition of STAT4 phosphorylation in PIBF-treated cells. The silencing of STAT6 by small interfering RNA reduced the cytokine effects. Because the activation of the STAT6 pathway depends on the ligation of IL-4R, we tested the involvement of IL-4R in PIBF-induced STAT6 activation. Although PIBF does not bind to IL-4R, the blocking of the latter with an Ab abolished PIBF-induced STAT6 activation, whereas the blocking of the IL-13R had no effect. PIBF activated suppressor of cytokine signaling-3 and inhibited IL-12-induced suppressor of cytokine signaling-1 activation. The blocking of IL-4R counteracted all the described effects, suggesting that the PIBF receptor interacts with IL-4R alpha-chain, allowing PIBF to activate the STAT6 pathway. PIBF did not phosphorylate Jak3, suggesting that the gamma-chain is not needed for PIBF signaling. Confocal microscopic analysis revealed a colocalization and at 37 degrees C a cocapping of the FITC PIBF-activated PIBF receptor and PE anti-IL-4R-labeled IL-4R. After the digestion of the cells with phosphatidylinositol-specific phospholipase C, the STAT6-activating effect of PIBF was lost, whereas that of IL-4 remained unaltered. These data suggest the existence of a novel type of IL-4R composed of the IL-4R alpha-chain and the GPI-anchored PIBF receptor.     

Pokeweed antiviral protein depurinates the sarcin/ricin loop of the rRNA prior to binding of aminoacyl-tRNA to the ribosomal A-site

Ribosome-inactivating proteins, such as the pokeweed antiviral protein (PAP), inhibit translation by depurinating the conserved sarcin/ricin loop of the large ribosomal RNA. Depurinated ribosomes are unable to bind elongation factor 2, and, thus, the translocation step of the elongation cycle is inhibited. Though the consequences of depurination are well characterized, the ribosome conformation required for depurination to take place has not been described. In this report, we correlate biochemical and genetic data to conclude that pokeweed antiviral protein depurinates the sarcin/ricin loop when the A-site of the ribosomal peptidyl-transferase center is unoccupied. We show that prior incubation of ribosomes with puromycin, an analog of the 3'-terminus of aminoacyl-tRNA, inhibits both binding and depurination by PAP in a concentration-dependent manner. Expression of PAP in the yeast strain mak8-1 results in little depurination unless the cells are lysed, a process that would promote loss of aminoacyl-tRNA from the ribosome. The mak8-1 strain is known to exhibit a higher affinity for aminoacyl-tRNA compared with wild-type cells, and therefore, its ribosomes are more resistant to PAP in vivo. These data contribute to the mechanism of action of pokeweed antiviral protein; specifically, they have uncovered the ribosomal conformation required for depurination that leads to subsequent translation inhibition.     

Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro

Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at -14 degrees C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium. These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.     

Chromatin compaction at the mononucleosome level

Using a previously described FRET technique, we measured the distance between the ends of DNA fragments on which nucleosomes were reconstituted from recombinant and native histones. This distance was analyzed in its dependence on the DNA fragment length, concentration of mono- and divalent counterions, presence of linker histone H1, and histone modifications. We found that the linker DNA arms do not cross under all conditions studied but diverge slightly as they leave the histone core surface. Histone H1 leads to a global approach of the linker DNA arms, confirming the notion of a "stem structure". Increasing salt concentration also leads to an approach of the linker DNAs. To study the effect of acetylation, we compared chemically acetylated recombinant histones with histones prepared from HeLa cells, characterizing the sites of acetylation by mass spectroscopy. Nucleosomes from chemically acetylated histones have few modifications in the core domain and form nucleosomes normally. Acetylating all histones or selectively only H3 causes an opening of the nucleosome structure, indicated by the larger distances between the linker DNA ends. Selective acetylation of H4 distances the linker ends for short fragments but causes them to approach each other for fragments longer than 180 bp.