Inositol phosphorylceramide synthase is located in the Golgi apparatus of Saccharomyces cerevisiae

The plasma membrane of eukaryotic cells differs in lipid composition from most of the internal organelles, presumably reflecting differences in many of its functions. In particular, the plasma membrane is rich in sphingolipids and sterols, one property of which is to decrease the permeability and increase the thickness of lipid bilayers. In this paper, we examine the length of transmembrane domains throughout the yeast secretory pathway. Although the transmembrane domains of cis and medial Golgi residents are similar to those of endoplasmic reticulum proteins, these domains lengthen substantially beyond the medial Golgi, suggesting a thickening of the bilayer. Yeast sphingolipids have particularly long acyl chains, and Aur1p, the inositol phosphorylceramide synthase that initiates yeast sphingolipid synthesis, was found to be located in the Golgi apparatus by both immunofluorescence and membrane fractionation, with its active site apparently in the Golgi lumen. Thus, it appears that sphingolipid synthesis in yeast takes place in the Golgi, separated from glycerophospholipid synthesis in the endoplasmic reticulum. A similar separation has been found in mammalian cells, and this conservation suggests that such an arrangement of enzymes within the secretory pathway could be important for the creation of bilayers of different thickness within the cell.     

Metal-catalyzed oxidation of alpha-synuclein: helping to define the relationship between oligomers, protofibrils, and filaments

Oxidative stress is implicated in a number of neuro-degenerative diseases and is associated with the selective loss of dopaminergic neurons of the substantia nigra in Parkinson's disease. The role of alpha-synuclein as a potential target of intracellular oxidants has been demonstrated by the identification of posttranslational modifications of synuclein within intracellular aggregates that accumulate in Parkinson's disease brains, as well as the ability of a number of oxidative insults to induce synuclein oligomerization. The relationship between these relatively small soluble oligomers, potentially neurotoxic synuclein protofibrils, and synuclein filaments remains unclear. We have found that metal-catalyzed oxidation of alpha-synuclein inhibited formation of synuclein filaments with a concomitant accumulation of beta sheet-rich oligomers that may represent synuclein protofibrils. Similar results with a number of oxidative and enzymatic treatments suggest that the covalent association of synuclein into higher molecular mass oligomers/protofibrils represents an alternate pathway from filament formation and renders synuclein less prone to proteasomal degradation.     

Toxicogenomics of resveratrol in rat liver

Resveratrol, a polyphenolic compound found in grape skin and peanuts has been shown to prevent many diseases including cardiovascular diseases and cancer. To better understand resveratrol's potential in vivo toxicity, we studied the dose response using cDNA stress arrays coupled with drug metabolizing enzymatic (DME) assays to investigate the expression of stress-responsive genes and Phase I and II detoxifying enzymes in rat livers. Male and female CD rats were treated with high doses of resveratrol (0.3, 1.0 and 3.0 gm/kg/day) for a period of 28 days. Total RNA from rat liver was reverse-transcribed using gene-specific primers and hybridized to stress-related cDNA arrays. Among female rats, Phase I DME genes were repressed at 0.3 and 1.0 gm/kg/day doses, while genes such as manganese superoxide dismutase, cytochrome P450 reductase, quinone oxidoreductase and thiosulfate sulfurtransferase demonstrated a dose-dependent increase in gene expression. The modulation of these liver genes may implicate the potential toxicity as observed among the rats at the highest dose level of resveratrol. Real-Time PCR was conducted on some of the Phase II DME genes and anti-oxidant genes to validate the cDNA array data. The gene expression from real-time PCR demonstrated good correlation with the cDNA array data. UGT1A genes were amongst the most robustly induced especially at the high doses of resveratrol. We next performed Phase I and Phase II enzymatic assays on cytochrome P450 2E1 (CYP2E1), cytochrome P450 1A1 (CYP1A1), NAD(P)H:quinone oxidoreductase (NQO1), glutathione S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Induction of Phase II detoxifying enzymes was most pronounced at the highest dose of resveratrol. CYP1A1 activity demonstrated a decreasing trend among the 3 dose groups and CYP2E1 activity increased marginally among female rats over controls. In summary, at lower doses of resveratrol there are few significant changes in gene expression whereas the modulation of liver genes at the high dose of resveratrol may implicate the potential toxicity observed.     

Cloning, sequencing, and characterization of alternatively spliced glutaredoxin 1 cDNA and its genomic gene: chromosomal localization, mrna stability, and origin of pseudogenes

Alternatively spliced human glutaredoxin (Grx1(as)) cDNA was isolated from a neutrophil cDNA library, using a (32)P-labeled human glutaredoxin (Grx1) cDNA probe under non-stringent conditions. The sequence of Grx1(as) cDNA indicated that the open reading frame of the gene was identical to the open reading frame of the previously reported first human glutaredoxin (Grx1) cDNA, but the 3'-untranslated region of Grx1(as) was not homologous to Grx1 cDNA. Northern blot and RT-PCR analyses showed Grx1(as) mRNA was expressed in normal human neutrophils and transformed cells including U937, HL-60, THP, and Jurkat cells. Cloning and sequencing of the genomic gene corresponding to Grx1(as) cDNA showed that two different glutaredoxin cDNAs (Grx1(as) and Grx1) were generated from the same genomic gene via alternative splicing. Origination of Grx1(as) and Grx1 from the same gene was confirmed by chromosomal localization of the Grx1(as) gene to chromosome 5q13, the same location where the Grx1 gene was localized previously. During screening of the Grx1(as) genomic gene, two additional glutaredoxin pseudogenes were also isolated. Surprisingly, these pseudogenes contained 3'-untranslated regions that were nearly identical to the 3'-untranslated regions of Grx1(as,) not Grx1, cDNA. Because 3'-untranslated regions may be important in stabilizing mRNAs, the effect of the two 3'-untranslated regions of Grx1 and Grx1(as) on mRNA stability was investigated using luciferase reporter vectors with the 3'-untranslated regions. Luciferase activity was 2.6-fold greater in cells transfected with the reporter vector containing the 3'-untranslated region of Grx1(as) cDNA compared with the 3'-untranslated region of Grx1 cDNA. These data indicate that Grx1(as) cDNA is an alternatively spliced human Grx1 cDNA and that the Grx1(as) 3'-untranslated region may have a role in stabilizing mRNA.     

Functionalizing electrospun fibers with biologically relevant macromolecules

The development of functionalized polymers that can elicit specific biological responses is of great interest in the biomedical community, as well as the development of methods to fabricate these biologically functionalized polymers. For example, the generation of fibrous matrices with biological properties and fiber diameters commensurate with those of the natural extracellular matrix (ECM) may permit the development of novel materials for use in wound healing or tissue engineering. The goal of this work is, therefore, to create a biologically active functionalized electrospun matrix to permit immobilization and long-term delivery of growth factors. In this work, poly(ethylene glycol) functionalized with low molecular weight heparin (PEG-LMWH) was fabricated into fibers for possible use in drug delivery, tissue engineering, or wound repair applications. Electrospinning was chosen to process the LMWH into fiber form due to the small fiber diameters and high degree of porosity that can be obtained relatively quickly and using small amounts of starting material. Both free LMWH and PEG-LMWH were investigated for their ability to be incorporated into electrospun fibers. Each of the samples were mixed with a carrier polymer consisting of either a 10 wt % poly(ethylene oxide) (PEO) or 45 wt % poly(lactide-co-glycolide) (PLGA). Field emission scanning electron microscopy (FESEM), energy-dispersive X-ray analysis (EDX), UV-vis spectroscopy, and multiphoton microscopy were used to characterize the electrospun matrices. The incorporation of heparin into the electrospun PEO and PLGA fibers did not affect the surface morphology or fiber diameters. The fibers produced had diameters ranging from approximately 100 to 400 nm. Toluidine blue assays of heparin suggest that it can be incorporated into an electrospun matrix at concentrations ranging from 3.5 to 85 mug per milligram of electrospun fibers. Multiphoton microscopy confirmed that incorporation of PEG-LMWH into the matrix permits retention of the heparin for at least 14 days. Improvements in the binding of basic fibroblast growth factor to the electrospun fibers were also observed for fibers functionalized with PEG-LMWH over those functionalized with LMWH alone. The combination of these results suggests the utility for producing electrospun fibers that are appropriately functionalized for use in biomaterials applications.     

Conformational behavior of chemically reactive alanine-rich repetitive protein polymers

The synthesis of protein-based polymers with controlled conformational properties and functional group placement offers many opportunities for the design of advanced materials. In this work, protein engineering methods have been used to produce repetitive alanine-rich protein polymers with the sequence [(AAAQ)(5)(AAAE)(AAAQ)(5)](x) (x = 2 and 6); these macromolecules may mimic architectural features of certain alanine-rich helical sequences found in natural proteins. Various proteins from this family can be readily expressed and purified from Escherichia coli. Circular dichroic spectroscopy (CD) characterization demonstrates that the purified proteins are highly helical under a variety of conditions. Thermal analysis of [(AAAQ)(5)(AAAE)(AAAQ)(5)](2) via differential scanning calorimetry (DSC) and CD indicates that the protein undergoes a reversible helix-coil transition at approximately 45 degrees C and that the protein conformation can be manipulated at elevated temperatures depending on solution conditions. The demonstrated conformational properties of these artificial proteins suggest that they may be excellent candidates for elucidating structure-function relationships in biopolymers for nanotechnology and biological applications.     

A mucosal IgA-mediated excretory immune system in vivo

The capacity of mucosal IgA Abs to serve as an excretory immune system in vivo was investigated. Mice expressing a transgenic TCR were immunized intragastrically with the cognate Ag to elicit a vigorous mucosal IgA Ab response. Soon after i.v. challenge, Ag was detected within the epithelial cells of the small intestinal crypts and to a lesser degree within the epithelial cells higher up the villi, paralleling the gradient in expression of the polymeric Ig receptor and the transport of its ligand, oligomeric IgA. Uptake of Ag into the epithelial cells occurred only from the basolateral aspect and only when Ag complexed to IgA Ab could be present in the lamina propria. The results support the concept that local IgA Abs can excrete Ags from the body by transporting them directly through mucosal epithelial cells, using the same mechanism that transports free IgA into the mucosal secretions.     

Effect of spaceflight on isoflavonoid accumulation in etiolated soybean seedlings

In order to explore the potential impact of microgravity on flavonoid biosynthesis, we examined isoflavonoid levels in soybean (Glycine max) tissues generated under both spaceflight and clinorotation conditions. A 6-day Space Shuttle-based microgravity exposure resulted in enhanced accumulation of isoflavone glycosides (daidzin, 6"-O-malonyl-7-O-glucosyl daidzein, genistin, 6"-O-malonyl-7-O-glucosyl genistein) in hypocotyl and root tissues, but reduced levels in cotyledons (relative to 1g controls on Earth). Soybean seedlings grown on a horizontally rotating clinostat for 3, 4 and 5 days exhibited (relative to a vertical clinorotation control) an isoflavonoid accumulation pattern similar to the space-grown tissues. Elevated isoflavonoid levels attributable to the clinorotation treatment were transient, with the greatest increase observed in the three-day-treated tissues and smaller increases in the four- and five-day-treated tissues. Differences between stresses presented by spaceflight and clinorotation and the resulting biochemical adaptations are discussed, as is whether the increase in isoflavonoid concentrations were due to differential rates of development under the "gravity" treatments employed. Results suggest that spaceflight exposure does not impair isoflavonoid accumulation in developing soybean tissues and that isoflavonoids respond positively to microgravity as a biochemical strategy of adaptation.     

Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.