Suppressor gene analysis reveals an essential role for sphingolipids in transport of glycosylphosphatidylinositol-anchored proteins in Saccharomyces cerevisiae

Sphingolipids are normally necessary for growth of Saccharomyces cerevisiae cells, but mutant strains that bypass the need for sphingolipids have been identified. Such bypass mutants fail to grow under stressful conditions, including low pH (pH 4.1), when they lack sphingolipids. To begin to understand why sphingolipids seem to be necessary for coping with low-pH stress, we screened a genomic library and selected a suppressor gene, CWP2 (cell wall protein 2), that when present in multiple copies partially compensates for the lack of sphingolipids and enhances survival at low pH. To explain these results, we present evidence that sphingolipids are required for a normal rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins, including Cwp2 and Gas1/Gpg1, from the endoplasmic reticulum (ER) to the Golgi apparatus. The effect of sphingolipids is specific for transport of GPI-anchored proteins because no effect on the rate of transport of carboxypeptidase Y, a non-GPI-anchored protein, was observed. Since the Gasl protein accumulated in the ER with a GPI anchor in cells lacking sphingolipids, we conclude that sphingolipids are not necessary for anchor attachment. Therefore, sphingolipids must be necessary for a step in formation of COPII vesicles or for their transport to the Golgi apparatus. Our data identify the Cwp2 protein as a vital component in protecting cells from the stress of low pH.     

Transmission Electron Microscopy-Based Analysis of Electrically Conductive Surface Defects in Large Area GaSb Homoepitaxial Diodes Grown Using Molecular Beam Epitaxy

We investigate a mechanism causing shorting of large area GaSb diodes grown on GaSb substrates using molecular beam epitaxy (MBE). The source of these shorts is determined to be large crystallographic defects on the surface of the diodes that are formed around droplets of gallium ejected from the gallium Knudsen cells during MBE. The gallium droplets cause defects in the crystal structure, and, as the epitaxy continues, the gallium is incorporated into the surrounding material. The shape of the defects is pyramidal with a central void extending from the epi-surface to the gallium core. Processing a GaSb diode with these surface defects results in the top-side contact metal migrating into the defect and shorting the diode. This prevents realization of large area diodes that are critical to applications such as photovoltaics and detectors. The diodes in this study are electrically characterized and the defect formation mechanism is investigated using cross-section transmission electron microscopy and electron dispersive spectroscopy.     

A method for improving global pyranometer measurements by modeling responsivity functions

Accurate global solar radiation measurements are crucial to climate change research and the development of solar energy technologies. Pyranometers produce an electrical signal proportional to global irradiance. The signal-to-irradiance ratio is the responsivity (RS) of the instrument (RS = signal/irradiance = microvolts/(W/m²)). Most engineering measurements are made using a constant RS. It is known that RS varies with day of year, zenith angle, and net infrared radiation. This study proposes a method to find an RS function to model a pyranometer’s changing RS. Using a reference irradiance calculated from direct and diffuse instruments, we found instantaneous RS for two global pyranometers over 31 sunny days in a two-year period. We performed successive independent regressions of the error between the constant and instantaneous RS with respect to zenith angle, day of year, and net infrared to obtain an RS function. An alternative method replaced the infrared regression with an independently developed technique to account for thermal offset. Results show improved uncertainties with the function method than with the single-calibration value. Lower uncertainties also occur using a black-and-white (8-48), rather than all-black (PSP), shaded pyranometer as the diffuse reference instrument. We conclude that the function method is extremely effective in reducing uncertainty in the irradiance measurements for global PSP pyranometers if they are calibrated at the deployment site. Furthermore, it was found that the function method accounts for the pyranometer’s thermal offset, rendering further corrections unnecessary. The improvements in irradiance data achieved in this study will serve to increase the accuracy of solar energy assessments and atmospheric research.     

X-ray diffraction from intact tau aggregates in human brain tissue

We describe an instrument to record x-ray diffraction patterns from diseased regions of human brain tissue by combining an in-line visible light fluorescence microscope with an x-ray diffraction microprobe. We use thiazine red fluorescence to specifically label and detect the filamentous tau protein pathology associated with Pick's disease, as several labs have done previously. We demonstrate that thiazine red-enhanced regions within the tissue show periodic structure in x-ray diffraction that is not observed in healthy tissue. One observed periodicity (4.2 ?) is characteristic of cross-beta sheet structure, consistent with previous results from powder diffraction studies performed on purified, dried tau protein.     

Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation

Single-channel activities were observed in outside-out patches excised from oocytes expressing a mammalian 5-hydroxytryptamine (5-HT) transporter. Channel conductance was larger for a mutant in which asparagine177 of the third putative transmembrane domain was replaced by glycine, suggesting that this residue lies within or near the permeation pathway. The N177G mutant enables quantitative single-channel measurements; it displays two conducting states. One state, with conductance of approximately 6 pS, is induced by 5-HT and is permeable to Na+. The other state (conductance of approximately 13 pS) is associated with substrate-independent leakage current and is permeable to both Na+ and Li+. Cl- is not a major current carrier. Channel lifetimes under all conditions measured are approximately 2.5 ms. The single-channel phenomena account for previously observed macroscopic electrophysiological phenomena, including 5-HT-induced transport-associated currents and substrate-independent leakage currents. The channel openings occur several orders of magnitude less frequently than would be expected if one such opening occurred for each transport cycle and therefore do not represent an obligatory step in transport. Nevertheless, single-channel events produced by neurotransmitter transporters indicate the functional and structural similarities between transporters and ion channels and provide a new tool, at single-molecule resolution, for detailed structure-function studies of transporters.     

The association of shame and guilt with suicidality

1,25-dihydroxyvitamin D3 (VD) is a modulator of growth and differentiation of many cell types, including keratinocytes. We have recently shown in cultured keratinocytes that VD induces tyrosine phosphorylation of proteins involved in signal transduction, such as Shc. In an attempt to identify VD-responsive tyrosine kinases, we studied the effects of VD on the activity of the nonreceptor tyrosine kinase Src. Although VD did not stimulate Src activity in keratinocytes cultured in standard media containing 0.15 mM calcium, preincubation of the cells with 1.8 mM Ca2+ caused a rapid activation of Src in response to VD (10(-8)-10(-7) M). Elevation of calcium concentration alone caused an increase in Src activity as well, but the peak of Src activity was delayed (60 min vs. 15 min) and approximately 2-fold lower in comparison with VD-treated cells. VD treatment also induced tyrosine dephosphorylation of Src and a formation of an Src-Shc-Grb2 complex. Taken together, these findings imply that Src is involved in VD signaling in keratinocytes.     

Ultrafast long-wavelength photodetectors fabricated onlow-temperature InGaAs on GaAs

The authors report the first successful demonstration of a metal-semiconductor-metal photodetector (MSMPD) fabricated on low-temperature InGaAs grown on GaAs by molecular beam epitaxy (MBE) for long-wavelength fiber optic applications. Interdigitated MSMPDs with finger widths and spacings of 0.2, 0.5, 1.0, and 2.0 μm were tested using a femtosecond pulsed laser and high-speed electrooptic sampling. A FWHM pulsed response of 2 and 1.3 ps was measured for low-temperature In _0.25Ga_0.75As and In_0.35Ga_0.65As, respectively. The latter is the fastest response reported to data for a photodetector capable of detection to wavelengths as long as 1.3 μm     

Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes

Xenopus oocytes expressed kappa-opioid specific binding sites after injection of cRNA prepared from a clone of the rat kappa-opioid receptor. Coinjection of kappa receptor cRNA with cRNA coding for a G protein-linked, inwardly rectifying, K+ channel (GIRK1, or KGA) resulted in oocytes that responded to the kappa agonist U-69593 by activating a large (1.0-1.5-microA) K+ current. U-69593 exhibited an EC50 of 260 +/- 50 nM and was blocked by the opioid antagonists norbinaltorphimine and naloxone. The kappa agonist bremazocine was 200-fold more potent than U-69593 in eliciting K+ current but exhibited a partial agonist profile in this expression system. The present results indicate that stimulation of inwardly rectifying K+ channels may be a potential effector mechanism for kappa-opioid receptors.     

Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis

Sphingolipid-related metabolites have been implicated as potential signaling molecules in many studies with mammalian cells as well as in some studies with yeast. Our previous work showed that sphingolipid-deficient strains of Saccharomyces cerevisiae are unable to resist a heat shock, indicating that sphingolipids are necessary for surviving heat stress. Recent evidence suggests that one role for the sphingolipid intermediate ceramide may be to act as a second messenger to signal accumulation of the thermoprotectant trehalose. We examine here the mechanism for generating the severalfold increase in ceramide observed during heat shock. As judged by compositional analysis and mass spectrometry, the major ceramides produced during heat shock are similar to those found in complex sphingolipids, a mixture of N-hydroxyhexacosanoyl C18 and C20 phytosphingosines. Since the most studied mechanism for ceramide generation in animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S. cerevisiae for an analogous enzyme. Using [3H]phytosphingosine and [3H]inositol-labeled yeast sphingolipids, a novel membrane-associated phospholipase C-type activity that generated ceramide from inositol-P-ceramide, mannosylinositol-P-ceramide, and mannose(inositol-P)2-ceramide was demonstrated. The sphingolipid head groups were concomitantly liberated with the expected stoichiometry. However, other data demonstrate that the ceramide generated during heat shock is not likely to be derived by breakdown of complex sphingolipids. For example, the water-soluble fraction of heat-shocked cells showed no increase in any of the sphingolipid head groups, which is inconsistent with complex sphingolipid hydrolysis. Rather, we find that de novo ceramide synthesis involving ceramide synthase appears to be responsible for heat-induced ceramide elevation. In support of this hypothesis, we find that the potent ceramide synthase inhibitor, australifungin, completely inhibits both the heat-induced increase in incorporation of [3H]sphinganine into ceramide as well as the heat-induced increase in ceramide as measured by mass. Thus, heat-induced ceramide most likely arises by temperature activation of the enzymes that generate ceramide precursors, activation of ceramide synthase itself, or both.