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.     

Predictors of mortality and the provision of dialysis in patients with acute tubular necrosis. The Auriculin Anaritide Acute Renal Failure Study Group

BACKGROUND: Improvement of angina pectoris symptoms after cholesterol lowering has raised questions as to the underlying mechanisms. METHODS: Rabbit experiment: We compared arterial blood samples from New Zealand White cholesterol-supplemented rabbits (n = 6) with nonsupplemented rabbit samples (n = 4) in a closed-loop circulation diffusion system. The pH and partial pressures of oxygen (pO2) and carbon dioxide (pCO2) were measured continuously. The samples were first oxygen (O2) saturated (pO2, 160 mm Hg; pCO2, 4 mm Hg) and then desaturated in 100% nitrogen. Cholesterol levels were determined in whole blood, plasma (P Chol), red blood cells (RBCs), and RBC membranes. Human experiment: We exposed quadruple desaturated venous blood samples (n = 4) with P Chol levels of 87 to 400 mg/dL in a gas exchanger to capillary gas conditions (pO2, 23 mm Hg; pCO2, 46 mm Hg). After 15 minutes we performed blood gas analyses and compared our results to baseline values. RESULTS: In the rabbit experiment the cholesterol-supplemented group as compared to the control group showed higher plasma pO2 levels during the saturation phase and lower plasma pO2 levels during the desaturation phase. It also had a markedly increased RBC membrane cholesterol content: 121 +/- 3 (standard error of the mean [SEM]) mg/dL versus 22 +/- 1.7 mg/dL in the control group (P < .05). This barrier to RBC membrane O2 diffusion caused delayed O2 entry into the RBCs during saturation, with a higher plasma pO2, and delayed O2 release from the RBCs during desaturation, with a lower plasma pO2. In the human experiment the P Chol level was inversely correlated with the percentage change of O2 content in milliliters of O2 per deciliter of blood (P < .05). CONCLUSIONS: Increased RBC membrane cholesterol in hypercholesterolemia appears to decrease the transmembrane O2 diffusion rate.     

Does stent placement improve the results of ineffective or complicated iliac artery angioplasty?

OBJECTIVE: This study was undertaken to determine the results and complications of stents placed for initially unsuccessful or complicated iliac percutaneous transluminal angioplasty (PTA), the effect of location (external iliac or common iliac) on outcome, and the influence of superficial femoral artery patency on benefit. DESIGN: From 1992 through 1997, 350 patients underwent iliac artery PTA at the authors' institutions. Of this group, 88 patients (88 arteries) had one or more stents placed after PTA (140 stents in total) for residual stenosis or pressure gradient (63 patients), iliac dissection (12 patients), long-segment occlusion (10 patients), or recurrent stenosis (3 patients). Thirty patients required the placement of more than one stent. The indications for PTA in these 88 patients were claudication (48 patients) and limb-threatening ischemia (40 patients). Forty-seven patients had stents placed in the common iliac, 29 patients had stents placed in the external iliac, and 12 patients had stents placed in both. Seventy-one arteries (81%) were stenotic, and 17 (19%) were occluded before PTA. Sixty-six arteries were treated by interventional radiologists, 15 by a vascular surgeon, and 7 jointly. MAIN OUTCOME MEASURE: Criteria for success included (1) increase of at least one clinical category of chronic limb ischemia from baseline or satisfactory wound healing, (2) maintenance of an ankle/brachial index increase of more than 0.10 above the preprocedure index, and (3) residual angiographic stenosis less than 25% and, for patients with pressure gradient measurements, a residual gradient less than 10 mm Hg. RESULTS: Stent placement was accomplished in all 88 patients with 16 (18%) major complications. Mean follow-up was 17 months (range, 3 to 48 months). By life-table analysis, success was 75% at 1 year, 62% at 2 years, and 57% at 3 years. No cardiovascular risk factor or independent variable was statistically significant in predicting success. There was no difference in success rates for common iliac or external iliac lesions. Superficial femoral artery patency did not correlate with outcome. CONCLUSIONS: Although stents can eliminate residual lesions and arterial dissection, these patients are likely to require adjuvant or subsequent procedures to attain clinical success. By controlling the PTA complication and treating the emergent problem, stents may allow for subsequent elective intervention.     

Growth factor regulation of insulin-like growth factor (IGF) binding proteins (IGFBP) and preadipocyte differentiation in porcine stromal-vascular cell cultures

The influence of anti-IGF-1 and anti-transforming growth factor beta (TGF-beta) neutralizing antibodies on preadipocyte differentiation and secretion of IGFBPs was examined in serum free porcine stromal-vascular cultures. Cultures were stained for morphological analysis and conditioned media were collected for: TGF-beta determination by ELISA, IGF-1 by RIA, and IGFBP analysis by ligand blotting. After 6 d of treatment, anti-TGF-beta increased fat proportions by 2.7 fold compared to controls. Anti-IGF-1 decreased fat cell proportions by 14-fold. Anti-TGF-beta increased concentrations of IGF-1 5.8-fold and IGFBP-2 and IGFBP-3 by 8- and 7-fold in conditioned media whereas IGFBP-4 decreased 5-fold. Anti-IGF-1 increased concentrations of IGFBP-2 and 3 by 9- and 35-fold, respectively. TGF-beta increased concentrations of IGFBP-1, 2 and 3 by 3-fold, 18-fold and 3-fold, respectively, after 9 d in culture (6 d of treatment). There was no change in TGF-beta levels in anti-IGF-1 treated cultures compared to controls. Control antibodies and negative controls had no effect. These results provide evidence that endogenously produced IGF-1 and TGF-beta has a major influence on preadipocyte differentiation in serum free media by modulating IGFBP production/secretion.     

Production and perception of final consonant voicing in speech produced by inexperienced signers during simultaneous communication

Simultaneous communications combines both spoken and manual modes to produce each word of an utterance. This study investigated the potential influence of alterations in the temporal structure of speech produced by inexperienced signers during simultaneous communication on the perception of final consonant voicing. Inexperienced signers recorded words that differed only in the voicing characteristic of the final consonant under two conditions: (1) speech alone and (2) simultaneous communication. The words were subsequently digitally edited to remove the final consonant and played to 20 listeners who, in a forced-choice paradigm, circled the word they thought they heard. Results indicated that accurate perception of final consonant voicing was not impaired by changes in the temporal structure of speech that accompanied the inexperienced signers' simultaneous communication.     

Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer

Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apol/CD95. One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are potentially harmful to the organism, such as virus-infected or tumour cells. Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was amplified in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL.     

Bilateral detection thresholds in dextrals and sinistrals reflect the more sensitive side of the nose, which is not lateralized

Recent studies have shed light on the different relationships between odorant receptor expression and the specification of neural identity in the olfactory systems of vertebrates and invertebrates. In mice, neuronal identity and axon guidance are specified by the single expressed olfactory receptor, whereas in C. elegans, neuronal identity appears to be independent of receptor expression.     

Establishment and characterization of a megakaryoblast cell line with amplification of MLL

A new cell line with megakaryoblastic features, designated UoC-M1, was established from the malignant cells of a 68-year-old patient with acute myeloid leukemia. The patient's leukemic cells reacted with alpha-naphthyl acetate esterase and acid phosphatase and expressed CD7, CD24, CD34, CD38, CD45, HLA-DR and CD61. Cytogenetic analysis of the patient's malignant cells (and of the UoC-M1 cells) showed a human, male hypodiploid karyotype with many chromosome rearrangements and marker chromosomes. Spectral karyotyping (SKY) analysis complemented the G-banded karyotyping and clarified several chromosomal translocations and identified the marker chromosomes. Fluorescence in situ hybridization (FISH) and SKY analysis demonstrated that one marker chromosome contained three segments of chromosome 9 interspersed with three segments of chromosome 11, as well as a portion of chromosome 19. FISH analysis with a probe for MLL revealed that the UoC-M1 cells contained four copies of the MLL gene. Southern blot analysis determined that the MLL gene had a germline profile while Northern and Western analyses showed that the MLL mRNAs and protein were of the appropriate sizes. This is the first report of amplification of the MLL gene which may be an additional mechanism of leukemogenesis or disease progression.     

Expression of platelet-derived growth factor (PDGF)-A, PDGF-B and the PDGF-alpha receptor, but not the PDGF-beta receptor, in human malignant melanoma in vivo

There has been considerable interest in the potential role of growth factors in the initiation and development of cutaneous malignant melanoma (CMM). Platelet-derived growth factor (PDGF) has been shown to be secreted by melanoma cell lines and by metastatic melanoma in vivo. PDGF also has been reported to stimulate the development of tumour stroma and new blood vessels. We studied the expression of PDGF and its receptors by both immunohistochemistry (IHC) and in situ hybridization (ISH) in primary and metastatic melanoma and in normal skin specimens. Cryostat sections were incubated with 35S-labelled riboprobes and antibodies for PDGF-AA, PDGF-alpha receptor, PDGF-BB and PDGF-beta receptor. Both primary and metastatic melanoma exhibited significant expression of PDGF-AA, PDGF-BB and PDGF-alpha receptor by both IHC and ISH, compared with only background expression in normal skin. We did not observe expression of PDGF-beta receptor in melanoma. Our results suggest that PDGF may function as an autocrine growth factor, as well as an angiogenesis factor, in CMM tumour development. This expression of the PDGF-alpha receptor rather than the beta receptor may be unique among solid tumours.