The tocopherol transfer protein mediates vitamin E trafficking between cerebellar astrocytes and neurons.

Alpha-tocopherol (vitamin E) is an essential nutrient that functions as a major lipid-soluble antioxidant in humans. The alpha-tocopherol transfer protein (TTP) binds α-tocopherol with high affinity and selectivity and regulates whole-body distribution of the vitamin. Heritable mutations in the TTPA gene result in familial vitamin E deficiency, elevated indices of oxidative stress, and progressive neurodegeneration that manifest primarily in spinocerebellar ataxia. Although the essential role of vitamin E in neurological health has been recognized for over 50 years, the mechanisms by which this essential nutrient is transported in the central nervous system are poorly understood. Here we found that, in the murine cerebellum, TTP is selectively expressed in glial fibrillary acidic protein-positive astrocytes, where it facilitates efflux of vitamin E to neighboring neurons. We also show that induction of oxidative stress enhances the transcription of the TtpA gene in cultured cerebellar astrocytes. Furthermore, secretion of vitamin E from astrocytes is mediated by an ABC-type transporter, and uptake of the vitamin into neurons involves the low-density lipoprotein receptor-related protein 1. Taken together, our data indicate that TTP-expressing astrocytes control the delivery of vitamin E from astrocytes to neurons, and that this process is homeostatically responsive to oxidative stress. These are the first observations that address the detailed molecular mechanisms of vitamin E transport in the central nervous system, and these results have important implications for understanding the molecular underpinnings of oxidative stress-related neurodegenerative diseases.

Altered vitamin E status in Niemann-Pick type C disease.

Vitamin E (α-tocopherol) is the major lipid-soluble antioxidant in many species. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by mutations in the NPC1 or NPC2 gene, which regulates lipid transport through the endocytic pathway. NPC disease is characterized by massive intracellular accumulation of unesterified cholesterol and other lipids in lysosomal vesicles. We examined the roles that NPC1/2 proteins play in the intracellular trafficking of tocopherol. Reduction of NPC1 or NPC2 expression or function in cultured cells caused a marked lysosomal accumulation of vitamin E in cultured cells. In vivo, tocopherol significantly accumulated in murine Npc1-null and Npc2-null livers, Npc2-null cerebella, and Npc1-null cerebral cortices. Plasma tocopherol levels were within the normal range in Npc1-null and Npc2-null mice, and in plasma samples from human NPC patients. The binding affinity of tocopherol to the purified sterol-binding domain of NPC1 and to purified NPC2 was significantly weaker than that of cholesterol (measurements kindly performed by R. Infante, University of Texas Southwestern Medical Center, Dallas, TX). Taken together, our observations indicate that functionality of NPC1/2 proteins is necessary for proper bioavailability of vitamin E and that the NPC pathology might involve tissue-specific perturbations of vitamin E status.

Requirement for Akt-mediated survival in cell transformation by the dbl oncogene.

The dbl oncogene product is the founding member of a large family of oncogenic proteins that function by activating the small GTP-binding proteins Cdc42, Rac and Rho. Through its substrate GTPases, Dbl transduces proliferative signals from cell-surface receptors to diverse cellular effectors and signaling pathways. The mechanisms by which these multiple signals are integrated, as well as their relative contribution to Dbl-induced cell transformation, are presently poorly understood. We investigated the role of the survival regulators PI3-kinase and Akt in Dbl-induced cell transformation. We found that Dbl induced the phosphorylation of Akt on threonine 308, through the GTPases Rac and Cdc42 and in a PI3-kinase dependent manner. Pharmacological or biochemical interference with this pathway lead to a marked, dose-dependent inhibition of the focus formation activity exhibited by Dbl-expressing cells. Dbl expression stimulated the phosphorylation of the anti-apoptotic Akt substrate Bad, and caused a marked decrease in basal levels of apoptosis. Finally, we found that activated Cdc42 existed in cells in complex with phosphoionositide-dependent kinase-1 (PDK1), the downstream mediator of PI3-kinase action. The data indicate that Dbl signaling stimulate the formation of a novel survival complex, through which anti-apoptotic signals are generated and propagated.

A novel Cdc42Hs mutant induces cellular transformation.

Cdc42Hs is a small GTPase of the Rho-subfamily, which regulates signaling pathways that influence cell morphology and polarity, cell-cycle progression and transcription. An essential role for Cdc42Hs in cell growth regulation has been suggested by the finding that the Dbl oncoprotein is an upstream activator-a guanine nucleotide exchange factor (GEF)-for Cdc42Hs, and that activated mutants of the closely related GTPases Rac and Rho are transforming. As we were unable to obtain significant over-expression of GTPase-defective Cdc42Hs mutants, we have generated a mutant, Cdc42Hs(F28L), which can undergo spontaneous GTP-GDP exchange while maintaining full GTPase activity, and thus should exhibit functional activities normally imparted by Dbl. In cultured fibroblasts, Cdc42Hs(F28L) activated the c-Jun kinase (JNK1) and stimulated filopodia formation. Cells stably expressing Cdc42Hs(F28L) also exhibited several hallmarks of transformation-reduced contact inhibition, lower dependence on serum for growth, and anchorage-independent growth. Our findings indicate that Cdc42Hs plays a role in cell proliferation, and is a likely physiological mediator of Dbl-induced transformation.

In vitro bioassay of erythropoietin using synchronized rabbit erythroid precursors.

Synchronized erythroid precursors obtained from the bone marrow of rabbits and plated in methyl-cellulose were used as a bioassay for the measurement of erythropoietin (Ep). Rabbits were given five daily injections of phenylhydrazine followed by a single dose of actinomycin-D. This treatment resulted in a rapid repopulation of bone marrow by synchronized erythroid precursors which can be stored at -180 degrees C for long periods. Grown in vitro for 2 days in the presence of added Ep these cells divided to form colonies (CFUE). The erythroid nature of these colonies was confirmed by 59Fe incorporation into heme. Preliminary studies indicate that this system is suitable for the measurement of Ep in human sera. It is simple, inexpensive, reproducible, and permits measurements at the physiologic range of Ep concentrations.

Enrichment for GM-CFU from human bone marrow using Sambucus nigra agglutinin: potential application to bone marrow transplantation.

A new lectin, purified from black elder-berries, Sambucus nigra L. (SNA I, hereafter called SNA), was used for fractionation of normal human marrow cells. The stem cell enrichment capability of SNA was investigated by comparing colony formation (GM-CFU) in unagglutinated cell fractions (SNA-) following agglutination with SNA with that with soybean agglutinin (SBA-). GM-CFU recovery in SNA- was equal or superior to that in the SBA- fraction. A modified procedure was developed to combine stem cell enrichment and depletion of E-rosette-forming T-lymphocytes. Bone marrow cells were exposed in one step to SNA and to untreated sheep red blood cells (SRBC). Nonagglutinated, nonrosetting cells were collected after Ficoll-Hypaque-gradient separation. The procedure, a modification of Reisner's multistep procedure involving the agglutination of SBA+ cells; separation of the unagglutinated SBA- cells from the top of a 5% bovine-serum-albumin gradient; the formation of E-rosettes with SRBC; and the separation of rosettes over a Ficoll-Hypaque gradient, is faster, simpler, and as effective for T-cell depletion and stem cell enrichment.

DNA synthesis by erythroid precursors in a completely defined medium: a rapid, sensitive, and convenient bioassay for erythropoietin.

We detail a novel, sensitive, and reproducible in vitro bioassay for erythropoietin that can be performed conveniently in any laboratory and is well suited for analysis of large numbers of samples. The assay measures DNA synthesis by a cohort of highly erythropoietin-responsive red cell precursors appearing in bone marrow of anemic rabbits after treatment with a single dose of actinomycin D. The assay is conducted in a completely defined culture medium that totally dispenses not only with serum but also with serum-replacing factors. Under well-defined conditions, incorporation of [3H]thymidine by the cells depends specifically on erythropoietin. A stimulation index of up to 40-fold is obtained at 50 mU/ml of the hormone. The assay is linear in the range 0-50 mU/ml and not saturated before 1 U/ml of erythropoietin. Sample volumes of 1-30 microliter suffice for assay. Assay cells can be frozen in aliquots that retain their viability and ability to respond to erythropoietin over extended periods. Using microtiter-plate techniques, cells from one rabbit suffice for over 5000 triplicate erythropoietin determinations. Concentrations of 0.1-0.2 mU/well of erythropoietin can be detected. Erythropoietin values determined in sera from a variety of patients correlate extremely well with values obtained by the colony formation method. The ability to follow erythropoietin-dependent DNA synthesis and multiple cell divisions by a cohort of erythroid precursors in completely defined culture conditions may find application in controlled studies of red cell development.

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic consequences of cerebral vasospasm on perforating arteries: a phantom model study.

BACKGROUND AND PURPOSE: Hemodynamics of cerebral vasospasm after subarachnoid hemorrhage remain unclear, and the discrepancy between ultrasonographic or angiographic evidence of arterial narrowing and neurological ischemic deficit is still debated. Most blood flow studies have been involved with large arteries, and thus, very little is known regarding the hemodynamic behavior of small perforating vessels. Patients with symptomatic vasospasm, however, often present with neurological signs suggesting involvement of deep-sited areas of the brain supplied by perforating arteries. METHODS: A pulsatile pump was set to provide an outflow of 350 mL/min through a 10-mm-diameter C-flex tube at a perfusion pressure of 130/80 mm Hg. The perfusion fluid used was prepared to approximate blood viscosity. Perforating arteries were simulated by a 1-mm tube connected to the parent tube at a 90 degrees angle. Cylindrical stenotic devices of decreasing diameters were then introduced into the parent tube at the level of the aperture of the secondary tube and 1.5 diameters upstream of it. Velocity profiles both proximal and distal to the stenosis in the parent tube were obtained with a newly developed ultrasonographic flowmeter that allows for high spatial resolution. RESULTS: Increasing stenosis resulted in decreased outflow in the main tube, although it was significant only with severe stenosis. Whenever the simulated stenosis was placed upstream of the secondary tube, flow reduction was associated with a progressive change in the velocity profile, which gradually changed from laminar conditions to a jet stream limited to the center of the lumen. Further diameter reduction was responsible for the occurrence of flow separation with retrograde flow velocities in the periphery of the lumen. In the secondary tube, flow reduction was much more pronounced and began at a lesser degree of stenosis. Increasing fluid viscosity and decreasing perfusion pressure enhanced flow separation and prominently affected the outflow in the secondary tube. Conversely, whenever the simulated stenosis involved the branching area of the secondary tube, there was a slightly progressive decrease in the relative flow in the main tube as the stenosis became tighter. When the stenosis equaled the diameter of the secondary tube, the relative contribution of the secondary tube increased markedly at the expense of the main tube outflow. CONCLUSIONS: The present results show that local cerebral vasospasm induces changes in postvasospastic velocity profile affecting the shear rate and may eventually lead to flow separation. This phenomenon may, in turn, result in a venturi-like effect over the aperture of perforating arteries branching out of the postvasospastic portion of the affected parent artery. These alterations of cerebral hemodynamics may account for at least part of the vasospasm symptomatology, especially in the vertebrobasilar system, where vasospasm is commonly focal rather than diffuse. Furthermore, these changes proved to be affected significantly by manipulations of pressure and viscosity, supporting the use of hyperdynamic therapy in the management of cerebral vasospasm.

Structure-function relationship in the tocopherol transfer protein.

The role of specific amino acid residues in mediating the biochemical functions of tocopherol transfer protein (TTP) was investigated using site-directed mutagenesis and functional assays. These findings further current understanding of TTP mechanism of action and its role in human health.

On the continuous culturing of B.95-8 cells in the presence of phosphonoacetic acid.

Lymphoblastoid B.95-8 cells were cultured for four months and three weeks in the presence of increasing concentrations (50--200 microgram/ml) of phosphonoacetic acid (PAA). Several weeks after removal of the PAA, the cultures, in parallel with untreated B.95-8 cells, were tested for the presence of: 1) Epstein-Barr virus (EBV) viral capsid antigen (VCA), and b) transformation of human cord blood lymphocytes. There was no difference in the percentage of cells exhibiting VCA in the B.95-8 PAA treated and untreated cells. However, transformation assays indicated 10 times less transforming virus in culture supernatant harvested from B.95-8 cultures treated with PAA, as compared with the control cultures. Electron microscopic studies indicated the presence of virus particles in B.95-8 control cells and their almost complete absence in the PAA-treated cells.

Decrease in GABA synthesis rate in rat cortex following GABA-transaminase inhibition correlates with the decrease in GAD(67) protein.

gamma-Aminobutyric acid (GABA) synthesis in the brain is mediated by two major isoforms of glutamic acid decarboxylase, GAD(65) and GAD(67). The contribution of these isoforms to GABA synthesis flux (V(GAD)) is not known quantitatively. In the present study we compared V(GAD) in cortex of control and vigabatrin-treated rats under alpha-chloralose/70% nitrous oxide anesthesia, with total GAD activity and GAD isoform composition (GAD(65) and GAD(67)) measured by enzymatic assay and quantitative immunoblotting. V(GAD) was determined by re-analysis of 13C NMR data obtained ex vivo and in vivo during infusions of [1-13C]glucose using an extension of a model of glutamate-glutamine cycling that included a discrete GABAergic neuronal compartment with relevant interconnecting fluxes. V(GAD) was significantly lower in vigabatrin-treated rats (0.030-0.05 micromol/min per g, P<0.003) compared to the non-treated control group (0.10-0.15 micromol/min per g). The 67-70% decrease in V(GAD) was associated with a 13% decrease in total GAD activity (P=0.01) and a selective 44+/-15% decrease in GAD(67) protein (from 0.63+/-0.10 to 0.35+/-0.08 microg protein/mg tissue, P<0.05); GAD(65) protein was unchanged. The reduction in GAD(67) protein could account for a maximum of approximately 65% of the decrease in V(GAD) in vigabatrin-treated animals suggesting that inhibition of GAD(65) must have also occurred in these experiments, although product inhibition of GAD(67) by increased GABA could play a role. GAD(67) could account for 56-85% of cortical GABA synthesis flux under basal conditions and the entire flux after vigabatrin treatment.

Discrete activation of transduction pathways associated with acetylcholine m1 receptor by several muscarinic ligands.

Activation of transfected muscarinic m1 acetylcholine receptors (m1AChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat m1AChR gene, carbachol elicited all three responses with EC50 values of 2.6, 3.8 and 76 microM, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca2+ in intact m1AChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent m1AChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimer's disease.

Human sperm and hamster oocyte interaction: a model system to assess sperm entry into the oocyte after partial zona dissection.

A model system of hamster oocyte and human sperm interaction was used to assess sperm entry into the perivitelline space after partial zona dissection. The procedure of zona dissection was standardized by creating slits which included one fourth, one eighth, one eighth X 2, or one sixteenth of the zonal circumference. Manipulated eggs were allowed to interact with 1 X 10(6) sperm/mL for 3 hours. A single large or medium slit was equally effective in permitting sperm entry into 46% and 47% of the manipulated eggs, respectively. However, the longer slit doubled the average number of sperm detected in the perivitelline space. A second medium-sized slit increased the rate of sperm entry into the perivitelline space to 76%, but the incidence of damaged oocytes also increased. A small slit did not permit sperm entry into any of the manipulated oocytes. This heterologous system of gamete interaction provides a model to evaluate requirements for successful partial zona dissection or other related procedures for assisted fertilization in the human.

The presence of a sponsoring embryo in a batch of poor quality thawed embryos significantly increases pregnancy and implantation rate.

OBJECTIVE: To evaluate quantitatively the effect of one good-quality (sponsoring) embryo in a batch of low-quality thawed embryos on the implantation and pregnancy rates (PR). DESIGN: Retrospective analysis of data. SETTING: Tertiary care center IVF clinic affiliated with a university medical school. PATIENT(S): Between March 1988 and April 1995, 392 IVF patients underwent a total of 440 thawing and ET cycles of 1,436 multicellular embryos. MAIN OUTCOME MEASURE(S): Implantation, clinical pregnancy, and multiple pregnancy rates. RESULT(S): In the absence of sponsoring embryos in the thawed batch of embryos, a PR of 9.8% with an implantation rate of 3.1% was achieved. In the presence of a single sponsoring embryo, the PR nearly doubled (18.2%), with a significantly higher implantation rate of 7.0%. Only singleton pregnancies were achieved in the absence of sponsoring embryos compared with 21.7% multiple pregnancies in the single sponsoring embryo group. CONCLUSION(S): The presence of a sponsoring embryo in a batch of poor quality thawed embryos is an important factor that significantly increased pregnancy and implantation rates. The optimal strategy for planning batches of multicellular frozen embryos is to include at least one sponsoring embryo in each batch when possible. We speculate that the sponsoring embryo may favorably influence the chances of low-quality embryos to undergo successful implantation.

A resonance Raman study of octopus bathorhodopsin with deuterium labeled retinal chromophores.

The resonance Raman spectrum of octopus bathorhodopsin in the fingerprint region and in the ethylenic-Schiff base region have been obtained at 80 K using the "pump-probe" technique as have its deuterated chromophore analogues at the C7D; C8D; C8,C7D2; C10D; C11D; C11, C12D2; C14D; C15D; C14, C15D2; and N16D positions. While these data are not sufficient to make definitive band assignments, many tentative assignments can be made. Because of the close spectral similarity between the octopus bathorhodopsin spectrum and that of bovine bathorhodopsin, we conclude that the essential configuration of octopus bathorhodopsin's chromophore is all-trans like. The data suggest that the Schiff base, C = N, configuration is trans (anti). The observed conformationally sensitive fingerprint bands show pronounced isotope shifts upon chromophore deuteration. The size of the shifts differ, in certain cases, from those found for bovine bathorhodopsin. Thus, the internal mode composition of the fingerprint bands differs somewhat from bovine bathorhodopsin, suggesting a somewhat different in situ chromophore conformation. An analysis of the NH bend frequency, the Schiff base C = N stretch frequency, and its shift upon Schiff base deuteration suggests that the hydrogen bonding between the protonated Schiff base with its protein binding pocket is weaker in octopus bathorhodopsin than in bovine bathorhodopsin but stronger than that found in bacteriorhodopsin's bR568 pigment.

Quantitative sorting of normal and abnormal coronary flow wave form shapes.

The normal phasic flow wave form in an epicardial coronary artery has a distinct characteristic shape, which reflects the interaction between the coronary tree, myocardial function and hemodynamic conditions. Since clinical measurements of phasic coronary wave forms are becoming available, determination of abnormal coronary flow wave forms is important. We suggest here an objective and automatic method to discriminate between normal and abnormal flow wave forms based on the Karhunen-Loève Transform (KLT), and experimentally tested it. The normal flow domain was represented by the resting flow waves measured in the left anterior descending arteries in 31 anesthetized dogs. The abnormal flow conditions, imposed and tested experimentally, were varying stenosis severity and severely reduced left ventricular pressure. In addition, the effects of reactive hyperemia on the shape of the flow were examined. The sorting index was based on the mean-square error (MSE) calculated for each flow signal based on a truncated KLT expansion. The results show excellent discrimination between the normal and the abnormal groups. During reactive hyperemia, however, MSE did not change significantly. These results indicate that the shape of abnormal coronary flow wave forms can be identified and discriminated from normal wave forms.

Is cord blood erythropoietin a marker of intrapartum hypoxia?

A sensitive assay was used to compare the biological activity of cord serum erythropoietin in two groups of infants born with or without labor-induced hypoxia. The mean cord serum erythropoietin activity in 161 infants delivered after vaginal labor was 116 +/- 36 mU/mL, and was indistinguishable from that observed in 23 infants delivered by preplanned, elective cesarean section, 114 +/- 12 mU/mL (P = .75). The bioassay measured effective erythropoietin activity, including the contribution of potentiators in serum. These results indicate that duration and intensity of labor are insufficient to cause a significant increase in effective erythropoietin activity.

Myocardial mechanics and coronary flow dynamics.

The interaction between cardiac mechanics and coronary flow is highlighted here. Left ventricular (LV) structure and geometry are related to coronary flow dynamics and used in the analysis of experimental coronary flow data. The important role of the collagen mesh in the generation of the intramyocardial pressure (IMP), the pressure in the interstitial fluid, at a wide range of loading conditions is emphasized. The calculated IMP, based on a structural model of the LV myocardium, can explain most of the observed coronary compression characteristics under a variety of loading and contractility conditions. A more general compression function, the extravascular compressive pressure (ECP), is suggested to define coronary compression and is presented here based on the dynamics of the coronary inflow under constant perfusion conditions. Coronary compression is shown to be affected by fluid transport and the bi-directional coupling of coronary hemodynamics and IMP dynamics.

The T-SCAN technology: electrical impedance as a diagnostic tool for breast cancer detection.

In this paper we present the T-SCAN technology and its use as a diagnostic tool for breast cancer detection. We show, using theoretical models with simplified geometries, that displaying planar two-dimensional maps of the currents detected at the breast's surface relate to the electric field distribution within the breast. This distribution is a manifestation of the bulk spatial inhomogeneities in the complex dielectric constant that represent the various tissue types. These differences may be used to discriminate between various pathological states. We furthermore illustrate a useful classifier, based on admittance data measured up to 2 kHz, and we argue that low frequency impedance measurements can be used successfully in breast cancer diagnosis.