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.

Vitamin E is essential for Purkinje neuron integrity.

α-Tocopherol (vitamin E) is an essential dietary antioxidant with important neuroprotective functions. α-Tocopherol deficiency manifests primarily in neurological pathologies, notably cerebellar dysfunctions such as spinocerebellar ataxia. To study the roles of α-tocopherol in the cerebellum, we used the α-tocopherol transfer protein for the murine version (Ttpa(-/)(-)) mice which lack the α-tocopherol transfer protein (TTP) and are a faithful model of vitamin E deficiency and oxidative stress. When fed vitamin E-deficient diet, Ttpa(-/)(-) mice had un-detectable levels of α-tocopherol in plasma and several brain regions. Dietary supplementation with α-tocopherol normalized plasma levels of the vitamin, but only modestly increased its levels in the cerebellum and prefrontal cortex, indicating a critical function of brain TTP. Vitamin E deficiency caused an increase in cerebellar oxidative stress evidenced by increased protein nitrosylation, which was prevented by dietary supplementation with the vitamin. Concomitantly, vitamin E deficiency precipitated cellular atrophy and diminished dendritic branching of Purkinje neurons, the predominant output regulator of the cerebellar cortex. The anatomic decline induced by vitamin E deficiency was paralleled by behavioral deficits in motor coordination and cognitive functions that were normalized upon vitamin E supplementation. These observations underscore the essential role of vitamin E and TTP in maintaining CNS function, and support the notion that α-tocopherol supplementation may comprise an effective intervention in oxidative stress-related neurological disorders.

Brain activation associated with practiced left hand mirror writing.

Mirror writing occurs in healthy children, in various pathologies and occasionally in healthy adults. There are only scant experimental data on the underlying brain processes. Eight, right-handed, healthy young adults were scanned (BOLD-fMRI) before and after practicing left-hand mirror-writing (lh-MW) over seven sessions. They wrote dictated words, using either the right hand with regularly oriented writing or lh-MW. An MRI compatible stylus-point recording system was used and online visual feedback was provided. Practice resulted in increased speed and readability of lh-MW but the number of movement segments was unchanged. Post-training signal increases occurred in visual, right lateral and medial premotor areas, and in right anterior and posterior peri-sylvian areas corresponding to language areas. These results suggest that lh-MW may constitute a latent ability that can be reinstated by a relatively brief practice experience. Concurrently, right hemisphere language processing areas may emerge, reflecting perhaps a reduction in trans-hemispheric suppression.

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.

The alpha-tocopherol transfer protein.

Almost a century ago, plant extracts were documented to be critical for the fertility of rodents. This activity was later ascribed to vitamin E, a term comprising a number of structurally related plant lipids that function as fat soluble antioxidants. The alpha-tocopherol transfer protein (TTP) is a critical regulator of vitamin E status that stimulates the movement of vitamin E between membrane vesicles in vitro and facilitates the secretion of tocopherol from hepatocytes. Heritable mutations in the ttpA gene cause ataxia with vitamin E deficiency (AVED), an autosomal recessive disorder characterized by low plasma vitamin E levels and progressive neurodegeneration. This chapter summarizes recent advances in our understanding of the molecular and physiological aspects of TTP activity.

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.

Derivation of a diploid human embryonic stem cell line from a mononuclear zygote.

BACKGROUND: IVF occasionally produces aneuploid zygotes with one or three pronuclei (PN). Routinely, these zygotes are discarded. The aim of this work was to establish human embryonic stem cell (hESC) lines from blastocysts resulting from abnormal fertilization. METHODS: Abnormally fertilized zygotes were cultured to the blastocyst stage and, following zona pellucida digestion, zona-free blastocysts were placed on a mouse feeder layer. Culture of hESCs was carried out as described earlier. RESULTS: Six out of the nine developing blastocysts attached to the feeder layer. One hESC line, originating from a mononuclear zygote following ICSI, was successfully derived. This line displayed typical phenotype and embryonic surface markers, and exhibited the potential to develop into all three embryonic germ layers both in vitro (by embryoid body formation) and in vivo (teratoma generation). Genetic examination revealed normal diploid karyotype and heterozygotic appearance for metachromatic leukodystrophy (MLD). CONCLUSION: This method, which requires neither immuno nor mechanical removal of the trophectoderm, may facilitate the derivation of hESC lines in general, and those from abnormal embryos in particular. Furthermore, it is shown that aneuploid zygotes can be used as a source for normal hESC derivation and hold the potential to generate aneuploid hESC lines for research purposes.

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.

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.

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.

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.

[Familiarizing medical students with hospital social worker's role].

Patient care in a general hospital is usually provided by a multi-professional team. Treatment is most effective when each professional understands the functions of the various other members of the team. Professional literature and research have highlighted differences in perception by social workers and physicians of the proper function of the medical social worker. Our social work department has developed a teaching program for medical students to enhance their knowledge with regard to this issue. It is presented at a single-session group meeting of an hour and a half, with structured content and goals.

Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation.

Dbl is a representative prototype of a growing family of oncogene products that contain the Dbl homology/pleckstrin homology elements in their primary structures and are associated with a variety of neoplastic pathologies. Members of the Dbl family have been shown to function as physiological activators (guanine nucleotide exchange factors) of the Rho-like small GTPases. Although the expression of GTPase-defective versions of Rho proteins has been shown to induce a transformed phenotype under different conditions, their transformation capacity has been typically weak and incomplete relative to that exhibited by dbl-like oncogenes. Moreover, in some cases (e.g. NIH3T3 fibroblasts), expression of GTPase-defective Cdc42 results in growth inhibition. Thus, in attempting to reconstitute dbl-induced transformation of NIH3T3 fibroblasts, we have generated spontaneously activated ("fast-cycling") mutants of Cdc42, Rac1, and RhoA that mimic the functional effects of activation by the Dbl oncoprotein. When stably expressed in NIH3T3 cells, all three mutants caused the loss of serum dependence and showed increased saturation density. Furthermore, all three stable cell lines were tumorigenic when injected into nude mice. Our data demonstrate that all three Dbl targets need to be activated to promote the full complement of Dbl effects. More importantly, activation of each of these GTP-binding proteins contributes to a different and distinct facet of cellular transformation.

Unequal pronuclear size--a powerful predictor of embryonic chromosome anomalies.

PURPOSE: Our purpose was to evaluate whether pronuclei of unequal size, observed in 13.7% of zygotes evaluated after in vitro fertilization (IVF), are predictive of chromosome anomalies in the developing embryo. METHODS: Five ploidy of 38 embryos grown from zygotes with unequal-sized pronuclei was assessed by fluorescent in situ hybridization (FISH). Twenty-six embryos developed after intracytoplasmic injection of sperm (ICSI) and 12 embryos were derived from conventional IVF. RESULTS: Chromosome anomalies were documented in the ICSI and IVF groups in 88.5 and 50% of cases, respectively. CONCLUSIONS: We suggest that FISH should be employed to examine the ploidy of zygotes with unequal pronuclei, prior to embryo transfer.

Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAP.

The Rho-related small GTP-binding protein Cdc42 has a low intrinsic GTPase activity that is significantly enhanced by its specific GTPase-activating protein, Cdc42GAP. In this report, we present the tertiary structure for the aluminum fluoride-promoted complex between Cdc42 and a catalytically active domain of Cdc42GAP as well as the complex between Cdc42 and the catalytically compromised Cdc42GAP(R305A) mutant. These structures, which mimic the transition state for the GTP hydrolytic reaction, show the presence of an AIF3 molecule, as was seen for the corresponding Ras-p120RasGAP complex, but in contrast to what has been reported for the Rho-Cdc42GAP complex or for heterotrimeric G protein alpha subunits, where AIF4- was observed. The Cdc42GAP stabilizes both the switch I and switch II domains of Cdc42 and contributes a highly conserved arginine (Arg 305) to the active site. Comparison of the structures for the wild type and mutant Cdc42GAP complexes provides important insights into the GAP-catalyzed GTP hydrolytic reaction.

Biochemical studies of the mechanism of action of the Cdc42-GTPase-activating protein.

The small GTP-binding proteins Rac, Rho, and Cdc42 were shown to mediate a variety of signaling pathways including cytoskeletal rearrangements, cell-cycle progression, and transformation. Key to the proper function of these GTP-binding proteins is an efficient shut-off mechanism that ensures the decay of the signal. Regulatory proteins termed GAPs (GTPase-activating proteins) enhance the intrinsic GTP hydrolysis of the GTP-binding proteins, thereby ensuring signal termination. We have used site-specific mutagenesis to elucidate the limit domain for GAP activity in Cdc42-GAP, and show that in addition to the known GAP-homology domain (three conserved boxes), a C-terminal region outside that domain is also essential for GAP activity. In addition, we have replaced the conserved arginine (Arg305), which was suggested by structural studies to be a key catalytic residue, with an alanine and found that the R305A Cdc42-GAP mutant has a greatly diminished catalytic capacity but is still able to bind Cdc42 with high affinity. Thus, a key catalytic role for this residue is confirmed. However, we also present evidence for the involvement of an additional residue(s), since the R305A Cdc42-GAP mutant still exhibits measurable activity. Some of this residual activity might result from a neighboring arginine, since a double mutant R305A/R306A shows a further decrease in catalytic activity.

Transformation activity of Cdc42 requires a region unique to Rho-related proteins.

The Rho subfamily GTP-binding protein Cdc42 mediates actin cytoskeletal rearrangements and cell cycle progression and is essential for Ras transformation. Expression of a Cdc42 mutant (Cdc42(F28L)) that undergoes spontaneous activation (guanine nucleotide exchange) results in transformation of NIH3T3 fibroblasts. In this report, we show that deletion of residues 120-139 from Cdc42(F28L), which comprise an insert region unique to Rho subfamily proteins but is missing in other GTP-binding proteins, yields a Cdc42 molecule that still undergoes spontaneous GTP-GDP exchange and stimulates both actin cytoskeletal changes and the activation of the cellular targets p21-activated kinase and the c-Jun kinase (JNK1). However, this Cdc42 mutant is unable to transform cells. These findings indicate that the Rho subfamily insert region is dispensable for many of the known signaling pathways initiated by activated Cdc42 but is essential for its regulation of cell growth.