Cellular Quantification of Tyrosine Hydroxylase in the Rat Brain by Immunoautoradiography

Abstract: We developed a rapid and sensitive radioimmunohistochemical method for the quantification of tyrosine hydroxylase (TH) at both the anatomical and cellular level. Coronal tissue sections from fresh-frozen rat brains were incubated in the presence of a TH monoclonal antibody. The reaction was revealed with a ³⁵S-labeled secondary antibody. TH content was quantified in catecholaminergic brain areas by measuring optical density on autoradiographic films or silver grain density on autoradiographic emulsion-coated sections. Regional TH concentrations determined in the locus ceruleus (LC), substantia nigra pars compacta (SNC), and ventral tegmental area (VTA) were significantly increased by 45% after reserpine treatment in the LC but unchanged in the SNC and VTA. Microscopic examination of TH radioimmunolabeling showed a heavy accumulation of silver grains over catecholaminergic cell bodies. In the LC, grain density per cell was heterogeneous and higher in the ventral than in the dorsal part of the structure. After reserpine treatment, TH levels were significantly increased (57%) in the neurons of the LC but not in those of the SNC or VTA. The data support the validity of this radioimmunohistochemical method as a tool for quantifying TH protein at the cellular level and they confirm that TH protein content is differentially regulated in noradrenergic and dopaminergic neurons in response to reserpine.     

Full-length prion protein aggregates to amyloid fibrils and spherical particles by distinct pathways

As limited structural information is available on prion protein (PrP) misfolding and aggregation, a causative link between the specific (supra)molecular structure of PrP and transmissible spongiform encephalopathies remains to be elucidated. In this study, high pressure was utilized, as an approach to perturb protein structure, to characterize different morphological and structural PrP aggregates. It was shown that full-length recombinant PrP undergoes beta-sheet aggregation on high-pressure-induced destabilization. By tuning the physicochemical conditions, the assembly process evolves through two distinct pathways leading to the irreversible formation of spherical particles or amyloid fibrils, respectively. When the PrP aggregation propensity is enhanced, high pressure induces the formation of a partially unfolded aggregated protein, Agg(HP), which relaxes at ambient pressure to form amorphous aggregates. The latter largely retain the native secondary structure. On prolonged incubation at high pressure, followed by depressurization, Agg(HP) transforms to a monodisperse population of spherical particles of about 20 nm in diameter, characterized by an essentially beta-sheet secondary structure. When the PrP aggregation propensity is decreased, an oligomeric reaction intermediate, I(HP), is formed under high pressure. After pressure release, I(HP) relaxes to the original native structure. However, on prolonged incubation at high pressure and subsequent depressurization, it transforms to amyloid fibrils. Structural evaluation, using optical spectroscopic methods, demonstrates that the conformation adopted by the subfibrillar oligomeric intermediate, I(HP), constitutes a necessary prerequisite for the formation of amyloids. The use of high-pressure perturbation thus provides an insight into the molecular mechanism of the first stages of PrP misfolding into amyloids.     

Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice

Unlike the adjustable gastric banding procedure (AGB), Roux-en-Y gastric bypass surgery (RYGBP) in humans has an intriguing effect: a rapid and substantial control of type 2 diabetes mellitus (T2DM). We performed gastric lap-band (GLB) and entero-gastro anastomosis (EGA) procedures in C57Bl6 mice that were fed a high-fat diet. The EGA procedure specifically reduced food intake and increased insulin sensitivity as measured by endogenous glucose production. Intestinal gluconeogenesis increased after the EGA procedure, but not after gastric banding. All EGA effects were abolished in GLUT-2 knockout mice and in mice with portal vein denervation. We thus provide mechanistic evidence that the beneficial effects of the EGA procedure on food intake and glucose homeostasis involve intestinal gluconeogenesis and its detection via a GLUT-2 and hepatoportal sensor pathway.     

Diet-induced epigenetic regulation in vivo of the intestinal fructose transporter Glut5 during development of rat small intestine

Metabolic complications arising from excessive fructose consumption are increasing dramatically even in young children, but little is known about ontogenetic mechanisms regulating Glut5 [glucose transporter 5; encoded by the Slc2a5 (solute carrier family 2 member 5) gene]. Glut5 expression is low postnatally and does not increase, unless luminal fructose and systemic glucocorticoids are present, until ≥ 14 days of age, suggesting substrate-inducible age- and hormone-sensitive regulation. In the present study, we perfused intestines of 10- and 20-day-old rats with either fructose or glucose then analysed the binding of Pol II (RNA polymerase II) and GR (glucocorticoid receptor), as well as acetylation of histones H3 and H4 by chromatin immunoprecipitation. Abundance of Glut5 mRNA increased only with fructose perfusion and age, a pattern that matched that of Pol II binding and histone H3 acetylation to the Glut5 promoter. Although many regions of the Glut5 promoter respond to developmental signals, fewer regions perceive dietary signals. Age- but not fructose-dependent expression of Sglt1 [sodium-dependent glucose co-transporter 1 encoded by the Slc5a1(solute carrier family 5 member 1) gene] also correlated with Pol II binding and histone H3 acetylation. In contrast, G6Pase (glucose-6-phosphatase; encoded by the G6pc gene) expression, which decreases with age and increases with fructose, is associated only with age-dependent changes in histone H4 acetylation. Induction of Glut5 during ontogenetic development appears to be specifically mediated by GR translocation to the nucleus and subsequent binding to the Glut5 promoter, whereas the glucocorticoid-independent regulation of Sglt1 by age was not associated with any GR binding to the Sglt1 promoter.     

An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.     

Hot fusion

A team from Harvard University has demonstrated that human embryonic stem cells have the capacity to reprogram an adult cell nucleus, leading to a new way of deriving human embryonic stem cell lines without using oocytes.     

Unraveling the mechanism of radiosensitization by gemcitabine: the role of TP53

Gemcitabine has excellent radiosensitizing properties, as shown in both preclinical and clinical studies. Radiosensitization correlated with the early S-phase block of gemcitabine. In the present study, we investigated the role of TP53 in the radiosensitizing effect of gemcitabine. Isogenic A549 cells differing in TP53 status were treated with gemcitabine during the 24 h prior to irradiation. Cell survival was determined 7 days after irradiation by the sulforhodamine B test. In addition, cell cycle perturbation was determined by flow cytometry and TP53 expression by Western blot analysis. Gemcitabine caused a concentration-dependent radiosensitizing effect in all cell lines. Transformed A549 cells were less sensitive to the cytotoxic effect of gemcitabine. The cell cycle arrest early in the S phase was dependent on the drug dose but was comparable in the different cell lines and was not related to functional TP53. Using isogenic cell lines, we have shown that neither TP53 status nor the transfection procedure influenced the radiosensitizing effect of gemcitabine. Since both the radiosensitizing effect at equitoxic concentrations and the cell cycle effect of gemcitabine were independent of TP53 expression, it is likely that TP53 protein does not play a crucial role in the radiosensitizing mechanism of gemcitabine.