Spatiotemporal resolution of BDNF neuroprotection against glutamate excitotoxicity in cultured hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) protects hippocampal neurons from glutamate excitotoxicity as determined by analysis of chromatin condensation, through activation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. However, it is still unknown whether BDNF also prevents the degeneration of axons and dendrites, and the functional demise of synapses, which would be required to preserve neuronal activity. Herein, we have studied the time-dependent changes in several neurobiological markers, and the regulation of proteolytic mechanisms in cultured rat hippocampal neurons, through quantitative western blot and immunocytochemistry. Calpain activation peaked immediately after the neurodegenerative input, followed by a transient increase in ubiquitin-conjugated proteins and increased abundance of cleaved-caspase-3. Proteasome and calpain inhibition did not reproduce the protective effect of BDNF and caspase inhibition in preventing chromatin condensation. However, proteasome and calpain inhibition did protect the neuronal markers for dendrites (MAP-2), axons (Neurofilament-H) and the vesicular glutamate transporters (VGLUT1-2), whereas caspase inhibition was unable to mimic the protective effect of BDNF on neurites and synaptic markers. BDNF partially prevented the downregulation of synaptic activity measured by the KCl-evoked glutamate release using a Förster (Fluorescence) resonance energy transfer (FRET) glutamate nanosensor. These results translate a time-dependent activation of proteases and spatial segregation of these mechanisms, where calpain activation is followed by proteasome deregulation, from neuronal processes to the soma, and finally by caspase activation in the cell body. Moreover, PI3-K and PLCγ small molecule inhibitors significantly blocked the protective action of BDNF, suggesting an activity-dependent mechanism of neuroprotection. Ultimately, we hypothesize that neuronal repair after a degenerative insult is initiated at the synaptic level.

Development and use of fluorescent nanosensors for metabolite imaging in living cells.

To understand metabolic networks, fluxes and regulation, it is crucial to be able to determine the cellular and subcellular levels of metabolites. Methods such as PET and NMR imaging have provided us with the possibility of studying metabolic processes in living organisms. However, at present these technologies do not permit measuring at the subcellular level. The cameleon, a fluorescence resonance energy transfer (FRET)-based nanosensor uses the ability of the calcium-bound form of calmodulin to interact with calmodulin binding polypeptides to turn the corresponding dramatic conformational change into a change in resonance energy transfer between two fluorescent proteins attached to the fusion protein. The cameleon and its derivatives were successfully used to follow calcium changes in real time not only in isolated cells, but also in living organisms. To provide a set of tools for real-time measurements of metabolite levels with subcellular resolution, protein-based nanosensors for various metabolites were developed. The metabolite nanosensors consist of two variants of the green fluorescent protein fused to bacterial periplasmic binding proteins. Different from the cameleon, a conformational change in the binding protein is directly detected as a change in FRET efficiency. The prototypes are able to detect various carbohydrates such as ribose, glucose and maltose as purified proteins in vitro. The nanosensors can be expressed in yeast and in mammalian cell cultures and were used to determine carbohydrate homeostasis in living cells with subcellular resolution. One future goal is to expand the set of sensors to cover a wider spectrum of metabolites by using the natural spectrum of bacterial periplasmic binding proteins and by computational design of the binding pockets of the prototype sensors.

Endothelial nitric oxide synthase intron 4 polymorphism influences the progression of renal disease.

BACKGROUND/AIM: Nitric oxide is a potent regulator of intrarenal hemodynamics and may influence the renal function. We investigated whether polymorphism of intron 4 of the endothelial constitutive nitric oxide synthase (ecNOS) gene is related to the progression of chronic renal failure. METHODS: Polymorphism of ecNOS intron 4 was studied in 1,005 hemodialysis patients (710 with nondiabetic nephropathy and 295 with diabetic nephropathy) and was compared with the findings in 189 healthy subjects. ecNOS genotypes were determined by the polymerase chain reaction, followed by agarose gel electrophoresis. RESULTS: The frequencies of ecNOS4a/a, ecNOS4a/b, and ecNOS4b/b genotypes were, respectively, 0% (0/189), 13.8% (26/189), and 86.2% (163/189) in the control group; 1.7% (12/710), 22.1% (157/710), and 76.2% (541/710) in the nondiabetic nephropathy group, and 1.0% (3/295), 22.7% (67/295), and 76.3% (225/295) in the diabetic nephropathy group. The frequency of ecNOS4a (ecNOSa/a and ecNOSa/b) was significantly higher in both the nondiabetic group and in the diabetic group than in the controls (p = 0.0025 and p = 0.0438, respectively). CONCLUSION: There was a significantly higher frequency of the a allele of intron 4 in both nondiabetic and diabetic hemodialysis patients, so the polymorphism of intron 4 of the ecNOS gene may have a wide influence on the progression of renal disease.

Transition Structures for the Aromatic Claisen Rearrangements by the Molecular Orbital Method.

Ab initio calculations were performed for eight Claisen rearrangements, eqs 1-8. Transition-state (TS) structures of [3,3] sigmatropic rearrangements of reactions 1-4 are similar, but their activation energies (E(a)'s) are different, E(a)(1) < E(a)(2) and E(a)(3) < E(a)(4). From the intermediate of reaction 3, a hydrogen is moved intermolecularly to form the product, o-allyl phenol. The lower reactivities of reactions 2 and 4 relative to reactions 1 and 3 are ascribed to large endothermicities in the sigmatropic rearrangements, respectively. Chair-type transition states are more favorable than boat-type transition states in reactions 1-4. The allyl group is released from the in-plane C-X (X = O or N) sigma bond and is captured by the pi-type lone-pair electrons. The sulfur- and phosphorus-containing rearrangements, reactions 5-8, are computed to have smaller activation energies but are to be less exothermic than those of oxgyen- and nitrogen-containing rearrangements.

[Latent impairment of left ventricular filling in hypertension without left ventricular hypertrophy and improvement by dipyridamole treatment: pulsed Doppler echocardiography study].

Left ventricular (LV) filling impairment in patients with hypertension (HT) not necessarily associated with LV hypertrophy has not been sufficiently investigated. Therefore, we examined the response of LV filling to isometric exercise in patients with HT without LV hypertrophy and LV filling abnormality at rest. We studied 25 patients (aged 40 to 66 years, mean 51 +/- 7 years) and 13 age-matched normal subjects. The HT patients were selected by the following criteria: 1) systolic blood pressure (sBP) over 160 mmHg and/or diastolic BP over 90 mmHg was observed at least three times during the last 6 months, 2) LV wall thickness was under 11 mm, and 3) the ratio of peak atrial LV inflow velocity (A) to peak early diastolic LV inflow velocity (E) was within the mean +/- SD of normal subjects. LV inflow was measured by pulsed Doppler flowmetry before and during handgrip exercise (50% maximal effort for one minute and a half) in the patients before [HT-D (-)] and after [HT-D (+)] dipyridamole (D) administration (0.28 mg/kg/4 min) and in the normal subjects (N). Doppler-derived indices were A, E, A/E, DR (the deceleration rate from peak to half of the early diastolic inflow velocity), % delta A/E (% change in A/E from baseline), and % delta DR (% change in DR from baseline). There was no significant difference in LV wall thickness between the HT and N groups. There was also no significant difference in A/E at rest between the three groups. Increase of sBP and heart rate were similar in all groups during handgrip exercise.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of gene expression for immunomodulating cytokines in peripheral blood mononuclear cells in response to orally administered PSK, an immunomodulating protein-bound polysaccharide.

The protein-bound polysaccharide extracted from a fungus, PSK, has been used as a biological response modifier in the treatment of cancer patients in Japan for over 16 years. The administration of PSK to tumor-bearing rodents inhibited tumor growth and modulated immune responses. Recently, an in vitro study has revealed that PSK is a strong inducer of cytokine gene expression and production in human peripheral blood mononuclear cells (PBMC). To establish whether PSK has cytokine-inducing activities in vivo, we have orally administered PSK (1 g, the clinical dose) to 12 healthy volunteers and 9 gastric cancer patients who had undergone gastrectomy, and assessed the gene expression for cytokines in PBMC of each subject. As determined by the reverse-transcribed polymerase chain reaction method, the induction of gene expression for both tumor necrosis factor alpha and interleukin-8 (IL-8) was detected in PBMC from 5 of the 12 healthy volunteers (42%) and 4 of the 9 patients (44%). Furthermore, the concentration of serum IL-8 was elevated in 5 healthy volunteers given PSK orally, who had shown induction of IL-8 gene expression, as detected by enzyme-linked immunosorbent assay. These findings indicate that responsiveness of PBMC to PSK, in terms of gene expression and production of cytokines, varies among individuals. Thus, when using PSK to treat cancer patients, it seems advisable to select patients on the basis of their responsiveness to PSK. We speculate that the cytokines induced by PSK might mediate the immunoenhancing action of this agent in vivo.

[Hepatic artery and portal vein infusion of adriamycin in experimental liver metastasis].

Hepatic artery and portal vein infusion of adriamycin (ADM) to normal rabbit and the experimental liver metastasis model of VX2 tumor were discussed in this study. The concentration of ADM in the peripheral blood, liver, myocardium, lung of normal rabbit and metastatic tumor were measured in the HPLC method. There was no difference between arterial infusion and portal infusion in the normal rabbits. In the metastatic tumor, one hour after the infusion, concentration of ADM showed no difference between arterial and portal infusion, but two and three hours later, the concentration was significantly higher after portal infusion than arterial infusion. It was suspected that portal infusion would be more effective for liver metastasis. The number of tumor nodules for estimation of the anti-tumor effect on metastatic models was decreased significantly after arterial and portal infusion compared with the controls, but there was no statistical difference between arterial and portal infusion.