Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the nerve growth factor (NGF) gene family, have been suggested to play a role in experience-dependent modification of neural networks in the developing nervous system. In this study we addressed the question of whether these neurotrophins are involved in long-term potentiation (LTP) in developing visual cortex. We recorded layer II/III field potentials and whole-cell currents evoked by test stimulation of layer IV at 0.1 Hz in visual cortical slices prepared from young rats (postnatal day 15-25) and observed effects of BDNF, NT-3, and NGF on these responses. Then we analyzed the effects of these neurotrophins on LTP induced by tetanic (⌰-burst type) stimulation of layer IV. We found that BDNF at 200 ng/ml potentiated field potentials and EPSCs in most cases and that this potentiation lasted after cessation of the BDNF application. At the concentration of 20 ng/ml, BDNF did not show such an effect, but it enhanced the magnitude of expressed LTP. On the other hand, NT-3 and NGF had none of these effects. Immunohistochemical staining of slices with antibody against BDNF showed that exogenous BDNF penetrated into the whole slice within ϳ5 min of its application. The actions of BDNF were blocked by preincubation of slices with TrkB-IgG fusion protein, a BDNF scavenger, or coapplication of K252a, an inhibitor for receptor tyrosine kinases. TrkB-IgG or K252a itself completely blocked LTP, suggesting that endogenous BDNF or another TrkB ligand plays a role in LTP in the developing visual cortex. Key words: brain-derived neurotrophic factor; long-term potentiation; nerve growth factor; neurotrophin-3; visual cortex; synaptic plasticityNeurotrophins of the nerve growth factor (NGF) family have been considered to play roles in the differentiation, neurite outgrowth, and survival of developing neurons and maintenance of a certain group of matured neurons (Levi-Montalcini, 1987;Barde, 1989;Davies, 1994). In addition to these well known functions, accumulating evidence from recent studies suggests that the neurotrophins are involved in more rapid changes in the CNS and peripheral nervous system L ohof et al., 1993; Kim et al., 1994; Le␤mann et al., 1994;Kang and Schuman, 1995;Levine et al., 1995;Wang et al., 1995;Stoop and Poo, 1996; Carmignoto et al., 1997; for reviews, see Bonhoeffer, 1996;Lewin and Barde, 1996). In particular, brain-derived neurotrophic factor (BDN F) is suggested to play a role in a form of synaptic plasticity, long-term potentiation (LTP), in the hippocampus (Castrén et al., 1992a;Patterson et al., 1992;Kang and Schuman, 1995;Figurov et al., 1996). Also, LTP in the hippocampus is reported to be impaired in BDN F knockout mice (Korte et al., 1995;Patterson et al., 1996) In the developing visual cortex, it has been reported that the experience-dependent plasticity of structure and f unction of neural circuits is influenced by the neurotrophins, although the involvement of NGF is a matter of controversy (Maffei et al., 1992;Car...

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The natriuretic effect of DA-1 agonists is less in the spontaneously hypertensive rat (SHR) than its normotensive control, the Wistar-Kyoto rat (WKY). To determine a mechanism of the decreased effect of DA-1 agonists on sodium transport, DA-1 receptors in renal proximal convoluted tubule (PCI) were studied by radioligand binding and by adenylate cyclase (AC) determinations. Specific binding of '25I-SCH 23982 (defined by 10 MM SCH 23390, a DA-1 antagonist) was concentration dependent, saturable, and stereoselective. The dissociation constant, maximum receptor density, and DA-1 antagonist inhibition constant were similar in SHR and WKY. The apparent molecular weight of the DA-1 receptor determined by the photoaffinity D1 probe '25I-MAB was also similar in WKY and SHR. However, DA-1 agonists competed more effectively for specific '25I-SCH 23982 binding sites in WKY than in SHR.Basal as well as forskolin, parathyroid hormone, GTP and Gpp(NH)p-stimulated-AC activities were similar. In contrast DA-1 agonists (fenoldopam, SKF 38393, SND 911C12) stimulated AC activity to a lesser extent in SHR. GTP and Gpp(NH)p enhanced the ability of DA-1 agonists to stimulate AC activity in WKY but not in SHR. These data suggest a defect in the DA-1 receptor-second messenger coupling mechanism in the PCI of the SHR.

The coupling between the dopamine1 (DA1) receptor and the G protein/adenylyl cyclase (AC) enzyme complex is defective in the proximal convoluted tubule (PCT) of 20-wk-old spontaneously hypertensive rats (SHRs). Because this coupling defect could have been due to desensitization secondary to elevated renal dopamine levels in the adult animal, we studied the interaction between DA1 receptors and AC in PCT of rats as early as 3 wk of age, a time when renal dopamine levels are similar in SHRs and their normotensive controls (Wistar-Kyoto rats, WKYs). Maximum receptor density did not change with age and was similar in WKYs and SHRs in all the age groups studied (3, 8, and 20 wk). Basal-, forskolin-, and guanyl nucleotide-stimulated AC activities were also similar in WKYs and SHRs and did not change with age. However, the DA1 agonist-stimulated AC activity was greater in WKYs than in SHRs and increased with age in WKYs but not in SHRs. Moreover, the ability of a nonhydrolyzable analogue of GTP, Gpp(NH)p, to enhance DA1 agonist (SND-919-C12, 1 microM)-stimulated AC activity increased with age in WKY but not in SHRs. To determine if the defect noted in the PCT of SHRs is due to a defective D1A receptor gene, parallel studies were performed in the striatum, since this receptor is expressed predominantly in the latter tissue. In contrast to the results in PCT, radioligand binding and AC studies in striatum revealed no differences between WKYs and SHRs.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain-derived neurotrophic factor (BDNF) is reported to enhance synaptic transmission and to play a role in long-term potentiation in hippocampus and neocortex. If so, a shortage or blockade of BDNF might lead to another form of synaptic plasticity, long-term depression (LTD). To test this possibility and to elucidate mechanisms if it is the case, EPSCs evoked by test stimulation of layer IV were recorded from layer II/III neurons in visual cortical slices of young rats in the whole-cell voltage-clamp mode. LTD was induced by low-frequency stimulation (LFS) at 1 Hz for 10-15 min if each pulse of the LFS was paired with depolarization of neurons to -30 mV but was not induced if their membrane potentials were kept at -70 mV. Such an LTD was blocked by exogenously applied BDNF, probably through presynaptic mechanisms. Suppression of endogenous BDNF activity by the anti-BDNF antibody or an inhibitor for BDNF receptors made otherwise ineffective stimuli (LFS without postsynaptic depolarization) effective for LTD induction, suggesting that endogenous BDNF may prevent low-frequency inputs from inducing LTD in the developing visual cortex.

We have previously reported a defect in the coupling of the renal dopamine-1 receptor (D1) to adenylate cyclase (AC) in the proximal convoluted tubule (PCT) of the spontaneously hypertensive rat (Okamoto-Aoki strain). To determine if this defect is present in another model of hypertension, we microdissected PCTs from Dahl salt-sensitive (DSS) and Dahl salt-resistant (DSR) rats on low- or high-NaCl diet. The ability of two selective D1 agonists, fenoldopam and SND-919-C12, and forskolin to stimulate AC activity in PCT was determined in each of the four groups of rats. Fenoldopam (10(-7) M) and SND-919-C12 (10(-6) M) failed to stimulate AC activity in the PCT of DSS rats whether on a low- or high-NaCl diet. In DSR rats, however, both fenoldopam and SND-919-C12 stimulated AC activity by 289-320% and 220-270%, respectively, whether on a low- or high-NaCl intake. Forskolin (10(-5) M), which directly stimulates AC activity, increased AC activity in all four groups. These studies show that in DSS rats the D1 receptor in the PCT fails to respond to D1 agonists. This defect is not a consequence of the hypertension because it was present in the DSS rats on a low-salt diet and before blood pressure elevation.

BackgroundThe yeast Saccharomyces cerevisiae is a promising host cell for producing a wide range of chemicals. However, attempts to metabolically engineer Crabtree-positive S. cerevisiae invariably face a common issue: how to reduce dominant ethanol production. Here, we propose a yeast metabolic engineering strategy for decreasing ethanol subgeneration involving tugging the carbon flux at an important hub branching point (e.g., pyruvate). Tugging flux at a central glycolytic overflow metabolism point arising from high glycolytic activity may substantially increase higher alcohol production in S. cerevisiae. We validated this possibility by testing 2,3-butanediol (2,3-BDO) production, which is routed via pyruvate as the important hub compound.ResultsBy searching for high-activity acetolactate synthase (ALS) enzymes that catalyze the important first-step reaction in 2,3-BDO biosynthesis, and tuning several fermentation conditions, we demonstrated that a stronger pyruvate pulling effect (tugging of pyruvate carbon flux) is very effective for increasing 2,3-BDO production and reducing ethanol subgeneration by S. cerevisiae. To further confirm the validity of the pyruvate carbon flux tugging strategy, we constructed an evolved pyruvate decarboxylase (PDC)-deficient yeast (PDCΔ) strain that lacked three isozymes of PDC. In parallel with re-sequencing to identify genomic mutations, liquid chromatography–tandem mass spectrometry analysis of intermediate metabolites revealed significant accumulation of pyruvate and NADH in the evolved PDCΔ strain. Harnessing the high-activity ALS and additional downstream enzymes in the evolved PDCΔ strain resulted in a high yield of 2,3-BDO (a maximum of 0.41 g g−1 glucose consumed) and no ethanol subgeneration, thereby confirming the utility of our strategy. Using this engineered strain, we demonstrated a high 2,3-BDO titer (81.0 g L−1) in a fed-batch fermentation using a high concentration of glucose as the sole carbon source.ConclusionsWe demonstrated that the pyruvate carbon flux tugging strategy is very effective for increasing 2,3-BDO production and decreasing ethanol subgeneration in Crabtree-positive S. cerevisiae. High activity of the common first-step enzyme for the conversion of pyruvate, which links to both the TCA cycle and amino acid biosynthesis, is likely important for the production of various chemicals by S. cerevisiae.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1176-y) contains supplementary material, which is available to authorized users.

Porcine dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin. It is processed by proteases into 3 independent proteins: dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We fractionated DPP and DSP along with TGF-β activity by ion exchange (IE) chromatography from developing pig molars and measured their alkaline phosphatase (ALP)-stimulating activity in human periodontal (HPDL) cells with or without TGF-β receptor inhibitor. We then purified TGF-β-unbound or -bound DPP and DSP by reversephase high-performance liquid chromatography (RP-HPLC) using the ALP-HPDL system. The TGF-β isoform bound to DPP and DSP was identified as being TGF-β1 by both ELISA and LC-MS/ MS analysis. We incubated carrier-free human recombinant TGF-β1 (CF-hTGF-β1) with TGF-β-unbound DPP or DSP and characterized the binding on IE-HPLC using the ALP-HPDL system. When only CF-hTGF-β1 was incubated, approximately 3.6% of the ALP-stimulating activity remained. DPP and DSP rescued the loss of TGF-β1 activity. Approximately 19% and 10% of the ALP stimulating activities were retained by the binding of TGF-β to DPP and DSP, respectively. The type I collagen infrequently bound to CF-hTGF-β1. We conclude that both DPP and DSP help retain TGF-β1 activity in porcine dentin.