Vascular endothelial growth factor (VEGF) is a potent regulator of placental vascular function. Endothelial dysfunction is a key factor associated with preeclampsia. In this study, we examined expression of VEGF, endocrine gland-derived VEGF (EG-VEGF), VEGF receptors 1 and 2 (VEGFR-1 and VEGFR-2), and neuropilin-1 and -2 (NP-1 and NP-2) in human placentas from women with normal and preeclamptic (PE) pregnancies using quantitative or semiquantitative PCR. We found that total VEGF mRNA expression was increased 2.8-fold (P < 0.05), along with increases in mRNA expression of VEGF121, 165, and 189 (P < 0.05; 1.7-, 1.9-, and 1.8-fold, respectively) in PE vs. normal placentas. Expression of VEGFR-1 mRNA, but not EG-VEGF and the other three VEGF receptors studied, was elevated (P < 0.05) 2.7-fold in PE vs. normal placentas. Protein expression of VEGF and its four receptors was determined using Western blot analysis. For VEGF, two major isoforms (VEGF165 and 189) were detected. For VEGFR-1, VEGFR-2, NP-1, and NP-2, one major band was observed at 180, 235, 130, and 130 kDa, respectively. All of these bands were corresponding to their positive controls. Of these five proteins studied, only VEGFR-1 levels were increased (P < 0.05; 1.7-fold) in PE placentas. The expression of VEGF and the four VEGF receptors was confirmed using immunohistochemistry. They were primarily present in syncytiotrophoblasts and endothelial cells of villous capillaries and large vessels. Thus, together with previous reports that VEGFR-1 mediates trophoblast function and inhibits VEGF-induced angiogenesis and endothelium-dependent vasodilation, these data suggest that the increased VEGFR-1 expression may alter VEGF- mediated function on trophoblast and endothelial cells in PE placentas.

In streptomycete anthracycline biosynthetic gene clusters, small open reading frames are located just upstream of minimal polyketide synthase genes. aknX is such a gene found in the aklavinone-aclacinomycin biosynthetic gene cluster of Streptomyces galilaeus. In order to identify its function, the aknX gene was expressed in Escherichia coli. The cell extract prepared from E. coli cells overexpressing AknX protein exhibited anthrone oxygenase activity, which converted emodinanthrone to anthraquinone emodin. This indicates that AknX and related gene products such as DnrG and SnoaB are involved in the formation of aklanonic acid from its anthrone precursor, as suggested by their homology with TcmH and ActVA6. The AknX protein fused with a His 6 tag was efficiently purified to homogeneity by Ni 2؉ affinity and anion-exchange column chromatography. The native molecular mass of AknX was estimated to be 42 kDa by gel filtration. Thus, native AknX is considered to have a homotrimeric subunit structure. AknX, like TcmH and ActVA6, possesses no apparent prosthetic group for oxygen activation. Site-directed mutagenesis was carried out to identify the key amino acid residue(s) involved in the oxygenation reaction. Of seven AknX mutants expressed, the W67F mutant showed significantly reduced oxygenase activity, suggesting the important role of the W67 residue in the AknX reaction. A possible mechanism for the reaction via peroxy anion intermediate is proposed.Aclacinomycins, which are anthracycline antibiotics produced by Streptomyces galilaeus, show potent antitumor activity by inhibiting complex formation of DNA and topoisomerase II and thus have lower cardiotoxicity than other anthracyclines that inhibit topoisomerase II by stabilization of cleavable complex (8). Aclacinomycin A, also called aclarubicin, has been clinically used in France, Japan, and other Asian countries for the treatment of carcinoma of the stomach, pulmonary carcinoma, oophoroma, malignant lymphadenoma, and acute leukemia. The aglycone moiety of aclacinomycins is aklavinone, which also serves as a common precursor for aglycones of other anthracyclines such as daunomycinone, pyrromycinone, and ε-rhodomycinone, etc. Figure 1 shows the chemical structures of representative anthracycline antibiotics.Previously, we cloned the aklavinone biosynthetic gene cluster from S. galilaeus strain 3AR-33, a mutant strain accumulating aklavinone (18). The 3.4-kb BamHI fragment complemented aklavinone production in the mutant strain ANR-58 as well as aclacinomycin production in the strain . Nucleotide sequence analysis of the fragment showed the presence of open reading frames (ORFs) most typical of bacterial type II polyketide synthase (PKS) genes that code for ␤-ketoacyl synthase (KS), the so-called chain-length factor, and acyl carrier protein (3). In addition to these minimal PKS genes, we noted the presence of a small ORF, aknX, just upstream of the KS gene aknB. Such genes are not found in typical type II PKS gene clusters like that of actinorhodin (2). However, s...

BackgroundAmyotrophic lateral sclerosis (ALS) is characterized by selective degeneration of motor neurons. The gene encoding Cu/Zn superoxide dismutase (SOD1) is responsible for 20% of familial ALS cases. Growth hormone (GH) concentrations are low in the cerebrospinal fluid of patients with ALS; however, its association with motoneuronal death is not known. We tested the neuroprotective effects of GH on human SOD-1-expressing cultured motor neurons and SOD1G93A transgenic mice.ResultsIn cultured motor neurons, cytotoxicity was induced by A23187, GNSO, or homocysteine, and the effects of GH were determined by MTT, bax, PARP cleavage pattern, Hoechst nuclear staining, MAPK, and PI3K assay. In SOD-1 transgenic mice, rotarod motor performance was evaluated. Survival analysis of motoneuronal loss was done using cresyl violet, GFAP, and Bcl-2 staining. GH prevents motorneuronal death caused by GSNO and homocysteine, but not that by A23187. It activates MAPK and PI3K. GH-treated mice showed prolonged survival with improved motor performance and weight loss. GH decreased cresyl violet positive motoneuronal loss with strong Bcl-2 and less GFAP immunoreactivity.ConclusionsOur results demonstrate that GH has a protective effect on mutant SOD-1-expressing motor neurons.

BackgroundNerve growth factor (NGF) is known not only as a major factor for neuronal plasticity but also as a pain stimulator. Although there have been several trials with NGF for its application in the regeneration or protection of the nervous system, the pain induced by NGF remains a challenge to be overcome. In this study, the pain induced by NGF gene therapy was evaluated.ResultsVehicle or recombinant dog NGF plasmid was administered into the intrathecal space of dogs. Twenty-four hours after the vehicle or NGF plasmid inoculation, dogs were subcutaneously treated with 150 mg/kg pyridoxine every day for 7 days. For pain assessment, physical examination and electrophysiological recording were performed. Only in the vehicle-treated group, weight loss occurred, while NGF plasmid inoculation significantly improved this physical abnormalities. In the vehicle-treated group, electrophysiological recordings showed that H-reflex disappeared at 24 h after the last pyridoxine treatment. However, in the NGF plasmid inoculated group, the H-reflex were normal. In the results of immunohistochemistry, the NGF plasmid administration efficiently expressed in the dorsal root ganglia and significantly increased the pyridoxine-induced reduction of calcitonin gene-related peptide (CGRP) immunoreactive neurons, but not in substance P immunoreactive neurons, in the dorsal root ganglia.ConclusionsGiven these results, we reason that NGF gene therapy in pyridoxine induced neuropathic dogs does not induce neuropathic pain with this dosage, even with increasing the expression of CGRP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-015-0236-5) contains supplementary material, which is available to authorized users.

To construct a sensory neuropathy model, excess pyridoxine (150 mg/kg s.i.d.) was injected subcutaneously in dogs over a period of 7 days. During the administrations period, the dogs experienced body weight reduction and proprioceptive loss involving the hindquarters. After pyridoxine administration was completed, electrophysiological recordings showed that the M wave remained at a normal state, but the H-reflex of the treated dogs disappeared at 7 days. The dorsal funiculus of L4 was disrupted irregularly in the axons and myelin with vacuolation. The dorsal root ganglia of L4, and sciatic and tibial nerves showed degenerative changes and vacuolation. However, the lateral and ventral funiculi of L4 showed a normal histopathologic pattern. Although this subcutaneous administration method did not cause systemic toxicity and effectively induced sensory neuropathy, this study confirmed the possibility of producing a pyridoxine-induced sensory neuropathy model in dogs with short-term administration.