Studies on expression of aldehyde dehydrogenase in normal and cancerous tissues of thyroids.

Recently published articles have reported the controversial data regarding expression of aldehyde dehydrogenase isozyme 1A1 (ALDH1A1), a potential candidate marker for normal and cancer stem cells (CSCs), in thyroid tissues. These data prompted us to re-evaluate expression of ALDH1A1 in normal and cancerous thyroid tissues by 2 different means. The first method was immunohistochemistry with 2 different anti-ALDH1A1 antibodies from distinct companies. Following validating the integrity of these 2 antibodies by Western blotting with ALDH-expressing and nonexpressing cancer cell lines and immunohistochemistry with breast and colon tissues, we report here significant and comparable expression of ALDH1A1 in both normal and cancerous thyroid tissues with both antibodies. Next, relative expression levels of ALDH isozymes were evaluated by reverse transcription-polymerase chain reaction (RT-PCR), revealing that ALDH1A1 was the most highly expressed isozyme followed by ALDH9A1 and relative expression patterns of isozymes were very similar in normal and cancerous tissues. All these data demonstrate that thyroid cells of normal and cancer origins do express ALDH1A1 and to a lesser extent 9A1. Further study will be necessary to study functional significance of ALDH1A1 in the function and behaviors of thyroid normal and cancer stem cells.

High-Fat Diet-Induced Diabetic Conditions Exacerbate Cognitive Impairment in a Mouse Model of Alzheimer's Disease Via a Specific Tau Phosphorylation Pattern.

BACKGROUND: Epidemiological evidence has demonstrated a clear association between diabetes mellitus and increased risk of Alzheimer's disease (AD). Cerebral accumulation of phosphorylated tau aggregates, a cardinal neuropathological feature of AD, is associated with neurodegeneration and cognitive decline. Clinical and experimental studies indicate that diabetes mellitus affects the development of tau pathology; however, the underlying molecular mechanisms remain unknown. OBJECTIVE: In the present study, we used a unique diabetic AD mouse model to investigate the changes in tau phosphorylation patterns occurring in the diabetic brain. DESIGN: Tau-transgenic mice were fed a high-fat diet (n = 24) to model diabetes mellitus. These mice developed prominent obesity, severe insulin resistance, and mild hyperglycemia, which led to early-onset neurodegeneration and behavioral impairment associated with the accumulation of hyperphosphorylated tau aggregates. RESULTS: Comprehensive phosphoproteomic analysis revealed a unique tau phosphorylation signature in the brains of mice with diabetic AD. Bioinformatic analysis of the phosphoproteomics data revealed putative tau-related kinases and cell signaling pathways involved in the interaction between diabetes mellitus and AD. CONCLUSION: These findings offer potential novel targets that can be used to develop tau-based therapies and biomarkers for use in AD.

Prophylactic and therapeutic efficacies of a selective inhibitor of the immunoproteasome for Hashimoto's thyroiditis, but not for Graves' hyperthyroidism, in mice.

Major histocompatibility complex (MHC) class I-restricted T cell epitopes are generated mainly by the immunoproteasome in antigen-presenting cells. Therefore, inhibition of activity of this proteolytic complex molecule is thought to be a potential treatment for cell-mediated autoimmune diseases. We therefore studied the efficacy of an immunoproteasome inhibitor, ONX 0914 (formerly PR-957), for the treatment of autoimmune thyroid diseases, including cell-mediated Hashimoto's thyroiditis and autoantibody-mediated Graves' hyperthyroidism using mouse models. Our data show that ONX 0914 was effective prophylactically and therapeutically at suppressing the degree of intrathyroidal lymphocyte infiltration and, to a lesser degree, the titres of anti-thyroglobulin autoantibodies in non-obese diabetic (NOD)-H2(h4) mice, an iodine-induced autoimmune thyroiditis model. It also inhibited differentiation of T cells to T helper type 1 (Th1) and Th17 cells, effector T cell subsets critical for development of thyroiditis in this mouse strain. In contrast, its effect on the Graves' model was negligible. Although ONX 0914 exerts its immune-suppressive effect through not only suppression of immune proteasome but also other mechanism(s), such as inhibition of T cell differentiation, the present results suggest that the immunoproteasome is a novel drug target in treatment of Hashimoto's thyroiditis in particular and cell-mediated autoimmune diseases in general.

Alleviation of Abeta-induced cognitive impairment by ultrasound-mediated gene transfer of HGF in a mouse model.

A new therapeutic approach to treat Alzheimer's disease (AD) is needed, and the use of growth factors is considered to be a candidate. Hepatocyte growth factor (HGF) is a unique multifunctional growth factor, which has the potential effect to exert neurotrophic action and induce angiogenesis. In this study, we examined the effects of overexpression of human HGF plasmid DNA using ultrasound-mediated gene transfer into the brain in an Abeta-infused cognitive dysfunction mouse model. We demonstrated that HGF gene transfer significantly alleviated Abeta-induced cognitive impairment in mice in behavioral tests. These beneficial effects of HGF might be due to (1) significant recovery of the vessel density in the dentate gyrus of the hippocampus, (2) upregulation of BDNF, (3) a significant decrease in oxidative stress and (4) synaptic enhancement. A pharmacological approach including gene therapy to increase the HGF level in combination with anti-Abeta therapy might be a new therapeutic option for the treatment of AD.

Role of interleukin-1beta and tumor necrosis factor-alpha-dependent expression of cyclooxygenase-2 mRNA in thermal hyperalgesia induced by chronic inflammation in mice.

The present study investigated whether the endogenous pro-inflammatory cytokines [interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha)]-dependent expression of cyclooxygenase-2 (COX-2) mRNA within the spinal cord could be involved in the development of chronic inflammatory pain-like behaviors in mice. We demonstrated that the expression of COX-2 mRNA on the ipsilateral side of the spinal cord was significantly increased 6 h and 3 days after intraplantar injection of complete Freund's adjuvant (CFA), compared with the expression in saline-treated mice. In addition, the chronic pain-like behaviors following CFA injection were markedly suppressed by repeated intrathecal (i.t.) pre-treatment with the COX-2 inhibitor etodolac, but not with the COX-1 inhibitor mofezolac. The cytosolic level of the activated form of nuclear factor-kappa B (NF-kappaB), which is a major contributor to the induction of COX-2, on the ipsilateral side of the mouse spinal cord was also increased compared with that in the saline-treated mice. The key finding in the present study was that a single i.t. injection with either IL-1beta or TNF-alpha induced a marked increase in spinal COX-2 mRNA and persistent thermal hyperalgesia in mice. Furthermore, CFA-induced hypersensitivity to inflammatory pain was significantly reduced by repeated i.t. pre-injection of the recombinant Fc chimera of IL-1 receptor I or soluble TNF receptor I, which sequesters endogenous IL-1beta or TNF-alpha, respectively. In contrast, the expression of spinal COX-2 mRNA in CFA-treated mice was similar to that in saline-treated mice at 7 days after CFA injection. The present findings strongly indicate the early intrathecal use of the COX-2 inhibitor for the relief of chronic inflammatory pain. Furthermore, together with the result in a previous study that pro-inflammatory cytokines lead to stimulation of a NF-kappaB-dependent transcriptional pathway, these findings suggest that a spinal cytokine/NF-kappaB/COX-2 pathway may play an important role in the development, but not maintenance, of chronic pain following peripheral tissue inflammation.

Glycolipid stimulators for NKT cells bearing invariant V alpha 19-J alpha 33 TCR alpha chains.

Attempts have been made to find specific antigens for a novel NKT cell subset bearing invariant V alpha 19-J alpha 33 TCR alpha chains (V alpha 19 NKT cell). Comprehensive examinations revealed substantial antigenic activity in synthetic alpha-mannosylceramide (ManCer) that was presented by MR1. Structural modification of the sphingosine moiety of alpha-ManCer improved antigenic activity to enhance either Th1 or Th2 -promoting cytokine production by V alpha 19 NKT cells. Such alpha-ManCer analogues will be useful for developing new therapies as immunomodulators.

L-3,4-dihydroxyphenylalanine-induced c-Fos expression in the CNS under inhibition of central aromatic L-amino acid decarboxylase.

L-3,4-dihydroxyphenylalanine (DOPA) is a neurotransmitter candidate. To map the DOPAergic system functionally, DOPA-induced c-Fos expression was detected under inhibition of central aromatic L-amino acid decarboxylase (AADC). In rats treated with a central AADC inhibitor, DOPA significantly increased the number of c-Fos-positive nuclei in the paraventricular nuclei (PVN) and the nucleus tractus solitarii (NTS), and showed a tendency to increase in the supraoptic nuclei (SON), but not in the striatum. On the other hand, DOPA with a peripheral AADC inhibitor elevated the level of c-Fos-positive nuclei in the four regions, suggesting that DOPA itself induces c-Fos expression in the SON, PVN and NTS. In rats treated with 6-hydroxydopamine (6-OHDA) to lesion the nigrostriatal dopamine (DA) pathway, DOPA significantly induced c-Fos expression in the four regions under the inhibition of peripheral AADC. However, under the inhibition of central AADC, DOPA did not significantly increase the number of c-Fos-positive nuclei in the four regions, suggesting that DOPA at least in part induces c-Fos expression through its conversion to DA. It was likely that the 6-OHDA lesion enhanced the response to DA, but attenuated that to DOPA itself. In conclusion, we proposed that the SON, PVN and NTS include target sites for DOPA itself.

Coexpression of an unusual form of the EWS-WT1 fusion transcript and interleukin 2/15 receptor betamRNA in a desmoplastic small round cell tumour.

BACKGROUND: The beta chain of the interleukin 2/15 receptor (IL-2/15Rbeta) is induced by the expression of the EWS-WT1. A case of desmoplastic small round cell tumour (DSRCT) expressing only an unusual EWS-WT1 treated by us is reported here. AIM: To characterise an unusual form of EWS-WT1. METHODS: Frozen tissue sections of the axillary tumour were examined using a laser-assisted microdissection technique and reverse transcriptase polymerase chain reaction. RESULTS: The novel fusion of exon 8 of EWS and the defective exon 10 of WT1 (-KTS) was detected. Although it was an unusual form, the coexpression of the present EWS-WT1, IL-2/15Rbeta and Janus kinase (JAK1) mRNA was detected in the tumour cells. IL-2 and signal transducers and activators of transcription (STAT5) mRNA were detected in both tumour and stromal cells. CONCLUSION: The induction of the IL-2/15 receptor signalling pathway may contribute to tumorigenesis in DSRCT through a paracrine or an autocrine system, even though the EWS-WT1 was an unusual form.

A membrane-bound aminopeptidase isolated from monkey brain and its action on enkephalin.

A membrane-bound aminopeptidase which cleaves the tyrosine-glycine bond of enkephalin was purified about 1600-fold from monkey brain. This aminopeptidase hydrolyzed Leu-enkephalin with a Km value of 35 microM and also hydrolyzed basic, neutral and aromatic amino acid beta-naphthylamides. An apparently homogeneous enzyme consisted of a single polypeptide chain with a molecular weight of approx. 100 000. The optimum pH was in the neutral region. From the analysis of the reaction products, only aminopeptidase activity was detected. The enzyme was inactivated by metal chelators, but the activity could be restored by the addition of divalent cations, such as Co2+, Mg2+ and Zn2+. Puromycin, bestatin and amastatin, which are aminopeptidase inhibitors derived from microorganism, showed strong competitive inhibition of the enzyme, the most potent being amastatin, with a Ki value of 0.02 microM.

Purification and characterization of an enkephalin-degrading aminopeptidase from guinea pig serum.

An enkephalin-degrading aminopeptidase using Leu-enkephalin as a substrate was purified about 4100-fold from guinea pig serum. The purified preparation was apparently homogenous, showing one band on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was approx. 92 000. The aminopeptidase had a pH optimum of 7.0 with Km values of 0.12 mM and 0.18 mM for Leu- and Met-enkephalin, respectively. The enzyme hydrolyzed neutral, basic and aromatic amino acid beta-naphthylamides, but did not the acidic one. The enzyme was inhibited strongly by metal-chelating agents, bestatin and amastatin and weakly by puromycin. Among several biologically active peptides, angiotensin III and substance P strongly inhibited the enzyme.

Analysis of long-term gene expression in neurons of the hippocampal subfields following traumatic brain injury in rats.

After experimental traumatic brain injury (TBI), widespread neuronal loss is progressive and continues in selectively vulnerable brain regions, such as the hippocampus, for months to years after the initial insult. To clarify the molecular mechanisms underlying secondary or delayed cell death in hippocampal neurons after TBI, we compared long-term changes in gene expression in the CA1, CA3 and dentate gyrus (DG) subfields of the rat hippocampus at 24 h and 3, 6, and 12 months after TBI with changes in gene expression in sham-operated rats. We used laser capture microdissection to collect several hundred hippocampal neurons from the CA1, CA3, and DG subfields and linearly amplified the nanogram samples of neuronal RNA with T7 RNA polymerase. Subsequent quantitative analysis of gene expression using ribonuclease protection assay revealed that mRNA expression of the anti-apoptotic gene, Bcl-2, and the chaperone heat shock protein 70 was significantly downregulated at 3, 6 (Bcl-2 only), and 12 months after TBI. Interestingly, the expression of the pro-apoptotic genes caspase-3 and caspase-9 was also significantly decreased at 3, 6 (caspase-9 only), and 12 months after TBI, suggesting that long-term neuronal loss after TBI is not mediated by increased expression of pro-apoptotic genes. The expression of two aging-related genes, p21 and integrin beta3 (ITbeta3), transiently increased 24 h after TBI, returned to baseline levels at 3 months and significantly decreased below sham levels at 12 months (ITbeta3 only). Expression of the gene for the antioxidant glutathione peroxidase-1 also significantly increased 6 months after TBI. These results suggest that decreased levels of neuroprotective genes may contribute to long-term neurodegeneration in animals and human patients after TBI. Conversely, long-term increases in antioxidant gene expression after TBI may be an endogenous neuroprotective response that compensates for the decrease in expression of other neuroprotective genes.

Partial prevention of compactin (ML-236B) inhibition of in vitro hematopoiesis by dolichol and dolichyl phosphate.

We have reported that the exogenous addition of dolichyl phosphate (Dol-P) enhances the colony-forming capacities of early erythroid progenitors (BFU-E), late erythroid progenitors (CFU-E), and granulocyte-macrophage progenitors (CFU-GM) in adult mouse bone marrow, and that dolichol (Dol) enhances that of only CFU-E (Int. J. Cell Cloning 3:313, 1985). Compactin (2.5-10 microM), a specific inhibitor of mevalonate biosynthesis that causes a decrease of endogenous Dol biosynthesis, inhibited colony formation of CFU-GM. Exogenous addition of Dol-P partially prevented this inhibition, but Dol and the other mevalonate metabolites, such as cholesterol, coenzyme Q10, and isopentenyladenine, could not. In addition, we have found that the colony-forming capacity of CFU-E in fetal mouse liver was not enhanced by exogenous Dol or Dol-P. But the decrease of colony formation or DNA synthesis of fetal CFU-E in the presence of compactin was prevented by the exogenous addition of Dol or Dol-P.

Synergistic effect of dolichyl phosphate and human recombinant granulocyte colony-stimulating factor on recovery from neutropenia in mice treated with anti-cancer drugs.

Severe hematopoietic injury in mice was induced by using either 5-fluorouracil, adriamycin, mitomycin C, or vinblastine. Daily subcutaneous administration of purified human recombinant granulocyte colony-stimulating factor (rG-CSF; 0.3-10.0 micrograms/day) markedly accelerated recovery from the drug-induced granulocytopenia in a dose-dependent manner, as reported previously. On the other hand, daily intraperitoneal administration of dolichyl phosphate (Dol-P) also enhanced granulopoiesis to accelerate recovery from granulocytopenia, although the effect of Dol-P was relatively moderate as compared with that of rG-CSF. A synergistic recovery of granulopoiesis was observed when Dol-P was administered together with rG-CSF to the mice treated with anti-cancer drugs. Joint use of Dol-P (1 mg/day) and rG-CSF (0.3 micrograms/day) was as effective as a higher dose of rG-CSF (3 micrograms/day). Joint use of Dol-P (1 mg/day) and rG-CSF (3 micrograms/day) was sometimes more effective.

Spinorphin as an endogenous inhibitor of enkephalin-degrading enzymes: roles in pain and inflammation.

It is possible that enkephalins are involved in the pain-modulating mechanism in the spinal cord. Enkephalins, however, are short-lived, being rapidly degraded by various endogenous enzymes. Many substances that inhibit enkephalin-degradation have been investigated and it has been reported that some inhibitors (e.g. kelatorphan and RB101) alone showed anti-nociceptive activity. We found an endogenous factor that modulated enkephalin-degrading activity and purified it from bovine spinal cord based on its inhibitory activity toward enkephalin-degrading enzymes. Structural analysis revealed the factor to be Leu-Val-Val-Tyr-Pro-Trp-Thr and it was named spinorphin. It has been found that spinorphin inhibited the activity toward various enkephalin-degrading enzymes from monkey brain, especially dipeptidyl peptidase III (DPPIII, Ki=5.1 x 10(-7) M). Recently we reported that this inhibitor significantly inhibited bradykinin (BK)-induced nociceptive flexor responses. Importantly, the mode of inhibition to BK-responses by spinorphin was different from the case with morphine. The morphine-induced blockade of BK-response was attenuated by pertussis toxin treatment, whereas that of spinorphin was not. We also have reported roles for spinorphin in inflammation. Spinorphin significantly inhibited the functions of polymorphonuclear neutrophils (PMNs) by suppressing the binding of fMLF to its receptor on PMNs. Further, this inhibitor suppressed the carrageenan-induced accumulation of PMN in mouse air pouches after intravenous administration. These results indicate that spinorphin may be an endogenous anti-inflammatory regulator. The possible role of spinorphin and its analog as regulators in pain and inflammation will be discussed.

The retarded rate of acid-catalyzed solvolysis of glycoside bonds between reducing-end glucose residue and ceramide in glycosphingolipids compared with that of glycoside bonds between hexopyranosides.

Rates of acid-catalyzed solvolysis of glycoside bonds in glycosphingolipids were compared to establish a basis for conducting saccharide analysis. Permethylated globotetraosylceramide and asialogangliotriaosylceramide as model compounds for methylation and sugar composition analysis, respectively, were solvolyzed under acidic conditions and the sugar components thus obtained were determined at specified times by gas liquid chromatography, after they had been derivatized. Reducing-end glucose residues in both compounds were liberated more slowly than other sugar residues. Glycoside bonds between reducing-end glucose and ceramide in glycosphingolipids would thus appear to be more resistant towards acid-catalysed solvolysis than other glycoside bonds between hexopyranosides.

Insight into the mechanism of trypanosome lytic factor-1 killing of Trypanosoma brucei brucei.

It has been known for almost a century that normal human serum can lyse the extracellular blood parasite Trypanosoma brucei brucei. This process is a result of a non-immune killing factor in human sera known as trypanosome lytic factor (TLF). In this work, we demonstrate that killing of T. b. brucei by trypanosome lytic factor-1 (TLF-1) in vitro is inhibited by the lipophyllic iron chelator, LI, the lipophyllic antioxidant DPPD, and the protease inhibitors antipain and E64. Thus TLF-1 killing likely requires iron, oxidants, and serine and cysteine proteases. Furthermore, we demonstrate that TLF-1 mediated lysis causes measurable peroxidation in T. brucei lipids via a reaction that is inhibited by DPPD, weak bases, and human haptoglobin. We hypothesize that TLF-1 lysis requires intracellular factors within the trypanosome including high intracellular H2O2 and high polyenoic lipid concentrations, lysosomal acidification and proteases, and intracellular iron sources. The data presented supports the hypothesis that the combination of these factors with TLF-1 inside the lysosome results in lysosomal membrane breakdown, release of the lysosomal contents, and subsequent autodigestion of the cell.

The lysosomal targeting and intracellular metabolism of trypanosome lytic factor by Trypanosoma brucei brucei.

Trypanosome lytic factor (TLF) provides innate protection for humans against infection by the animal pathogen Trypanosoma brucei brucei but not against the agent of human African sleeping sickness, Trypanosoma brucei rhodesiense. TLF exists in two forms, TLF-1 and TLF-2. Prior studies suggested that TLF-1 causes lysosomal disruption and subsequent cell death in T. b. brucei. Here we confirm the lysosomal targeting of TLF-1 by immunolocalization with the trypanosome lysosomal membrane protein p67, and by co-fractionation of radiolabelled TLF-1 with lysosomal enzymes. In addition, pulse-chase studies indicate that TLF-1 is not degraded within the lysosome as compared to the host protein transferrin. In TLF-1 treated cells, transferrin is degraded normally, indicating that lysosomal proteases remain active during the early phase of TLF-1 treatment but fail to degrade TLF-1. Following endocytosis a TLF lipoprotein appears to undergo disulfide bond reduction prior to entering the lysosome. Results presented here indicate that TLF-1 lipoproteins are targeted to the lysosome but are resistant to trypanosome lysosomal proteases.

1,3-Disubstituted benzazepines as novel, potent, selective neuropeptide Y Y1 receptor antagonists.

A novel series of potent and selective non-peptide neuropeptide Y (NPY) Y1 receptor antagonists, having benzazepine nuclei, have been designed, synthesized, and evaluated for activity. Chemical modification of the R(1) and R(3) substituents in structure 1 (Chart 1) yields several compounds that show high affinity for the Y1 receptor (K(i) values of less than 10 nM). SAR studies revealed that introduction of an isopropylurea group at R(1) and a 3-(benzo-condensed-urea) group, 3-(fluorophenylurea) group, or a 3-(N-(4-hydroxyphenyl)guanidine) group at R(3) in structure 1 afforded potent and subtype-selective NPY Y1 receptor antagonists. 3-(3-(Benzothiazol-6-yl)ureido)-1-N-(3-(N'-(3-isopropylureido++ +))benzyl )-2,3,4,5-tetrahydro-1H-1-benzazepin-2-one (21), which was one of the most potent derivatives, competitively inhibited specific [(125)I]peptide YY (PYY) binding to Y1 receptors in human neuroblastoma SK-N-MC cells (K(i) = 5.1 nM). 21 not only inhibited the Y1 receptor-mediated increase in cytosolic free Ca(2+) concentration in SK-N-MC cells but also antagonized the Y1 receptor-mediated inhibitory effect of peptide YY on gastrin-induced histamine release in rat enterochromaffin-like cells. 21 showed no significant affinity in 17 receptor binding assays including Y2, Y4, and Y5 receptors.

A novel angiogenic inhibitor derived from Japanese shark cartilage (I). Extraction and estimation of inhibitory activities toward tumor and embryonic angiogenesis.

Guanidine extraction and crude fractionation of Japanese shark cartilage by ultrafiltration on a molecular weight basis were conducted and the antiangiogenic activities were assayed as to the inhibitions of tumor and embryonic angiogenesis. Significant inhibition of angiogenesis was found, and there was a linear relationship between the results of the two assays. The inhibitory activities were concentrated in the fraction in the molecular weight range of 103 to 104, and were resistant to heat treatment.

Inhibition by 9alpha-fluoromedoroxyprogesterone acetate (FMPA) against mammary carcinoma induced by dimethylbenz[a]anthracene in rats and angiogenesis in the rabbit cornea - comparison with medroxyprogesterone acetate (MPA).

Medroxyprogesterone acetate (MPA) is currently used therapeutically in the treatment of mammary and endometrial carcinomas. In order to develop a more potent and useful drug, we synthesized the novel compound, 9alpha-fluoromedoroxyprogesterone acetate (FMPA), by fluorinating MPA, and we also previously reported that FMPA displays more potent anti-angiogenic activity in the chorioallantoic membrane assay than MPA. In the present study, we investigated (1) the effects of FMPA on rat mammary carcinomas induced by dimethylbenz[a]anthracene (DMBA) to determine the anti-tumor activity, (2) the effect on angiogenesis in rabbit corneal assays, and (3) compared these results with those for MPA. FMPA inhibited the growth of mammary carcinomas in a dose-dependent manner (7.5, 30 and 120 mg/kg). Almost complete involution of the carcinomas was observed at doses of 30 and 120 mg/kg. MPA also inhibited the growth of carcinomas at doses of 30 and 120 mg/kg, but no involution of carcinomas was observed even at 120 mg/kg. FMPA significantly and MPA to a lesser degree inhibited carcinogenesis at 120 mg/kg within their treatments. In rabbit corneal assays, FMPA significantly inhibited angiogenesis (IC50 value=0.085 microg/pellet). MPA also significantly inhibited angiogenesis (IC50 value=0.60 microg/pellet). From these results, we conclude that FMPA is potentially more effective in the treatment of mammary carcinomas than MPA.