Inhibition of actin polymerization by marine toxin pectenotoxin-2.

Pectenotoxin-2 (PCTX-2) is one of the polyether macrolide toxins isolated from scallops involved in diarrheic shellfish poisoning via actin depolymerization. In the present study, we examined the bioactive mechanism of PCTX-2 in smooth muscle cells and clarify mode of action of the PCTX-2-induced actin depolymerization using purified skeletal actin. PCTX-2 (300 nM-3 µM) non-selectively inhibited vascular smooth muscle contractions elicited by high K+ or phenylephrine in a dose-dependent manner. However, elevated cytosolic Ca2+ and myosin light chain phosphorylation stimulated by high K+ were only slightly inhibited by PCTX-2. By monitoring the fluorescent intensity of pyrenyl-actin, PCTX-2 was found to inhibit both the velocity and degree of actin polymerization. The critical concentration of G-actin was linearly increased in accordance with the concentration of PCTX-2, indicating sequestration of G-actin with 1 to 1 ratio. The kinetics of F-actin depolymerization by dilution assay indicated that PCTX-2 does not sever F-actin. Transmission electron microscopic and confocal microscopic observations demonstrated that PCTX-2 selectively depolymerized filamentous actin without affecting tublin. In conclusion, PCTX-2 is a potent natural actin depolymerizer which sequesters G-actin without severing F-actin.

Malfunction of spermatogenesis in experimental ischemic mice.

An experimental ischemia (EI)-induced mouse model was used to analyze pathological and biochemical alterations in testes. Initial morphological changes were observed in Sertoli cells of EI testes at the light microscopic level. Examination of the ultrastructure using transmission electron microscopy confirmed that Sertoli cells were partially detached from the basement membrane of the seminiferous epithelium and that the cell membranes of adjacent Sertoli cells were not joined. The functional integrity of the blood-testis barrier (BTB) was assessed using the lanthanum tracer technique. Lanthanum had penetrated into the spaces between adjacent Sertoli cells in the adluminal compartment up to the lumen of the seminiferous epithelium in EI testes. Proteome analysis showed that the expression of heat shock protein (HSP) 70 was significantly upregulated in EI testes. Western blot analysis confirmed that the expression of HSP70 increased in a time-dependent manner after the EI procedure. HSP70 immunostaining was observed in spermatocytes and in round and elongated spermatids in EI testes. Our results suggest that a change in the junctions between adjacent Sertoli cells on the basal compartment is involved in the BTB disruption in EI testes. Therefore, male infertility caused by the BTB disruption could be associated with heat stress induced by ischemia.

Oxidative stress-mediated antitumor activity of erythorbic acid in high doses.

Intravenous (iv) infusion of high-dose ascorbic acid (AA) has been used as a treatment for cancer patients. The tumoricidal action of AA occurs due to its prooxidant effect. Erythorbic acid (EA), one of the AA epimers, has reduced vitamin C activity, while the antioxidant activity of EA is similar to that of AA. Currently, other physiological and pharmacological functions of EA are not well known. We examined the cytotoxicity of EA to murine colon carcinoma (colon-26) cells and the antitumor activity of EA in tumor-bearing mice. Cytotoxic activity of EA to colon-26 cells was evaluated by using the calcein-AM assay. EA showed the same cytotoxic activity to colon-26 cells as that of AA. The cytotoxicity of EA was shown to be caused by oxidative stress. Next, colon-26 tumor-bearing mice were iv administered EA and AA on alternate days for 4 times, and tumor growth rates were measured. Tumor growth was significantly inhibited by administration of high-dose EA in vivo as well as AA. Finally, the in vivo biodistribution and clearance of EA and AA were investigated in tumor-bearing mice. Endogenous AA in the tumor was consumed to resist oxidative stress caused by reactive oxygen species that was generated by administered EA. These results indicated that the oxidative stress-mediated antitumor activity is one of the pharmacological functions of high-dose iv EA.

tRNA modifying enzymes, NSUN2 and METTL1, determine sensitivity to 5-fluorouracil in HeLa cells.

Nonessential tRNA modifications by methyltransferases are evolutionarily conserved and have been reported to stabilize mature tRNA molecules and prevent rapid tRNA decay (RTD). The tRNA modifying enzymes, NSUN2 and METTL1, are mammalian orthologs of yeast Trm4 and Trm8, which are required for protecting tRNA against RTD. A simultaneous overexpression of NSUN2 and METTL1 is widely observed among human cancers suggesting that targeting of both proteins provides a novel powerful strategy for cancer chemotherapy. Here, we show that combined knockdown of NSUN2 and METTL1 in HeLa cells drastically potentiate sensitivity of cells to 5-fluorouracil (5-FU) whereas heat stress of cells revealed no effects. Since NSUN2 and METTL1 are phosphorylated by Aurora-B and Akt, respectively, and their tRNA modifying activities are suppressed by phosphorylation, overexpression of constitutively dephosphorylated forms of both methyltransferases is able to suppress 5-FU sensitivity. Thus, NSUN2 and METTL1 are implicated in 5-FU sensitivity in HeLa cells. Interfering with methylation of tRNAs might provide a promising rationale to improve 5-FU chemotherapy of cancer.

Synthesis and biological evaluation of new BSH-conjugated chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer.

New disodium mercaptoundecahydro-closo-dodecaborate (BSH)-conjugated chlorin derivatives 11, 12, 16 and 20 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized. The in vivo biodistribution and clearance of 11, 12, 16 and 20 were investigated in tumor-bearing mice. Compounds 12 and 16 showed good tumor-selective accumulation among the four derivatives. The time to maximum accumulation of compound 16 in tumor tissue was one-fourth of that of compound 12, and clearance from normal tissues of compound 16 was similar to that of compound 12. The in vivo therapeutic efficacy of PDT using 16, which has twice as many boron atoms as 12, was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 6h after injection of 16. Tumor growth was significantly inhibited by PDT using 16. These results suggested that 16 is a good candidate for both PDT and BNCT of cancer.

Synthesis and biological evaluation of new boron-containing chlorin derivatives as agents for both photodynamic therapy and boron neutron capture therapy of cancer.

New boron-containing chlorin derivatives 9 and 13 as agents for both photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of cancer were synthesized from photoprotoporphyrin IX dimethyl ester (2) and L-4-boronophenylalanine-related compounds. The in vivo biodistribution and clearance of 9 and 13 were investigated in tumor-bearing mice. The time to maximum accumulation of compound 13 in tumor tissue was one-fourth of that of compound 9, and compound 13 showed rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 13 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 13. Tumor growth was significantly inhibited by PDT using 13. These results suggested that 13 might be a good candidate for both PDT and BNCT of cancer.

Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy.

Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3h after intravenous injection and rapid clearance from normal tissues within 24h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use.

The detergent-soluble cytoplasmic pool of survivin suppresses anoikis and its expression is associated with metastatic disease of human colon cancer.

Survivin is a component of the chromosomal passenger complex (CPC) that is essential for accurate chromosome segregation. Interfering with the function of Survivin in mitosis leads to chromosome segregation errors and defective cytokinesis. Survivin contains a Baculovirus IAP Repeat (BIR) and therefore was originally classified as inhibitor of apopotosis protein (IAP), yet its role in apoptosis after cellular stress remains largely unknown. We demonstrate here, that Survivin predominantly suppresses anoikis, a form of programmed cell death induced by loss of cellular adhesion to extracellular matrix. Interestingly, cells ectopically overexpressing EGFP-Survivin showed after loss of cell-matrix-interaction a decreased expression of IκB-α. Subsequent subcellular protein fractionation and immunoprecipitation experiments revealed that XIAP interacts with detergent-soluble Survivin which is known to cooperatively activate NF-κB signaling. Examination of the expression levels of detergent soluble Survivin in colorectal cancer cell lines and in colorectal cancerous tissues revealed that detergent soluble cytoplasmic Survivin levels correlated inversely with anoikis susceptibility in colorectal cancer. Therefore, the detergent soluble cytoplasmic Survivin might be a promising predictive biomarker for lymph node and distant metastases of colorectal cancer. We conclude that an anti-apoptotic function of detergent-soluble Survivin in interphase cells experiencing anoikis is mediated at least via XIAP/IκB-α/NF-κB signaling.

Staining internal limiting membrane with a mixture of BBG and sodium hyaluronate.

AIMS: To evaluate the intraoperative applicability and safety of a mixture of brilliant blue G and sodium hyaluronate (visco-BBG) for staining the inner limiting membrane (ILM). METHODS: A retrospective consecutive case series. Seventy-four eyes that had undergone ILM peeling were studied. During vitrectomy, ILM peeling with visco-BBG (visco-BBG group) was performed on 40 eyes; 12 with a macular hole (MH), 26 with an epimacular membrane (ERM) and 2 with a retinal detachment due to a MH (MHRD). ILM peeling with BBG dissolved in balanced salt solution (BSS-BBG group) was performed on 34 eyes; 9 with a MH, 23 with an ERM and 2 with a MHRD. The main outcome measures were the distribution of the dye within the vitreous cavity and the retinal sensitivity in the MH patients of the two groups by microperimetry. RESULTS: The visco-BBG was injected over the retina where the ILM was intended to be peeled, and it stained the ILM in all cases. It did not disperse throughout the vitreous cavity or into the subretinal space. The BSS-BBG dispersed throughout the vitreous cavity, and its distribution was difficult to control. The two solutions did not stain the epiretinal membranes or any residual posterior hyaloid membrane. The difference in the retinal sensitivity between the two patients with MH of two groups was not significant. No complications were found in the visco-BBG group, although an accidental retinal perforation was found in one eye of the BSS-BBG group. Transmission electron microscopy confirmed that the membrane peeled was the ILM. CONCLUSIONS: Visco-BBG can be a useful method to assist macular surgery and can overcome some of the disadvantages of conventional BBG solutions dissolved in BSS.

Intramolecular acyl migration and enzymatic hydrolysis of 2-O-α-D-glucopyranosyl-6-O-(2-pentylheptanoyl)-L-ascorbic acid.

2-O-α-D-Glucopyranosyl-6-O-(2-pentylheptanoyl)-L-ascorbic acid (6-bDode-AA-2G) underwent an intramolecular acyl migration to yield approximately 12% of 2-O-α-D-glucopyranosyl-5-O-(2-pentylheptanoyl)-L-ascorbic acid (5-bDode-AA-2G) in neutral solutions for 3 days. In small intestine homogenate from guinea pigs for 12h, 6-bDode-AA-2G, which hardly underwent acyl migration to give 5-bDode-AA-2G, was predominantly hydrolyzed with α-glucosidase and then with esterase to ascorbic acid.

Antioxidant properties of ethyl vanillin in vitro and in vivo.

We systematically evaluated the antioxidant activity of ethyl vanillin, a vanillin analog, as compared with the activities of vanillin and other vanillin analogs using multiple assay systems. Ethyl vanillin and vanillin exerted stronger antioxidant effects than did vanillyl alcohol or vanillic acid in the oxygen radical absorbance capacity (ORAC) assay, although the antioxidant activities of vanillyl alcohol and vanillic acid were clearly superior to those of ethyl vanillin and vanillin in the three model radical assays. The antioxidant activity of ethyl vanillin was much stronger than that of vanillin in the oxidative hemolysis inhibition assay, but was the same as that of vanillin in the ORAC assay. Oral administration of ethyl vanillin to mice increased the concentration of ethyl vanillic acid, and effectively raised antioxidant activity in the plasma as compared to the effect of vanillin. These data suggest that the antioxidant activity of ethyl vanillin might be more beneficial than has been thought in daily health practice.

Unexpected role of α-fetoprotein in spermatogenesis.

BACKGROUND: Heat shock severely affects sperm production (spermatogenesis) and results in a rapid loss of haploid germ cells, or in other words, sperm formation (spermiogenesis) is inhibited. However, the mechanisms behind the effects of heat shock on spermatogenesis are obscure. METHODOLOGY/PRINCIPAL FINDINGS: To identify the inhibitory factor of spermiogenesis, experimental cryptorchid (EC) mice were used in this study. Here we show that α-fetoprotein (AFP) is specifically expressed in the testes of EC mice by proteome analysis. AFP was also specifically localized spermatocytes by immunohistochemical analysis and was secreted into the circulation system of EC mice by immunoblot analysis. Since spermatogenesis of an advanced mammal cannot be reproduced with in vitro, we performed the microinjection of AFP into the seminiferous tubules of normal mice to determine whether AFP inhibits spermiogenesis in vivo. AFP was directly responsible for the block in spermiogenesis of normal mice. To investigate whether AFP inhibits cell differentiation in other models, using EC mice we performed a partial hepatectomy (PH) that triggers a rapid regenerative response in the remnant liver tissue. We also found that liver regeneration is inhibited in EC mice with PH. The result suggests that AFP released into the blood of EC mice regulates liver regeneration by inhibiting the cell division of hepatocytes. CONCLUSIONS/SIGNIFICANCE: AFP is a well-known cancer-specific marker, but AFP has no known function in healthy human beings. Our findings indicate that AFP expressed under EC conditions plays a role as a regulatory factor in spermatogenesis and in hepatic generation.

Design and synthesis of novel cyclooxygenase-1 inhibitors as analgesics: 5-amino-2-ethoxy-N-(substituted-phenyl)benzamides.

We previously found that N-(4-aminophenyl)-4-trifluoromethylbenzamide (TFAP), a COX-1 inhibitor, exhibits an analgesic effect without causing gastric damage. Unfortunately, TFAP causes reddish purple coloration of urine, and its analgesic effect is less potent than that of indomethacin. Herein we describe our study focusing on the development of 4- and 5-amino-2-alkoxy-N-phenylbenzamide scaffolds, designed on the basis of the structures of TFAP and parsalmide, another known COX-1 inhibitory analgesic agent. 5-Amino-2-ethoxy-N-(2- or 3-substituted phenyl)benzamide derivatives exhibited analgesic activity in a murine acetic acid induced writhing test. Among these compounds, 5-amino-2-ethoxy-N-(2-methoxyphenyl)benzamide (9 v) possesses potent COX-1 inhibitory and analgesic activities, similar to those of indomethacin. In addition, 5-amino-2-ethoxy-N-(3-trifluoromethylphenyl)benzamide (9 g) showed a more potent analgesic effect than indomethacin or 9 v without causing apparent gastric damage or coloration of urine, although its COX-1 inhibitory activity was weaker than that of indomethacin or 9 v. Thus, 9 g and 9 v appear to be promising candidates for analgesic agents and are attractive lead compounds for further development of COX-1 inhibitors.

Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays.

BACKGROUND: Vanillin, a compound widely used in foods, beverages, cosmetics and drugs, has been reported to exhibit multifunctional effects such as antimutagenic, antiangiogenetic, anti-colitis, anti-sickling, and antianalgesic effects. However, results of studies on the antioxidant activity of vanillin are not consistent. METHODS: We systematically evaluated the antioxidant activity of vanillin using multiple assay systems. DPPH radical-, galvinoxyl radical-, and ABTS(+)-scavenging assays, ORAC assay and an oxidative hemolysis inhibition assay (OxHLIA) were used for determining the antioxidant activity. RESULTS AND CONCLUSION: Vanillin showed stronger activity than did ascorbic acid and Trolox in the ABTS(+)-scavenging assay but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays. Vanillin showed much stronger antioxidant activity than did ascorbic acid and Trolox in the ORAC assay and OxHLIA. In the ABTS(+)-scavenging assay, ORAC assay and OxHLIA, vanillin reacted with radicals via a self-dimerization mechanism. The dimerization contributed to the high reaction stoichiometry against ABTS(+) and AAPH-derived radicals to result in the strong effect of vanillin. Oral administration of vanillin to mice increased the vanillin concentration and the antioxidant activity in plasma. These data suggested that antioxidant activity of vanillin might be more beneficial than has been thought for daily health care. GENERAL SIGNIFICANCE: Based on the results of the present study, we propose the addition of antioxidant capacity to the multifunctionality of vanillin.

Continual maintenance of the blood-testis barrier during spermatogenesis: the intermediate compartment theory revisited.

Tight junctions occur between the lateral processes of neighboring Sertoli cells that divide the seminiferous epithelium into two compartments: basal and adluminal compartments. These tight junctions constitute the blood-testis barrier (BTB). The established theory that the BTB must open when spermatocytes translocate from the basal compartment to the adluminal compartment is marked by one contradiction, that is, normal spermatogenesis occurs in the testis because the BTB is expected to constantly seclude the adluminal compartment from the basal compartment in order to protect haploid germ cells from the autoimmune system. Subsequently, another concept was proposed in which two BTBs divide the seminiferous epithelium into three compartments: basal, intermediate and adluminal compartments. It has been suggested that the transition from the basal region to the adluminal region without the BTB open occurs through the agency of a short-lived intermediate compartment embodying some primary spermatocytes. In contrast, the results of recent findings in the molecular architecture of the BTB suggest that the BTB in the seminiferous epithelium must "open". In this paper, I re-examine the BTBs of boar and experimental cryptorchid mouse testes by transmission electron microscope (TEM). TEM analysis showed that an atypical basal compartment existed in the thin seminiferous epithelium of 14-day post-cryptorchid mice testes. In developmental boar testes, ectoplasmic specialization (ES) of the seminiferous epithelium showed dynamic behavior. The intermediate compartment was clearly observed between the basal and adluminal compartments of the mature boar seminiferous epithelium. ESs were observed between Sertoli cells and spermatids at all developmental stages, including early, late and mature. Furthermore, ESs were situated on the apical surface of the seminiferous epithelium. From these results, I propose that the BTB is continually maintained during spermatogenesis and suggest a model of ES circulation in the seminiferous epithelium.

Binding of glutamate receptor delta2 to its scaffold protein, Delphilin, is regulated by PKA.

The glutamate receptor delta2 (GluRdelta2) is selectively expressed in cerebellar Purkinje cells and plays an important role in motor learning, motor coordination, and long-term depression. Delphilin is identified as a GluRdelta2-interacting protein, selectively expressed in Purkinje cell-parallel fiber synapses, and specifically interacts with the GluRdelta2 C-terminus via its PDZ domain. Here, surface plasmon resonance analyses showed that Delphilin PDZ bound to GluRdelta2 C-terminal peptide (DPDRGTSI), but not to its phosphopeptides (DPDRGphosphoTSI and DPDRGTphosphoSI). We showed the incorporation of phosphate into threonine at -2 (-2T) and serine at -1 (-1S) of GluRdelta2 C-terminus by cAMP-dependent protein kinase (PKA) in vitro. In the experiments using heterologous expression system, Delphilin coimmunoprecipitated with GluRdelta2 was dramatically decreased under the condition with forskolin and isobutylmethylxanthine, which led to cAMP-dependent phosphorylation by PKA. Thus, phosphorylation of -2T and/or -1S of GluRdelta2 C-terminus by PKA may regulate the binding of GluRdelta2 to its scaffolding protein, Delphilin.

Abnormal spermatogenesis in mice unable to synthesize ascorbic acid.

Although exposure to environmental toxicants, including endocrine-disrupting chemicals, is thought to be a possible cause of male infertility, the pathogenesis of male reproductive disorders remains unclear. In the present study, we used Gulo-/- mutant mice, which are unable to synthesize ascorbic acid, to study the importance of dietary vitamin C (VC) on spermatogenesis. Regular chow containing approximately 110 mg/kg VC is unable to support the growth of these mutant mice, but a VC supplement in their drinking water (330 mg/L) is able to ameliorate the VC deficiency. Testes of Gulo-/- mutants born from heterozygous mothers without VC supplement (VC-deficient mice) and those born from mothers given a VC supplement (VC-sufficient mice) were examined by morphological and biochemical analyses. Morphological analysis revealed that apoptosis of spermatocytes occurred frequently in VC-deficient mice at 20 days of age. Two-dimensional electrophoresis analysis revealed the specific disappearance of heat-shock protein (Hsp) 70 in the testes of 20-day-old VC-deficient mice. In the present study, the relationship between the apoptosis of spermatocytes and Hsp70 in VC-deficient mice is discussed.

Differential development of TRPV1-expressing sensory nerves in peripheral organs.

In mouse ontogeny, neurons immunoreactive for transient receptor potential vanilloid receptor 1 (TRPV1) were observed primarily in the dorsal root ganglia (DRG) at embryonic day 13 (E13). In the embryonic period, the number of TRPV1(+) neurons decreased, but then gradually increased postnatally. Some of TRPV1(+) neurons were also immunoreactive for calcitonin gene-related peptide (CGRP). At postnatal day 7 (P7), 66% of CGRP(+) neurons were TRPV1(+), and 55% of TRPV1(+) neurons were also CGRP(+) in the L4 DRG. In the peripheral organs, TRPV1-immunorective nerve fibers were transiently observed in the skin at E14. They were also observed in the urinary tract at E14, and in the rectum at E15. Many TRPV1(+) nerve fibers in these organs were also CGRP(+). At P1, TRPV1(+) nerve fibers were observed in the respiratory organs, and to a lesser extent in the stomach, colon, skin, and skeletal muscles. The number of TRPV1(+) nerve fibers on each organ gradually increased postnatally. At P7, TRPV1(+) nerve fibers were also observed in the small intestine and kidneys. The percentage of total TRPV1(+) nerve fibers that co-localized with CGRP was greater in most organs at P7 than at P1. The present results indicate that TRPV1 expression on peripheral processes differs among organs. The differential time course of TRPV1 expression in the cell bodies might be related to the organs to which they project. Co-localization of TRPV1 with CGRP on nerve fibers also varies among organs. This suggests that the TRPV1-mediated neuropeptide release that occurs in certain pathophysiologic conditions also varies among organs.

Full-term development of hamster embryos produced by injection of round spermatids into oocytes.

The golden hamster is a mammal in which microinjection of round spermatids into oocytes (ROSI) was first attempted. However, no live ROSI offspring have ever been obtained in this species. This is the first report of live hamster offspring obtained by round spermatid injection. Over 90% of oocytes, injected with round spermatids, were activated without any additional stimulation. The proportion of the oocytes that were fertilized normally and that developed to morulae and blastocysts was higher when the plasma membranes of the spermatids were broken before injection, as compared with when the membranes were left intact. Five percent of 57 ROSI morulae/blastocysts developed into live offspring after transfer to foster mothers.

Bilateral persistent sciatic arteries in a Japanese man.

During student dissecting practice, a rare developmental anomaly showing persistent sciatic artery (PSA) was found in the bilateral lower limbs of a 74-year-old Japanese male cadaver. The PSA was a continuation of the internal iliac artery on both sides, did not anastomose with the perforating arteries and ended by anastomosing with the popliteal artery on both sides. The course and distribution of the PSA were relatively consistent with previous reports. In the present case, however, the PSA and inferior gluteal arteries existed simultaneously on both sides, despite the general assumption that the inferior gluteal artery is a remnant of sciatic artery regression.