Protein-bound 4-hydroxy-2-hexenal as a marker of oxidized n-3 polyunsaturated fatty acids

In the present study, to investigate the contribution of n-3 PUFAs in the oxidative modification of protein in vivo, we characterize the covalent binding of 4-hydroxy-2-hexenal (HHE), a potent cytotoxic aldehyde originating from the peroxidation of n-3 PUFAs, to protein and describe the production of this aldehyde in oxidatively modified LDL and in human atherosclerotic lesions. Upon incubation with BSA, HHE was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a potential marker of oxidatively modified proteins under oxidative stress. To detect the protein-bound HHE in vivo, we raised monoclonal antibody HHE53 (MAb HHE53) directed to the HHE-modified protein and identified the Michael addition-type HHE-histidine adduct as the major epitope. This antibody reacted with copper-oxidized LDL, suggesting that HHE was produced during the oxidative modification of LDL. In addition, we demonstrated that the materials immunoreactive to MAb HHE53 indeed constituted the atherosclerotic lesions, in which intense positivity was associated primarily with macrophage-derived foam cells. The results of this study suggest that the reaction between oxidized n-3 PUFAs and protein might represent a process common to the formation of degenerative proteins during aging and its related diseases.     

Peritoneal injection of fucoidan suppresses the increase of plasma IgE induced by OVA-sensitization

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-γ in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in vivo is crucially dependent on the route and timing of its administration.     

Ceramide Stimulates ABCA12 Expression via Peroxisome Proliferator-activated Receptor �� in Human Keratinocytes

ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise ∼50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C₂-Cer and C₆-Cer), but not C₈-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C₆-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARδ expression (but not other PPARs or liver X receptors), whereas PPARδ knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARδ is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARδ-mediated signaling pathway, providing a substrate-driven, feed-forward mechanism for regulating this key lipid transporter.The outermost layer of mammalian epidermis, the stratum corneum, is essential for permeability barrier function and critical for terrestrial life. The stratum corneum consists of terminally differentiated, anucleate keratinocytes, or corneocytes, surrounded by lipid-enriched lamellar membranes composed of three major lipids, ceramides, cholesterol, and free fatty acids (1). These lipids are delivered to the extracellular spaces of the stratum corneum through exocytosis of lamellar body contents from outermost stratum granulosum cells (2). Mature lamellar bodies contain primarily cholesterol, phospholipids, and glucosylceramides (3). Following lamellar body secretion, the secreted phospholipids and glucosylceramides are converted to free fatty acids and ceramides by phospholipases and β-glucocerebrosidase, respectively (1, 4). ABCA12 (ATP binding cassette transporter, family 12), a lipid transporter predominantly expressed in epidermis, has been shown to play a vital role in the formation of mature lamellar bodies (5, 6), although how this transporter is regulated remains unresolved.ABCA12 is a member of the ABCA subfamily of transporters, which are involved in the transport of a variety of lipids (7). Mutations in ABCA1 cause Tangier disease, which is due to a defect in transporting cholesterol and phospholipids from intracellular lipid stores to apolipoproteins, particularly apolipoprotein A-I (8–11). Mutations in ABCA3 cause neonatal respiratory failure due to a defect in surfactant transport from alveolar type II cells into the alveolar space (12). Mutations in ABCA4 cause Stargardt''s macular degeneration, with visual loss due to a defect in transporting phosphatidylethanolamine-retinylidene out of retinal pigment cells (13).Recently, mutations in ABCA12 have been shown to cause harlequin ichthyosis and a subgroup of lamellar ichthyosis, two disorders of keratinization (5, 14, 15). ABCA12 mutations lead to an abnormality in lamellar body formation, a decrease in lamellar membranes in the extracellular spaces of the stratum corneum, an accumulation of glucosylceramide in the epidermis with a reduction in ceramide (16), and ultimately loss of permeability barrier function (17), which in harlequin ichthyosis can result in neonatal lethality (5, 15). Strikingly, genetic correction of ABCA12 deficiency in patients'' keratinocytes by gene transfer normalized loading of glucosylceramides into lamellar bodies (5). These studies demonstrate a critical role for ABCA12 in epidermal physiology, specifically in the formation of mature lamellar bodies and subsequent permeability barrier homeostasis. Hence, it is crucial to understand how ABCA12 is regulated.Our laboratory recently demonstrated that activation of peroxisome proliferator-activated receptor (PPARδ and PPARγ) or liver X receptor (LXR) stimulates ABCA12 expression in cultured human keratinocytes (18). Both PPARs and LXR are important lipid sensors that stimulate keratinocyte differentiation and enhance permeability barrier function (19). Additionally, PPARα and -δ as well as LXR activators stimulate ceramide synthesis in keratinocytes (20, 21). Likewise, ceramide synthesis increases in keratinocytes during differentiation, foreshadowing the formation of lamellar bodies (22, 23).In addition to serving as structural membrane components, ceramides are also important signaling molecules that can induce growth arrest, differentiation, and apoptosis in various cells, including keratinocytes (24–26). Moreover, distal ceramide metabolites, sphingosine and sphingosine-1-phosphate (Fig. 1), are also important signaling molecules (27).Open in a separate windowFIGURE 1.The central role of ceramide in sphingolipid metabolism in keratinocytes. C1P, ceramide 1-phosphate; Sph, sphingosine; S1P, sphingosine-1-phosphate; GlcCer, glucosylceramide; SM, sphingomyelin.It is well established that the expression of ABCA1 is regulated by cellular cholesterol levels in many cell types, including keratinocytes (28). Cholesterol, if metabolized to certain oxysterols, can activate LXR, which then stimulates ABCA1 expression and the transport of cholesterol out of cells (29). This example of feed-forward regulation leads us to hypothesize that either ceramide or a metabolite of ceramide might stimulate ABCA12 expression, thereby leading to an increase in the transport of glucosylceramides into maturing lamellar bodies. Here, we provide evidence that ceramide stimulates ABCA12 expression in keratinocytes via a mechanism involving PPARδ signaling.     

Profiling of BoNT/C3-reversible gene expression induced by lysophosphatidic acid: ephrinB1 gene up-regulation underlying neuropathic hyperalgesia and allodynia

Lysophosphatidic acid (LPA) signaling, through LPA₁ receptor and its downstream RhoA, has been reported to initiate nerve injury-induced neuropathic pain. In the present study, we performed gene expression profiling of the dorsal root ganglion (DRG) to identify genes induced by intrathecal injection of LPA in a botulinum toxin C3 (BoNT/C3)-reversible manner. We selected and functionally characterized ephrinB1 from 82 identified genes as a potential gene involved in pain transmission, since ephrinB1 is implicated to modulate N-methyl-d-aspartate (NMDA) receptor functions in spinal pain transmission. The LPA-induced and BoNT/C3-reversible ephrinB1 gene expression was confirmed by quantitative real-time PCR. Furthermore, treatments with an antisense oligodeoxynucleotide for ephrinB1 largely abolished the LPA-induced thermal hyperalgesia and allodynia in response to mechanical or Aβ-fiber-mediated electrical stimuli on day 1 after the injection. In addition, intrathecal treatment with a soluble ligand, ephrinB1-Fc, caused similar neuropathic pain-like behaviors in a manner that was reversible by the NMDA receptor antagonist MK-801. These results suggest that ephrinB1 plays a crucial role in LPA-induced neuropathic pain. In addition, the present study may provide a new strategy to identify unique neuropathic pain-related genes.     

Liposome-coupled peptides induce long-lived memory CD8 T cells without CD4 T cells

CD8(+) T cells provide broad immunity to viruses, because they are able to recognize all types of viral proteins. Therefore, the development of vaccines capable of inducing long-lived memory CD8(+) T cells is desired to prevent diseases, especially those for which no vaccines currently exist. However, in designing CD8(+) T cell vaccines, the role of CD4(+) T cells in the induction and maintenance of memory CD8(+) T cells remains uncertain. In the present study, the necessity or not of CD4(+) T cells in the induction and maintenance of memory CD8(+) T cells was investigated in mice immunized with liposome-coupled CTL epitope peptides. When OVA-derived CTL epitope peptides were chemically coupled to the surfaces of liposomes and inoculated into mice, both primary and secondary CTL responses were successfully induced. The results were further confirmed in CD4(+) T cell-eliminated mice, suggesting that CD4(+) T cells were not required for the generation of memory CD8(+) T cells in the case of immunization with liposome-coupled peptides. Thus, surface-linked liposomal antigens, capable of inducing long-lived memory CD8(+) T cells without the contribution of CD4(+) T cells, might be applicable for the development of vaccines to prevent viral infection, especially for those viruses that evade humoral immunity by varying their surface proteins, such as influenza viruses, HIV, HCV, SARS coronaviruses, and Ebola viruses.     

Effectiveness of the 1RM estimation method based on isometric squat using a back-dynamometer

This study aimed to clarify the relationships between isometric squat (IS) using a back dynamometer and 1 repetition maximum (1RM) squat for maximum force and muscle activities and to examine the effectiveness of a 1RM estimation method based on IS. The subjects were 15 young men with weight training experience (mean age 20.7 ± 0.8 years, mean height 171.3 ± 4.4 cm, mean weight 64.4 ± 8.4 kg). They performed the IS with various stance widths and squat depths. The measured data of exerted maximum force and the action potential of the agonist muscles were compared with the 1RM squat data. The exerted maximum force during IS was significantly larger in wide stance (140% shoulder width) than in narrow stance (5-cm width). The maximum force was significantly larger with decreased knee flexion. As for muscle activity, the % root mean square value of muscle electric potential of the rectus femoris and the vastus lateralis tended to be higher in wide stance. As for exerted maximum force, wide stance and parallel depth in IS showed a significant and high correlation (r = 0.73) with 1RM squat. Simple linear regression analysis revealed a significant estimated regression equation [Y = 0.992X + 30.3 (Y:1RM, X:IS)]. However, the standard error of an estimate value obtained by the regression equation was very large (11.19 kg). In conclusion, IS with wide stance and parallel depth may be useful for the estimation of 1RM squat. However, estimating a 1RM by IS using a back dynamometer may be difficult.     

Structure–activity relationship of marinostatin,a serine protease inhibitor isolated from a marine organism

A 12‐residue MST isolated from a marine organism is a potent serine protease inhibitor that has a double cyclic structure composed of two ester linkages formed between the β‐hydroxyl and β‐carboxyl groups, Thr³‐Asp⁹ and Ser⁸‐Asp¹¹. MST was synthesized by a regioselective esterification procedure employing two sets of orthogonally removable side‐chain protecting groups for the Asp and Ser/Thr residues. In the MST molecule, there were no significant changes observed in yield by changing the order of esterification. SAR study of MST revealed that the minimum required structure for expressing the inhibitory activity is the sequence (1–9) in a monocyclic structure where Pro⁷ located in the ring plays a crucial role in keeping the structural rigidity. By applying the structural motif of MST, we rationally designed protease inhibitory specificities that differ from those of the natural product. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Foraminiferal oxygen and carbon isotopes during the last 34 kyr off northern Japan, northwestern Pacific

Oxygen and carbon isotopes of foraminifera were analyzed in core PC4, water depth 1366 m, off northern Japan, near the east side of the Tsugaru Strait (130 m depth) between the open northwestern Pacific Ocean and the Japan Sea. At present, the site is at the confluence of the Tsugaru Warm Current which flows eastwards out of the Sea of Japan through the Tsugaru Strait, the subarctic Oyashio Current and the subtropic Kuroshio Current. During the Last Glacial Maximum (LGM), the Oyashio Current penetrated further to the South and outflow from the Japan Sea was restricted by glacio-eustatic sea level lowering.The isotopic values of the planktic foraminifer Neogloboquadrina pachyderma (sinistral) and the benthic foraminifer Uvigerina akitaensis reflect rapid millennial-scale paleoceanographic changes between 34 and 6 ka. Hydrographic changes during deglaciation were related to events at high northern latitudes, but Holocene hydrographic changes were dominated by local effects, such as the development of the outflow of the Tsugaru Warm Current. High values of planktic δ¹⁸O during the LGM reflect the southward advance of the Oyashio Current. These values decreased by 0.3‰ from 19.4 to 18.9 ka, then increased by 0.5‰ at 18 ka, with highest values between 17.5 and 15 ka. The δ¹⁸O oscillations between 19.4 and 15 ka may reflect millennial-scale warm–cold oscillations during Heinrich event 1. Planktic microfossil data indicate that cold Oyashio waters flowed from the northwestern Pacific into the Japan Sea via the Tsugaru Strait between 17 and 16 ka, consistent with the occurrence of the highest planktic δ¹⁸O values in core PC4. Planktic δ¹⁸O values rapidly decreased by 0.9‰ at 15 ka, possibly reflecting the effects of both a rapid increase in fresh water flux and rising temperatures in the subarctic North Pacific. During the Younger Dryas, cold event planktic δ¹⁸O values increased by 0.5‰, followed by a gradual decrease by 1‰ from the early to middle Holocene, reflecting a gradual increase in eastward outflow via the Tsugaru Strait with sea level rise. Both planktic and benthic foraminiferal δ¹³C values oscillated between 34 and 10 ka, at relatively large amplitudes (about 0.5‰), then remained relatively stable during the last 10 kyr. Several negative planktic and benthic ( − 0.7‰) δ¹³C excursions were present in sediment dated between the precipitation of secondary carbonates during episodic methane release possibly associated with methane release from continental margin sediments.     

Fucose removal from complex-type oligosaccharide enhances the antibody-dependent cellular cytotoxicity of single-gene-encoded bispecific antibody comprising of two single-chain antibodies linked to the antibody constant region

Bispecific antibodies (bsAbs) have the potential to extend binding selectivity, increase avidity and exert potent cytotoxicity due to the combination of dual specificities. scFv2-Fc type of single-gene-encoded bispecific antibody, composed of two different single-chain Fvs and an Fc, has been reported to be capable of binding to different antigens. The aim of this study was to determine the effect of fucose removal on effector functions of scFv2-Fc since fucose depletion from oligosaccharide of human IgG1 and scFv-Fc results in significant enhancement of ADCC. We generated novel single-gene-encoded bsAb with dual specificity against tumor associated glycoprotein (TAG)-72 and MUC1 mucin as fucose-negative scFv2-Fc from alpha-1,6-fucosyltransferase knock-out CHO cells and a highly fucosylated scFv2-Fc comparator from parental CHO cells. Expression, assembly and the antigen-binding activity of the scFv2-Fc were not influenced by removal of fucose. The fucose negative scFv2-Fc bound with higher avidity to FcgammaRIIIa and enhanced ADCC compared to the highly fucosylated scFv2-Fc. These results demonstrate that ADCC-enhancement by removal of fucose is effective in not only whole IgG1 and scFv-Fc, but also scFv2-Fc targeting two different antigens, and thus increases the potential of fucose-negative scFv2-Fcs as novel therapeutic candidates.