Glycation exacerbates the neuronal toxicity of β-amyloid

Accumulation evidence shows that β-amyloid (Aβ) is a neurotoxic and accumulation of Aβ is responsible for the pathology of Alzheimer''s disease (AD). However, it is currently not fully understood what makes Aβ toxic and accumulated. Previous studies demonstrate that Aβ is a suitable substrate for glycation, producing one form of the advanced glycation endproducts (AGEs). We speculated that Aβ-AGE formation may exacerbate the neurotoxicity. To explore whether the Aβ-AGE is more toxic than the authentic Aβ and to understand the molecular mechanisms, we synthesized glycated Aβ by incubating Aβ with methylglyoxal (MG) in vitro and identified the formation of glycated Aβ by fluorescence spectrophotometer. Then, we treated the primary hippocampal neurons cultured 8 days in vitro with Aβ-AGE or Aβ for 24 h. We observed that glycation exacerbated neurotoxicity of Aβ with upregulation of receptor for AGE (RAGE) and activation of glycogen synthase kinase-3 (GSK-3), whereas simultaneous application of RAGE antibody or GSK-3 inhibitor reversed the neuronal damages aggravated by glycated Aβ. Thereafter, we found that Aβ is also glycated with an age-dependent elevation of AGEs in Tg2576 mice, whereas inhibition of Aβ-AGE formation by subcutaneously infusion of aminoguanidine for 3 months significantly rescued the early cognitive deficit in mice. Our data reveal for the first time that the glycated Aβ is more toxic. We propose that the glycated Aβ with the altered secondary structure may be a more suitable ligand than Aβ for RAGE and subsequent activation of GSK-3 that can lead to cascade pathologies of AD, therefore glycated Aβ may be a new therapeutic target for AD.     

Genetic relationships among Chinese pigs and other pig populations from Hunan Province, China

In this study, protein-level polymorphisms of transferrin, pre-albumin, hemopexin, ceruloplasmin and amylase were investigated in Hunan native pigs and Large Yorkshire pigs collected from Hunan (a province of China), allowing calculations of allele frequencies, average heterozygosities, inbreeding coefficients and genetic distances. The genetic relationship between Southeast Asian native pigs and American pigs was more distant than those among Southeast Asian native pig breeds. The genetic relationship between Southeast Asian native pig breeds and Hampshire pigs was the most distant.     

Carcass composition and meat quality of indigenous Yanan pigs of China

The Yanan (YN) pig is a traditional Chinese indigenous breed that is raised in southwest China in the Sichuan Province, but there is little data on the germplasm characteristics of this breed. To evaluate carcass characteristics and meat quality of the YN pig, we compared carcass and meat quality of YN pigs and Landrace × Yanan (CY) hybrid pigs; 30 YN pigs and 30 CY pigs weighing 20 ± 2 kg were reared and slaughtered at the normal slaughter weight (100-120 kg). The carcasses were chilled and the left carcass side was dissected into bone, lean meat, fat, and skin; meat quality parameters were measured. Carcasses of YN pigs were lighter (88.85 vs 90.05 kg, P < 0.05) and shorter (71.88 vs 77.61 cm, P < 0.001); they contained less lean meat (41.60 vs 49.25%, P < 0.001), less ham and breech (25.93 vs 27.53%, P < 0.001) and less carcass bone (9.83 vs 10.53%, P < 0.01) than did carcasses of CY pigs. On the other hand, YN pigs had more carcass subcutaneous fat and skin (48.58 vs 40.23%, P < 0.001), thicker backfat (3.67 vs 3.43 cm, P < 0.001) and smaller loin muscle area (9.83 vs 26.91 cm(2), P < 0.001) compared with CY pigs. Among meat quality parameters, YN pigs had higher pH(1) (6.41 vs 6.17, P < 0.001), higher color score(u) (3.86 vs 3.36, P < 0.001) and lower Minolta L(u) values (40.89 vs 45.32, P < 0.01) than CY pigs. On the other hand, YN pigs had lower drip loss (1.31 vs 2.26%, P < 0.05) and lesser fiber area (2351.34 vs 3025.43 μm(2), P < 0.01) than CY pigs. Both breeds had high intramuscular fat (4.46% in YN and 4.45% in CY). No significant differences in other carcass traits and meat quality were found in the two populations. We conclude that YN pigs could be used in commercial pig production to provide good tasting and high-quality niche products.     

L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs

The objective was to determine whether adding L-carnitine in IVM/IVC medium enhanced maturation and developmental competence of porcine oocytes in vitro. Oocyte maturation rates did not differ significantly among groups supplemented with 0, 0.25, 0.5, or 1 mg/mL of L-carnitine added during IVM (although 2 mg/mL of L-carnitine reduced maturation rate). Compared with control oocytes, those treated with 0.5 mg/mL of L-carnitine during IVM had greater (P < 0.05) rates of blastocyst formation after parthenogenetic activation, and these blastocysts had less (P < 0.05) apoptosis. Adding 0.5 mg/mL of L-carnitine during IVM also significantly reduced intracellular reactive oxygen species (ROS), and increased glutathione (GSH) concentrations. With or without glucose supplementation, 0.5 mg/mL of L-carnitine in the IVM medium significantly hastened nuclear maturation of oocytes. Moreover, supplementing the IVM medium with either glucose or L-carnitine increased (P < 0.05) percentages of oocytes that reached the metaphase II (MII) stage, relative to a control group. Final maturation rates in IVM medium containing either glucose or L-carnitine were not significantly different. Adding L-carnitine (0 to 2 mg/mL) to IVC medium for activated porcine oocytes did not significantly affect development. However, 0.5 mg/mL of L-carnitine in IVC medium significantly reduced reactive oxygen species levels and apoptosis in activated blastocysts, although glutathione concentrations were not significantly altered. In conclusion, adding L-carnitine during IVM/IVC improved developmental potential of porcine oocytes, and also the quality of parthenogenetic embryos, probably by accelerating nuclear maturation, and preventing oxidative damage and apoptosis.     

Simultaneous synthesis of luciferase and human nerve growth factor in caterpillars infected with a recombinant baculovirus

Human nerve growth factor (hNGF) gene was proliferated with human leucocyte DNA as template by PCR. Then a fusion gene coding hNGF and luciferase (Luc) cDNAs was inserted into transfer vector pSXIVVI⁺X3/3 with the control of Syn XIV promoter. Luc and hNGF were simultaneously synthesized in Spodoptera larvae upon infection with a recombinant baculovirus, TnNPV-Luc-NGF-OCC⁺. Densitometric scanning of SDS-PAGE revealed that ∼18% of the total Coomassie blue-stainable protein of the infected larvae was represented by Luc protein, while the hNGF level was ∼8%. Both proteins were similar to their authentic counterparts in terms of immunoreactivity. Received 16 August 1998/ Accepted in revised form 14 October 1998     

PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy

CD8⁺ T cells are critical in the balance between fetal tolerance and antiviral immunity. T-cell immunoglobulin mucin-3 (Tim-3) and programmed cell death-1 (PD-1) are important negative immune regulatory molecules involved in viral persistence and tumor metastasis. Here, we demonstrate that Tim-3⁺PD-1⁺CD8⁺ T cells from decidua greatly outnumbered those from peripheral blood during human early pregnancy. Co-culture of trophoblasts with CD8⁺ T cells upregulated PD-1⁺ and/or Tim-3⁺ immune cells. Furthermore, the population of CD8⁺ T cells co-expressing PD-1 and Tim-3 was enriched within the intermediate memory subset in decidua. This population exhibited high proliferative activity and Th2-type cytokine producing capacity. Blockade of Tim-3 and PD-1 resulted in decreased in vitro proliferation and Th2-type cytokine production while increased trophoblast killing and IFN-γ producing capacities of CD8⁺ T cells. Pregnant CBA/J females challenged with Tim-3 and/or PD-1 blocking antibodies were more susceptible to fetal loss, which was associated with CD8⁺ T-cell dysfunction. Importantly, the number and function of Tim-3⁺PD-1⁺CD8⁺ T cells in decidua were significantly impaired in miscarriage. These findings underline the important roles of Tim-3 and PD-1 pathways in regulating decidual CD8⁺ T-cell function and maintaining normal pregnancy.Successful pregnancy requires the maternal immune system to tolerate the semi-allogeneic fetus. A failure in immune tolerance may result in abnormal pregnancies, such as recurrent spontaneous abortion. For many years, the model of immune regulation during pregnancy has been based on a shift in the maternal immune response towards a Th2 bias. The shift from producing inflammatory Th1-type cytokines toward Th2-type cytokines promotes maternal–fetal tolerance.^{1, 2} In addition, maternal administration of the Th2-type cytokine interleukin (IL)-10 or blockade of the Th1-type cytokine tumor necrosis factor (TNF)-α is known to prevent pregnancy loss induced by lipopolysaccharide.^{3, 4}Compared with CD4⁺ T cells, our understanding of the role of CD8⁺ T cells during pregnancy remains poorly understood. CD8⁺ T cells, which directly recognize allogeneic major histocompatibility complex (MHC) class I molecules, have important roles in defense against viral infections. Studies on several murine models have demonstrated the existence of CD8⁺ T cells at the maternal–fetal interface.⁵ During normal pregnancy, the major antigen present is the embryo-derived paternal antigen expressed on extravillous trophoblast (EVT) cells. These cells do not express MHC class I human leukocyte antigens (HLA)-A and HLA-B,⁶ which are the main causes of CD8⁺ T cell-mediated rejection. However, HLA-C and HLA-G, highly expressed on EVT cells,⁶ can elicit a direct cytotoxic response by CD8⁺ T cells during hematopoietic stem cell and allogeneic organ transplantation.^{7, 8} Therefore, whether suppressor or regulatory CD8⁺ T cells are present at the maternal–fetal interface, and how they function to maintain normal pregnancy, remain to be explored.Inhibitory co-stimulatory signals have crucial roles in regulating CD8⁺ T-cell activation or tolerance. It has been shown that exhausted T cells express up to seven different inhibitory molecules,⁹ including PD-1 and Tim-3. PD-1 has been identified as a marker for dysfunctional T cells, and blockade of PD-1 signals has been shown to revert the dysfunctional state of exhausted CD8⁺ T cells in most cases.^{10, 11} Tim-3 has been similarly associated with CD8⁺ T-cell exhaustion as Tim-3 blockade restores proliferation and cytokine production.^{12, 13} Tim-3 and PD-1 co-expression on T cells characterizes the most severely exhausted CD8⁺ T-cell subset, and combined blockade of Tim-3 and PD-1 restores the function of exhausted CD8⁺ T cells.^{14, 15, 16} However, much less is known about the functional regulation of Tim-3 and PD-1 on CD8⁺ T cells during pregnancy.In this study, we investigated Tim-3 and PD-1 expression on CD8⁺ T cells from decidua and peripheral blood in normal pregnant women and those who underwent miscarriage. In particular, we used surface and intracellular phenotype analysis, as well as multifunctional assays, to study the role of Tim-3 and PD-1 signaling pathways in regulating decidual CD8⁺ (dCD8⁺) T-cell function and maintenance of pregnancy. Our data indicate that Tim-3 and PD-1 co-expression on CD8⁺ T cells might be important in maintaining maternal–fetal immune tolerance and successful pregnancy. These results could provide a strategy for developing novel therapies that enhance Tim-3 and PD-1 signals to promote maternal–fetal tolerance and prevent pregnancy loss.     

Are bone mineral density loci associated with hip osteoporotic fractures? A validation study on previously reported genome-wide association loci in a Chinese population

Osteoporosis is a heritable disease characterized mainly by low bone mineral density (BMD) and/or osteoporotic fractures (OF). Most genome-wide association studies on osteoporosis have focused on BMD, whereas little effort has been expended to identify genetic variants directly linked to OF. To determine whether BMD-loci are also associated with OF risk, we performed a validation study to examine 23 BMD-loci reported by recent genome-wide association studies for association with hip OF risk. Our sample consisted of 700 elderly Chinese Han subjects, 350 with hip OF and 350 healthy matched controls. We identified four BMD-loci that were significantly associated with hip OF in this Chinese population, including 7q21 (FLJ42280, P = 1.17 × 10(-4) for rs4729260; P = 0.008 for rs7781370), 6p21 (MHC, P = 0.004 for rs3130340), 13q14 (TNFSF11, P = 0.012 for rs9533090; P = 0.018 for rs9594759; P = 0.020 for rs9594738; P = 0.044 for rs9594751), and 18q21 (TNFRSF11A, P = 0.015 for rs884205). The SNP rs4729260 at 7q21 remained significantly associated, even after conservative Bonferroni's correction. Our results further highlight the importance of these loci in the pathogenesis of osteoporosis, and demonstrate that it is feasible and useful to use OF as the direct phenotype to conduct genetic studies, to enhance our understanding of the genetic architecture of osteoporosis.     

Dynamics and stability analysis of the growth and astaxanthin production system of Haematococcus pluvialis

This paper investigated high cell density cultivation of Haematococcus pluvialis for astaxanthin production in 3.7-L bioreactors. A biomass concentration of 2.74 g L⁻¹and an astaxanthin yield of 64.4 mg L⁻¹ were obtained. Based on the experimental results, a new and simple dynamic model is proposed, differing from Monod kinetics, to describe cell growth, product formation and substrate consumption. Good agreement was found between the model predictions and experimental data. The model revealed that there was cell growth inhibition on product formation and product feedback compensation for substrate consumption, but no substrate inhibition or product inhibition of cell growth. Stability analysis demonstrated that no multiplicity of steady states was observed; the unique positive steady state was locally asymptotically stable; and the effect of dilution rate on steady states was greater than that of the initial substrate concentration. Received 23 February 1999/ Accepted in revised form 08 June 1999     

Orexin affects dorsal root ganglion neurons: a mechanism for regulating the spinal nociceptive processing

Orexins (orexin A and B) are initially known to be a hypothalamic peptide critical for feeding and normal wakefulness. In addition, emerging evidence from behavioral tests suggests that orexins are also involved in the regulation of nociceptive processing, suggesting a novel potential therapeutic approach for pain treatment. Both spinal and supraspinal mechanisms appear to contribute to the role of orexin in nociception. In the spinal cord, dorsal root ganglion (DRG) neurons are primary afferent neurons that transmit peripheral stimuli to the pain-processing areas. Morphological results show that both orexin A and orexin-1 receptor are distributed in DRG neurons. Moreover, by using whole-cell patch-clamp recordings and calcium imaging measurements we found that orexin A induced excitability and intracellular calcium concentration elevation in the isolated rat DRG neurons, which was mainly dependent on the activation of spinal orexin-1 receptor. Based on these findings, we propose a hypothesis that the direct effect of orexin A on DRG neurons would represent a possible mechanism for the orexinergic modulation of spinal nociceptive transmission.     

Expression of human PAI-2 in the baculovirus expression system

Using pSXIVVI⁺X3 as an expressing vector, an occluded recombinant Trichoplusia ni nuclear polyhedrosis virus carrying the cDNA encoding plasminogen activators inhibitor-2 (PAI-2) under the control of the Syn and XIV promoters, has been constructed. SDS-PAGE and immunoblot analysis revealed that the virus-mediated PAI-2, with a molecular weight of ∼45 kDa, was synthesized in the Sf cells at a level of ∼16% of total intracellular protein and in the supernatant phase at a level of ∼64% of total extracellular protein secreted into the hemolymph of infected larvae. The expressed protein was similar to its authentic counterpart in terms of immunoreactivity and bioactivity. Received 5 May 1998/ Accepted in revised form 15 July 1998