The Neuroprotective Effects of Reg‐2 Following Spinal Cord Transection Injury

This study was designed to elucidate the potential neuroprotective effects of Reg‐2 (regeneration gene protein 2) in a rodent model of spinal cord transection injury at the ninth thoracic level. Reg‐2 at 100 and 500 μg, recombinant rat ciliary neurotrophic factor, or vehicle were delivered intrathecally using Alzet miniosmotic pumps. We found that Reg‐2 treatment significantly reduced neuronal death in the spinal cord. There was also an attenuation of inflammation at the injury site and an increase in white matter sparing and retained myelination. Retrograde tracing revealed that Reg‐2 protected axons of long descending pathways at 6 weeks post‐SCI, and the number of FluoroGold‐labeled neurons in spinal and supraspinal regions was also significantly increased. Immunofluorescent staining confirmed that the spared white matter contained neurofilament‐positive axons. Moreover, behavioral improvements were revealed by Basso Beattie Bresnahan locomotor rating scores and grid‐walk analysis. These results suggest that Reg‐2 might promote functional recovery by increasing axonal growth, inhibiting neuronal apoptosis, and attenuating spinal cord secondary injury after SCI. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

The Effects of Serotonin on Functionally Diverse Isolated Lamprey Spinal Cord Neurons

The experiments reported here showed that application of serotonin (5-hydroxytryptamine, 5-HT) (100 M) did not induce any significant current through the membranes of any of the spinal neurons studied (n= 62). At the same time, the membranes of most motoneurons and interneurons (15 of 18) underwent slight depolarization (2–6 mV) in the presence of 5-HT, which was not accompanied by any change in the input resistance of the cells. Depolarization to 10–20 mV occurred in some cells (3 of 18) of these functional groups, this being accompanied by 20–60% decreases in input resistance. The same concentration of 5-HT induced transient low-amplitude depolarization of most sensory spinal neurons (dorsal sensory cells), this changing smoothly to long-term hyperpolarization by 2–7 mV. The input resistance of the cell membranes in these cases showed no significant change (n= 8). Data were obtained which provided a better understanding of the mechanism by which 5-HT modulates the activity of spinal neurons. Thus, 5-HT facilitates chemoreceptive currents induced by application of NMDA to motoneurons and interneurons, while the NMDA responses of dorsal sensory cells were decreased by 5-HT. 5-HT affected the post-spike afterresponses of neurons. 5-HT significantly decreased the amplitude of afterhyperpolarization arising at the end of the descending phase of action potentials in motoneurons and interneurons and increased the amplitude of afterdepolarization in these types of cells. In sensory spinal neurons, 5-HT had no great effect on post-spike afterresponses. The results obtained here support the suggestion that 5-HT significantly modulates the activity of spinal neurons of different functional types. 5-HT facilitates excitation induced by subthreshold depolarization in motoneurons and some interneurons, facilitating the generation of rhythmic discharges by decreasing afterhyperpolarization. In sensory cells, 5-HT enhances inhibition due to hyperpolarization, suppressing NMDA currents. The differences in the effects of 5-HT on functionally diverse neurons are presumed to be associated with the combination of different types of 5-HT receptors on the membranes of these types of spinal neurons.     

Temporal Changes of Astrocyte Activation and Glutamate Transporter‐1 Expression in the Spinal Cord After Spinal Nerve Ligation‐Induced Neuropathic Pain

Astrocyte activation is involved in the neuropathic pain. As a glutamate scavenger, the glutamate transporter‐1 (GLT‐1) is exclusively expressed on the astrocytes and probably correlates with astrocyte activation. In the present study, we attempted to clarify the temporal changing courses of astrocyte activation and GLT‐1 expression, as well as their correlations induced by a neuropathic pain model, namely, spinal nerve ligation (SNL) in which rapidly appearing (<3 days) and persistent (>21 days) mechanical allodynia and thermal hyperalgesia were presented. Immunofluorescent staining showed that GLT‐1 was expressed exclusively in most (not all) of the astrocytes, even when the GLT‐1 expression reached its peak. The expression of GLT‐1 displayed an interesting biphasic change, with an initial up‐regulation followed by a down‐regulation after SNL. Our results also demonstrated that SNL induced a marked and long‐term (>21 days) activation of astrocytes in the ipsilateral spinal dorsal horn. These results suggest that astrocyte activation, the change of GLT‐1 expression and the potential relationship between them might play key roles in the induction and/or maintenance of neuropathic pain. The present results provide novel clues in understanding the mechanisms underlying the involvement of astrocytes and GLT‐1 in the neuropathic pain. Anat Rec, 291:513–518, 2008. © 2008 Wiley‐Liss, Inc.     

嗅鞘细胞培养上清液对体外培养脊髓神经元存活及活性的影响

目的:通过检测鞘嗅细胞培养上清液,研究其是否能够分泌促进神经元存活及活性的物质,以便为CNS神经损伤修复提供相关移植的理论依据。方法:分离培养嗅神经及嗅球颗粒层(ONGL)细胞,经过传代培养进行纯化嗅鞘细胞,最后收集其培养上清液。浓缩并检测其蛋白浓度,分别以10ug/ml、20ug/ml、40ug/ml、80ug/ml、160ug/ml的浓度加到培养的脊髓神经元的存活及活性指标。结果:嗅鞘细胞培养上清的蛋白浓度为40ug/ml、80ug/ml时对神经元夏活具有促进作用(与对照组相比P<0.01),而80ug/ml时对神经元活性具有促进作用(P<0.01)。结论:嗅鞘细胞在培养状态能够分泌一些活性物质,这些活性物质对神经元的存活和活性有明显的促进作用。     

Correlation of the Mitochondrial Activity of Two‐Cell Embryos Produced In Vitro and the Two‐Cell Block In Kunming and B6C3F1 Mice

The correlation between the early embryonic block to development and mitochondrial activity was investigated comparing two‐cell embryos produced in vitro from Kunming (KM) and B6C3F1 mice. One‐cell embryos were obtained from two species of hybrids (female KM mice mated with KM males and female B6C3F1 mice mated with KM males) and cultured for 84 hr in M16 media. The mitochondrial membrane potential, ATP content, and reactive oxygen species levels were measured in the resulting KM and B6C3F1 two‐cell embryos. Mitochondrial membrane potential and ATP content were also determined in KM and B6C3F1 metaphase II eggs. The results showed that the two‐cell block was observed in cultured KM embryos but not in B6C3F1 embryos. Mitochondrial membrane potential and ATP content of KM two‐cell embryos were significantly lower than in B6C3F1 two‐cell embryos (P < 0.01). Interestingly, the reactive oxygen species levels of KM two‐cell embryos were significantly lower than their B6C3F1 counterparts (P < 0.01). There was no difference in mitochondrial membrane potential and ATP content between KM and B6C3F1 metaphase II eggs. It is concluded that KM mice have an early two‐cell embryo block and that a possible “blocking” mechanism is the lower mitochondrial membrane potential and ATP content in these embryos. The results suggest a new approach for overcoming early embryonic development block, that of manipulating mitochondrial activity. Anat Rec, 292:661–669, 2009. © 2009 Wiley‐Liss, Inc.     

脱细胞异体神经基质移植物对脊髓前角运动神经元的保护作用

目的探讨脱细胞神经基质移植物异体移植对脊髓前角运动神经元的保护作用。方法W ist-ar大鼠30只,10只用化学提取法制备坐骨神经脱细胞基质移植物;另20只分为实验组(坐骨神经缺损移植物桥接组)和对照组(坐骨神经缺损组),每组10只。动物饲养3到4个月后取移植物、脊髓腰6-骶1节段和术侧腓肠肌,做HE、尼氏和AchE结合镀银染色以及电镜标本,进行组织学和超微结构观察,并对脊髓前角细胞的数量做统计学分析。结果实验组动物移植术后3到4个月术侧下肢行走时,后蹬动作有力,足趾能分开,步态趋于正常;针刺足底有逃避动作。对照组动物术侧下肢的运动和感觉功能未见恢复。实验组动物的移植物内见有大量再生的神经纤维,髓鞘结构清晰,腓肠肌内见有再生的神经纤维束和运动终板。实验组移植侧和对照组缺损侧脊髓腰6到骶1节段前角外侧群细胞的数量比正常侧减少,尤以对照组为甚。实验组移植侧前角细胞比正常侧的前角细胞数减少而移植侧前角细胞数比对照组缺损侧明显减少(P<0.01)。结论脱细胞异体神经基质移植物桥接周围神经缺损对运动神经元胞体的存活具有良好的保护作用。     

Ca2+与NO在过氧化氢所致正常成年大鼠肠系膜微血管通透性增高中的作用

 目的 观察内皮细胞Ca2+浓度及NO生成在过氧化氢所致正常成年大鼠肠系膜微血管通透性增高中的作用。方法 通过测定在体大鼠肠系膜微血管静水传导性观察微血管通透性变化。采用钙荧光指示剂(Fura 2-AM)、NO荧光指示剂(DAF-2 DA)标记在体微血管内皮细胞,并应用荧光显微镜检测细胞内钙或NO的荧光信号,观察H2O2作用下内皮细胞内钙离子浓度([Ca2+]i)、NO的变化。结果 H2O2可增加正常成年大鼠微血管通透性（正常对照的6.13±0.87倍,P<0.01）,同时增加微血管内皮细胞[Ca2+]i（714.58±144.70 nmol/L,P<0.01）,并促进内皮细胞NO的生成（正常对照荧光强度的1034.3%±44.3%,P<0.01）。Ca2+通道阻滞剂氯化镧可抑制H2O2所引起的微血管通透性增加（P<0.01）及内皮细胞[Ca2+]i升高（P<0.01）。NOS抑制剂AP-Cav-1可抑制H2O2所引起的微血管通透性增加（P<0.01）,但对H2O2的Ca2+增加作用无影响。结论 H2O2所致的通透性增加与细胞内Ca2+增加、NO的产生增多有关。     

In Vitro Effects of Sodium Benzoate on Th1/Th2 Deviation in Patients with Multiple Sclerosis

Interleukin 4 (IL-4) can improve the clinical manifestations in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Sodium benzoate (NaB) deviates the cytokine profile to Th2 (or IL-4 producing) cells in EAE and thus might be effective in the treatment of MS. Therefore, in this study the effect of different concentrations of NaB on the percentage and mRNA levels of IL-4 and interferon gamma (IFN-γ)-producing peripheral blood mononuclear cells (PBMCs) of 20 Relapsing-remitting multiple sclerosis (RR-MS) patients and eight healthy controls was evaluated in the presence of mitogen (phytohemagglutinin, PHA) or specific antigen (myelin basic protein, MBP). Our results showed that in the patient’s group the percentage of CD4⁺IL-4⁺ cells was significantly increased in the presence of all concentrations of NaB when PBMCs were stimulated by MBP (p = 0.001) or PHA (p < 0.03). The same results were obtained for normal donors in the highest concentration of NaB, 1000 µg/ml (p = 0.02). Moreover, in the patient’s group the percentage of CD4⁺IFN-γ⁺ cells was decreased significantly when the PBMCs were stimulated by PHA and NaB (p < 0.004) or by MBP and 1000 µg/ml of NaB (p < 0.03). The effect of NaB on IL-4 and IFN-γ production was also documented at the mRNA levels. In conclusion, our data suggest that NaB is able to induce IL-4 production by human PBMCs and therefore might be a useful candidate for conjunctive therapy in RR-MS.     

Bcl‐2 protects TK6 cells against hydroquinone‐induced apoptosis through PARP‐1 cytoplasm translocation and stabilizing mitochondrial membrane potential

B cell leukemia/lymphoma‐2 (Bcl‐2) suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating mitochondrial membrane potential (MMP). This study aimed to investigate the role of Bcl‐2 in controlling the mitochondrial pathway of apoptosis during hydroquinone (HQ)‐induced TK6 cytotoxicity. In this study, HQ, one metabolite of benzene, decreased the MMP in a concentration‐dependent manner and induced the generation of reactive oxygen species (ROS), the activation of the DNA damage marker γ‐H2AX, and production of the DNA damage‐responsive enzyme poly(ADP‐ribose)polymerase‐1 (PARP‐1). Exposure of TK6 cells to HQ leads to an increase in Bcl‐2 and co‐localization with PARP‐1 in the cytoplasm. Inhibition of Bcl‐2 using the BH3 mimetic, ABT‐737, suppressed the PARP‐1 nuclear to cytoplasm translocation and sensitized TK6 cells to HQ‐induced apoptosis through depolarization of the MMP. Western blot analysis indicated that ABT‐737 combined with HQ increased the levels of cleaved PARP and γ‐H2AX, but significantly decreased the level of P53. Thus, ABT‐737 can influence PARP‐1 translocation and induce apoptosis via mitochondria‐mediated apoptotic pathway, independently of P53. In addition, we found that knockdown of PARP‐1 attenuated the HQ‐induced production of cleaved PARP and P53. These results identify Bcl‐2 as a protective mediator of HQ‐induced apoptosis and show that upregulation of Bcl‐2 helps to localize PARP‐1 to the cytoplasm and stabilize MMP. Environ. Mol. Mutagen. 59:49–59, 2018. © 2017 Wiley Periodicals, Inc.     

Metabolism of hyperpolarized 13C‐acetoacetate to β‐hydroxybutyrate detects real‐time mitochondrial redox state and dysfunction in heart tissue

Mitochondrial dysfunction is considered to be an important component of many metabolic diseases yet there is no reliable imaging biomarker for monitoring mitochondrial damage in vivo. A large prior literature on inter‐conversion of β‐hydroxybutyrate and acetoacetate indicates that the process is mitochondrial and that the ratio reflects a specifically mitochondrial redox state. Therefore, the conversion of [1,3‐¹³C]acetoacetate to [1,3‐¹³C]β‐hydroxybutyrate is expected to be sensitive to the abnormal redox state present in dysfunctional mitochondria. In this study, we examined the conversion of hyperpolarized (HP) ¹³C‐acetoacetate (AcAc) to ¹³C‐β‐hydroxybutyrate (β‐HB) as a potential imaging biomarker for mitochondrial redox and dysfunction in perfused rat hearts. Conversion of HP‐AcAc to β‐HB was investigated using ¹³C magnetic resonance spectroscopy in Langendorff‐perfused rat hearts in four groups: control, global ischemic reperfusion, low‐flow ischemic, and rotenone (mitochondrial complex‐I inhibitor)‐treated hearts. We observed that more β‐HB was produced from AcAc in ischemic hearts and the hearts exposed to complex I inhibitor rotenone compared with controls, consistent with the accumulation of excess mitochondrial NADH. The increase in β‐HB, as detected by ¹³C MRS, was validated by a direct measure of tissue β‐HB by ¹H nuclear magnetic resonance in tissue extracts. The redox ratio, NAD⁺/NADH, measured by enzyme assays of homogenized tissue, also paralleled production of β‐HB from AcAc. Transmission electron microscopy of tissues provided direct evidence for abnormal mitochondrial structure in each ischemic tissue model. The results suggest that conversion of HP‐AcAc to HP‐β‐HB detected by ¹³C‐MRS may serve as a useful diagnostic marker of mitochondrial redox and dysfunction in heart tissue in vivo.     

The expression of Bcl‐2 by proliferating cells varies in different categories of B‐cell lymphoma

Masir N, Jones M, Lee A M, Goff L K, Clear A J, Lister A, Marafioti T & Mason D Y
(2010) Histopathology 56 , 617–626 The expression of Bcl‐2 by proliferating cells varies in different categories of B‐cell lymphoma Aims: To investigate the relationship between Bcl‐2 protein expression and cell proliferation at single‐cell level in B‐cell lymphomas using double‐labelling techniques. Methods and results: The relationship between Bcl‐2 protein expression and cell proliferation was explored in 124 cases of B‐cell lymphoma using double immunofluorescence labelling for Bcl‐2 and Ki67. In follicular lymphoma, marginal zone lymphoma and a subset of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL), neoplastic cells tended to lose Bcl‐2 when they are in cell cycle. This pattern is usually maintained in both follicular lymphoma and CLL/SLL when they undergo high‐grade transformation. In mantle cell lymphoma, diffuse large B‐cell lymphoma and a subset of CLL/SLL, the inverse relationship (between Bcl‐2 and Ki67) was not observed, i.e. the proliferating cells tended to show co‐expression of Bcl‐2. Conclusions: In low‐grade lymphomas, including those that are transformed, Bcl‐2 expression is lost when cell proliferate. However, in more aggressive tumours (i.e. mantle cell and de novo diffuse large B‐cell lymphomas) the inverse Bcl‐2/Ki67 relationship was not observed. It would be of interest to explore the clinical implications in lymphoma of the presence and absence of the inverse Bcl‐2/Ki67 pattern.     

Protease‐activated receptor‐2 signalling by tissue factor on dendritic cells suppresses antigen‐specific CD4+ T‐cell priming

The precise function of tissue factor (TF) expressed by dendritic cells (DC) is uncertain. As well as initiating thrombin generation it can signal through protease‐activated receptor 2 (PAR‐2) when complexed with factor VIIa. We investigated the expression and function of TF on mouse bone marrow (BM) ‐derived DC; 20% of BM‐derived DC expressed TF, which did not vary after incubation with lipopolysaccharide (LPS) or dexamethasone (DEX). However, the pro‐coagulant activity of DEX‐treated DC in recalcified plasma was 30‐fold less than LPS‐treated DC. In antigen‐specific and allogeneic T‐cell culture experiments, the TF on DEX‐treated DC provided a signal through PAR‐2, which contributed to the reduced ability of these cells to stimulate CD4⁺ T‐cell proliferation and cytokine production. In vivo, an inhibitory anti‐TF antibody and a PAR‐2 antagonist enhanced antigen‐specific priming in two models where antigen was given without adjuvant, with an effect approximately 50% that seen with LPS, suggesting that a similar mechanism was operational physiologically. These data suggest a novel TF and PAR‐2‐dependent mechanism on DEX‐DC in vitro and unprimed DC in vivo that contributes to the low immunogenicity of these cells. Targeting this pathway has the potential to influence antigen‐specific CD4⁺ T‐cell activation.     

Ligation of human Fc receptor like‐2 by monoclonal antibodies down‐regulates B‐cell receptor‐mediated signalling

B‐cell antigen receptor (BCR) signalling and its regulation through negative and positive regulators are critical for balancing B‐cell response and function. Human Fc receptor like‐2 (FCRL2), a member of the newly identified FCRL family, could influence B‐cell signalling due to possession of both immunoreceptor tyrosine‐based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified, we generated FCRL2‐specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signalling in an FCRL2‐expressing B‐cell line. Two anti‐FCRL2 mAb‐producing hybridoma clones (5A7‐E7 and 3D8‐G8) were selected. None of the mAbs displayed any cross‐reactivity with the other members of the FCRL family including recombinant FCRL1, ‐3, ‐4 and ‐5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signalling mediators such as calcium mobilization and phosphorylation of the mitogen‐activated protein kinases Erk, p38 and Jnk. These findings indicate that the FCRL2 ITIM motifs are functional and the anti‐FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells.     

Differential regulation and impact of fucosyltransferase VII and core 2 β1,6‐N‐acetyl‐glycosaminyltransferase for generation of E‐selectin and P‐selectin ligands in murine CD4+ T cells

Ligands for E‐selectin and P‐selectin (E‐lig and P‐lig) are induced on CD4⁺ T cells upon differentiation into effector T cells. Glycosyltransferases, especially α 1,3‐fucosyltransferase VII (FucT‐VII) and core 2 β1,6‐N‐acetyl‐glycosaminyltransferase I (C2GlcNAcT‐I), are critical for their synthesis. We here analysed the signals that control the expression of E‐lig, P‐lig and mRNA coding for FucT‐VII and C2GlcNAcT‐I. In line with previous reports, we found that P‐lig expression correlates with the regulation of C2GlcNAcT‐I, whereas E‐lig expression can occur at low levels of C2GlcNAcT‐I mRNA but requires high FucT‐VII mRNA expression. Interestingly, the two enzymes are regulated by different signals. Activation‐induced C2GlcNAcT‐I up‐regulation under permissive (T helper type 1) conditions was strongly reduced by cyclosporin A (CsA), suggesting the involvement of T‐cell receptor‐dependent, calcineurin/NFAT‐dependent signals in combination with interleukin‐12 (IL‐12) ‐mediated signals in the regulation of C2GlcNAcT‐I. In contrast, expression of FucT‐VII mRNA was not significantly inhibited by CsA. Interleukin‐4 inhibited the expression of FucT‐VII but IL‐2 and IL‐7 were found to support induction of FucT‐VII and E‐lig. E‐selectin, P‐selectin and their ligands initially appeared to have rather overlapping functions. These findings however, unravel striking differences in the regulation of E‐lig and P‐lig expression, dictated by the dominance of FucT‐VII and C2GlcNAcT‐I, respectively, and their dependency on signals from either promiscuous or homeostatic cytokines (FucT‐VII) or a strong T‐cell receptor signal in combination with inflammatory cytokines in case of C2GlcNAcT‐I.     

Use of the human colorectal adenocarcinoma (Caco‐2) cell line for isolating respiratory viruses from nasopharyngeal aspirates

The human colorectal adenocarcinoma‐derived Caco‐2 cell line was evaluated as a means isolating common respiratory viruses from nasopharyngeal aspirates for the diagnosis of respiratory diseases. One hundred eighty‐nine direct immunofluorescence positive nasopharyngeal aspirates obtained from patients with various viral respiratory diseases were cultured in the presence of Caco‐2 cells or the following conventional cell lines: LLC‐MK2, MDCK, HEp‐2, and A549. Caco‐2 cell cultures effectively propagated the majority (84%) of the viruses present in nasopharyngeal aspirate samples compared with any positive cultures obtained using the panel cells (78%) or individual cell line MDCK (38%), HEp‐2 (21%), LLC‐MK2 (27%), or A549 (37%) cell lines. The differences against individual cell line were statistically significant (P = < 0.000001). Culture in Caco‐2 cells resulted in the isolation of 85% (36/42) of viruses which were not cultivated in conventional cell lines. By contrast, 80% (24/30) of viruses not cultivated in Caco‐2 cells were isolated using the conventional panel. The findings indicated that Caco‐2 cells were sensitive to a wide range of viruses and can be used to culture a broad range of respiratory viruses. J. Med. Virol. 85:874–879, 2013. © 2013 Wiley Periodicals, Inc.     

Spatio‐Temporal Development of Axonopathy in Canine Intervertebral Disc Disease as a Translational Large Animal Model for Nonexperimental Spinal Cord Injury

Spinal cord injury (SCI) represents a devastating central nervous system disease that still lacks sufficient therapies. Here, dogs are increasingly recognized as a preclinical animal model for the development of future therapies. The aim of this study was a detailed characterization of axonopathy in canine intervertebral disc disease, which produces a mixed contusive and compressive injury and functions as a spontaneous translational animal model for human SCI. The results revealed an early occurrence of ultrastructurally distinct axonal swelling. Immunohistochemically, enhanced axonal expression of β‐amyloid precursor protein, non‐phosphorylated neurofilament (n‐NF) and growth‐associated protein‐43 was detected in the epicenter during acute canine SCI. Indicative of a progressive axonopathy, these changes showed a cranial and caudally accentuated spatial progression in the subacute disease phase. In canine spinal cord slice cultures, immunoreactivity of axons was confined to n‐NF. Real‐time quantitative polymerase chain reaction of naturally traumatized tissue and slice cultures revealed a temporally distinct dysregulation of the matrix metalloproteinases (MMP)‐2 and MMP‐9 with a dominating expression of the latter. Contrasting to early axonopathy, diminished myelin basic protein immunoreactivity and phagocytosis were delayed. The results present a basis for assessing new therapies in the canine animal model for translational research that might allow partial extrapolation to human SCI.     

Preproenkephalin mRNA is Expressed in a Subpopulation of GABAergic Neurons in the Spinal Dorsal Horn of the GAD67‐GFP Knock‐In Mouse

GABA (γ‐aminobutyric acid)ergic neurons in the spinal dorsal horn have been reported to be divided into distinctive populations, with different cotransmitters and neuropeptides. In this study, we examined the colocalization of enkephalin (ENK) mRNA with GABA in the spinal dorsal horn using the glutamic acid decarboxylase (GAD)₆₇‐green fluorescence protein (GFP) knock‐in mouse. Our approach was to perform in situ hybridization histochemistry to detect mRNA for preproenkephalin (PPE, the precursor protein for ENK), combined with immunohistochemistry for GFP to reveal GABAergic neurons. Quantitative analysis indicated that more than 44.4% (2967/6681) of GFP‐immunoreactive neurons showed signals for PPE mRNA in the spinal dorsal horn. While 53.9% (2967/5501) of PPE mRNA‐expressing neurons were immunoreactive for GFP. The double‐labeled neurons were observed throughout the spinal dorsal horn, although they had a preferential localization in superficial layers. The present results provide a detailed morphological evidence that ENK and GABA colocalized in a subpopulation of neurons in the spinal dorsal horn, which are likely to represent local inhibitory dorsal horn interneurons involved in the modulation of pain transmission. Anat Rec, 291:1334–1341, 2008. © 2008 Wiley‐Liss, Inc.     

Growth‐Inhibitory Activity of Melatonin on Murine Foregastric Carcinoma Cells In Vitro and the Underlying Molecular Mechanism

Melatonin (MLT) is an indolic hormone produced mainly by the pineal gland. Recent human and animal studies have shown that MLT exerts obvious oncostatic activity both in vitro and in vivo. The purpose of this study was to investigate the antiproliferative effect of MLT on the murine foregastric carcinoma (MFC) cell and to determine the underlying molecular mechanism. Cell viability was determined using the Cell Counting Kit‐8 (CCK‐8) and the results revealed that MLT exhibited a dose‐ and time‐dependent inhibitory effect on MFC cell growth. Our studies also demonstrated upregulation of p21 and Bax and downregulation of Bcl‐2 at both the mRNA and the protein levels in response to MLT treatment of MFC cells. These changes in the expression of these molecules were consistent with the results of the CCK‐8. Furthermore, the mRNA and protein expression of membranous MLT receptors was also upregulated. Taken together, these results confirm the oncostatic effect of MLT in MFC cells and the expression of membranous MLT receptors is a potential approach to tumor cells in gastric cancer therapeutic treatment. Anat Rec, 296:914–920, 2013. © 2013 Wiley Periodicals, Inc.     

Effects of the differential expression of ZO‐1 and ZO‐2 on podocyte structure and function

Glomerular podocytes in the kidney originate from columnar epithelial cells possessing tight junctions. During podocyte differentiation, tight junctions are replaced by slit diaphragms, which are formed between foot processes and function as a blood filtration barrier. Although the expression of most tight junction components is suppressed during podocyte differentiation, several components, including ZO‐1 and ZO‐2, are consistently expressed. We recently showed that podocyte‐specific deletion of ZO‐1 gene impaired slit diaphragm formation, leading to proteinuria and glomerular sclerosis. Here, we address the relevance of ZO‐2, whose sequence is highly similar to ZO‐1, in the maintenance of the structure and function of podocytes. In glomerular development, the spatiotemporal expression of ZO‐2 was similar to that of ZO‐1 until the capillary loop stage. Subsequently, the distribution patterns of ZO‐1 and ZO‐2 diverged at the maturation stage, when slit diaphragms are formed. This divergence could partly rely on the ability of ZO‐2 to interact with the slit diaphragm membrane proteins. Podocyte‐specific deletion of the ZO‐2 gene did not cause overt defects; however, double knockout of ZO‐1 and ZO‐2 genes accelerated the defects observed in ZO‐1 knockout mice. These results suggest that ZO‐2 plays supportive roles in the ZO‐1‐dependent regulation of podocyte filtration barrier.