Alterations in synapses and mitochondria induced by acute or chronic intermittent hypoxia in the pre-Bötzinger complex of rats: an ultrastructural triple-labeling study with immunocytochemistry and histochemistry.

Introduction: The pre-Bötzinger complex (pre-BötC), a kernel of inspiratory rhythmogenesis, is a heterogeneous network with excitatory glutamatergic and inhibitory GABAergic and glycinergic neurons. Inspiratory rhythm generation relies on synchronous activation of glutamatergic neuron, whilst inhibitory neurons play a critical role in shaping the breathing pattern, endowing the rhythm with flexibility in adapting to environmental, metabolic, and behavioral needs. Here we report ultrastructural alterations in excitatory, asymmetric synapses (AS) and inhibitory, symmetric synapses (SS), especially perforated synapses with discontinuous postsynaptic densities (PSDs) in the pre-BötC in rats exposed to daily acute intermittent hypoxia (dAIH) or chronic (C) IH. Methods: We utilized for the first time a combination of somatostatin (SST) and neurokinin 1 receptor (NK1R) double immunocytochemistry with cytochrome oxidase histochemistry, to reveal synaptic characteristics and mitochondrial dynamic in the pre-BötC. Results: We found perforated synapses with synaptic vesicles accumulated in distinct pools in apposition to each discrete PSD segments. dAIH induced significant increases in the PSD size of macular AS, and the proportion of perforated synapses. AS were predominant in the dAIH group, whereas SS were in a high proportion in the CIH group. dAIH significantly increased SST and NK1R expressions, whereas CIH led to a decrease. Desmosome-like contacts (DLC) were characterized for the first time in the pre-BötC. They were distributed alongside of synapses, especially SS. Mitochondria appeared in more proximity to DLC than synapses, suggestive of a higher energy demand of the DLC. Findings of single spines with dual AS and SS innervation provide morphological evidence of excitation-inhibition interplay within a single spine in the pre-BötC. In particular, we characterized spine-shaft microdomains of concentrated synapses coupled with mitochondrial positioning that could serve as a structural basis for synchrony of spine-shaft communication. Mitochondria were found within spines and ultrastructural features of mitochondrial fusion and fission were depicted for the first time in the pre-BötC. Conclusion: We provide ultrastructural evidence of excitation-inhibition synapses in shafts and spines, and DLC in association with synapses that coincide with mitochondrial dynamic in their contribution to respiratory plasticity in the pre-BötC.

Chronic intermittent hypoxia alters the dendritic mitochondrial structure and activity in the pre-Bötzinger complex of rats.

Mitochondrial bioenergetics is dynamically coupled with neuronal activities, which are altered by hypoxia-induced respiratory neuroplasticity. Here we report structural features of postsynaptic mitochondria in the pre-Bötzinger complex (pre-BötC) of rats treated with chronic intermittent hypoxia (CIH) simulating a severe condition of obstructive sleep apnea. The subcellular changes in dendritic mitochondria and histochemistry of cytochrome c oxidase (CO) activity were examined in pre-BötC neurons localized by immunoreactivity of neurokinin 1 receptors. Assays of mitochondrial electron transport chain (ETC) complex I, IV, V activities, and membrane potential were performed in the ventrolateral medulla containing the pre-BötC region. We found significant decreases in the mean length and area of dendritic mitochondria in the pre-BötC of CIH rats, when compared to the normoxic control and hypoxic group with daily acute intermittent hypoxia (dAIH) that evokes robust synaptic plasticity. Notably, these morphological alterations were mainly observed in the mitochondria in close proximity to the synapses. In addition, the proportion of mitochondria presented with enlarged compartments and filamentous cytoskeletal elements in the CIH group was less than the control and dAIH groups. Intriguingly, these distinct characteristics of structural adaptability were observed in the mitochondria within spatially restricted dendritic spines. Furthermore, the proportion of moderately to darkly CO-reactive mitochondria was reduced in the CIH group, indicating reduced mitochondrial activity. Consistently, mitochondrial ETC enzyme activities and membrane potential were lowered in the CIH group. These findings suggest that hypoxia-induced respiratory plasticity was characterized by spatially confined mitochondrial alterations within postsynaptic spines in the pre-BötC neurons. In contrast to the robust plasticity evoked by dAIH preconditioning, a severe CIH challenge may weaken the local mitochondrial bioenergetics that the fuel postsynaptic activities of the respiratory motor drive.

Catecholaminergic neurons in synaptic connections with pre-Bötzinger complex neurons in the rostral ventrolateral medulla in normoxic and daily acute intermittent hypoxic rats.

The rostral ventrolateral medulla (RVLM) contains cardiovascular-related catecholaminergic neurons and respiratory-related pre-Bötzinger complex (pre-BötC) neurons, which are intermingled and functionally connected for coordinating cardiorespiratory activities. Daily acute intermittent hypoxia (dAIH) is known to elicit respiratory plasticity. However, it is unclear if the catecholaminergic neurons directly synapse onto pre-BötC neurons, and if the local circuitry exhibits plasticity when exposed to dAIH. The present study was aimed to determine the synaptic phenotypes between dopamine-ß-hydroxylase (DßH)-immunoreactive (ir) catecholaminergic neurons and neurokinin 1 receptor (NK1R)-ir pre-BötC neurons, and the effect of dAIH on the neuronal network. Immunofluorescence histochemistry was used to reveal immunoreactivities of DßH and NK1R in the RVLM of normoxic and dAIH rats. Synaptic phenotypes were examined with double-labeling immunoelectron microscopy. We found that DßH immunoreactivity was expressed in somata and processes, some of which were in close apposition to NK1R-ir pre-BötC neurons. DßH-ir gold particles were localized to somata, dendrites, and axonal terminals. DßH-ir terminals formed asymmetric synapses, and occasionally, symmetric synapses in the pre-BötC, featuring the local circuitry. Of the synapses, 28% in normoxic and 29.6% in dAIH groups were apposed to NK1R-ir dendrites. Significant increases in DßH expression and NK1R-ir processes were found in the dAIH group. Moreover, the area and number of processes in close appositions were significantly elevated, strongly suggesting that dAIH induced plasticity with increased connections and interactions between the cardiovascular- and respiratory-related neurons in the local circuitry. In conclusion, asymmetric synapses are predominant in the crosstalk between catecholaminergic and pre-BötC neurons in the RVLM, elaborating excitatory transmission driving the coupling of cardiorespiratory activities. The neural network manifests plasticity in response to dAIH challenge.

[Extraskeletal myxoid chondrosarcoma: a report of 5 cases and review of literature].

OBJECTIVE: To study the clinicopathologic features, immunophenotype and differential diagnosis of extraskeletal myxoid chondrosarcoma (EMC). METHODS: The clinicopathologic features of 5 cases of EMC (during the period from 2008 to 2013) were retrospectively analyzed. Immunohistochemical study (EnVision method) was carried out using the archival material. The literature was reviewed. RESULTS: There were altogether 3 female patients and 2 male patients. Their age ranged from 38 to 63 years (average = 51 years). The patients primarily presented with a tender soft tissue mass. All the tumors studied were solitary and the duration of disease onset varied from 3 months to 1 year. The sites of involvement included toe (number = 2), intracranial (number = 1), thigh (number = 1) and shoulder (number = 1). Gross examination showed white nodular masses with a gelatinous cut surface. The average tumor size measured 5.2 cm in greatest dimension. Histologically, a multinodular architecture with fibrous or loose fibrovascular septa separating lobules of tumor cells was identified. The lobules contained abundant myxoid stroma, with peripheral accentuation of tumor cellularity. Two cases were diagnosed as cellular variant of EMC, with invasive growth pattern and hemorrhage. The tumor cells in cellular EMC were arranged in solid nodules, with rare myxoid matrix in between. The nuclei were relatively uniform, round to oval and contained prominent nucleoli. The mitotic figure ranged from 5 to 10 per 10 high-power fields. Immunohistochemical study showed that all of the 5 cases were positive for vimentin, mitochondria and CD56. Two cases expressed synaptophysin and NSE. Focal positivity for these neuroendocrine markers was detected in the other 2 cases. Chromogranin and S-100 protein expression was demonstrated in 2 cases. The staining for epithelial membrane antigen was positive in case 2 and negative in the other 4 cases. CD117 showed diffuse positivity in case 1, the other 4 cases were not expressed. CONCLUSIONS: EMC is a rare soft tissue sarcoma characterized by distinctive histopathologic features and often shows neuroendocrine differentiation. Although EMC is a slow-growing tumor, it carries a high local recurrence rate and even metastases, warranting long-term follow up.

[The colocalized relationship between p53 mutants 249M, 273H and hsp70 in HepG2 cells].

AIM: To synthesize p53 mutants 249M, 273H with site-directed mutagenesis and construct the relative GFP expression vectors, transfect HepG2 cells and then detect the co-localization of p53 and hsp70. METHODS: Two sets of primers were designed according to the gene sequence of p53, and mismatches were introduced into primers. Mutagenesis was performed in a two-step PCR. The amplified fragments containing the mutation site from the second PCR were subcloned into the pEGFP-C3 vectors. The localization of p53 and hsp70 was observed in transfected HepG2 cells using confocal microscopy. RESULTS: The sequencing analysis showed that the mutated sites were correct. The expression vectors were constructed successfully. Endogenous hsp70 co-localized in cytoplasm with 273H p53, wt p53 and 249M p53 localized in nucleus. CONCLUSION: We have found p53 and hsp70 positive products were colocalized in nuclei of human HCC, while the endogenous hsp70 were colocalized in cytoplasm with 273H p53 in HepG2 cells. This contradictory phenomenon suggests that perhaps there are unclear mechanisms existed between the relationship of hsp70 and p53.

Construction and expression of a eukaryotic expression vector containing a fusion gene of the Hantaan virus S gene and hsp70 gene.

Hantavirus (HV) infection leads to a kind of severe systematic syndrome, hemorrhagic fever with renal syndrome (HFRS). Heat shock proteins (HSPs) can be used as adjuvants assisting soluble antigens to produce specific targets which can be attacked by cytotoxic T lymphocytes. For further research on HFRS vaccine, this study aimed to express Hantaan virus nucleocapsid protein (HTNV NP)-HSP70 fusion protein in COS-7 cells. First, an HTNV S gene encoding NP was amplified by PCR with a mutated termination code and cloned into eukaryotic expression vector pCDNA3.1(+), into which the full-length hsp70 gene had already been inserted, to form the S-hsp70 fusion expression vector pCDNA3.1(+)/S-hsp70. Then this recombinant plasmid was transfected into COS-7 cells by liposome, and eukaryotic expression of NP-HSP70 fusion protein was detected by immunocytochemistry and western blot. The results show that the eukaryotic expression vector pCDNA3.1(+)/S-hsp70 was successfully constructed and the NP-HSP70 fusion protein was effectively expressed in COS-7 cells. This study demonstrates that the NP-HSP70 fusion protein was expressed effectively from the pCDNA3.1(+)/S-hsp70 vector in a eukaryotic system and thus provides a basis for using this plasmid as a new DNA vaccine against HV infection.

[Significant increase of glucose transport activity in breast cancer].

OBJECTIVE: To study the expression level and significance of glucose transporter 1 (Glut-1) in normal breast tissue, adenosis, adenoma and breast carcinoma. METHODS: A total of 147 cases of female breast tissue samples, including 92 cases of invasive ductal carcinoma, 26 cases of breast fibroadenoma, 24 cases of breast adenosis and 5 cases of normal breast tissues, were collected for quantitative detection of the expression of Glut-1 protein by immunohistochemistry (EnVision method) and Western blot, and its mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In normal breast tissue and benign lesions of the breast, Glut-1 was undetectable or only weakly detectable in cytoplasm of ductal and acinar epithelia. In contrast, the intensity of Glut-1 staining was significantly higher in invasive ductal carcinomas (P = 0.0002) with protein expression predominantly in cellular membrane and lesser in cytoplasm. Western blot and RT-PCR analyses showed that the expression of Glut-1 protein and mRNA were significantly increased in invasive ductal carcinoma than fibroadenoma (P =0.001 for protein; P <0.05 for mRNA) and adenosis (P =0.001 for protein; P < 0.05 for mRNA). There was a significant difference among groups (P = 0.0002 for protein; P = 0.0001 for mRNA). CONCLUSIONS: Glucose transport activity, as indicated by Glut-1 protein and its mRNA expression, significantly increases in breast carcinoma than non-cancerous lesions. The over-expression of Glut-1 in breast carcinoma is tightly coupled with tumor cell proliferation, invasion and metastasis, implying that Glut-1 may serve as a new marker in the early diagnosis and prognostication of breast malignancy as well as a new therapeutic target.

[Expression of Hsp70 Grp94 and IgG in human lung carcinoma].

AIM: To explore the expression and significance of heat shock protein 70 (Hsp70), glucose regulated protein 94 (Grp94) and immunoglobulin G (IgG) in human lung carcinoma. METHODS: The expression of Hsp70, Grp94 and IgG in 40 human lung carcinomas was studied using immunohistochemical technique and image analysis. The localization among Hsp70, Grp94 and IgG was analyzed by double labeling immunofluorescent staining and laser scanning confocal microscopy. RESULTS: Hsp70, Grp94 and IgG in Human lung carcinomas showed high expression. The positive rate of Hsp70, Grp94 and IgG was 65% (26/40), 45% (18/40), and 82.5% (33/40), respectively. The average value of optical density was 5.10 +/- 0.32, 3.52 +/- 0.35, and 8.12 +/- 0.31, respectively. Hsp70 was localized in nucleus and cellular cytoplasm while Grp94 and IgG were mainly localized in cellular cytoplasm. Ten cases showed Hsp70 was co-localized with IgG and Eighteen cases showed Grp94 was co-localized with IgG. CONCLUSION: High expression of Hsp70, Grp94 and IgG in Human lung carcinomas suggested that Hsp70, Grp94 and IgG might play an important role in the development of human lung carcinoma. IgG is co-localized with Hsp70 or Grp94. The study will lay a theorical basis for further research on anti-tumor immunotherapy.

Preparation and targeted delivery of immunoliposomes bearing poly(ethylene glycol)-coupled humanized anti-hepatoma disulfide-stabilized Fv (hdsFv25) in vitro.

Peptides in bee venom (PBV) have attracted considerable attention for anti-cancer therapy. In this study, sterically stabilized liposomal PBV (PBV-SL) was prepared using Soybean phosphatidylcholine, cholesterol, and the cholesterol derivatives of PEG with terminal COOH groups. The humanized anti-hepatoma disulfide-stabilized Fv (hdsFv25) was coupled to sterically stabilized liposomes. The hdsFv25-immunoliposome has strong affinity and specificity to SMMC-7721 cells in vitro. PBV-loaded sterically stabilized liposomes modified with the hdsFv25 can kill SMMC-7721 cells in vitro with higher efficiency than non-targeted liposomes. These results demonstrate that this strategy should also be applicable to immunotherapy for other cancers.

Target ability and therapy efficacy of immunoliposomes using a humanized antihepatoma disulfide-stabilized Fv fragment on tumor cells.

Recently the use of peptides in bee venom (PBV) for cancer therapy has attracted considerable attention. However, PBV's extensive use is prohibited by its intense hemolytic activity. In this study, the sterically stabilized liposomal PBV (PBV-SL) was prepared using soybean phosphatidylcholine, cholesterol, and cholesterol-PEG-COOH. The humanized antihepatoma disulfide-stabilized Fv (hdsFv25) was reengineered, expressed, and coupled to sterically stabilized liposomes using the N-hydroxysuccinimide ester method. The hdsFv25-immunoliposomes (SIL [hdscFv25]) were immunoreactive as determined by ELISA assay. PBV-SIL [hdscFv25] can kill SMMC-7721 cells in vitro with higher efficiency than nontargeted liposomes. PBV-SIL [hdsFv25] displayed high antitumor activity and resulted in a significant reduction in tumor size compared to nontargeted liposomes and PBV. These results indicated that this strategy should be applicable to applicable in the treatment of other cancers.

[Expression of HSP70 in human umbilical vein endothelial cells induced by Hantavirus].

AIM: To study stress response in human umbilical vein endothelial cells (HUVEC) infected with Hantavirus (HTV). METHODS: The expression of HSP70 was detected by immunocytochemical staining, in situ nucleic acid hybridization and RT-PCR. RESULTS: After being infected with HTV, the expression of HSP70 in HUVEC increased. RT-PCR showed that the level of HSP70 mRNA increased rapidly and kept at a high level in comparison with the normal control (P<0.05). CONCLUSION: The over-expression of HSP70 in HUVECs can be induced by Hantavirus infection, which may play a role in the inhibition of virus reproduction and the protection of cells from virus infection.

Primary targeting of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha against hepatocellular carcinoma.

AIM: To obtain human recombinant Fv-immunotoxin hscFv(25)-mTNFalpha (mutant human TNFalpha fused to human scFv(25)) against hepatocellular carcinoma (HCC). METHODS: Two relevant sites of enzymatic digestion were added to mTNFalpha by PCR. MTNFalpha was linked to the 3' end of hscFv(25) in pGEX4T-1 vector. This anti-HCC recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was expressed in Escherichia coli and purified from inclusions. After purified by glutathione-S-transferase affinity chromatography and thrombin digestion, it was identified by electrophoresis and Western blot. And then, the purified recombinant Fv-immunotoxin was injected into nude mice with HCC xenografts through their tail veins. MTNFalpha protein and PBS were used as control at the same time. After treated for two weeks, nude mice were executed. The bulk and weight of tumors were observed. The tumor tissues were stained by immunohistochemical method with TNFalpha antibody. RESULTS: The expression ratio of recombinant Fv-immunotoxin hscFv(25)-mTNFalpha was 12% of bacterial protein. The result of tumor restraining trials of hscFv(25)-mTNFalpha showed 2/5 complete remission and 3/5 partial remission. mTNFalpha restraining trials showed 5/5 partial remission. The therapeutic result of hscFv(25)-mTNFalpha was better than that of mTNFalpha (F=8.70, P<0.05). The hscFv(25)-mTNFalpha remedial tumor tissues were positive for TNFalpha by immunohistochemical staining. The positive granules mainly existed in the cytoplasm of tumor cell. CONCLUSION: Recombinant Fv-immunotoxin hscFv(25)-mTNFalpha has better therapeutic effect than mTNFalpha. It can inhibit the cellular growth of HCC and has some potential of clinical application.