Distribution and Dynamics of Gap Junction Channels Revealed in Living Cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

Distribution and Dynamics of Gap Junction Channels Revealed in Living Cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

Identification and Functional Expression of HCx31.9, a Novel Gap Junction Gene

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.     

Mass Spectrometric Identification and Quantification of Hemorphins Extracted from Human Adrenal and Pheochromocytoma Tissue

Abstract: The hemorphins are a family of recently identified opioid receptor binding peptides derived from the proteolytic processing of the β, γ, δ, and ε chains of hemoglobin. They have previously been identified at high concentration in human pituitary glands and in the CSF of patients with cerebral bleeding. Hemorphins are potent inhibitors of angiotensin converting enzyme and therefore possibly have a role to play in blood pressure regulation. We report the presence of four hemorphin peptides in extracts of normal adrenal tissue and in pheochromocytoma tumors. The hemorphins were quantified and structurally characterized using mass spectrometry. High concentrations of hemorphins were found in all samples, comparable with the levels reported in the literature for pituitary and brain tissue.     

间质热疗法组织中温度分布与光纤加入端的关系

主要利用了有限元方法,模拟研究了在激光间质热疗法中,被加热区域组织中温度的分布情况。组织的温度对组织中细胞有着极其重要的影响,对温度的控制是间质热疗法成败的关键因素。到目前为止,在激光间质热疗法中,还没有一种很精确方法能够实时测量激光辐射后组织中温度分布。为了能为临床处理提供一些有用参考,利用Ansys对不同的光纤加入端所形成的温度场进行了模拟,不失为一种好的方法。     

Genetic Recombination between Human and Animal Parasites Creates Novel Strains of Human Pathogen

Genetic recombination between pathogens derived from humans and livestock has the potential to create novel pathogen strains, highlighted by the influenza pandemic H1N1/09, which was derived from a re-assortment of swine, avian and human influenza A viruses. Here we investigated whether genetic recombination between subspecies of the protozoan parasite, Trypanosoma brucei, from humans and animals can generate new strains of human pathogen, T. b. rhodesiense (Tbr) responsible for sleeping sickness (Human African Trypanosomiasis, HAT) in East Africa. The trait of human infectivity in Tbr is conferred by a single gene, SRA, which is potentially transferable to the animal pathogen Tbb by sexual reproduction. We tracked the inheritance of SRA in crosses of Tbr and Tbb set up by co-transmitting genetically-engineered fluorescent parental trypanosome lines through tsetse flies. SRA was readily transferred into new genetic backgrounds by sexual reproduction between Tbr and Tbb, thus creating new strains of the human pathogen, Tbr. There was no evidence of diminished growth or transmissibility of hybrid trypanosomes carrying SRA. Although expression of SRA is critical to survival of Tbr in the human host, we show that the gene exists as a single copy in a representative collection of Tbr strains. SRA was found on one homologue of chromosome IV in the majority of Tbr isolates examined, but some Ugandan Tbr had SRA on both homologues. The mobility of SRA by genetic recombination readily explains the observed genetic variability of Tbr in East Africa. We conclude that new strains of the human pathogen Tbr are being generated continuously by recombination with the much larger pool of animal-infective trypanosomes. Such novel recombinants present a risk for future outbreaks of HAT.     

The Carboxyl Tail of Connexin32 Regulates Gap Junction Assembly in Human Prostate and Pancreatic Cancer Cells

Connexins, the constituent proteins of gap junctions, are transmembrane proteins. A connexin (Cx) traverses the membrane four times and has one intracellular and two extracellular loops with the amino and carboxyl termini facing the cytoplasm. The transmembrane and the extracellular loop domains are highly conserved among different Cxs, whereas the carboxyl termini, often called the cytoplasmic tails, are highly divergent. We have explored the role of the cytoplasmic tail of Cx32, a Cx expressed in polarized and differentiated cells, in regulating gap junction assembly. Our results demonstrate that compared with the full-length Cx32, the cytoplasmic tail-deleted Cx32 is assembled into small gap junctions in human pancreatic and prostatic cancer cells. Our results further document that the expression of the full-length Cx32 in cells, which express the tail-deleted Cx32, increases the size of gap junctions, whereas the expression of the tail-deleted Cx32 in cells, which express the full-length Cx32, has the opposite effect. Moreover, we show that the tail is required for the clustering of cell-cell channels and that in cells expressing the tail-deleted Cx32, the expression of cell surface-targeted cytoplasmic tail alone is sufficient to enhance the size of gap junctions. Our live-cell imaging data further demonstrate that gap junctions formed of the tail-deleted Cx32 are highly mobile compared with those formed of full-length Cx32. Our results suggest that the cytoplasmic tail of Cx32 is not required to initiate the assembly of gap junctions but for their subsequent growth and stability. Our findings suggest that the cytoplasmic tail of Cx32 may be involved in regulating the permeability of gap junctions by regulating their size.     

人类生殖相关新基因的定位和组织表达

基因定位对研究基因之间以及基因与疾病之间的相互关系具有重要意义。应用辐射杂种细胞系技术（RH）对我们克隆的人类新基因HBRP（Human BSP-Related Protein）进行了染色体定位,结果将该基因定位于19q13.2～13.3,同时应用生物信息学方法在人类基因组重叠片段数据库进行该基因的定位,结果相吻合。研究证明,RH技术具有快速、精确、简便等优点,是基因定位研究中一强有力的技术。同时通过RT-PCR方法研究了HBRP基因在人体各组织中的表达分布,结果显示该基因在睾丸、肠、肾、肝、脾、胃、胰腺组织有较高的表达,而在检测的脑、肺、骨骼肌、心肌组织中表达较弱。 Abstract:Gene localization is significant in elucidating the interaction between genes,gene and diseases.Using radiation hybrid (RH) technique,we cloned and localized a novel gene,designated human BSP-related protein (HBRP) on 19q13.2～13.3,in line with its localization in data bank of overlapping fragment of human genome through bioinformatics method.It is suggested RH is rapid,precise,simple and powerful in gene localization.In addition,we detected the expression and distribution of HBRP in human tissues by RT-PCR.The results showed HBRP was highly expressed in intestine,kidney,liver,spleen,stomach and pancreas,whereas lowly in brain,lung,muscle and heart.     

Cell Type- and Tissue Context-dependent Nuclear Distribution of Human Ago2

Argonaute-2 protein (Ago2), a major component of RNA-induced silencing complex (RISC), has been viewed as a cytoplasmic protein. In this study, we demonstrated by immunofluorescence confocal microscopy that Ago2 is distributed mainly as a nuclear protein in primary human foreskin keratinocytes in monolayer cultures and their derived organotypic (raft) cultures, although it exhibits only a minimal level of nuclear distribution in continuous cell lines such as HeLa and HaCaT cells. Oncogenic human papillomavirus type 16 (HPV16) or type 18 (HPV18) infection of the keratinocytes does not affect the nuclear Ago2 distribution. Examination of human tissues reveals that Ago2 exhibits primarily as a nuclear protein in skin, normal cervix, and cervical cancer tissues, but not in larynx. Together, our data provide the first convincing evidence that the subcellular distribution of Ago2 occurs in a cell type- and tissue context-dependent manner and may correlate with its various functions in regulation of gene expression.     

人分化相关基因Ndr2的克隆与组织表达谱研究

人Ndr1基因参与细胞终末分化 ,并且对肿瘤细胞增殖和肿瘤转移具有抑制作用 .从人 2 2周孕龄胎肝cDNA文库中获得与人Ndr1基因同源的一段表达性序列标签 ,继而从成人脑cDNA文库分离出其全长cDNA(2 12 1bp) ,并将该基因命名为Ndr2 .其染色体定位为 14q11 1- 11 2 ,开放阅读框编码 371个氨基酸 ,且与NDR1蛋白一样 ,含有一个典型的α β水解酶折叠类结构域 (α βhydrolasefold) .Northern杂交和点杂交分析显示 ,该基因与Ndr1一样 ,在脑中高表达 ,在胚胎组织的表达较低 ,在 8种人肿瘤细胞中的表达极低 .然而 ,Ndr2基因的组织表达谱与Ndr1又有鲜明的差异 :其在成人骨骼肌和脑等神经组织中表达最高 ,在唾液腺、肝、肾、心肌和气管中的表达次之 .结果提示 ,NDR2具有与NDR1相似或相关的重要功能 .     

A Mitosis-specific Phosphorylation of the Gap Junction Protein Connexin43 in Human Vascular Cells: Biochemical Characterization and Localization

Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43_m. Cx43_m was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all ³²P_i from Cx43_m by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell–cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.     

Regulation of Connexin43 Gap Junction Protein Triggers Vascular Recovery and Healing in Human Ocular Persistent Epithelial Defect Wounds

Transiently blocking the expression of the gap junction protein connexin43 using antisense oligodeoxynucleotides or blocking hemichannels with connexin mimetic peptides has been shown to significantly improve outcomes in a range of acute wound models. Less is known about their likely effects in nonhealing wounds. In the eye, prolonged inflammation and lack of epithelial recovery in nonhealing corneal epithelial wounds may lead to corneal opacity, blindness or enucleation. We report here the first human applications of antisense oligodeoxynucleotides that transiently block translation of connexin43 in a prospective study of five eyes with severe ocular surface burns (persistent epithelial defects), which were unresponsive to established therapy for 7 days to 8 weeks prior to treatment. Connexin43-specific antisense oligodeoxynucleotide was delivered in cold, thermoreversible Poloxamer407 gel under either an amniotic membrane graft or a bandage contact lens. The connexin43-specific antisense application reduced inflammation within 1–2 days, and in all five eyes complete and stable corneal reepithelialization was obtained. Recovery of the vascular bed and limbal reperfusion appeared to precede corneal epithelial recovery. We conclude that connexin modulation provides a number of benefits for nonhealing ocular burn wounds, one of which is to promote vascular recovery.     

Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues

DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the “gold standard” of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.     

Bactericidal Activity and Synergy Studies of Peptide AP-CECT7121 Against Multi-resistant Bacteria Isolated from Human and Animal Soft Tissue Infections

AP-CECT7121 is an antimicrobial peptide, produced by Enterococcus faecalis CECT7121, with bactericidal activity against Gram-positive bacteria. The aim of this study was to evaluate the bactericidal activity of AP-CECT7121, alone and with gentamicin, against multi-resistant bacteria isolated from human and animals with soft tissue infections. During the period 2014–2015, bacterial strains producing human and animal soft tissue infections were studied. Samples from patients attended at a general hospital and cattle from four dairies in the Province of Buenos Aires (Argentina) were included. Twenty-two methicillin-resistant Staphylococcus aureus (11, human blood samples; 11, cow milk) and five vancomycin-resistant Ent. faecium strains isolated from four mastitic dairy cows were tested. AP-CECT7121 (12 mg/L) potency was assessed by time-kill curves alone or with sub-inhibitory concentrations of gentamicin. All staphylococcal strains were susceptible to gentamicin; enterococci did not show high-level gentamicin resistance. Colony counts were carried out at 0, 2, 4, 8, and 24 h of incubation. AP-CECT7121 showed bactericidal activity against all the enterococcal strains. In addition, AP-CECT7121 had a bactericidal effect on most staphylococci (16/22). Early AP-CECT7121/gentamicin synergy (4–8 h) for all staphylococci was detected. At 24 h, synergy (19/22) and indifference (3/22) were observed. Synergy with gentamicin was detected for staphylococci. AP-CECT7121 constitutes an attractive candidate for its use as a natural therapeutic tool for the treatment of infections produced by multi-resistant Staph. aureus and vancomycin-resistant Ent. faecium isolated from humans and animals.     

Differential Distribution of Elastic Tissue in Human Natural Skin and Tissue-Engineered Skin

Tissue-engineered skins (TES), manufactured by epidermal and dermal equivalents, are now being used in biological, pharmacotoxicological and clinical applications. It is thus interesting to know to what extent artificial organs are similar to natural counterparts. Elastic fibres are important constituents of the extracellular matrix of natural skin (NS). The aim of our study was to investigate the possible occurrence and distribution of elastic tissue in a model of human TES using different histochemical techniques, including classical Orcein and Fuchsin-Resorcin methods and immunohistochemistry, at both light and electron microscopical levels. Immunoperoxidase and high resolution immunogold methods were used. In NS, classical staining techniques and elastin-immunohistochemistry revealed a well-organized network of elastic fibres. High resolution immunocytochemistry revealed an intense labelling in the amorphous component of elastic fibres. Fibres of different diameters were immunostained. In TES, no stained elastic fibres were observed using classical staining techniques, and the interpretation of immunoperoxidase observations was not clear-cut. In contrast, immunogold staining at the electron microscopical level provided specific labelling of elastin-like immunoreactive material in the dermal equivalent. However, ultrastructural immunocytochemistry revealed that elastic tissue organization in TES was poor compared to that in NS. This study demonstrates that elastic fibres are a component of the extracellular matrix in this model of TES and suggests that fibroblasts of the dermal equivalent are engaged in matrix secretion. Nevertheless, the level of extracellular matrix organization in TES is low compared to NS. Moreover, this study also suggests that different models of bilayered TES may differ with respect to extracellular matrix organization. These aspects should be considered when TES is used in biological and pharmacotoxicological studies. A better understanding of the factors influencing extracellular matrix formation in TES is necessary to achieve further development of skin generation in vitro.     

Chemical Elements of Human Bone Tissue Investigated by Nuclear Analytical and Related Methods

The mass fractions (in milligrams per kilogram given on a dry mass basis) of 69 chemical elements in the intact rib bone of 84 apparently healthy 15- to 58-year-old citizens (38 females and 46 males) of a nonindustrial region were investigated using five instrumental analytical methods: neutron activation analysis with high-resolution spectrometry of short- and of long-lived radionuclides, particle-induced gamma-ray emission spectrometry, inductively coupled plasma atomic emission, and mass spectrometry. The mass fractions of chemical elements were measured for rib bone of both males and females, taken separately and together. The present results indicate that rib bone can be used as exposure monitors in occupational medicine and environmental health studies to assess, or indicate, the body burden of Al, B, Ba, Be, Ca, Cr, F, Li, Mg, Na, Ni, P, Pb, rare-earth elements (REEs), Sr, and Zn. Our data show an exponential increase with the age of the content of REEs in the rib of people living in an ecologically safe region.     

胚胎骨Ⅰ型胶原的提取与鉴定

应用酸性、中性交互提取法从人胚骨中提取Ⅰ型胶原,经SDS-PAGE电泳,氨基酸分析和免疫学方法鉴定．结果表明所提胶原电泳区带与Ⅰ型标准品相同,环状沉淀反应阳性,氨基酸分析甘氨酸占1000个氨基酸总残基的1/3,羟脯氨酸与脯氨酸之比为0.65,符合Ⅰ型胶原特征,并显示有较高的纯度,可用于胶原制品的制作．