ZnT-8, A Pancreatic Beta-Cell-Specific Zinc Transporter

The zinc content in the pancreatic beta cell is among the highest of the body. Zinc appears to be an important metal for insulin-secreting cells as insulin is stored inside secretory vesicles as a solid hexamer bound with two Zn²⁺ ions per hexamer. Zinc is also an important component of insulin secretion mechanisms and is likely to modulate the function of neighbouring cells via paracrine/autocrine interactions. Therefore beta cells undoubtedly need very efficient and specialized transporters to accumulate sufficient amounts of zinc in secretion vesicles. We report here the discovery and the characteristics of a new zinc transporter, ZnT-8, belonging to the CDF (Cation Diffusion Facilitator) family and expressed only in pancreatic beta cells. This transporter, localized in secretion vesicles membrane, facilitates the accumulation of zinc from the cytoplasm into intracellular insulin-containing vesicles and is a major component for providing zinc to insulin maturation and/or storage processes in insulin-secreting pancreatic beta cells. We discovered mammalian orthologs (rat, mouse, chimpanzee, and dog) and found these ZnT-8 proteins very similar (98% conserved amino acids) to human ZnT-8, indicating a high conservation during evolution.     

Pancreatic fate of a (125)I-labelled mouse monoclonal antibody directed against pancreatic B-cell surface ganglioside(s) in control and diabetic rats

The possible use of a mouse monoclonal antibody directed against rat pancreatic B-cell surface ganglioside(s) and labelled with radioactive iodine for selective imaging of the endocrine pancreas by a non-invasive procedure was investigated by following its pancreatic fate in experiments conducted either in vitro by incubation of rat isolated pancreatic islets, acinar tissue and pancreatic pieces or in vivo after intravenous injection of the (125)I-labelled antibodies ([(125)I]gamma-G). Although the binding of [(125)I]gamma-G per microg protein was about one order of magnitude higher in isolated islets than in acinar tissue, no significant difference was detected when comparing pancreatic pieces or isolated islets from control animals and rats rendered diabetic by one or two prior administrations of streptozotocin (STZ rats). Likewise, except in one set of experiments, no significant difference was found between control animals and STZ rats, when measuring the radioactive content of the pancreatic gland, relative to that of plasma, 1-4 days after the intravenous injection of [(125)I]gamma-G. These findings indicate that under the present experimental conditions, the mouse monoclonal antibody labelled with radioactive iodine does not appear to be a promising tool for selective imaging of the endocrine pancreas, e.g. by single photon emission computerized tomography.     

Neural Cell Adhesion Molecule (N-CAM) Is Required for Cell Type Segregation and Normal Ultrastructure in Pancreatic Islets

Classical cell dissociation/reaggregation experiments with embryonic tissue and cultured cells have established that cellular cohesiveness, mediated by cell adhesion molecules, is important in determining the organization of cells within tissue and organs. We have employed N-CAM-deficient mice to determine whether N-CAM plays a functional role in the proper segregation of cells during the development of islets of Langerhans. In N-CAM-deficient mice the normal localization of glucagon-producing α cells in the periphery of pancreatic islets is lost, resulting in a more randomized cell distribution. In contrast to the expected reduction of cell–cell adhesion in N-CAM-deficient mice, a significant increase in the clustering of cadherins, F-actin, and cell–cell junctions is observed suggesting enhanced cadherin-mediated adhesion in the absence of proper N-CAM function. These data together with the polarized distribution of islet cell nuclei and Na⁺/K⁺-ATPase indicate that islet cell polarity is also affected. Finally, degranulation of β cells suggests that N-CAM is required for normal turnover of insulin-containing secretory granules. Taken together, our results confirm in vivo the hypothesis that a cell adhesion molecule, in this case N-CAM, is required for cell type segregation during organogenesis. Possible mechanisms underlying this phenomenon may include changes in cadherin-mediated adhesion and cell polarity.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

Decreased Expression of Cilia Genes in Pancreatic Islets as a Risk Factor for Type 2 Diabetes in Mice and Humans

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Comprehensive Proteomics Analysis of Stressed Human Islets Identifies GDF15 as a Target for Type 1 Diabetes Intervention

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Fluorescence Microscopy of Fresh Tissue as a Rapid Technique for Assessing Early Injury to Mesophyll

A technique was developed for sectioning fresh red spruce foliage (Picea rubens Sarg.) for use in fluorescence microscopy. This allowed rapid examination of mesophyll in 3-5 mm needle sections. Healthy, ozone treated and cold stressed needles were examined to assess the utility of this technique for early detection of damage. Healthy mesophyll cells fluoresced bright red, while injured cells fluoresced yellow-green in ozone treated needles, and yellow-orange in frozen needles. Shifts in fluorescence wavelengths may be useful for early detection of injury to mesophyll before it is evident by standard light or electron microscopy.     

用绿色荧光蛋白（GFP）作为报告分子筛选有效的siRNA

 建立一种利用绿色荧光蛋白（GFP）作为报告分子筛选能有效抑制目的基因表达的siRNA的方法.以巨噬细胞移动抑制因子（MIF）基因为研究对象,筛选能有效沉默MIF表达的质粒载体介导的siRNA.构建拥有同一Kozak共有翻译启始序列、翻译启始密码子ATG的MIF-GFP融合表达载体pEGFP-MIF.分别将3个靶向MIF的siRNA表达质粒与pEGFP-MIF共转化HEK293细胞,在荧光显微镜下观察HEK293细胞中GFP的表达,并用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.同时,将MIF siRNA表达质粒分别与MIF表达载体共转化HEK293细胞,用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.定量PCR结果显示,GFP表达低的细胞中,MIF mRNA的表达也明显降低;利用pEGFP-MIF和MIF表达载体筛选到的有效MIF siRNA的结果一致.因此,建立了目的基因与GFP融合表达,以GFP作为报告分子来筛选抑制目的基因表达siRNA的方法,并为进行多个基因的有效siRNA的筛选提供解决方案.     

Flow Cytometric Applications of Sulforhodamine 101 as a Fluorescent Stain for Total Cellular Protein

In this paper, the use of Sulforhodamine 101 (SR 101; C.I. 14318) as a fluorescent stain for flow cytometric determinations of total cellular protein (TCP) is described. Flow cytometric quantification of TCP fluorescence can provide a valuable analytical parameter for assessing both changes occurring in overall cellular protein content, such as in response to blast transformation, and heterogeneity in cellular size within a specimen, such as a tumor. Very little information is available in the literature pertaining to the use of SR 101 as a protein stain. Like fluorescein isothiocyanate (FITC), SR 101 can be excited at 488 nm; however, it binds ionically and has an emission maximum at 600 nm, which is advantageous in certain staining and filter combinations. In this report, the utility of SR 101 staining is demonstrated using pokeweed mitogen-stimu-lated lymphocytes and cycloheximide- and di-methylsufloxide-treated cells. Single, two- and three-color flow cytometric applications are possible, using SR 101 in combination with 4',6-diamidino-2-phenylindole (DAPI) and/or FITC.     

苜蓿种质间染色体多态性的荧光原位杂交检测

利用染色体荧光原位杂交技术(FISH),将3种重复序列5S rDNA、45S rDNA和C0t-1 DNA用不同荧光物进行标记,对我国10个不同地理来源的苜蓿种质(Medicago sativa L.;2n=4X=32)进行了染色体多态性检测。结果表明,利用以上重复序列可以较好地将苜蓿32条染色体区分为16对特征不同的染色体,10份不同种质材料FISH带纹特征表现高度相似,比较不同种质间同源染色体重复序列杂交特征,揭示出种质群体内和群体间多态性染色体的存在,其中不同的同源染色体多态性表现不尽一致,1号染色体(随体染色体)多态性最高,10份材料中检出7个多态型,3、4、15号染色体保守性较强,在不同种质间表现为单态,其他染色体多态性居中。对在地理分布上自西向东的10个材料进行染色体多态性比较,结果显示分布于西藏、新疆以及分布在辽宁的材料部分染色体多态型显著区别于其他材料。     

Synthesis,biological assays and QSAR studies of N-(9-benzyl-2-phenyl-8-azapurin-6-yl)-amides as ligands for A1 adenosine receptors

2-Phenyl-9-benzyl-8-azapurines, bearing at the 6 position an amido group interposed between the 8-azapurine moiety and an alkyl or a substituted phenyl group, have been synthesised and assayed as ligands for adenosine receptors. All the compounds show high affinity for the A₁ adenosine receptor, and many of them also show a good selectivity for A₁ with respect to A_2A and A₃ adenosine receptors. Based on the quite rich library containing such compounds and relevant biological data, QSAR models, able to rationalise the results and to give a quantitative estimate of the observed trends were also developed. The obtained models can assist in the design of new compounds selectively active on A₁ adenosine receptor.     

DiOC6(3): a Useful Dye for Staining the Endoplasmic Reticulum

The present review discusses the fluorescent organelle probe, DiOC₆(3), with reference to its structure, chemistry, availability, spectral properties, labeling procedures, vital staining characteristics, and major applications in cellular and molecular biology. The specificity of dye for endoplasmic reticulum is summarized. We examine the simplicity and advantages of the fluorescent dye system for evaluating structure and function of endoplasmic reticulum. Other significant uses of the dye are also discussed.     

Uptake and intracellular distribution of labile and total Zn(II) in C6 rat glioma cells investigated with fluorescent probes and atomic absorption

The uptake, intracellular distribution and cytotoxicity of high doses of extracellular zinc was investigated in C6 rat glioma cells. Net zinc uptake occurred only above certain thresholds in time and concentration, below them no alterations of the intracellular zinc level were observed. These results were obtained by measurements with the fluorescent dye Zinquin and by atomic absorption spectrometry, yielding similar results with both methods. Sequestration of zinc in intracellular vesicles was observed by fluorescence microscopy. A protective effect of vesicular sequestration is indicated, because increased levels of intracellular zinc located in vesicles did not necessarily lead to an increase in cytotoxicity. We were able to show that in C6 cells, in contrast to other cell lines, zinc that is released from proteins by the NO donor SNOC is also sequestered in vesicular structures. These zinc-carrying vesicles showed to be constitutive and are assumed to have a function in the maintainance of the cytosolic content of Zn²⁺ ions.     

6-Aminohexanoic acid as a chemical chaperone for apolipoprotein(a)

Apolipoprotein (a) (apo(a)) is a component of the atherogenic lipoprotein, Lp(a). The efficiency with which apo(a) escapes the endoplasmic reticulum (ER) and is secreted by the liver is a major determinant of plasma Lp(a) levels. Apo(a) contains a series of domains homologous to plasminogen kringle (K) 4, each of which possesses a potential lysine-binding site. By using primary mouse hepatocytes expressing a 17K4 human apo(a) protein, we found that high concentrations (25-200 mM) of the lysine analog, 6-aminohexanoic acid (6AHA), increased apo(a) secretion 8-14-fold. This was accompanied by a decrease in apo(a) presecretory degradation. 6AHA inhibited accumulation of apo(a) in the ER induced by the proteasome inhibitor, lactacystin. Thus, 6AHA appeared to inhibit degradation by increasing apo(a) export from the ER. Significantly, 6AHA overcame the block in apo(a) secretion induced by the ER glucosidase inhibitor, castanospermine. 6AHA may therefore circumvent the requirement for calnexin and calreticulin interaction in apo(a) secretion. Sucrose gradients and a gel-based folding assay were unable to detect any influence of 6AHA on apo(a) folding. However, non-covalent or small, disulfide-dependent changes in apo(a) conformation would not be detected in these assays. Proline also increased the efficiency of apo(a) secretion. We propose that 6AHA and proline can act as chemical chaperones for apo(a).     

Specificity of mutagenesis by 4-aminobiphenyl. A possible role for N-(deoxyadenosin-8-yl)-4-aminobiphenyl as a premutational lesion

Mutagenesis by N-acetoxy-N-trifluoroacetyl-4-aminobiphenyl, a reactive form of the human bladder carcinogen 4-aminobiphenyl (ABP), was studied in Escherichia coli virus M13mp10. N-acetoxy-N-trifluoroacetyl-4-ABP-treated DNA containing 140 lesions/duplex genome, when introduced into excision repair-competent cells induced for SOS mutagenic processing, resulted in a 40-fold increase in mutation frequency over background in the lacZ alpha gene fragment. DNA sequence changes were determined for 20 independent mutants. G-C base pairs were the major targets for base pair substitution mutations, although significant mutagenic activity was also observed at certain A-T base pairs. Deletion and frameshift mutations also were found in this sample. The salient feature of this partial "mutational spectrum" was a hotspot that occurred at position 6357 (amino acid 30 of the beta-galactosidase fragment encoded by M13mp10); this A-T to T-A transversion appeared in 6 of the 20 mutants. The property of ABP to mutate A-T base pairs was consistent with the result that N-hydroxy-ABP reverted Salmonella typhimurium strain TA104, which is presumed to revert primarily due to mutations at these sites. The ability of the major carcinogen-DNA adduct formed by ABP in vivo and in vitro, N-(deoxyguanosin-8-yl)-4-aminobiphenyl, to cause base pair substitution mutations was also investigated. This adduct was positioned specifically in the minus strand at position 6270 in duplex M13mp10 DNA. In the presence of the mutagenesis-enhancing plasmid pGW16 and UV induction of SOS mutagenic processing, it was shown that fewer than 0.02% of the adducts resulted in transition or transversion mutations following transfection of DNA into excision-repair competent cells. Similar results were obtained in uvrA and uvrC backgrounds. Although the major adduct did not cause base substitution mutations under these experimental conditions, the contribution of this lesion to the entire spectrum of mutations in the lacZ alpha fragment seems likely.     

N-(1,3-Diaryl-3-oxopropyl)amides as a new template for xanthine oxidase inhibitors

A series of forty two N-(1,3-diaryl-3-oxopropyl)amides were synthesized via an efficient, modified Dakin-West reaction and were evaluated for in vitro xanthine oxidase inhibitory activity for the first time. Structure-activity relationship analyses have been presented. Selected active xanthine oxidase inhibitors (3r, 3s, and 3zh) were assessed in vivo to study their anti-hyperuricemic effect in potassium oxonate induced hyperuricemic mice model. Compound 3s emerged as the most potent xanthine oxidase inhibitor (IC(50)=2.45 μM) as well as the most potent anti-hyperuricemic agent. The basis of significant inhibition of xanthine oxidase by 3s was rationalized by its molecular docking into catalytic site of xanthine oxidase.     

Camwood (Baphia nitida) Extract as a Histological Stain for Interglobular Dentine, Striated Muscle, and Counterstaining

The shavings of the dried heartwood of the tree Baphia nitida are ground to a fine powder, and 6 gm of the powder are extracted in 100 ml absolute ethanol at 27-30 for 6-24 hr. The extract is filtered with Whatman No. 1 paper and stored in a screw-capped bottle. For staining the interglobular dentine of nondecalcified sections of formlin-fixed teeth, sawed cross sections 20-30 μ thick were dehydrated in ethanol and stained in the undiluted extract for 6-12 hr at room temperature. The interglobular dentine was stained a bright golden brown on a pale brown background. For staining striated muscle, the extract was diluted 1:1 with distilled water and filtered. After mordanting formalin-fixed paraffin sections with 0.25% KMnO₄ for 5 min, and bleaching with 5% oxalic acid for 10 min, they were washed in water and stained for 2-24 hr at room temperature. The striations were stained light to deep golden brown. For use as a counterstain, a 1:6 dilution of the original extract was required. When applied after haematoxylin for 15-30 min, it stained tissue components in varying shades of golden brown with distribution comparable to that produced by 1% eosin.     

Creatine transporter (CrT; Slc6a8) knockout mice as a model of human CrT deficiency

Mutations in the creatine (Cr) transporter (CrT; Slc6a8) gene lead to absence of brain Cr and intellectual disabilities, loss of speech, and behavioral abnormalities. To date, no mouse model of CrT deficiency exists in which to understand and develop treatments for this condition. The purpose of this study was to generate a mouse model of human CrT deficiency. We created mice with exons 2–4 of Slc6a8 flanked by loxP sites and crossed these to Cre:CMV mice to create a line of ubiquitous CrT knockout expressing mice. Mice were tested for learning and memory deficits and assayed for Cr and neurotransmitter levels. Male CrT^−/y (affected) mice lack Cr in the brain and muscle with significant reductions of Cr in other tissues including heart and testes. CrT^−/y mice showed increased path length during acquisition and reversal learning in the Morris water maze. During probe trials, CrT^−/y mice showed increased average distance from the platform site. CrT^−/y mice showed reduced novel object recognition and conditioned fear memory compared to CrT^+/y. CrT^−/y mice had increased serotonin and 5-hydroxyindole acetic acid in the hippocampus and prefrontal cortex. Ubiquitous CrT knockout mice have learning and memory deficits resembling human CrT deficiency and this model should be useful in understanding this disorder.     

Novel enzyme immunoassay for thyrotropin-releasing hormone using N-(4-diazophenyl)maleimide as a coupling agent

A novel enzyme immunoassay (EIA) for thyrotropin-releasing hormone (TRH) was developed which used N-(4-diazophenyl)maleimide (DPM) as a new heterobifunctional agent capable of cross-linking TRH to mercaptosuccinyl bovine serum albumin and to beta-D-galactosidase. The resulting conjugates act as the immunogen producing anti-TRH serum in rabbits and the enzyme marker of TRH in the EIA, respectively. This EIA with a double-antibody technique was sensitive and reproducible in measuring TRH at concentrations as low as 50 pg per tube, and monospecific to the hormone showing no cross-reactivity with the hormone analogue L-pGlu-L-His-L-Pro and TRH constituents. Using this assay, the distribution of immunoreactive TRH in the brain was determined easily in rats. The use of DPM should provide a valuable new method for developing EIA hitherto possible for other peptide hormones containing neither a free carboxy nor a free amino group, using imidazole, phenolic, and indole group(s) of the amino acid as a reaction site.