Quantitative correlative proton and electron microprobe analysis of biological specimens

Summary To investigate the possibility of quantitative correlative proton microprobe (PMP) and electron microprobe (EMP) analysis of biological soft tissue, model specimens were analyzed by both techniques. The specimens consisted of freeze-dried sections of gelatin containing known concentrations of nickel chloride. Both for PMP and for EMP, the signal was expressed as the ratio of the characteristic intensity and the continuum intensity in a peak-free region of the spectrum. With both techniques, calibration curves (signal versus known concentration) obtained, showed a deviation from linearity at high nickel concentrations. However, a linear relation (correlation coefficient 0.996) was obtained in a plot of EMP signal versus PMP signal. This indicates that quantitative correlative PMP and EMP analysis can be carried out by using the same standard for both analytical techniques.     

The use of PIXE in experimental studies of the physiology of human skin epidermis

In order to understand the normal and pathological physiologies of the epidermal cells, the simultaneous determination of several elements in the different cellular strata is of crucial importance. In recent years the electron microprobe (EMP) has become an established technique in this field. Its high spatial resolution, in principle, allows measurements of various cell organelles. However, the limited (intrinsic) sensitivity of the EMP represents a serious drawback to the technique. The introduction of the proton microprobe (PMP) has significantly improved the sensitivity, although the ultimate spatial resolution of the PMP is much less than that of the EMP. When studying the elemental profiles in skin epidermis, it is possible to use skin sections with a thickness of the order of 10 μm, then the spatial resolution of the PMP is equal to or better than that of the EMP since the electrons are scattered to a significant degree in the sample. The characteristics of the two methods have been compared by analysis of parallel duplicate freeze-dried sections of normal human skin. The distributions of the elements P, S, Cl, and K, obtained with the two techniques, were in good agreement. In addition, the PMP provided distributions of the important elements Ca, Fe, and Zn. In a recently started study, the useful features of the PMP will be used for studying how efficient a barrier the skin is to nickel and chromate ions. A preliminary experiment has been performed by exposing cadaverous skin, not older than 24-h postmortem, to solutions of the two ions. After an 18-h exposure, samples were prepared by shock-freezing and sectioning. The first results from PMP analysis of these samples demonstrate the presence of a nickel and chromium gradient in the outer strata in the epidermis (mainly stratum corneum). A third experiment deals with the physiology of psoriatic skin. Calcium is an important element in the differentiation. Hence, the higher sensitivity of the PMP has been used in analysis of sections from psoriatic skin epidermis. Preliminary results are presented.     

Practical notes on the production of thin aminoplastic standards for quantitative X-ray microanalysis

Detailed, step-by-step, practical instructions are provided for the production of homogeneous aminoplastic standards containing known concentrations of light and heavy elements. These can be used for the fully quantitative microprobe analyses of both plastic-embedded and cryosectioned biological soft-tissue specimens.     

The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion

The growth arrest-specific-3 (GAS3)/PMP22 proteins are members of the four-transmembrane (tetraspan) superfamily. Although the function of these proteins is poorly understood, GAS3/PMP22 proteins have been implicated in the control of growth and progression of certain cancers. Epithelial membrane protein-2 (EMP2), a GAS3/PMP22 family member, was recently identified as a putative tumor suppressor gene. Here, we addressed the normal function of EMP2 by testing the prediction that it influences integrin-related cell functions. We observed that EMP2 associates with the beta(1) integrin subunit. Co-immunoprecipitation and immunodepletion experiments indicated that approximately 60% of beta(1) integrins and EMP2 can be isolated in common protein complexes. Whereas this association between EMP2 and beta(1) integrin may be direct or indirect, it has features of integrin heterodimer selectivity. Thus, by laser confocal microscopy, EMP2 colocalized with alpha(6)beta(1) but not alpha(5)beta(1) integrin. Increased expression of EMP2 also influenced the integrin heterodimer repertoire present on the plasma membrane. EMP2 specifically increased the surface expression of the alpha(6)beta(1) integrin while decreasing that of the alpha(5)beta(1) protein. Reciprocally, reduction in EMP2 expression using a specific ribozyme decreased surface expression of alpha(6)beta(1) integrin. Accordingly, these EMP2-mediated changes resulted in a dramatic alteration in cellular adhesion to extracellular matrix proteins. This study demonstrates for the first time the interaction of a GAS3/PMP22 family member with an integrin protein and suggests that such interactions and their functional consequences are a physiologic role of GAS3/PMP22 proteins.     

Plasminogen activator inhibitor-1 fused with erythropoietin (EPO) mimetic peptide (EMP) enhances the EPO activity of EMP.

Erythropoietin (EPO) mimetic peptide (EMP) encoding sequence was inserted into the gene of plasminogen activator inhibitor-1 (PAI-1) between Ala348 and Pro349 (P2'-P3'), generating a novel gene, PAI-1/EMP (PMP). This was cloned into pET32a expression vector, fused with TrxA peptide in the vector, and a 63-kDa protein was expressed in inclusion bodies with an expression level >50%. The TrxA/PMP protein was purified by Ni-NTA-agarose metal-ligand affinity chromatography to a purity >90%, showing a single, silver-stained band on SDS-PAGE. Using a reticulocyte counting assay, the EPO activity of PMP was determined to be 5,000 IU/mg, 2,500-fold that of EMP.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Identification and Characterization of a cDNA and the Structural Gene Encoding the Mouse Epithelial Membrane Protein-1

The PMP22/EMP/MP20 gene family includes four closely related proteins, peripheral myelin protein-22 (PMP22), epithelial membrane protein-1 (EMP-1), epithelial membrane protein-2 (EMP-2), and epithelial membrane protein-3 (EMP-3), which share amino acid identities ranging from 33 to 43%. In addition, the lens-specific membrane protein MP20 represents a more distant relative. Functionally, this family of proteins is likely to play important roles in the control of cell proliferation, cell differentiation, and cell death. In particular, mutations affecting thePMP22gene are responsible for various hereditary peripheral neuropathies in humans and mice. We report the isolation and characterization of a mouse EMP-1 cDNA and the correspondingemp-1gene. Mouse EMP-1 displays 93% amino acid identity to rat EMP-1 and 39% identity to mouse PMP22. The cDNA-predicted EMP-1 protein contains four putative membrane-associated domains and can beN-linked glycosylatedin vitro.EMP-1 is encoded by a single-copy gene with the positions of introns exactly conserved betweenemp-1andPMP22,corroborating the hypothesis that both genes belong to the same family. Computer-predicted structural domains of EMP-1 are partially mirrored by the exon/intron structure ofemp-1.Most interestingly, exon 4, which covers the potential second transmembrane domain, a small intracellular loop, and half of the third transmembrane domain, encodes the most highly conserved regions between the EMP-1 and PMP22 proteins and is also remarkably conserved in the MP20 gene, indicating some shared functional significance for this module in the PMP22/EMP/MP20 family.     

Improved detection and quantification of the (immuno) peroxidase product using reflection contrast microscopy

Reflection contrast microscopy (RCM) is a sensitive tool to detect minor amounts of precipitated diaminobenzidine (DABox) in immunoperoxidase stained specimens. One of the main issues in immunocytochemistry is the ongoing need for more sensitive and quantitative techniques. Therefore we applied RCM, using a new simple model system, to methods previously described for increased sensitivity in immunocytochemistry with bright field microscopy. Addition of imidazole was found the most sensitive method and addition of Nickel and Cobalt ions gave the most enhanced colour intensity. Variation of the enzyme reaction parameters yielded a continuous increase in reflection with time. This was then discussed in view of other model studies of peroxidase kinetics. A quantitative relationship between the amount of peroxidase and the reflection of DABox was observed, indicating that quantitative immunoperoxidase studies with RCM are feasible. In situ hybridization (ISH) was then used as a useful biological model for RCM to test the optimal conditions for DAB staining found in the model system (high concentrations of DAB and peroxidase and 2 h incubation time). There was no background staining in the model system, also after prolonged incubation time. The ISH experiments showed that the contrast (ratio) between specific signal and chromosome background did not increase in time, whereas only the use of high avPO concentrations yielded the highest contrast.     

Improved detection and quantification of the (immuno) peroxidase product using reflection contrast microscopy

Summary Reflection contrast microscopy (RCM) is a sensitive tool to detect minor amounts of precipitated diaminobenzidine (DABox) in immunoperoxidase stained specimens. One of the main issues in immunocytochemistry is the ongoing need for more sensitive and quantitative techniques. Therefore we applied RCM, using a new simple model system, to methods previously described for increased sensitivity in immunocytochemistry with bright field microscopy. Addition of imidazole was found the most sensitive method and addition of Nickel and Cobalt ions gave the most enhanced colour intensity. Variation of the enzyme reaction parameters yielded a continuous increase in reflection with time. This was then discussed in view of other model studies of peroxidase kinetics. A quantitative relationship between the amount of peroxidase and the reflection of DABox was observed, indicating that quantitative immunoperoxidase studies with RCM are feasible.In situ hybridization (ISH) was then used as a useful biological model for RCM to test the optimal conditions for DAB staining found in the model system (high concentrations of DAB and peroxidase and 2 h incubation time). There was no background staining in the model system, also after prolonged incubation time. The ISH experiments showed that the contrast (ratio) between specific signal and chromosome background did not increase in time, whereas only the use of high avPO concentrations yielded the highest contrast.     

Correlation of behavior changes and BOLD signal in Alzheimer-like rat model

Memory impairment is usually the early and most promi-nent clinical manifestation of Alzheimer disease (AD), aprogressive neurodegenerative illness characterized bygradual deposition of neuritic plaques and neurofibrillarytangles in the brain of the patie…     

Mouse liver PMP70 and ALDP: homomeric interactions prevail in vivo

ALDP, ALDPR, PMP70 and PMP70R are half ATP-binding cassette (ABC) transporters of the mammalian peroxisomal membrane. By analogy with other members of this family, it is assumed that peroxisomal ABC transporters must dimerize to become functional units. However, not much is known regarding the type of dimers (i.e., homodimers and/or heterodimers) that are formed in vivo under normal expression conditions. In this work, we have characterized the quaternary structure of mouse liver PMP70 and ALDP. The PMP70 protein complex was purified to apparent homogeneity using a two-step purification protocol. The ALDP-containing protein complex was characterized by preparative immunoprecipitation experiments. In both cases, no evidence for the existence of heteromeric interactions or for the presence of accessory proteins in these ABC transporter protein complexes could be obtained. Our data indicate that the majority (if not all) of mouse liver PMP70 and ALDP are homomeric proteins.     

Developmental analysis of a putative ATP/ADP carrier protein localized on glyoxysomal membranes during the peroxisome transition in pumpkin cotyledons

In order to clarify the peroxisomal membrane proteins (PMPs), we characterized one of the major PMPs, PMP38. The deduced amino acid sequence for its cDNA in Arabidopsis thaliana contained polypeptides with 331 amino acids and had high similarity with those of Homo sapiens PMP34 and Candida boidinii PMP47 known as homologues of mitochondrial ATP/ADP carrier protein. We expected PMP38 to be localized on peroxisomal membranes, because it had the membrane peroxisomal targeting signal. Cell fractionation and immunocytochemical analysis using pumpkin cotyledons revealed that PMP38 is localized on peroxisomal membranes as an integral membrane protein. The amount of PMP38 in pumpkin cotyledons increased and reached the maximum protein level after 6 d in the dark but decreased thereafter. Illumination of the seedlings caused a significant decrease in the amount of the protein. These results clearly showed that the membrane protein PMP38 in glyoxysomes changes dramatically during the transformation of glyoxysomes to leaf peroxisomes, as do the other glyoxysomal enzymes, especially enzymes of the fatty acid beta-oxidation cycle, that are localized in the matrix of glyoxysomes.     

Dual fluorophore-nitroxide probes for analysis of vitamin C in biological liquids

A new method for quantitative analysis of vitamin C in biological and chemical liquids was proposed. The method is based on the use of dual molecule consisting of a fluorescent chromophore and a nitroxide radical. In the dual molecule, the nitroxide acts as a quencher of the fluorescence of the chromophore fragment. Reduction of the nitroxide fragment by ascorbic acid results in decay of ESR signal and enhancement of the fluorescence. By performing the series of pseudo-first-order reactions between the dual molecule and ascorbic acid and consequent plotting rate constants versus ascorbic acid concentrations the calibration curves for the vitamin C analysis were obtained. Variations of chemical structure of fluorophore and nitroxide fragments allow to regulate fluorescent properties and redox potentials of the dual molecules. The proposed fluorophore-nitroxide hybrids retain all features of the spin labels and fluorescence probes gaining new advantages for monitoring redox reactions and radical processes by two independent techniques: ESR and steady-state fluorescent spectroscopy. The method was applied to the vitamin C analysis in commercial fruit juices.     

Microprobe quantitation procedure that calculates concentrations of chemical elements in soft biological tissue sections

In quantitative studies of soft biological tissues with the use of electron-probe analysis, standards similar to the sample by chemical content, preparation and elemental concentration are used. However, since X-ray intensity and stability affect the measurement accuracy, in practice massive mineralogical standards are used. The aim of the present study was to develop an X-ray microprobe quantitation procedure that calculates element concentrations in sections of soft biological tissues with reference to peculiar features of the object under study using conventional mineralogical standards.     

Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists

A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a Solid-Phase Extraction column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post exercise versus pre exercise condition in cyclists (F: 136+/-93 nmol/l versus 67+/-50 nmol/l (p<0.001); E: 797+/-400 nmol/l versus 408+/-252 nmol/l (p<0.001)). The F/E ratio was 0.18+/-0.11 versus 0.16+/-0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11+/-0.07) and cyclist individuals at rest (p<0.05).     

Protein interactomics based on direct molecular fishing on paramagnetic particles: Experimental simulation and SPR validation

We describe an experimental approach for direct molecular fishing of prey protein on the surface of two types of paramagnetic particles (PMP) having different size and composition. Human microsomal cytochrome b₅ (b₅) and its known partner human cytochrome P450 3A5 (CYP3A5) were used as bait and prey proteins, respectively. For assessing the level of unspecific binding of background proteins, α‐fetoprotein (aFP) was used. SPR measurements were applied for quantitative analysis of trapped proteins (CYP3A5 and aFP) after fishing on PMP. It was shown that the described approach of molecular fishing on micro‐PMP provides enough prey proteins for LC‐MS/MS identification and SPR validation, so this approach can be used for discovery of new protein–protein interactions in the framework of Human Proteome Project.     

辽东半岛结缕草群落分类及生态的研究

本文用群丛表法和群落数量分类法以东半岛的结缕草群落进行分析,把该地结缕草群系划分四个群丛,分别是：结缕草群丛,结缕草＋矮丛苔草群丛,结缕草＋矮丛苔草－花木蓝灌草丛和结缕草＋矮丛苔草＋多年生杂类草群丛,并讨论了这些群丛的种类和生活型组成,群落结构和生物量垂分布等订特征。用主分量分析法（ＰＣＡ）对５８个样地进行排序,经分析得出：群落草本层受光率和土壤有机质含量是决定群落类型主自然生态因子。相关分析的结     

How often is dysplasia diagnosed by biopsy or smear examination? Application of a maximum likelihood based method to the assessment of detection rates in the nasal mucosa of nickel workers.

In view of the known increased risk of nasal carcinoma and the high prevalence of dysplastic lesions of the nasal mucosa among nickel workers, regular screening for the existence of possibly precancerous dysplastic lesions is offered to workers in a Norwegian nickel refinery. Unfortunately, available sampling techniques do not allow the identification of all subjects in whom dysplastic changes are present. Independent histological and cytological (brush cytology) diagnoses, obtained for each of a group of 90 workers, have been used to estimate, by a maximum likelihood method, the probabilities that existing dysplastic lesions will be detected by each of these two screening methods. In the group studied, cytology performed rather less well than histology in unambiguously detecting dysplasia. However, when cytological specimens showing irregular (possibly dysplastic) epithelial cells were grouped with those showing clear dysplastic changes, detection probabilities were estimated at 0.52 by histology and 0.57 by cytology. Detection probabilities were estimated to be higher among subjects with a previously known history of dysplasia, particularly by histology (P < 0.01), probably due to larger dysplastic areas. In view of both its greater facility and speed of sampling, and its greater acceptability, brush cytology may be preferable to biopsy sampling for the screening of large numbers of workers at risk.