Unique gene structure and paralogy define the 7D-cadherin family.

Cadherins are Ca2+-dependent transmembrane glycoproteins crucial for cell-cell adhesion in vertebrates and invertebrates. Classification of this superfamily due to their phylogenetic relationship is currently restricted to three major subfamilies: classical, desmosomal and protocadherins. Here we report evidence for a common phylogenetic origin of the kidney-specific Ksp- (Cdh16) and the intestine-specific LI-cadherin (Cdh17). Both genes consist of 18 exons and the positions of their exon-intron boundaries as well as their intron phases are perfectly conserved. We found an extensive paralogy of more than 40 megabases in mammals as well as teleost fish species encompassing the Ksp- and LI-cadherin genes. A comparable paralogy was not detected for other cadherin gene loci. These findings suggest that the Ksp- and LI-cadherin genes originated by chromosomal duplication early during vertebrate evolution and support our assumption that both proteins are paralogues within a separate cadherin family that we have termed 7D-cadherins.

The application of a SERS fiber probe for the investigation of sensitive biological samples.

The applicability of an etched and silver or gold coated SERS fiber probe in combination with a commercially available laboratory micro-Raman setup or a home built mobile micro-Raman setup to perform on-site field measurements was evaluated and successfully tested on different biological samples. The SERS fiber probe allows one to perform measurements with high spatial resolution. Simultaneously, the laser power used for Raman spectroscopy on biological samples as compared with conventional Raman experiments can be reduced by more than two orders of magnitude. This experimental arrangement was tested to investigate sensitive biological samples like mint plants (Bergamot mint, spear mint) and citrus fruits (kumquat). Furthermore, traces of fungicides on wine leaves were detected by means of such a SERS fiber probe setup.

Identification of biotic and abiotic particles by using a combination of optical tweezers and in situ Raman spectroscopy.

A highly versatile setup, which introduces an optical gradient trap into a Raman spectrometer, is presented. The particular configuration, which consists of two lasers, makes trapping independent from the Raman excitation laser and allows a separate adjustment of the trapping and excitation wavelengths. Thus, the excitation wavelength can be chosen according to the needs of the application. We describe the successful application of an optical gradient trap on transparent as well as on reflective, metal-coated microparticles. Raman spectra were recorded from optically trapped polystyrene beads and from single biological cells (e.g., erythrocytes, yeast cells). Also, metal-coated microparticles were trapped and used as surface enhanced Raman spectroscopy (SERS) substrates for tests on yeast cells. Furthermore, the optical gradient trap was combined with a SERS fiber probe. Raman spectra were recorded from trapped red blood cells using the SERS fiber probe for excitation.

Phylogenetic origin of LI-cadherin revealed by protein and gene structure analysis.

The intestine specific LI-cadherin differs in its overall structure from classical and desmosomal cadherins by the presence of seven instead of five cadherin repeats and a short cytoplasmic domain. Despite the low sequence similarity, a comparative protein structure analysis revealed that LI-cadherin may have originated from a five-repeat predecessor cadherin by a duplication of the first two aminoterminal repeats. To test this hypothesis, we cloned the murine LI-cadherin gene and compared its structure to that of other cadherins. The intron-exon organization, including the intron positions and phases, is perfectly conserved between repeats 3-7 of LI-cadherin and 1-5 of classical cadherins. Moreover, the genomic structure of the repeats 1-2 and 3-4 is identical for LI-cadherin and highly similar to that of the repeats 1-2 of classical cadherins. These findings strengthen our assumption that LI-cadherin originated from an ancestral cadherin with five domains by a partial gene duplication event.

Ksp-cadherin is a functional cell-cell adhesion molecule related to LI-cadherin.

Ksp- and LI-cadherin are structurally homologous proteins coexpressed with E-cadherin in renal and intestinal epithelia, respectively. Whereas LI-cadherin has been shown to mediate Ca2+-dependent homotypic cell-cell adhesion independent of stable interactions with the cytoskeleton, little is known about the physiological role of Ksp-cadherin. To analyze its potential adhesive and morphoregulatory functions, we expressed murine Ksp-cadherin in CHO cells. In this report, we show that Ksp-cadherin induces homotypic and Ca2+-dependent cell-cell adhesion that can be specifically blocked with antibodies raised against the cadherin repeats EC1 and EC2. Ksp-cadherin mediates about the same quantitative adhesive effect (aggregation index) as LI- and E-cadherin. However, the cellular phenotype induced by Ksp-cadherin resembles more closely that of LI- than E-cadherin. This could reflect our observation, that Ksp-cadherin, as well as LI-cadherin, does not directly interact with beta-catenin. In conclusion, both cadherins are thus not only structurally but also functionally related and may share other functions within their respective epithelia.

Raman spectroscopy investigation of biological materials by use of etched and silver coated glass fiber tips.

The results for surface enhanced Raman scattering (SERS) studies on biological samples are reported. Etched and silver coated glass fiber tips were used as a SERS substrate. This method enabled the recording of spectra of biological samples, such as plant tissue or microbiological cells, with a high spatial resolution. Because of the low laser power used with the fiber tips, it was even possible to investigate tissues that are very sensitive toward laser power as it is used in a common micro-Raman setup.

The result of equilibrium-constant calculations strongly depends on the evaluation method used and on the type of experimental errors.

The determination of equilibrium constants is a widespread tool both to understand and to characterize protein-protein interactions. A variety of different methods, among them Scatchard analysis, is used to calculate these constants. Although more than 1000 articles dealing with equilibrium constants are published every year, the effects of experimental errors on the results are often disregarded when interpreting the data. In the present study we theoretically analysed the effect of various types of experimental errors on equilibrium constants derived by three different methods. A computer simulation clearly showed that certain experimental errors, namely inaccurate background correction, inexact calibration, saturation effects, slow kinetics and simple scattering, can adversely affect the result. The analysis further revealed that, for a given type of error, the same data set can produce different results depending on the method used.

Determination of optimal non-denaturing elution conditions from affinity columns by a solid-phase screen.

The purification of biological macromolecules by affinity chromatography is a widespread technique used to separate a protein from other biological components. However, this method may destroy the protein's physiological activity because elution conditions aimed to dissociate the protein of interest from the high-affinity matrix often irreversibly denature it. In the present work, we have developed a solid-phase assay to determine the optimal elution conditions for any buffer (in two steps) by determining (i) the lowest buffer concentration yielding maximum dissociation from the immobilized component and (ii) the highest buffer concentration that can be used without the loss of the protein's binding activity. Any buffer that can be reasonably used between these defined concentrations is suitable for elution within this interval. The screen is easily performed within a few hours and only requires nanograms to a few micrograms of protein. As an example, we demonstrate that more than 95% of the human transferrin receptor bound to a transferrin-sepharose ligand affinity column can be eluted with full binding activity at KSCN concentrations between 232 and 414 nM, whereas elution with urea is not suitable to purify fully functional protein.

LI-cadherin: a marker of gastric metaplasia and neoplasia.

BACKGROUND: Intestinal metaplasia is considered a risk factor for the development of gastric adenocarcinomas of the intestinal type and is found in approximately 20% of gastric biopsies. Conventional histology only detects advanced stages of intestinal metaplasia. AIMS: To study expression of the enterocyte specific adhesion molecule liver-intestinal (LI)-cadherin in intestinal metaplasia as well as in gastric cancer, and to evaluate its use as a diagnostic marker molecule. PATIENTS: Gastric biopsies (n=77) from 30 consecutive patients (n=30; aged 28-90 years) as well as surgically resected tissue samples (n=24) of all types of gastric carcinomas were analysed. METHODS: Single and double label immunofluorescence detection on cryosections of gastric biopsies; alkaline phosphatase antialkaline phosphatase method on paraffin embedded carcinoma tissue sections. RESULTS: Of 77 biopsies (from 30 patients), 12 (from 10 patients) stained positive for LI-cadherin. LI-cadherin staining correlated with the presence of intestinal metaplasia. Conventional histological diagnosis however failed to detect subtle gastric intestinal metaplasia (three of 10 patients). In contrast, only LI-cadherin and villin were positive in these cases whereas sucrase-isomaltase also failed to detect intestinal metaplasia in four of 10 patients. Well differentiated gastric carcinomas showed intense staining for LI-cadherin while undifferentiated carcinomas showed only weak diffuse cytoplasmic staining. CONCLUSIONS: To detect early metaplastic changes in the gastric mucosa, LI-cadherin has a sensitivity superior to sucrase-isomaltase and conventional histology and comparable with that of villin. Its specificity exceeds that of villin. Thus LI-cadherin represents a new, reliable, and powerful marker molecule for early detection of gastric intestinal metaplasia and well differentiated adenocarcinomas.

Structural and functional stability of the mature transferrin receptor from human placenta.

The transferrin receptor (TfR) is a N- and O-glycosylated transmembrane protein mediating the cellular iron uptake by binding and internalization of diferric transferrin. In this study, rate constants and dissociation constants of 125I-ferri-transferrin binding to the human TfR were examined dependent on receptor glycan composition, pH, bivalent cations, and temperature. To do so, purified human placental TfR was noncovalently immobilized to polystyrene surfaces and subjected to alterations in various parameters. We found that transferrin binding was clearly dependent on a receptor pretreatment with buffers of various pH in that most of the TfR molecules irreversibly lost transferrin binding activity below pH 6.5. However, the dissociation constant of the remaining active binding sites was not affected. Similarly, we were able to define the thermal stability of the receptor as a function of transferrin binding ability. Binding of transferrin was completely lost provided that the receptor was pretreated at temperatures of at least 65 degrees C. Treatment with EDTA also caused an irreversible loss of transferrin binding activity, indicating that the functionally active conformation of the mature TfR depends on bivalent cations. In order to examine the role of the receptor glycans, we enzymatically removed the sialic acid residues, the hybrid and oligomannosidic N-glycans, or all types of N-glycans. In contrast to the parameters described above, all desialylated and N-deglycosylated TfR variants had exactly the same transferrin binding properties as the native TfR. To assess changes in the secondary structure of the receptor, circular dichroic spectra were recorded from TfR at pH 5.0, from heat pretreated receptor and from deglycosylated TfR. Since the receptor did not exhibit detectable changes in the CD spectrum of the deglycosylated receptor, it can be concluded that the N-linked carbohydrates of the mature, fully processed TfR are not essential for transferrin binding and conformational stability.

LI-cadherin gene expression during mouse intestinal development.

LI-cadherin (Liver-Intestine cadherin) is a member of a subclass (7-D cadherins) within the cadherin superfamily. Although its cellular function as a cell-cell adhesion molecule has been demonstrated in cell culture studies, its physiological function still needs to be explored in the intact organism. After isolating the cDNA for mouse LI-cadherin, we generated specific antibodies against the overexpressed protein and studied its expression pattern in adult mouse tissues and mouse embryos. The mouse LI-cadherin sequence is 91% identical to the sequence of rat LI-cadherin and exhibits the same structural features described for rat LI-cadherin. In mouse adult tissue, LI-cadherin is expressed in the intestine and in small amounts in the spleen. In contrast to rat, Mouse LI-cadherin was not expressed in liver. During mouse embryogenesis, LI-cadherin expression begins at embryonic day 12.5. With the exception of transient expression in the urogenital sinus and the common bile duct on day 13.5, LI-cadherin was found exclusively in the intestinal epithelium. Its expression coincides with the formation of intestinal villi, a developmental stage that includes major tissue remodeling, growth, and differentiation. LI-cadherin is expressed along the entire anterior-posterior axis of the developing intestine as well as along the entire villus axis once villi begin to form. LI-cadherin occupies all cell surfaces of the deeper layers of the epithelium, distributing to basolateral surfaces only in the cells of the outer epithelial layer. LI-cadherin was found to be always co-expressed with E-cadherin.

Short telomeres in patients with vascular dementia: an indicator of low antioxidative capacity and a possible risk factor?

Progressive cerebrovascular atherosclerosis and consecutive stroke are among the most common causes of dementia. However, specific risk factors for vascular dementia are still not known. Human telomeres shorten with each cell division in vitro and with donor age in vivo. In human fibroblasts in vitro, the telomere shortening rate decreased with increasing antioxidative capacity. There was a good intra-individual correlation between the age-corrected telomere lengths in fibroblasts and peripheral blood mononuclear cells. In 186 individuals including 149 geriatric patients (age range, 55-98 yr), leukocyte telomeres in patients with probable or possible vascular dementia were significantly shorter than in three age-matched control groups, namely in cognitively competent patients suffering from cerebrovascular or cardiovascular disease alone, in patients with probable Alzheimer's dementia, and in apparently healthy control subjects. No correlation was found to polymorphisms in the apolipoprotein E and glutathione-S-transferase genes. Telomere length may be an independent predictor for the risk of vascular dementia.

Experiences with external quality assessment (EQA) in molecular diagnostics in clinical laboratories in Germany. Working Group of the German Societies for Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM).

The German Societies for Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM) have established an official working group on molecular diagnostics in the field of laboratory medicine. The group's objectives are to support the establishment of molecular biology methods for the use in diagnostics in German clinical laboratories. Towards this end, we have defined specific aims, which are 1) the implementation and extension of external quality assessment (EQA) schemes and methodological exercises offered to clinical diagnostic laboratories, 2) the establishment of a proficiency network and data base within the societies, 3) the implementation of an educational program in molecular diagnostic procedures for clinical chemists and laboratory physicians through the organisation of symposia and workshops and 4) the cooperation with other DGKC/DGLM working groups on shared aspects of laboratory analysis, e.g. preanalytics. The focus of this presentation is to introduce some of these goals in more detail with particular emphasis on the first two program aspects and to discuss experiences with these activities.

Intestinal cell adhesion molecules. Liver-intestine cadherin.

The cadherin superfamily comprises a large number of cell adhesion molecules, several of which are expressed in the gastrointestinal tract. LI-cadherin represents a novel type of cadherin within the cadherin superfamily distinguished from other cadherins by structural and functional features described in this review. In the mouse and human, LI-cadherin is selectively expressed on the basolateral surface of enterocytes and goblet cells in the small and large intestine, whereas in the rat this cadherin is additionally detectable in hepatocytes. LI-cadherin is capable of mediating Ca(2+)-dependent homophilic cell-cell adhesion independent of interactions with the cytoskeleton, indicating that the adhesive function of this novel cadherin is complementary to that of E-cadherin and desmosomal cadherins co expressed in the intestinal mucosa.

Differential modulation of human immunoglobulin isotype production by the neuropeptides substance P, NKA and NKB.

The modifying effects of tachykinins substance P, neurokinin A and neurokinin B on immunoglobulin production were analyzed in an in vitro culture system. Purified human T- and B-cells were stimulated with TGFbeta2 and IL-5 to induce preferential IgA production. Neuropeptides had the following effects. (1) The levels of IgA and IgG4 production were enhanced by IL-5 and TGFbeta2; IgA levels remained constant or were slightly augmented by neuropeptides, whereas IgG4 was further augmented. (2) IL-5 and TGFbeta2 did not alter IgG3 production, but neuropeptides stimulated secretion of this subclass. (3) IgG1 and IgM production were inhibited by IL-5 and TGFbeta2. This effect was prevented by neuropeptides. (4) Other isotypes including IgG2 and IgE remained unaffected. Except for IgM, these effects were blocked by specific receptor antagonists indicating specificity. The tachykinin receptor NK-1 mRNA was detected in B- and T-cells, whereas NK-3 mRNA was only present in T- and B-cell coculture following activation. Furthermore, neuropeptide effects depended on cytokine co-stimulation and the presence of T-cells. These results suggest that neuropeptides are potent modifiers of preferential IgA synthesis.

Age and dementia effect on neuropsychological test performance in very old age--influence of risk factors for dementia.

In old age a large part of the variance in cognitive performance in population samples is explained by normal aging; in addition many subjects over 80 years are demented and therefore dementia also explains a part of cognitive variability. The question is whether the different factors for dementia (such as ApoE4, external atrophy parameter of the cranial computer tomography [cCT], education, sex or serum zinc level) influence the relation between age or dementia and Mini Mental State (MMSE) performance. In an epidemiological study data were analyzed of N = 239 subjects for the above factors. Most statistically significant variables of the MMSE do not change the amount of the partial correlation coefficient between the parameters age or dementia and MMSE. The external atrophy, however, diminishes the magnitude of the partial correlation between age and MMSE. In contrast the dementia-MMSE relation is unchanged. This points to a generally similar factor structure of cognitive aging and dementia in old age, but differences exist with respect to the importance of the external atrophy parameter of the brain. Most factors investigated explain separate parts of variance of cognitive performance in old age.

Structural model of phospholipid-reconstituted human transferrin receptor derived by electron microscopy.

BACKGROUND: The transferrin receptor (TfR) regulates the cellular uptake of serum iron. Although the TfR serves as a model system for endocytosis receptors, neither crystal structure analysis nor electron microscopy has yet revealed the molecular dimensions of the TfR. To derive the first molecular model, we analyzed purified, lipid-reconstituted human TfR by high-resolution electron microscopy. RESULTS: A structural model of phospholipid-reconstituted TfR was derived from 72 cryo-electron microscopic images. The TfR dimer consists of a large extracellular globular domain (6.4 x 7.5 x 10.5 nm) separated from the membrane by a thin molecular stalk (2.9 nm). A comparative protein sequence analysis suggests that the stalk corresponds to amino acid residues 89-126. Under phospholipid-reconstitution conditions, the human TfR not only integrates into vesicles, but also forms rosette-like structures called proteoparticles. Scanning transmission electron microscopy revealed an overall diameter of 31.5 nm and a molecular mass of 1669 +/- 26 kDa for the proteoparticles, corresponding to nine TfR dimers. The average mass of a single receptor dimer was determined as being 186 +/- 4 kDa. CONCLUSIONS: Proteoparticles resemble TfR exosomes that are expelled by sheep reticulocytes upon maturation. The structure of proteoparticles in vitro is thus interpreted as being the result of the TfR's strong self-association potential, which might facilitate the endosomal sequestration of the TfR away from other membrane proteins and its subsequent return to the cell surface within tubular structures. The stalk is assumed to facilitate the tight packing of receptor molecules in coated pits and recycling tubuli.

p53 mutagenesis in Klatskin tumors.

Mutagenesis of the p53 tumor-suppressor gene represents the most common genetic alteration in human malignancies but has not yet been investigated in Klatskin tumors. Cancerous and normal liver tissues were obtained from 12 patients after surgical resection of Klatsin tumors. Genomic DNA was extracted and served as a template for PCR amplification and sequencing of a 1,574-bp fragment of the p53 gene comprising the exons 5 through 8. Immunohistochemical expression analysis was performed using five different antibodies. Missense mutations were detected in 2 of 12 patients--one transversion on codon 273 (Arg --> Leu) and a transition on codon 168 (His --> Arg). In all specimens, immunohistochemistry was negative regarding a nuclear overexpression. An apparent clinicopathologic impact of p53 mutations was not observed. This report on mutagenesis of the p53 gene in Klatskin tumors shows that the most commonly mutated tumor suppressor gene in human cancers is also mutated in a subset of patients with Klatskin tumors. Assessment of a clinical or pathological impact of p53 mutagenesis on Klatskin tumors requires evaluation in larger studies.

Human transferrin receptor is active and plasma membrane-targeted in yeast.

The human transferrin receptor, a type II plasma membrane protein which mediates iron transport in human cells, was expressed in the yeast Saccharomyces cerevisiae. The transferrin receptor synthesized by yeast cells was posttranslationally modified comparable to the native receptor with respect to glycosylation and dimer formation. The location of the expressed receptor in the yeast plasma membrane indicates that the targeting of this type II membrane protein shares similar mechanisms in yeast and mammalian cells. The yeast-expressed transferrin receptor showed binding activity towards its natural ligand, transferrin in an ELISA binding assay.