LI-cadherin-mediated cell-cell adhesion does not require cytoplasmic interactions.

The adhesive function of classical cadherins depends on the association with cytoplasmic proteins, termed catenins, which serve as a link between cadherins and the actin cytoskeleton. LI-cadherin, a structurally different member of the cadherin family, mediates Ca2+-dependent cell-cell adhesion, although its markedly short cytoplasmic domain exhibits no homology to this highly conserved region of classical cadherins. We now examined whether the adhesive function of LI-cadherin depends on the interaction with catenins, the actin cytoskeleton or other cytoplasmic components. In contrast to classical cadherins, LI-cadherin, when expressed in mouse L cells, was neither associated with catenins nor did it induce an upregulation of beta-catenin. Consistent with these findings, LI-cadherin was not resistant to detergent extraction and did not induce a reorganization of the actin cytoskeleton. However, LI-cadherin was still able to mediate Ca2+-dependent cell-cell adhesion. To analyze whether this function requires any interaction with proteins other than catenins, a glycosyl phosphatidylinositol-anchored form of LI-cadherin (LI-cadherin(GPI)) was constructed and expressed in Drosophila S2 cells. The mutant protein was able to induce Ca2+-dependent, homophilic cell-cell adhesion, and its adhesive properties were indistinguishable from those of wild type LI-cadherin. These findings indicate that the adhesive function of LI-cadherin is independent of any interaction with cytoplasmic components, and consequently should not be sensitive to regulatory mechanisms affecting the binding of classical cadherins to catenins and to the cytoskeleton. Thus, we postulate that the adhesive function of LI-cadherin is complementary to that of coexpressed classical cadherins ensuring cell-cell contacts even under conditions that downregulate the function of classical cadherins.

Liver-intestine cadherin: molecular cloning and characterization of a novel Ca(2+)-dependent cell adhesion molecule expressed in liver and intestine.

A novel member of the cadherin family of cell adhesion molecules has been characterized by cloning from rat liver, sequencing of the corresponding cDNA, and functional analysis after heterologous expression in nonadhesive S2 cells. cDNA clones were isolated using a polyclonal antibody inhibiting Ca(2+)-dependent intercellular adhesion of hepatoma cells. As inferred from the deduced amino acid sequence, the novel molecule has homologies with E-, P-, and N-cadherins, but differs from these classical cadherins in four characteristics. Its extracellular domain is composed of five homologous repeated domains instead of four characteristic for the classical cadherins. Four of the five domains are characterized by the sequence motifs DXNDN and DXD or modifications thereof representing putative Ca(2+)-binding sites of classical cadherins. In its NH2-terminal region, this cadherin lacks both the precursor segment and the endogenous protease cleavage site RXKR found in classical cadherins. In the extracellular EC1 domain, the novel cadherin contains an AAL sequence in place of the HAV sequence motif representing the common cell adhesion recognition sequence of E-, P-, and N-cadherin. In contrast to the conserved cytoplasmic domain of classical cadherins with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. Examination of transfected S2 cells showed that despite these structural differences, this cadherin mediates intercellular adhesion in a Ca(2+)-dependent manner. The novel cadherin is solely expressed in liver and intestine and was, hence, assigned the name LI-cadherin. In these tissues, LI-cadherin is localized to the basolateral domain of hepatocytes and enterocytes. These results suggest that LI-cadherin represents a new cadherin subtype and may have a role in the morphological organization of liver and intestine.

Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury.

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins.

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.

Comparative studies of high resolution Z-DNA crystal structures. Part 1: Common hydration patterns of alternating dC-dG.

The water structure in three crystal forms of the left-handed Z-DNA hexamer [d(CGCGCG)]2 has been analyzed. Several common motifs have been found in the first hydration shells. On the convex surface, the major groove of the left-handed conformation, water molecules bridge the guanine O-6 keto groups at GpC steps. Cytosine N-4 nitrogens of opposite strands are hydrated by tandem water molecules. At the bottom of the minor groove, a string of water molecules connects the cytosine O-2 keto groups. Across the minor groove guanine N-2 nitrogens are bridged to phosphate oxygens of cytosine and guanine residues by one or two water molecules. In contrast to the very regular geometry of the water structure around the bases, the arrangement of water molecules between phosphate groups appears to be less ordered. However, there is a strong correlation between the interphosphate distances and the number of water molecules or ions which link the phosphate groups. In all three structures various ions, such as sodium and magnesium ions, as well as the protonated amino and imino groups of the polycation spermine displace and replace water molecules in the first hydration shell. Nevertheless, the analysis reveals that numerous first hydration shell water molecules in Z-DNA crystals can be regarded as part of the DNA structure. Their positions and thermal parameters are generally independent of changes in the local crystallographic environment.

Direct calibration ELISA: a rapid method for the simplified determination of association constants of unlabeled biological molecules.

We present a novel method for the rapid determination of association constants. The method is based on the direct calibration of an enzyme-linked immunosorbent assay (dcELISA) and does not require any external calibration. It combines kinetic and equilibrium binding experiments and can be performed on a single microtiter plate. The absorbance data are evaluated by several linearized plots without the need for sophisticated computations. The dcELISA has been used to analyze the binding of a monoclonal antibody, OKT9, to its cognate antigen, the human transferrin receptor, and yielded an association constant of Ka = 2.2 x 10(9) l/mol and a complex formation rate constant of kc = 2.7 x 10(-4) s-1. A 26% larger association constant was obtained with a radioimmunoassay (RIA)-based Scatchard analysis using 125I-labeled OKT9. By quantifying the binding of the same iodinated antibody with the dcELISA we were able to verify that the iodination modifies the binding properties of the antibody. The dcELISA thus appears to be superior to all methods requiring covalent modifications. In principle, the direct calibration method can also be combined with all other solid phase assays. It should thus expand their scope in quantifying the binding properties of biologically important molecules.

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.

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.

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.

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.

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.

In an epidemiological sample the apolipoprotein E4 allele is associated to dementia and loss of memory function only in the very old.

The apolipoprotein E4 allele has been reported to be associated with late onset Alzheimer's disease. Here we report the relation of several neuropsychological test parameters and the diagnosis of dementia to the apolipoprotein E polymorphism in an epidemiological sample of 477 subjects aged 70-103 years. The apolipoprotein E4 allele was found to be associated with reduced performance in several sensitive neuropsychological memory tests and with diagnosis of dementia only in the oldest subjects (> 84 years). The association with dementia in this population based sample was much weaker than previously described and became only significant in a logistic regression analysis when age was included in the model.

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.

Anatomical basis for congenital deformities of the lower extremities. Part I. The hip and thigh.

Congenital deformities frequently produce problems not always discernible at birth. Often, a period of time is required for the development of signs and symptoms. The present discussion presents the intrauterine anatomy of a midterm fetus relative to conditions of the hip and thigh. Cryomicrotomy is used in this study to present the best anatomical evidence of the morphology involved.

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 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.