The Alzheimer disease-related calcium-binding protein Calmyrin is present in human forebrain with an altered distribution in Alzheimer's as compared to normal ageing brains.

The EF-hand calcium binding protein Calmyrin (also called CIB-1) was shown to interact with presenilin-2 (PS-2), suggesting that this interaction might play a role in the pathogenesis of Alzheimer's disease (AD). Here we have investigated the distribution of Calmyrin in normal human and AD brain. In normal brain Calmyrin immunoreactivity was unevenly distributed with immunostaining in pyramidal neurones and interneurones of the palaeo-cortex and neocortex, cerebellar granule cells and hypothalamic neurones of the paraventricular, ventromedial and arcuate nuclei. Moderate immunoreactivity was present in hippocampal pyramidal cells and stronger in dentate gyrus neurones. Thalamic and septal neurones were devoid of immunoreactivity. No apparent differences were visible between stainings of brain sections from younger and older nondemented patients. In AD brain a substantial loss of Calmyrin-immunopositive neurones was observed in all regions, especially in cortical areas. Still immunoreactive neurones, however, displayed stronger staining that was especially concentrated in perinuclear regions. Calmyrin immunosignals were in part associated with diffuse and senile plaques. Thus, although protein levels of Calmyrin are low in human forebrain, its cellular localization as well as its altered distribution in AD brain suggest that it may be involved in the pathogenesis of AD.

Transcription patterning of uncoupled proliferation and differentiation in myelodysplastic bone marrow with erythroid-focused arrays.

Because abnormal erythroid differentiation is the most common manifestation of the myelodysplastic syndromes (MDS), it was hypothesized that erythroid gene expression may be used to illustrate myelodysplastic transcription patterns. Ten normal bone marrow aspirates (NBM) were first analyzed using an erythroid-focused cDNA array to define steady-state transcription levels. Proliferation and differentiation gene subsets were identified by statistically significant differences between NBM and erythroleukemia gene expression. Next, cDNAs from 5 separate MDS aspirates were studied: refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation (RAEB-T), and RAEB-T/secondary MDS. A distinct pattern of significantly increased proliferation-associated and reduced differentiation-associated gene activity was established for MDS.

Detection of cytomegalovirus in infant cerebrospinal fluid by conventional PCR, nested PCR and PCR-Digene.

The possibility of amplification of human cytomegalovirus (HCMV) DNA in cerebrospinal fluid (CSF) for the diagnosis of HCMV central nervous system (CNS) infection in infants was studied. Single-step PCR, nested PCR and PCR-Digene were used to assay CSF specimens from 37 patients. Criteria for patient inclusion in the study were: 1. clinical manifestations suggesting CMV neuroinfection such as seizures, hypertonia, hypotonia, intracranial calcification, microcephaly, chorioretinitis; 2. any of the following symptoms: anaemia, hepetomegaly, prolonged cholestatic jaundice, or hepatitis, splenomegaly, thrombocytopenia, intrauterine hypotrophy; 3. serologic presentation, and/or positive results for CMV infection obtained by single-step PCR and PCR-Digene in urine and/or blood. PCR-Digene results were positive in 6 CSF samples. Four CSF samples were positive by nested PCR and 1 CSF sample by single step PCR. We found that the double PCR was about ten or more times more sensitive than single PCR and the PCR-Digene was only three times more sensitive than nested-PCR. The results were correlated with serology. Thirty-three out of 37 examined patients were seropositive (ELISA IgG); ELISA IgM gave positive results in 9 patients. In control studies, cells infected with other members of the herpes virus family were negative with these methods, which suggest that amplification combined with primers from the IE and the L-region of CMV is specific. In conclusion, nested-PCR seems to be the best method for early diagnosis of CMV infection in CSF due to an absence of false positive results and its high specificity and sensitivity.

Identification of the dombrock blood group glycoprotein as a polymorphic member of the ADP-ribosyltransferase gene family.

Identification of the 25 known human blood group molecules is of fundamental importance for the fields of erythroid cell biology and transfusion medicine. Here we provide the first molecular description of the "Dombrock" blood group system. A candidate gene was identified by in silico analyses of approximately 5000 expressed sequence tags (ESTs) from terminally differentiating human erythroid cells. Transfection experiments demonstrated specific binding of anti-Dombrock and confirmed glycosylphosphatidylinositol membrane attachment. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Homology studies suggest that the Dombrock molecule is a member of the adenosine 5'-diphosphate (ADP)-ribosyltransferase ectoenzyme gene family. Genotypic comparisons suggest Do(a) versus Do(b) antigenicity results from a single amino acid substitution within an encoded arginine-glycine-aspartic acid (RGD) motif of the molecule.

Glycosylphosphatidylinositol-anchored proteins are not required for crosslinking-mediated endocytosis or transfection of avidin bioconjugates into biotinylated cells.

Even though glycosylphosphatidylinositol (GPI)-anchored proteins lack direct structural contact with the intracellular space, these ubiquitously expressed surface receptors activate signaling cascades and endocytosis when crosslinked by extracellular ligands. Such properties may be due to their association with membrane microdomains composed of glycosphingolipids, cholesterol and some signaling proteins. In this study, we hypothesize that GPI proteins may be required for crosslinking-mediated endocytosis of extracellular bioconjugates. To test this hypothesis, we first biotinylated the surface membranes of native K562 erythroleukemia cells versus K562 cells incapable of surface GPI protein expression. We then compared the entry of fluorescently labeled avidin or DNA condensed on polyethylenimine-avidin bioconjugates into the two biotinylated cell populations. Using fluorescence microscopy, nearly 100% efficiency of fluorescent avidin endocytosis was demonstrated in both cell types over a 24 h period. Surprisingly, plasmid DNA transfer was slightly more efficient among the biotinylated GPI-negative cells as measured by the expression of green fluorescence protein. Our findings that GPI proteins are not required for the endocytosis of avidin bioconjugates into biotinylated cells suggest that endocytosis associated with general membrane crosslinking may be due to overall reorganization of the membrane domains rather than GPI protein-specific interactions.

Targeted transfer of polyethylenimine-avidin-DNA bioconjugates to hematopoietic cells using biotinylated monoclonal antibodies.

Here we examine whether attachment of biotinylated antibodies to proteins on the cell surface increases the transfection efficiency of polyethylenimine-avidin-DNA bioconjugate gene transfer. Preliminary experiments were performed to compare avidin endocytosis into cells incubated with biotinylated antibodies. Antibody biotinylation resulted in the endocytosis of avidin-FITC into nearly 100% of cells compared with no detectable binding or entry into unbiotinylated cells. Gene transfer was accomplished with avidin conjugated to polyethylenimine (PEI) at a molar ratio of 4:1 (PA4). Plasmid DNA encoding the green fluorescent protein (GFP) gene was condensed on the PA4, and transfection efficiencies were measured by flow cytometry as the percentage of cells that fluoresced at levels greater than two standard deviations above the negative control. Gene transfer efficiencies were compared among K562, HEL, and Jurkat leukemia cell lines. Control transfections with DNA alone or untargeted PEI-DNA resulted in

Surface membrane biotinylation efficiently mediates the endocytosis of avidin bioconjugates into nucleated cells.

Here we demonstrate that biotin covalently attached to cell surface obligates existing receptors to endocytose avidin bioconjugates into nucleated cells. Incubation of fluorescein-labeled avidin with biotinylated cell lines resulted in uniform and rapid surface attachment and endocytosis compared with no detectable association of the avidin-conjugated dye with unbiotinylated cells. Uptake was detected within minutes with efficiencies approaching 100% in cell lines and freshly obtained peripheral blood mononuclear cells. After 24 h, avidin was barely detectable on the surface of the nucleated cells. In marked contrast, fluorescent avidin remained exclusively on the external membrane of erythrocytes after 24 h. To investigate biotin-mediated endocytosis for the delivery of DNA, we prepared polyethylenimine-avidin (PEI-avidin) conjugates. Surface biotinylation significantly increased the transfection efficiencies of PEI-avidin condensed plasmid DNA coding green fluorescent protein (GFP) to the level of transferrin-receptor targeted gene delivery (15-20% GFP positive cells in culture after 48 h). The increase in transfection efficiency was blocked by the addition of free avidin or biotin to the culture medium. Biotin covalently bound to cell surface membrane proteins efficiently mediates the entry of avidin bioconjugates into nucleated cells.

Protection of cells from complement-mediated attack: CD59 receptor clustering for the entry of macromolecules into hematopoietic cells.

CD59 is a glycosylphosphatidylinositol anchored protein (GPI-protein) that is expressed on surface membranes to protect host cells from complement-mediated attack. CD59 may also serve as a receptor for the endocytosis of macromolecules into nucleated cells. Here we investigate the effects of primary clustering of CD59 with anti-CD59 monoclonal antibody and the secondary clustering of biotinylated anti-CD59 with avidin on red blood cells and erythroleukemic K562 cells. On red blood cells, CD59-targeted antibodies remained evenly distributed on the external membranes. In contrast, clustering, capping and endocytosis of the CD59-targeted complexes was detected on K562 cells. Secondary clustering appeared more efficient and resulted in endosomal localization of the fluorescently labeled complexes within 2 hours. The endocytosis of CD59-bound complexes did not affect K562 cell viability or growth and the surface level of CD59 was constant during the process. These result suggest clustering and subsequent endocytosis of CD59 may enable the entry of macromolecules to the endosomal compartments of hematopoietic cells.

Biotinylation modifies red cell antigens.

BACKGROUND: Chemical biotinylation of red cell membranes may be useful for several clinical applications, including red cell survival studies. STUDY DESIGN AND METHODS: To examine the possible effects of biotinylation on red cell antigens, standard hemagglutination assays were performed on matched sets of control and biotinylated red cells. The red cells were biotinylated at a final concentration of 2.0 pg of sulfo-N-hydroxysuccinimide-biotin per cell, and antigen-negative cells were directly compared to antigen-positive cells when possible. The hemagglutination assays were graded in a blinded fashion. Forty-one red cell antigens from 21 of the 23 established blood group systems were tested. RESULTS: Hemagglutination based upon antibody binding to A, A1, M, N, S, s, P1, D, C, E, c, e, C(w), Lu(b), K, k, Kp(b), Le(a), Le(b), Fy(a), Fy(b), Jk(a), Jk(b), Di(a), Wr(a), Wr(b), Yt(a), Xg(a), Sc1, Do(b), Co(a), Ch, H, Ge2, Cr(a), Kn(a), I, and P was not affected by biotinylation. Unexpectedly, the hemagglutination of Di(b+) and LW(a+) red cells was blocked after biotinylation. Conversely, MH04 monoclonal anti-A agglutinated red cells expressing B only after biotinylation. BIRMA-1 monoclonal anti-A and polyclonal anti-A from sera did not agglutinate the biotinylated B red cells. CONCLUSION: Biotinylation of human red cells specifically modified their antigenicity, as measured by standard hemagglutination assays.

Topography of Helicobacter pylori infection in gastric cancer patients.

The role of Helicobacter pylori in gastric carcinogenesis is a subject of an increasing interest. In this report we describe a prospective study on the resected stomachs to establish the prevalence of H. pylori in different types of gastric carcinoma. The material consisted of 62 consecutive patients operated on stomach adenocarcinomas Fifty six percent of the patients were intestinal type, 34%--diffuse type and 10%--mixed type. The presence of H. pylori was studied in specimens from surgically removed stomachs. The conformation of the bacterial infection was done by means of rapid urease test, microbiological culture, Warthin-Starry and immunohistochemical staining. The overall prevalence of H. pylori infection was 69% (43/62). There was a statistically significant difference in the infection rates between the types of carcinoma--75% in the intestinal type and 62% in the diffuse type. The most sensitive was immunohistochemical staining. The bacterial colonies were cumulated far from the tumor tissue. In cardiac cancer the most intense of infection was an antrum and lower part of gastric body. In opposite; in antrum and pylorus cancer the scope of colonisation increased in fundus and subcardiac region with statistical signification. We could not detect H. pylori in the tumor tissue itself as in the normal mucosa of the stomach. In gastric antrum the most intense colonisation was detected on mucosal atrophy, but in the upper part of the stomach--on the mucosal metaplasia.

p30, a novel protein target of mouse calcyclin (S100A6).

A novel protein target of mouse calcyclin (S100A6) was detected by a gel overlay method with 125I-labelled calcyclin. Interaction of calcyclin with its 30 kDa target protein (p30) present in Ehrlich ascites tumour (EAT) cells depended on the presence of Ca2+ ions. The binding of p30, evidenced by the reaction with 125I-labelled calcyclin, was found to be of higher affinity than the binding between mouse calcyclin and annexin II or glyceraldehyde-3-phosphate dehydrogenase. Examination of tissue extracts by the gel overlay method has shown that p30 is present not only in the EAT cells but also in mouse brain and spleen. This novel target protein of mouse calcyclin was purified to homogeneity from EAT cells by means of Phenyl-Sepharose chromatography, affinity chromatography and CM-cellulose chromatography. Purified p30 was digested with alpha-chymotrypsin and a partial amino acid sequence of one of the resulting peptides was established. A database search analysis revealed that the sequence is unique, with a similarity of less than 55% to any other known protein sequence.

Chicken gizzard calcyclin--distribution and potential target proteins.

The distribution of calcyclin in some chicken tissues was studied by Western blotting using polyclonal antibodies raised against calcyclin purified from chicken gizzard. The protein was found in gizzard muscle and in a lesser amount in skeletal and cardiac muscle. No immunological reaction was observed in chicken liver. Immunohistochemical studies of chicken gizzard tissue revealed the presence of calcyclin only in muscle fibers. Ca(2+)-dependent interaction of chicken gizzard calcyclin with potential protein targets was also examined. By gel overlay method it was found that calcyclin bound to three proteins with molecular masses of approximately 35 kDa, 25 kDa and 15 kDa present in the cytosolic fraction derived from chicken gizzard muscle. The chicken gizzard calcyclin was also shown to interact with lysozyme.

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon.

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound 45Ca2+ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca2+ concentration. Crosslinking experiments with BS3 showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg(2+)-ATPase activity of myosin in the presence of Ca2+.

PCR for identification and typing of Helicobacter pylori isolated from children.

From 191 children with upper gastroduodenal disease 236 gastric biopsy specimens were taken. H. pylori was detected by use of culture Gram staining histological examination and PCR technique. A segment of DNA coding protein synthesis of 26 kDa or urease A and B gene were used for PCR amplification. PCR technique was also used for determination of the presence of Cag A gene in 72 strains of H. pylori isolated from children. Genetic typing of H. pylori strains by RFLP analysis of PCR amplified urease B gene 933 bp fragment and RAPD were performed. Biopsy specimens taken from children with gastritis were in 52% H. pylori culture and PCR positive, while 18.1% PCR positive only. Similarly, specimens taken from children with duodenal ulcer were in 50% H. pylori culture and PCR positive, while 12.5% PCR positive only. Fifty one (70.8%) from 72 strains of H. pylori were Cag A positive. Molecular typing of the strains isolated during first and follow-up endoscopy allowed the differentiation between reinfection or new infection and coinfection. It was shown that RAPD typing had better discrimination power in comparison to PCR--RFLP method.

Interaction of calcyclin and its cyanogen bromide fragments with annexin II and glyceraldehyde 3-phosphate dehydrogenase.

The structural properties of calcyclin protein are quite well characterized but its function remains obscure. To help elucidate the biological role of calcyclin we have performed the in vitro studies of the Ca(2+)-dependent interaction of Ehrlich ascites tumor cells calcyclin and its cyanogen bromide fragments with two potential calcyclin targets: annexin II and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The binding of annexin II, evidenced by the reaction with 125I-calcyclin, was found to be very weak and occurred only for intact calcyclin. On the other hand the interaction between calcyclin and GAPDH was of high affinity and could be assigned to the N-terminal region of calcyclin. Intact calcyclin and its N-terminal fragment bound to GAPDH in the gel overlay and affinity chromatography assay. When examined in the presence of a crosslinking agent the interaction resulted in the formation of 46K covalent adduct between calcyclin monomer and GAPDH subunit. Fluorescence of 5-iodoacetamido-fluorescein-labelled calcyclin was efficiently quenched by GAPDH in the presence of Ca2+. Titration experiments revealed the stoichiometry of one calcyclin monomer binding to each of GAPDH subunits with a binding constant of 10(8) M-1. The results of this work suggest that the binding between calcyclin and GAPDH may have bearing on calcyclin function.

Calcyclin from mouse Ehrlich ascites tumor cells and rabbit lung form non-covalent dimers.

Crosslinking treatments of fresh cytosol from mouse Ehrlich ascites tumor (EAT) cells revealed the existence of calcyclin dimers which were sensitive to SDS, but not to reducing agents, which suggests the existence of non-covalent dimers. In stored EAT cell cytosol and preparations of purified calcyclin dimers were also formed by S-S bridging (covalent dimers). The S-S dimers did not bind to organomercurial Agarose and could be separated from reduced forms of calcyclin that bound to the resin. Calcyclin eluted from the resin with DTT was a mixture of monomers and non-covalent dimers as shown by crosslinking and subsequent immunoblotting. Calcyclin from rabbit lung, lacking a cysteine residue, could also be crosslinked as a dimer. It is suggested that the ability of calcyclin to form non-covalent dimers is of physiological significance.

Characterization of calcyclin fragments obtained by CNBr-cleavage.

1. Two calcyclin fragments were obtained by CNBr-cleavage. 2. One fragment represented N-terminal end of a molecule (residues 1-56), and another one a C-terminal end (residues 57-89). 3. Properties of intact calcyclin such as binding of calcium, binding to hydrophobic resins and interaction with calcyclin specific antibodies were not retained by these fragments. 4. However, both fragments were able to form dimers and higher forms of aggregates as seen for uncleaved calcyclin. 5. This indicates that both halves of the molecule contain the regions responsible for non-covalent interaction which might participate in dimer formation.

Effects of plant transfer ribonucleic acids on interferon production.

An optimal interferon (IFN) production was obtained at concentrations of 50 micrograms/ml poly I:C and 2000 micrograms/ml DEAE dextran in Lpa cells. It was shown that methionine initiator tRNA (tRNAiMet) in a dose 50 micrograms/ml or crude tRNA (tRNAc) applied to Lpa cell during the stages of IFN induction, IFN induction and synthesis, as well as during IFN synthesis resulted in a continuous IFN production for up to 24 hr. Exposure of the cells to 150 micrograms/ml tRNAiMet during the stages of IFN induction and IFN induction and synthesis caused total inhibition of IFN production. This effect was partially observed only after the high dose of tRNAc. Addition of a high dose of tRNAiMet or tRNAc to cells during the synthesis stage caused no inhibition but prolongation of IFN production.