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Three types of covalently crosslinked assemblies consisting of multiple (1) molecular layers of human serum albumin (HSA); (2) alternating layers of HSA and unfractionated heparin; and (3) alternating layers of HSA and partly depolymerized heparin fixed with one end to HSA were prepared on various surfaces. Adsorption of fibrinogen, IgG, and antithrombin (ATIII) from human citrated plasma on coated surfaces was evaluated by ELISA. Fibrinogen adsorption on coated ELISA plates was lower than that on bare polystyrene. There was no IgG adsorption on the HSA coating alone, but considerably high IgG adsorption was detected on the heparin-containing surface. The adsorption of ATIII increased with increasing heparin on the surface. The effect of multilayer coatings on platelets was tested by incubation of modified vascular prostheses with citrated blood. The most favorable interaction with platelets was observed on the HSA assembly. The interaction of platelets with the surface bearing unfractionated heparin was higher than that of the surface covered with partly depolymerized heparin. The long-term durability of the HSA-heparin coating was proven by a 21-day implantation of coated polyurethane plates in goat heart.

In a continuing search for proteins that target calcium/calmodulin-dependent protein kinase II (CaMKII) to postsynaptic density (PSD) substrates important in synaptic plasticity, we showed that the PSD protein densin-180 binds CaMKII. Four putative splice variants (A-D) of the cytosolic tail of densin-180 are shown to be differentially expressed during brain development. Densin-180 splicing affects CaMKII phosphorylation of specific serine residues. Variants A, B, and D, but not C, bind CaMKII stoichiometrically and with high affinity, mediated by a differentially spliced domain. Densin-180 differs from the previously identified CaMKII-binding protein NR2B in that binding does not strictly require CaMKII autophosphorylation. Binding of densin-180 and NR2B to CaMKII is noncompetitive, indicating different interaction sites on CaMKII. Expression of the membrane-targeted CaMKII-binding domain of densin-180 confers membrane localization to coexpressed CaMKII without requiring calcium mobilization, suggesting that densin-180 plays a role in the constitutive association of CaMKII with PSDs.

The vasopressin (AVP) and oxytocin (OXT) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei (PVN) display reversible structural plasticity of neurons and glial cells under different conditions of neuropeptide secretion. In the present study, we investigated the expression of two immunoglobulin superfamily (IgSF) proteins, Kilon and OBCAM, in the magnocellular neurons by using monoclonal antibodies. Anti-Kilon antibody reacted specifically with the bacterially expressed recombinant Kilon but not with the recombinant OBCAM, and similarly anti-OBCAM antibody specifically recognized the recombinant OBCAM. Western blotting analysis revealed the specific expression of Kilon and OBCAM in the SON homogenates. Although Kilon and OBCAM of the SON homogenates were present as the insoluble form, most Kilon was present in the Triton-insoluble fraction, and OBCAM was localized mainly in the Triton-soluble fraction. Immunocytochemistry revealed Kilon and OBCAM immunoreactivity in the magnocellular neurons of the SON and PVN of the rat hypothalamus compared with outside of the SON and PVN in the hypothalamus. The double-labeling study with confocal microscopy further demonstrated that Kilon immunoreactivity was observed mainly in the dendrites of AVP-secreting neurons and also occasionally OXT-secreting neurons. However, OBCAM immunoreactivity was exclusively seen in the dendrites of AVP-secreting magnocellular neurons. Chronic physiological stimulation by 2% NaCl had no effect on the expression levels of either IgLON protein in the SON. Our study thus demonstrated specific expression of Kilon and OBCAM in the hypothalamic magnocellular neurons, particularly in dendrites, suggesting that they confer on magnocellular neurons the ability to rearrange dendritic connectivity.

Mouse Staufen (mStau) is a double-stranded RNA-binding protein associated with polysomes and the rough endoplasmic reticulum (RER). We describe a novel endogenous isoform of mStau (termed mStau(i)) which has an insertion of six amino acids within dsRBD3, the major double-stranded RNA (dsRNA)-binding domain. With a structural change of the RNA-binding domain, this conserved and widely distributed isoform showed strongly impaired dsRNA-binding ability. In transfected cells, mStau(i) exhibited the same tubulovesicular distribution (RER) as mStau when weakly expressed; however, when overexpressed, mStau(i) was found in large cytoplasmic granules. Markers of the RER colocalized with mStau(i)-containing granules, showing that overexpressed mStau(i) could still be associated with the RER. Cotransfection of mStau(i) with mStau relocalized overexpressed mStau(i) to the reticular RER, suggesting that they can form a complex on the RER and that a balance between these isoforms is important to achieve proper localization. Coimmunoprecipitation demonstrated that the two mStau isoforms are components of the same complex in vivo. Analysis of the immunoprecipitates showed that mStau is a component of an RNA-protein complex and that the association with mStau(i) drastically reduces the RNA content of the complex. We propose that this new isoform, by forming a multiple-isoform complex, regulates the amount of RNA in mStau complexes in mammalian cells.

Within the 5.9-kb DNA region between the tfdR and tfdK genes on the 2,4-dichlorophenoxyacetic acid (2,4-D) catabolic plasmid pJP4 from Ralstonia eutropha JMP134, we identified five open reading frames (ORFs) with significant homology to the genes for chlorocatechol and chlorophenol metabolism (tfdCDEF and tfdB) already present elsewhere on pJP4. The five ORFs were organized and assigned as follows: tfdD(II)C(II)E(II)F(II) and tfdB(II) (in short, the tfd(II) cluster), by analogy to tfdCDEF and tfdB (the tfd(I) cluster). Primer extension analysis of mRNA isolated from 2,4-D-grown R. eutropha JMP134 identified a single transcription start site in front of the first gene of the cluster, tfdD(II), suggesting an operon-like organization for the tfd(II) genes. By expressing each ORF in Escherichia coli, we confirmed that tfdD(II) coded for a chloromuconate cycloisomerase, tfdC(II) coded for a chlorocatechol 1, 2-dioxygenase, tfdE(II) coded for a dienelactone hydrolase, tfdF(II) coded for a maleylacetate reductase, and tfdB(II) coded for a chlorophenol hydroxylase. Dot blot hybridizations of mRNA isolated from R. eutropha JMP134 showed that both tfd(I) and tfd(II) genes are transcribed upon induction with 2,4-D. Thus, the functions encoded by the tfd(II) genes seem to be redundant with respect to those of the tfd(I) cluster. One reason why the tfd(II) genes do not disappear from plasmid pJP4 might be the necessity for keeping the regulatory genes for the 2,4-D pathway expression tfdR and tfdS.

<h4>Background</h4>In patients with Peutz-Jeghers syndrome (PJS), causative germline mutations in the LKB1/STK11 gene on chromosome 19p13.3 have been identified. Because of the loss of heterozygosity (LOH) at 19p13.3 in hamartomas and the cancer susceptibility of patients with PJS, LKB1/STK11 is suggested to act as a tumour suppressor. However, the frequency of genetic and epigenetic inactivation of LKB1/STK11 in sporadic tumours is unclear.<h4>Aims</h4>To investigate the LKB1/STK11 gene for promoter hypermethylation and allelic loss in tumour specimens of patients with sporadic colorectal cancer.<h4>Methods</h4>DNA from 50 consecutive paraffin embedded sporadic colorectal adenocarcinomas and corresponding normal epithelium was extracted. After bisulphite treatment, specimens were analysed for methylation of the LKB1/STK11 promoter 5'-CpG island by methylation specific polymerase chain reaction (MSP). In addition, tumours were analysed for LOH of chromosome 19p13.3. In tumours exhibiting LOH, LKB1/STK11 was sequenced.<h4>Results</h4>MSP was successful in 48 of 50 tumour specimens. Of those, four (8%) demonstrated hypermethylation of the LKB1/STK11 promoter 5'-CpG island. Moreover, LOH at either D19S886 or D19S878 was observed in five of 38 (13%) informative tumours. All five tumours showing LOH at 19p13.3 were advanced and four of five were located in the left sided colon. There was no correlation between LOH and LKB1/STK11 promoter hypermethylation or somatic mutation.<h4>Conclusions</h4>In sporadic colorectal cancer, hypermethylation of the LKB1/STK11 promoter and allelic loss at the STK 11 gene locus are rare events. LOH at 19p13.3 was associated with advanced tumour stage and left sided location but not with LKB1/STK11 promoter hypermethylation or somatic mutation.

The thalamocortical axon (TCA) projection originates in dorsal thalamus, conveys sensory input to the neocortex, and has a critical role in cortical development. We show that the secreted axon guidance molecule netrin-1 acts in vitro as an attractant and growth promoter for dorsal thalamic axons and is required for the proper development of the TCA projection in vivo. As TCAs approach the hypothalamus, they turn laterally into the ventral telencephalon and extend toward the cortex through a population of netrin-1-expressing cells. DCC and neogenin, receptors implicated in mediating the attractant effects of netrin-1, are expressed in dorsal thalamus, whereas unc5h2 and unc5h3, netrin-1 receptors implicated in repulsion, are not. In vitro, dorsal thalamic axons show biased growth toward a source of netrin-1, which can be abolished by netrin-1-blocking antibodies. Netrin-1 also enhances overall axon outgrowth from explants of dorsal thalamus. The biased growth of dorsal thalamic axons toward the internal capsule zone of ventral telencephalic explants is attenuated, but not significantly, by netrin-1-blocking antibodies, suggesting that it releases another attractant activity for TCAs in addition to netrin-1. Analyses of netrin-1 -/- mice reveal that the TCA projection through the ventral telencephalon is disorganized, their pathway is abnormally restricted, and fewer dorsal thalamic axons reach cortex. These findings demonstrate that netrin-1 promotes the growth of TCAs through the ventral telencephalon and cooperates with other guidance cues to control their pathfinding from dorsal thalamus to cortex.

A striking feature of early embryogenesis in a number of organisms is the use of embryonic linker histones or high mobility group proteins in place of somatic histone H1. The transition in chromatin composition towards somatic H1 appears to be correlated with a major increase in transcription at the activation of the zygotic genome. Previous studies have supported the idea that the mouse embryo essentially follows this pattern, with the significant difference that the substitute linker histone might be the differentiation variant H1 degrees, rather than an embryonic variant. We show that histone H1 degrees is not a major linker histone during early mouse development. Instead, somatic H1 was present throughout this period. Though present in mature oocytes, somatic H1 was not found on maternal metaphase II chromatin. Upon formation of pronuclear envelopes, somatic H1 was rapidly incorporated onto maternal and paternal chromatin, and the amount of somatic H1 steadily increased on embryonic chromatin through to the 8-cell stage. Microinjection of somatic H1 into oocytes, and nuclear transfer experiments, demonstrated that factors in the oocyte cytoplasm and the nuclear envelope, played central roles in regulating the loading of H1 onto chromatin. Exchange of H1 from transferred nuclei onto maternal chromatin required breakdown of the nuclear envelope and the extent of exchange was inversely correlated with the developmental advancement of the donor nucleus.

Hypotransferrinemic (Trf(hpx/hpx)) mice have a severe deficiency in serum transferrin (Trf) as the result of a spontaneous mutation linked to the murine Trf locus. They are born alive, but before weaning, die from severe anemia if they are not treated with exogenous Trf or red blood cell transfusions. We have determined the molecular basis of the hpx mutation. It results from a single point mutation, which alters an invariable nucleotide in the splice donor site after exon 16 of the Trf gene. No normal Trf messenger RNA (mRNA) is made from the hpx allele. A small amount of mRNA results from the usage of cryptic splice sites within exon 16. The predominant cryptic splice site produces a Trf mRNA carrying a 27-base pair (bp), in-frame deletion. Less than 1% of normal levels of a Trf-like protein is found in the serum of Trf(hpx/hpx) mice, most likely resulting from translation of the internally deleted mRNA. Despite their severe Trf deficiency, however, Trf(hpx/hpx) mice initially treated with transferrin injections can survive after weaning without any further treatment. They have massive tissue iron overload develop in all nonhematopoietic tissues, while they continue to have severe iron deficiency anemia. Their liver iron burden is 100-fold greater than that of wild-type mice and 15- to 20-fold more than that of mice lacking the hemochromatosis gene, Hfe. Trf(hpx/hpx) mice thus provide an additional model with a defined molecular defect for the study of genetic iron disorders.

Hereditary hemochromatosis (HC) is one of the most common single-gene hereditary diseases. A phenotypic hallmark of HC is low iron in reticuloendothelial cells in spite of body iron overload. Most patients with HC have the same mutation, a change of cysteine at position 282 to tyrosine (C282Y) in the HFE protein. The role of HFE in iron metabolism and the basis for the phenotypic abnormalities of HC are not understood. To clarify the role of HFE in the phenotypic expression of HC, we studied monocytes-macrophages from subjects carrying the C282Y mutation in the HFE protein and clinically expressing HC and transfected them with wild-type HFE by using an attenuated Salmonella typhimurium strain as a gene carrier. The Salmonella system allowed us to deliver genes of interest specifically to monocytes-macrophages with high transduction efficiency. The accumulation of (55)Fe delivered by (55)Fe-Tf was significantly lower in macrophages from patients with HC than from controls expressing wild-type HFE. Transfection of HC macrophages with the HFE gene resulted in a high level of expression of HFE protein at the cell surface. The accumulation of (55)Fe delivered by (55)Fe-Tf was raised by 40% to 60%, and this was reflected by an increase in the (55)Fe-ferritin pool within the HFE-transfected cells. These results suggest that the iron-deficient phenotype of HC macrophages is a direct effect of the HFE mutation, and they demonstrate a role for HFE in the accumulation of iron in these cells.

Metallothioneins (MTs), a group of ubiquitous metalloproteins, comprise isoforms encoded by ten functional genes in humans. Different MT isoforms possibly play different functional roles during development or under various physiological conditions. The MT-1E isoform mRNA has been recently shown to be differentially expressed in oestrogen receptor (OR)-positive and OR-negative breast cancer cell lines. In this study, we evaluated MT-1E mRNA expression via semi-quantitative RT-PCR in 51 primary invasive ductal breast cancer tissues, concurrently with OR-positive and progesterone receptor (PR)-positive MCF7 cells, OR-negative and PR-negative MDA-MB-231 cells and PR-transfected MDA-MB-231 breast cancer cells (ABC28). We demonstrated significantly higher MT-1E mRNA expression in OR-negative compared with OR-positive breast cancer tissues (P = 0.026). MCF7 cells lacked MT-1E mRNA expression, while both OR- and PR-negative MDA-MD-231 cells exhibited a high level of MT-1E mRNA expression. The level of MT-1E mRNA expression in progesterone-treated and -untreated ABC28 cells remained similar as the parental cell line MDA-MB-231-C2 cells. The results suggest that MT-1E may have specific and functional roles in OR-negative invasive ductal breast cancers, possibly mediated via effector genes downstream of the oestrogen receptor, but not through the PR pathway.

Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC(50)) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (DeltaE*) below 1.1 were measured for most dyes.

Identifying translocations of the MLL gene at chromosome band 11q23 is important for the characterization and treatment of leukemia. However, cytogenetic analysis does not always find the translocations and the many partner genes of MLL make molecular detection difficult. We developed cDNA panhandle PCR to identify der(11) transcripts regardless of the partner gene. By reverse transcribing first-strand cDNAs with oligonucleotides containing coding sequence from the 5' MLL breakpoint cluster region at the 5' ends and random hexamers at the 3' ends, known MLL sequence was attached to the unknown partner sequence. This enabled the formation of stem-loop templates with the fusion point of the chimeric transcript in the loop and the use of MLL primers in two-sided PCR. The assay was validated by detection of the known fusion transcript and the transcript from the normal MLL allele in the cell line MV4-11. cDNA panhandle PCR then was used to identify the fusion transcripts in two cases of treatment-related acute myeloid leukemia where the karyotypes were normal and the partner genes unknown. cDNA panhandle PCR revealed a fusion of MLL with AF-10 in one case and a fusion of MLL with ELL in the other. Alternatively spliced transcripts and exon scrambling were detectable by the method. Leukemias with normal karyotypes may contain cryptic translocations of MLL with a variety of partner genes. cDNA panhandle PCR is useful for identifying MLL translocations and determining unknown partner sequences in the fusion transcripts.

<h4>Background</h4>Major depression and suicide are associated with fewer serotonin transporter (5-HTT) sites. The 5'-flanking promoter region of the 5-HTT gene has a biallelic insertion/deletion (5-HTTLPR). We assayed prefrontal cortical (PFC) 5-HTT binding in major depression and suicide and examine the relationship to the 5-HTTLPR allele.<h4>Methods</h4>Postmortem brain samples from 220 individuals were genotyped for the 5-HTTLPR polymorphism. Binding of 5-HTT was assayed by quantitative autoradiography in the PFC of a subset of subjects (n = 159). Clinical information, including DSM-III-R Axis I diagnoses, was obtained by psychological autopsy and medical chart review.<h4>Results</h4>Binding to 5-HTT was lower in the ventral PFC of suicides compared with nonsuicides and was lower throughout the PFC of subjects with a history of major depression. The 5-HTTLPR genotype was associated with major depression but not with suicide or 5-HTT binding.<h4>Conclusions</h4>A diffuse reduction of 5-HTT binding in the PFC of individuals with major depression may reflect a widespread impairment of serotonergic function consistent with the range of psychopathologic features in major depression. The localized reduction in 5-HTT binding in the ventral PFC of suicides may reflect reduced serotonin input to that brain region, underlying the predisposition to act on suicidal thoughts. The 5-HTTLPR genotype was not related to the level of 5-HTT binding and does not explain why 5-HTT binding is lower in major depression or suicide. Arch Gen Psychiatry. 2000;57:729-738

The RET (rearranged during transfection) proto-oncogene encodes a tyrosine kinase receptor involved in both multiple endocrine neoplasia type 2 (MEN 2), an inherited cancer syndrome, and Hirschsprung disease (HSCR), a developmental defect of enteric neurons. We report here that the expression of RET receptor induces apoptosis. This pro-apoptotic effect of RET is inhibited in the presence of its ligand glial cell line-derived neurotrophic factor (GDNF). Furthermore, we present evidence that RET induces apoptosis via its own cleavage by caspases, a phenomenon allowing the liberation/exposure of a pro-apoptotic domain of RET. In addition, we report that Hirschsprung-associated RET mutations impair GDNF control of RET pro-apoptotic activity. These results indicate that HSCR may result from apoptosis of RET-expressing enteric neuroblasts.

<h4>Background & aims</h4>Most hemochromatosis patients of Northern European descent are homozygous for the C282Y mutation of HFE gene. In Italy, many patients with iron overload are not homozygous for C282Y, and the presence of other mutations or other genetic determinant has been suggested.<h4>Methods</h4>Five unrelated Italian patients heterozygous for C282Y with the classic hemochromatosis phenotype were studied. The entire coding sequence and the exon-intron boundaries of the HFE gene were analyzed. Chromosome 6p haplotypes were defined in each patient by analysis of D6S265, D6S105, and D6S1281 microsatellites.<h4>Results</h4>Two novel nonsense HFE mutations were identified in exon 3 in the C282Y negative chromosome. The first one, a G-to-T transition at codon 168, was detected in 3 probands; the second, a G-to-A transition at codon 169, was detected in the others.<h4>Conclusions</h4>The 2 nonsense mutations in the compound heterozygous state with C282Y result in the classic hemochromatosis phenotype in several unrelated Italian patients. This confirms that hemochromatosis in Italy is not as homogeneous as in northern Europe and suggests that other mutations can exist in C282Y or H63D heterozygotes with iron overload. These findings have practical implications for diagnostic and screening strategies for hemochromatosis.

Sox10 is an important transcriptional regulator in the neural crest and various neural-crest derived lineages, such as the Schwann cells of the peripheral nervous system. Recently, we identified the gene for myelin Protein zero (P(0)) as a transcriptional target of Sox10 in Schwann cells, allowing for the first time a detailed analysis of Sox10 responsive elements and their functional interaction with Sox10. Here we show that Sox10 functions through two different types of DNA response elements, one that allows binding of monomers, and a second that favors cooperative binding of two molecules. This dimeric binding required the presence of two heptameric Sox binding sites in a specific orientation and spacing, and was mediated by an N-terminal region of Sox10 with high conservation in the related Sox9, which also exhibited dimeric binding. This argues that the conserved region has the capacity to function as a DNA-dependent dimerization domain. The interaction between Sox10 dimers and DNA differed dramatically from that of Sox10 monomers, as it drastically reduced the protein's off-rate and increased the protein-induced angle of DNA bending. These results indicate that functionally relevant interactions between Sox10 and DNA occur through completely different modes of binding.

DNase I was used to probe the higher order chromatin structure in whole nuclei. The digestion profiles obtained were the result of single-stranded cuts and were independent of pH, type of divalent ion and chromatin repeat length. Furthermore, the protection from digestion of the DNA at the entry/exit points on the nucleosome was found to be caused not by the H1/H5 histone tails, but by the compact structure that these proteins support. In order to resolve symmetry ambiguities, DNase I digestion fragments over several nucleosome repeat lengths were analysed quantitatively and compared with computer simulations using combinations of the experimentally obtained rate constants (some of which were converted to 0 to simulate steric protection from DNase I digestion). A clear picture of precisely defined, alternating, asymmetrically protected nucleosomes emerged. The linker DNA is inside the fibre, while the nucleosomes are positioned above and below a helical path and/or with alternating orientation towards the dyad axis. The dinucleosomal modulation of the digestion patterns comes from alternate protection of cutting sites inside the nucleosome and not from alternating exposure to the enzyme of the linker DNA.

The subset of dendritic cells (DCs) and the nature of the signal inducing DC maturation determine the capacity of DCs to generate polarized immune responses. In this study, we show that the ability of human monocyte-derived DCs (myeloid DC(1)) to promote T helper type 1 (Th1) or Th2 differentiation was also found to be critically dependent on stimulator/responder ratio. At a low ratio (1:300), mature DCs that have been differentiated after inflammatory (Staphylococcus aureus Cowan 1 or lipopolysaccharide) or T cell-dependent (CD40 ligand) stimulation induced naive T cells to become Th2 (interleukin [IL]-4(+), IL-5(+), interferon gamma) effectors. Th2 differentiation was dependent on B7-CD28 costimulation and enhanced by OX40-OX40 ligand interactions. However, high DC/T cell ratio (1:4) favored a mixed Th1/Th2 cell development. Thus, the fact that the same DC lineage stimulates polarized Th1 or Th2 responses may be relevant since it allows the antigen-presenting cells to initiate an appropriate response for the signal received at the peripheral sites. Controlling the number and the rate of DC migration to the T cell areas in lymphoid tissues may be important for the therapeutic use of DCs.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by polyglutamine expansion in the disease protein, huntingtin. In HD patients and transgenic mice, the affected neurons form characteristic ubiquitin-positive nuclear inclusions (NIs). We have established ecdysone-inducible stable mouse Neuro2a cell lines that express truncated N-terminal huntingtin (tNhtt) with different polyglutamine lengths which form both cytoplasmic and nuclear aggregates in a polyglutamine length- and inducer dose-dependent manner. Here we demonstrate that newly synthesized polyglutamine-expanded truncated huntingtin interacts with members of Hsp40 and Hsp70 families of chaperones in a polyglutamine length-dependent manner. Of these interacting chaperones, only Hdj-2 and Hsc70 frequently (Hdj-2 > Hsc70) co-localize with both the aggregates in the cellular model and with the NIs in the brains of HD exon 1 transgenic mice. However, Hdj-2 and Hsc70 do not co-localize with cytoplasmic aggregates in the brains of transgenic mice despite these chaperones being primarily localized in the cytoplasmic compartment. This strongly suggests that the chaperone interaction and their redistribution to the aggregates are two completely different phenomena of the cellular unfolded protein response. This unfolded protein response is also evident from the dramatic induction of Hsp70 on expression of polyglutamine-expanded protein in the cellular model. Transient overexpression of either Hdj-1 or Hsc70 suppresses the aggregate formation; however, suppression efficiency is much higher in Hdj-1 compared with Hsc70. Overexpression of Hdj-1 and Hsc70 is also able to protect cell death caused by polyglutamine-expanded tNhtt and their combination proved to be most effective.

<h4>Background and objectives</h4>Hereditary hemochromatosis (HHC) is a common, recessively inherited, genetic disorder associated with an abnormality of the HFE gene. Subjects homozygous for a point mutation in the gene coding sequence, leading to the amino acid substitution C282Y, are usually affected by the disease. A second point mutation, causing the amino acid substitution H63D, has been described, and compound heterozygotes for the two mutations or homozygotes for the H63D mutation are at risk of developing a milder form of HHC. In populations of northern European origin the C282Y substitution accounts for more than 90% of cases of HHC. In Italy, however, fewer than 70% of patients with HHC are homozygous or compound heterozygous for HFE mutations. Even in the absence of mutations in its coding region, the HFE gene might be involved in the pathogenesis of HHC through inhibition of transcription of the gene or reduced stability of its mRNA.<h4>Design and methods</h4>Since little is known about the regulation of HFE expression, we investigated 17 subjects heterozygous for one of the HFE mutations and with biochemical evidence of iron overload and compared the levels of wild type and mutated mRNAs in their peripheral blood cells. c-DNA regions flanking the mutated codons were amplified by reverse transcriptase polymerase chain reaction (PCR). PCR products derived from the two alleles were differentiated and quantified by digestion with restriction enzymes, electrophoresis in an agarose gel stained with ethidium bromide and densitometric scanning of the gel.<h4>Results</h4>In all cases wild type and mutated mRNAs were expressed at similar levels, suggesting that reduced expression of an HFE allele coding a normal protein is not involved in the pathogenesis of iron overload. However, we can not rule out that a tissue specific regulation of HFE expression in the cells directly involved in iron absorption is altered and contributes to the pathogenesis of the disease. E INTERPRETATION AND CONCLUSIONS: Our results suggest that primary iron overload is a multigenic syndrome; this hypothesis is strongly supported by the recent demonstration that the juvenile hemochromatosis locus maps to human chromosome 1q.

<h4>Background</h4>To clarify the cell adhesion status in ulcerative colitis (UC)-associated colon neoplasm, expression of cell adhesion molecules were investigated and compared with that of sporadic colon neoplasm.<h4>Methods</h4>A total of 14 low grade dysplasias, 16 high grade dysplasias, and 8 adenocarcinomas associated with UC and 17 sporadic adenomas with mild to moderate dysplasia, 22 adenomas with severe dysplasia, and 15 invasive adenocarcinomas were immunohistochemically examined using monoclonal antibodies against CD44, E-cadherin, alpha- and beta-catenin, and deleted colon carcinoma (DCC).<h4>Results</h4>CD44, especially its standard form, and DCC expression was stronger in the sporadic colon neoplasms than in the UC-associated lesions. Although E-cadherin did not show significant differences between the two cases, alpha-catenin was more expressed in sporadic colon adenomas with severe dysplasia and carcinomas than in their UC-associated counterparts. Membranous beta-catenin staining was stronger in UC-associated neoplasms, whereas sporadic lesions had greater cytoplasmic and nuclear expression.<h4>Conclusions</h4>The differences in cell adhesion molecule expression suggests that UC-associated and sporadic colon neoplasms arise from different pathways of tumorigenesis.

The axonal chemoattractant netrin-1 guides spinal commissural axons by activating its receptor DCC (Deleted in Colorectal Cancer). We have found that chemical inhibitors of metalloproteases potentiate netrin-mediated axon outgrowth in vitro. We have also found that DCC is a substrate for metalloprotease-dependent ectodomain shedding, and that the inhibitors block proteolytic processing of DCC and cause an increase in DCC protein levels on axons within spinal cord explants. Thus, potentiation of netrin activity by inhibitors may result from stabilization of DCC on the axons, and proteolytic activity may regulate axon migration by controlling the number of functional extracellular axon guidance receptors.

<h4>Background</h4>Genes involved in the serotonin system are major candidates in association studies of suicidal behavior. In this case-control study we investigated whether the serotonin transporter (5-HTT) gene encoding the protein responsible for the reuptake of serotonin from the synapse after its release from serotonergic neurons is a susceptibility factor for suicidal behavior.<h4>Methods</h4>A functional polymorphism of the 5-HTT gene (a 44-base pair insertion/deletion in the 5-HTT-linked polymorphic region [5-HTTLPR]) was studied in a population of 237 consecutive patients with affective disorder (unipolar or bipolar) and 187 control subjects. Ninety-nine patients had attempted suicide at least once, of whom 26 made a violent attempt.<h4>Results</h4>No association was found between the "s" allele of the 5-HTTLPR and suicide attempt; however, there was a significant difference in allele distributions between patients who had made violent suicide attempts and control subjects.<h4>Conclusions</h4>A genetic variant of the 5-HTT gene may predispose individuals to violent suicidal behavior. The precise phenotype associated with the 5-HTT gene is unclear, and therefore further studies are required to replicate these findings.

In the rodent primary somatosensory cortex, the configuration of whiskers and sinus hairs on the snout and of receptor-dense zones on the paws is topographically represented as discrete modules of layer IV granule cells (barrels) and thalamocortical afferent terminals. The role of neural activity, particularly activity mediated by NMDARs (N-methyl-D-aspartate receptors), in patterning of the somatosensory cortex has been a subject of debate. We have generated mice in which deletion of the NMDAR1 (NR1) gene is restricted to excitatory cortical neurons, and here we show that sensory periphery-related patterns develop normally in the brainstem and thalamic somatosensory relay stations of these mice. In the somatosensory cortex, thalamocortical afferents corresponding to large whiskers form patterns and display critical period plasticity, but their patterning is not as distinct as that seen in the cortex of normal mice. Other thalamocortical patterns corresponding to sinus hairs and digits are mostly absent. The cellular aggregates known as barrels and barrel boundaries do not develop even at sites where thalamocortical afferents cluster. Our findings indicate that cortical NMDARs are essential for the aggregation of layer IV cells into barrels and for development of the full complement of thalamocortical patterns.

A series of 11 novel 3beta-substituted biphenyltropanes was synthesized and evaluated by selective radioligand binding assays for affinity to monoamine transporters. Both 5-HTT potency and selectivity for 5-HTT over DAT was greatest with electron withdrawing group at the 3''-position.

Netrin is a secreted protein that can act as a chemotropic axon guidance cue. Two classes of Netrin receptor, DCC and UNC-5 (refs 6-9), are required for axon guidance and are thought to mediate Netrin signals in growth cones through their cytoplasmic domains. However, in the guidance of Drosophila photoreceptor axons, the DCC orthologue Frazzled is required not in the photoreceptor neurons but instead in their targets, indicating that Frazzled also has a non-cell-autonomous function. Here we show that Frazzled can capture Netrin and 'present' it for recognition by other receptors. Moreover, Frazzled itself is actively localized within the axon through its cytoplasmic domain, and thereby rearranges Netrin protein into a spatial pattern completely different from the pattern of Netrin gene expression. Frazzled-dependent guidance of one pioneer neuron in the central nervous system can be accounted for solely on the basis of this ability of Frazzled to control Netrin distribution, and not by Frazzled signalling. We propose a model of patterning mechanism in which a receptor rearranges secreted ligand molecules, thereby creating positional information for other receptors.

The C282Y mutation of the HFE gene has been reported to be present in most of the patients with hereditary haemochromatosis (HH) of Northern European ancestry. HH affects approximately 1/300 individuals, but it is not evenly distributed in the different European countries. In the present study, polymerase chain reaction (PCR) and restriction-enzyme digestion were used to analyse the frequency of the most important mutation in haemochromatosis (C282Y) in subjects from Majorca (Balearic Islands, Spain) and patients with haemochromatosis. The results were compared with other studies from Spain and Europe. A total of 420 Majorcan chromosomes were analysed and the C282Y mutation was observed at a frequency of 2.62%+/-0.8 (11 heterozygotes: eight men and three women). In the group of hereditary haemochromatosis probands, 13 out of 14 were homozygous for the C282Y mutation. In the distribution of the C282Y mutation, a north-west to south-east cline was detected, supporting the Celtic origin of this mutation.

Olfactomedin-related proteins are secreted glycoproteins with conserved C-terminal motifs. Olfactomedin was originally identified as the major component of the mucus layer that surrounds the chemosensory dendrites of olfactory neurons. Homologues were subsequently found also in other tissues, including the brain and in species ranging from Caenorhabditis elegans to Homo sapiens. Most importantly, the TIGR/myocilin protein, expressed in the eye and associated with the pathogenesis of glaucoma, is an olfactomedin-related protein. The prevalence of olfactomedin-related proteins among species and their identification in different tissues prompted us to investigate whether a gene family exists within a species, specifically Homo sapiens. A GenBank search indeed revealed an entire human gene family of olfactomedin-related proteins with at least five members, designated hOlfA through hOlfD and the TIGR/myocilin protein. hOlfA corresponds to the rat neuronal AMZ protein. Phylogenetic analyses of 18 olfactomedin-related sequences resolved four distinct subfamilies. Among the human proteins, hOlfA and hOlfC, both expressed in brain, are most closely related. Northern blot analyses of 16 human tissues demonstrated highly specific expression patterns: hOlfA is expressed in brain, hOlfB in pancreas and prostate, hOlfC in cerebellum, hOlfD in colon, small intestine and prostate and TIGR/myocilin in heart and skeletal muscle. The link between TIGR/myocilin and ocular hypertension and the expression of several of these proteins in mucus-lined tissues suggest that they play an important role in regulating physical properties of the extracellular environment. Future studies can now assess whether other members of this gene family, like TIGR/myocilin, are also associated with human disease processes.

Hereditary haemochromatosis is one of the most common genetic disorders affecting populations of European ancestry. Isolation of a strong candidate gene, the HFE gene, allows genetic diagnosis in a large number of cases. However, different mutation frequencies have been reported in hereditary haemochromatosis patient populations from various geographic regions. Such data and phenotype-genotype correlations from Swiss patients with hereditary haemochromatosis are lacking. The objective of our study was to determine the frequency of HFE gene mutations in Swiss patients with hereditary haemochromatosis and to describe the clinical phenotype of patients with either a homozygous C282Y mutation or compound heterozygotes. 71 patients with a clinical diagnosis of hereditary haemochromatosis were identified through a questionnaire sent to physicians caring for hereditary haemochromatosis patients. Pertinent clinical data, in particular those reflecting iron body stores, were collected. Genotyping for the C282Y and H63D mutation of the HFE gene was performed. In 90% of the cases a mutation of the HFE gene was found. 86% of the patients were homozygous for the C282Y mutation, 4% were compound heterozygotes for the C282Y and the H63D mutation. Patients with the homozygous C282Y mutation showed a broad phenotypic spectrum that could not be accounted for by age or sex differences only. Our results demonstrate that within the Swiss population genetic testing can also identify the vast majority of patients with hereditary haemochromatosis. However, the diagnosis is not ruled out by a negative genetic test. Furthermore, a broad phenotypic spectrum is associated with the homozygous C282Y mutation in Swiss hereditary haemochromatosis patients. The implications of these findings for planning of widespread genetic screening for hereditary haemochromatosis in the general population are discussed.

Oral squamous carcinogenesis is a multistep process in which multiple genetic events occur that alter the normal functions of oncogenes and tumour suppressor genes. This can result in increased production of growth factors or numbers of cell surface receptors, enhanced intracellular messenger messenger signalling, and/or increased production of transcription factors. In combination with the loss of tumour suppressor activity, this leads to a cell phenotype capable of increased cell proliferation, with loss of cell cohesion, and the ability to infiltrate local tissue and spread to distant sites. Recent advances in the understanding of the molecular control of these various pathways will allow more accurate diagnosis and assessment of prognosis, and might lead the way for more novel approaches to treatment and prevention.

The mechanism that leads to iron overload in hereditary hemochromatosis is not yet fully understood and genes other than HFE may be involved. Nramp2 is an intestinal iron transporter, upregulated by dietary iron deficiency, which also colocalizes with transferrin in recycling endosomes. The purpose of the present study was to analyze the coding region of the Nramp2 gene in 14 hemochromatosis probands which did not carry any HFE mutations on both chromosomes. We confirmed the existence of a polymorphism (1254 T --> C), which presumably is not associated with hereditary hemochromatosis, but we did not find any mutation. On the other hand, we identified 17 splice variants of the Nramp2 mRNA. Eight corresponded to activation of cryptic splicing sequences between exons 3 and 4. They were observed in a majority of hemochromatosis probands and control subjects. This indicates the existence of an important splicing instability in this region. At this stage, the biological significance of these variants is unclear. Our study did not find evidence for the involvement of the Nramp2 gene in hereditary hemochromatosis. The remaining question is whether hemochromatosis probands in our study have iron overload because of environmental factors or due to mutation in gene(s) other than HFE and Nramp2.

A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against alpha1-proteinase inhibitor (human and sheep), inter-alpha-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, alpha2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus

The selective serotonin re-uptake inhibitors (SSRIs) which modulate serotonergic activity are effective in the treatment of serotonin-related mental disorders, such as depression and anxiety. These agents bind to the serotonin transporter (5-HTT) and inhibit its capacity to transport serotonin (5-hydroxytryptamine; 5-HT). A functional polymorphism in the promoter region of 5-HTT (5-HTTLPR) has been described. The insertion variant of this polymorphism (long allele) is associated with higher expression of brain 5-HTT compared to the deletion variant (short allele). An association between the 5-HTTLPR polymorphism and mental disorders has been reported by some, but not all, investigators. In addition, the 5-HTT gene polymorphisms were found to be associated with a better and faster response to SSRIs with or without pindolol augmentation in depressed patients. Further studies are needed to clarify the relationship between the 5-HTT genotype, the susceptibility to mental disorders, the response to serotonergic agents and the side effect profile.

No abstract available.

<h4>Objective</h4>To investigate the antithrombotic mechanism of glycosaminoglycan (GAG) extracted from sea cucumber.<h4>Methods</h4>We studied the effects of GAG on the coagulant pathway by measuring cloting time. The antithrombin mechanism of GAG was checked by assaying its effects on the thrombin activity in normal human pooled plasma, purified human heparin cofactor II system and antithrombin III system. The effects of GAG on the assembly, dispersion, and structure of fibrin gels as well as on the activity of plasmin were studied by means of turbidimetry, electron microscopy, and chromogenic substrate assay. We studied the effect of GAG on the expression and transcription of tissue factor (TF) and thrombomodulin (TM) in LPS (lipopolysaccharide)-stimulated human umbilical vein endothelial cells (HUVECs), and used heparin as a control. HUVECs were treated with different concentrations of GAG (1 microgram/ml, 5 micrograms/ml, and 10 micrograms/ml respectively) and 5 micrograms/ml heparin as a control together with LPS (1 microgram/ml). After incubation for 6 hours, TF and TM were investigated by ELISA and the mRNA study was carried out by RT-PCR. In a clinical trail, a series of variables were observed before and after treatment with GAG in patients recovering from cerebral ischemic stroke or suffering from ischemic heart disease.<h4>Results</h4>The TT and APTT were significantly prolonged by GAG (0.1 microgram/ml). GAG inhibited thrombin activity in the presence of HCII with a second order rate constant of 1.14 x 10(7) m-1.min-1, which was 4.6 times higher than that of ATIII. GAG significantly inhibited the polymerization of fibrin monomer and enhanced the activity of plasmin in a concentration dependent manner. GAG could impair TF mRNA expression and up-regulate TM mRNA expression. The result of clinical trail showed that the fat metabolism was enhanced in addition to the anticoagulant and the blood viscosity reducing effects. No side-effect was found.<h4>Conclusions</h4>GAG mainly affected on the intrinsic pathway of blood coagulation. GAG was similar to dermatan sulfate both in the efficiency and in the mechanism of antithrombin. The acceleration of colt lysis by GAG depended on its ability to increase the activity of plasmin, to inhibit the polymerizing of fibrin monomer, and consequently, to alter the architecture of the fibrin net work. This effect on HUVECs appears to be at a transcriptional level and might be relevant for the antithrombotic action of GAG. GAG possess anticoagulant activity in vivo and it is a promising drug for antithrombotic therapy.

Two different methods to determine steroid receptors were analysed with respect to their ability to estimate prognosis in primary breast cancer patients. The immunohistochemical assay (IHA) was compared with the dextran-coated charcoal (DCC) method of receptor determination. A random sample of 281 patients with invasive ductal carcinoma was drawn from 841 consecutive patients with primary breast carcinoma treated at Odense University Hospital between 1 January 1980 and 31 December 1990. Receptor determination by the DCC method had been carried out previously in 164 patients for the oestrogen receptor and in 132 patients for the progesterone receptor. The former group was reassessed by IHA with the antibody ER1D5, and the latter with the antibody PgR-ICA. The median follow-up time was 8.3 years (range 2.9-12.9 years). A cutoff of zero was used for the DCC method. Immunohistochemical results were quantified by counting in systematically random sampled fields of vision and values above zero were considered to be positive. Overall agreement of positive and negative cases was 86% for the oestrogen receptor and 83% for the progesterone receptor. Although the study included a limited number of patients, receptor positive cases fared better than negative cases in all situations. Investigation of the prognostic power revealed that classification based on IHA allowed better discrimination of patients than classification based on the DCC method. The reason for this difference might be because distinction between benign and malignant tissue is possible using the IHAmethod. Thus, IHAresults appear to be more clinically relevant.

Advanced DNA level studies based on HLA class II sequence analysis have revealed considerable diversity in HLA among Asian Indians. High resolution typing of specific alleles such as DR2 and DR4 in the HLA class II region by PCR-SSP or SSOP hybridization and their associated DR-DQ haplotypes have helped to detect unique haplotypes and novel alleles which have subsequently been confirmed by sequencing. Incidentally, remarkable stability has been maintained in several other DRB1 alleles viz. DR1, DR7, DR9 and DR10. The ARMS-PCR technology has been found to be particularly useful for typing HLA-A, HLA-B and HLA-Cw alleles. These technologies are far superior over serological methods. Our studies have shown remarkable heterogeneity of common HLA-A and B alleles in Asian Indians. Molecular subtyping of HLA-A2 revealed that subtype A(*)0211 is found only in Indian population and may be the result of selection pressure in this population. Investigations into polymorphism in the HLA-B27 gene revealed that subtypes common both to the western caucasians and orientals occur in the Indian population. It is apparent that the population of the Indian subcontinent, placed as it is between the Caucasoids and Negroids on one hand and Australoids and Mongoloids on the other, provides a rich source of many HLA haplotypes. While the most frequent Caucasian haplotypes occur with a reasonable frequency in Asian Indians, those found predominantly in other ethnic groups (e.g., australian Aborigines and populations of Oceania, China and Japan) are also detected. Knowledge on this is most important for donor selection during organ and bone marrow transplantation and for designing MHC targeted vaccines in specific diseases.